WO2019030007A1

WO2019030007A1 - Process for the recovery of fermentation products

Info

Publication number: WO2019030007A1
Application number: PCT/EP2018/070315
Authority: WO
Inventors: Nicolas SALAVIN; Julien MAIRESSE; Li Chen; Luis A. ROBLES MACÍAS
Original assignee: Total Raffinage Chimie; Amyris, Inc.
Priority date: 2017-08-07
Filing date: 2018-07-26
Publication date: 2019-02-14

Abstract

The present application relates to methods for the recovery of fermentation products from a fermentation mixture. Methods are provided for the recovery of cell-associated fermentation products, which methods rely on a drying process, and do not require the addition of surfactants. More particularly, methods are provided comprising the drying of a fermentation mixture or a cellular fraction thereof, and the recovery of fermentation products from the gaseous phase obtained by said drying. Also provided herein are methods for the recovery of fermentation products from emulsions, which methods rely on the use of a membrane, more particularly a membrane that is permeable for the fermentation medium.

Description

PROCESS FOR THE RECOVERY OF FERMENTATION PRODUCTS

FIELD OF THE INVENTION

The present invention is directed to a method for recovering (cell-associated) fermentation products such as (cell-associated) farnesene from a fermentation mixture.

BACKGROUND

The microbial production of bio-organic compounds such as farnesene is known in the art. The fermentation products produced in the microbial cells may be present in the culture supernatant and/or associated with the microbial cells. A problem encountered in the microbial fermentation of bio-organic compounds is that it is difficult to recover the fermentation products, and more particularly the cell-associated fermentation products, from the complex fermentation mixture, which lowers the overall efficiency of the microbial production process.

Nowadays, fermentation products associated to the microbial cells are typically lost with the microbial waste stream, or require further downstream processing steps of permeabilizing or lysing the cells, and the use of chemical treatment (e.g. solvent extraction or use of detergent) or other extraction steps for selectively recovering the product of interest.

Also the recovery of bio-organic compounds from the fermentation mixture may be difficult. Separation of the bio-organic compound from the fermentation mixture often relies on the use of a surfactant to break emulsions comprising the bio-organic compound. However, adding a surfactant has a cost and the surfactant becomes an impurity that has to be removed downstream in the process. Moreover, not all emulsions are broken by addition of a surfactant.

Accordingly, there remains a need in the art for more efficient and cost-effective methods for obtaining bio-organic compounds from microbial fermentation.

SUMMARY OF THE INVENTION

It is an object of the present invention to improve the recovery of fermentation products from a fermentation mixture, more particularly to recover cell-associated fermentation products with a low solubility in water from the fermentation mixture, thereby increasing the overall recovery yield of the microbial fermentation process. Indeed, it has been found that that the yield of fermentation products can be increased by (additionally) recovering the product from the fermentation medium, whereby the fermentation product is recovered from a gaseous phase. This method is particularly suitable for fermentation products which have a low solubility in water, such as a solubility of 2g/L, more particularly of less than 1 g/L. Indeed it is observed that a significant fraction of these compounds is not efficiently extracted but remains associated with the cells in the fermentation broth. The methods comprise a drying step of the fermentation medium, wherein a gaseous phase is generated from which the fermentation product can be recuperated. It is a further object of the present invention to reduce the use of chemicals in recovery processes by way of the methods provided.

More particularly, the invention provides methods wherein the cell-bound fraction of the bio- organic fermentation product is recovered by drying and/or wherein membranes are used to avoid the necessity of chemicals.

The present invention is in particular captured by any one or any combination of one or more of the below numbered aspects and embodiments (i) to (xvii) wherein:

(i) A method for the recovery of one or more fermentation products from a fermentation medium comprising the following steps:

(a) optionally concentrating the fermentation medium or a cellular fraction thereof by removal of free solvent;

(b) drying the fermentation medium or a cellular fraction thereof or the concentrate obtained in step (a), thereby generating a gaseous phase; and

(c) recovering the one or more fermentation products from the gaseous phase obtained in step (b).

(ii) The method according to (i), wherein the fermentation product as a low solubility in water, such as a solubility of 2g/L or less, such as a solubility of 1 g/L or less in water.

(iii) The method according to (i) or (ii), wherein the fermentation product is a hydrocarbon, a fatty acid or a polyalkanoate or (poly)hydroxyalkanoate, such as a hyaluronic acid (HA)- conjugated C3-C5 hydrocarbon.

(iv) The method according to (i) to (iii), wherein the one or more fermentation products are hydrocarbons, preferably terpenes, more preferably sesquiterpenes.

(v) The method according to (i) to (iv), wherein the fermentation product is farnesene. (vi) The method according to any one of (i) to (v), wherein the one or more fermentation products are cell-associated fermentation products.

(vii) The method according to any one of (i) to (vi), wherein said concentrate contains less than 3 wt% (v/v) free solvent, preferably less than 1 wt% (v/v) free solvent.

(viii) The method according to any one of (i) to (vii), wherein the concentration step (a) is performed using a decanter centrifuge.

(ix) The method according to any one of (i) to (viii), wherein step (b) is performed using a combination of contact and fluidized bed drying.

(x) The method according to any one of (i) to (ix), wherein step (c) is performed by condensing the gaseous phase and separating the fermentation product from the condensate.

(xi) The method according to (x), wherein said separation of the one or more fermentation product from the condensate is performed by decanting the condensate.

(xii) The method according to any one of (i) to (xi), wherein said cellular fraction is obtained following solid/liquid separation of the fermentation mixture into a solid/liquid heavy phase and a solid/liquid light phase, wherein said solid/liquid heavy phase comprises the cellular fraction.

(xiii) The method according to (xii), wherein said solid/liquid separation is performed by means of a centrifuge.

(xiv) The method according to any one of (x) or (xiii), further comprising recovering one or more of said fermentation products from the solid/liquid light phase.

