WO2020016363A1 - Procédé par voie humide de récupération d'une huile produite par des microorganismes - Google Patents

Procédé par voie humide de récupération d'une huile produite par des microorganismes Download PDF

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Publication number
WO2020016363A1
WO2020016363A1 PCT/EP2019/069383 EP2019069383W WO2020016363A1 WO 2020016363 A1 WO2020016363 A1 WO 2020016363A1 EP 2019069383 W EP2019069383 W EP 2019069383W WO 2020016363 A1 WO2020016363 A1 WO 2020016363A1
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liquid
lipids
solid
fermentation medium
phase containing
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PCT/EP2019/069383
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English (en)
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Sébastien RIFFLART
Maha BAHI
Anthony HUTIN
Fernando Leal-Calderon
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Total Raffinage Chimie
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Publication of WO2020016363A1 publication Critical patent/WO2020016363A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/006—Refining fats or fatty oils by extraction

Definitions

  • the present invention relates to the recovery of oil produced by microorganisms.
  • the invention provides methods for recovering oil produced by a microorganism through a wet process.
  • the invention accordingly relates to the fields of biology, microbiology, fermentation technology and oil and fuel production technology.
  • Lipids for use in biofuels can be produced in microorganisms, such as algae, fungi and bacteria.
  • oleaginous microorganisms including the well characterized yeast Yarrowia lipolytica, produce lipids.
  • Microorganisms synthesize lipids with distinct carbon chain lengths and degrees of unsaturation.
  • the lipid may be excreted by the microorganism or may remain inside the cell, stored in organelles, termed lipid bodies or lipid droplets, as storage lipids.
  • the lipids can be produced as fatty acids, triacylglycerides (TAG) or ester of fatty acids.
  • TAG triacylglycerides
  • the lipid profile of a cell i.e., the relative amounts of fatty acid species that make up the total lipids in the cell, is determined by the activities and substrate specificities of various enzymes that synthesize fatty acids (fatty acid synthase, elongase, desaturase), various enzymes that stabilize fatty acids by incorporating them into storage lipids (acyltransferases), and various enzymes that degrade fatty acids and storage lipids (e.g. lipases).
  • the lipid yield of oleaginous organisms can be increased by the up-regulation, down-regulation, or deletion of genes implicated in a lipid pathway.
  • producing a lipid using a microorganism involves growing microorganisms which are capable of producing a desired lipid in a fermentor or bioreactor, lysing the microorganisms if the lipids are intracellular products, and recovering the lipids produced by breaking the emulsion obtained.
  • US201 1295028A1 discloses processes for obtaining a lipid from a cell by lysing the cell, contacting the cell with a base and/or salt, and separating the lipid.
  • the disclosed processes include raising the pH of the cell composition to 8 or above and separating lipid from the cell composition.
  • the microbial cells suitable are organisms such as algae, bacteria, fungi and protist. Yeast such as Ascomycetes or Basidiomycetes are mentioned.
  • the salt used is selected from alkali metal salts, alkali earth metal salts, sulfate salts and their combination. In the examples, NaCI and Na2(S0 4 )2 salts have been tested on microalgae.
  • lipids could be recovered at pH above 9.5 from an emulsion containing lipids obtained from microorganisms producing mainly C18 and/or C19 fatty acids, in particular oleic acid (C18:1 ).
  • WO2015/095696 discloses processes for obtaining microbial oil from microbial cells by lysing the cells to form a lysed cell composition, treating the lysed cell composition to form an oil-containing emulsion and then recovering the oil from the oil-containing emulsion.
  • the heating to at least 50°C is provided for demulsifying the oil-containing emulsion.
  • the processes disclosed still provide addition of salts and of acid or basis for either lysing or demulsifying. Such addition of chemicals increases considerably the cost of the process and also requires treatment of the waste waters produced, which can be complicated and costly.
  • W02018013670A1 discloses a method for extracting a microbial oil comprising polyunsaturated fatty acids from a ferment broth containing oleaginous microorganism.
  • the fatty acids produced include C18 and C20 fatty acids. No mention of production of C19 fatty acids is made.
  • the method disclosed concerns only oils recovered by a lysing step. To improve demulsification, the method provides a dewatering step before extraction.
  • a typical solventless extraction method involves the following steps : pasteurizing or heating the cell- containing broth; lysing the cells to release microbial oil from the cells to form a lysed cell composition, which is in the from of a solution; treating the lysed cell solution by heat, salt and pH adjustment in order to coalesce the oil droplets and remove emulsion from the solution; centrifuging the demulsified solution.
  • W02015095690A2 discloses processes for obtaining microbial oil from microbial cells without using solvents
  • the fatty acids produced include C18, C20, C22 fatty acids. No mention of production of C19 fatty acids is made.
  • the methods disclosed concern only oils recovered by a lysing step. An emulsifier is added prior to, during or after the lysing of the cells to improve demulsification.
  • a process has here been found able to recover lipids containing mainly one or several fatty acids chosen from C18 and/or C19 fatty acids, without addition of any chemical compound(s), contrarily to the above prior art disclosure.
  • the process disclosed is a solventless extraction process.
  • An object of the present invention is a process for recovering lipids produced by fermentation of microbial cells from a fermentation medium, said lipids comprising mainly one or several fatty acids chosen from C18 and C19 fatty acids, comprising:
  • step (c) demulsifying the fermentation medium of step (a), the lysed fermentation medium of step (b) or the solid/liquid light phase containing lipids of step (b)(i), wherein said fermentation medium of step (a) or solid/liquid light phase containing lipids of step (b)(i) is heated at a temperature from 30 to 80°C under agitation for at least 2 hours, thereby generating a stream having a first phase containing the lipids and a second phase containing water, and
  • step (d) separating the first phase containing the lipids from the stream obtained in step (c),
  • chemical compound include organic and inorganic compound.
  • the present invention is useful for recovering at least 25wt%, 30 wt%, 35wt%, 40 wt%, 45wt%, 50wt%, 55wt%, 60wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt%, 95 wt%, or higher, of lipids as measured by % dry cell weight.
  • 45wt%, 55wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt%, 95 wt% or higher of lipids are recovered.
  • a recovery as high as 100wt%, 99wt%, 98wt%, 97 wt%, 96 wt%, 95 wt% of lipids as measured by % dry cell weight can be obtained by the process of the invention.
  • from 45 to 100wt% or from 60 to 99wt% or from 65 to 99wt% of lipids as measured by % dry cell weight are recovered.
  • the present invention allows recovering lipids comprising mainly C18 and/or C19 fatty acids.
