Method for obtaining a solution of volatile fatty acids, solution obtained, and use for the production of polyhydroxyalkanoates

Abstract

The invention relates to a method for treating a mixture obtained from organic waste by anaerobic digestion, making it possible to obtain a particular volatile fatty acid (VFA) solution. The invention also relates to the VFA solution obtained and to the use thereof for producing PHAs, in particular high-quality PHBVs.

Description

[0001] PROCESS FOR OBTAINING A SOLUTION OF VOLATILE FATTY ACIDS, SOLUTION OBTAINED AND USE FOR THE PRODUCTION OF POLYHYDROXYALKANOATES

[0002] Technical field

[0003] The present invention relates to the use of organic waste to produce a solution of volatile fatty acids, particularly suitable for the manufacture of quality polyhydroxyalkanoates.

[0004] The invention relates in particular to a method for treating a mixture obtained by anaerobic digestion of organic waste to obtain a solution of volatile fatty acids. The invention also relates to the solution of volatile fatty acids obtained and its use for the production of polyhydroxyalkanoates.

[0005] Prior art

[0006] Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters that can be produced by fermentation by microorganisms, including bacteria. Depending on the microorganisms used and the processes implemented, PHAs can be produced from a variety of raw materials, including sugars, starches, and lipids, but also from volatile fatty acids (VFAs), which are primarily produced petrochemically.

[0007] PHAs have a wide range of potential applications, including the production of bioplastics, packaging materials, medical devices, textile fibers, plastic parts, cosmetic products, objects or parts manufactured by extrusion, injection molding or 3D printing, or in the field of encapsulation.

[0008] However, current PHA manufacturing processes are not suitable and have many problems that prevent their large-scale implementation.

[0009] One obstacle is the low yield of these processes, which makes it difficult to produce large volumes of PHA at a competitive cost compared to traditional petroleum-based plastics.

[0010] In addition, the organic materials used for fermentation by microorganisms intended to produce PHAs generally come from areas and crops dedicated to human food (soy, sugar cane, rapeseed, etc.), which makes the production of PHAs unviable for meeting global demand and sustainably replacing petrochemicals.

[0011] More recently, solutions have been proposed to produce PHAs from volatile fatty acids obtained by anaerobic digestion of organic waste. Since organic waste is a local, renewable, and abundant raw material at very low cost, these solutions represent a more sustainable way to produce PHAs.

[0012] However, currently, there are very few cost-effective commercial technologies implementing the production of PHA from organic waste. The main reason is the fact that organic waste is an inpure raw material, which leads to poor yields, the production of impure PHA of poor quality and / or color, and high production costs. Furthermore, recent processes existing on the market have problems of quality, yield and reproducibility, particularly related to the fact that these processes do not take into account the fact that organic waste is a very heterogeneous fermentation substrate.

[0013] Therefore, while PHAs have the potential to be a sustainable and environmentally friendly alternative to traditional plastics, there remains a significant need for a solution that enables the cost-effective production of high-quality, sustainable PHAs.

[0014] The objective of the invention is therefore to meet all of these needs and to overcome the drawbacks and limitations of the prior art.

[0015] Summary of the invention

[0016] To meet this objective, the invention proposes a method making it possible in particular to manage the heterogeneity of organic waste in order to improve the quality of the products obtained and to reduce the production cost of AGVs and PHAs.

[0017] To this end, the invention relates to a method for treating a mixture obtained by anaerobic digestion of organic waste, comprising a step of treating said mixture by resin filtration and / or by activated carbon filtration and / or by membrane filtration so as to obtain a colorless liquid solution comprising volatile fatty acids (VFAs).

[0018] Preferably, the treatment by resin filtration and / or activated carbon and / or membrane filtration is carried out at a pH greater than or equal to 6, in particular greater than or equal to 6.8. Such a pH is important to obtain better filtration by resin and / or activated carbon and / or membrane filtration. It also has the advantage of being able to more easily concentrate the solution in VFA.

[0019] Advantageously, the treatment method according to the invention makes it possible to obtain a liquid solution comprising at least 10 g / L of AGV, colorless and stable regardless of the organic waste used.

[0020] The invention also relates to a colorless liquid solution having a pH greater than or equal to 6 and comprising at least 10 g / L of VFA, capable of being obtained by the treatment method according to the invention. The VFA solution according to the invention preferably comprises at least one VFA chosen from acetic, propionic, butyric, isobutyric, isovaleric, valeric, caproic acids and mixtures thereof.

[0021] According to one embodiment, the AGV solution according to the invention preferably comprises at least the following AGVs: acetic acid, propionic acid and butyric acid.

[0022] Advantageously, the method according to the invention makes it possible to treat any type of organic waste to obtain a colorless VFA solution having characteristics allowing it to be used in particular for the large-scale production of quality PHA. The yield and properties of the PHA depend on the quality of the VFA solution used to produce them, and the invention thus makes it possible to remove the problems of quality defects as well as the financial and supply risks linked to the raw materials of existing processes.

[0023] The invention therefore also relates to a process for producing polyhydroxyalkanoates (PHA) from organic waste comprising the implementation of the following steps:

[0024] *obtaining a mixture by anaerobic digestion of organic waste,

[0025] *treatment of the mixture obtained in step 1) by the process of treating a mixture obtained by anaerobic digestion of organic waste, and

[0026] *transformation of the AGVs in the solution obtained in step 2) into PHA by at least one halophilic bacterium.

[0027] Advantageously, halophilic bacteria are bacteria that do not require a sterile medium, which also helps reduce costs. In addition, the use of halophilic bacteria allows for simpler and less expensive PHA extraction than conventional technologies. Cell lysis is achieved by a simple osmotic shock with water (and possibly a surfactant to reduce the viscosity of the medium), whereas conventional PHA extraction methods, such as:

[0028] - Solubilization / filtration / precipitation with the use of large volumes of organic solvents (e.g. chloroform, dichloromethane, ethanol, methanol, dimethyl carbonate, etc.)

[0029] - Mechanical extraction using ball mills, high pressure homogenizers, ultrasonication,

[0030] - Use of surfactant, enzymes, acids / bases (e.g. sodium hypochlorite), hydrogen peroxide; or

[0031] - Use of supercritical fluid, are expensive in electricity and / or chemicals. The methods for treating a mixture of AGV and obtaining PHA according to the invention are implemented from a mixture obtained by anaerobic digestion of organic waste. According to a variant of the invention, it is preferred that the organic waste undergoes a pre-treatment before the anaerobic digestion step. This pre-treatment can be physical and / or chemical and / or biological. It can be chosen from thermal, ultrasound, microwave, high-pressure homogenizer, acid, basic, enzymatic, fungal, lactic pre-fermentation and the combination of at least two of these treatments. According to a preferred embodiment, it is a pre-treatment by lactic pre-fermentation.

[0032] Also, according to a particular aspect, the invention also relates to a process for producing AGV by anaerobic digestion comprising a lactic pre-fermentation step.

[0033] The invention is now described in detail with reference to the figures and non-limiting test results.

[0034] Brief description of the figures

[0035] [Fig. 1a] is a photograph of an AGV solution obtained by anaerobic digestion of grape marc without treatment according to the invention.

[0036] [Fig.lb] is a photograph of an AGV solution obtained by anaerobic digestion of grape marc after treatment according to the invention (pHIO treatment).

[0037] [Fig.2] is a representation of the comparison of two HPLC chromatograms: a VFA solution obtained by anaerobic digestion of grape marc without treatment according to the invention (bottom chromatogram) and a VFA solution obtained by anaerobic digestion of grape marc after treatment according to the invention (pHIO treatment) (top chromatogram) (ordinate = mAU intensity, abscissa = time in min).

[0038] [Fig.3] represents the monitoring of the growth of Haloferax mediterranei (by monitoring the Optical Density of the bacteria) using as culture medium a VFA solution obtained by anaerobic digestion of grape marc without treatment according to the invention, compared to the monitoring of the growth of Haloferax mediterranei (by monitoring the Optical Density of the bacteria) using as culture medium a VFA solution obtained by anaerobic digestion of grape marc after treatment according to the invention (treatment pH 10).