(xv) The method according to (xiv), wherein said one or more fermentation products are recovered from the solid/liquid light phase by the following steps:

optionally clarifying the solid/liquid light phase,

- concentrating the solid/liquid light phase or the clarified solid/liquid light phase to form a concentrated solid/liquid light phase,

evaporating the concentrated solid/liquid light phase in a first evaporation, evaporating the residual concentrated solid/liquid light phase from the first evaporation in a second evaporation,

- recovering the one or more fermentation products from the gaseous phase from the second evaporation, and

optionally recovering fermentation product from the gaseous phase from the first evaporation. (xvi) The method according to (xv), wherein the residual concentrated solid/liquid light phase from the second evaporation is subjected to the drying step (b).

(xvii) The method according to (xv) or (xvi), wherein the solid/liquid light phase or the clarified solid/liquid light phase is concentrated by means of one or more membranes, preferably ceramic membranes.

(xviii) A method for the recovery of one or more fermentation products from a fermentation medium comprising the following steps:

optionally separating the fermentation medium into a solid/liquid heavy phase and a solid/liquid light phase,

- optionally clarifying the fermentation medium or the solid/liquid light phase,

concentrating the fermentation medium, the solid/liquid light phase, the clarified fermentation medium or the clarified solid/liquid light phase into a concentrate and a permeate,

evaporating the concentrate in a first evaporation,

- evaporating the residual concentrate in the first evaporation in a second evaporation,

recovering the fermentation product from the gaseous phase from the second evaporation, and

optionally recovering one or more fermentation products from the gaseous phase from the first evaporation,

wherein said concentration step is performed by means of one or more membranes, wherein said membrane is permeable for the fermentation medium.

(xix) A method for the production of an organic compound, comprising the following steps:

(a) providing a genetically engineered micro-organism capable of producing the organic compound;

(b) fermenting a fermentable medium using said micro-organism; and

(c) recovering the fermentation product from the fermentation mixture by a method according to any one of (i) to (xviii). 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 does not in any way limit the scope of the claims.

Figure 1 : Schematic representation of a process for the recovery of farnesene 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.

The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-10% or less, preferably +/-5% or less, more preferably or less, and still more preferably +/-0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

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-associated" as used herein in connection to fermentation products refers to fermentation products that are associated to the host cell or host cell debris.

The term "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 terms "free solvent" and "bound solvent" as used herein in connection to cellular material refer respectively, to solvent that is not associated and associated with the cellular material through Van der Waals forces.

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 wildtype host cell which affects the production of the bio-organic compound. However, also considered host cells are cells naturally producing a bio-organic compound of interest. In particular embodiments, the host cell is a microbial cell.

The term "fraction" as used herein refers to a (further defined) part of a whole or whole amount.

The present application generally relates to the recovery of organic compounds, more particularly organic compounds with a low solubility in water, that are produced by host cells, more particularly microbial host cells, from a fermentation mixture. More particularly, the present inventors have realized methods for increasing the yield of organic compounds that are recovered from the fermentation medium. Thus the application generally provides methods for improving the recovery of organic compounds with a low solubility in water from a fermentation medium. In particular embodiments, the invention relates to recovering bio-organic compounds from different fractions of the fermentation broth. In particular embodiments, the one or more fermentation products refer to the same bio-organic product that is recovered from different fractions of the fermentation broth. In alternative embodiments, the one or more fermentation products refer to different bio-organic compounds that are recovered from different fractions of the fermentation broth. Also, the methods of the invention may be used for the recovery of both the same and different bio- organic products. Typically however, in the context of the present invention when referring to different fractions of a bio-organic product, this will refer to the recovery of different fractions of the same bio-organic product. Irrespective thereof, reference will generally be made herein to "bio-organic product", "bio-organic products" or "one or more bio-organic products". More particularly, the invention provides methods for recovering organic compounds produced and secreted by host cells of which a fraction may remain associated to the host cells and/or a fraction may be present in emulsions in the fermentation medium. More particularly, the invention provides methods for recovering organic compounds that are associated to the host cells (i.e. cell-associated bio-organic compounds). The invention further provides methods for recovering fermentation products that are contained in emulsions in the fermentation mixture. In particular embodiments, methods are provided for the recovery of a bio-organic compound which are aimed at the recovery of the bio-organic compound which is cell-bound as well as recovering the bio-organic compound from the fermentation medium, in particular bio-organic compounds that are contained in emulsions. The methods provided herein both individually and together increase the overall yield of the microbial production process.

The recovery processes envisaged herein may further address one or more of the drawbacks of the prior art processes in that they do not require the addition of chemicals, and hence, are environmentally friendly and more economic.

The different aspects and embodiments of the invention are detailed herein below.

Host cell fermentation

The bio-organic compounds envisaged herein are produced by host cell fermentation, typically 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. 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, 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/C0₂ mixture or other suitable non-oxygen gas or gasses. In further embodiments, the culture medium is adapted to ensure cultivation of the host cell and/or bioproduction of the bio-organic compound by the host cell of interest. The fermentation can be conducted continuously, batch-wise, or some combination thereof.

Suitable micro-organisms for fermentation are known in the art. Non-limiting examples of suitable micro-organisms include bacteria such as Escherichia (e.g. E. coli), Bacillus or Lactobacillus species, fungi, in particular yeasts such as Saccharomyces (e.g. S. cerevisiae) or Pichia 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.

The invention provided herein is applicable to a broad range of bio-organic compounds. The bio-organic compounds or fermentation products for which the methods described herein are of interest are poorly miscible or immiscible with water, and can evaporate before they chemically degrade. The hydrophobicity of a compound can be expressed by the octanol-water partition coefficient (Kow). Preferably, the bio-organic compounds or fermentation products have log Kow higher than 3, more preferably higher than 5. Preferably, the solubility in water of the bio-organic compounds or fermentation products is low, for instance lower than 3 g/L, 2g/L or less more particularly lower than 1 g/L, more preferably lower than 10 mg/L, even more preferably lower than 1 mg/L, such as about 0.1 mg/L or 0.01 mg/L. Solubility in water in this context generally refers to solubility at room temperature, i.e. 20°C. The methods of the invention are of interest for all compounds having low solubility in water.

Non-limiting examples of bio-organic compounds for which the methods described herein are particularly suitable include isoprenoids such as farnesene, which can be produced using genetically modified host cells as described in the international applications WO2013/071 172 and WO2014/144135, which are incorporated by reference herein.