  • “comprising mainly C18 and/or C19 fatty acids” refers to a C18 or C19 or C18+C19 fatty acids proportion of at least 75% or higher as a weight percentage of total fatty acids.
  • the separation step (d) includes a liquid/liquid separation, a solid/liquid separation, or the both.
  • the process further comprises, prior to the demulsification step
  • This separation step can be performed by a solid/liquid separation. This embodiment is particularly advantageous for a process recovering an extracellular production of lipids, in which the lysing step (b) is not performed.
  • the first phase containing the lipids may be separated without addition of chemical compound(s) into a liquid/liquid light phase containing the lipids and a liquid/liquid heavy phase by a liquid/liquid separation.
  • no solid/liquid separation is performed on the fermentation medium or on the lysed fermentation medium. In such a case, in the separation step
  • the first phase containing the lipids may be separated without addition of chemical compound(s) into a solid/liquid light phase containing the lipids and a solid/liquid heavy phase by a solid/liquid separation, and the solid/liquid light phase is separated without addition of chemical compound(s) into a liquid/liquid light phase containing the lipids and a liquid/liquid heavy phase by a liquid/liquid separation.
  • the fermentation medium provided at step (a) is submitted to a dewatering step without addition of chemical compound(s) prior the lysing step.
  • the dewatering step may be a solid/liquid separation step selected from centrifugation, filtration, and decantation.
  • the dewatering step may be a solid/liquid separation step selected from centrifugation or cross flow filtration.
  • cross flow filtration is performed, at least one tubular inorganic membrane having a pore size of 0.5pm at most is used to separate the microbial cells from the fermentation medium or the lysed fermentation medium.
  • the stream obtained in step (c) is heated at a temperature from 30 to 90°C prior to the separation step (d).
  • Figure 1 Schematic representation of a process for the recovery of lipids from a fermentation mixture according to an embodiment of the method disclosed herein.
  • bio-organic compound is meant herein an organic compound that is made by microbial cells, including recombinant microbial cells as well as naturally occurring microbial cells.
  • free bio-organic compound refers to bio-organic compound which is not in an emulsion and forms a continuous phase, usually supernatant.
  • cell refers to a microorganism, capable of being grown in a liquid growth medium.
  • dry weight or“dry matter” means weight determined in the relative absence of water.
  • reference to oleaginous cells as comprising a specified percentage of a particular component by dry weight means that the percentage is calculated based on the weight of the cell after substantially all water has been removed (until constant weight).
  • The“dry cell weight” or“dry cell matter” means weight determined in the relative absence of water after sample washing for insoluble solids removal.
  • microbial cell refers to organisms such as algae, bacteria, fungi, protest and combinations thereof, e.g. unicellular organisms.
  • cell-associated as used herein in connection to fermentation products refers to fermentation products that are associated to the host cell or host cell debris.
  • emulsion generally refers to a mixture of two immiscible liquids, such as water and an oil. As used herein, it particularly refers to a mixture of a bio-organic compound envisaged herein and water.
  • the term“host cell” as used herein refers to a microbial cell which is used for the production of a bio-organic compound.
  • the host cell may be a recombinant cell, which implies that is has been genetically modified to induce or increase the production of the bio-organic compound.
  • the host cell contains a foreign DNA and/or has one or more genetic modifications compared to the wild type organism which affects the production of the bio-organic compound.
  • host cells are microbial cells naturally producing a bio- organic compound of interest.
  • the bio-organic compounds envisaged herein are produced by microbial fermentation.
  • Microbial production of organic compounds is well known in the art, and the invention is applicable to any technique deemed suitable by a skilled person involving microbial fermentation.
  • micro-organisms are cultured under conditions suitable for the production of the organic compounds by the microbial host cells. Suitable conditions include many parameters, such as temperature ranges, levels of aeration, pH and media composition. Each of these conditions, individually and in combination, is typically optimized to allow the host cell to grow and/or to ensure optimal production of the organic compound of interest.
  • Exemplary culture media include broths or gels.
  • the host cells may be grown in a culture medium comprising a carbon source to be used for growth of the host cell.
  • exemplary carbon sources include carbohydrates, such as glucose, fructose, cellulose, or the like, that can be directly metabolized by the host cell.
  • enzymes can be added to the culture medium to facilitate the mobilization (e.g., the depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source.
  • a culture medium may optionally contain further nutrients as required by the particular microbial strain, including inorganic nitrogen sources such as ammonia or ammonium salts like ammonium sulfate, and minerals like phosphates, potassium salts, magnesium salts, manganese salts, iron salts, copper salts, zinc salts, calcium salts or sodium salts .
  • Other growth conditions such as temperature, cell density, and the like are generally selected to provide an economical process. Temperatures during each of the growth phase and the production phase may range from above the freezing temperature of the medium to about 50°C.
  • the fermentation may be conducted aerobically, anaerobically, or substantially anaerobically. Briefly, anaerobic conditions refer to an environment devoid of oxygen.
  • Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation.
  • Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1 % oxygen.
  • the percent of oxygen can be maintained by, for example, sparging the culture with an N2/CO2 mixture or other suitable non- oxygen gas or gasses.
  • the fermentation can be conducted continuously, batch-wise, or some combination thereof. Conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates can be used.
  • this step does not require addition of any chemical compound(s) such as a surfactant, an emulsifier, a solvent, a basic compound, an acid compound.
  • Suitable micro-organisms for fermentation are known in the art.
  • suitable micro-organisms include bacteria such as Escherichia (e.g. £. coli), Bacillus or Lactobacillus species, fungi, in particular yeasts such as Saccharomyces (e.g. S. cerevisiae) species, or algae such as Chlorella species.
  • the microbial host cell is a fungus, preferably a yeast.
  • the micro-organisms may naturally produce the bio-organic compound of interest, or they may have been genetically modified (i.e. recombinant micro-organisms) to ensure production of the bio-organic compound of interest.
  • Suitable micro-organisms for use in the present invention are capable to produce lipids comprising mainly C18 and/or C19 fatty acids; in particular, the micro-organism is capable to excrete (extracellular production) or secrete (intracellular production) the lipids.
  • the microbial cell is selected from the group consisting of algae, bacteria, molds, fungi, plants, yeasts and combination thereof.
  • a microbial cell is an eukaryotic cell, such as a yeast cell, a fungi cell, an algae cell.
  • the microbial cell is a yeast or an algae.
  • suitable cells include, but are not limited to, fungal or yeast species, such as Arxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, and Yarrowia.
