WO2022090960A1

WO2022090960A1 - Process for extraction and purification of polyhydroxyalkanoates

Info

Publication number: WO2022090960A1
Application number: PCT/IB2021/059931
Authority: WO
Inventors: Bruno Sommer Ferreira; João Manuel BEIRÃO TORNEIRO CAVALHEIRO; Hugo FERRÃO DIAS DE ALMEIDA; Joshua ANJOS
Original assignee: Biotrend - Inovação E Engenharia Em Biotecnologia, S.A.
Priority date: 2020-10-30
Filing date: 2021-10-27
Publication date: 2022-05-05
Also published as: EP4237467A1

Abstract

The present invention relates to a process for extraction and purification of polyhydroxyalkanoates (PHAs) from PHA-containing material obtained from different sources. The process comprises reacting PHA-containing material with H₂O₂ and adding an alkali up to a pH value equal to 9 or higher.

Description

DESCRIPTION

"PROCESS FOR EXTRACTION AND PURIFICATION OF

POLYHYDROXYALKANOATE S "

FIELD OF THE INVENTION

The present invention relates to a process for extraction and puri fication of polyhydroxyalkanoates ( PHAs ) from PHA- containing material obtained from di f ferent sources .

BACKGROUND OF THE INVENTION

The non-renewable origin of most currently used polymers and their lack of biodegradability in short time spans is behind their excessive carbon and water footprints and waste management concerns , resulting in an increased research interest in the development of more environmentally compatible and economically sustainable materials , particularly for single-use applications .

Biodegradable natural polymers extracted or derived from renewable resources are steadily replacing the crude oil-based polymers . Three families of biopolymers are usually researched and used : polymers directly extracted from biomass (biomass can comprise polysaccharides , proteins and various lipids ) , biomass-derived monomers treated with classical chemical and manufacturing routes to obtain biodegradable and/or renewable polymers (polylactate , bio-polyethylene ) , and polymers produced by natural or genetically modi fied micro-organisms (polyhydroxyalkanoates , PHAs ) . PHAs have attracted special interest because they comprise a group of naturally derived polyesters synthesi zed by a wide range of microorganisms as intracellular carbon and energy materials . They are usually accumulated within cells when growth is l imited by nutrients such as nitrogen, oxygen, phosphorous and other essential elements , while in the presence of excess carbon . Instead of being consumed for the cellular growth, the excess of carbon is taken into the cells and stored in the form of PHA granules .

PHAs production at large scale from microorganisms usually involves fermentation, isolation and puri fication processes , which imply higher production costs . For example , manufacturers in Brazil and China have used sugarcane bagasse and cornstarch as a renewable resource to produce PHA inexpensively . In this context , pioneering and significant research ef forts on the use of mixed microbial cultures from wastes as a cheaper alternative to pure microbial culture has been carried out , enabling the use of renewable carbon which does not compete with food or feed applications . Such consortia of PHA-producing microorganisms can adapt to changes in the substrate and enable the use of cheap mixed substrates such as low cost agricultural or industrial waste feedstock and even municipal wastes .

The most common PHA is a homopolymer, the polyhydroxybutyrate ( PHB ) . It has properties quite similar to polypropylene , albeit sti f fer and much more brittle . The copolymers , poly ( p-hydroxybutyrate-co-valerate ) ( PHBV) , are ideal for packaging as they are less sti f f and much tougher than PHB but they have poorer barrier properties to gases . PHB, PHBV and other short-chain length co-polymers are important biodegradable plastics that are becoming of signi ficant interest in food packaging applications .

The downstream processing for PHAs recovery and puri fication from microbial biomass plays a vital role in the PHAs manufacturing process . It is a maj or contributor to the product production cost , it defines the material speci fications and quality, and may have a significant impact on the environmental sustainability of the product and of its production process . The PHA puri fication step is often recogni zed as the most critical when assessing the process feasibility and sustainability . The traditional puri fication processes , relying on organic solvents , including halogenated, are being banned from the industrial setting, while new hopes of using microbes that spontaneously release the PHA to the medium ( ex . upon osmotic shock) have not yet been realized due to their low productivity .