[0039] [Fig.4a] is a representation of the comparison of two IR spectra of PHBV obtained with a VFA solution obtained by anaerobic digestion of grape marc after treatment according to the invention (treatment pH 10) (Bottom spectrum) and reference PHBV (Top spectrum) (ordinate = %T, abscissa = cm-1). [Fig.4b] is a representation of the comparison of two IR spectra of PHBV obtained with a VFA solution obtained by anaerobic digestion of grape marc without treatment according to the invention (bottom spectrum) and PHBV obtained from a VFA solution obtained by anaerobic digestion of grape marc after treatment according to the invention (treatment pH 10) (Top spectrum) (ordinate = %T, abscissa = cm-1).

[0040] [Fig.5] is a photograph comparing PHBVs obtained with an AGV solution obtained by anaerobic digestion of grape marc without treatment according to the invention (bottom photograph) and PHBVs obtained from an AGV solution obtained by anaerobic digestion of grape marc after treatment according to the invention (pH 10 treatment) (top photograph).

[0041] [Fig.6] is a representation of the comparison of two HPLC chromatograms: a VFA solution obtained by anaerobic digestion of grape marc without treatment according to the invention (bottom chromatogram) and a VFA solution obtained by anaerobic digestion of grape marc after treatment according to the invention (pH9 treatment) (bottom chromatogram) (ordinate = mAU intensity, abscissa = time in min).

[0042] [Fig.7] represents the monitoring of the growth of Haloferax mediterranei (by monitoring the Optical Density of the bacteria) using as culture medium a VFA solution obtained by anaerobic digestion of grape marc without treatment according to the invention, compared to the monitoring of the growth of Haloferax mediterranei (by monitoring the Optical Density of the bacteria) using as culture medium a VFA solution obtained by anaerobic digestion of grape marc after treatment according to the invention (treatment pH 9).

[0043] [Fig.8a] is a representation of the comparison of two IR spectra of PHBV obtained with a VFA solution obtained by anaerobic digestion of grape marc after treatment according to the invention (pH 9 treatment) (lower spectrum) and reference PHBV (upper spectrum) (ordinate = %T, abscissa = cm-1).

[0044] [Fig.8b] is a representation of the comparison of two IR spectra of PHBV obtained with a VFA solution obtained by anaerobic digestion of grape marc without treatment according to the invention (lower spectrum) and of PHBV obtained from a VFA solution obtained by anaerobic digestion of grape marc after treatment according to the invention (upper spectrum) (treatment pH 9) (ordinate = %T, abscissa = cm-1).

[0045] [Fig.9] is a photograph comparing PHBVs obtained with an AGV solution obtained by anaerobic digestion of grape marc without treatment according to the invention (left photograph) and PHBVs obtained from an AGV solution obtained by anaerobic digestion of grape marc after treatment according to the invention (pH9 treatment) (right photograph).

[0046] [Fig.10] is a representation of the comparison of two HPLC chromatograms: a VFA solution obtained by anaerobic digestion of food waste without treatment according to the invention (bottom chromatogram) and a VFA solution obtained by anaerobic digestion of food waste after treatment according to the invention (pH9.5 treatment) (top chromatogram) (ordinate = mAU intensity, abscissa = time in min).

[0047] [Fig.11] is a photograph comparing PHBVs obtained with an AGV solution obtained by anaerobic digestion of food waste without treatment according to the invention (right photograph) and PHBVs obtained from an AGV solution obtained by anaerobic digestion of food waste after treatment according to the invention (pH9.5 treatment) (left photograph).

[0048] Detailed description of the invention

[0049] Definitions

[0050] “Volatile fatty acids or VFAs” means fatty acids with short carbon chains (ranging from 1 to 6 carbons).

[0051] For the purposes of the invention, “Acidogenesis” means a stage of anaerobic digestion, during which the amino acids and / or sugars and / or fats of the organic matter are transformed into VFAs.

[0052] For the purposes of the invention, "activated carbon" means a material with a porous structure consisting essentially of carbonaceous matter. The material has a strong adsorbent power due to its high specific surface area.

[0053] For the purposes of the invention, “organic waste” means waste comprising organic materials.

[0054] Anaerobic digestion is the natural process in which microorganisms break down organic matter in the absence of oxygen. It generally occurs in four phases: hydrolysis, acidogenesis, acetogenesis, and methanogenesis.

[0055] For the purposes of the invention, "activated carbon filtration" means a filtration process (e.g. sintered, scraper filter, vacuum and / or pressure filtration, etc.) where the AGV solution to be treated is in contact with activated carbon particles.

[0056] For the purposes of the invention, “lactic pre-fermentation” means a natural process during which microorganisms break down organic matter (particularly sugars) into lactic acid.

[0057] For the purposes of the invention, the term "absorbent resin" means a type of macroporous polymer. Commonly used for plant extraction, pharmaceutical purification and juice decolorization, these resins are designed to have a large exchange surface area as well as high porosity. For the purposes of the invention, the term "ion exchange resin" means a type of polymer that allows ion exchange. This type of resin is generally in the form of insoluble, porous microbeads with a large exchange surface area. There are four types of ion exchange resins, distinguished by the functional groups they carry, namely:

[0058] *Strongly acidic - carrying sulfonate group;

[0059] *Strongly basic - carrying quaternary ammonium group;

[0060] * Weakly acidic - carboxylate group carrier; and

[0061] *Weakly basic - carries primary / secondary / tertiary amine groups.

[0062] Process for treating a mixture obtained by anaerobic digestion of organic waste

[0063] The subject of the invention is therefore a method for treating a mixture obtained by anaerobic digestion of organic waste, comprising a step of treating said mixture by filtration through resin and / or activated carbon and / or by membrane filtration, so as to obtain a colorless liquid solution comprising VFAs.

[0064] The mixture obtained by anaerobic digestion of organic waste can be obtained by any means of implementing anaerobic digestion on organic waste.

[0065] Organic waste can be chosen from all organic waste. It is preferably chosen from the following organic waste: grape marc, organic food waste, organic agricultural waste, organic waste from the agri-food industry, carbon-containing organic waste and their mixtures. This may include in particular:

[0066] -household and restaurant food waste, such as fruit, starchy foods and vegetables,

[0067] - organic waste from agriculture or the agri-food industry such as brewery grains, fruit marc, particularly grape marc, green waste, waste from the dairy industry, harvest by-products,

[0068] -carbonaceous industrial waste such as paper pulp, organic effluents

[0069] -or combinations of these wastes.

[0070] The organic waste is preferably pre-ground before the anaerobic digestion step and / or undergoes one or more pre-treatment steps. This may be, for example, at least one pre-treatment chosen from thermal, ultrasound, microwave, high-pressure homogenizer, acid, basic, enzymatic, fungal treatment, lactic fermentation and the combination of at least two of these treatments. Preferably, before anaerobic digestion (before or after grinding), the organic waste is pre-treated by lactic pre-fermentation. Such a step advantageously makes it possible to increase the VFA content in the VFA solution and to reduce or eliminate problems of heterogeneity of the organic waste.

[0071] The invention therefore also relates to a method for obtaining an AGV solution from organic waste comprising an anaerobic digestion step and at least one step of pre-treatment of the organic waste by lactic pre-fermentation. The lactic pre-fermentation may be preceded or followed by a step of grinding the waste and / or by at least one other pre-treatment.

[0072] Pre-treatment by lactic pre-fermentation can be carried out with suitable microorganisms, in particular lactic acid bacteria. These can preferably be lactic acid bacteria such as lactobacilli and / or bifidobacter bacteria.

[0073] Pre-treatment by lactic fermentation can be carried out in a suitable device, preferably in an anaerobic digester. Pre-treatment by lactic pre-fermentation can be carried out with one or more of the following conditions, preferably all of them:

[0074] - under agitation,

[0075] - at a redox potential lower than -300mV, preferably between -550 mV and -400 mV,

[0076] - between 20 and 60°C,

[0077] - at a pH between 3 and 5,

[0078] - for at least 24 hours, preferably between 1 and 6 days.

[0079] Preferably, pre-treatment by lactic pre-fermentation includes a step of adjusting the pH between 3 and 5. This step makes it possible to stabilize the pH between 3 and 5, because the pH of the solution naturally becomes too acidic (less than 3) during the fermentation of sugars into lactic acid.

