Method for recovering and purifying polyhydroxyalkanoates from fermentation broth

Abstract

A process for the recovery and purification of polyhydroxyalkanoates (PHAs) from a fermentation broth derived from microbial fermentation, comprising the steps of: (a) subjecting the fermentation broth itself to a heat treatment in the presence of at least one acid; (b) separating the fermentation broth obtained in step (a) directly or after storage to obtain an aqueous suspension of cellular biomass containing polyhydroxyalkanoate (PHA) and an aqueous phase containing fermentation residues; (c) subjecting the aqueous suspension of cellular biomass comprising polyhydroxyalkanoate (PHA) obtained in step (b) to extraction, purification and drying; characterized in that the step (a) is carried out at a pH of 4 to 6, preferably 4.5 to 5.5, more preferably at a pH equal to 5, at a temperature of 50 DEG C to 70 DEG C, preferably 55 DEG C to 65 DEG C, more preferably at a temperature equal to 60 DEG C for a time of 5 to 30 minutes, preferably 8 to 20 minutes, more preferably equal to 10 minutes. The above polyhydroxyalkanoates (PHA) can be advantageously used in a variety of applications, in particular in the fields of medicine, pharmacology, cosmetics, agriculture, engineering and food packaging.

Description

[0001] The present invention relates to a method for recovering and purifying polyhydroxyalkanoates (PHA) from fermentation broth.

[0002] More specifically, the present invention relates to a method for recovering and purifying polyhydroxyalkanoate (PHA) from a fermentation broth derived from microbial fermentation, comprising the following steps: (a) heat-treating the fermentation broth in the presence of at least one acid; (b) separating the fermentation broth obtained in step (a) directly or after storage to obtain an aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) and an aqueous phase containing fermentation residues; (c) extracting, purifying and drying the aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) obtained in step (b); characterized in that step (a) is carried out at a specific pH value, temperature and time.

[0003] The above-mentioned polyhydroxyalkanoates (PHAs) can be advantageously used in various applications, particularly in the fields of medicine, pharmacology, cosmetics, agriculture, engineering and food packaging.

[0004] Polyhydroxyalkanoates (PHA) are homopolymers or copolymers of hydroxyalkanoates, such as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxyvalerate (4HV), 3-hydroxyhexanoate (3HH). The homopolymers or copolymers obtained from renewable resources are completely biodegradable and are synthesized and accumulated by various prokaryotic microorganisms, especially bacteria, as carbon and energy reserves for cell metabolism and, in most cases, under growth conditions where essential nutrients are found in limited concentrations. Polyhydroxyalkanoates (PHA) are formed as intracellular granules and can accumulate up to 80% by weight of the cell mass, which must then be extracted from them by manipulation in such a way as to obtain polyhydroxyalkanoates (PHA) of sufficient purity.

[0005] Polyhydroxyalkanoates (PHAs) have many advantages over synthetic polymers and other biopolymers obtained from renewable resources [e.g., polylactic acid (PLA)], especially in terms of biodegradability, recyclability and hydrophobicity, which make them particularly promising products as biodegradable alternatives to petrochemical-derived polymers.

[0006] Furthermore, polyhydroxyalkanoates (PHAs) are of industrial interest because they can be processed with conventional equipment and can therefore be developed in a wide variety of applications, even with high added value. Among the applications, the main properties that distinguish them, especially compared to other biopolymers, are good mechanical and barrier properties (e.g., in the field of food packaging) and biocompatibility (e.g., in the fields of medicine, pharmacology, cosmetics).

[0007] The production of polyhydroxyalkanoates (PHA) is known in the art. However, it should be noted that the production methods of polyhydroxyalkanoates (PHA) on an industrial scale are generally more complicated than those used for other biopolymers, and therefore the production time and costs are high. In particular, fermentation is a rather cumbersome process economically speaking, on the one hand because of the cost of the raw materials used, and on the other hand because it is characterized by a rather low final productivity. Therefore, at the end of the process, the recovery and purification of the polyhydroxyalkanoates (PHA) must be carried out in a manner with the greatest possible efficiency, without affecting the quality of the polyhydroxyalkanoates (PHA) produced.

[0008] As mentioned above, polyhydroxyalkanoates (PHA) are synthesized by specific prokaryotic microorganisms, in particular bacteria, as an additional source of nutrition used when they have to survive under unfavorable conditions (cellular stress). Therefore, the same microorganisms that synthesize and accumulate polyhydroxyalkanoates (PHA) are able to activate metabolic processes that lead to the depolymerization of the produced polyhydroxyalkanoates (PHA) if they are subjected to unfavorable growth conditions. Therefore, in the process for the production of polyhydroxyalkanoates (PHA) by fermentation, it is important not to trigger said metabolic processes in order to maximize the yield of the desired end product.

[0009] The literature describes laboratory and industrial methods for the recovery and purification of the different types of polyhydroxyalkanoates (PHA) produced by the above-mentioned microorganisms via biosynthetic pathways. It is known that the extraction process of the produced polyhydroxyalkanoates (PHA) plays a crucial role in determining their final properties, both in terms of crystallinity and purity.

[0010] For example, Pagliano G. et al. reported in "Recovery of Polyhydroxyalkanoates From Single and Mixed Microbial Cultures: A Review", "Frontiers in Bioengineering and Biotechnology" (2021), Vol. 9, Article 624021 that the main parameters that must be analyzed in order to evaluate the production method of polyhydroxyalkanoates (PHA) are:

[0011] - the recovery efficiency of the polyhydroxyalkanoates (PHA) obtained, i.e. the ratio between the polyhydroxyalkanoates (PHA) recovered at the end of the extraction process and the polyhydroxyalkanoates (PHA) accumulated in the microorganism at the end of the fermentation, measured by laboratory analysis such as solvent extraction, gas chromatography (GC) or thermogravimetric analysis (TGA);

[0012] - the purity of the polyhydroxyalkanoates (PHAs) obtained, usually determined by thermogravimetric analysis (TGA) (e.g. measuring the immobilization residue at 600° C.), or by looking for typical contaminants, in particular lipids and proteins;

[0013] The quality of the obtained polyhydroxyalkanoate (PHA) is usually evaluated by measuring the molecular weight and polydispersity index of the polyhydroxyalkanoate (PHA) by gel permeation chromatography (GPC).

[0014] In the case of intracellular biopolymer production, such as that of polyhydroxyalkanoates (PHA), once the synthesis is complete and the fermentation broth has been discharged from the fermentation reactor, said fermentation broth comprising water, fermentation residues (metabolites and organic and / or inorganic compounds used to promote growth) and cell biomass comprising polyhydroxyalkanoates (PHA) undergoes a separation phase, which is carried out using known techniques, such as filtration, centrifugation, flocculation, and subsequent washing, for example by dilution with deionized water, to obtain an aqueous suspension of cell biomass comprising polyhydroxyalkanoates (PHA) and an aqueous phase comprising fermentation residues. Said separation and washing steps can be repeated several times.

[0015] The aqueous phase is removed as an aqueous stream, while the aqueous suspension of cell biomass containing polyhydroxyalkanoates (PHA) is subjected to extraction, which can be performed in the absence of a solvent by mechanical cell lysis and / or chemical cell lysis, during which the cell walls are destroyed to obtain an aqueous suspension containing organic matter derived from cell wall destruction, wherein the organic matter contains polypeptides, phospholipids, DNA, RNA, peptidoglycan (referred to as non-PHA cellular material-NPCM) and polyhydroxyalkanoates (PHA) in solid form.

[0016] Subsequently, in order to separate the organic matter derived from the disruption of the cell walls (referred to as non-PHA cell matter-NPCM) from the polyhydroxyalkanoates (PHA), the aqueous suspension comprising the organic matter derived from the disruption of the cell walls (referred to as non-PHA cell matter-NPCM) and the polyhydroxyalkanoates (PHA) is again subjected to a separation and washing step, for example by dilution with demineralized water (as described above), to obtain an aqueous suspension comprising polyhydroxyalkanoates (PHA).

[0017] Alternatively, the extraction may be performed by freezing and freeze-drying in the presence of a solvent to obtain a dehydrated cell powder comprising polyhydroxyalkanoate (PHA), which is subsequently dissolved in the presence of a solvent.

[0018] At the end of the above-mentioned extraction, a purification step is carried out so as to obtain a precipitate of the polyhydroxyalkanoate (PHA) in order to remove the impurities still present and to increase the purity of the polyhydroxyalkanoate (PHA) obtained. Generally, the purification step is carried out by using an antisolvent in the case of extraction in the presence of a solvent, or by washing one or more times, for example with demineralized water, optionally with the addition of an oxidizing agent, in the case of extraction in the absence of a solvent, so as to obtain a solid residue comprising the purified polyhydroxyalkanoate (PHA).

