WO2019030072A1 - Dry process for extraction of oil produced by microorganisms - Google Patents

Abstract

The present invention is a process for extracting lipids produced by fermentation of microbial cells from a fermentation medium comprising : • (a) providing a fermentation medium from a fermentor having a moisture content of 85%wt or more, • (b) dewatering the fermentation medium by a tangential flow filtration performed under conditions suitable 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, • (c) drying the dewatered fermentation medium of step (b) under conditions suitable to obtain a dried microbial biomass having a moisture content of at most 3%wt, • (d) pressing the dried microbial biomass of step (c) to extract lipids therefrom,

Description

DRY PROCESS FOR EXTRACTION OF OIL PRODUCED BY

MICROORGANISMS

[Field of the invention]

The present invention relates to the production and extraction of oil from microorganisms. In particular, the invention provides methods for extracting and recovering oil obtained from a microorganism by a dry process. The invention accordingly relates to the fields of biology, microbiology, fermentation technology and oil and fuel production technology.

[Background of the invention]

Lipids for use in biofuels can be produced in microorganisms, such as algae, fungi and bacteria.

Many oleaginous microorganisms, including the well characterized yeast Yarrowia lipolytica, produce lipids.

Microorganisms synthesize lipids with distinct carbon chain lengths and degrees of unsaturation. These fatty acids can be stored in organelles, termed lipid bodies or lipid droplets, as storage lipids, for example as triacylglycerides (TAG). 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.

Typically, manufacturing a lipid in a microorganism involves growing microorganisms which are capable of producing a desired lipid in a fermentor or bioreactor, isolating the microbial biomass, drying it, and extracting the intracellular lipids which are a form of oil.

US2012130099A1 discloses to extract the intracellular lipids by subjecting a dried microbial biomass to pressure, the biomass being heated before and/or during the pressing step. The moisture content of the dried microbial biomass must be of less than 6% by weight. The pressing step, conducted with an expeller in a continuous flow mode allows cell lysis to occur. More than 75% of the oil by weight in the dry microbial biomass can be extracted from the biomass in the pressing step. At the outlet of the fermentor, a cross-flow filtration can be performed on hollow fibers to dewater the cultured biomass before the drying step. This document also provides to perform a conditioning step before the pressing step and after the drying step, to change the physical and/or physicochemical properties (including the moisture content) of the biomass.

Although efficient, such process necessitates numerous steps prior the pressing steps which are costly in terms of energy consumption.

Moreover, micro-organisms filtration leads to internal fouling such that severe washings are generally needed to recover the initial permeability of membranes. However, hollow fibers are typically made from organic materials which are more sensitive to chemical washings. Thus, the use of organic membranes, such as the ones mentioned in WO2012135756A2, may render difficult the washing.

There is therefore a need for providing a process for the extraction of oil from microorganisms which allows saving energy while recovering high amounts of oil.

[Brief summary of the invention]

An object of the present invention is a process for extracting lipids produced by fermentation of microbial cells from a fermentation medium comprising :

(a) providing a fermentation medium from a fermentor having a moisture content of 85%wt or more,

(b) dewatering the fermentation medium by a tangential flow filtration performed at conditions effective 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,

(c) drying the dewatered fermentation medium of step (b) at conditions effective to obtain a dried microbial biomass having a moisture content of at most 3%wt,

(d) pressing the dried microbial biomass of step (c) to extract lipids therefrom, wherein the dewatering step is performed using at least one tubular inorganic membrane having a pore size of 0.5μηη at most and the pressing step is performed at a temperature of 95 to 130°C.

The process according to the invention allows recovering lipids at high yield (higher than 80%wt) with a reduced number of steps, the required steps being dewatering, drying and pressing without intermediate steps. The particular membrane(s) used for the dewatering step allows relatively high steady flow rates for several hours, allowing treating great quantities of broth. In particular, no clogging is observed. Only two steps require heating (drying and pressing) which allows for energy economy.

In some embodiments, the inorganic membrane has a pore size from 0.1 to 0.5μηη.

In some embodiments, the inorganic membrane is made of a material selected from titanium oxide, zirconium oxide, alumina, silicon carbide, boron carbide, silicon nitride, aluminium nitride, boron nitride, agglomerated carbon or mixtures thereof

In some embodiments, the pressing step (d) is performed at a temperature from 100 to 125°C.

In some embodiments, in the pressing step, the dried microbial biomass of step (c) is passed through an expeller press.

In some embodiments, the dried microbial biomass from which lipids have been extracted by the pressing step d) is submitted to solvent extraction for further recovery of lipids.

[Brief description of the figures]

The teaching of the application is illustrated by the following Figure which is to be considered as illustrative only and do not in any way limit the scope of the claims.

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.

[Detailed description of the invention]

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. Where reference is made to embodiments as comprising certain elements or steps, this encompasses also embodiments which consist essentially of the recited elements or steps.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.

With "bio-organic compound" or "microbial-derived 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.

The term "cell" refers to a microorganism, capable of being grown in a liquid growth medium.

The term "dry weight" or "dry matter" means weight determined in the relative absence of water. For example, 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" or "total suspended solids" means weight determined in the relative absence of water after sample washing for insoluble solids removal.

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. In particular embodiments, 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. However, also considered host cells are microbial cells naturally producing a bio- organic compound of interest.