[0080] According to a particular embodiment, the lactic pre-fermentation step comprises the following steps:

[0081] - grinding, preferably in water, to obtain an aqueous solution comprising between 5 and 20% dry matter, in particular between 10 and 12% dry matter,

[0082] - introduction into an anaerobic digester for pre-fermentation,

[0083] - possibly inoculation of the reactor using lactic bacteria,

[0084] - adjustment of the pH between 3 and 5, preferably using sodium hydroxide solution, for example sodium hydroxide solution NaOH 30% v / v, - heating the medium between 20 and 40°C,

[0085] - after 2 to 8 days, preferably 3 to 5 days, recovery of a lactic acid solution containing pre-treated organic waste.

[0086] According to one variant, before anaerobic digestion, the process comprises at least one step of pre-treatment of organic waste by lactic pre-fermentation and at least one other pre-treatment step.

[0087] According to one embodiment, in particular if the organic waste has been pretreated, in particular by lactic pre-fermentation, a solid-liquid separation step can be implemented before anaerobic digestion to recover the liquid phase and eliminate the solid phase.

[0088] Anaerobic digestion can be carried out by inoculating organic waste, preferably previously ground and / or pretreated, with any type of suitable anaerobic microorganism, preferably any type of suitable anaerobic bacteria. These may include bacteria chosen from Clostridia (Clostridiaceae), Bacilli (Lactobacillaceae, Leuconostocacea, Streptococcacea), Propionobacter, Acetobacter and mixtures thereof. These may include, for example, Butirybacterium rettgeri, Pseudomonas aeruginosa, Clostridium acetobutycilium, Acetobacter woodii or a mixture of at least two of these bacteria. It is also possible to use anaerobic bacteria naturally present in existing mixtures, such as sludge from sewage treatment plants and / or animal manure such as cow manure and / or ruminant ruminant liquid.

[0089] According to one embodiment of the invention, the anaerobic digestion of organic waste is carried out by inoculating the organic waste, preferably previously crushed and / or pretreated, with a mixture of sludge from treatment plants and / or animal manure, preferably from cows.

[0090] Preferably, the pH and / or temperature of the mixture may be adjusted before and / or during the anaerobic digestion step, to be adapted to an ideal pH and / or temperature for the bacteria used. The pH of the solution may be adjusted by any suitable means, in particular by adding at least one acid such as hydrochloric acid and / or at least one base such as sodium hydroxide. The pH adjustment may be facilitated by introducing air into the solution in order to evaporate the dissolved CO2 and / or by aerating the solution by stirring in order to evaporate the dissolved CO2.

[0091] Anaerobic digestion is carried out at a redox potential below -300mV, preferably between -550mV and -400mV.

[0092] Anaerobic digestion can be carried out in any suitable device capable of achieving anaerobic conditions, preferably in a stirred anaerobic digester. The resulting mixture includes digested organic waste and VFAs.

[0093] According to the invention, this mixture is then treated by filtration through resin and / or activated carbon and / or by membrane filtration, so as to obtain a colorless liquid solution comprising VFAs.

[0094] According to one embodiment, the method according to the invention comprises a step of treating said mixture by membrane filtration and by filtration on resin and / or activated carbon, so as to obtain a colorless liquid solution comprising VFAs.

[0095] Preferably, the step of filtration on resin and / or activated carbon and / or by membrane filtration is carried out at a pH greater than or equal to 6, in particular a pH greater than or equal to 6.8, preferably greater than or equal to 7. It may be in particular between 6 and 12, between 6 and 11, between 6 and 10.5, between 6.8 and 12, between 6.8 and 11, between 6.8 and 10.5, between 7 and 12, between 7 and 11, between 7 and 10.5.

[0096] According to a preferred embodiment, the filtration step on resin and / or activated carbon and / or by membrane filtration is carried out at a pH between 6 and 9, in particular between 6 and 8.5, between 6 and 8.

[0097] Advantageously, such a pH allows the VFAs to be less easily captured by the resin and / or by the activated carbon and / or by membrane filtration while promoting the capture of other compounds, in particular compounds participating in the coloring of the medium.

[0098] Indeed, according to the invention, it is preferable to have such a pH because:

[0099] - at a pH greater than or equal to the pKa of the AGVs present in the mixture increased by 2 (pka AGV +2), the carboxylic acids are in the carboxylate form and therefore:

[0100] *VFAs are little or not captured by activated carbon

[0101] *AGVs are larger and therefore more easily concentrated during membrane filtration,

[0102] *AGVs are heavier and can therefore be more easily concentrated, particularly by evapoconcentration

[0103] - at a pH lower than or equal to the average pka value of the phenolic compounds present in the mixture reduced by 2 (pka Phenols -2), the phenols are in -OH form (and not in phenolate form) and are therefore more easily captured during filtration, particularly during filtration with activated carbon.

[0104] Thus, the method according to the invention may comprise, prior to resin filtration and / or activated carbon and / or membrane filtration, a step of adjusting the pH of the mixture obtained by anaerobic digestion of organic waste to a desired pH, said pH being greater than or equal to 6, preferably greater than or equal to 6.8, even more preferably greater than 7, in particular between 6 and 9.

[0105] The pH of the mixture may be adjusted by any suitable means, in particular by adding at least one acid such as hydrochloric acid and / or at least one base such as sodium hydroxide. The pH adjustment may be facilitated by introducing air into the mixture in order to evaporate the dissolved CO2 and / or by aerating the mixture by stirring in order to evaporate the dissolved CO2.

[0106] According to one embodiment, the resin filtration and / or activated carbon filtration and / or membrane filtration step is carried out at a pH greater than or equal to the average pKa value of the VFAs present in the mixture increased by 2 (pka VFA +2) and less than or equal to the average pka value of the phenolic compounds present in the mixture decreased by 2 (pka Phenols -2).

[0107] When the method comprises a step of treatment by activated carbon filtration, this step may consist of introducing activated carbon in powder or granular form. According to a suitable embodiment, the treatment by activated carbon filtration is carried out by adding between 0.1 and 5% by mass (weight / weight) of activated carbon in powder or granular form to the mixture, in particular between 0.2 and 5%, between 0.3 and 5%, between 0.5 and 5%, between 0.8 and 5%, between 1 and 5%.

[0108] According to one embodiment, the activated carbon filtration step is carried out by implementing the following sub-steps:

[0109] - adjustment of the pH of the mixture obtained by anaerobic digestion of organic waste, to a pH greater than or equal to 6,

[0110] - addition of activated carbon in powder form to the mixture, preferably between 0.1 and 5%,

[0111] - stirring, preferably between 100 and 1500 revolutions per minute, for a period of at least 1 minute, preferably for at least 5 minutes, in particular at least 10 minutes, in particular between 1 and 60 minutes,

[0112] - centrifugation, preferably between 5000 and 15,000g,

[0113] - possibly filtration, for example on Buchner or sintered glass, to eliminate any remaining suspended particles.

[0114] When the process includes an activated carbon filtration treatment step, this step may consist of using one or more activated carbon cartridges in the mixture. This may in particular be a carbon filter used in water treatment. The use of a cartridge or a carbon filter allows for faster in-line filtration. When the process includes a membrane filtration treatment step, it is preferably carried out by microfiltration and / or ultrafiltration and / or nanofiltration and / or reverse osmosis.

[0115] When the process includes a resin filtration treatment step, it is preferably carried out using an absorbent resin or an ion exchange resin.

[0116] Advantageously, the use of resin allows the specific capture of impurities present in the mixture obtained by anaerobic digestion of organic waste. In this way, the use of resin allows the said mixture to be filtered, eliminating the impurities.

[0117] The method of treatment according to the invention of a mixture obtained by anaerobic digestion of organic waste may comprise a step prior to resin filtration and / or activated carbon filtration and / or membrane filtration, which consists of separating the solid phase from the liquid phase of the mixture obtained by anaerobic digestion of organic waste. Thus the treatment method according to the invention may comprise, before resin filtration and / or activated carbon filtration and / or membrane filtration, a step of solid / liquid separation of a mixture obtained by anaerobic digestion of organic waste, to recover the liquid phase and eliminate the solid phase.

[0118] The method of treating according to the invention a mixture obtained by anaerobic digestion of organic waste may also comprise a solid / liquid separation step after the resin filtration step and / or activated carbon filtration and / or membrane filtration. This step is particularly useful during treatment with activated carbon if the activated carbon is in powder form, so as to remove the activated carbon from the AGV solution. Thus, the treatment method according to the invention may comprise, after resin filtration and / or activated carbon filtration and / or membrane filtration, in particular after filtration with activated carbon in powder form, a solid / liquid separation step, to recover the liquid phase and remove the solid phase.