In certain embodiments, the bio-organic compounds are hydrocarbons or hydrocarbon-rich molecules. In a further embodiment the hydrocarbons are preferably C₅-C₃₀ hydrocarbons. In certain embodiments, the hydrocarbons are terpenes, which refer to a class of unsaturated hydrocarbons that are composed of one or more isoprene (C₅H₈) units. In further embodiments, the terpenes are sesquiterpenes, which contain three isoprene units. In further embodiments, the bio-organic compounds are isoprenoids, preferably C₅-C₂o isoprenoids, more preferably Ci₀-C₂o isoprenoids. In certain embodiments, the isoprenoid is a Ci5 isoprenoid. These compounds are derived from terpenes containing three isoprene units (also called sesquiterpenes). Non-limting examples of sesquiterpenes are farnesene, periplanone B, gingkolide B, amorphadiene, artemisinin, artemisinic acid, valencene, nootkatone, epi-cedrol, epi-aristolochene, farnesol, gossypol, sanonin, periplanone, forskolin, and patchoulol (which is also known as patchouli alcohol). In preferred embodiments, the bio-organic compound is farnesene. In further embodiments, the bio- organic compound is a-farnesene, β-farnesene or a mixture thereof. Other embodiments of the bio-organic compounds for which the methods of the invention are suited are aldehydes, diglycerides, triglycerides, free fatty acids, in particular Ci₆-Ci₇ free fatty acids, or hydroxyalkanoates, e.g. polyhydroxyalkanoates or polyesters .

Overall recovery process

Provided herein are methods for recovering bio-organic compounds from a fermentation mixture. A fermentation mixture (also referred to herein as fermentor broth or whole cell broth (WCB)) typically comprises host cells such as micro-organisms, a culture medium and, once cultivation has started, the fermentation products or bio-organic compounds produced by the micro-organisms. These bio-organic compounds are preferably released or secreted by the micro-organisms in the culture medium as free bio-organic compounds, from which they may be recovered using conventional recovery processes including, but not limited to, chromatography, extraction, solvent extraction, membrane separation, electrodialysis, reverse osmosis, distillation, chemical derivatization and crystallization. Typically, the methods of recovery involve separation of the cellular fraction from the rest of the fermentation medium. This is in the art typically ensured by centrifugation, whereby a microbial pellet is generated and discarded, while the supernatant is used for further extraction of the bio-organic compound. Some of the bio-organic compounds of interest will remain at least in part associated to the microbial host cell during the standard recovery processes (referred to herein as cell-associated bio-organic compounds or cell-associated bio-organic compound fraction). These are typically lost in current microbial production processes with the microbial pellet.

Another source of loss of bio-organic compounds is due to emulsion formation, which is inherent to microbial production systems. Indeed, 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 (also referred to as the emulsion bio-organic compounds or emulsion bio-organic compound fraction).

Provided herein are recovery processes for bio-organic compounds from a fermentation mixture characterized in that they comprise the recovery of said cell-associated bio-organic compounds and/or said bio-organic compounds comprised in emulsions.

Accordingly, in an aspect, the invention comprises methods for the (improved) recovery of bio-organic compounds from a fermentation mixture, which methods comprise recovering the fraction of bio-organic compounds which are cell-associated bio-organic compounds and/or recovering the bio-organic compounds which are present in the fermentation medium in emulsions. Typically, as will be understood by the skilled person, these methods also comprise the recovery of the free bio-organic compounds or free bio-organic compound fraction by traditional methods, though this will not be considered in detail herein.

In particular embodiments, the methods of the invention comprise a solid/liquid separation step.

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 host cells such as the micro-organisms and cell-associated bio-organic compounds, and may further comprise host cell debris, culture medium and bio-organic compounds. The solid/liquid heavy phase is preferably a liquid stream. The supernatant or light phase obtained by solid/liquid separation of the fermentation mixture, also referred to herein as "concentrated clarified broth (CCB)", 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. 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.

In embodiments, solid/liquid separation separates the fermentation mixture into a solid/liquid heavy phase comprising less than 3 vol%, preferably less than 1 vol%, free bio- organic compounds and a solid/liquid light phase. In embodiments, solid/liquid separation separates the fermentation mixture into a solid/liquid light phase comprising less than 10 vol%, preferably less than 1 vol% host cells (micro-organisms) and a solid/liquid heavy phase. In embodiments, solid/liquid separation separates the fermentation mixture into a solid/liquid heavy phase comprising less than 3 vol%, preferably less than 1 vol%, free bio- organic compounds and a solid/liquid light phase comprising less than 10 vol%, preferably less than 1 vol%, host cells. The volumetric composition of the light and/or heavy phases produced can be suitably determined by the skilled person, e.g. by taking a sample and spinning it in a capillary tube in a laboratory centrifuge.

In particular embodiments, the methods further involve recovering cell-associated bio- organic compounds from the solid/liquid heavy phase, and/or recovering bio-organic compounds comprised in an emulsion from the solid/liquid light phase.

In particular embodiments, the methods comprise the steps of providing a fermentation mixture comprising host cells (micro-organisms), a culture medium and the fermentation products or bio-organic compounds produced by the host cells (micro-organisms); performing a solid/liquid separation of the fermentation mixture into a heavy phase and a light phase; and recovering cell-associated bio-organic compounds from the solid/liquid heavy phase, and/or recovering bio-organic compounds comprised in an emulsion from the solid/liquid light phase. It will however be understood by the skilled person that, while it is of general interest to separate the host cells (micro-organisms) from the fermentation mixture for the recuperation of the free bio-organic compounds, it can be envisaged that the methods of the present invention could also be carried out without separating the micro-organisms from the fermentation medium. More particularly, the methods of the present invention, in particular the methods relating to the recovery of bio-organic compounds contained in an emulsion, may also be considered on the whole fermentation medium.

