"Process for producing polyhydroxyalkanoates for food packages" of: W.H.IN S.r.l., Italian nationality, Vicolo San Tiburzio 3, Parma designated inventors: Simone GIORGINI Filed on:

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DESCRIPTION TEXT FIELD OF THE INVENTION

The present description relates to a new process for producing polyhydroxyalkanoates (PHA) for food packages.

BACKGROUND OF THE INVENTION

Notwithstanding the wide use made in past years of polymers derived from oil for infinite applications and not, the use of these polymers involves considerable problems due to their slow degradation and the expected depletion of world oil reserves. Therefore, the need is felt for having recourse to new materials which - by guaranteeing the same physical features of the polymers derived from oil - could be eco- friendly and produced starting from raw materials other than oil.

Polyhydroxyalkanoates (PHA) are thermoplastic polyester polymers synthetized by various kinds of microorganisms through the fermentation of sugars or lipids. PHA are biodegradable and they are used in the production of bioplastics. PHA are linear macromolecules, produced by the microorganisms under the form of granules: under particular culture conditions, such as the absence of determined nutrients such as nitrogen, phosphorus and/or sulphur, the microorganisms accumulate at inter- and intra-cellular level as carbonaceous reserve source, in inclusion bodies. PHA dissolve in organic solvents but they are poorly soluble in water.

The composition of polyhydroxyalkanoates is very varied and it depends upon the type of microorganisms therefrom they are synthetized and upon the culture matrix. The formula of general structure is the following: where n is the number of groups CH2, y is the number of monomers which can vary from 100 to 30,000, and R is the lateral chain. The group R is an alkyl group with a number of carbon atoms going from 1 to 15, it can be branched or linear and contain substituents of various type. One speaks about short-chain polyhydroxyalkanoates when there are 1-5 atoms of carbon, one speaks about medium-chain polyhydroxyalkanoates when there are 6-14 atoms of carbon. The great variability of lateral chains and of monomers provides to such materials as much variability of physical features, with melting points going from 60 to 140°C. In fact, PHA can be found which are mainly thermoplastic polymers, such as for example polyhydroxybutyrate, or gums or elastomers such as for example polyhydroxyoctanoate.

The Regulation (EU) 10/2011 of 14 January 2011 relating the materials and the objects of plastics intended to come in contact with the food products establishes that PHA result to be not toxic if in contact with food. For this reason, PHA result to be suitable to the use as materials for packaging food products since they have features such as high biodegradability, low permeability to oxygen, to water and to carbon dioxide and a very low migration rate. The scientific article by Pradhan et al. with title "Microbial production, ultrasound-assisted extraction and characterization of biopolymer polyhydroxybutyrate (PHB) from terrestrial (P. hysterophorus ) and aquatic (E. crassipes) invasive weeds" describes a method for producing a biopolymer of polyhydroxybutyrate (PHB) from water hyacinth (E. crassipes) and from P. hysterophorus. In the document, the bacterium used for fermentation is Ralstonia eutropha .

The scientific article by Radhika et al. with title "Bioproduction, statistical optimization and characterization of microbial plastic (poly 3-hydroxy butyrate) employing various hydrolysates of water hyacinth (Eichhornia crassipes) as sole carbon source" describes a method and relative optimizations for the bioproduction of poly 3-hydroxy butyrate (PHB) by using different hydrolyzed water hyacinths as source of carbon. Hydrolysis is implemented with cellulase enzyme Aspergillus niger, whereas the bacterium used for fermentation is Cupravidus necator.

The scientific article by Saratale et al. with title "Utilization of Noxious Weed Water Hyacinth Biomass as a Potential Feedstock for Biopolymers Production: A Novel Approach" describes a method for producing biopolymers (PHB) by using water hyacinth as biomass. The bacterium used for fermentation is Ralstonia eutropha.

None of the detected documents shows experiments of characterization of a PHA film in terms of mechanical and chemical properties meeting the parameters for using PHA produced with the described method for food packages, and does not teach a method of producing PHA for a food package.

Many researchers have been attracted by the innumerable applications of polyhydroxyalkanoates as alternative to the polymers derived from oil since biodegradable and obtainable from sources other than oil. Nevertheless, processes for producing PHA which are economically and technically advantageous are not yet available.