[0119] According to one embodiment, the treatment of a mixture obtained by anaerobic digestion of organic waste according to the invention may comprise at least the following steps: a) solid / liquid separation of a mixture obtained by anaerobic digestion of organic waste, to recover the liquid phase and eliminate the solid phase, b) treatment of the liquid phase obtained in step a) by resin filtration and / or activated carbon filtration and / or by membrane filtration, c) optionally solid / liquid separation of the mixture obtained in step b), to recover the liquid phase and eliminate the solid phase, d) recovery of a colorless liquid solution comprising VFAs.The solid / liquid separation steps of the process, in particular step a) and / or step c), are preferably carried out by a method chosen from centrifugation, flocculation, the use of a filter press, the use of a manual and hydraulic press, the use of a rotary drum filter, filtration, centrifugal decantation or a combination of at least two of these methods.

[0120] The solid phases removed during these separation steps can be recycled. For example, the digestate removed during step a) can be used to produce biogas through methanization or composting. Similarly, the solid phase of activated carbon recovered in step c) can be used to regenerate new activated carbon or used to produce biogas through methanization or composting.

[0121] According to one embodiment, the treatment of a mixture obtained by anaerobic digestion of organic waste according to the invention may comprise at least the following steps: a) solid / liquid separation of a mixture obtained by anaerobic digestion of organic waste, to recover the liquid phase and eliminate the solid phase; b1) treatment of the liquid phase obtained in step a) by resin filtration b2) treatment of the liquid phase obtained in step b1) by activated carbon filtration and / or by membrane filtration, c) optionally solid / liquid separation of the mixture obtained in step b2), to recover the liquid phase and eliminate the solid phase, d) recovery of a colorless liquid solution comprising VFAs.

[0122] According to another embodiment, the treatment of a mixture obtained by anaerobic digestion of organic waste according to the invention may comprise at least the following steps: a) solid / liquid separation of a mixture obtained by anaerobic digestion of organic waste, to recover the liquid phase and eliminate the solid phase; b1) treatment of the liquid phase obtained in step a) by activated carbon filtration and / or by membrane filtration; b2) treatment of the liquid phase obtained in step b1) by resin filtration; c) optionally solid / liquid separation of the mixture obtained in step b2), to recover the liquid phase and eliminate the solid phase, d) recovery of a colorless liquid solution comprising VFAs. Thus, according to particular embodiments of the invention, the resin filtration step may be carried out upstream or downstream of activated carbon filtration and / or by membrane filtration.

[0123] The method for treating a mixture obtained by anaerobic digestion of organic waste according to the invention may also comprise a step of concentrating the colorless solution in VFA. This step is preferably carried out concomitantly with step b) / or after step b), preferably after step d). This concentration may in particular be carried out during membrane filtration (in step b), the membrane filtration advantageously making it possible, in addition to filtering, to concentrate the solution in VFA. This concentration may also be carried out concomitantly with step b) and / or after step b), preferably after step d) by at least one method chosen from evapoconcentration, electrodialysis, liquid / liquid extraction, pervaporation, adsorption, precipitation, distillation, membrane filtration and a combination of at least two of these methods.

[0124] The method may also include a step of adding NaCl and any other minerals to the VFA solution. NaCl is preferably added at a concentration of at least 5% (by weight / volume of the VFA solution), preferably between 5 and 30%. The presence of NaCl allows the medium to be stored without contamination problems while facilitating the use of the solution, particularly for producing PHAs. NaCl may be added alone or with other compounds.

[0125] The solution obtained by implementing the method according to the invention is colorless and comprises VFAs, preferably at least 10 g / L of VFAs, in particular at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 g / L of VFAs.

[0126] Preferably in the solution obtained by the process of the invention, the percentage of impurities relative to the total of molecules present in the solution (impurities + VFA) measured by HPLC, is less than 10%. This ratio is calculated by taking the total of the area represented by the impurities and the total of the air represented by all the molecules (impurities and VFA) on the chromatogram obtained by HPLC. Thus the VFA represent at least 90% of the molecules present in the solution obtained according to the invention.

[0127] The VFAs present in the solution obtained by the treatment method according to the invention are preferably chosen from formic acid, acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, isobutyric acid and isovaleric acid and mixtures thereof. Preferably, the colorless VFA solution according to the invention comprises at least acetic acid, propionic acid and butyric acid.

[0128] According to a particular embodiment, the solution obtained comprises (percentages given in % by mass): - Acetic: between 30 and 90%, preferably between 40 and 80%

[0129] - Propionic: between 10 and 70%, preferably between 20 and 50%

[0130] - Butyric: between 0 and 30%, preferably between 0 and 10%.

[0131] The method of treating a mixture obtained by anaerobic digestion of organic waste according to the invention can be implemented alone or in a broader method of using AGV such as a method of manufacturing PHA from organic waste.

[0132] Colorless AGV solution

[0133] The invention also relates to a colorless liquid solution obtained from organic waste. This solution is preferably obtained by implementing a method for treating a mixture obtained from anaerobic digestion of organic waste.

[0134] The AGV solution according to the invention is colorless and comprises at least 10 g / L of AGV, preferably at least 10 g / L of AGV, in particular at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 g / L of AGV.

[0135] Preferably in the solution according to the invention, the percentage of impurities relative to the total of molecules present in the solution (impurities + VFA) measured by HPLC, is less than 10%. This ratio is calculated by taking the total of the area represented by the impurities and the total of the air represented by all the molecules (impurities and VFA) on the chromatogram obtained by HPLC. Thus the VFAs represent at least 90% of the molecules present in the solution according to the invention.

[0136] The VFAs present in the solution obtained according to the invention are preferably chosen from formic acid, acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid and mixtures thereof. Preferably, the colorless VFA solution according to the invention comprises at least acetic acid, propionic acid and butyric acid.

[0137] According to a particular embodiment, the AGV solution according to the invention comprises (percentages given in % by mass):

[0138] - Acetic: between 30 and 90%, preferably between 40 and 80%

[0139] - Propionic: between 10 and 70%, preferably between 20 and 50%

[0140] - Butyric: between 0 and 30%, preferably between 0 and 10%.

[0141] The AGV solution according to the invention preferably comprises at least 5% NaCl (by weight / volume of the AGV solution), preferably between 5 and 30%.

[0142] The AGV solution according to the invention preferably has a pH greater than or equal to 6, in particular a pH greater than or equal to 6.8, preferably greater than or equal to 7. It may be in particular between 6 and 12, between 6 and 11, between 6 and 10.5, between 6.8 and 12, between 6.8 and 11, between 6.8 and 10.5, between 7 and 12, between 7 and 11, between 7 and 10.5.

[0143] If the VFA solution is to be used to manufacture PHAs by fermentation with microorganisms, the pH of the VFA solution can be adjusted to suit

[0144] The AGV solution according to the invention can be stored in any suitable container or used without storage for different applications, in particular for the manufacture of PHA.

[0145] Process for obtaining PHA from organic waste

[0146] The invention also relates to a process for manufacturing polyhydroxyalkanoates (PHA) from organic waste, in particular a process comprising the implementation of the following steps:

[0147] *obtaining a mixture by anaerobic digestion of organic waste,

[0148] *treatment of the mixture obtained in step 1) by the treatment process to obtain a colorless solution containing AGVs according to the invention,

[0149] *transformation of the AGVs in the solution obtained in step 2) into PHA by at least one halophilic bacterium.

[0150] The first step of the process consists of obtaining a mixture by anaerobic digestion of organic waste.

[0151] The mixture obtained by anaerobic digestion of organic waste can be obtained by any means of implementing anaerobic digestion on organic waste.

[0152] Organic waste can be chosen from all organic waste. It is preferably chosen from the following organic waste: grape marc, organic food waste, organic agricultural waste, organic waste from the agri-food industry, carbon-containing organic waste and their mixtures. This may include in particular:

[0153] -household and restaurant food waste, such as fruit, starchy foods and vegetables,

[0154] - organic waste from agriculture or the agri-food industry such as brewery grains, fruit marc, particularly grape marc, green waste, waste from the dairy industry, harvest by-products,

[0155] -carbonaceous industrial waste such as paper pulp, organic effluents

[0156] -or combinations of these wastes.