Recovery of cell-associated bio-organic compounds

The present inventors have realized a new method for recovering bio-organic compounds that are associated to a host cell producing the organic compound (e.g. located in an inner cell compartment or adsorbed on the cell wall), which method does not require the use of chemicals, but relies on a drying process. Also advantageously, the method allows recovering cellular material as a dried stream with potential commercial value, and drastically reduces the amount of solids and bio-organic compounds in waste streams (such as in vinasse, which is a by-product of the sugar industry), which may help meet environmental regulations.

This aspect focuses on the recovery of bio-organic compounds from a cellular fraction of the fermentation medium, i.e. a fraction which contains the host cells such as the microorganisms. It will be understood that the methods can be performed either on the fermentation medium as such, i.e. comprising the cellular material as well as the non- cellular material, or on a cellular fraction of the fermentation medium, i.e. comprising a concentrated amount of cellular material. For instance, the cellular fraction can be the solid/liquid heavy phase of a solid/liquid separation carried out on the fermentation medium.

The methods for the recovery of cell-associated bio-organic compounds envisaged herein comprise drying the fermentation mixture comprising the cellular material or a cellular fraction thereof to a gaseous phase and recovering the bio-organic product therefrom. Accordingly, in an aspect, the invention provides methods for the recovery of cell- associated bio-organic compounds from a fermentation mixture, comprising the steps of (a) drying a fermentation mixture or a cellular fraction thereof, thereby generating a gaseous phase; and (b) recovering the cell-associated fermentation product from the gaseous phase obtained in step (a).

The drying step in the methods envisaged herein is performed under conditions suitable for evaporating the solvent (e.g. water) and cell-associated bio-organic compound from the fermentation mixture or a cellular fraction thereof. Thus, the drying step comprises the transition of the solvent and the cell-associated bio-organic compound from a liquid to a gas phase. More particularly, the drying step is performed under conditions that not only free solvent, but also bound solvent, and cell-associated bio-organic compound are removed from the fermentation mixture or the cellular fraction thereof. Suitable drying conditions can be determined by the skilled person.

Preferably, the drying step results in a gaseous phase and a dried phase, the dried phase having a residual moisture content below 10 wt%, more preferably below 5 wt%.

In particular embodiments, a two-step drying process is applied, wherein the feed stream, in particular the fermentation mixture or a cellular fraction thereof, is first dried through application of direct heat transfer, and subsequently subjected to indirect heat transfer- based drying. In particular embodiments, the drying step is performed using a combination of contact drying and fluidized bed drying.

For contact drying, a thin film dryer can be used, such as a horizontal thin film dryer, wherein heat transfer is direct from a heated shell to a feed stream that is applied as a thin film along the heated dryer wall. The dryer may be provided with a screw to help solids to be easily dried and pushed out of the vessel once this done and to help solids not to be stuck on the dryer shell.

For fluidized bed drying, the CFT dryer of Buss-SMS-Canzler can be used. The dryer is filled with dry, pre-loaded cellular material containing feed, which is fluidized, e.g. by a rotor. At the beginning of the process, the CFT vessel or dryer is nearly full of dry cellular material containing feed. The shell is heated, and heat is transferred to the dry, pre-loaded cellular material containing feed. Once the vessel and the dry cellular material are at the right temperature and pressure; wet, fresh feed (i.e. fermentation mixture comprising the cellular material or a cellular fraction thereof) injection starts. The wet feed is encapsulated by and distributed throughout the hot dry feed and dried efficiently. Thus, heat transfer is indirect from the shell to the dry, pre-loaded cellular material containing feed, which transfers heat to the fresh, wet feed. Residence time, temperature and pressure determine the final moisture content and suitable conditions can be determined by the skilled person.

As the gaseous phase contains the organic compound of interest, a next step comprises the recovery of the organic compound from the gaseous phase. In particular embodiments, the recovery of the bio-organic compound from the gaseous phase obtained in the drying step, is performed by condensing the gaseous phase and separating the bio-organic compound from the condensate. Techniques for the condensation of a gaseous phase are well known in the art, and include, for example, cooling the gas. The temperature for the condensation process depends on the physical properties of the bio-organic compound. In certain embodiments, the pressure of the gaseous phase is increased for the condensation. The condensation process may be facilitated at higher gas pressure.

Recovery of the bio-organic compound from the condensate may be achieved through classical recovery processes. The absence of surfactants or other chemicals in the condensate makes it easier to recover the fermentation product from the condensate. For fermentation products that are immiscible with the solvent (e.g. farnesene and water), the condensate typically generates a biphasic liquid system, which allows easy separation, e.g. by decantation. Accordingly, in embodiments, the bio-organic compound, in particular a bio- organic compound that is poorly miscible or immiscible with the solvent, is separated from the condensate by decantation. The bio-organic compound may also be selectively removed, e.g. by using a membrane, adsorption on a solid, absorption with a liquid solvent, etc.

In particular embodiments, the fermentation mixture or the cellular fraction thereof is concentrated prior to the drying step by (partial) removal of free solvent (e.g. water), and the concentrate is subjected to the drying step. Such concentration step advantageously reduces the amount of energy required for the drying process.

Accordingly, in embodiments a method is provided for the recovery of cell-associated bio- organic compounds from a fermentation mixture, comprising the following steps:

(a) concentrating a fermentation mixture or a cellular fraction thereof; (b) drying the concentrate obtained in step (a), thereby generating a gaseous phase; and (c) recovering the cell-associated fermentation product from the gaseous phase obtained in step (b).

Preferably, the concentration step is performed under conditions suitable for removing free solvent (e.g. water) from the fermentation mixture or the cellular fraction thereof, without losing cell-associated fermentation product. As used herein, "free solvent" refers to solvent that can be easily removed e.g. by decanting. In embodiments, the concentration step is performed under conditions suitable for separating the fermentation mixture or the cellular fraction thereof into a liquid or light phase containing less than 5% (v/v) solids, preferably less than 3% (v/v) solids, and a solid phase (i.e. the concentrate). In embodiments, the concentration step results in a concentrate having a free solvent content below 3 wt%, more preferably below 1 wt%, most preferably a concentrate without free solvent. In embodiments, the concentration step results in a concentrate having a free solvent content below 3 wt%, more preferably below 1 wt%, most preferably a concentrate without free solvent, and a liquid or light phase containing less than 5% (v/v) solids, preferably less than 3% (v/v) solids.