The steps of extraction and puri fication of PHAs from producing biomass are commonly regarded as among those that contribute the most to the final price of the polymer . This is more so for the extraction of PHAs from biomass used in the treatment of municipal wastes , due to the complex matrix of materials that can adsorb the biomass and the heterogeneous nature of the mixed culture . Further, some applications of the puri fied polymer may require obtaining stringent speci fication, whereas less demanding applications may exist that can eventually use non- fully puri fied material s while complying with the required functional features and relevant regulatory framework .

A number of companies produce polyhydroxyalkanoates in industrial scale , all using pure cultures fermented on mostly refined raw materials . Although for high-purity and low volume applications the use of organic solvents , mostly fossil-based, is still considered, the production of bulk quantities of commodity products will inevitably be increasingly performed with aqueous-based extraction and puri fication processes .

EP 1705250 Al , entitled "A method for separating, extracting and puri fying poly-beta-hydroxyalkanoates ( PHAs ) directly from bacterial fermented broth" discloses a method for directly separating and purifying polyhydroxyal kanoates in cells from fermentation liquid and it relates to the field of post-treatment technology of biological engineering . The method includes the steps of : pretreating fermentation liquid with physical method for breaking cell wall ; adj usting the pH value of the pretreated fermentation liquid to alkaline ; adding an anionic surfactant ; reacting under agitation; separating and extracting the coagulated precipitate from the reaction liquid; washing and drying . The invention was so far only validated on pure cultures grown on standard industrial fermentation media, which entrains much less contaminants than the challenging conditions of the streams emerging from the mixed culture fermentations .

EP 2606080 Bl , entitled "Method for recovery of stabili zed polyhydroxyalkanoates from biomass that has been used to treat organic waste" relates to a method of increasing the chemical and/or thermal stability of PHA in biomass where the biomass is contained within mixed liquor, and wherein the mixed liquor is treated by a combination of removing water from the mixed liquor and pH adj ustment of the mixed liquor or maintenance of the pH of the mixed liquor within a selected pH range , and wherein the method includes reducing the pH of the mixed liquor below 6 , or maintaining the pH o f the mixed liquor below 6 for a selected period of time , and wherein the pH adj ustment of the mixed liquor to below 6 or the maintenance of the pH of the mixed liquor below 6 gives rise to an increase in chemical and/or thermal stability of the PHA in the biomass . After drying the mixed liquor, the PHA is extracted with a non-chlorinated organic solvent at temperatures above 100 ° C . The process was designed to provide high purity PHAs with enhanced thermal stability . However, such process relies on the use of organic solvents processed at high temperature ( typically at more than 100 ° C ) , which add on cost and would require equipment and safety features which may not be usually employed in the plants producing the PHA- containing biomass , namely wastewater treatment plants .

EP 3287526 Bl , entitled "Method of manufacturing microbially produced plastic and microbially produced plastic" , relates to a method of manufacturing a microbially produced plastic comprising a step of applying heat treatment to fat containing hydrogen peroxide and a step of culturing microbes in a culture liquid containing the fat that has been subj ected to the heat treatment .

Further, for the feasible application at decentrali zed locations complying with the regulatory environment and permits of operation of plants other than chemical companies , such as wastewater treatment plants , the extraction process should use standard and readily available equipment , preferably equipment that can be routinely found on-site and not requiring special training for the plant operators . This precludes the use of processes using organic solvents .

Further, there is still the need to provide an alternative process for extracting PHAs without using organic solvents and sti ll allowing to obtain PHAs with a high level of purity . SUMMARY OF THE INVENTION

Surprisingly, the aforementioned problem was solved by the present invention.

The present invention provides a process for extraction and purification of polyhydroxyalkanoates (PHA) characterized by reacting PHA-containing material with H2O2 and adding an alkali up to a pH value equal to 9 or higher.

In a preferred embodiment, the reaction is carried out at a pH value above 11, more preferably above 11,4 and most preferably above 11,5.