[0157] According to a particularly suitable embodiment, step 1) comprises the following steps:

[0158] - crushing of organic waste, - possibly pre-treatment of crushed organic waste, said pre-treatment being able to be carried out before or after crushing,

[0159] - anaerobic digestion and recovery of a solid / liquid mixture including VFAs.

[0160] Organic waste is therefore preferably ground before the anaerobic digestion stage.

[0161] The grinding step is preferably carried out so as to obtain an aqueous solution comprising at least 5% dry matter, preferably at least 10% dry matter, in particular between 10 and 20%.

[0162] Preferably, the grinding is carried out to a size less than 100 mm, preferably less than 50 mm, in particular to a size between 0.1 and 100 mm, in particular between 0.1 and 50 mm.

[0163] Grinding can be carried out using any suitable device. It can be carried out in particular in the presence of liquid and / or by mechanical mixing, for example using a homogenizer, a food mixer, a mill, a grinder, a hammer mill.

[0164] After or before grinding, organic waste may undergo one or more pre-treatment steps. This may include, for example, at least one pre-treatment chosen from heat treatment, ultrasound, microwave, high-pressure homogenizer, acid, basic, enzymatic, fungal, lactic pre-fermentation and the combination of at least two of these treatments.

[0165] According to one embodiment, step 1 comprises at least one step of pre-treatment of organic waste by lactic pre-fermentation. Such a step advantageously makes it possible to increase the VFA content in the VFA solution and to reduce or eliminate problems of heterogeneity of organic waste.

[0166] The invention also relates to a process for manufacturing PHA from organic waste, comprising at least one step of pre-treatment of the organic waste by lactic pre-fermentation. The pre-fermentation may be preceded or followed by a step of grinding the waste and / or by at least one other pre-treatment.

[0167] In the PHA manufacturing process according to the invention, the pre-treatment by lactic prefermentation can be carried out with suitable microorganisms, in particular lactic acid bacteria. These can preferably be lactic acid bacteria such as lactobacilli and / or bifidobacter bacteria.

[0168] Pre-treatment by lactic pre-fermentation can be carried out in a suitable device, preferably in an anaerobic digester. Pre-treatment by lactic pre-fermentation can be carried out with one or more of the following conditions, preferably all of them:

[0169] - under agitation,

[0170] - at a redox potential lower than -300mV, preferably between -550 mV and -400 mV,

[0171] - between 20 and 60°C,

[0172] - at a pH between 3 and 5,

[0173] - for at least 24 hours, preferably between 1 and 6 days.

[0174] Preferably, pre-treatment by lactic pre-fermentation includes a step of adjusting the pH between 3 and 5. This step makes it possible to stabilize the pH between 3 and 5, because the pH of the solution naturally becomes too acidic (less than 3) during the fermentation of sugars into lactic acid.

[0175] According to a particular embodiment, in the PHA manufacturing process according to the invention, the lactic pre-fermentation step comprises the following steps:

[0176] - grinding, preferably in water, to obtain an aqueous solution comprising between 5 and 20% dry matter, in particular between 10 and 12% dry matter,

[0177] - introduction into an anaerobic digester for pre-fermentation,

[0178] - possibly inoculation of the reactor using lactic bacteria,

[0179] - pH adjustment between 3 and 5, preferably using sodium hydroxide solution, for example sodium hydroxide solution NaOH 30% v / v,

[0180] - heating the environment between 20 and 40°C,

[0181] - after 2 to 8 days, preferably 3 to 5 days, recovery of a lactic acid solution containing pre-treated organic waste.

[0182] According to a variant, step 1 of the PHA production process according to the invention comprises at least one step of pre-treatment of organic waste by lactic pre-fermentation and at least one other pre-treatment step.

[0183] According to one embodiment, in particular if the organic waste has been pretreated, in particular by lactic pre-fermentation, a solid-liquid separation step can be implemented before anaerobic digestion to recover the liquid phase and eliminate the solid phase.

[0184] In the PHA manufacturing process according to the invention, anaerobic digestion can be carried out by inoculating the organic waste, preferably previously ground and / or pretreated, with any type of suitable anaerobic microorganism, preferably any type of suitable anaerobic bacteria. These may in particular be bacteria chosen from Clostridia (Clostridiaceae), Bacilli (Lactobacillaceae, Leuconostocacea, Streptococcacea), Propionobacter, Acetobacter and mixtures thereof. These may be, for example, Butirybacterium rettgeri, Pseudomonas aeruginosa, Clostridium acetobutycilium, acetobacter woodii or the mixture of at least two of these bacteria. It is also possible to use anaerobic bacteria naturally present in existing mixtures, such as sludge from sewage treatment plants and / or animal manure such as cow manure and / or ruminant ruminal fluid.

[0185] According to one embodiment of the invention, the anaerobic digestion of organic waste is carried out by inoculating the organic waste, preferably previously crushed and / or pretreated, with a mixture of sludge from treatment plants and / or animal manure, preferably from cows.

[0186] Preferably, the pH and / or temperature of the mixture can be adjusted before and / or during the anaerobic digestion step, to be adapted to an ideal pH and / or temperature for the bacteria used. The pH of the solution can be adjusted by any suitable means, in particular by adding at least one acid such as hydrochloric acid and / or at least one base such as sodium hydroxide. The pH adjustment can be facilitated by introducing air into the solution in order to evaporate the dissolved CO2 and / or by aerating the solution by stirring in order to evaporate the dissolved CO2.

[0187] Preferably, in the PHA manufacturing process according to the invention, the anaerobic digestion is carried out at a redox potential lower than -300 mV, preferably between -550 mV and -400 mV.

[0188] Anaerobic digestion can be carried out in any suitable device capable of achieving anaerobic conditions, preferably in an agitated anaerobic digester.

[0189] The mixture obtained at the end of step 1) includes digested organic waste and VFAs.

[0190] Step 2) of the PHA production process consists of implementing the process for treating a mixture containing AGVs according to the invention as described in the present application and makes it possible to obtain an AGV solution optionally containing NaCl.

[0191] Step 3) of the PHA production process is the transformation of the VFAs from the solution obtained in step 2) into PHA by at least one halophilic bacterium.

[0192] Prior to inoculating the AGV solution with halophilic bacteria to produce PHAs, the process preferably comprises:

[0193] - supplementing the AGV solution with elements necessary for the culture of halophilic bacteria used to produce PHAs, and / or

[0194] - pH adjustment to a pH necessary for the cultivation of halophilic bacteria used to produce PHAs. The culture medium for halophilic bacteria requires a NaCl concentration of between 5 and 30% (weight / volume) for optimal growth and production. To this NaCl it is important to add other nutrients for the growth of halophilic bacteria. These may include KCl, MgCl26H2O, MgSO47H2O, NaHCO3, NaBr, NH4Cl, KH2PO4, CaCl2, FeCl3 and / or trace element solutions (trace elements are generally in trace amounts). It is possible to use artificial synthetic media known for halophilic bacteria, in particular those containing yeast extracts as a source of nutrients. According to a particularly suitable variant, the VFA solution is supplemented with artificial seawater or with a mixture obtained after evaporation of seawater.

[0195] The pH is preferably adjusted between 6 and 8. The pH adjustment can be carried out using acids or bases or with a suitable buffer such as HEPES, MOPS, Tris, NaHCO3 and / or PIPES.

[0196] The solution can then be inoculated with halophilic bacteria to produce PHAs from VFAs.

[0197] The halophilic bacteria are preferentially chosen from bacteria of the genus Halomonas, Haloarcula, Halococcus, Halobacterium, Natrinema, Halogeometricum, Halorubrum, Halobifoma, Natronobacterium, Natronococcus, Halorhabdus, Haloquadratum, Halopiger, Halogranum, Haloterringena, and mixtures thereof.

[0198] Preferably, the bacteria are chosen from the species Haloferax mediterranei, Haloferax volcanii, Haloferax gibbonsii, Halomonas boliviensis, Halomonas halophila, Halomonas bluphagenesis and mixtures thereof. According to a particularly suitable embodiment, the bacteria used comprise at least Haloferax mediterranei.