The concentration step may be performed using a centrifuge such as a decanter centrifuge, by evaporation, by using membranes as described elsewhere herein, by drying, etc. In embodiments, the concentration step is performed using a decanter centrifuge. The operation conditions, e.g. flow rate of the fermentation mixture or a cellular fraction thereof, of the decanter centrifuge depend on the bio-organic compound that is produced and can be determined by the skilled person.

In particular embodiments, the methods envisaged herein above may also comprise the step of recovering free bio-organic compounds from the light phase obtained by solid/liquid separation of the fermentation mixture as described above. Methods for recovering free bio-organic compounds from the light phase are known in the art as detailed above. In addition, according to particular embodiments, the methods for recovering the cell- associated bio-organic products as taught herein are combined with methods for the recovery of bio-organic compounds from the light phase with a higher yield (i.e. including recovery from emulsion) such as those described herein below. In still further embodiments, the methods for recovering the cell-associated bio-organic products as taught herein are combined with methods for the recovery of free bio-organic compounds and bio-organic compounds contained in an emulsion such as those described herein below

Recovery of host cell material

The dried phase, which typically comprises host cells and host cell debris, obtained by drying a fermentation mixture or a cellular fraction thereof, or a concentrate of said fermentation mixture or said cellular fraction thereof, is a valuable co-product of the methods envisaged herein. The dried host cell material, e.g. the dried yeast, can be used as animal feed, in particular cattle feed. The dried host cell material may also be processed further, e.g. to extract bio-organic compounds. Accordingly, also disclosed herein is a method for recovering host cell material from a fermentation mixture comprising drying a fermentation mixture or a cellular fraction thereof under conditions suitable for removing solvent and cell-associated fermentation product from the fermentation mixture or the cellular fraction thereof, wherein the dried fermentation mixture or the cellular fraction thereof comprises the host cell material. More particularly in these methods, the host cell material is microbial material In further embodiments, a method for recovering microbial material from a fermentation mixture is provided comprising the following steps:

(a) concentrating a fermentation mixture or a cellular fraction thereof by removal of free solvent; and

(b) drying the concentrate obtained in step (a) under conditions suitable for removing solvent and cell-associated fermentation product from the concentrate,

wherein the dried concentrate obtained in step (b) comprises the microbial material.

Further disclosed herein is (dried) microbial material obtainable by such method. Said microbial material preferably contains less than 5 wt% solvent, and less than 1 wt% fermentation product, and is more preferably free of solvent and fermentation product.

Recovery of bio-organic compounds from an emulsion

The present inventors have further realized methods for (improving the) recovery of bio- organic products from a fermentation mixture by recovering bio-organic compounds present in emulsions. More particularly, the methods provided herein involve destabilizing emulsions that may be present in the fermentation mixture thereby enabling recovery of fermentation product comprised in said emulsions, without the need of chemicals, in particular surfactants. This reduces costs, and eliminates downstream processing to remove the surfactant. Moreover, the methods of the invention destabilize emulsions that are breakable with a surfactant as applied in current processes, as wells as emulsions that are not breakable with a surfactant, and hence, allow to recover more fermentation product compared to current processes. Indeed, the inventors have found that bio-organic compounds can be readily recovered from emulsions by the combination of a dewatering step with one or more evaporation steps.

According to this aspect the methods comprise a step of dewatering the fermentation mixture or fraction thereof to form a concentrated fermentation mixture or fraction thereof and evaporating the concentrated fermentation mixture in such a way as to obtain a distillate comprising the bio-organic compounds. It will be understood that the methods according to this aspect can be performed on complete fermentation mixture or on a fraction thereof, such as the solid/liquid light phase of a separation step as described above, which is essentially a non-cellular fraction.

Accordingly, in particular embodiments, the invention provides methods for recovering bio- organic compounds from a fermentation mixture, said method comprising the steps of concentrating or dewatering the fermentation mixture to form a concentrated fermentation mixture, evaporating the concentrated fermentation mixture in an evaporator, and recovering the distillate from the evaporator to obtain the bio-organic compound.

Optionally, the methods described herein may further comprise steps to remove products which will interfere with the efficient recovery of the bio-organic compounds from the emulsions. More particularly these steps are the clarification of the fermentation mixture prior to the dewatering step. A clarifying step may be performed to remove (residual) host cells from the fermentation mixture or the fraction thereof. Accordingly, a clarified fermentation mixture as referred to herein is substantially free of host cells. The clarifying step may occur by liquid/solid separation, e.g. liquid/solid separation as described elsewhere herein, by sedimentation followed by decantation, by filtration, by centrifugation, in a continuous disk stack nozzle centrifuge, or any combination thereof.

The methods envisaged herein comprise concentrating the fermentation mixture or a fraction thereof into a concentrated fermentation mixture or fraction thereof, thereby reducing the volume for subsequent downstream processing. In embodiments, the concentrated fermentation mixture or fraction thereof comprises about 50% of the volume of the fermentation mixture or the fraction thereof, preferably the concentrated fermentation mixture or fraction thereof is at most about 40%, 35%, or 30%, more preferably at most about 25%, 20%, or 15%, still more preferably at most about 10%, 5%, 4%, 3%, 2% or 1 % of the volume of the fermentation mixture or the fraction thereof. The concentration step may occur for example, but without limitation, by means of one or more membranes, by centrifugation, or by means of a decanter.

In embodiments, the concentration step occurs by means of one or more membranes, in particular water-permeable membranes. In particular embodiments, the concentration step occurs by means of one or more ceramic membranes, in particular water-permeable ceramic membranes. In particular embodiments, the concentration step occurs by means of one or more membranes, in particular ceramic membranes, more particularly water- permeable (ceramic) membranes, with a pore size between about 20 nm and about 1000 nm, preferably between about 40 nm and about 800 nm, more preferably between about 40 nm and about 200 nm, such as about 50 nm, about 100 nm, or about 200 nm.