The methods of the present invention are applicable for extracting a variety of lipids from a variety of microorganisms. In the methods of the present invention, the lipid- producing microorganism (e.g., a yeast) is first cultivated under conditions that allows for lipid production to generate oil-containing microbial biomass. The oil- containing biomass is then, depending on the method employed, dewatered, dried, and lysed by mechanical methods to extract the oil.

A. Microbial Cell

Suitable microbial cells used in the present invention are lipid-containing cells. Suitable micro-organisms for fermentation are known in the art. Non-limiting examples of 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. In particular embodiments, 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.

Any species of organism that produces suitable lipid or hydrocarbon can be used in the methods of the invention, although microorganisms that naturally produce high levels of suitable lipid or hydrocarbon are preferred.

Suitable micro-organisms for use in the present invention are capable to produce lipids comprising mainly C12-C30 fatty acids.

Those fatty acids can be present in the lipids in one or more forms such as triglycerides (also named triacylglycerides or triacylglycerols), diglycerides (also named diacylglycerols), monoglycerides (also named monoacylglycerols) and free fatty acids, the majority of the fatty acids being present in the form of triglycerides. In particular, the content of triglycerides can be of 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 lipids. Preferably, the content of triglycerides is of 95% or higher as a weight percentage of lipids. The content of triglycerides can be determined using ASTM D6584-17.

In preferred embodiments, the fatty acids in the form of triglycerides have a number of unsaturations such that the triglycerides are liquid in the conditions of the process. Preferably, these fatty acids are monounsaturated (they have a single double bond between the carbon atoms).

Although particularly adapted to recover lipids from micro-organisms producing mainly C18 fatty acids, in particular monounsaturated C18 fatty acids, the invention may also be used to recover lipids from micro-organisms producing one or several fatty acids chosen from C12, C14, C16, C18 and branched C16-C30 fatty acids. In particular, these fatty acids alone or in mixture represent the majority of the fatty acids. For example, the concentration of these fatty acids alone or in mixture is of at least 50%wt or 60w% or 70w% or 75% or higher as a weight percentage of total fatty acids.

In particular, the micro-organism is capable to secrete (intracellular production) the lipids.

In certain embodiments, 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.

In some embodiments, the microbial cell comprises mainly C18 fatty acids, in particular monounsaturated C18 fatty acids. In the present invention "comprising mainly C18 fatty acids" refers to a C18 fatty acids concentration within the cell of at least 75% or higher as a weight percentage of total C16 and C18 fatty acids.

In some embodiments, the microbial cell comprises C18 fatty acids, in particular monounsaturated C18 fatty acids, at a concentration of 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 C16 and C18 fatty acids in the cell. In some embodiments, the microbial cell comprises 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%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or higher as a weight percentage of total C16 and C18 fatty acids in the cell.

Lipid content of a microbial cell can be measured by drying a weighted amount of microbial cells, lysing the dried microbial cells, for example by milling, lyophilisation or any other lysing method, and extracting the lipids released by solvent extraction, for example by hexane extraction. The solvent-lipid mixture recovered can then be heated until complete solvent evaporation, and the remaining lipid weighted.

The nature and proportions of lipids can be determined by chromatography techniques, in particular gas chromatography or by NMR.

In some embodiments, the microbial cell is selected from the group consisting of algae, bacteria, molds, fungi, plants, yeasts and combination thereof.

In some embodiments, a microbial cell is an eukaryotic cell, such as a yeast cell, a fungi cell, an algae cell. Advantageously, the cell is a yeast or an algae.

Examples of 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.

In some embodiments, the cell is Saccharomyces cerevisiae, Yarrowia lipolytica, or Arxula adeninivorans.

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).

Thus, a wide variety of microbial biomass is suitable for use in the methods of the invention. In accordance with these methods, the oil-containing biomass is typically dewatered, dried, and then mechanically lysed to extract the oil. B. Step (a) for providing a fermentation medium

The fermentation medium is liquid. It is a solution containing microbial cells in a liquid growth medium.

In some embodiments, this step includes culturing microbial cells under conditions suitable for the production of the organic compounds by the microbial cells.

Typically, the liquid growth medium contains an aqueous medium.

Suitable culture conditions for a microbial cell for use with the invention include, but are not limited to, suitable media, bioreactor, temperature, pH, light and oxygen conditions that permit lipid production. A suitable medium refers to any medium in which a microbial cell is typically cultured. Such media typically comprises an aqueous medium having assimilable carbon, nitrogen, and phosphate sources, as well as appropriate salts, minerals, metals, and other nutrients, such as vitamins.

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. Typically, 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. In addition, 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, and the like, and minerals and the like. 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. In a preferred embodiment, the fermentation is conducted aerobically.

The fermentation can be conducted continuously, batch-wise, or some combination thereof. Microbial cells for use with the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates.

Examples of culture conditions are provided in US2012130099A1 , incorporated therein by reference.

The fermentation medium provided in step (a) has a moisture content of 85%wt or more. Typically, the moisture content is not more than 95%wt. Typically, the moisture content is measured by gravimetric analysis at a temperature sufficient to evaporate the water.

C. Dewatering step (b)

The objective of this step is to reduce the moisture content of the fermentation medium provided by step (a). This dewatering step is performed using one or more tubular inorganic membranes, in other words, non-organic membrane(s).