[0199] Before being introduced into the AGV solution according to the invention to produce PHAs, it is possible to provide a pre-culture step for the halophilic bacteria. This pre-culture step may consist of an accumulation of halophilic bacteria biomass on a culture medium (for example a minimal synthetic medium) until the end of the exponential growth phase, as defined by the culture conditions

[0200] The halophilic bacteria, after or without a prior pre-culture step, are introduced into the VFA solution. Inoculation is preferably carried out at an optical density between 0.1 and 2. The VFA solution containing the halophilic bacteria is then incubated preferentially:

[0201] - at a temperature between 30 and 60°C and / or

[0202] - at a rotation speed between 50 and 1000 rpm. The incubation stage preferably lasts 24 hours to 6 days, particularly between 3 and 5 days.

[0203] During this step, halophilic bacteria use VFAs to produce PHAs.

[0204] The biomass containing the PHAs is then recovered, preferably after total consumption of the AGVs. The biomass recovery can be done by solid / liquid extraction, in particular for example by centrifugation.

[0205] The PHAs are then extracted from the biomass by any suitable means. A particularly suitable method is the lysis of bacteria by osmotic shock, followed by solid / liquid separation.

[0206] According to a particular embodiment, the extraction of PHAs is carried out by implementing the following steps:

[0207] - lysis by osmotic shock,

[0208] - at least one solid-liquid separation, for example by centrifugation, preferably several solid-liquid separations separated by washing steps with water and / or ethanol.

[0209] Extraction volumes are preferably between 25 and 200% of the culture volume.

[0210] The PHAs are then dried by any suitable method, for example by spray-drying and / or by oven drying, which can be carried out between 30 and 120°C until a constant mass is obtained.

[0211] Thus, step 3) of the PHA production process according to the invention may comprise the implementation of the following steps:

[0212] - adjustment of the pH of the solution from step 2 to a pH between 6 and 8,

[0213] - addition of NaCI and any nutrients necessary for the culture of halophilic bacteria,

[0214] - transformation of the AGVs of the solution obtained in step 2) into PHA by at least one halophilic bacterium, said halophilic bacterium having possibly undergone a prior pre-culture step,

[0215] - recovery of biomass containing PHAs,

[0216] - extraction of PHAs from biomass,

[0217] - possibly drying of the PHAs obtained.

[0218] According to a particular embodiment of the invention, the PHA production method comprises the implementation of the following steps:

[0219] - crushing of organic waste, - at least one pre-treatment, including at least one pre-treatment by lactic pre-fermentation,

[0220] - optionally a solid / liquid separation step,

[0221] - anaerobic digestion,

[0222] - solid / liquid separation to recover the liquid fraction and eliminate the solid fraction,

[0223] - pH adjustment above 6 and carbon filtration and / or membrane filtration, and recovery of a colorless AGV solution,

[0224] - addition of NaCl and any other elements necessary for the halophilic bacteria used in the process,

[0225] - adjustment of the pH to a pH suitable for the halophilic bacteria used in the process,

[0226] - transformation of the AGVs of the solution obtained in step 2) into PHA by at least one halophilic bacterium, said halophilic bacterium having possibly undergone a prior pre-culture step,

[0227] - recovery of biomass containing PHAs,

[0228] - extraction of PHAs from the biomass containing them,

[0229] - possibly drying of PHAs.

[0230] The PHAs obtained by implementing the process according to the invention are in the form of a white powder.

[0231] The PHAs obtained according to the invention are preferably poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). PHBVs are copolymers comprising two units: HV and HB. Preferably, the PHBVs obtained according to the invention comprise between 1 and 70 mol% of hydroxyvalerate (HV) units, preferably at least 10%, at least 15%, at least 20%, in particular between 10 and 30%.

[0232] The PHA powder obtained by the process according to the invention comprises at least 90% by weight of PHBV, preferably at least 92%, even more preferably at least 95%. The remainder of the powder is preferably made up of at least:

[0233] - cellular residues, and / or

[0234] - proteins, and / or

[0235] - extracellular polymers, and / or

[0236] - pigments, and / or

[0237] - residual salts, and / or

[0238] - residual solvents. According to a variant, the PHBV powder obtained according to the invention comprises at least 90% by weight of PHBV comprising at least 10 mol% of HV units, preferably at least 20%.

[0239] The obtained PHBV powder can be used for many applications such as the following applications:

[0240] - Encapsulation (biodegradable matrix, controlled release)

[0241] - Cosmetic (Binder, Gelling agent, mattifying agent, exfoliating agent, etc.)

[0242] - Textile fiber (biodegradable textile fiber)

[0243] - Applications in plastics: plastic parts and flexible and rigid packaging

[0244] - Biomedical devices

[0245] - extrusion

[0246] - injection molding

[0247] - 3D printing.

[0248] The invention is now illustrated by non-limiting examples and test results illustrating the invention.

[0249] Anaerobic examples of grape marc (step 1 of the process of of PHA

[0250] An example of a process for the anaerobic digestion of grape marc corresponding to step 1 of the PHA production process according to the invention is presented below. It comprises the implementation of the following steps:

[0251] - Grinding (mixer) of the marc in water to obtain an aqueous solution of grape marc between 10 and 15% dry matter (DM).

[0252] - Introduction into a 5L agitated anaerobic digester

[0253] - Inoculation using WWTP sludge or animal manure (enriched and acclimatized)

[0254] - Adjustment and regulation of pH between 8 and 10 using sodium hydroxide solution (NaOH 30% v / v).

[0255] - Heating the environment to 35°C

[0256] - After 7 days (residence time) Subtitration of the AGV solution containing digested grape marc

[0257] - Solid / liquid separation (press) Recovery of the AGV solution to be purified. food with pre-treatment

[0258] An example of a process for the anaerobic digestion of organic food waste corresponding to step 1 of the PHA production process according to the invention is presented below. It comprises the implementation of the following steps:

[0259] - Grinding (mixer) of food waste in water to obtain an aqueous solution between 10-12% dry matter (DM),

[0260] - Introduction into a 5L agitated anaerobic digester for lactic pre-fermentation,

[0261] - Optional: Inoculation of the reactor with lactobacillus and bifidobacter bacteria

[0262] - Adjust the pH between 3-5 using sodium hydroxide solution (NaOH 30% v / v)

[0263] - Heating the environment to 35°C

[0264] - After 4 days (residence time) Subtraction of 5L of lactic acid (LA) solution containing digested food waste.

[0265] - solid-liquid separation to obtain a liquid AL solution,

[0266] - The liquid AL solution ([AL] = 15-30 g / L) is introduced into a 5L stirred anaerobic digester.

[0267] - Inoculation using WWTP sludge or animal manure (enriched and acclimatized)

[0268] - Optional: Introduction of air and / or mixing of the medium for evaporation of dissolved CO2

[0269] - Automatic pH adjustment and regulation between 7-10 (VFA composition varies depending on conditions).

[0270] - Heating to 35°C

[0271] - After 7 days, subtraction of the 5L digester Recovery of the AGV solution to be purified. ([AGV] = 15-50 g / L) food (step 1 of the process of

[0272] An example of a process for the anaerobic digestion of organic food waste corresponding to step 1 of the PHA production process according to the invention is presented below. It comprises the implementation of the following steps:

[0273] - Grinding (mixer) of food waste in water to obtain an aqueous solution between 5 and 15% dry matter (DM)

[0274] - Introduction into a 5L agitated anaerobic digester.

[0275] - Inoculation using WWTP sludge or animal manure (enriched and acclimatized) - Adjustment and regulation of pH between 9 and 10 using sodium hydroxide solution (NaOH 30% v / v).

[0276] - Heating the environment to 35°C

[0277] - After 7 days (stay time) Subtraction of the AGV solution containing digested food waste.