Without wanting to be bound by any theory, it is contemplated that water permeation through the membrane decreases water content upstream of the membrane, which leads to (i) progressive apparition of a phase enriched in bio-organic compound, such as farnesene, upstream of the membrane, (ii) collapse of the emulsion and (iii) subsequent separation of water downstream of the membrane and bio-organic compound, such as farnesene, upstream of the membrane. Hence, emulsion breaking by means of a membrane relies on a succession of (i) application of physical forces that lead to (ii) change in chemical composition, which in turn leads to a collapse of the emulsion. In contrast, conventional processes to break emulsions rely on either (i) chemical interaction (e.g. use of surfactants), (ii) application of physical forces, or (iii) chemical interaction followed by application of physical forces.

Accordingly, a related aspect disclosed herein is directed to the use of a membrane for breaking an emulsion, wherein the membrane is permeable for one of the phases of the emulsion. The membrane may be permeable for the continuous phase of the emulsion or for the dispersed phase.

In other embodiments, the concentration step of the methods disclosed herein occurs by centrifugation.

In particular embodiments of the methods provided herein, the clarification and concentration step occurs simultaneously. For instance, in embodiments wherein the concentration step occurs by means of membranes, the concentrated fermentation mixture or fraction thereof is substantially free of host cells (i.e. is clarified). Advantageously, this allows to obtain waste water streams without host cells, which may be desirable in order to meet environmental regulations.

The methods provided herein may further comprise the step of evaporating the residual concentrated fermentation mixture or fraction thereof so as to obtain a distillate containing the bio-organic compounds. The evaporation is preferably performed in a two-steps process, wherein first the solvent, e.g. water, is evaporated, followed by evaporation of the bio-organic compound. These steps can be subsequently carried out in a single evaporator, or two different evaporators can be used.

A non-limiting example of an evaporator that is suitable for use in the methods described herein is a wiped film evaporator. The evaporation conditions depend on the type of solvent and bio-organic compound and can be determined by the skilled person. For example, evaporation of water can be conducted at a pressure between about 40 mbar and about 55 mbar, preferably between about 45 mbar and about 50 mbar, more preferably at a pressure of about 50 mbar, and a temperature of between about 50 °C and about 120 °C, preferably between about 60 °C and about 1 10 °C, more preferably at a temperature of about 1 10 °C. Evaporation of farnesene can be conducted at a pressure between about 1 mbar and about 5 mbar, preferably at a pressure of about 2 mbar, and a temperature of between about 50 °C and about 120 °C, preferably between about 60 °C and about 1 10 °C, more preferably at a temperature of about 1 10 °C.

In said first evaporation (residual) solvent (e.g. water) is removed from the concentrated fermentation mixture or fraction thereof. Preferably, the first evaporation results in a non- evaporated fermentation mixture or fraction thereof with a solvent content below 1 wt%, more preferably below 0.5 wt%. The solvent content can be measured by well-known methods, such as by Karl Fischer titration. The distillate of the first evaporation is thus mainly composed of solvent (e.g. water). The distillate may further comprise some bio- organic compound, and is further characterized by the absence of surfactants or other emulsifying agents, which allows easy recovery of the bio-organic compounds contained therein. The residual or non-evaporated concentrated fermentation mixture or fraction thereof is then subjected to a second evaporation, wherein the bio-organic compound is evaporated. The distillate of the second evaporator evaporation is mainly composed of bio- organic compound, and is further characterized by the absence of surfactants or other emulsifying agents, which allows easy recovery of the bio-organic compounds contained therein. In embodiments, bio-organic compound is recovered from the distillate of the second evaporation. In embodiments, bio-organic compound is recovered from the distillate of the first evaporation and the second evaporation. In certain embodiments, the distillates of the first and the second evaporation are mixed and the bio-organic compound is recovered from said mixture of distillates.

Recovery of bio-organic compounds from the distillate(s) of the evaporation(s) can be achieved by means of a condenser, a coalescer, a decanter, or using a membrane, adsorption on a solid, absorption with a liquid solvent, etc.

Any residual bio-organic compound that remains non-evaporated in the (second) evaporator (i.e. present in the bottom of said evaporator), may be recovered by feeding said stream to a dryer, where residual bio-organic compound can be recovered. Said drying step comprises the transition of the solvent and the bio-organic compound from a liquid to a gas phase, and may be performed e.g. using a combination of contact and fluidized bed drying as described elsewhere herein. The recovery of the bio-organic compound from the gaseous phase obtained in the drying step may be performed by condensing the gaseous phase and separating the bio-organic compound from the condensate, e.g. through classical recovery processes as indicated before. For fermentation products that are immiscible with the solvent (e.g. farnesene in water), the condensate typically generates a biphasic liquid system, which allows easy separation, e.g. by decantation or cooling. Accordingly, in particular embodiments, methods are provided which comprise the steps of: providing a fermentation mixture or a fraction thereof, said fermentation mixture or said fraction thereof comprising an emulsion comprising bio-organic compounds; optionally clarifying the fermentation mixture or the fraction thereof,

- concentrating or dewatering the (clarified) fermentation mixture or fraction thereof to form a concentrated fermentation mixture or fraction thereof,

evaporating the concentrated fermentation mixture or fraction thereof in a first evaporation,

evaporating the residual concentrated fermentation mixture or fraction thereof from the first evaporation in a second evaporation, and

recovering the distillate from the second evaporation to obtain the bio-organic compound, and

optionally recovering bio-organic compound from the distillate from the first evaporation.

As detailed above, the methods provided herein may be performed on a liquid phase of a liquid/solid separation step performed on the fermentation mixture.