  • fungal or yeast species such as Arxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Leucosporidiella, Lipomyces, Mortierella, Ogat
  • the microbial cell is Saccharomyces cerevisiae, Yarrowia lipolytica, or Arxula adeninivorans.
  • the host cell is a microbial cell selected from the genus Escherichia, Bacillus, Lactobacillus, Pantoea, Zymomonas, Rhodococcus, Pseudomonas, Chroococcales, Chroococcidiopsidales, Chroococcidiopsidales, Chroococcidiopsidales, Gloeobacterales, Nostocales, Oscillatoriales, Pleurocapsales, Spirulinales, Synechococcales, Incertae sedis.
  • the microbial cell comprises at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, or more lipid as measured by % dry cell weight.
  • Example of suitable genetically modified cells include, but are not limited to, the cells obtained by the processes disclosed in WO2016/094520 A1 or WO2016/014900 A2 (both documents incorporated therein by reference).
  • the bio-organic compound envisaged therein is a lipid containing mainly C18 and/or C19 fatty acids.
  • the lipid may also contain other fatty acids such as C16 fatty acidsln some embodiments, the proportion of C18 and C19 fatty acids is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, or higher as a weight percentage of total fatty acids produced by the microbial cell.
  • the lipids produced contain mainly C18 fatty acids.
  • the proportion of C18 fatty acids is at least 75wt% or higher as a weight percentage of total fatty acids produced by the microbial cells.
  • the proportion of C18 fatty acids is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, or higher as a weight percentage of total fatty acids produced by the microbial cell.
  • the proportion of C18 fatty acids is at least 85% or higher as a weight percentage of total fatty acids produced by the microbial cell.
  • C18 fatty acids include oleic acid.
  • the microbial cell produces oleic acid at a concentration of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%,
  • the proportion of C19 fatty acids is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, or higher as a weight percentage of total fatty acids produced by the microbial cell.
  • C19 fatty acids include 10-methyloctadecanoic acid.
  • a fermentation medium also referred to herein as fermentation mixture, fermentor broth or whole cell broth (WCB)
  • WBC whole cell broth
  • bio-organic compounds can be intracellular products or can be released by the micro-organisms in the culture medium from which they may be recovered.
  • Emulsion formation can be promoted in the fermentation medium by the mechanical energy from fermentation (e.g. from agitators or fermentation gases produced by the microbial host cells), or by the microbial host cells or various bio-molecules therein.
  • the mechanical energy from fermentation e.g. from agitators or fermentation gases produced by the microbial host cells
  • the microbial host cells or various bio-molecules therein In the case of excreted bio-organic compounds, formation of an emulsion is observed along with production of bio-organic compounds.
  • emulsion can occur in the recovery process once the cells are lysed and the bio-compounds freed into the fermentation medium.
  • Emulsion formation which is inherent to microbial production systems, is a source of loss of bio-organic compounds to recover.
  • recovery processes for bio-organic compounds from a fermentation medium characterized in that they comprise the recovery of said bio- organic compounds comprised in emulsions without addition of any chemical compound(s) in particular for lysing if required, for demulsification and for separation of the lipids from the demulsified medium.
  • the invention comprises methods for the (improved) recovery of lipids containing mainly C18 and/or C19 fatty acids from a fermentation mixture or a lysed fermentation mixture, which methods comprise recovering the lipids which are present in the fermentation medium in emulsions without addition of any chemical compound(s) in particular for lysing if required, for demulsification and for separation of the lipids from the demulsified medium.
  • the objective of this step is to reduce the moisture content of the fermentation medium provided by step (a), prior a lysing step, thereby generating a dewatered fermentation medium. In other words, this step performs a concentration of the fermentation medium.
  • This step applies for intracellular production of bio-organic compounds. This step does not require addition of any chemical compound(s).
  • This step also generates a water stream which can be used for the fermentation.
  • this dewatering step is a solid/liquid separation step.
  • the solid/liquid separation of the fermentation mixture may be achieved by well-known techniques, including, without limitation, centrifugation, filtration, and decantation, preferably by centrifugation and filtration, in particular cross flow filtration.
  • a centrifuge can separate the fermentation mixture in batch or on a continuous flow basis. Preferably continuous flow centrifugation is used in the methods described herein.
  • a non-limiting example of a centrifuge suitable for solid/liquid separation of a fermentation mixture as taught herein is a disk stack centrifuge, such as a disk stack centrifuge with nozzles. Centrifugation conditions can be suitably determined by the skilled person to achieve the desired solid/liquid separation.
  • Tangential flow filtration also known as cross-flow filtration
  • TFF Tangential flow filtration
  • membrane system(s) membrane system(s) and flow force to purify solids from liquids.
  • TFF generates a retentate and a permeate.
  • the dewatered fermentation medium is recovered as the retentate.
  • the retentate should not pass through the membrane system(s) at a significant level.
  • the retentate also should not adhere significantly to the membrane system(s) material.
  • the dewatering step is performed by cross flow filtration using one or more tubular inorganic membranes, in other words, non-organic membrane(s).
  • the inorganic membrane is a ceramic membrane.
  • Non-limiting examples of tangential flow filtration include those involving the use of a membrane with a pore size of at most 0.5 micrometer, at most 0.4 micrometer, at most 0.3 micrometer, at most 0.2 micrometer, at most 0.18 micrometer.
  • the pore size of the membrane is of 0.1 micrometer or more.
  • the pore size may be within a range defined by any combination of the above limits. Preferred pore sizes of TFF allow solutes and debris in the fermentation broth to flow through, but not microbial cells.
  • a membrane has at least one channel of tubular shape.
  • the surface of the membrane in contact with the fluid has an active layer which determines the porosity of the membrane.
  • the active layer is full of holes, the diameters of which do not allow the pass through of the microbial cells.
  • a membrane with a multi-layer configuration for filtration of a medium is provided with at least one first layer which is a carrier layer and a second layer which is a separation layer that filters the medium and generates a retentate and a permeate.
  • the separation layer is a ceramic or carbon material, in other word a non organic material.
  • the inorganic material of the membrane is a ceramic.
  • the inorganic material may be selected from titanium oxide (Ti0 2 ), zirconium oxide (Zr0 2 ), alumina (Al 2 0 3 ), silicon carbide (SiC), boron carbide (B 4 C), silicon nitride (Si 3 N 4 ), aluminium nitride (AIN), boron nitride (BN), agglomerated carbone or mixtures thereof.
  • the material of the membrane is selected from titanium oxide (Ti0 2 ), zirconium oxide (Zr0 2 ), alumina (Al 2 0 3 ).