[0019] Subsequently, the solid residue obtained, containing the purified polyhydroxyalkanoate (PHA), is dried in order to remove any anti-solvent and / or water that may be present, thereby obtaining a minimal residual moisture content (i.e. a moisture content lower than or equal to 0.5%), thus allowing the use of the polyhydroxyalkanoate (PHA) in subsequent applications.

[0020] It is known in the literature how it is possible and useful to insert between the separation and washing stage and the extraction stage an intermediate pretreatment stage, the purpose of which is to precondition the aqueous suspension of cell biomass comprising polyhydroxyalkanoates (PHA) to the subsequent extraction stage.

[0021] For example, Koller M. et al., in "Strategies for recovery and purification of poly[(R)-3-hydroxyalkanoates](PHA)biopolyesters from surrounding biomass", "Engineering in Life Sciences" (2013), Vol. 13, pp. 549-562, reported the possibility of drying an aqueous suspension of cell biomass containing polyhydroxyalkanoates (PHA) by freeze drying or by heat treatment (e.g., by drying with hot air) before a cell lysis step. The obtained dried cell biomass containing polyhydroxyalkanoates (PHA) can then be washed with a polar solvent such as methanol, ethanol, acetone to remove lipids and subjected to a cell lysis step using a solvent such as chloroform to obtain polyhydroxyalkanoates (PHA) with an increased purity. The authors also report the possibility of performing mechanical cell lysis, wherein an aqueous suspension of cell biomass containing polyhydroxyalkanoates (PHA) is introduced into a homogenizer (in two steps at 800 bar) and subsequently subjected to chemical cell lysis in the presence of a surfactant (sodium dodecyl sulfate - SDS) (1% relative to the aqueous suspension of cell biomass) and a base (sodium hydroxide - NaOH) (pH 12).

[0022] Pérez-Rivero C. et al., in "A Sustainable Approach for the Downstream Processing of Bacterial polyhydroxyalkanoates: State-of-the-art and latest developments", "Biochemical Engineering Journal" (2019), Article 107283, reported that before the cell lysis step, an aqueous suspension of cell biomass containing polyhydroxyalkanoates (PHA) was pretreated by heating, freezing, adding salt, grinding in liquid nitrogen or using hot compressed water to increase the permeability of the microbial cells and thereby increase the extraction yield and purity of polyhydroxyalkanoates (PHA).

[0023] Kunasundari B. in "Isolation and recovery of microbialpolyhydroxyalkanoates", "eXPRESS Polymer Letters" (2011), Vol. 5, No. 7, pp. 620-634, reported that in the pretreatment technology of the aqueous suspension of the cell biomass containing polyhydroxyalkanoates (PHA), a precipitant, particularly sodium dihydrogen phosphate, calcium chloride and polyacrylamide, was added, which allowed the cells of the microorganism to be easily separated from the remaining components of the fermentation broth by filtration (e.g., by a filter press).

[0024] Jiang Y. et al. reported the efficacy of the cell lysis process produced by the addition of alkali and surfactants in “Feasibility study of an alkaline-based chemical treatment for the purification of polyhydroxybutyrate produced by a mixed enriched culture”, “AMB Express” (2015) 5:5.

[0025] Yu J. et al., in "Generation and Utilization of Microbial Biomass Hydrolysates in Recovery and Production of Poly(3-hydroxybutyrate)", "Intech Open - Biomass Now - Cultivation and Utilization" (2013), Chapter 2, pages 33-48, http: / / dx.doi.org / 10.5772 / 52940, reported a method for extracting poly-3-hydroxybutyrate (PHB) from a fermentation broth, which comprises centrifuging the fermentation broth, treating it with an acid, treating it with an alkali, preferably in the presence of a surfactant, treating it with hypochlorite, and then washing and drying it.

[0026] Patent US 9,683,076 relates to a method for recovering and purifying polyhydroxyalkanoate (PHA) from cell culture, which comprises:

[0027] (a) acidifying the cell culture by adding an aqueous solution of an inorganic or organic acid selected from sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, acetic acid, citric acid or a mixture thereof so as to obtain a pH value of less than or equal to 6, and subjecting the cell culture to cell fractionation using high pressure homogenization at a temperature greater than or equal to 10° C. and less than or equal to 80° C. so as to obtain a PHA suspension;

[0028] (b) alkalizing the PHA suspension thus obtained so as to obtain a pH value greater than or equal to 8;

[0029] (c) diluting the PHA suspension and subjecting the diluted PHA suspension to a first tangential filtration to obtain a concentrated PHA suspension as a first retentate and a first aqueous phase as a first permeate;

[0030] (d) bleaching the concentrated PHA suspension;

[0031] (e) diluting the PHA suspension after bleaching and subjecting the diluted bleached PHA suspension to a second cross-flow filtration to obtain a concentrated bleached PHA suspension as a second retentate and a second aqueous phase as a second permeate; and

[0032] (f) drying the concentrated bleached PHA suspension.

[0033] The above process is said to be advantageous because it is carried out continuously, ie without batch steps, separates the PHA from the suspension and is free of solvent, and enables the PHA to be obtained in a form as pure as possible without causing a reduction in molecular weight.

[0034] Patent EP 1,705,250 relates to a method for directly isolating and purifying polyhydroxyalkanoates from cells derived from bacterial fermentation broth, which comprises the following steps:

[0035] (1) Pre-treating the fermentation broth by physical methods to achieve cell membrane destruction;

[0036] (2) adjusting the pH value of the pretreated fermentation broth to an alkaline value;

[0037] (3) adding anionic surfactant and reacting under stirring;

[0038] (4) separating and extracting the precipitate from the reaction solution;

[0039] (5) washing and drying;

[0040] The steps of adjusting pH and adding surfactant are interchangeable.

[0041] It is said that the above method can effectively reduce separation and purification costs, reduce pollution, and can be applied in industry.

[0042] US Patent No. 7,393,668 relates to a method for recovering polyhydroxyalkanoate from microbial cells containing polyhydroxyalkanoate, which comprises the following steps (a) and (b):

[0043] (a) comprising the steps of adding an alkali to an aqueous suspension of microbial cells containing polyhydroxyalkanoate while stirring, and subjecting the suspension to a physical disruption treatment to disrupt the cells, dissolve or emulsify cellular substances other than polyhydroxyalkanoate in the cells, and then separating the polyhydroxyalkanoate from the aqueous suspension, and

[0044] (b) comprising the steps of treating the separated polyhydroxyalkanoate with an enzyme and / or a surfactant to dissolve impurities adhering to the polyhydroxyalkanoate or dissolve them after decomposition, and then washing the polyhydroxyalkanoate with a hydrophilic solvent and / or water.

[0045] The prior art reported above describes methods for recovering and purifying polyhydroxyalkanoates (PHA), but does not focus on the development of methods involving a stabilization step of the fermentation broth itself and its possible combination with subsequent extraction and purification steps. In particular, there is no analysis of the criticalities that occur when the cells of the microorganisms used for the fermentation are subjected to unfavorable environmental conditions, although the cells are in an active life state (i.e. without substrate and aeration), which usually occurs at the end of the fermentation but before the cell lysis step. In fact, under these conditions, the cells of the microorganisms that are still alive activate metabolic processes that, due to the action of enzymes (such as depolymerases), consist in consuming the polyhydroxyalkanoates (PHA) accumulated therein during the fermentation, which are therefore detrimental to both the quality and the quantity of the polyhydroxyalkanoates (PHA) that can be obtained. Specifically, the depolymerization process of polyhydroxyalkanoate (PHA) is of intracellular type and includes the hydrolysis of polyhydroxyalkanoate (PHA) accumulated in the cells of microorganisms during fermentation, which is broken down into simpler elements useful for cellular metabolism as described above, for example, Knoll M. et al. in "The PHA Depolymerase Engineering Database: A systematic analysis tool for the diverse family of polyhydroxyalkanoate (PHA) depolymerases" "BMC Bioinformatics" (2009), doi: 10.1186 / 1471-2105-10-89.

[0046] Furthermore, in the prior art reported above, there is no description of a treatment applicable to the fermentation broth itself which does not affect the quality of the final product and which is capable of reducing the loss of polyhydroxyalkanoates (PHA) which usually occurs during the extraction phase due to the residual activity of the cells of the microorganisms used or also due to changes in the rheological properties of the fermentation broth (e.g., changes in the viscosity of the fermentation broth due to autolysis phenomena of the cells of the microorganisms). Furthermore, the prior art reported above does not describe how to properly apply the intermediate pretreatment step at the moment when the conditions favorable to the growth of the cells of the microorganisms cease, so as to quickly suppress any metabolic processes capable of changing the quality and quantity of polyhydroxyalkanoates (PHA) present in the cells of the microorganisms, without having to wait for the technical time required for the first treatment operation of the fermentation broth, such as separation and washing. However, in industrial practice, as well as in experimental practice, in the case where it is not possible to immediately treat the fermentation broth, it is always advantageous to stabilize the fermentation broth immediately after fermentation and therefore at the moment when the conditions favorable to the growth of the cells of the microorganisms cease, in order to avoid the start of the above-mentioned metabolic processes that affect the quality of the final product.