In some embodiments, the dewatering step is performed at a temperature suitable for reducing the viscosity of the fermentation medium provided by step (a). The temperature may for example be raised up to 90°C.

Tangential flow filtration (TFF), also known as cross-flow filtration, is a separation technique that uses membrane system(s) and flow force to purify solids from liquids. It generates a retentate and a permeate. Typically, 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. TFF can also be performed using hollow fiber filtration systems.

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. In some embodiments, 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.

In some embodiments, a membrane has at least one channel of tubular shape.

In some embodiments, 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.

In some embodiments, 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. In such a case, the separation layer is a ceramic or carbon material, in other word a non organic material.

In some embodiment, the inorganic material of the membrane is a ceramic.

In some embodiments, the inorganic material of the membrane is selected from titanium oxide (T1O2), zirconium oxide (Zr02 ), alumina (AI2O3), silicon carbide (SiC), boron carbide (B₄C), silicon nitride (Si₃N₄), aluminium nitride (AIN), boron nitride (BN), agglomerated carbon or mixtures thereof. In some embodiments, the material of the membrane is selected from titanium oxide (T1O2), zirconium oxide (Zr0₂), alumina (Al₂0₃).

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.

The use of inorganic membrane(s) allows steady flow rate without clogging despite fouling.

The at least one tubular inorganic membrane can be provided in a filtration module. In particular, a filtration module may comprise one or more tubular inorganic membranes, each membrane may itself comprise several tubular channels arranged in a bundle. By way of example, 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 broth can then be performed. The cleaning treatment to recover some or all of the initial permeability of the membrane(s) include or 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, acid solution, or solutions usually used for washing bioreactors, or combinations thereof. Solutions used for washing bioreactors (also named "Clean In Place" (CIP) solutions) are either acidic or alkaline solutions including surfactants and chelators. Cleaning treatment can advantageously be improved by raising the temperature, for example up to 50°C. In other words, in one embodiment, the temperature is raised during the cleaning treatment, for example up to 50°C.

D. drying step (c)

The objective of the drying step (c) is to reduce moisture content in the microbial biomass, here the dewatered fermentation medium obtained at step (b). Drying, as referred to herein, refers to the removal of some or all of the free moisture or surface moisture of the microbial biomass. Like dewatering, the drying process should not result in significant loss of oil from the microbial biomass. Thus, the drying step should typically not cause lysis of a significant number of the microbial cells, because in most cases, the lipids are located in intracellular compartments of the microbial biomass. Several methods of drying microbial biomass known in the art for other purposes are suitable for use in the methods of the present invention. Microbial biomass after the free moisture or surface moisture has been removed is referred to as dried microbial biomass.

The drying step allows recovering a dried microbial biomass containing less than 3% moisture by weight.

In various embodiments, the dry microbial biomass has a moisture content in the range of 0.1 % to 3% by weight. In various embodiments, the dry microbial biomass has a moisture content of less than 2.9% by weight. In various embodiments, the dry microbial biomass has a moisture content in the range of 0.5% to 2.8% by weight. The moisture content may be in a range defined by any combination of the above limits.

Non-limiting examples of drying methods suitable for use in preparing dry microbial biomass in accordance with the methods of the invention include the use of dryers such as a drum dryer, spray dryer, and a tray dryer, each of which is described below.

Drum dryers are one of the most economical methods for drying large amounts of microbial biomass. Drum dryers, or roller dryers, consist of two large steel cylinders that turn toward each other and are heated from the inside by steam. In some embodiments, the microbial biomass is applied to the outside of the large cylinders in thin sheets. Through the heat from the steam, the microbial biomass is then dried, typically in less than one revolution of the large cylinders, and the resulting dry microbial biomass is scraped off of the cylinders by a steel blade. The resulting dry microbial biomass has a flaky or powder consistency.

In one embodiment, the microbial biomass is first dewatered and then dried using a drum dryer, in particular, a double drum drier. Spray drying is a commonly used method of drying a liquid feed using a hot gas. A spray dryer takes a liquid stream (e.g., containing the microbial biomass) and separates the solute as a solid and the liquid into a vapor. The liquid input stream is sprayed through a nozzle into a hot vapor stream and vaporized. Solids form as moisture quickly leaves the droplets. The nozzle of the spray dryer is adjustable, and typically is adjusted to make the droplets as small as possible to maximize heat transfer and the rate of water vaporization. The resulting dry solids may have a fine, powdery consistency, depending on the size of the nozzle used.

Tray dryers are typically used for laboratory work and small pilot scale drying operations. Tray dryers work on the basis of convection heating and evaporation. Fermentation broth containing the microbial biomass can be dried effectively from a wide range of cell concentrations using heat and an air vent to remove evaporated water.

Flash dryers are typically used for drying solids that have been de-watered or inherently have a low moisture content. Also known as "pneumatic dryers", these dryers typically disperse wet material into a stream of heated air (or gas) which conveys it through a drying duct. The heat from the airstream (or gas stream) dries the material as it is conveyed through the drying duct. The dried product is then separated using cyclones and/or bag filters. Elevated drying temperatures can be used with many products, because the flashing off of surface moisture instantly cools the drying gas/air without appreciably increasing the product temperature.

E. extraction of lipids

The objective of the pressing step (d) is to extract lipids from the dried microbial biomass. In this step, a lysing of the cell occurs which allows rupturing the cell wall and/or cell membrane of a cell to release their cytoplasmic content.