In another embodiment, the ratio of H2O2 to PHA-containing material in the process of the invention is within a range of 20 mL H2O2 30 vol/g of biomass (dry weight) to 0.1 mL H2O2 30vol/g of biomass (dry weight) .

In one aspect of the invention, the alkali can be added simultaneously with or sequentially to H2O2.

In a preferred embodiment, the alkali of the reaction is selected from a group consisting of NaOH, KOH and Ca(OH)2, with NaOH being preferred.

In another embodiment the PHA-containing material is a mixed microbial culture or a pure culture containing intracellular PHAs .

In another aspect, the process of the invention further comprises a pre-treatment step which may be chemical, mechanical, electric or enzymatic. DETAILED DESCRIPTION OF THE INVENTION

To solve the problems identi fied above , the present invention provides a process for extraction and puri fication of polyhydroxyalkanoates ( PHA) characteri zed by reacting PHA- containing material with H2O2 and adding an alkali up to a pH value equal to 9 or higher .

Particularly, the process of the invention comprises the steps of : a ) adding a PHA-containing material to a reaction vessel b ) adj usting the pH to a value of 9 or higher by adding an alkali to the PHA-containing material under stirring; c ) adding H2O2 and alkali over 30 to 90 minutes under stirring; d) submitting the mixture to a digestion period by stirring for 2 , 5 to 36 hours followed by separation, washing and drying steps .

Within the context of the present invention, the term "PHA-containing material" means any PHA-containing microbial biomass or streams containing or derived from PHA-containing microbial biomass , including pre-treated microbial biomass . "PHA-containing microbial biomass" means any microbial biomass containing intracellular PHA, including microbial biomass consisting on or derived from pure microbial cultures , microbial biomass consisting on or derived from mixed microbial cultures , microbial biomass consisting on or derived from naturally occurring microorganisms , microbial biomass consisting on or derived from genetically modified microbial cultures, or any combination thereof.

Preferably, the reaction is carried out at a pH value above 11, more preferably above 11,4 and most preferably above 11,5.

In one embodiment, the ratio of H2O2 to PHA-containing material in the process of the invention is within a range of 20 mL H2O2 30 vol/g of biomass (dry weight) to 0.1 mL H2O2 30vol/g of biomass (dry weight) .

In the process of the invention the alkali can be added simultaneously with or sequentially to H2O2.

The alkali of the reaction is selected from a group consisting of NaOH, KOH and Ca(OH)2, with NaOH being preferred .

In a preferred embodiment, PHA-containing material is a mixed microbial culture or a pure culture containing intracellular PHAs .

The process of the invention may further comprise a pre-treatment step of which may be chemical, mechanical, electric or enzymatic.

The chemical pre-treatment comprises the pre-treatment of the microbial biomass with a surfactant and/or with a bleaching agent and/or with an alkali. The surfactant can be any surfactant which interferes with the integrity of the cellular membrane of microbial cells such as, for example, sodium dodecyl sulphate and similar. The bleaching agent is selected from sodium hypochlorite or hydrogen peroxide and the alkali is selected from the group consisting of NaOH, KOH and

Ca ( OH) ₂ .

The enzymatic pre-treatment i s any treatment using enzymes involved in hydrolysing components of the microbial cell walls , either isolated or in combination, including, without limitation, proteases , lipases , carbohydrases or the like .

The mechanical pre-treatment is selected from any treatment allowing cell disruption, such as sonication, high pressure homogenisation, milling, and the like .

The electric pre-treatment is selected from any treatment allowing cell disruption, such as pulsed electric f ield processing, and the like .

Surprisingly, the slow addition of H2O2 and NaOH to the PHA-containing material leads to the formation of superoxide (O2 ~ ) when the pH of the reaction is equal or higher than 9 . This superoxide reacts with PHA-containing material and leads to digestion/decomposition of the biomass . This slow addition approach avoids that pH drops below 9 when H2O2 is added and that pH goes to values higher than 12 . 5 when NaOH is added and provides PHAs with high purity levels .

The process of the present invention was found to be suitable to obtain a viable product and contrary to known processes , it is easy to be controlled without violent reactions which generally occurs due to high temperatures and/or the release of gases .