[0047] The above-mentioned stabilization stage is particularly important in cases where the first steps in the treatment of the fermentation broth consist of laborious or complex operations which take time to carry out, especially on a large scale, such as separation and washing stages which, as mentioned above, can provide for repeated concentration and dilution of the fermentation broth and which, at an industrial level, may require processing times of even tens of hours.

[0048] Another common problem in the known technology is the concentration of microbial cells in the fermentation broth subjected to chemical and / or mechanical lysis: in fact, it is usually necessary to operate at a cell concentration in the fermentation broth below 200 g / l (dry weight), since increasing the cell concentration reduces the efficiency and efficacy of the process.

[0049] The Applicant was therefore faced with the problem of finding a process for recovering polyhydroxyalkanoates (PHA) from fermentation broths originating from microbial fermentations which overcomes the above-mentioned disadvantages.

[0050] The applicant has now found that the recovery of polyhydroxyalkanoates (PHA) from a fermentation broth originating from a microbial fermentation can be advantageously carried out by a method comprising the following steps: (a) heat-treating the fermentation broth itself in the presence of at least one acid; (b) separating the fermentation broth obtained in step (a) directly or after storage and then separating, obtaining an aqueous suspension of cell biomass containing polyhydroxyalkanoates (PHA) and an aqueous phase containing fermentation residues; (c) extracting, purifying and drying the aqueous suspension of cell biomass containing polyhydroxyalkanoates (PHA) obtained in step (b); characterized in that step (a) is carried out at a specific pH value, temperature and time. The method allows the inhibition of the cell activity of the cells of the microorganisms used in the fermentation, and thus inhibits the self-consumption of the produced polyhydroxyalkanoates (PHA). In this way, the subsequent separation, extraction, purification and drying steps can also be delayed, and the yield and quality of the polyhydroxyalkanoates (PHA) obtained are consistent with the yield and quality of the polyhydroxyalkanoates (PHA) that can be obtained by instantaneous treatment of the fermentation broth. Furthermore, the process object of the present invention allows obtaining changes in the rheological properties of the fermentation broth, which facilitates and makes more efficient the polyhydroxyalkanoate (PHA) extraction process, which can be simpler and carried out at higher concentrations than those reported in the known art. Furthermore, the process object of the present invention can be advantageously applied to extraction processes in the presence of solvents and in the absence of solvents, and therefore also to already existing processes, improving their industrial feasibility. Last but not least, the process object of the present invention allows carrying out the extraction step in the absence of solvents at high cell concentrations [i.e. at cell concentrations greater than or equal to 200 g / l (dry weight)], maintaining high efficiency.

[0051] The object of the present invention is therefore a method for recovering polyhydroxyalkanoates (PHA) from a fermentation broth originating from a microbial fermentation, comprising the following steps:

[0052] (a) subjecting the fermentation broth itself to a heat treatment in the presence of at least one acid;

[0053] (b) directly separating or storing the fermentation broth obtained in step (a) and then separating to obtain an aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) and an aqueous phase containing fermentation residues;

[0054] (c) extracting, purifying and drying the aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) obtained in step (b);

[0055] Characterized in that step (a) is carried out at a pH of 4 to 6, preferably 4.5 to 5.5, more preferably at a pH of 5, at a temperature of 50°C to 70°C, preferably 55°C to 65°C, more preferably at a temperature of 60°C, for a time of 5 minutes to 30 minutes, preferably 8 minutes to 20 minutes, more preferably equal to 10 minutes.

[0056] For the purposes of the present description and of the following claims, the definitions of numerical ranges always include the endpoints, unless stated otherwise.

[0057] For the purposes of this specification and the following claims, the term "comprising" also encompasses the terms "consisting essentially of" or "consisting of.

[0058] The fermentation broth that can be used for the purpose of the present invention may originate from the fermentation of any microorganism capable of producing polyhydroxyalkanoates (PHA), in particular capable of producing intracellular polyhydroxyalkanoates (PHA).

[0059] Specific examples of microorganisms that can be advantageously used for the purposes of the present invention belong to the genera Cupriavidus, Pseudomonas, Bacillus, Ralstonia, Halomonas, Alcaligenes, Escherichia, Azotobacter, Aeromonas, Nocardia, Methylobaterium, Burkholderia, Hydrogenophaga, preferably Cupriavidus necator.

[0060] The nutrient substrate may be any type of substrate that can be metabolized by the above-mentioned microorganisms to produce polyhydroxyalkanoates (PHA), and may be selected from, for example, the following: a culture medium containing sugars (e.g., glucose); juice, molasses or pulp obtained from processing of plant products such as fruits, beets, sugar cane, oilseeds, etc.; wastewater, such as wastewater from water treatment or processing of juice; or the like. In addition to carbohydrates and proteins, the substrate generally contains various growth factors, such as compounds containing nitrogen and / or phosphorus, and other elements useful for cell growth of the above-mentioned microorganisms.

[0061] For the purposes of the present invention, the fermentation can be carried out batchwise, or as a discontinuous culture (fed-batch fermentation) or as a continuous culture.

[0062] According to a preferred embodiment of the present invention, the at least one acid may be selected from, for example, an inorganic acid or an organic acid, such as: sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, citric acid, preferably sulfuric acid.

[0063] At the end of step (a), the fermentation broth obtained is cooled (for example, by letting the temperature drop naturally, or by means of an air cooling jacket or a water cooling jacket present in the fermentation reactor).

[0064] According to a preferred embodiment of the present invention, the fermentation broth obtained in step (a) can be cooled to a temperature of 5°C to 40°C, preferably 10°C to 30°C, within 5 minutes to 2 hours, preferably 30 minutes to 1 hour.

[0065] According to a preferred embodiment of the present invention, the fermentation broth obtained in step (a) may be stored for 4 to 24 hours, preferably 10 to 22 hours.

[0066] According to a preferred embodiment of the present invention, in the step (b), the fermentation broth obtained in step (a) can be directly or stored and then subjected to tangential filtration and / or centrifugation, which can be carried out in batches or continuously, more preferably by continuous centrifugation, to obtain an aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) and an aqueous phase containing fermentation residues. Preferably, at the end of the filtration and / or centrifugation, a cell concentration of 200 g / l (dry weight) to 800 g / l (dry weight), more preferably 250 g / l (dry weight) to 500 g / l (dry weight) is obtained.

[0067] According to a preferred embodiment of the present invention, the aqueous suspension of cell biomass containing polyhydroxyalkanoates (PHA) can be subjected to a washing step by diluting with demineralized water or a diluted aqueous solution containing trace cell debris (i.e. the diluted aqueous solution of the aqueous phase containing the fermentation residue as described above) before the extraction, purification and drying step (c), followed by a concentration stage by tangential filtration and / or centrifugation, which can be carried out batchwise or continuously, preferably by continuous centrifugation. Preferably, at the end of the washing step, a cell concentration of 100 g / l (dry weight) to 400 g / l (dry weight), preferably 150 g / l (dry weight) to 250 g / l (dry weight) is obtained. l (dry weight), while at the end of the concentration step, a cell concentration of 200 g / l (dry weight) to 800 g / l (dry weight), preferably 250 g / l (dry weight) to 500 g / l (dry weight) is obtained.

[0068] The above washing and concentration steps can be repeated multiple times.

[0069] The cell concentration of the obtained aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) can be measured by determining the dry weight of the cells of the microorganism used (in grams per liter of fermentation broth), the cells of the microorganism being derived from a known volume of fermentation broth sample collected at preset time intervals (e.g., during fermentation and / or at the end of fermentation, and / or at the end of the washing and concentration steps). Specifically, the dry weight of the cell biomass refers to the weight of the cells of the microorganism contained in a known volume of fermentation broth, which is determined by filtering on Whatman GF filters / F (0.7 μm) to remove all moisture and then weighing the cells of the above-mentioned microorganism after heat treatment in a ventilated oven at 105°C to constant weight (about 24 hours).

[0070] The aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) obtained in the separation step (b) is subjected to the step (c) of extraction, purification and drying.

[0071] Step (c) of extraction, purification and drying, object of the process of the present invention, can be carried out in the presence or absence of a solvent.

[0072] According to a preferred embodiment of the present invention, if said step (c) is carried out in the absence of a solvent, the aqueous suspension of cell biomass comprising polyhydroxyalkanoates (PHA) obtained in separation (b) may be subjected to mechanical cell lysis and / or chemical cell lysis.

[0073] According to a preferred embodiment of the present invention, the aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) can be subjected to cell lysis by mechanical treatment, which can be carried out using a high-pressure homogenizer (e.g., Gea's homogenizer Mod. Panda PLUS 2000) at a pressure of 500 bar to 2000 bar, preferably 800 bar to 1500 bar, at a temperature of 10° C. to 80° C., preferably 20° C. to 50° C. Typically, the mechanical treatment requires 1 to 5 steps in the homogenizer and is carried out at a temperature and pressure that avoids possible degradation of polyhydroxyalkanoate (PHA).