  • conditions of filtrations are chosen to reduce the moisture content of the fermentation medium so as to obtain a dewatered fermentation medium having a moisture content of 80%wt or less, of 78%wt or less, of 75%wt or less, or of 70%wt or less or even of 65%wt or less.
  • Those conditions will be determined by the man skilled in the art by controlling the moisture content while monitoring one or several of the following parameters: the transmembrane pressure (TMP), the differential pressure, the feed flowrate, the cross flow velocity.
  • TMP transmembrane pressure
  • the moisture content / dry matter can be measured by monitoring the weight in function of time at a temperature suitable for water evaporation (e.g. 100-1 10°C) until the weight is constant.
  • the at least one tubular inorganic membrane can be provided in a filtration module.
  • a filtration module may comprise one or more tubular inorganic membranes, each membrane may itself comprise several tubular channels arranged in a bundle.
  • membranes having from 4 to 10 tubular channels may be used, but the invention is not limited by a number of tubular channels in a tubular membrane or by the number of the tubular membranes used.
  • the man skilled in the art knows how to choose the number of membranes and tubular channels in the membrane used depending on the filtration surface required to perform the filtration.
  • Several filtration modules may be provided, some of which being cleaned so as to restore their permeability while the others are used for filtration. A continuous treatment of the dewatered broth can then be performed.
  • the cleaning treatment to recover some or all of the initial permeability of the membrane(s) may include one or several of the following actions: flushing the membranes with water, rinsing the membrane(s) with water, washing the membrane(s) using alkaline and/or acid solution.
  • the temperature is raised during the cleaning treatment, for example up to 50°C.
  • Such lysing step is performed when the lipids are intracellular products. Lysing may be performed on the fermentation medium obtained from the fermentor. In a preferred embodiment, the lysing step is performed on a dewatered fermentation medium.
  • the lysing step which is a widely established step in the extraction of bio-organic compounds from fermentation medium, ruptures the cell wall and/or cell membrane of a cell to release their cytoplasmic content into the fermentation medium.
  • a lysed fermentation medium is obtained by lysing the microbial cells contained in the fermentation medium or the dewatered fermentation medium.
  • Such lysed fermentation medium comprises lysed cells, including cell debris, lipids (initially comprised in the cells), other natural contents of the cells and optionally, aqueous components from the fermentation broth.
  • lysing is performed by mechanically treating without addition of chemical compound(s).
  • mechanically treating includes, but is not limited to, homogenizing a cell, applying ultrasound to a cell, cold-pressing a cell, milling a cell or the like, and combinations thereof.
  • Mechanical devices for treating a cell can include, but is not limited to, processes utilizing a French pressure cell press, a sonicator, a homogenizer, a ball mill, a rod mill, a pebble mill, a bead mill, a high pressure grinding roll, a vertical shaft impactor, an industrial blender, a high shear mixer, a paddle mixer, a polytron homogenizer or the like, and combinations thereof.
  • lysing is performed by milling, in particular using a bead or ball mill, for example an agitator bead or ball mill or an accelerator bead or ball mill, or using a homogenizer.
  • An agitator bead or ball mill has separating system formed from a separating component which has two circular discs co-axial to the chamber axis, and between which are installed several transporting or vane elements distributed symmetrically around the disc center point and leading inwards from the disc edge.
  • the transporting or vane elements create a back pressure on the material and milling body mix so by centrifugal force and different specific densities the milling bodies are separated from the product and transported back to the inner chamber of the mill.
  • a suitable agitator bead or ball mill is for example described in EP1468739.
  • An accelerator bead or ball mill has a milling chamber and an agitator having a rotatably mounted and driven agitator shaft and accelerators arranged thereupon.
  • a suitable agitator bead or ball mill is disclosed in W020101 12274A1 , its accelerator that is arranged furthest downstream, that is, the accelerator closest to the milling material outlet, lengthens axially and extends along the axial length of the sieve.
  • lysing is performed by milling using an accelerator bead or ball mill, in particular an accelerator bead mill.
  • the lysed fermentation medium resulting from the lysing step (b) may then be submitted to a solid/liquid separation step prior to the demulsification step.
  • the solid/liquid light phase has a dry matter content from 25 to 50wt% or from 30 to 45wt%. The dry matter content may be within a range defined by any combination of these limits.
  • this separation is performed by a solid/liquid separation.
  • This optional separation step separates the fermentation medium or the lysed fermentation medium into a solid/liquid light phase containing the lipids and a solid/liquid heavy phase containing the microbial cells.
  • the dewatering step is not necessary.
  • the solid/liquid light phase obtained is submitted directly to the demulsification step (c).
  • the solid/liquid separation step which is a widely established step in the extraction of bio-organic compounds from fermentation medium, separates the micro- organisms from the fermentation mixture.
  • the stream comprising the micro- organisms is also referred to herein as“microbial pellet”,“cell slurry”,“solid/liquid underflow”,“solid/liquid centrifuge waste stream” or“solid/liquid heavy phase”.
  • This stream comprises the micro-organisms and cell-associated bio-organic compounds, and may further comprise host cell debris, culture medium and some bio-organic compounds.
  • the solid/liquid heavy phase is preferably a liquid stream.
  • the generally supernatant or light phase obtained by solid/liquid separation of the fermentation mixture also referred to herein as“solid/liquid light phase”, comprises the culture medium, free bio-organic compounds and bio-organic compounds comprised in an emulsion, and may further comprise host cell debris.
  • the solid/liquid separation of the fermentation mixture may be achieved by well-known techniques, including, without limitation, centrifugation, filtration, and decantation, preferably by centrifugation.
  • the fermentation medium of step (a), the lysed fermentation medium of step (b) or the solid/liquid light phase containing lipids of step (b)(i) can be submitted to an aging step of at least 48 hours, without agitation. This step does not require either addition of chemical compound(s).
  • the aging step may last from 48 hours to 5 days.
  • the aging step is preferably performed at a positive ambient temperature, for example from 0°C to 40°C depending on the season.
  • the aging step may be performed at a temperature from 0 to 40°C or from 0 to 30°C. Such aging step is therefore performed without any heating.
  • This aging step is not a pasteurization step in which heat is applied to inactivate undesirable enzymes such as the ones that might degrade the oil.
  • the fermentation medium of step (a), the lysed fermentation medium of step (b) or the solid/liquid light phase containing lipids of step (b)(i) is left at rest, without any heating, agitation and addition of chemical compounds.
  • the demulsification step generates a stream having a first phase containing the lipids and a second phase containing water by breaking the emulsions containing the lipids previously formed.