The pressing step involves subjecting pressure sufficient to extract oil from the dried microbial biomass of step (c). Thus, the feedstock that is pressed in the pressing step comprises oil predominantly or completely encapsulated in cells of the biomass.

The pressing step is performed at a temperature from 95 to 130°C, or from 100 to 125°C. The temperature used may be in a range defined by any of the above limits. An appropriate pressure can be applied continuously or for one or more cycles. Extracted lipids may contain up to 15wt% of solids.

In some embodiments, the extracted lipids are submitted to a solid/liquid separation. This allows reducing partly or completely the solid amount in the extracted lipids. This solid/lipid separation can be performed by filtration, such as for example press filtration, by centrifugation or by decantation.

The invention allows recovering from 80 to 99 wt % or from 80 to 95wt% or from 81 to 94 wt % of lipids after the solid/liquid separation.

In some embodiments, the pressing step is conducted with an expeller press. In various embodiments, the pressing step is conducted in a continuous flow mode.

The expeller press is a device comprising a continuously rotating worm shaft within a cage having a feeder at one end and a choke at the opposite end, and having openings within the cage.

The dried microbial biomass enters the cage through the feeder, and rotation of the worm shaft advances the dried microbial biomass along the cage and applies pressure to the dried microbial biomass disposed between the cage and the choke, the pressure releasing oil through the openings of cage and extruding spent biomass from the choke end of the cage.

Expeller presses (screw presses) are routinely used for mechanical extraction of oil from soybeans and oil seeds. Generally, the main sections of an expeller press include an intake, a rotating feeder screw, a cage or barrel, a worm shaft and an oil pan. The expeller press is a continuous cage press, in which pressure is developed by a continuously rotating worm shaft. An extremely high pressure is built up in the cage or barrel through the action of the worm working against an adjustable choke, which constricts the discharge of the pressed cake (spent cells) from the end of the barrel.

The expeller press may be configured to allow pressure control, for example by adjusting rotational velocity of the worm shaft.

The cage on the expeller press can be heated using steam or any hot fluid. The cage can optionally be preheated. In the present invention, the cage is held to a temperature of between 95 to 130°C or 100 to 125°C or at a temperature in a range defined by any combination of these limits.

The other operating conditions of the expeller press will be determined by the man skilled in the art by controlling the oil recovery at the above mentioned temperature while monitoring one or several of the following parameters : rotation velocity of the worm shaft, feeding rate, feed moisture, nozzle diameter.

The resulting pressed solids or cake (spent biomass of reduced oil content relative to the feedstock supplied to the screw press) is expelled from the expeller press through the discharge cone at the end of the barrel/shaft. The choke utilizes a hydraulic system to control the exit aperture on the expeller press. A fully optimized oil press operation can extract most of the available oil in the oil-bearing material. A variety of factors can affect the residual oil content in the pressed cake. These factors include, but are not limited to, the ability of the press to rupture oil- containing cells, material moisture and cellular compartments and the composition of the oil-bearing material itself, which can have an affinity for the expelled oil. In some cases, the oil-bearing material may have a high affinity for the expelled oil and can absorb the expelled oil back into the material, thereby trapping it.

In that event, the oil remaining in the spent biomass (in the pressed cake) can be re-pressed or subjected to solvent extraction to recover the oil. Solvent extraction may be performed by usual solvent extractions techniques. Suitable solvents for performing extraction include, but are not limited to, alkanes, alcohols, in particular anhydrous alcohols, aromatic compounds, chlorinated compounds, ethers, ketones, esters, aldehyde, sulfides.

Suitable alkanes include hexane, propane, butane, pentane, heptane, cyclohexane, methyl pentane, isopentane, 2-methyl pentane, 3-methyl pentane, preferably hexane. Suitable alcohols include ethanol, n-propanol, isopropanol, n- butanol, isobutanol, allyl alcohol. Suitable aromatics include benzene, toluene, xylene. Suitable chlorinated compounds include chloroform, dichloromethane, trichloromethane. Suitable ethers include ethyl ether, dialkyl ethers such as diethyl ether. Suitable ketones include acetone, butanone. Suitable esters include methyl acetate ester, ethyl acetate ester. Suitable aldehydes include furfural. Suitable sulfides include alkyl sulfides, n-alkyl mercaptans, carbon disulfide. In one embodiment, suitable solvents include alkanes, alkyl sulfides, n-alkyl mercaptans, dialkyl ether and aromatics.

It is not necessary to use biological agents to extract oil using an expeller press, ie: agents such as enzymes that are produced independently of the microbial cells. Optionally, bulking agents (also named "press aids") may be used. The bulking agent can have an average particle size of less than 1 .5 mm. In various embodiments, the bulking agent is selected from the group consisting of cellulose, corn stover, dried rosemary, soybean hulls, spent biomass (biomass of reduced lipid content relative to the biomass from which it was prepared), sugar cane bagasse, and switchgrass.

The invention will be further understood with reference to the following non-limiting examples.

[Examples]

Example 1 : description of the process according to an embodiment

Figure 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 passed through a cross-filtration device 1 12, comprising non organic membranes, for reduction of its moisture content. The membranes are as disclosed above in the present specification. The permeate (stream #3) obtained is discarded or preferably recycled to the bioreactor or for other application within the process, while the retentate (stream #2) is conducted to a drier device 1 14, for example a double drum drier, for further reduction of its moisture content to less than 3%wt. The dried stream (stream #4) is then introduced into a pressing device 1 16, here an expeller press (screw oil expeller), for releasing the lipids contained in the cells contained in the dried stream. Crude oil containing the lipids released by the cells (stream #6) is then recovered.