Further, it was confirmed that the present process is ef fective for extraction of PHAs from very diverse microbial biomass , including pure cultures , and mixed cultures obtained from the conversion of di f ferent wastes .

The process of the invention exhibits further advantages : Processing of both cleaner biomasses ( ex . pure microbial cultures obtained from the fermentation o f refined raw materials ) and crude biomasses ( ex . pure microbial cultures obtained from the fermentation of industrial sidestreams or mixed cultures obtained from the conversion of municipal or agro-industrial wastes ) ;

The digestion reaction can be performed without temperature control ;

The conditions can be set in order to minimise the ef fect of the digestion reaction on the polymer molecular weight ;

Obtention of high purity polymers , i . e . with a purity of more than 98 % ;

The polymer allows obtaining white formulations and white plastic parts (very important to allow the incorporation of coloured pigments in the plastics ) , while the solutions currently on the market originate beige products ;

The final product does not contain a chlorinated smell ; by using the processes of prior art , a typical chlorine smell was detected on the resulting polymer powder even after multiple washing steps and this is very relevant for processing, final use and product stability, since when residual chlorinated compounds remained on the f inal product , chlorine vapors were released when processing the polymer and corrosion issues on the processing equipment could arise ;

A reduction of chemicals is achieved as compared to the benchmark bleach-based process . EXAMPLES

The present invention is described in more detail and speci fically with reference to the examples , which are not intended to be limitative .

Reference Example 1 - PHA Production

PHA-containing microbial biomass was produced from the fermentation of fruit pulp, an industrial by-product of the j uice industry, using mixed microbial cultures . Briefly, a first stage of the process consists of acidogenic fermentation in which the available feedstock is converted into volatile fatty acids , such as , but not limited to , acetic acid, lactic acid, propionic acid, butyric acid, valeric acid, caproic acid and also ethanol . In the second stage of the process , a cyclic regime of carbon excess and carbon limitation was established to enhance PHA storage capacity ( generic information on PHA production using mixed microbial cultures can be obtained, for example , in Serafim et al . , 2008 and speci fic information of the system used for PHA production using fermentation of fruit pulp in Melendez-Rodriguez et al . , 2018 ) .

Reference Example 2 - PHA extraction : Benchmark process using sodium hypochlorite

220 g of PHA-containing microbial biomass , with a dry weight of 9% on total wet microbial biomass and 45% of PHA on dry weight , was mixed with di f ferent amounts of domestic bleach, i . e . sodium hypochlorite ( 400 g, 300 g and 200 g) and the mixture was stirred at room temperature for 3 hours . After this digestion period, the mixture was centri fuged and the solids were recovered . The thus recovered solids were resuspended in water, using the same volume as the digestion reaction volume , and the wash water discarded . The washed and recovered solids were oven dried at 60 ° C until constant weight and the recovered material was ground to a powder in a mortar . The PHA recovery yield decreased with decreasing amounts of sodium hypochlorite ( 100% , 58 % and 32 % when 400 g, 300 g and 200 g were used, respectively) , suggesting that the reduction of the quantities of sodium hypochlorite did not allow the ef fective digestion of the non-PHA microbial biomas s components . In order to swi ftly assess the purity of the recovered PHA powder, a small amount was placed on a glass slide which was slowly heated in a heating plate up to a maximum of 180 ° C . Most of the material recovered us ing 200 g of bleach did not melt and became brown upon heating . In the trial using 300 g of bleach a higher amount of molten material is observed, but still a signi ficant amount of the material did not melt and became brown . Most of the material recovered from the trial using 400 g of bleach melted, but a number of yellowish or brown spots of unmolten material remain . These yellowish or brown spots are a strong indication that the recovered material still retains non-PHA microbial biomass components , and hence does not has the purity required for most potential applications . Thermogravimetric analysis of the material with best melting behaviour yielded a purity of 99 . 5% , which indicates that even a small amount of impurities will have an impact on the polymer functional speci fications . Additionally, the recovered powder, despite the wash cycles , still retained a strong chlorine smell .