[0074] According to a preferred embodiment of the present invention, at the end of the mechanical treatment, the obtained aqueous suspension containing polyhydroxyalkanoate (PHA) can be subjected to chemical cell lysis, wherein the chemical cell lysis comprises:

[0075] - alkalizing the aqueous suspension containing polyhydroxyalkanoate (PHA) by adding at least one strong base, preferably at least one inorganic strong base such as an aqueous solution of sodium hydroxide, potassium hydroxide or a mixture thereof, until a pH value greater than or equal to 9, preferably between 9.5 and 10, is obtained, obtaining an alkalized aqueous suspension containing polyhydroxyalkanoate (PHA);

[0076] - adding at least one surfactant to the alkalized aqueous suspension comprising polyhydroxyalkanoate (PHA) in an amount of 0.05 to 1 g, preferably 0.1 to 0.5 g, per gram of dry weight of the aqueous suspension comprising polyhydroxyalkanoate (PHA).

[0077] According to a preferred embodiment of the present invention, the chemical cell lysis may be performed at a temperature of 10°C to 80°C, preferably 20°C to 50°C, for a time of 20 minutes to 120 minutes, preferably 50 minutes to 70 minutes.

[0078] The surfactant may be selected, for example, from anionic surfactants, cationic surfactants or nonionic surfactants. The surfactant is preferably selected from those having a low environmental impact in order to avoid disposal problems.

[0079] The anionic surfactants useful for the purposes of the present invention may be selected, for example, from the following: alkyl or alkenyl sulfates, alkyl or alkenyl benzene sulfonates, alkyl or alkenyl ether sulfates, alkyl or alkenyl carboxylates, alkyl or alkenyl ether carboxylates or mixtures thereof. Particularly preferred are C 10 -C 18 Alkyl sulfate, more preferably sodium dodecyl sulfate (SDS).

[0080] Cationic surfactants suitable for the purposes of the present invention may be chosen, for example, from alkyltrimethylammonium or dialkyldimethylammonium salts, or mixtures thereof.

[0081] Nonionic surfactants suitable for the purpose of the present invention may be selected, for example, from polyoxyalkylene (preferably polyoxyethylene) alkyl or alkenyl ethers, alkyl or alkenyl phenyl ethers, polyoxyethylene / polyoxypropylene copolymers or mixtures thereof.

[0082] According to a preferred embodiment of the present invention, the aqueous suspension containing polyhydroxyalkanoates (PHA) obtained from chemical cell lysis can be subjected to a first concentration step by tangential filtration and / or centrifugation, which can be performed batchwise or continuously, preferably by continuous centrifugation, followed by a washing phase by dilution with demineralized water or with a diluted aqueous solution containing traces of cell debris, followed by a second concentration step by tangential filtration and / or centrifugation, which can be performed batchwise or continuously, preferably by continuous centrifugation. Preferably, at the end of the first concentration step, a cell concentration of 200 g / l (dry weight) to 800 g / l (dry weight), more preferably 250 g / l (dry weight) to 500 g / l (dry weight) is obtained, and at the end of the washing step, a cell concentration of 100 g / l (dry weight) to 400 g / l, more preferably 150 g / l (dry weight) to 200 g / l (dry weight) is obtained, and at the end of the second concentration step, a cell concentration of 200 g / l (dry weight) to 800 g / l (dry weight), preferably 250 g / l (dry weight) to 500 g / l (dry weight) is obtained.

[0083] The above concentration and dilution steps may be repeated multiple times.

[0084] It should be noted that the term "cell concentration" refers to the concentration of organic matter derived from the destruction of the cell wall after mechanical cell lysis and / or chemical cell lysis, said organic matter comprising polypeptides, phospholipids, DNA, RNA, peptidoglycan (referred to as non-PHA cellular matter-NPCM) and polyhydroxyalkanoates (PHA) in solid form. In particular, dry weight refers to the weight of said organic matter and said polyhydroxyalkanoates (PHA) contained in a known volume of an aqueous suspension containing polyhydroxyalkanoates (PHA), which is determined by weighing said organic matter and polyhydroxyalkanoates (PHA) after filtering on a Whatman GF / F filter (0.7 μm) to remove all water and then heat treating in a ventilated oven at 105°C until constant weight (about 24 hours).

[0085] According to a preferred embodiment of the present invention, the aqueous suspension comprising polyhydroxyalkanoates (PHA) obtained from chemical cell lysis can be dried, preferably in a ventilated oven, at a temperature greater than or equal to 50° C., preferably from 55° C. to 70° C., until a solid residue comprising polyhydroxyalkanoates (PHA) having a residual humidity of less than 0.5% is obtained.

[0086] According to a preferred embodiment of the present invention, if said step (c) is carried out in the presence of a solvent, the aqueous suspension of the cell biomass containing polyhydroxyalkanoate (PHA) obtained in the separation step (b) may be frozen and freeze-dried.

[0087] According to a preferred embodiment of the present invention, the aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) obtained in step (b) can be subjected to a process comprising:

[0088] - freezing the aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) at -20°C and subsequently freeze-drying at -46°C and 0.15 mbar for a period of greater than or equal to 12 hours, preferably 18 to 24 hours, until a dehydrated cell powder containing polyhydroxyalkanoate (PHA) is obtained;

[0089] - washing the dehydrated cell powder containing polyhydroxyalkanoate (PHA) with ethanol at a temperature of 30°C to 60°C, preferably 50°C, for a period of 30 minutes to 6 hours, preferably 4 hours, by operating at a ratio of dehydrated cell powder:ethanol of 1:10 to 1:40, preferably 1:20, to obtain a suspension containing polyhydroxyalkanoate (PHA);

[0090] - filtering the obtained suspension containing polyhydroxyalkanoate (PHA) through a porous membrane filter or a cellulose filter and drying the solid residue containing polyhydroxyalkanoate (PHA) remaining on the filter at room temperature (25° C.) for a period of greater than or equal to 12 hours, preferably 18 to 24 hours;

[0091] - dissolving the solid residue obtained comprising dried polyhydroxyalkanoate (PHA) in a stirred vessel or reflux reactor, in the presence of at least one solvent (which may be chosen, for example, from chloroform, 1,2-dichloroethane, dichloromethane, preferably chloroform), at a temperature ranging from 40° C. to 70° C., preferably 60° C., operating at a ratio of dried residue:solvent ranging from 1:100 to 1:500, preferably equal to 1:200, for a period ranging from 4 hours to 24 hours, preferably 8 hours, and optionally repeating said dissolution up to 3 times.

[0092] - filtering the solution containing polyhydroxyalkanoate (PHA) obtained after the dissolution using a membrane filter or a cellulose filter to obtain a solid residue containing no polyhydroxyalkanoate (PHA) and a solution containing polyhydroxyalkanoate (PHA);

[0093] - optionally, concentrating the solution comprising polyhydroxyalkanoate (PHA) obtained by operating at a temperature lower than or equal to 70° C., preferably between 40° C. and 60° C., at a pressure lower than atmospheric pressure, preferably equal to 0.5 bar;

[0094] - contacting the solution comprising polyhydroxyalkanoate (PHA) in a stirred vessel with an excess of ethanol cooled to -20°C for a period of 0.1 to 60 minutes, preferably 0.5 to 25 minutes, before or after possible concentration, obtaining precipitation of a solid residue comprising polyhydroxyalkanoate (PHA);

[0095] - Mechanically collecting the solid residue containing polyhydroxyalkanoate (PHA) obtained and drying it at room temperature (25° C.) for a period of greater than or equal to 12 hours, preferably between 20 and 30 hours, or drying it in a ventilated oven at a temperature equal to 50° C. and under low pressure (for example, 11 absolute mabr) until a solid residue containing polyhydroxyalkanoate (PHA) having a residual humidity less than 0.5% is obtained.

[0096] As regards the polyhydroxyalkanoates (PHAs) which can be produced according to the present invention, these are generally polymers containing repeating units having the general formula (I):

[0097] -O-CHR1-(CH2) n -CO-(I)

[0098] Wherein R1 represents a hydrogen atom, or is selected from C1-C 12 Alkyl, C4-C 16 Cycloalkyl, C2-C 12 Alkenyl, optionally substituted by at least one group selected from halogen such as fluorine, chlorine, bromine, -CN, -OH, -COOH, -OR3, -COOR3, wherein R3 represents C1-C4 alkyl or benzyl, and n is an integer of 1-6, preferably 1 or 2. Preferably, R1 is methyl or ethyl, and n is 1 or 2.