  • the breaking of the emulsions is performed without addition of any chemical compound(s), thus without addition of any surfactant compounds, salt, alkaline compound, acid compound.
  • any surfactant compounds, salt, alkaline compound, acid compound there is no need to control the pH of the emulsion.
  • the fermentation medium of step (a), or solid/liquid light phase containing lipids of step (b)(i) is heated for demulsifying.
  • the heating is from 30 to 80°C or from 35°C to 70 °C or from 40°C to 60°C, or within a range defined by any combination of these limits.
  • Such heating at any of the above mentioned temperature ranges is performed under agitation for at least 2 hours, preferably for at least 3 hours.
  • heating may be performed from 2 hours to 24 hours or from 3 hours to 23 hours or within a time range defined by any combination of these limits.
  • agitating and“agitation” refer to a process to impart motion within a medium through application of a force.
  • the agitation may be performed by stirring, mixing, blending, shaking, vibrating or combination thereof.
  • the agitation may be performed by a rotational stirring, an impeller or a spiral stirrer.
  • impellers examples include propellers with blades, propellers with blades and counter blades, propellers with blades inside a guide tube.
  • Counter blades are usually fixed at a predetermined distance of the walls of a tank, whereas the blades rotate around a central axis of the tank.
  • the rotation speed, and eventually the features of impellers to use, can be determined by the man skilled in the art by monitoring the quantity of oil recovered.
  • Demulsification step may be performed in a container, for example in a container equipped with heating means.
  • heating means include, without limitation, a double wall for hot fluid circulation, double wall containing electric heating, impellers with heating blades.
  • a stream having a first phase containing the lipids and a second phase containing the water is generated.
  • the first phase is supernatant and the second phase is heavier than the supernatant phase.
  • the second phase may also contain cell debris and/or some left over lipids.
  • the demulsified stream may contain: an oil light phase containing the lipids (generally supernatant phase) and, in the second phase (generally heavier than the first phase), eventually an emulsion phase, a heavy phase containing mainly water, and eventually, at the bottom, deposits (debris if any and dead cells).
  • the second phase may be recycled until it contains no more left over lipids, such recycle being send to any step prior to the demulsification step or at the beginning of the demulsification step.
  • Optional heating step
  • the separation step (d) is preceded by a heating step in which the stream generated by the demulsifying step is heated at a temperature from 30 to 90°C, or from 30 to 85 °C or from 40 to 70°C to improve the oil recovery.
  • the temperature may be within a range defined by any combination of the above limits. This step does not require addition of any chemical compound(s).
  • the heating is performed on the stream generated by the demulsifying step. This heating can be performed by passing the stream in a heat exchanger. Separation step (d)
  • This step allows separation of the first phase containing the lipids from the stream generated at the demulsifying step, eventually heated by the optional heating step, and thus the recovery of the lipids.
  • This step does not require addition of any chemical compound(s).
  • This separation step (d) may include a solid/liquid separation, a liquid/liquid separation, or a solid/liquid separation followed by a liquid/liquid separation.
  • the first phase containing the lipids is separated by a liquid- liquid separation.
  • the liquid/liquid separation step which is a widely established step in the extraction of bio-organic compounds, separates the bio-organic compound from the second phase.
  • the light phase obtained comprising the bio-organic compound is also referred to herein as“crude”,“supernatant phase” or“liquid/liquid light phase”.
  • This stream comprises the bio-organic compounds produced by the fermentation and may further comprise some dead cells.
  • the heavy phase recovered also referred to herein as“phase heavier than the supernatant phase“ or“liquid/liquid heavy phase”, comprises the culture medium, dead cells and may further comprise host-cell debris, eventually some free bio-organic compounds and bio-organic compounds comprised in an emulsion.
  • a further solid phase may be obtained, also referred to herein as“discharged phase”,“discharge composition” which comprises the culture medium, host-cell debris, dead cells and may further comprise free bio-organic compounds and bio-organic compounds comprised in an emulsion.
  • the liquid/liquid separation may be achieved by well-known techniques, including, without limitation, centrifugation, filtration, and decantation, preferably centrifugation.
  • a centrifuge can separate liquid/liquid light phase in batch or on a continuous flow basis. Preferably continuous flow centrifugation is used in the methods described herein.
  • a non-limiting example of a centrifuge suitable for liquid/liquid separation of a fermentation mixture as taught herein is a disk stack centrifuge. Centrifugation conditions can be suitably determined by the skilled person to achieve the desired liquid/liquid separation.
  • the separation step (d) is a liquid/liquid separation
  • the fermentation medium or the lysed fermentation has undergone a solid/liquid separation into a solid/liquid light phase containing the lipids and a solid/liquid heavy phase containing the microbial cells, and the solid/liquid light phase is further submitted to the demulsification step (c).
  • the first phase containing the lipids is separated by a solid/liquid separation followed by a liquid/liquid separation.
  • This solid/liquid separation may be performed as described above for the optional solid/liquid separation. This is particularly advantageous when the fermentation medium or the lysed fermentation medium has not been submitted to a solid/liquid separation prior to the demulsification step (c).
  • the first phase containing the lipids is separated into a solid/liquid light phase containing the lipids and a solid/liquid heavy phase by a solid/liquid separation, and the solid/liquid light phase is separated into a liquid/liquid light phase containing the lipids and a liquid/liquid heavy phase.
  • the heavy phases recovered by the solid/liquid and/or liquid/liquid separations can be recycled in the process partly or completely.
  • the heavy phase of the liquid/liquid separation which is aqueous can be submitted, partly or completely, to a chemical or coalescence treatment in a further step (e) to recover residual lipids droplets and then recycled into the fermentor of the fermentation step.
  • the heavy phase of the solid/liquid separation can be recycled partly or completely to the optional lysing step and/or submitted to a solvent extraction for more lipids recovery, in a further step (f).
  • Example 1 description of the process according to an embodiment
  • FIG. 1 shows a schematic representation of a process for the recovery of lipids from a fermentation mixture according to an embodiment of the present invention.
  • a fermentation mixture whole cell broth, stream #1 ) prepared in a bioreactor 1 10 is fed into in a dewatering device 1 12, such as a centrifuge or a cross flow filtration device for reduction of its moisture.
  • the permeate (stream #3) obtained is discarded or preferably recycled partly or in totality in the process, for example in the bioreactor 1 10.
  • the retentate dewatered fermentation medium-stream #2
  • the lysed fermentation medium (stream #4) is then separated in a separation solid-liquid separation device 1 16 into a solid/liquid light phase (stream #5) and a solid/liquid heavy phase (stream #6).