[0278] - Solid / liquid separation (press) Recovery of the AGV solution to be purified

[0279] 4: Activated carbon filtration treatment according to the invention 2 of the ed of PHA ion according to the i

[0280] An example of a process according to the invention for treating a mixture obtained by anaerobic digestion of organic waste (as obtained in Examples 1 to 3), corresponding to step 2 of the PHA production process according to the invention, is presented below. It comprises the implementation of the following steps:

[0281] - Adjustment of the pH of the AGV solution between 7-10,

[0282] - Addition of 1 - 5% by mass of activated carbon in powder form in the AGV solution to be treated

[0283] - Stir at 500-1500 rpm for 1-60 minutes

[0284] - Centrifugation between 5000 and 15,000g

[0285] - Option: Filtration to eliminate the last suspended particles waste disposal

[0286] An example of a PHBV production process corresponding to step 3 of the PHA production process according to the invention is presented below. It comprises the implementation of the following steps:

[0287] - a step of accumulation of biomass (preculture) on minimal synthetic medium from a cryobank of halophilic microorganisms:

[0288] * Incubation of the preculture between 30 and 45°C and rotation between 100 and 200 revolutions per minute

[0289] * Recovery of biomass for production when it is at the end of the exponential phase

[0290] - Recovery by centrifugation (between 5000 and 15000g)

[0291] - the addition of nutrients and salts, necessary for the growth and optimal production of the microorganism, in the AGV solution to constitute the culture medium for the production of PHBV:

[0292] * Volume of 400mL * Addition of salts (powder) directly to the AGV solution from organic waste acidogenesis

[0293] * Addition of the buffer via concentrated solution at 200g / L of PIPES

[0294] * Adjust pH between 6 and 8.

[0295] - inoculation of the culture medium with Haloferax mediterranei at an optical density between 0.1 and 2 via the preculture biomass

[0296] - incubation of the Haloferax mediterranei culture (for intracellular PHA production), at temperatures between 30°C and 45°C and rotation speeds of 100 rpm to 200 rpm

[0297] * Total consumption of AGVs for the concentrations cited above

[0298] - recovery of biomass after total consumption of the substrate (AGV) by solid / liquid separation (centrifugation speeds between 5000g and 15000g for 10 to 20min at 20°C)

[0299] - extraction of PHBV in successive stages:

[0300] * lysis by osmotic shock by resuspending the biomass in a volume of an aqueous solution of SDS at 0.1% (weight / weight), stirring at 900 rpm via motor for 1 hour.

[0301] * solid liquid separation (centrifugation 6000g 20min 20°C)

[0302] * aqueous wash with agitation at 900 rpm for 30 min

[0303] * solid liquid separation (centrifugation 6000g 20min 20°C)

[0304] * ethanol wash with agitation at 900 rpm for 30 min

[0305] * solid liquid separation (centrifugation 6000g 20min 20°C)

[0306] * drying in an oven at 60°C for 72 hours of the white powder obtained.

[0307] The extraction volumes used can be between 25% and 200% of the culture volume. of PHBV from organic waste in bioreactor of PHA according to the i

[0308] An example of a PHBV production process corresponding to step 3 of the PHA production process according to the invention is presented below. It comprises the implementation of the following steps:

[0309] - a step of accumulation of biomass (preculture) on minimal synthetic medium from a cryobank of halophilic microorganism Tl

[0310] * Incubation of the preculture between 30 and 45°C and rotation between 100 and 200 revolutions per minute

[0311] * Recovery of biomass for production when it is at the end of the exponential phase (~70h at 37°C 140 revolutions per minute)

[0312] - the addition of nutrients and salts, necessary for the growth and optimal production of the microorganism, in the AGV solution to constitute the culture medium for the production of PHBV

[0313] * Volume from 2 to 5L

[0314] * Addition of salts (powder) directly to the AGV solution resulting from acidogenesis of organic waste

[0315] * pH adjustment between 6 and 8 via automatic regulation

[0316] - inoculation of the culture medium with Haloferax mediterranei at an optical density between 0.1 and 2 via the preculture biomass

[0317] - start-up of the culture in bioreactor with the following parameters:

[0318] * pO2 regulation (minimum value) between 5% and 30%

[0319] * agitation (regulation cascade) between 100 and 900 revolutions per minute

[0320] * ventilation (regulation cascade) between 0.1vvm and 2vvm

[0321] * pH regulated via 2mol / L HCI solution and 2mol / L NaOH

[0322] * temperature between 30°C and 45°C

[0323] * Total consumption of AGVs for the concentrations cited above in ~80h at 37°C and pO2 > 20%

[0324] - recovery of biomass after total consumption of the substrate (AGV) by solid / liquid separation (centrifugation speeds between 5000g and 15000g for 10 to 20min at 20°C)

[0325] - extraction of PHBV in successive stages:

[0326] * lysis by osmotic shock by resuspending the biomass in a volume of an aqueous solution of SDS at 0.1% (weight / weight), stirring at 900 rpm via motor for 1 hour

[0327] * solid liquid separation (centrifugation 6000g 20min 20°C)

[0328] * aqueous wash with agitation at 900 rpm for 30 min

[0329] * solid liquid separation (centrifugation 6000g 20min 20°C)

[0330] * ethanol wash with stirring at 900 rpm for 30 min * solid liquid separation (centrifugation 6000g 20 min 20°C)

[0331] * drying in an oven at 60°C for 72 hours of the white powder obtained.

[0332] Comparative tests

[0333] The methods of Examples 1 and 4 were combined to obtain a VFA solution from grape marc by varying the pH at the stage before activated carbon filtration:

[0334] - pHIO = 1T solution

[0335] - pH9 = 2T solution.

[0336] The processes of Examples 2 and 4 were combined to obtain a VFA solution from food waste with a pH of 9.5 before activated carbon filtration (3T solution).

[0337] The same processes without activated carbon filtration were implemented to obtain an AGV solution without treatment according to the invention:

[0338] - pHIO grape marc = 1ST solution

[0339] - grape marc pH9 = 2ST solution

[0340] - food waste pH9.5 = 3ST solution.

[0341] The photos of the AGV solutions without treatment (solution 1ST) presented in Figure 1a, and with treatment according to the invention (solution 1T) presented in Figure 1b, clearly show that the invention makes it possible to obtain a colorless AGV solution unlike the process without treatment according to the invention.

[0342] Effect of the treatment according to the invention on the AGV solution obtained

[0343] Furthermore, the AGV solutions were analyzed by HPLC under the following conditions:

[0344] - HPLC: Hitachi Chromaster model (VWR)

[0345] - Column: Hi-Plex H

[0346] - Conditions: 60°C - flow of 0.2 mL of LC quality water containing 0.005M H2SO4

[0347] The chromatograms in Figure 2 (solution 1T and solution 1ST), Figure 6 (solution 2T, solution 2ST) and Figure 10 (solution 3T, solution 3ST) also clearly show that the solution according to the invention does indeed include VFAs but only includes very few impurities and other constituents present in the solution without treatment according to the invention.

[0348] For each of the three examples, the ratios calculated by taking the total area represented by the impurities and the total air represented by all molecules (impurities and VFAs) are presented in the tables below (Table 1 corresponding to Figure 2, Table 2 corresponding to Figure 6, Table 3 corresponding to Figure 10). [Table 1]

[0349] [Table 2] [Table 3]

[0350] It is found that impurities represent less than 10% in the solutions according to the invention, whereas they represent more than 29% without treatment in the case of food waste, and more than 52% without treatment in the case of grape marc. Thus, VFAs represent at least 90% of the molecules present in the solution according to the invention. Influence of pH on the treatment process according to the invention and on the VFA solution obtained.

[0351] Furthermore, comparative tests were carried out by varying, before filtration with activated carbon according to the invention, the pH of the mixture obtained by anaerobic digestion of grape marc using the methods of examples 1 and 4. 3 pH values ​​were tested and the results obtained were compared to those of the mixture before filtration.

[0352] AGV solutions were analyzed by HPLC under the following conditions:

[0353] - HPLC: Hitachi Chromaster model (VWR)

[0354] - Column: Hi-Plex H

[0355] - Conditions: 60°C - flow of 0.2 mL of LC quality water containing 0.005M H2SO4

[0356] The following calculations were then carried out from the HPLC results obtained (the areas are the areas on the chromatograms):

[0357] - elimination of impurities: 1- (Impurity area after) / (Impurity area before)

[0358] - loss of AGVs: 1- (AGV area after) / (AGV area before).

[0359] The results are presented in Table 4.

[0360] [Table 4]

[0361] It is found that the process according to the invention makes it possible to eliminate a very large part of the impurities present in the mixture before filtration. It is also found that with an acidic pH (pH2), filtration with activated carbon results in a significant loss of the VFAs present in the mixture before filtration, whereas with a pH greater than or equal to 6, almost all of the VFAs present in the mixture before filtration are found in the VFA solutions according to the invention. Advantages of the VFA solution according to the invention for bacterial growth

[0362] Figure 3 (solution T1 and solution STI) and Figure 7 (solution T2 and solution ST2) compare the monitoring of the optical density after inoculation of a halophilic microorganism (Haloferax mediterranei) in a solution treated according to the invention and in an untreated solution. It can be seen that the growth of the bacteria is much faster with the solution treated according to the invention.