The crude oil stream obtained may then be purified by appropriate treatments such as filtration, refining, bleaching and deodorization. These treatments are usual and not described herein. The lipid stream may further be submitted to chemical reactions. A pressed cake (stream #7) is also recovered, which can be further treated to recover more oil phase using conventional techniques, such as solvent extraction followed by a separation step and solvent distillation.

Tests have been performed using a process as the one disclosed into figure 1. These tests are detailed in examples 2-6. Examples 2-4 refer to the cross filtration step, example 5 to the drying step and example 6 to the oil extraction step.

In the examples, unless otherwise indicated :

- the 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 or dry cell weight 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 composition of fatty acids, mostly in the form of TAG, has been determined using a method similar to ISO 12966 by reacting the lipids with methanol in presence of hydrochloric acid to form methyl esters of the fatty acids followed by gas chromatography analysis of the esters. Heptadecanoic acid or tridecanoic acid are used as internal standards and added to the lipid sample before transformation into methyl esters. Broth used in the filtration examples

A 20 L broth of genetically modified Yarrowia Iipoiytica has been fermented in a bioreactor for 5 days. The features of the broth are collected in the below table 1 .

Table 1 :

Parameter Unit Fermentation broth lipid content %wt 66,2

Total suspended solids (TSS) (NF T 90-105-2) g/kg 88.7

Dry matter (DM) (EN 12880) Wt% 1 1.3

The fatty acids (in the form of TAG) content of the broth is given below in table 2. Table 2

Nd : non detected

Filtration Tests : Tubular ceramic membranes have been tested in a cross-flow filtration bench.

Membranes INSIDE CeRAM™ 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.

Table 3

Membrane Material Properties

M1 T1O2 and ZrO2 1 single channel

Hydraulic diameter : 6mm

Area : 47 cm²

Pore size : 0.14μηη

M2 T1O2 and ZrO2 1 single channel Hydraulic diameter : 6mm

Area : 47 cm²

Pore size : Ο.δμηη

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). Then, the instantaneous and average permeate flowrate per filtration area is calculated as well as, for tests with permeate production, the mass concentration factor (MCF = initial weight of sample / retentate weight = initial weight of sample / (initial weight of sample - permeate weight)).

The transmembrane pressure is calculated with the pressure sensors: TMP = (retentate pressure before membrane + retentate pressure after membrane) / 2 - permeate pressure (which can be taken equal to 0). The concentration factor (CF) for TSS can be also calculated as CF (TSS) = retentate TSS / broth TSS.

Example 2- cross-flow filtration -

The test is performed with the membrane 0,14μηη (M1 ) with the below operating conditions :

Membrane : M1

Mass concentration factor MCF = 1 (no production of permeate, recycling of permeate)

Initial weight : 3730.9 g

Average TMP : 1.3 bar

Feed Flowrate : 450 kg/h

Cross flow velocity : 4.4 m/s

Temperature : 25°C

The results are shown in the below table 4. With these operating conditions, the permeate has a good quality (low turbididy, except in the first minutes) and the instantaneous flowrate is around 140 kg/h/m² at the beginning to around 1 10 kg/h/m².

Table 4

Example 3 - cross-flow filtration - effect of pressure

The TMP is increased incrementally and for each TMP, the instantaneous flowrate is measured, and then the same is performed by decreasing value of TMP.

Operating conditions :

Membrane :M1

Mass concentration factor MCF = 1 (no production of permeate, recycling of permeate)

Initial weight : 3730.9 g

Feed Flowrate : 450 kg/h

Cross flow velocity : 4.4 m/s

The results are shown in the below table 5. The results show that with a cross-flow velocity of 4,4 m/s, the TMP should not exceed 2,1 bar. With a higher TMP, the flowrate is not significantly higher and the fouling seems to be more important. Table 5

Duration Temperature TMP (bar) Instantaneous Permeate

(min) CO permeate flowrate turbidity (NTU)

(kg/h/m²)

0 25.6 1.31 1 13.8 0.828

19 25 2.1 15 139.6 9.17

31 25 3.325 142.1 8.57

39 24 2.1 107.2 3.48

44 23 1.315 75.1 1 .36 Example 4 - cross-flow filtration - effect ofMCF

The two membranes M1 and M2 have been tested. The test with the membrane M2 has been conducted at the lowest TMP possible so as to reduce the permeate flowrate and to avoid the formation of deposit along the membrane : the ratio tangential velocity (related to the retentate flow rate) / permeate flow rate is kept as high as possible without modifying the tangential velocity.

Table 6 : Operating conditions

The results are shown in the below table 7.

For the membrane Ο,δμηη (M2), 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%.

For the membrane 0,14μηη (M1 ), the permeate flowrate is steady during the increase of MCF (until 2,5) and the permeate has a good quality (TSS equal to 0). The final DM of retentate reaches 23,3wt% which corresponds to a water content of 76.7 %wt.