  • the solid/liquid light phase (stream #5) containing the lipids is then demulsified by heating at a temperature from 30 to 80°C under agitation for at least 2 hours.
  • this demulsification is performed in a device 1 17, such as a tank, reactor or similar, equipped with an agitating element (not represented) and a heating device (not represented).
  • the stream #7 generated is then separated in a liquid-liquid separation device 1 18 into a liquid/liquid light phase (stream #8 crude containing the lipids) and a liquid/liquid heavy phase (stream #9). The process thus allows recovering the lipids (stream #7).
  • the light phase (crude containing most of the lipids, stream #8) can be further treated. It can for example be submitted to a polishing centrifuge to remove some dead cells and dissolved lipids. The lipid stream obtained may then be purified appropriate treatments such as refining, bleaching and deodorization. These treatments are usual and not described herein. The lipid stream may further be submitted to chemical reactions.
  • the liquid/liquid heavy phase #9 which is an aqueous phase can be recycled partly or in totality in the process, for example in the bioreactor 1 10.
  • the lysed fermentation medium #4 or the solid/liquid light phase containing lipids #5 can be optionally submitted to an aging step without addition of chemical compound(s).
  • Such aging step can be performed in a dedicated tank for example placed between devices 1 14 and 1 16 or between devices 1 16 and 1 17. This aging step can also be performed in the tank 1 17 used for demulsification.
  • the solid/liquid heavy phase #6 may be recycled to the lysed tank 1 14 or may be submitted to a further treatment such as a solvent extraction using a suitable solvent, such as alkanes, alcohols, in particular anhydrous alcohols, aromatic compounds, chlorinated compounds, ethers, ketones, esters, aldehyde, sulfides.
  • a suitable solvent such as alkanes, alcohols, in particular anhydrous alcohols, aromatic compounds, chlorinated compounds, ethers, ketones, esters, aldehyde, sulfides.
  • the process described in figure 1 is particularly adapted for intracellular production of lipids. In case of extracellular production, the dewatering and lysing steps (devices 1 12 and 1 14) should be omitted.
  • Example 2 Tangential flow filtration test.
  • a 20 L broth of genetically modified Yarrowia lipolytica has been fermented in a bioreactor for 5 days.
  • lipid content has been determined by gravimetric analysis of extracted lipids. Lipids have been extracted by lyophilisation until weight stabilization followed by disruption and then solvent extraction in hexane.
  • the dry matter (DM) content has been measured by gravimetric analysis until weight stabilization,
  • the moisture content has been determined from the dry matter content
  • the turbidity has been measured by a turbidimeter which sends a beam of light through a water sample and measures the amount of light passing through the water in relation to the amount of light that is reflected by the particles in the water, The density at 20°C using an oscillating tube density meter,
  • the fatty acids by reacting the fatty acids with methanol and heptadecanoic acid or tridecanoic acid as internal standard, to form methyl esters of the fatty acid to measure and the ester of heptadecanoic acid or of tridecanoic acid, followed by gas chromatography analysis of the esters using the ester of heptadecanoic acid or of tridecanoic acid as internal standard.
  • the features of the broth are collected in the below table 1.
  • the fatty acids content of the broth is given below in table 2.
  • Membranes INSIDE CeRAMTM from Tami Industries have been tested, both of which have an external diameter of 10mm and a length of 250mm. Their properties are presented in the below table 3.
  • the retentate flowrate is determined by volume sampling (retentate weight sampled during a determined period of time); this measurement has been performed with water at a determined set of parameters (of the pump and the back pressure valve), before the tests and it has been performed for different set of parameters during the tests.
  • the permeate flowrate is measured, either by a balance (and recorded with time) for the tests with permeate production, or by volume sampling (permeate weight sampled during a determined period of time) for the tests with no permeate production (recycling of permeate; variations vs time or operating conditions).
  • the permeate flowrate decreases a lot (the test is stopped before reaching a MCF of 2,5): internal fouling have probably occurred. Moreover, the permeate is not clear at the beginning (as shown by TSS values).
  • the water content for the retentate is 80.9wt%.
  • the permeate flowrate is steady during the increase of MCF (until 2,5) and the permeate has a good quality (TSS equal to 0).
  • TSS good quality
  • the final DM of retentate reaches 23,3wt% which corresponds to a water content of 76.7 %wt.
  • the results show fouling of the membranes leading to a loss of permeability.
  • a washing step allows recovering the permeability.
  • An alkaline washing enables to recover 67% of the initial water permeability.
  • Ultrasil P1 1 is a chlorinated, powdery alkaline detergent composed of emulsifiers, degreasers and surfactants for the cleaning of membrane systems.
  • a broth containing Yarrowia Lipolytica cells has been prepared in a fermentor.
  • the cell used is a genetically modified cell, which produces oil having a typical composition shown in the below table 1.
  • Table 6 Typical composition of oil produced by Yarrowia Lipolytica genetically modified
  • the broth has been dewatered to obtain a dewatered broth B1.
  • the dewatered broth B1 corresponds to stream #2 in figure 1.
  • this dewatered broth B1 has been submitted to a cell disruption by bead milling in a DYNO-MILL MULTI LAB®, in the following conditions :
  • LC1 A lysed cell composition LC1 is obtained.
  • LC1 corresponds to stream #4 in figure 1.
  • LC1 lysed cell composition
  • LC2 corresponds to stream #5 in figure 1.
  • the dewatered broth B1 has been submitted to a cell disruption by bead milling in a DYNO-MILL MULTI LAB®, in the following conditions :
  • a lysed cell composition LC’1 is obtained.
  • the oil recovery yield (wt%) is calculated as the ratio between free oil (supernatant oil) weight (g) and total oil weight in the emulsion (g).
  • the total oil weight in the emulsion is obtained by assuming that the oil accounts for all the dry matter of the emulsion, even when the emulsion contains debris. Real yields are therefore higher than calculated yields.
  • the measured error on the yields values is about 5%.
  • the oil recovery yield has been calculated from measured volume, considering an oil density of about 912g/L at 20°C.
  • each of the samples has been centrifuged at 4500g (g being the earth gravity constant) at 25°C for 10 min using a Rotanta 460 RF Hettich centrifuge, before measuring the oil recovery yield.
  • Example 3 comparison of lysing using bead mill
  • the dewatered broth B1 has been submitted to a cell disruption by bead milling in a DYNO-MILL MULTI LAB®, with two different milling tools, as shown in table 7.
  • Milling tool of process#1 is an agitator bead mill whereas the milling tool of process #2 is an accelerator bead mill.