Reference Example 3 - PHA extraction : Combining the use of surfactant in alkaline conditions with sodium hypochlorite

220 g of PHA-containing microbial biomass , produced as in Reference Example 1 , with a dry weight of 9% on total wet microbial biomass and 45% of PHA on dry weight , was mixed with di f ferent amounts of sodium dodecyl sulphate and sodium hydroxide ( 5 N solution) and stirred at room temperature during 30 to 90 minutes . After this stage , di f ferent amounts of domestic bleach were added and the mixture was stirred at room temperature during 1 to 3 hours . After this digestion period, the mixture was centri fuged and the solids were recovered . The thus recovered solids were resuspended in water, using the same volume as the digestion reaction volume , and the wash water was discarded . The washed and recovered solids were oven dried at 60 ° C until constant weight and the recovered material was ground to a powder in a mortar . Table 1 summarises the conditions and the results .

Table 1 - Results of PHAs extraction using surfactant in alkaline conditions with sodium hypochlorite

The milder conditions 1- 1 , using a lower concentration of SDS and remaining reagents , resulted in a very low PHA recovery and the recovered material did not melt properly, generating a mostly brown residue when heating . The remaining conditions resulted in higher yield and the recovered material melted upon heating without generating brown or even yellowish residues , a strong indication that the recovered material had a good purity . Despite the reduced amount of bleach used in these examples , the total consumption is still very high ( 5- 10 times the amount of PHA-containing microbial biomas s on dry weight ) and the final product still retained a strong chlorine smell .

Reference Example 4 - PHA extraction : Combining the use of sodium hypochlorite and hydrogen peroxide

220 g of PHA-containing microbial biomass , produced as in Reference Example 1 , with a dry weight of 9% on total wet microbial biomass and 45% of PHA on dry weight , was mixed with 200 g of domestic bleach and the mixture was stirred for 1 hour at room temperature . After this digestion period, 200 g of 30 volume strength hydrogen peroxide were added and mixed for another hour at room temperature . The mixture was centri fuged and the solids were recovered . The thus recovered solids were resuspended in water, using the same volume as the digestion reaction volume , and the wash water di scarded . The washed and recovered solids were oven dried at 60 ° C until constant weight and the recovered material was ground to a powder in a mortar . The PHA recovery yield decreased was 51 % and a signi ficant amount of non-molten yellowish and brown material was obtained, suggesting that the used conditions did not allow the e f fective digestion of the non-PHA microbial biomass components .

Reference Example 5 - PHA extraction : Combining the use of surfactant and hydrogen peroxide

220 g of PHA-containing microbial biomass , produced as in Reference Example 1 , with a dry weight of 9% on total wet microbial biomass and 45% of PHA on dry weight , was mixed with di f ferent amounts of sodium dodecyl sulphate and sodium hydroxide ( 5 N solution) and incubated during 30- 90 minutes at room temperature . After this stage , di f ferent amounts of 30 volume strength hydrogen peroxide were added and the mixture was stirred at room temperature during 1 to 3 hours . After this digestion period, the mixture was centri fuged and the solids were recovered . The thus recovered solids were resuspended in water, using the same volume as the digestion reaction volume , and the wash water discarded . The washed and recovered solids were oven dried at 60 ° C until constant weight and the recovered material was ground to a powder in a mortar . Table 2 summarises the conditions and the results .

Table 2 - Results of PHAs extraction using surfactant and hydrogen peroxide

The milder conditions 2- 1 , using a lower concentration of SDS and remaining reagents , resulted in a very low PHA recovery and the recovered material did not melt properly, generating a mostly brown residue when heating . Trial 2-2 , resulted in a higher polymer recovery, but a mostly brown residue was also obtained when heating . The remaining conditions resulted in a similar yield as for 2-2 , but the recovered material melted upon heating, albeit generating some yellowish or brown residues , a strong indication that the recovered material did not have a good purity . Reference Example 6 - PHA extraction: Combining the use of sodium hydroxide and hydrogen peroxide