[0363] Influence of the invention on the PHBVs obtained

[0364] The method of example 5 is then implemented on the AGV solutions previously observed (on AGV solution treated according to the invention and on untreated AGV solution).

[0365] Figures 4a (PHBV obtained from solution T1) and 8a (PHBV obtained from solution T2) compare the infrared spectrum of the PHBV obtained according to the invention compared to those of reference PHBV. A spectrum very close to the reference PHBV is observed for the PHBV obtained according to the invention.

[0366] Furthermore, the purity of the PHBVs obtained was measured by methanolysis of PHBV (CHCI3 - MeOH H2SO4 3%v / v) at 100°C for 5h and GC analysis with internal standard (methyl benzoate). The purity of the PHBVs obtained according to the invention from the AGV solutions of these examples is at least 90%.

[0367] Figures 4b (PHBV obtained from solution T1 and from solution STI) and 8b (PHBV obtained from solution T2 and from solution ST2) compare the infrared spectrum of PHBV obtained according to the invention compared to those of PHBV obtained from AGV not treated according to the invention. It is observed that the IR spectra are very different.

[0368] Furthermore, the purity of the obtained PHBVs was measured by methanolysis of PHBV (CHCI3 - MeOH H2SO4 3%v / v) at 100°C for 5h and GC analysis with internal standard (methyl benzoate). The purity results of PHBVs are shown in Table 5.

[0369] [Table 5]

[0370] It can be seen that the treatment according to the invention makes it possible to very significantly increase the purity of PHBV obtained. Finally, the photos of the PHBV powders obtained are presented in Figure 9 (PHBV obtained from solution T1) and in Figure 11 (PHBV obtained from solution T3). It can be seen that the PHBV powder obtained according to the invention is white, which allows its use in many applications, whereas the PHA powder obtained without treatment of AGVs is colored (whether it is obtained from grape marc or food waste) and can therefore hardly be used as is.

[0371] The treatment of AGVs according to the invention, and the PHA manufacturing process according to the invention therefore have a major effect on the purity of the PHAs obtained, on the coloring of the powder and on the yields obtained. The invention therefore makes it possible to make accessible the large-scale manufacturing of PHAs from organic waste.

Claims

CLAIMS

1. A method of treating a mixture obtained by anaerobic digestion of organic waste, comprising a step of treating said mixture by resin filtration and / or by activated carbon filtration and / or by membrane filtration at a pH greater than or equal to 6, so as to obtain a colorless liquid solution comprising volatile fatty acids (VFAs).

2. Treatment method according to claim 1, characterized in that it comprises at least the following steps: -a) solid / liquid separation of a mixture obtained by anaerobic digestion of organic waste, to recover the liquid phase and eliminate the solid phase, - b) treatment of the liquid phase obtained in step a) by resin filtration and / or by activated carbon filtration and / or by membrane filtration, - c) optionally solid / liquid separation of the mixture obtained in step b), to recover the liquid phase and eliminate the solid phase, - d) recovery of a colorless liquid solution comprising VFAs.

3. Treatment method according to claim 2, characterized in that step a) and / or step c) are carried out by a method chosen from centrifugation, flocculation, the use of a filter press, the use of a manual and hydraulic press, the use of a rotary drum filter, filtration, centrifugal decantation or a combination of at least two of these methods.

4. Treatment method according to one of claims 2 or 3, characterized in that it comprises a step making it possible to concentrate in AGV the colorless solution comprising AGV, said step being carried out concomitantly with step b) and / or after step d).

5. Treatment method according to one of the preceding claims, characterized in that the step of treatment by resin filtration and / or by activated carbon filtration and / or by membrane filtration comprises, prior to filtration, a step of adjusting the pH of the mixture obtained by anaerobic digestion of organic waste to a desired pH, said pH being greater than or equal to 6, preferably greater than or equal to 7.

6. Treatment method according to the preceding claim, characterized in that the step of concentrating the solution in AGV is carried out during membrane filtration and / or by at least one method chosen from evapoconcentration, electrodialysis, liquid / liquid extraction, pervaporation, adsorption, precipitation, distillation and a combination of at least two of these methods.

7. Treatment method according to one of the preceding claims, characterized in that the treatment by activated carbon filtration is carried out by adding activated carbon powder or granules to the mixture containing AGVs and / or to an activated carbon cartridge.

8. Treatment method according to one of the preceding claims, characterized in that the treatment by filtration with activated carbon is carried out by adding between 0.1 and 5% by mass of activated carbon in the form of powder or granules in the mixture.

9. Treatment method according to one of the preceding claims, characterized in that the treatment by activated carbon filtration is carried out by implementing the following sub-steps: - adjustment of the pH of the mixture to a pH greater than or equal to 6, preferably greater than or equal to 6.8, - addition of activated carbon in powder form to the mixture, - hustle, - centrifugation.

10. Treatment method according to one of the preceding claims, characterized in that the treatment by membrane filtration is carried out by microfiltration and / or ultrafiltration and / or nanofiltration and / or reverse osmosis.

11. Treatment method according to one of the preceding claims, characterized in that the treatment by resin filtration is carried out using an absorbent resin and / or an ion exchange resin.

12. Treatment method according to one of the preceding claims, characterized in that the organic waste is chosen from grape marc, organic food waste, organic agricultural waste, organic waste from the agri-food industry, carbon-containing organic waste and mixtures thereof.

13. Treatment method according to one of the preceding claims, characterized in that it also comprises a step of adding NaCl and possibly other minerals.

14. Colorless liquid solution comprising volatile fatty acids obtainable by a treatment method according to one of claims 1 to 13, characterized in that it has a pH of between 6 and 8 and in that it comprises at least 10 g / L of VFA.

15. Colorless liquid solution according to the preceding claim, characterized in that it comprises at least acetic acid, propionic acid and butyric acid.

16. Colorless liquid solution according to one of claims 14 or 15, characterized in that it also comprises at least 5% NaCl (weight / volume).

17. Colorless solution according to one of claims 14 to 16, characterized in that the percentage of impurities present in the solution relative to the total of molecules present in the solution, measured by HPLC, is less than 10%.

18. A method of producing polyhydroxyalkanoates (PHA) from organic waste comprising carrying out the following steps: 1) obtaining a mixture by anaerobic digestion of organic waste, 2) treatment of the mixture obtained in step 1) by the method according to one of claims 1 to 13 to obtain a colorless solution containing AGVs, 3) transformation of the AGVs of the solution obtained in step 2) into PHA by at least one halophilic bacterium.

19. A method of producing PHA according to claim 18, characterized in that step 1) comprises the following steps: - crushing of organic waste, - possibly pre-treatment of crushed organic waste, - anaerobic digestion and recovery of a solid / liquid mixture including VFAs.

20. Process for producing PHA according to the preceding claim, characterized in that the pre-treatment of the organic waste is chosen from thermal, ultrasound, microwave, high pressure homogenizer, acid, basic, enzymatic, fungal treatment, lactic pre-fermentation and the combination of at least two of these treatments.

21. A method of producing PHA according to one of claims 18 to 20, characterized in that step 3 comprises the following steps: - adjusting the pH of the solution from step 2 to a pH between 6 and 8, - addition of NaCI and any nutrients necessary for the culture of halophilic bacteria, - transformation of the AGVs of the solution obtained in step 2) into PHA by at least one halophilic bacterium, - recovery of biomass containing PHAs, - extraction of PHAs from biomass, - possibly drying of PHAs.

22. Process for producing PHA according to one of claims 18 to 21, characterized in that the bacteria used in step 3) are bacteria chosen from Haloferax mediterranei, Haloferax volcanii, Haloferax gibbonsii, Halomonas boliviensis, Halomonas halophila, Halomonas bluphagenesis and mixtures thereof.

23. Process for producing PHA according to one of claims 18 to 22, characterized in that the PHAs obtained in step 3 are poly(3- hydroxybutyrate-co-3-hydroxyva lerate) (PH BV).

24. Process for producing PHA according to one of claims 18 to 23, characterized in that the PHAs obtained in step 3 are PHBVs comprising between 1 and 70 mol% of hydroxyvalerate (HV) units, preferably 10 to 30%.

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