OBJECT AND SUMMARY OF THE INVENTION

The object of the present invention is to implement a process for producing a polyhydroxyalkanoate-based material for food packages suitable to contain food, which is economically advantageous, technically easy to be implemented. Polyhydroxyalkanoates in fact are biodegradable polymers, not toxic to human health and little pollutant.

According to the invention, the above-mentioned object is reached thanks to the subject mentioned specifically in the following claims, meant as integrating portion of the present description.

The present invention relates to a process for producing a PHA-based material for food packages providing the use of a low-cost plant biomass as organic matrix to be subjected to fermentation and a bacterium capable of producing high amounts of PHA starting from the above-mentioned plant biomass. The so-implemented food packages are safe since they do not release toxic substances, thus being able to be put in direct contact with various food, such as for example: dried fruits, berries, vegetables, citrus fruits, fresh and cold processed products such as meat and fish.

The solution proposed by the present invention consists in a specific process wherein as biomass the water hyacinth is used, which is subjected to an enzymatic hydrolysis process without requiring a pre-treatment, and subsequent fermentation in presence of Pseudomonas Putida KT2440, obtaining production yields of PHA higher than 80% by weight with respect to the weight of the starting plant biomass. Subsequently, the method provides the extraction and the processing of PHA so as to obtain a food package.

As clearly expressed in the description, the PHA-based film implemented with the method of the present invention has particularly advantageous properties for the implementation of food packages, such as:

- an angle of contact with water comprised between 97 and 105°, preferably equal to 101.8°; a permeability to oxygen and to carbon dioxide respectively comprised between 2 and 14 barrer and between 32 and 60 barrer, preferably respectively equal to approximately 11.5 and 53.9 barrer;

- a high flexibility and deformability with a tension to effort comprised between 1 and 10 MPa, preferably equal to approximately 4.86 MPa and a percentage to deformation comprised between 300 and 600%, preferably equal to approximately 349%.

The chemical features of the polyhydroxyalkanoate-based film make it particularly useful for implementing food packages, since capable of guaranteeing the preservation of food by limiting the entrance of oxygen and carbon dioxide, as well as having a high impermeability to water. The film mechanical features also guarantee the resistance of the package which will not be damaged during the respective use. Still considering PHA as material which does not release toxic substances to food.

Additional advantage of the present invention lies in the optimization of fermentative step.

In the planning step of the present invention the best growing conditions of the microorganism were selected, such as the selections of media for the growth of strain in plate, during the step of pre-inoculation in the fermenter; The correct mixture of pre-treated plant and culture medium, and such conditions allow to obtain the high yields reported in the patent application text.

The method of the present invention advantageously allows to obtain production yields of a mixture of several PHA, higher than 80% by weight with respect to the weight of the starting plant biomass by using a method which does not require biomass pre-treatment steps before the enzymatic hydrolysis, thus resulting economically advantageous with respect to those shown in the prior art, as well as more bio-sustainable (absence of use of acids and alkaline substances for the pre-treatment), and allowing to obtain a mixture of PHA in solid form with high energy yields thanks to the optimization of the fermentation process, subsequently processable to obtain food packages with optimum chemical and mechanical properties for the intended use.

In an embodiment, the present invention relates to a process for the production of a polyhydroxyalkanoate-based material for a food package, wherein the process comprises the following steps:

(a) providing a shredded lignocellulosic biomass consisting of water hyacinth;

(b) hydrolyzing the shredded lignocellulosic biomass by enzymatic treatment with the at least one cellulase and/or the at least one hemicellulase obtaining a lignocellulosic hydrolysate, wherein the at least one cellulase is selected from cellulases extracted from Aspergillus niger and Hypocrea jecorina and/or the at least one hemicellulase is selected from hemicellulases extracted from Aspergillus niger and Trichoderma viride; (c) fermenting the lignocellulosic hydrolysate in presence of a bacterium capable of producing polyhydroxyalkanoates obtaining a fermentation product containing PHA, wherein the bacterium is Pseudomonas putida, preferably the bacterium is Pseudomonas putida strain KT2440;

(d) extracting polyhydroxyalkanoates from the fermentation product obtaining PHA in solid form,

(e) subjecting polyhydroxyalkanoates in solid form to a processing for the manufacture of a PHA-based food package.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, several specific details are provided to allow a thorough understanding of the embodiments. The embodiments can be implemented without one or more specific details, or with other processes, components, materials, etc. In other cases, well-known structures, materials or procedures are not shown or described in details to avoid to confuse aspects of the embodiments .