Accordingly, in particular embodiments, methods for the recovery of bio-organic compounds from a fermentation mixture, said method comprising the following steps:

providing a fermentation mixture comprising micro-organisms, a culture medium and the fermentation products or bio-organic compounds produced by the microorganisms;

performing a solid/liquid separation of the fermentation mixture into a heavy phase comprising the micro-organisms and a light phase;

optionally clarifying the solid/liquid light phase;

- concentrating or dewatering the (clarified) solid/liquid light phase to form a concentrated solid/liquid light phase, preferably by means of one or more membranes as taught herein;

evaporating the concentrated solid/liquid light phase in a first evaporation;

evaporating the residual concentrated solid/liquid light phase from the first evaporator in a second evaporation;

recovering the distillate from the second evaporation to obtain the bio-organic compound; and

optionally, recovering bio-organic compound from the distillate from the first evaporation. Again, it will be understood that the methods provided herein for the recovery of bio-organic compounds present in emulsions of fermentation mixture may be combined with the classical methods to recover free bio-organic compounds from the fermentation mixture and/or with methods for recovering cell-bound bio-organic compounds from the fermentation mixture. In these embodiments, the methods may comprise the step of combining said bio-organic compound recovered from the different fractions.

Accordingly, in particular embodiments methods are provided for the recovery of bio- organic compounds from a fermentation mixture, said method comprising the following steps:

- providing a fermentation mixture comprising micro-organisms, a culture medium and the fermentation products or bio-organic compounds produced by the microorganisms;

- optionally clarifying the solid/liquid light phase;

concentrating or dewatering the (clarified) solid/liquid light phase to form a concentrated solid/liquid light phase, preferably by means of one or more membranes;

evaporating the concentrated solid/liquid light phase in a first evaporation;

- evaporating the residual concentrated solid/liquid light phase from the first evaporation in a second evaporation;

recovering the distillate from the second evaporation to obtain the bio-organic compound;

optionally recovering bio-organic compound from the distillate from the first evaporation;

optionally concentrating the solid/liquid heavy phase by removal of free solvent; drying the solid/liquid heavy phase or the concentrated solid/liquid heavy phase, thereby generating a gaseous phase;

recovering the bio-organic compound, in particular cell-associated bio-organic compound, from the gaseous phase.

In embodiments, the residual or non-evaporated concentrated solid/liquid light phase from the second evaporation is also subjected to the drying step. This allows the recovery of any residual fermentation product that may still be present, and further enhancing the efficacy of the recovery process. Indeed, the integrated process combining the recovery of cell- associated bio-organic compounds from the solid/liquid heavy phase, and the recovery of bio-organic compounds, including bio-organic compounds comprised in an emulsion, from the solid/liquid light phase provides synergy as compared to the separate recovery processes.

In particular embodiments, the method is for the recovery of bio-organic compounds from a fermentation mixture, wherein the fermentation mixture (whole cell broth, WCB) is separated in a centrifuge in a liquid/solid light phase (concentrated, clarified broth, CCB) and a liquid/solid heavy phase (cell slurry). The method then comprises recovering the bio- organic compound from the light phase and/or the heavy phase. To recover the bio-organic product from the heavy phase, the liquid/solid heavy phase or cell slurry, which comprises yeast cells, cell-associated bio-organic product, water and water solubles, is concentrated to remove water and water solubles from the liquid/solid heavy phase, without loss of the bio-organic product of interest. The concentrate is then processed on a dryer. The gaseous phase composed of water and the bio-organic product of interest is recovered (and optionally mixed with the gas outlets of evaporators of the liquid/solid light phase, see below).

To recover the bio-organic compound form the liquid/solid light phase, the liquid/solid light phase (CCB) stream, which is composed of free bio-organic compound, water, water solubles, including emulsions comprising the bio-organic compound of interest, and some yeast, is dewatered using water-permeable ceramic membrane(s) or a centrifuge to form a concentrated liquid/solid light phase. The use of the membrane(s) allows for obtaining a waste water stream (the permeate) containing water and water solubles, but without yeast nor bio-organic product of interest, which is advantageous to meet environmental regulations and to reach maximal recovery yield of the bio-organic product of interest. The concentrated liquid/solid light phase is then fed to a (first) evaporator or pre-evaporator for evaporation. The distillate from said first evaporator is mainly composed of water, and may comprise some bio-organic product of interest. The bottom of said first evaporator (i.e. non- evaporated concentrated liquid/solid light phase) is fed to a second evaporator. Optionally, the bottom of said first evaporator may be fed to a polishing centrifuge to remove solids prior to said second evaporation. The gaseous phase from said second evaporator comprises the bio-organic product of interest. The bottom of said second evaporator is fed to a dryer (optionally the same dryer as above) so as to recover any residual bio-organic product that may still be trapped in it. In particular embodiments, the gaseous phases from the dryer, the first and second evaporator are combined and subjected to condensation, to recover the bio-organic product. In particular embodiments, the condensation results in a biphasic liquid system, from which the bio-organic product can easily be separated by decantation. Dried yeast cells (e.g. yeast cell wall and yeast debris) can be isolated from the solid outlet of the dryer. This stream can be used as cattle feed.

Accordingly, in particular embodiments, methods are provided for the recovery of bio- organic compounds from a fermentation mixture, said method comprising the following steps:

optionally clarifying the solid/liquid light phase;

- evaporating the concentrated solid/liquid light phase in a first evaporation; and recovering bio-organic compound from the distillate from the first evaporation;

evaporating the residual (or non-evaporated) concentrated solid/liquid light phase from the first evaporation in a second evaporation; and recovering the distillate from the second evaporation to obtain the bio-organic compound;

and

concentrating the solid/liquid heavy phase by removal of free solvent;

drying the solid/liquid heavy phase or the concentrated solid/liquid heavy phase in a drying step, thereby generating a gaseous phase; and recovering the bio-organic compound, in particular cell-associated bio-organic compound, from the gaseous phase.

In particular embodiments, the distillate from the first and second evaporation step and the gaseous phase from the drying step are combined to recover the bio-organic product.

In particular embodiments of the methods described above, the bio-organic product is a hydrocarbon or hydrocarbon-rich molecule, such as a C₅-C₃o hydrocarbons or C₅-C₃o hydrocarbon-rich molecules, more particularly a C₅-C₂o isoprenoid or a Ci₀-C₂o isoprenoid. In certain embodiments, the bio-organic product is an isoprenoid, in particular a Ci₅ isoprenoid. In certain embodiments, the bio-organic product is a sesquiterpene. In further particular embodiments, the bio-organic compound is farnesene, such as a-farnesene, β- farnesene or a mixture thereof. The bio-organic compound may also be a free fatty acid, in particular a Ci₆-Ci₇ free fatty acid, or a hydroxyalkanoate.