Test with membrane M2 was expected to have better filtration behavior. In particular, higher flowrates were expected due to the greatest pore size. Surprisingly, the membrane with the smallest pore size (M1 ) gives the higher steady permeate flowrate, along with the lower water content. It would not be surprising to obtain much lower water content, in particular less than 75%wt or 70%wt or 65%wt, by optimizing the filtration conditions. Table 7

The lipid analysis for the test 1 is given in table 8. The final concentration factor (for TSS) is equal to 2.2. The measurement uncertainties can explain, in part, the loss of solids (if the membrane retains all the solids, the concentration factor should be, in theory, equal to the MCF); the other explanation should be the loss in the fouling on the membrane. That agrees with the loss of water permeability after the test 2 (95%). That means that some fouling occurs on the membrane.

The results show that the loss of total fatty acid in permeate is very low (lower than 0,01 %wt). We have to specify that for the ratio it has been calculated with the sum of total fatty acid in retentate and permeate.

The total fatty acid of the retentate is equal to 83%wt of total fatty acid from the fermentation broth (the sample). Table 8 : total fatty acid in retentate and total fatty acid, Ί l^~AG, FFA from Test 1.

Fatty acid Retentate in %wt Permeate in mg/L

TSS

Total fatty acid Total TAG FFA fatty

acid

lauric acid C12:0 nd nd nd nd myristic acid C14:0 nd nd nd nd palmitic acid C16:0 5.547 0.86 0.02 0.4 palmitoleic acid C16:1A9 7.412 1.22 0.005 0.04 stearic acid C18:0 2.319 0.47 0.02 0.49 oleic acid C18:1A9 74.239 0.75 0.14 0.01 linoleic acid C18:2 nd nd nd nd

Total fatty 89.46 3.30 0.19 0.94 acids/TAG/FFA

Measured TSS : 205.212g/L for the retentate

Nd : non detected

FFA : Free fatty acids

TAG : Triacylglycerides

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.

The following washing has been performed on the membranes :

- Water flushing (non recycling) and water rinsing (retentate recycling/5 min/1.2 bar/ feed circulation 450kg/h)

Washing : Ultrasil P1 1 (0.5wt%) at 66°C during 30min with feed circulation 300-350kg/h and TMP : 0.7 bar

Ultrasil P1 1 is a chlorinated, powdery alkaline detergent composed of emulsifiers, degreasers and surfactants for the cleaning of membrane systems.

Analysis of the flushing, rinsing and washing waters have shown that about 8%wt fatty acids from the initial sample are found in these streams, which should therefore be recycled to recover the maximum of lipids, at least for the flushing and rinsing streams. Example 5 -Drying

Drying has been performed using a double drum drying method. Product is fed into the nip between the pair of drums which always rotate in opposite directions. The counter-rotation of the drums toward each other draws the liquid pool into the nip and spreads it into a thin sheet that adheres to the hot drum dryer. The spreadsheet is split into two sheets that adhere on both hot drums.

Tests A-C have been performed with a 24"x24"atmospheric double drum dryer (ADDD). The pilot dryer is equipped with chrome plated, cast iron drums that are 24" in diameter and 24" long and ¾" thick. The drying area is 25 ft2.

Tests D-l have been performed with a 12"x18" double carbon steel drum dryer. The total drying surface area of the drums is 9.4ft2. The drums are adjusted manually with two hand wheels and the end boards are adjusted using a pneumatic valve. A variable frequency drive allows controlling the drum speed within the range 1 to 10 rpm. Dried product is scraped off the drum surface by two stainless steel knives which are controlled pneumatically. The pilot has a two point feed system in vapor hood.

The same Yarrowia lipolytics yeast has been tested in tests A-l.

Broths used for tests A-C

A 1000 L broth of genetically modified Yarrowia lipolytica has been fermented in a bioreactor for 5 days to produce oleic acid. The fermentation is run at 30°C and pH 3.5; with air flow 242 LPM at 14.7 psi fermentor back pressure with glucose as sugar feedstock. 670kg of broth at 65,7g/kg dry cell weight (DCW) have been harvested by the end of the fermentation and concentrated to obtain 160 liters of concentrated fermentation broth at 265g/L dry cell weight.

Characteristics of the concentrated broth dryed in tests A-C :

Yeast : Yarrowia lipolytica

Lipid content : 42%wt (83%wt of oleic acid and 8.9%wt of palmitoleic acid)

Moisture content : 73.5%wt

Broths used for tests D-G :

A 1000 L broth of genetically modified Yarrowia lipolytica has been fermented in a bioreactor for 5 days to produce oleic acid with glucose as sugar feedstock. 750kg of broth at 60.8g/kg dry cell weight (DCW) have been harvested by the end of the fermentation and concentrated to obtain 158kg of concentrated fermentation broth at 272g/L dry cell weight.

Yeast : Yarrowia Iipoiytica

Lipid content : 51.2%wt (85.8%wt of oleic acid and 0.3%wt of palmitoleic acid) Moisture content : 72,8%wt

Broths used for tests H- 1:

A 1000 L broth of genetically modified Yarrowia Iipoiytica has been fermented in a bioreactor for 5 days to produce oleic acid with glucose as sugar feedstock. 755kg of broth at 61.6g/kg dry cell weight (DCW) have been harvested by the end of the fermentation and concentrated to obtain 158kg of concentrated fermentation broth at 272g/L dry cell weight.