The reference in the whole present description to "one only embodiment" or "an embodiment" means that a particular aspect, structure or features described in relation to the embodiment is included in at least an embodiment. Therefore, the forms of expressions "in one only embodiment" or "in an embodiment" in several points in whole present description are not necessarily all referred to the same embodiment. Moreover, the particular aspects, structures or features can be combined in any suitable way in one or more embodiments.

The herein provided titles are only for convenience and do not interpret the scope or the meaning of the embodiments.

Hereinafter in the present description specific reference will be made to the production of food package under the form of a polyhydroxyalkanoate-based film the peculiar and surprising features thereof will be demonstrated. It is evident that the food packages, the present description relates to, are not in any way limited to the above-mentioned embodiment, but could assume any structure, such as for example containers, food trays, etc., which will be implemented according to the common production techniques of such articles. The food packages - thanks to the peculiar features provided thereto by the polyhydroxyalkanoates produced according to the process the present application relates to - are intended to be put in direct contact with food products, such as for example dried fruits, berries, vegetables, citrus fruits, fresh and cold processed products such as meat and fish, since they do not release toxic substances.

As herein used, under the term "lignocellulosic biomass" woody plants or non-woody herbaceous plants are meant. In the present description, the lignocellulosic biomass mainly consists of water hyacinth (Eichhornia crassipes (Mart.) Solms), a floating water plant belonging to the family of Pontederiaceae, which grows on the surface of rivers, channels and lakes of the tropical areas and it is native of Amazon basin. The water hyacinth is one of the most infesting and harmful plants in the world and then it is intended to destruction.

As herein used, the term "hydrolysis" relates to depolymerization, by adding water, not monomeric carbohydrates in oligomers and monomers of sugar or carboxylic acids.

As herein used, the term "enzymatic hydrolysis" relates to an enzymatic treatment of the lignocellulosic biomass which comprises cellulose and/or hemicellulose e oligosaccharides, wherein the enzymes ease hydrolysis of cellulose and/or hemicellulose and oligosaccharides to obtain mono- and/or di-saccharides. Typically, the treatment of enzymatic hydrolysis of the lignocellulosic biomass is performed by administering to the lignocellulosic biomass one or more enzymes capable of hydrolyzing the glycosidic bonds of cellulose and hemicellulose in presence of water or a buffer.

As herein used, the term "lignocellulosic hydrolysate" relates to the products of hydrolysis of the lignocellulosic biomass. The lignocellulosic hydrolysate mainly comprises lignocellulosic sugars derived from cellulose and/or hemicellulose .

As herein used, the term "aerobic fermentation" relates to the cultivation of a microorganism in a culture broth wherein the microorganism uses aerobic breathing. Typically, the aerobic fermentation is performed by adding oxygen or a mixture of gas containing oxygen (typically air) to the fermenter.

As herein used, the term "fermentation in a semi- discontinuous fed-batch mode" relates to a fermentative step performed in a semi-open system, that is open on entry but not on exit. This type of productive process provides in the initial step the administration of a limited volume of culture medium, generally equal to approximately 1/10 of the maximum volume. When the logarithmic step of the growth curve of the bacterium is reached, a constant flow of fresh and sterile culture medium is introduced without discharging contemporarily the fermented effluent. In such step there is an increase in the concentration of the bacterial biomass due to the production of polyhydroxyalkanoates by the bacteria. The discharge is performed when a pre-established filling-in volume of the bioreactor is reached.

The present invention relates to a process for producing a polyhydroxyalkanoate (PHA)-based material for food packages, where the process provides the use of a specific plant biomass and specific reagents. Such process represents an example of circular economy since it uses a raw material destined to waste, that is a lignocellulosic biomass mainly consisting of water hyacinth, and reagents having a relatively reduced purchasing cost and a particularly high production yield, in particular the enzymes required for hydrolysis of the lignocellulosic biomass and the bacterium used for the fermentation of the lignocellulosic biomass. The process the present invention relates to then is particularly advantageous from an economic point of view with respect to what illustrated sofar in the prior art, where the raw materials and the reagents used for producing PHA result to be particularly expensive and/or characterized by relatively low production yields.