220 g of PHA-containing microbial biomass, produced as in Reference Example 1, with a dry weight of 9% on total wet microbial biomass and 45% of PHA on dry weight, was mixed with different amounts of 30 volume strength hydrogen peroxide and the mixture was stirred during 0.5 to 16 hours at room temperature. Following this, sodium hydroxide (5 N solution) was added and the mixture was stirred at room temperature between 1.5 and 16 hours. After this digestion period, the mixture was centrifuged and the solids were recovered. The thus recovered solids were resuspended in water, using the same volume as the digestion reaction volume, and the wash water discarded. The washed and recovered solids were oven dried at 60°C until constant weight and the recovered material was ground to a powder in a mortar. Table 3 summarises the conditions and the results.

Table 3 - Results of PHAs extraction using sodium hydroxide and hydrogen peroxide

In experiment 3-1, an unexpectedly fast expansion of the reaction medium occurred due to vigorous gas release and which was very difficult to contain even through the addition of silicone-based anti-foaming agent (Simethicone 30% emulsion, from Dow-Corning) . In experiment 3-2 , the volume expansion also occurred vigorously, but could be managed with the addition of anti- foaming agent . In experiment 3-3 , only a slight volume expansion occurred, and the addition of antifoaming agent was not required . In all experiments , although a signi ficant amount of the recovered material did not melt , no signi ficant browning was observed upon melting . This indicates that most organic non-PHA microbial biomass components have been digested, but the recovered polymer is contaminated by a signi ficant amount of inorganics which were not fully removed in the washing process .

J .M . Gould on "Studies on the mechanism of alkaline peroxide deligni fication of agricultural residues . Biotechnology and Bioengineering" , 1985 , postulated that the conditions that were tested in Reference Example 6 caused the violent production and release of molecular oxygen and of these species .

Example 1A - PHA extraction : Controlled use of sodium hydroxide and hydrogen peroxide - pH 12 . 5

In this experiment , to 20 g of PHA-containing microbial biomass ( dry weight ) , produced as in Reference Example 1 , an initial amount o f sodium hydroxide was added in order to bring the pH of the reaction medium at around 12 . 5 . After that , hydrogen peroxide and sodium hydroxide were slowly added over 60 minutes without temperature control . A total of 20 mL of 30 volume strength hydrogen peroxide and 25 mL of sodium hydroxide ( 5 N solution) were added, after which the mixture was stirred for 16 hours . After this digestion period, the mixture was centri fuged and the solids were recovered . The thus recovered solids were resuspended in water, using the same volume as the digestion reaction volume , and the wash water discarded. Four additional wash cycles were carried out. The washed and recovered solids were oven dried at 60°C until constant weight and the recovered material was ground to a powder in a mortar. The PHA recovery yield was 81% and upon heating a mostly transparent melt was obtained at 175°C with rare yellowish spots, indicating that the material is compatible with many of the envisaged applications.

Example IB - PHA extraction: Controlled use of sodium hydroxide and hydrogen peroxide - pH 9.0

In this experiment, to 20 g of PHA-containing microbial biomass (dry weight) , produced as in Reference Example 1, an initial amount of sodium hydroxide is added in order to bring the pH of the reaction medium at around 9.0. After that, hydrogen peroxide and sodium hydroxide are slowly added over 30 minutes without temperature control. A total of 40 mL of 30 volume strength hydrogen peroxide and 50 mL of sodium hydroxide (5 N solution) are added, after which the mixture is stirred for 36 hours. After this digestion period, the mixture is centrifuged and the solids are recovered. The thus recovered solids are resuspended in water, using the same volume as the digestion reaction volume, and the wash water discarded. Four additional wash cycles are carried out. The washed and recovered solids are oven dried at 60°C until constant weight and the recovered material is ground to a powder in a mortar. Upon heating the powder, a mostly molten material was obtained at 175°C with a number of brownish spots .