[0099] The polyhydroxyalkanoates (PHA) may be homopolymers and copolymers or terpolymers. In the case of copolymers or terpolymers, they may be composed of different repeating units of formula (I) and at least one repeating unit derived from a comonomer capable of copolymerizing with hydroxyalkanoates, such as a lactone or a lactam. In the latter case, the repeating units of formula (I) are present in an amount equal to at least 10 mol % relative to the total number of moles of repeating units.

[0100] The repeating units of the general formula (I) are repeating units derived from 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxyundec-10-enoate, 4-hydroxyvalerate.

[0101] Particularly preferred polyhydroxyalkanoates (PHAs) are: poly-3-hydroxybutyrate (PHB), poly-3-hydroxyvalerate (PHV), poly-3-hydroxyhexanoate (PHH), poly-3-hydroxyoctanoate (PHO), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (PHBB), poly(3-hydroxyoctanoate-co-3-hydroxy-undecene-10-enoate) (PHOU), poly(3-hydroxybutyrate-co-3-hydroxyvalerate-c-4-hydroxyvalerate) (PHBVV), or mixtures thereof. Poly-3-hydroxybutyrate (PHB) is particularly preferred.

[0102] The following shows Figure 1 and Figure 2 Two embodiments of the method object of the present invention are shown for illustrative purposes only and not for limiting the present invention.

[0103] Specifically, Figure 1 An embodiment of the method object of the present invention is shown, in which the extraction, purification and drying steps (c) are carried out in the absence of a solvent. To this end, the fermentation broth is subjected to a heat treatment [step (a)] in the presence of at least one acid (e.g. sulfuric acid). Subsequently, the fermentation broth obtained in step (a) is directly or after storage ( Figure 1 The aqueous phase (indicated by the dashed line in the figure) is separated (e.g., by continuous centrifugation) and subsequently washed (e.g., by dilution with distilled water) (the separation / washing stage may be repeated several times) to obtain an aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) and an aqueous phase ( Figure 1 The aqueous suspension of cell biomass obtained containing polyhydroxyalkanoates (PHA) is subjected to mechanical treatment (i.e. mechanical cell lysis) (e.g. in a homogenizer) and subsequently to chemical cell lysis (e.g. in the presence of at least one base and at least one surfactant). The aqueous suspension containing polyhydroxyalkanoates (PHA) obtained at the end of the chemical cell lysis is Figure 1 The aqueous suspension containing polyhydroxyalkanoate (PHA) is obtained by separation (e.g., by continuous centrifugation) and subsequent washing (e.g., by dilution with distilled water) (the separation / washing steps can optionally be repeated multiple times) (the separation / washing steps can optionally be repeated multiple times) to obtain an aqueous suspension containing polyhydroxyalkanoate (PHA), which is dried (e.g., in a ventilated oven) to obtain a solid residue containing polyhydroxyalkanoate (PHA).

[0104] Figure 2An embodiment of the process object of the invention is shown, in which the extraction, purification and drying steps (c) are carried out in the presence of a solvent. To this end, the fermentation broth is subjected to a heat treatment in the presence of at least one acid (e.g. sulfuric acid) [step (a)]. Subsequently, the fermentation broth obtained in step (a) is directly or after storage ( Figure 2 The aqueous phase (indicated by the dashed line in the figure) is separated (e.g., by continuous centrifugation) and subsequently washed (e.g., by dilution with distilled water) (the separation / washing stage may be repeated several times) to obtain an aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) and an aqueous phase ( Figure 2 The obtained aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) was frozen / freeze-dried to obtain dehydrated cell powder containing polyhydroxyalkanoate (PHA) ( Figure 2 ), which is washed (e.g., with ethanol) to obtain a suspension containing polyhydroxyalkanoate (PHA) ( Figure 2 ), which was filtered to obtain a solid residue ( Figure 2 The solid residue containing the dried polyhydroxyalkanoate (PHA) is dissolved in the presence of at least one solvent (e.g., chloroform), and the obtained solution containing the polyhydroxyalkanoate (PHA) is Figure 2 The solid residue (not shown) was filtered to obtain a solid residue ( Figure 2 ) and a solution containing polyhydroxyalkanoate (PHA) ( Figure 2 The solution containing the polyhydroxyalkanoate (PHA) is precipitated in the presence of excess cold ethanol to obtain a solid residue containing the polyhydroxyalkanoate (PHA), which is dried (e.g., at room temperature or in a ventilated oven).

[0105] In order to better understand the present invention and put it into practice, some illustrative and non-limiting examples of the present invention are given below.

[0106] Example 1 (Present invention) [Step (c) in the absence of solvent]

[0107] For this purpose, fermentations were carried out with cells of Cupravidus Necator DSM 545, starting from a substrate of glucose and salts [(NH4)2SO4, MgSO4, KH2PO4, Na2HPO4], citric acid and trace metals, obtaining a cell concentration equal to 150 g / l (dry weight), containing 70% by weight of poly-3-hydroxybutyrate (PHB).

[0108] The content of poly-3-hydroxybutyrate (PHB) was determined by thermogravimetric analysis (TGA) according to UNI EN ISO 11358-1:2014 standard.

[0109] 2 liters of the obtained fermentation broth were placed in a 5 liter beaker, and a 10% aqueous sulfuric acid solution (previously prepared from 95%-97% sulfuric acid ( ISO-Merck), the whole was heated to 60°C and kept at said temperature and under stirring (250 rpm) for 10 minutes. Subsequently, the fermentation broth was cooled by letting the temperature drop naturally to room temperature (25°C) and centrifuged in a fixed rotor laboratory centrifuge (6000 rpm, 10 minutes), and the aqueous suspension of cell biomass (pellets) obtained was diluted to the initial cell concentration (i.e. 150 g / l) (dry weight) by adding demineralized water.

[0110] Subsequently, the diluted aqueous suspension of cell biomass (particles) was centrifuged again in a fixed rotor laboratory centrifuge (6000 rpm, 10 minutes), and the concentrated aqueous suspension of cell biomass (particles) obtained was diluted by adding demineralized water to reach a cell concentration of 250 g / l (dry weight).

[0111] The obtained diluted aqueous suspension of cell biomass (granules) was subjected to mechanical lysis at 1000 bar and room temperature (25° C.) by passage in a homogenizer (Mod. Panda PLUS 2000 from Gea) and subsequently diluted by adding demineralized water to reach a cell concentration of 100 g / l (dry weight). The aqueous suspension thus obtained was subjected to chemical lysis by adding a 20% aqueous sodium hydroxide solution (previously prepared from sodium hydroxide (purity ≥ 98% - Merck)) in an amount such that the pH reached a value equal to 9.6, and 0.15 g of sodium dodecyl sulfate (SDS) (purity 99% - Thermo Fisher Scientific) per gram of dry weight: the whole was kept at room temperature (25° C.) for 1 hour under stirring (250 rpm).

[0112] Subsequently, the whole was centrifuged using a fixed rotor laboratory centrifuge (6000 rpm, 20 minutes), and the obtained aqueous suspension (particles) was diluted to the initial cell concentration (i.e. 100 g / l) (dry weight) by adding demineralized water, and centrifuged again using a fixed rotor laboratory centrifuge (6000 rpm, 20 minutes). The concentrated aqueous suspension (particles) obtained was dried in a ventilated oven at 65° C. for 12 hours to obtain a solid residue containing poly-3-hydroxybutyrate (PHB).

[0113] The obtained solid residue containing poly-3-hydroxybutyrate (PHB) was subjected to thermogravimetric analysis (TGA) according to UNI EN ISO 11358-1:2014 TGA standard, obtaining at 600° C. a solid residue equal to 0.4% [solid residue not containing poly-3-hydroxybutyrate-(PHB)].

[0114] The weight average molecular weight (M) of the obtained poly-3-hydroxybutyrate (PHB) was measured by subjecting the solid residue containing poly-3-hydroxybutyrate (PHB) to GPC (gel permeation chromatography) using UltiMate 3000 Dionex manufactured by Thermo Fischer Scientific using a refractive index (RI) and operating under the following conditions: w ):

[0115] - TSKgel GMHHr-M column with pre-column (300x7,8 mm);

[0116] -Solvent / eluent: chloroform;

[0117] -Flow rate: 0.5ml / min;

[0118] - Temperature: 35°C;

[0119] -Polystyrene (PS) Standard Kit [Peak Molecular Weight (M p ) is 10 PS polymers ranging from 580 g / mol to 6570000 g / mol];

[0120] The result is equal to 1.6x10 6 Dalton.

[0121] The extraction yield of the whole process is expressed as follows: grams of poly-3-hydroxybutyrate (PHB) obtained at the end of the process / grams of poly-3-hydroxybutyrate (PHB) synthesized by the microorganism used and available upon discharge from the fermentation equipment was found to be >98%.