In an embodiment, the present invention relates to a process for the production of a polyhydroxyalkanoate-based material for food package, wherein the process comprises the following steps:

(a) providing a shredded lignocellulosic biomass mainly consisting of water hyacinth;

(b) hydrolyzing the shredded lignocellulosic biomass by enzymatic treatment with the at least one cellulase and/or the at least one hemicellulase obtaining a lignocellulosic hydrolysate, wherein the at least one cellulase is selected from cellulases extracted from Aspergillus niger and Hypocrea jecorina and/or the at least one hemicellulase is selected from hemicellulases extracted from Aspergillus niger and Trichoderma viride;

(c) fermenting the lignocellulosic hydrolysate in presence of a bacterium capable of producing polyhydroxyalkanoates obtaining a fermentation product containing polyhydroxyalkanoates, wherein the bacterium is Pseudomonas putida, preferably the bacterium is Pseudomonas putida strain KT2440;

(d) extracting polyhydroxyalkanoates from the fermentation product obtaining polyhydroxyalkanoates in solid form,

(e) subjecting polyhydroxyalkanoates in solid form to a processing for the manufacture of a polyhydroxyalkanoate- based food package.

The plant of water hyacinth (comprising stem, leaves, flowers and fruits) is shredded finely with the purpose of making the step of enzymatic hydrolysis more effective.

In an embodiment, the shredded plant biomass is subjected to a drying step, preferably performed at a temperature comprised between 40° and 100°C (preferably at about 100°C) for a period of time comprised between 24 and 72 hours (preferably about 24 h).

In an embodiment, the enzymatic hydrolysis of step (b) is performed in aqueous environment and provides the administration to the shredded lignocellulosic biomass of at least one cellulase selected among the cellulase extracted from Aspergillus niger and Hypocrea jecorina and/or the at least one hemicellulase selected among hemicellulases extracted from Aspergillus niger and Trichoderma viride. The hydrolysis involves the depolymerization of cellulose, of hemicellulose and of oligosaccharides of the plant of water hyacinth in simpler sugars which are easily processable by the microorganism used in the fermentation step. In an embodiment, the at least one cellulase and/or the at least one hemicellulase is/are added at a concentration comprised between 60 and 80% by weight with respect to the dry weight of the shredded plant biomass.

In an embodiment, the enzymatic hydrolysis of step (b) is performed under the following operating conditions: in aqueous environment at a pH comprised between 4.5 and 6, preferably equal to approximately 5.4, for a period of time comprised between 6 and 10 hours, preferably equal to approximately 8 hours, at a temperature comprised between 20 and 30°C, preferably equal to approximately 25°C. Preferably, the ratio by weight

(distilled)water :lignocellulosic biomass is equal to approximately 0.5:1. The enzymatic hydrolysis is preferably performed at rest, under maceration conditions.

In an embodiment, the bacterium Pseudomonas putida strain KT2440 before being inoculated in the fermenter is subjected to several growth steps:

- a first step, wherein the bacterium in lyophilized form is inoculated in a first flask containing a minimum culture medium (comprising Na2HPC>4,KH2PO4,NaCl and NH4CI and having a pH equal to approximately 6.8) and kept at about 30°C under stirring for approximately 72 h;

- a second step, wherein the culture broth of the preceding step is inoculated in a second flask (having a higher volume than the first flask) containing a minimum culture medium (comprising Na2HPC>4,KH2PO4,NaCl and NH4CI and having a pH equal to approximately 6.8) and kept at about 30°C under stirring for about 72 h, and

- a third step of scale-up, wherein the culture broth of the preceding step is inoculated in a third flask (having a higher volume than the second flask) containing a culture medium M9 and kept at about 30°C under stirring for about 72 h and subsequently placed under stirring for 12 h with an aeration level of 0.5 vvm, obtaining a fermentation broth suitable to be inserted in the fermenter to implement the fermentation step (c) of the lignocellulosic biomass.

In an embodiment, the fermentation of step (c) provides inoculating the lignocellulosic hydrolysate with a fermentation broth comprising the bacterium at a concentration comprised between 10 g/L and 50 g/L. The fermentation broth is inoculated into the lignocellulosic hydrolysate in a concentration comprised between 10% and 50% by weight with respect to the weight of the shredded plant biomass.

In an embodiment, at the beginning of fermentation step (c) a fermentation medium containing glucose, peptone, yeast extract, NaCl and a mixture of water and shredded plant biomass is added. Preferably, the mixture of water and shredded lignocellulosic biomass provides a ratio water:shredded biomass equal to approximately 1:0.5 g/L.