  • the lysed cells have been submitted to a liquid-solid separation using centrifuge Rotanta 460 RF Hettich at 4600 G for 10 min.
  • Three different phases are observed from up to down: a cream, an aqueous phase and a debris phase.
  • the oil titer of the different phases is measured, which makes it possible to quantify the oil distribution from the volume of each phase.
  • the lysis with process#2 allows recovering about 6 wt% more oil than the process #1. This extra-recovered oil comes from the debris phase. Whatever the process, the amount of oil in the aqueous phase remains the same.
  • a batch of LC2 (without cell debris) has been separated in several samples for which the pH has been adjusted at different pH ranging from 4.5 to 1 1 by addition of appropriate amounts of NaOH.
  • Each of the samples has been centrifuged at 4500g (g being the earth gravity constant) for 10 min or heated at 50 °C for at least 60 min and then centrifuged at 4500g, for 10min, using a Rotanta 460 RF Hettich centrifuge.
  • the characteristics of the tested LC2 emulsion are the following :
  • Mean diameter of oil droplets 3 microns (size range : 0.5 to 20 microns) - Dry matter : 40wt% (wt% of solid, corresponds to the wt% of oil)
  • LC2 has been separated into several samples for which the pH has been set to 8 and 9 by addition of an appropriate amount of NaOH.
  • LC2 has the same characteristics as in example 4.
  • TA1 Sorbitan monoleate, a non-ionic surfactant commercialized under the name Span® 80.
  • TA1 is an oil-soluble surfactant. It dissolves in organic solvents including ethanol, toluene and xylene. TA1 is dispersible in water.
  • TA2 is a polyether polyol (non-ionic surfactant) with a molecular weight of 2750g/mol, commercialized under the name TERGITOLTM L-62.
  • TA2 has a large hydrophobic section surrounded by two hydrophilic sections.
  • TA2 is soluble in water and soluble in organic solvents, including ethanol, toluene and xylene.
  • TA3 Alkyldiphenyloxide disulfonate, anionic surfactant, commercialized under the name of DOWFAXTM-2A1. This compound comprises two sulfonated aromatic rings linked by an ether function. Two C6 to C16 hydrocarbon chains are branched on these aromatic rings. At least 20% of the chains are C16 chains.
  • TA3 is a water- soluble surfactant. TA3 is highly soluble in strong acid and alkali solutions.
  • Each of the samples has been mixed for two hours using a magnetic bar and then centrifuged at 4500g for 10min or heated at 50°C for 60 to 120 min and centrifuged at 4500g, for 10min, using a Rotanta 460 RF Hettich centrifuge.
  • LC2 has been separated into several samples for which the pH has been set to 8 and 9 by addition of an appropriate amount of NaOH.
  • LC2 has the same characteristics as in example 4.
  • Table 12 collects the results obtained for different salts with a pH set at 8 by addition of NaOH.
  • the salt concentration is 0.01 mol/L
  • Each of the samples has been mixed for two hours using a magnetic bar and then centrifuged at 4500g for 10 min, using a Rotanta 460 RF Hettich centrifuge.
  • Salts with divalent cations, CaCI 2 and MgCI 2 are the most efficient salts for oil recovery.
  • LC2 has been separated into several samples for which the pH has been set to 8 or 9 by addition of an appropriate amount of NaOH.
  • LC2 has the same characteristics as in example 4.
  • the oil has been recovered after mixing with a magnetic bar for 2 hours followed by centrifugation at 4500g, for 10min, optionally after heating at 50°C for at least 60 min.
  • the oil yields recovered are shown in table 13.
  • Table 13 effects of pH, salts and surfactant TA3 on oil recovery yield (wt%)
  • the characteristics of the tested LC2 emulsion are the following:
  • Mean diameter of oil droplets 4 microns (size range : 0.5 to 20 microns)
  • Dry matter 31wt%(wt% of solid, corresponds to the wt% of oil)
  • LC’1 samples are kept cold at 4°C in order to be used later for other measurements.
  • a batch of LC’1 has been separated in several samples, 30ml_ of LC’1 in 50ml_ Falcon centrifugation tubes. These tubes have been submitted to a rotational stirring using a Stuart Rotator SB3 at three different rotation speeds 10; 20 and 40 rpm at room temperature (between 20 and 25°C).
  • Table 15 and table 16 show the difference in oil recovery yields between samples stored less than 2 days and more than 4 days.
  • Table 15 effects of rotation speed on a freshly lysed sample, stored less than 2 days at 4 ° C.
  • Table 16 effects of rotation speed on a lysed sample, stored more than 4 days at 4 °C).
  • a batch of LC’1 has been filled in a tank equipped with a spiral stirrer.
  • the spiral stirrer (R 3003.1 , IKA) has been used at 1 m/s.
  • the ratio D/T of the diameter of the stirrer (D) to the width of the tank (T) is of 0.9.
  • the ratio H/T of the height (H) of liquid to the width of the tank (T) is equal to 1.
  • Table 17 Effects of aging for lysates stored without agitation at 2 different temperatures on oil recovery yield (wt%) after a stirring at 20°C for 6 hours
  • Example 10 Effect of time, temperature and agitation on oil recovery
  • Table 18 Oil recovery yields (in wt%) as a function of time, agitation and temperature on freshly lysed samples.
  • a batch of LC’1 has been separated in several samples, 30ml_ of LC’1 in 50ml_ Falcon centrifugation tubes. These tubes have been submitted to a rotational stirring using a Stuart Rotator SB3 at a rotation speed of 20 rpm. Different agitation durations at different temperatures have been monitored. The oil recovery yields are collected in table 19.
  • Example 12 Effect of time and temperature on oil recovery - stirring with propeller
  • a batch of LC’1 has been filled in a tank comprising a 4-blades propeller (R 1342, IKA) with the following characteristics:
  • a batch of LC’1 has been filled in a tank equipped with a spiral stirrer which generates an axial flow.
  • a spiral stirrer (R 3003.1 , IKA) has been used.
  • LC lysed cell composition
  • a fermentation broth from a culture of oleaginous yeast Yarrowia lipolytica has been harvested and sent to a centrifuge (Alfa Laval model DX-203) to increase the cell concentration from an initial Dry Cell Weight of 84,2 (g/kg) out of the fermentor to 212,4 (g/kg) after concentration by centrifugation.
  • a centrifuge Alfa Laval model DX-203
  • a tangential flow filtration unit could be used instead of a centrifuge for this first step of concentration.