Example 1C - PHA extraction: Controlled use of sodium hydroxide and hydrogen peroxide - pH 13.0 In this experiment, to 20 g of PHA-containing microbial biomass (dry weight) , produced as in Reference Example 1, an initial amount of sodium hydroxide is added in order to bring the pH of the reaction medium at around 13.5. After that, hydrogen peroxide and sodium hydroxide are slowly added over 90 minutes without temperature control. A total of 15 mL of 30 volume strength hydrogen peroxide and 20 mL of sodium hydroxide (5 N solution) are added, after which the mixture is stirred for 2.5 hours. After this digestion period, the mixture is centrifuged and the solids are recovered. The thus recovered solids are resuspended in water, using the same volume as the digestion reaction volume, and the wash water discarded. Four additional wash cycles are carried out. The washed and recovered solids are oven dried at 60°C until constant weight and the recovered material is ground to a powder in a mortar. The PHA recovery yield is 67% and upon heating a mostly transparent melt was obtained at 175°C with rare yellowish spots, indicating that the material is compatible with many of the envisaged applications.

Example 2 - PHA extraction: Combined use of surfactant, sodium hypochlorite, and controlled use of sodium hydroxide and hydrogen peroxide

In this experiment, to PHA-containing microbial biomass, produced as in Reference Example 1, with a dry weight of 3612% on total wet microbial biomass and 5813% of PHA on dry weight, sodium dodecyl sulphate (SDS) was added and stirred for 30 min. Domestic bleach was then added and the mixture was stirred for 1 hour After this digestion period, the solids were recovered by centrifugation. The thus recovered solids were resuspended in water, using the same volume as the digestion reaction volume, and the wash water discarded. To the wet washed and recovered solids, NaOH solution was added to adj ust the solution pH to 12 . 5 . Next , 30 volume strength hydrogen peroxide and NaOH 5N solution were slowly added without temperature control and upon completion of the addition of these chemicals , the mixture was stirred without temperature control for a speci fied duration . The mixture was centri fuged and the solids were recovered . The thus recovered solids were resuspended in water, using the same volume as the digestion reaction volume , and the wash water discarded . This wash cycle was repeated four more times . The washed and recovered solids were oven dried at 60 ° C until constant weight and the recovered material was ground to a powder in a mortar . Table 4 summarises the conditions and the results .

Table 4 - Results of PHAs extraction using surfactant , sodium hypochlorite , sodium hydroxide and hydrogen peroxide

In all experiments , volume expansion was observed in the beginning of the 2nd stage , but manageable with the addition of minute amounts of anti- foaming agent . In all experiments , the recovered material melted without any browning or yellowing being observed on the molten material . In order to assess the potential ef fects of the PHA extraction protocol on the molecular weight of the polymer, the molecular weight of the extracted material was measured by gel permeation chromatography ( GPC ) and compared to the molecular weight of the native polymer, extracted from a sample of freeze-dried microbial biomass using chloroform, a method known not to af fect the molecular weight of the extracted and solubilised polymer . The results on Table 4 show a relatively small reduction in average molecular weight , particularly when conditions using low amounts of raw material . Further, in experiments 4- 1 and 4-2 , the scale of extraction was increased signi ficantly, using 1 . 3 kg and 4 . 1 kg of dry weight , instead of 150 g for experiments 4-3 and 4-4 , or 20 g used in the previous examples . When the 2nd stage is carried out , a temperature increase at the beginning throughout the reagent addition, with production of the oxidative species and their reaction with the microbial biomass . Such temperature increase was more pronounced in experiments 4- 1 and 4-2 due to the lower heat diss ipation capacity through the walls of the vessel since the larger the vessel , the lower the speci fic heat trans fer area available . It is well known that the ef ficiency of chemical digestion methods depends on the temperature , with an increase of the digestion performance for higher temperatures . It is likely that the temperature increase at larger scale may have contributed to a higher digestion of the biomass , but also that the process attacked the polymer as well , resulting in an average lower molecular weight . These findings clearly suggest that the implementation of this process at large scale , at higher temperature due to the exothermic reaction, may enable the reduction o f the amounts of chemicals used in the 2nd stage where hydrogen peroxide and sodium hydroxide are added in a controlled way, and may also enable reducing the holding time in the vessel and hence contribute to a signi ficant reduction in operational and investment costs . The polymer obtained in experiment 4- 1 was used in a formulation to produce a melt which was then extruded and success fully produced PHA pellets . Example 3A - Extraction of polyhYdroxybutyrate (PHB) from pure microbial cultures