In an embodiment, the fermentation of step (c) is performed under aerobic conditions. The bacterium Pseudomonas putida strain KT2440 in fact is an aerobic bacterium, requiring oxygen to perform its vital cycle. Under such conditions the bacterium reproduces and is capable of converting - during fermentation - the lignocellulosic sugars into polyhydroxyalkanoates, which accumulate both at intra-cellular level and at inter-cellular level. The aerobic fermentation is performed thanks to an aeration circuit ending in the fermenter's chamber.

In an embodiment, the fermentation of step (c) is performed for a period of time comprised between 72 and 96 h at a temperature comprised between 30 and 37°C. During such period the bacterium grows exponentially until reaching a stationary step.

In an embodiment, the fermentation of step (c) is performed in a semi-discontinuous fed-batch mode.

In an embodiment, during the fermentation of step (c) glucose is added. The addition of glucose to the bacterial biomass allows a greater growth of the bacterium. Preferably, glucose is added in a ratio by weight with respect to the fermentation product equal to 1.25:1.

In an embodiment, when in the fermentation step (c) a bacterial growth rate equal to approximately 0.15 h is reached, a mixture of acrylic acid, nonanoic acid and glucose is added in a ratio in mass equal to 1.25:1:0.05.

In an embodiment, the step of extracting the polyhydroxyalkanoates produced by the bacterium provides treating the product of the fermentation with at least one extraction solvent, wherein the extraction solvent is preferably selected from (i) halogenated organic compounds capable of solubilizing PHA and (ii) anionic surfactants capable of dissolving the cell biomass, to obtain polyhydroxyalkanoates in solid form, for example under form of granules.

According to a first embodiment of extraction step, the product of the fermentation is treated (i) with at least a halogenated organic compound (selected for example among chloroform, dichloroethane, dichloromethane and trichloromethane) at a higher temperature than room temperature (preferably at about 30°C), preferably under stirring conditions, and (ii) a mixture of water and an alkyl alcohol is dried or added, so as to move away the halogenated organic compound, obtaining polyhydroxyalkanoates in solid form (for example under the form of granules). Preferably, the ratio alkyl alcohol:water is equal to 7:3 in volume. The alkyl alcohol is preferably methanol. Polyhydroxyalkanoates are then further washed and dried.

According to a second embodiment of extraction step, (i) the product of the fermentation is treated with an anionic surfactant (for example sodium lauryl sulfate, ammonium laurate) at a temperature comprised between 80 and 100°C (preferably about 90°C) and (ii) the polyhydroxyalkanoates are separated from the fermentation product by centrifugation obtaining in the end polyhydroxyalkanoates in solid form, for example under form of granules. Polyhydroxyalkanoates are then further washed and dried.

The production process the present description relates to allows to obtain a production yield of polyhydroxyalkanoates equal to approximately 80% by weight with respect to the weight of the starting plant biomass.

In an embodiment, the step (e) of processing polyhydroxyalkanoates in solid form is selected from extrusion, thermoforming, blow moulding, injection moulding, calendering, transfer moulding, extrusion and blow moulding, direct compression moulding so as to obtain a food packaging.

In an embodiment, the extrusion is implemented by having recourse to a single screw extruder. During extrusion the processing temperature is preferably comprised between 60 and 120°C; under such processing conditions, PHA in solid form transform in a paste having plastic nature, which is subsequently subjected to pressing obtaining a polyhydroxyalkanoate-based film. The so-obtained film - depending upon the provided thickness - will be processed to implement pellicles or containers intended to be in direct contact with food products.

The film has particularly advantageous properties for the implementation of food packages such as: an angle of contact with water comprised between 97 and 105°, preferably equal to 101.8°; a permeability to oxygen and to carbon dioxide respectively comprised between 2 and 14 barrer and between 32 and 60 barrer, preferably respectively equal to approximately 11.5 and 53.9 barrer; a high flexibility and deformability with a tension to effort comprised between 1 and 10 MPa, preferably equal to approximately 4.86 MPa and a percentage to deformation comprised between 300 and 600%, preferably equal to approximately 349%.

The chemical features of the polyhydroxyalkanoate-based film make it particularly useful for the implementation of food packages, since it is capable of guaranteeing the preservation of food by limiting the entrance of oxygen and carbon dioxide, as well as having a high impermeability to water. The mechanical features of the film also guarantee the resistance of the package which will not be damaged during the respective use. Still considering PHA as material which does not release toxic substances to food.