[0122] Example 2 (Present invention) [Step (c) in the presence of a solvent]

[0123] For this purpose, fermentations were carried out with cells of Cupravidus Necator DSM 545, starting from a substrate of glucose and salts [(NH4)2SO4, MgSO4, KH2PO4, Na2HPO4], citric acid and trace metals, obtaining a concentration of cell biomass equal to 150 g / l (dry weight) containing 70% by weight of poly-3-hydroxybutyrate (PHB).

[0124] The content of poly-3-hydroxybutyrate (PHB) was determined by thermogravimetric analysis (TGA) according to UNI EN ISO 11358-1:2014 standard.

[0125] 2 liters of the obtained fermentation broth was placed in a 5 liter beaker and a 10% sulfuric acid aqueous solution (previously prepared from 95%-97% sulfuric acid) was added. ISO-Merck) (in an amount such that the pH reaches a value equal to 5), the whole is heated to 60° C. and maintained at said temperature and under stirring (250 rpm) for 10 minutes. Subsequently, the fermentation broth is cooled by letting the temperature drop naturally to room temperature (25° C.) and centrifuged in a fixed-rotor laboratory centrifuge (6000 rpm, 10 minutes), and the aqueous suspension of cell biomass (pellets) obtained is diluted to the initial cell concentration (i.e. 150 g / l) (dry weight) by adding demineralized water and centrifuged again in a fixed-rotor laboratory centrifuge (6000 rpm, 10 minutes).

[0126] Subsequently, the aqueous suspension of concentrated cell biomass (granules) obtained was frozen at -20°C and dried in a freeze dryer at -46°C, 0.15 mbar for 24 hours. The dehydrated cell powder obtained was washed with ethanol (96°-Carlo Erba)) (ratio of dehydrated cell powder: ethanol was 1:20) at a temperature of 50°C for 4 hours: then, filtered through a porous membrane filter to obtain a solid residue containing poly-3-hydroxybutyrate (PHB), which remained on the filter. The solid residue obtained was dried at room temperature (25°C) for 24 hours.

[0127] Subsequently, the dry solid residue was dissolved in chloroform (purity ≥ 99.8% - HiPerSolv -VWR) (ratio of dried solid residue:chloroform equal to 1:200) for 8 hours: the dissolution was repeated 3 times.

[0128] Subsequently, the whole is filtered through a porous membrane filter to obtain a solid residue not containing poly-3-hydroxybutyrate (PHB) and a solution containing poly-3-hydroxybutyrate (PHB) retained on the filter, which is contacted with an excess of ethanol (96°-Carlo Erba) at -20°C for 10 minutes: the obtained precipitate containing poly-3-hydroxybutyrate (PHB) is dried at room temperature (25°C) for 12 hours to obtain a solid residue containing poly-3-hydroxybutyrate (PHB).

[0129] The obtained solid residue containing poly-3-hydroxybutyrate (PHB) was subjected to thermogravimetric analysis (TGA) according to UNI EN ISO 11358-1:2014 TGA standard, obtaining at 600° C. a solid residue equal to 0.1% [solid residue not containing poly-3-hydroxybutyrate-(PHB)].

[0130] The weight average molecular weight (M) of the obtained poly-3-hydroxybutyrate (PHB) was measured by GPC (gel permeation chromatography). w ) was measured as in Example 1 above, and the result was equal to 1.6x10 6 Dalton.

[0131] The extraction yield of the whole process is expressed as follows: grams of poly-3-hydroxybutyrate (PHB) obtained at the end of the process / grams of poly-3-hydroxybutyrate (PHB) synthesized by the microorganism used and available upon discharge from the fermentation equipment was found to be >99%.

[0132] Example 3 (Comparison [without step (a) of treating the fermentation broth])

[0133] For this purpose, fermentations were carried out with cells of Cupravidus Necator DSM 545, starting from a substrate of glucose and salts [(NH4)2SO4, MgSO4, KH2PO4, Na2HPO4], citric acid and trace metals, obtaining a concentration of cell biomass equal to 150 g / l (dry weight) containing 70% poly-3-hydroxybutyrate (PHB).

[0134] The content of poly-3-hydroxybutyrate (PHB) was determined by thermogravimetric analysis (TGA) according to UNI EN ISO 11358-1:2014 standard.

[0135] 5 hours after the end of the fermentation, in which the fermentation broth was kept at room temperature (25°C), 2 liters of the fermentation broth were centrifuged in a fixed rotor laboratory centrifuge (6000 rpm, 10 minutes): in this case, it was not possible to obtain a clear separation of the solid and liquid phases, even by increasing the centrifugation speed and the centrifugation time.

[0136] In view of this result, it was decided not to carry out the extraction step and to estimate the content of poly-3-hydroxybutyrate (PHB) present in the cells in the partially separated solid phase by thermogravimetric analysis (TGA) according to the UNI EN ISO 11358-1:2014 standard.

[0137] Evaluating the differences in the results of two thermogravimetric analyses (TGA) performed, the first immediately after the cellular broth was discharged from the fermentation apparatus and the second on the cell material recovered after centrifugation starting 5 hours after fermentation, it was estimated that about 10% by weight of the poly-3-hydroxybutyrate (PHB) originally contained in the cells was no longer available.

[0138] Example 4 (Present invention) [Step (c) in the presence of solvent - extraction for 20 hours]

[0139] For this purpose, fermentations were carried out with cells of Cupravidus Necator DSM 545, starting from a substrate of glucose and salts [(NH4)2SO4, MgSO4, KH2PO4, Na2HPO4], citric acid and trace metals, obtaining a concentration of cell biomass equal to 150 g / l (dry weight) containing 70% by weight of poly-3-hydroxybutyrate (PHB).

[0140] The content of poly-3-hydroxybutyrate (PHB) was determined by thermogravimetric analysis (TGA) according to UNI EN ISO 11358-1:2014 standard.

[0141] 2 liters of the obtained fermentation broth was placed in a 5 liter beaker and a 10% sulfuric acid aqueous solution (previously prepared from 95%-97% sulfuric acid ( ISO-Merck) (in an amount such that the pH value is equal to 5), the whole is heated to 60°C and maintained at said temperature and under stirring (250 rpm) for 10 minutes. Subsequently, the fermentation broth is cooled and the temperature is allowed to rise naturally to room temperature (25°C). After storage at room temperature (25°C) for 20 hours, the fermentation broth is centrifuged in a fixed rotor laboratory centrifuge (6000 rpm, 10 minutes), and the aqueous suspension of concentrated cell biomass (granules) obtained is diluted to the initial cell concentration (i.e. 150 g / l) (dry weight) by adding demineralized water and centrifuged again in a fixed rotor laboratory centrifuge (6000 rpm, 10 minutes).

[0142] Subsequently, the aqueous suspension of concentrated cell biomass (granules) obtained was frozen at -20°C and dried in a freeze dryer at -46°C, 0.15 mbar for 24 hours. The dehydrated cell powder obtained was washed with ethanol (96°-Carlo Erba) (ratio of dehydrated cell powder: ethanol was 1:20) at a temperature of 50°C for 4 hours: then, filtered through a porous membrane filter to obtain a solid residue containing poly-3-hydroxybutyrate (PHB), which remained on the filter. The solid residue obtained was dried at room temperature (25°C) for 24 hours.

[0143] Subsequently, the dry solid residue was dissolved in chloroform (purity ≥ 99.8% - HiPerSolv -VWR) (ratio of dried solid residue:chloroform equal to 1:200) was dissolved for 8 hours: the dissolution was repeated 3 times.

[0144] Subsequently, the whole is filtered through a porous membrane filter to obtain a solid residue not containing poly-3-hydroxybutyrate (PHB) and a solution containing poly-3-hydroxybutyrate (PHB) retained on the filter, which is contacted with an excess of ethanol (96°-Carlo Erba) at -20°C for 10 minutes: the obtained precipitate containing poly-3-hydroxybutyrate (PHB) is dried at room temperature (25°C) for 12 hours to obtain a solid residue containing poly-3-hydroxybutyrate (PHB).

[0145] The solid residue obtained, which contained poly-3-hydroxybutyrate (PHB), was subjected to thermogravimetric analysis (TGA) according to UNI EN ISO 11358-1:2014 TGA standard, obtaining at 600° C. a solid residue equal to 0.1% by weight [the solid residue did not contain poly-3-hydroxybutyrate-(PHB)].

[0146] The weight average molecular weight (M) of the obtained poly-3-hydroxybutyrate (PHB) was measured by GPC (gel permeation chromatography). w ), the operation is performed as in the above embodiment 1, and the result is equal to 1.6x10 6 Dalton.

[0147] The extraction yield of the whole process is expressed as follows: grams of poly-3-hydroxybutyrate (PHB) obtained at the end of the process / grams of poly-3-hydroxybutyrate (PHB) synthesized by the microorganism used and available upon discharge from the fermentation equipment was found to be >99%.