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

EXAMPLES

Example 1 : Recovery of farnesene from a fermentation mixture

Figure 1 shows a schematic representation of a process for the recovery of farnesene from a fermentation mixture according to an embodiment of the present invention. The fermentation mixture (whole cell broth, WCB) is separated in a disk stack centrifuge with nozzles in a light phase (concentrated, clarified broth, CCB) and a heavy phase (cell slurry).

The liquid/solid heavy phase or cell slurry, which comprises yeast cells, cell-associated farnesene, water and water solubles, is concentrated on a decanter centrifuge to remove water and water solubles from the liquid/solid heavy phase, without farnesene losses. The concentrate of the decanter is then processed on a dryer, e.g. using the Combi Fluidization Technology (CFT, Buss-SMS-Canzler, Pratteln, Switzerland). The gaseous phase composed of water and farnesene is then mixed with the gas outlets of the evaporators, which mixture is condensed, resulting in a biphasic liquid system, from which the farnesene can easily be separated by decantation. Dried yeast cells (e.g. yeast cell wall and yeast debris) are isolated from the solid outlet of the dryer. This stream can be used as cattle feed.

The liquid/solid light phase (CCB) is composed of free farnesene, water, water solubles, including emulsions comprising farnesene, and some yeast. Said liquid/solid light phase stream is dewatered using water-permeable ceramic membranes to form a concentrated liquid/solid light phase. The use of the membrane allows for obtaining a waste water stream (the permeate) containing water and water solubles, but without yeast nor farnesene, which is advantageous to meet environmental regulations and to reach maximal farnesene recovery yield. The concentrated liquid/solid light phase is then fed to a first evaporator or pre-evaporator, in particular a wiped-film evaporator, for evaporation. The distillate from said first evaporator is mainly composed of water, and may comprise some farnesene. The bottom of said first evaporator is fed to a second evaporator, in particular a wiped-film evaporator. Optionally, the bottom of said first evaporator may be fed to a polishing centrifuge to remove solids prior to said second evaporation. The gaseous phase from said second evaporator comprises farnesene. The bottom of said second evaporator is fed to the CFT dryer so as to recover any residual farnesene that may still be trapped in it. The gaseous phases from the CFT dryer, the first and second evaporator are combined and subjected to condensation, e.g. by means of a coalescer and decanter, to recover the farnesene.

It is observed that a significant portion of farnesene is recovered from this fraction. Indeed, a mass balance calculation (using a computer simulation) shows that the process can provide 95 wt% farnesene recovery yield or higher, whereas a yield of between 70 and 90% is achieved with current processes. A similar calculation can be made for other terpenes, polyhydroxyalkanoids and free fatty acids, of which a significant fraction which similarly adheres to the cells during production. The process not only allows to increase yield of these products but eliminates the consumption of surfactants, which reduces costs. Furthermore, the dried host cells obtained from the drying step may be a valuable co- product of the process.

Claims

1 . A method for the recovery of one or more fermentation products with a solubility in water lower than 1 g/L from a fermentation medium comprising the following steps:

2. The method of according to claim 1 , wherein the one or more fermentation products are hydrocarbons, fatty acids or polyalkanoates.

3. The method according to claim 1 or 2, wherein the one or more fermentation products are hydrocarbons, preferably terpenes, more preferably sesquiterpenes.

4. The method according to claim 3, wherein the fermentation product is farnesene.

5. The method according to any one of claims 1 to 4, wherein the one or more fermentation products are cell-associated fermentation products.

6. The method according to any one of claims 1 to 5, wherein said concentrate contains less than 3 wt% (v/v) free solvent, preferably less than 1 wt% (v/v) free solvent.

7. The method according to any one of claims 1 to 6, wherein the concentration step (a) is performed using a decanter centrifuge.

8. The method according to any one of claims 1 to 7, wherein step (b) is performed using a combination of contact and fluidized bed drying.

9. The method according to any one of claims 1 to 8, wherein step (c) is performed by condensing the gaseous phase and separating the one or more fermentation products from the condensate.

10. The method according to claim 9, wherein said separation of the one or more fermentation products from the condensate is performed by decanting the condensate.

1 1 . The method according to any one of claims 1 to 10, wherein said cellular fraction is obtained following solid/liquid separation of the fermentation mixture into a solid/liquid heavy phase and a solid/liquid light phase, wherein said solid/liquid heavy phase comprises the cellular fraction.

12. The method according to claim 1 1 , wherein said solid/liquid separation is performed by means of a centrifuge.

13. The method according to any one of claims 1 1 or 12, further comprising recovering said one or more fermentation products from the solid/liquid light phase.

14. The method according to claim 13, wherein said one or more fermentation products are recovered from the solid/liquid light phase by the following steps:

optionally clarifying the solid/liquid light phase,

concentrating the solid/liquid light phase or the clarified solid/liquid light phase to form a concentrated solid/liquid light phase,

- evaporating the concentrated solid/liquid light phase in a first evaporation,

evaporating the residual concentrated solid/liquid light phase from the first evaporation in a second evaporation,

recovering the one or more fermentation products from the gaseous phase from the second evaporation, and

- optionally recovering said one or more fermentation products from the gaseous phase from the first evaporation.

15. The method according to claim 14, wherein the residual concentrated solid/liquid light phase from the second evaporation is subjected to the drying step (b).

16. The method according to claim 14 or 15, wherein the solid/liquid light phase or the clarified solid/liquid light phase is concentrated by means of one or more membranes, preferably ceramic membranes.

17. A method for the recovery of one or more fermentation products from a fermentation medium comprising the following steps:

evaporating the concentrate in a first evaporation,

optionally recovering the one or more fermentation products from the gaseous phase from the first evaporation;

18. A method for the production of an organic compound, comprising the following steps:

(i) providing a genetically engineered micro-organism capable of producing the organic compound;

(ii) fermenting a fermentable medium using said micro-organism; and

(iii) recovering the fermentation product from the fermentation mixture by a method according to any one of claims 1 to 17.