Materials and Methods

Pseudomonas putida strain KT2440

Pseudomonas putida is a bacterium of Gram-negative saprotrophic soil, shaped like a rod, obligate aerobe. Such bacterium is known and widely studied since it is capable of converting styrene into PHA.

Pseudomonas putida strain KT2440 is commercially available from the International collections ATCC, UKNCC, DSMZ, VKPM and NBRC. The Productive process

Plant preparation

The water hyacinth (Eichhornia crassipes (Mart.) Solms) consists by dry weight of 17.7% of lignin, 23.3% of hemicellulose and 25% of cellulose.

The water hyacinth is collected from riparian and river areas and can be dried naturally or with industrial driers then with drying temperatures going from 40° up to 100° for a period of time varying from a minimum of 24 hours to a maximum of 72 hours, then it is subjected to shredding.

Enzymatic hydrolysis of Water hyacinth

The enzymatic hydrolysis allows the conversion of the complex carbohydrates (cellulose and hemicellulose) of the plant into simpler structures (mono- and di-saccharides) which can be used by the bacterium for producing PHA. Such step allows an increase in the yield in terms of produced kg/L of PHA.

The enzymatic hydrolysis is performed by administering to the shredded plant biomass a mixture of water and one or more enzymes capable of converting the complex carbohydrates of the plant biomass into simpler sugars. Such enzymes are selected among cellulase and hemicellulose. Preferably, the cellulase(s) is(are) selected among cellulases extracted from Aspergillus niger and Hypocrea jecorina. Preferably, the hemicellulose(s) is(are) selected among hemicellulases extracted from Aspergillus niger and Trichoderma viride.

The at least one cellulase and/or the at least one hemicellulase are added to the shredded plant biomass in a concentration comprised between 60 and 80%, preferably about 72% with respect to the dry weight of the shredded plant biomass.

Growth in flask of Pseudomonas putida strain KT2440 25g of lyophilized bacterium are collected which are poured into a 100-ml flask with a minimum medium. The minimum medium is of the type M9 Minimal Salts Base, 5x, the composition thereof is shown in table 1. The growth of the bacterium is performed for 72 h, 30°C under stirring at about 250 rpm.

Table 1

In order to evaluate the strain growth, one has recourse to the spectrophotometer which measures the optical density at 600nm of wavelength; the searched value is of OD 6.00.

Scale-Up of growth of Pseudomonas putida strain KT2440

50 ml of broth are collected and inoculated in a 500- ml flask with a minimum medium of the type M9 Minimal Salts Base, 5x, the composition thereof is shown in table 1; while keeping the conditions of 72 h, 30°C and 250 rpm.

Subsequently, 250 ml of broth obtained from the preceding step are inoculated, in 5000-ml flasks with a medium M9, at 30°C under stirring at 250rpm for 72h.

Afterwards, one inoculates the bacterium in the fermenter.

Fermentation

The cultures of Pseudomonas putida are made to grow in the fermenter in a semi-discontinuous fed-batch mode: in the first step, growth is performed under optimum conditions of nutrients in presence of the hydrolized shredded plant biomass and of a fermentation medium, in the second step a situation of lack of an essential nutrient is generated (such as for example a source of nitrogen). In this second step of the fermentation the number of bacterial cells remains almost constant, whereas the concentration (g/litre culture) grows due to the effect of PHA production.

In the fermenter the medium consists of a mixture of finely shredded and hydrolized Eichhornia crassipes and an average fermentation medium, the composition thereof is given in table 2.

Table 2

10 μg/ml of the broth obtained from the preceding scale- up step are inoculated in the fermenter, where the broth was placed previously under stirring for 12 h at 250 rpm with a ventilation level of 0.5 vvm of aeration.

When the bacterial growth in the fermenter has reached a growth rate of about 0.15 h, a mixture of acrylic acid, nonanoic acid and glucose is added in a ratio between the supply masses of about 1.25:1:0.05.

The fermentation step is considered complete when the concentration of 60 g/1 includes 37.8 ± 2% g/1 of PHA. Extraction of polyhydroxyalkanoates from the fermenter

At the end of the fermentation step, one proceeds with extracting polyhydroxyalkanoates, which are soluble in organic solvents and insoluble in water.

Two extraction protocols were developed, the first one based upon PHA solubilization in a halogenated organic solvent, the second one based upon the selective dissolution of the cell biomass.