[0148] Example 5 (Invention) [Step (c) in the absence of solvent - high cell concentration]

[0149] For this purpose, fermentations were carried out with cells of Cupravidus Necator DSM 545, starting from a substrate of glucose and salts [(NH4)2SO4, MgSO4, KH2PO4, Na2HPO4], citric acid and trace metals, obtaining a concentration of cell biomass equal to 150 g / l (dry weight) containing 70% poly-3-hydroxybutyrate (PHB).

[0150] The content of poly-3-hydroxybutyrate (PHB) was determined by thermogravimetric analysis (TGA) according to UNI EN ISO 11358-1:2014 standard.

[0151] 2 liters of the fermentation broth obtained were placed in a 5 liter beaker and, after adding a 10% aqueous sulfuric acid solution (previously prepared from 95%-97% sulfuric acid (EMSURE ISO-Merck)) in an amount such that the pH reached a value equal to 5, the whole was heated to 60° C. and maintained at said temperature for 10 minutes under stirring (250 rpm). Subsequently, the fermentation broth was cooled by letting the temperature drop naturally to room temperature (25° C.) and centrifuged in a fixed rotor laboratory centrifuge (6000 rpm, 10 minutes), and the aqueous suspension of cell biomass (granules) obtained was diluted to the initial cell concentration (i.e. 150 g / l) (dry weight) by adding demineralized water.

[0152] Subsequently, the obtained diluted aqueous suspension of cell biomass (pellets) was centrifuged again in a fixed rotor laboratory centrifuge (6000 rpm, 10 minutes), and the obtained concentrated aqueous suspension of cell biomass (pellets) was diluted to a cell concentration of 250 g / l (dry weight) by adding demineralized water.

[0153] The obtained diluted aqueous suspension of cell biomass (granules) was subjected to mechanical lysis at 1000 bar and room temperature (25° C.) by passage in a homogenizer (Mod. Panda PLUS 2000 from Gea) and then diluted with demineralized water to a cell concentration of 200 g / l (dry weight). The obtained aqueous suspension was subjected to chemical lysis by adding a 20% aqueous sodium hydroxide solution (previously prepared from sodium hydroxide (purity ≥ 98% - Merck)) in an amount such that the pH reached a value equal to 9.6 and 0.15 g of sodium dodecyl sulfate (SDS) (purity 99% - Thermo Fisher Scientific) per gram of dry weight: the whole was kept at room temperature (25° C.) and under stirring for 1 hour.

[0154] Subsequently, the whole was centrifuged by a fixed rotor laboratory centrifuge (6000 rpm, 20 minutes), and the concentrated aqueous suspension (granules) obtained was diluted to the initial cell concentration (i.e. 200 g / l) (dry weight) by adding demineralized water, and centrifuged again using a fixed rotor laboratory centrifuge (6000 rpm, 20 minutes). The concentrated aqueous suspension (granules) obtained was dried in a ventilated oven at 65° C. for 12 hours to obtain a solid residue containing poly-3-hydroxybutyrate (PHB).

[0155] The solid residue obtained, comprising poly-3-hydroxybutyrate (PHB), was characterized by thermogravimetric analysis (TGA) according to UNI EN ISO 11358-1:2014 TGA, obtaining a solid residue equal to 0.4% by weight at 600° C. [the solid residue did not comprise poly-3-hydroxybutyrate-(PHB)].

[0156] The weight average molecular weight (M) of the obtained poly-3-hydroxybutyrate (PHB) was measured by GPC (gel permeation chromatography). w ) was measured as in Example 1 above, and the result was equal to 1.6×10 6 Dalton.

[0157] The extraction yield of the whole process is expressed as follows: the grams of poly-3-hydroxybutyrate (PHB) obtained at the end of the process / grams of poly-3-hydroxybutyrate (PHB) synthesized by the microorganism used and available upon discharge from the fermentation equipment was found to be >84%.

[0158] Example 6 (Comparison [without step (a) of treating the fermentation broth])

[0159] For this purpose, fermentations were carried out with cells of Cupravidus Necator DSM 545, starting from a substrate of glucose and salts [(NH4)2SO4, MgSO4, KH2PO4, Na2HPO4], citric acid and trace metals, obtaining a concentration of cell biomass equal to 150 g / l (dry weight) containing 70% by weight of poly-3-hydroxybutyrate (PHB).

[0160] The content of poly-3-hydroxybutyrate (PHB) was determined by thermogravimetric analysis (TGA) according to UNI EN ISO 11358-1:2014 standard.

[0161] 2 liters of the obtained fermentation broth were placed in a 5 liter beaker, and a 10% aqueous sulfuric acid solution (previously prepared from 95%-97% sulfuric acid ( ISO-Merck), the whole was heated to 60°C and kept at said temperature and under stirring (250 rpm) for 10 minutes. Subsequently, the fermentation broth was cooled by letting the temperature drop naturally to room temperature (25°C) and centrifuged in a fixed rotor laboratory centrifuge (6000 rpm, 10 minutes), the aqueous suspension of cell biomass (pellets) obtained was diluted to the initial cell concentration (i.e. 150 g / l) (dry weight) by adding demineralized water and centrifuged again in a fixed rotor laboratory centrifuge (6000 rpm, 10 minutes).

[0162] Subsequently, the aqueous suspension of concentrated cell biomass (granules) obtained was frozen at -20°C and dried in a freeze dryer at -46°C, 0.15 mbar for 24 hours. The dehydrated cell powder obtained was washed with ethanol (96°-Carlo Erba) (dehydrated cell powder: ethanol ratio of 1:20) at a temperature of 50°C for 4 hours: then filtered through a porous membrane filter to obtain a solid residue containing poly-3-hydroxybutyrate (PHB), which remained on the filter. The solid residue obtained was dried at room temperature (25°C) for 24 hours.

[0163] Subsequently, the dry solid residue was dissolved in chloroform (purity ≥ 99.8% - HiPerSolv -VWR) (ratio of dried solid residue:chloroform equal to 1:200) was dissolved for 8 hours: the dissolution was repeated 3 times.

[0164] Subsequently, the whole is filtered through a porous membrane filter to obtain a solid residue not containing poly-3-hydroxybutyrate (PHB) and a solution containing poly-3-hydroxybutyrate (PHB) retained on the filter, and the solution is contacted with an excess of ethanol (96°-Carlo Erba) at -20°C for 10 minutes: the obtained precipitate containing poly-3-hydroxybutyrate (PHB) is dried at room temperature (25°C) for 12 hours to obtain a solid residue containing poly-3-hydroxybutyrate (PHB).

[0165] The solid residue obtained, which contained poly-3-hydroxybutyrate (PHB), was subjected to thermogravimetric analysis (TGA) according to UNI EN ISO 11358-1:2014 TGA standard, obtaining at 600° C. a solid residue equal to 0.1% [the solid residue did not contain poly-3-hydroxybutyrate-(PHB)].

[0166] The weight average molecular weight (M) of the obtained poly-3-hydroxybutyrate (PHB) was measured by GPC (gel permeation chromatography). w ) was measured as in Example 1 above, and the result was equal to 1.6×10 6 Dalton.

[0167] The extraction yield of the whole process is expressed as follows: the grams of poly-3-hydroxybutyrate (PHB) obtained at the end of the process / grams of poly-3-hydroxybutyrate (PHB) synthesized by the microorganism used and available upon discharge from the fermentation equipment was found to be >99%.

Claims

1. A method for recovering polyhydroxyalkanoate (PHA) from a fermentation broth derived from microbial fermentation, comprising the following steps: (a) subjecting the fermentation broth itself to a heat treatment in the presence of at least one acid; (b) directly separating or storing the fermentation broth obtained in step (a) and then separating to obtain an aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) and an aqueous phase containing fermentation residues; (c) extracting, purifying and drying the aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) obtained in step (b); Characterized in that step (a) is carried out at a pH of 4 to 6, preferably 4.5 to 5.5, more preferably at a pH of 5, at a temperature of 50°C to 70°C, preferably 55°C to 65°C, more preferably at a temperature of 60°C, for a time of 5 minutes to 30 minutes, preferably 8 minutes to 20 minutes, more preferably 10 minutes.

2. The method for recovering polyhydroxyalkanoate (PHA) from a fermentation broth derived from microbial fermentation according to claim 1, wherein the at least one acid is selected from an inorganic acid or an organic acid, such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, citric acid, preferably sulfuric acid.

3. The method for recovering polyhydroxyalkanoate (PHA) from a fermentation broth derived from microbial fermentation according to claim 1 or 2, wherein the fermentation broth obtained in step (a) is cooled to a temperature of 5°C to 40°C, preferably 10°C to 30°C, within a period of 5 minutes to 2 hours, preferably 30 minutes to 1 hour.

4. The method for recovering polyhydroxyalkanoate (PHA) from a fermentation broth derived from microbial fermentation according to any one of the preceding claims, wherein the fermentation broth obtained in step (a) is stored for a period of 4 to 24 hours, preferably 10 to 22 hours.