  • the dry cell weight has been measured with a moisture analyzer (Metier Toledo model HB43): a sample is placed on a Glass Fiber 1 pm Filters (Millipore APFD9050) and weighted . The filter is then placed on an Erlenmeyer vacuum flask equipped with a fritted glass funnel and rinsed 3 times with deionized water to eliminate any residual soluble salts or sugar. The filter is then placed in the moisture analyzer where an infrared lamp is drying the sample up to 130°C until weight of the filter is reaching an asymptote. The Dry Cell Weight concentration is then calculated from the difference with the initial sample weight.
  • a moisture analyzer Metal Toledo model HB43
  • a sample of the concentrated broth was analyzed to measure a cell lipid content of 65.5% w/w.
  • the analytical procedure used can be summarized as follows: The sample is first dried by lyophilization and then subjected to acid -catalyzed transesterification with a solution of FICI/Methanol. After the transesterification is completed, the lipid-soluble components of the reaction mixture are separated from the water-soluble components using a two-phase liquid extraction and subsequently analyzed with a capillary gas chromatograph (GC) equipped with a robotic injector and a flame ionization detector (FID). Quantification of the methyl-ester products is achieved with use of both an internal standard and various concentrations of an external standard mixture of fatty acid methyl esters (FAMEs).
  • GC capillary gas chromatograph
  • FID flame ionization detector
  • a bead mill DYNO®- MILL Model KDL-Pilot
  • the lysed material has then been placed in a mixing tank equipped with a mixer (Feldmeier Farmamixer with 16” diameter impeller) and a double jacked for temperature control. The sample was let overnight under moderate mixing (90 RPM) at 60°C for a 14 Hr.
  • a mixer Feldmeier Farmamixer with 16” diameter impeller
  • the lysed material was diluted with 50L of deionized water and sent to a centrifuge Alfa Laval model DX-203 equipped with 3 nozzles of 0.5 mm fed with a gear pump (max feed rate of 4.5 L/min).
  • the feed rate of the pump is first manually adjusted so that only oil is getting out of the light phase outlet (during this phase all the outlets are recirculated to the feed tank).
  • the oil phase is then collected and the heavy phase (containing water, cell debris and some left over lipids) is recirculated to the feed tank until no oil is coming out of the light phase outlet.

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Abstract

L'invention concerne un procédé de récupération de lipides produits par fermentation de cellules microbiennes, comprenant : a) la fourniture d'un milieu de fermentation contenant des cellules microbiennes capables de produire des lipides comprenant principalement un ou plusieurs acides gras choisis parmi les acides gras en C18 et C19, (b) la lyse mécanique éventuelle des cellules microbiennes conduisant à un milieu de fermentation lysé, éventuellement séparé en une phase solide/liquide légère contenant des lipides et une phase solide/liquide lourde contenant les cellules lysées, (c) la désémulsion du milieu de fermentation, du milieu de fermentation lysé ou de la phase solide/liquide légère par chauffage à une température de 30 à 80°C sous agitation pendant au moins 2 heures, générant ainsi un flux ayant une première phase contenant principalement les lipides et une seconde phase contenant de l'eau, et (d) la séparation la première phase contenant les lipides. Le procédé est réalisé sans ajout de composé(s) chimique(s) à n'importe quel stade du procédé de l'étape (b) à l'étape (d).
PCT/EP2019/069383 2018-07-20 2019-07-18 Procédé par voie humide de récupération d'une huile produite par des microorganismes WO2020016363A1 (fr)

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Citations (9)

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Publication number Priority date Publication date Assignee Title
EP1468739A1 (fr) 2003-04-15 2004-10-20 Willy A. Bachofen AG Broyeur agitateur à billes
WO2010112274A1 (fr) 2009-02-24 2010-10-07 Willy A. Bachofen Ag Broyeur à billes à agitateur
US20110295028A1 (en) 2010-06-01 2011-12-01 Stephen Robert Cherinko Extraction of Lipid From Cells and Products Therefrom
US20120130099A1 (en) 2008-10-14 2012-05-24 Solazyme, Inc. Methods of microbial oil extraction and separation
WO2015095690A2 (fr) 2013-12-20 2015-06-25 Dsm Ip Assets B.V. Procédés d'obtention d'huile microbienne à partir de cellules microbiennes
WO2015095696A1 (fr) 2013-12-20 2015-06-25 Dsm Ip Assets B.V. Procédés pour l'obtention d'huile microbienne à partir de cellules microbiennes
WO2016014900A2 (fr) 2014-07-25 2016-01-28 Novogy, Inc. Promoteurs issus de yarrowia lipolytica et arxula adeninivorans et leurs procédés d'utilisation
WO2016094520A1 (fr) 2014-12-10 2016-06-16 Novogy, Inc. Production d'acide oléique dans une levure
WO2018013670A1 (fr) 2016-07-13 2018-01-18 Dsm Ip Assets B.V. Procédé d'extraction d'une huile microbienne comprenant des acides gras polyinsaturés à partir d'un bouillon de fermentation contenant des micro-organismes oléagineux

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Publication number Priority date Publication date Assignee Title
EP1468739A1 (fr) 2003-04-15 2004-10-20 Willy A. Bachofen AG Broyeur agitateur à billes
US20120130099A1 (en) 2008-10-14 2012-05-24 Solazyme, Inc. Methods of microbial oil extraction and separation
WO2010112274A1 (fr) 2009-02-24 2010-10-07 Willy A. Bachofen Ag Broyeur à billes à agitateur
US20110295028A1 (en) 2010-06-01 2011-12-01 Stephen Robert Cherinko Extraction of Lipid From Cells and Products Therefrom
WO2015095690A2 (fr) 2013-12-20 2015-06-25 Dsm Ip Assets B.V. Procédés d'obtention d'huile microbienne à partir de cellules microbiennes
WO2015095696A1 (fr) 2013-12-20 2015-06-25 Dsm Ip Assets B.V. Procédés pour l'obtention d'huile microbienne à partir de cellules microbiennes
WO2016014900A2 (fr) 2014-07-25 2016-01-28 Novogy, Inc. Promoteurs issus de yarrowia lipolytica et arxula adeninivorans et leurs procédés d'utilisation
WO2016094520A1 (fr) 2014-12-10 2016-06-16 Novogy, Inc. Production d'acide oléique dans une levure
WO2018013670A1 (fr) 2016-07-13 2018-01-18 Dsm Ip Assets B.V. Procédé d'extraction d'une huile microbienne comprenant des acides gras polyinsaturés à partir d'un bouillon de fermentation contenant des micro-organismes oléagineux

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