Protocol 1: solubilization of PHA in a halogenated organic compound

A: extraction with dichloroethane

The process, described in US 4324907 (Senior et al., 1982), provides treating the product of the fermentation with 1,2-dichloroethane at 83°C to solubilize PHA. PHA are precipitated by adding a mixture of methanol/water and, after filtration, the residue is addressed to a drying process to recover part of dichloroethane and to re-use it.

B: extraction with dichloromethane

The microbic biomass going out from the fermenter is mixed with dichloromethane and stirred for 4 hours at the temperature of 90°C. Under these conditions PHA within the biomass tend to solubilize in the dichloromethane. Afterwards, the organic step is centrifuged to separate the biomass from the solution containing DMC and PHA. The extracted polymer is recovered through evaporation of dichloromethane (Samori et al., 2015).

Protocol 2: selective dissolution of the cell biomass

A: extraction with sodium laurylsulfate (SDS)

The selective dissolution of non-PHA cell biomass is performed by adding SDS to the microbic biomass in ratio 2:1 by weight. The mixture then is stirred for 3 hours at 90°C. PHA are recovered after a passage in centrifuge for 10 minutes, then washed twice with water and one with ethanol, at last they are dried at 60°C under vacuum for 12 hours (Samorl et al., 2015).

B: extraction with ammonium-laurate

The protocol provides the selective dissolution of non- PHA cell biomass in a solution with pH 10 containing ammonium laurate in ratio 2:1 by weight. The mixture is stirred for 3 hours at 90°C. PHA are recovered after a passage in centrifuge, then washed with ammonium hydroxide and with a solution of water and ethanol.

For the recovery of ammonium laurate, to the aqueous step CO2 is added, so as to make the pH to diminish, so that the lauric acid precipitates and then is recovered by centrifugation. To the recovered lauric acid a new solution of ammonium hydroxide is added, so as to obtain again ammonium laurate for a subsequent reuse (Samori et al., 2015).

PRODUCTION_ AND_ CHARACTERIZATION_ OF

POLYHYDROXYALKANOATE-BASED FILM

Single screw extrusion

The extrusion by means of a single screw extruder represents the simplest and most used industrial process for producing packages made of polymeric material for food.

PHA granules are inserted in the extruder through a hopper provided with a system for blowing hot air, since PHA granules require a pre-heating for the subsequent processing. The extruder screw, being heated at 40°C, deforms the granules until making them to become a plastic paste. The paste at this point is pushed towards rollers which press such paste until the required thickness (for example 100 pm); thus, by making a PHA-based film to come out.

The so-obtained film could be processed to implement a film or a container for food. The film has features of high biodegradability, low permeability for O ₂ , H ₂ O and CO ₂ and it does not release toxic substances.

Water contact angle of film

The measurement of the water contact angle of film is useful to determine the wettability of film and then its permeability.

The used method consists in making drops of distilled water to fall from a syringe on the film surface, then evaluating if such drop crosses the surface itself.

The contact angle determined on PHA film subjected to analysis produced a really excellent result, being equal to 101.8 +/- 0.8°.

Gas permeation of film

Permeability to pure gases of CO ₂ and O ₂ was calculated according to the following equation: where P is permeability to gases, corresponds to the pressure difference (expressed in bar) in feeding and permeate compartments, t is time (expressed in seconds) and

1 is the film thickness (expressed in metres), whereas is the geometric parameter characteristic of the cell (expressed in m), obtained by the equation: where A is the film area (expressed in cm ² ) and V _feed and V _perm are the volumes (bar) of feeding and permeate.

Permeability to gases (P) is obtained by the slope during the representation of in function of t/1.

The results obtained on PHA film are respectively of 11.5 +/- 0,05 barrer for O2, and 53.9 +/- 0.05 barrer for C0 ₂ .

Film mechanical properties

The films were cut into 2.5 x 1.5 cm rectangular strips with an average thickness of 100 μm (the thickness being measured by using a micrometre). The traction tests were performed at room temperature (25°C) with a deformation speed of 0.5 mm/s.

The applied unit of measurement is Pascal applied to Young elasticity module which expresses the ratio between tension and deformation in a material.

The tension to effort of PHA film is equal to 4.86 +/- 0.60 Mpa with a % to deformation of 349 +/-12.

The results demonstrate high tension to effort and to deformation, useful for different and new uses in handling food packages.

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