Yeast : Yarrowia Iipoiytica

Lipid content : 49.9%wt (86.2%wt of oleic acid and 0.3%wt of palmitoleic acid) Moisture content : 72,8%wt

The results of tests A-C are collected in table 9. The results for tests D-l are collected in table 10.

Trial A showed that 60 psi is sufficient to dry concentrated broth. Chosen conditions led to a smooth uniform drum coating powder at the knives. At the end of the trial, 8.6 lbs of dry product was recovered in 10 minutes at 2% moisture. The dry product rate is 2.05 Ibs/hr/ft² and dry product temperature was measured at 134°C.

The dry product rate of test B is lower than for Trial A. Regarding moisture content of the dry product (1 .6%), the drum speed was a bit too low.

Table 9: results tests A-C

Test A B C

Steam Pressure (Psi) 60 55 82

Drum gap (initial - final) (inches) 0.012-0.17 0.008-0.10 0.014

Nip level (inches) 2 2 ¾

Drum speed (rpm) 3 2.2 4

Dry product rate (Ibs/hr/ft²) 2.05 1.67 1.85

Dry material moisture (wt%) 2 1.6 1.8 Table 10: results tests D-l

Tests D-l show that moisture contents of less than 3%wt are obtained.

The dry product of test D has good moisture content around 2.2% and the fine gap resulted in powder dry material. Test E was set up to recover more flakes by increasing the gap, however the final moisture decreased to 1 %. During test F, the drum speed and then the feed flow rate have been increased to increase the final moisture. The dry product has 1.8% moisture but in globally we recovered more powder than flakes. Test G was started with an even higher gap, the trial was successful with a good final moisture. Test H on a different broth was started with same operating conditions than test G, but nip level decrease was observed during the trial and ended up by spilling through the drums. Test I was then started with lower gap and allowed to dry the concentrated broth at 1.5% final moisture.

Example 6 - Oil extraction - effect of Temperature

A dried microbial biomass having the following composition was provided :

- dry matter (DM) content : 97,3 wt%,

oil content : 54.3 wt% (with respect to DM),

moisture content : 2.7 wt%.

This dried microbial biomass has been prepared from a genetically modified Yarrowia lipolytics which produces oil having a typical composition shown in the below table 1 1.

Table 1 1 : Typical composition of oil produced by Yarrowia Lipolytica genetically modified (fatty acids in the form of TAG)

% (wt)

Palmitic acid C16:0 3.5-6 Palmitoleic acid C16: 1 A9 6.5-8

Stearic acid C18:0 2.5-3

Oleic acid C18: 1 A9 85-87

A Komet press (from IBG Monforts Oekotec) has been used. A 4mm diameter nozzle has been used. A heating collar has been provided around the head of the press. The higher flow obtained was 3kg/h.

Pressing tests have been performed at 80, 1 10, 125 and 140°C.

The dried biomass passes in the press a first time to recover oil and a press cake. About 16g of the press cake has been collected for measurement. The rest of the press cake has been pressed to recover more oil. The oil content in the press cakes was measured by NMR. The crude oil has further been filtrated under vacuum over a Buchner device, using a filter paper with a 5-13μηη pore size. The oil content in the solid samples has been measured by NMR according to NF EN ISO 10565. Calibration of the NMR has been performed using a sunflower seeds sample containing 49%wt of oil or with samples containing 8% of oil.

Dry matter : measurement by weight difference after 13 hours at 103.5°C.

The yield is calculated from the oil contents in oil of the press cake using the following equation :

Where H_L and H_tx are the oil content of the cells and of the press cake respectively (over the matter, without drying).

And S_L and S_tx are the dry matter content of the cells and the press cake.

The dry matter content is from 95 to 95.6%wt.

The results are reported in the below table 12. Pressing at 80°C does not allow oil extraction even after two pass through the press. At 1 10°C, an extraction yield of 89,0 ±1 .4%wt can be obtained after the 1 ^st pass (tests 2-5). A second pass of the press cake allows recovering slightly more oil (tests 2-3, 5). At 125°C and 140°C, we note a lower efficiency of the pressing on the 1^st pass. A second pass allows obtaining an oil yield similar to the one obtained at 1 10°C.

Table 12 :

Test 1 2 3

Pressing 80°C 1 10°C 1 10°C temperature

Pass 1^st Pass 2^na Pass 1 ^st Pass 2^na Pass 1^st Pass 2^na Pass

Press entry

Input Material 354 327 354 151 354 160 Mass (g)

Press output

press cake (g) 327 315 167 138 176 147

Mean wt% 53.4 10.3 10.4 1 1.6 10.8 oil

content

in the

press

wt% 54.9 10.9 10.9 12.1 1 1.5 cake

over

DM)

Crude oil 0 0 169 181 164 182 Mass (g)

Fill er output

Filtered Oil (g) 0 0 152 153

Extraction 0 0 90.2 90.2 88.4 89.1 yield (%wt) Table 12

Test 4 5 6

Pressing 1 10°C 1 10°C 125°C temperature

Pass 1^st Pass 1^st Pass 1 Pass 1^st Pass 2^na Pass

Press entry

Press entry

Input Material 354 353 218 353 155 Mass (g)

Press output

press cake (g) 177 173 91 173 150

Mean wt% 10.0 12.5 12.5 12.1 10.2 oil

content

wt% 10.5 13.0 12.5 10.5 in the

over

press

DM)

cake

Crude oil 173 176 106 176 183 Mass (g)

Fil ter output

Oil extracted 86 163

(g)

Extraction 90.1 87.7 87.4 87.7 89.9 yield (%wt) Table 12

Example 7 - Solvent extraction

Press cakes resulting from pressing using the Komet press mentioned in example 6 have been processed by solvent extraction to recover more lipids. 2300 g of press cake containing 377 g of oil have been submitted to solvent extraction. The solvent extraction was carried out in an extractor by 6 successive washings by percolation of 1 .97-2.05 kg of hexane through the press cake maintained at 50 ° - 55 ° C. Such extraction is a counter current extraction. The portion of hexane used in the extraction tests was 5.2 kg per kg of press cake.