5. The method for recovering polyhydroxyalkanoate (PHA) from a fermentation broth derived from microbial fermentation according to any one of the preceding claims, wherein in the step (b), the fermentation broth obtained in step (a) is directly or after storage subjected to tangential filtration and / or centrifugation in batches or continuously, preferably continuous centrifugation, to obtain an aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) and an aqueous phase containing the fermentation residue.

6. A method for recovering polyhydroxyalkanoates (PHA) from a fermentation broth derived from microbial fermentation according to claim 5, wherein at the end of the filtration and / or centrifugation, a cell concentration of 200 g / l (dry weight) to 800 g / l (dry weight), preferably 250 g / l (dry weight) to 500 g / l (dry weight) is obtained.

7. A method for recovering polyhydroxyalkanoates (PHA) from a fermentation broth derived from a microbial fermentation according to any one of the preceding claims, wherein, prior to the extraction, purification and drying step (c), the aqueous suspension of cell biomass comprising polyhydroxyalkanoates (PHA) is subjected to a washing step by diluting with demineralized water or a diluted aqueous solution containing traces of cell debris, followed by a concentration step by tangential filtration and / or centrifugation carried out in batches or continuously, preferably by continuous centrifugation.

8. A method for recovering polyhydroxyalkanoates (PHA) from a fermentation broth derived from microbial fermentation according to claim 7, wherein at the end of the washing step, a cell concentration of 100 g / l (dry weight) to 400 g / l (dry weight), preferably 150 g / l (dry weight) to 250 g / l (dry weight) is obtained, and at the end of the concentration step, a cell concentration of 200 g / l (dry weight) to 800 g / l (dry weight), preferably 250 g / l (dry weight) to 500 g / l (dry weight) is obtained.

9. A method for recovering polyhydroxyalkanoate (PHA) from a fermentation broth derived from microbial fermentation according to any one of the preceding claims, wherein: If said step (c) is carried out in the absence of a solvent, the aqueous suspension of the cell biomass comprising polyhydroxyalkanoates (PHA) obtained in separation (b) is subjected to mechanical cell lysis and / or chemical cell lysis.

10. The method for recovering polyhydroxyalkanoate (PHA) from a fermentation broth derived from microbial fermentation according to claim 9, wherein the aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) is subjected to cell lysis by mechanical treatment, wherein the mechanical treatment is carried out using a high-pressure homogenizer at a pressure of 500 bar to 2000 bar, preferably 800 bar to 1500 bar and at a temperature of 10° C. to 80° C., preferably 20° C. to 50° C.

11. The method for recovering polyhydroxyalkanoate (PHA) from a fermentation broth derived from microbial fermentation according to claim 10, wherein at the end of the mechanical treatment, the obtained aqueous suspension containing polyhydroxyalkanoate (PHA) is subjected to chemical cell lysis, the chemical cell lysis comprising: - alkalizing the aqueous suspension comprising polyhydroxyalkanoate (PHA) by adding a strong base, preferably an aqueous solution of at least one inorganic strong base such as sodium hydroxide, potassium hydroxide or a mixture thereof, to obtain a pH value greater than or equal to 9, preferably between 9.5 and 10, to obtain an alkalized aqueous suspension comprising polyhydroxyalkanoate (PHA); - adding to the alkalized aqueous suspension comprising polyhydroxyalkanoate (PHA) at least one surfactant in an amount ranging from 0.05 g to 1 g, preferably from 0.1 g to 0.5 g, of surfactant per gram dry weight of the alkalized aqueous suspension comprising polyhydroxyalkanoate (PHA).

12. The method for recovering polyhydroxyalkanoate (PHA) from a fermentation broth derived from microbial fermentation according to claim 11, wherein the chemical cell lysis is carried out at a temperature of 10°C to 80°C, preferably 20°C to 50°C, for a time of 20 minutes to 120 minutes, preferably 50 minutes to 70 minutes.

13. A method for recovering polyhydroxyalkanoates (PHA) from a fermentation broth derived from microbial fermentation according to claim 11 or 12, wherein the aqueous suspension containing polyhydroxyalkanoates (PHA) obtained from chemical cell lysis is subjected to a first concentration step by tangential filtration and / or centrifugation performed batchwise or continuously, preferably by continuous centrifugation, followed by a washing step by dilution with demineralized water or a diluted aqueous solution containing trace amounts of cell debris, followed by a second concentration step by tangential filtration and / or centrifugation performed batchwise or continuously, preferably by continuous centrifugation.

14. A method for recovering polyhydroxyalkanoates (PHA) from a fermentation broth derived from microbial fermentation according to claim 13, wherein at the end of the first concentration step, a cell concentration of 200 g / l (dry weight) to 800 g / l (dry weight), more preferably 250 g / l (dry weight) to 500 g / l (dry weight) is obtained, at the end of the washing step, a cell concentration of 100 g / l (dry weight) to 400 g / l, more preferably 150 g / l (dry weight) to 200 g / l (dry weight) is obtained, and at the end of the second concentration step, a cell concentration of 200 g / l (dry weight) to 800 g / l (dry weight), preferably 250 g / l (dry weight) to 500 g / l (dry weight) is obtained.

15. A method for recovering polyhydroxyalkanoates (PHA) from a fermentation broth derived from microbial fermentation according to any one of claims 11 to 14, wherein the aqueous suspension comprising polyhydroxyalkanoates (PHA) obtained from chemical cell lysis is dried at a temperature greater than or equal to 50° C., preferably from 55° C. to 70° C., preferably in a ventilated oven, until a solid residue comprising polyhydroxyalkanoates (PHA) having a residual moisture content of less than 0.5% is obtained.

16. The method for recovering polyhydroxyalkanoate (PHA) from a fermentation broth derived from microbial fermentation according to any one of claims 1 to 8, wherein: If the step (c) is carried out in the presence of a solvent, the aqueous suspension of the cell biomass containing polyhydroxyalkanoate (PHA) obtained in the separation step (b) is frozen and freeze-dried.

17. The method for recovering polyhydroxyalkanoate (PHA) from a fermentation broth derived from microbial fermentation according to claim 16, wherein the aqueous suspension of cell biomass containing polyhydroxyalkanoate (PHA) obtained in step (b) is subjected to the following steps: - freezing the aqueous suspension of cell biomass comprising polyhydroxyalkanoate (PHA) at -20°C and subsequently freeze-drying at -46°C and 0.15 mbar for a period of greater than or equal to 12 hours, preferably 18 hours to 24 hours, until a dehydrated cell powder comprising polyhydroxyalkanoate (PHA) is obtained; - washing the dehydrated cell powder containing polyhydroxyalkanoate (PHA) with ethanol at a temperature of 30°C to 60°C, preferably 50°C, for a period of 30 minutes to 6 hours, preferably 4 hours, by operating at a ratio of dehydrated cell powder:ethanol of 1:10 to 1:40, preferably a ratio of 1:20, to obtain a suspension containing polyhydroxyalkanoate (PHA); - filtering the obtained suspension containing polyhydroxyalkanoate (PHA) through a porous membrane filter or a cellulose filter and drying the solid residue containing polyhydroxyalkanoate (PHA) remaining on the filter at room temperature (25° C.) for a period of greater than or equal to 12 hours, preferably 18 to 24 hours; - dissolving the solid residue obtained comprising dried polyhydroxyalkanoate (PHA) at a ratio of dried solid residue:solvent of 1:100 to 1:500, preferably equal to 1:200, at a temperature of 40° C. to 70° C., preferably 60° C., in a stirred vessel or reflux reactor, in the presence of at least one solvent chosen from chloroform, 1,2-dichloroethane, dichloromethane, preferably chloroform, for a period of 4 hours to 24 hours, preferably 8 hours, and optionally repeating said dissolution up to 3 times; - filtering the solution containing polyhydroxyalkanoate (PHA) obtained after the dissolution through a membrane filter or a cellulose filter to obtain a solid residue containing no polyhydroxyalkanoate (PHA) and a solution containing polyhydroxyalkanoate (PHA); - optionally, concentrating the solution comprising polyhydroxyalkanoate (PHA) obtained by operating at a temperature lower than or equal to 70° C., preferably between 40° C. and 60° C., and at a pressure lower than atmospheric pressure, preferably equal to 0.5 bar; - before or after any concentration, contacting the solution comprising polyhydroxyalkanoate (PHA) in a stirred vessel with an excess of ethanol cooled to -20°C for a period of 0.1 to 60 minutes, preferably 0.5 to 25 minutes, to obtain precipitation of a solid residue comprising polyhydroxyalkanoate (PHA); - The solid residue containing polyhydroxyalkanoate (PHA) obtained is collected mechanically and dried at room temperature (25° C.) for a period of greater than or equal to 12 hours, preferably for a period of 20 to 30 hours, or in a ventilated oven at a temperature of 50° C. and low pressure, until a solid residue containing polyhydroxyalkanoate (PHA) having a residual humidity of less than 0.5% is obtained.

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