At the end of each wash, the miscella (oil-hexane mixture) was evacuated by draining the marc. Table 13 summarizes the quantities of materials used for each wash. The dry matter content of each miscella was measured. This represents the solubilized oil and the fine suspended solids particles entrained by the solvent.

At the opening of the extractor, the dry matter content of the marc is 77.4± 1.3%wt, its water content is about 4.5%wt and its hexane content of about 18.1 %wt. The marc has then been placed in a hood to remove hexane. A dry matter content of 93,3%wt. of the marc is measured. The marc is then placed 2 hours in an oven at 50-60°C to eliminate the last traces of hexane. The final dry matter content of the marc reached 95.6%wt. The residual oil content of the marc is 1.12%wt on the material as such (or 1.18%wt on dry matter).

The miscellas were distilled at rotavap at 50-55 ° C under vacuum and further aerated by injection of nitrogen for 4 hours to eliminate traces of hexane. The final dry matter content of the oil was 99%wt. The oil was filtered through Buchner to remove the particles. The mass of extracted oil is 357 g (based on the oil content of the press cake by NMR).

Table 13

Washing # 1 2 3 4 5 6

Press cake mass (g) 2300 - - - - -

Hexane mass (g) 2005 2045 1970 2000 2015 1970

Miscella mass (g) 1596 1956 1820 1755 1989 1799

Dry matter content of miscella 12.45 5.10 2.36 0.97 0.49 0.22 (%wt)

Marc mass (g) 2370

Dry matter content in the marc 77.4 (%wt)

Claims

1. Process for extracting lipids produced by fermentation of microbial cells from a fermentation medium comprising :

(a) providing a fermentation medium from a fermentor having a moisture content of 85%wt or more,

(b) dewatering the fermentation medium by a tangential flow filtration performed under conditions suitable 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,

(c) drying the dewatered fermentation medium of step (b) under conditions suitable to obtain a dried microbial biomass having a moisture content of at most 3%wt,

(d) pressing the dried microbial biomass of step (c) to extract lipids therefrom,

wherein the dewatering step is performed using at least one tubular inorganic membrane having a pore size of 0.5μηη at most and the pressing step is performed at a temperature of 95 to 130°C.

2. Process according to claim 1 , wherein the inorganic membrane has a pore size from 0.1 to 0.5μηη.

3. Process according to claim 1 or claim 2, wherein the inorganic membrane is made of a material selected from titanium oxide, zirconium oxide, alumina, silicon carbide, boron carbide, silicon nitride, aluminium nitride, boron nitride, agglomerated carbone or mixtures thereof.

4. Process according to claim 3, wherein the inorganic membrane is made of a material selected from titanium oxide, zirconium oxide, alumina.

5. Process according to any one of claims 1 to 4, wherein a dewatered fermentation medium having a moisture content of 78%wt or less is obtained.

6. Process according to any one of claims 1 to 5, wherein the pressing step (d) is performed at a temperature from 100 to 125°C.

7. Process according to any one of claims 1 to 6, wherein, in the pressing step, the dried microbial biomass of step (c) is passed through an expeller press.

8. Process according to any one of claims 1 to 7, wherein the microbial cell is selected from the group consisting of algae, bacteria, molds, fungi, plants, yeasts and combination thereof, and is capable to produce lipids comprising mainly one or several fatty acids chosen from C12, C14, C16, C18 and branched C16- C30 fatty acids, in particular C18 fatty acids, the concentration of these fatty acids alone or in mixture being of at least 50%wt as a weight percentage of total fatty acids.

9. Process according to claim 8, wherein C18 fatty acids concentration within the cell is of at least 75% or higher as a weight percentage of total C16 and C18 fatty acids.

10. Process according to any one of claims 1 to 9, wherein the microbial cell comprises oleic acid at a concentration of at least 50% or higher as a weight percentage of total C16 and C18 fatty acids in the cell.

1 1. Process according to any one of claims 1 to 10, wherein the dried microbial biomass from which lipids have been extracted by the pressing step d) is submitted to solvent extraction for further recovery of lipids.

12. Process according to any one of claims 1 to 1 1 , wherein the at least one tubular inorganic membrane is provided in a filtration module and several filtration modules are provided, some of which being cleaned so as to restore their permeability while the others are used for tangential filtration.

13. Process according to claim 12, wherein the cleaning treatment 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 solution, acid solution, or acidic or alkaline solutions used for washing bioreactors including surfactants and chelators, or combinations thereof.

14. Process according to claim 12 or 13, wherein the temperature is raised during the cleaning treatment.

15. Process according to claim 14, wherein the temperature is raised up to 50°C.