PHB-containing microbial biomass was produced from the aerobic fermentation of sugars using a Burkholderla saccharl strain ( for details see Cesario et al . , 2014 ) . The PHB- containing microbial biomass , with a dry weight of 35% on total wet microbial biomass and 49% of PHB on dry weight , was diluted with water to a 20% dry cell weight and mixed with SDS and a solution of NaOH 5N, both to 5% of the total volume . The lower amount of chemicals used is due to the nature of the treated microbial biomass , which is more homogenous ( a pure microbial culture instead of mixed microbial culture ) and which was fermented on a cleaner culture medium ( a defined fermentation medium instead of industrial organic residues ) . After mixing for 30 minutes , domestic bleach was added to 20% of the volume and mixing continued for 60 minutes After this digestion period, the solids were recovered by centri fugation . The thus recovered solids were resuspended in water, using the same volume as the digestion reaction volume , and the wash water discarded . The washed and recovered solids were oven dried at 60 ° C until constant weight and the recovered material was ground to a powder in a mortar . A recovery yield of 95% was obtained, but the purity as assessed by the melting behaviour of the recovered powder was not satis factory . Next , 500 g of the recovered polymer powder was resuspended in water to a total of 2 . 5 kg mixture . 625 mL of NaOH 5N solution and 500 mL of 30 volume strength hydrogen peroxide were slowly added as described before and mixed without temperature control for a total of 3 hours . The thus recovered solids were resuspended in water, using the same volume as the digestion reaction volume , and the wash water discarded . Four additional washing steps were carried out . The washed and recovered solids were oven dried at 60°C until constant weight and the recovered material was ground to a powder in a mortar. A recovery yield of 91% was obtained, and the purity as assessed by the melting behaviour of the recovered powder was excellent, with a homogenous melt, without any brown or yellowish spots. The thus obtained polymer was used in a formulation to produce a melt which was then extruded (Haake Rheocord 90 single screw extruder) , and successfully produced a filament of 1.810.1 mm with adequate quality for 3D printing tests. Further, the polymer originated a white extruded filament that could be used to produce a white plastic part.

Example 3B - Extraction of polyhYdroxybutYrate (PHB) from pure microbial cultures

4.5 kg of PHB-containing microbial biomass described in Example 3A, was adjusted to a pH of 11.9 with NaOH 5N. Subsequently, 30 volume strength hydrogen peroxide were slowly added at a rate of 40 mL/h. The pH of the reactor was allowed to decrease as hydrogen peroxide was added. When the pH reached 11.2, the automated addition of NaOH was implemented in order to control the pH at that setpoint. A total of 320 mL of hydrogen peroxide and 75 mL of NaOH were added and 1.1 kg of wet pellet was recovered. A second digestion step was carried out using the same conditions as for the first digestion step, with a total addition of 125 mL hydrogen peroxide and 90 mL of NaOH. The controlled addition of the chemicals enabled to avoid extensive foam formation and also to control the temperature of the reaction, which did not exceed 37°C. The thus recovered solids were resuspended in water, using the same volume as the digestion reaction volume, and the wash water discarded. Two additional washing steps were carried out. The washed and recovered solids were oven dried at 60°C until constant weight and the recovered material was ground to a powder in a mortar, resulting in a white material with good melting properties.

Example 4 - Effect of extraction on the polymer

In this experiment, PHA-containing microbial biomass, produced as in Reference Example 1, but using cheese whey as raw material instead of fruit pulp, was used. Through the use of different culture parameters in the acidogenic reactor, the profile of the volatile fatty acids was varied and as result the produced polymers had different monomeric compositions. The extraction process was carried out as in Example 4-4. The monomeric composition of the polymer (hydroxybutyrate (HB) vs. hydroxyvalerate (HV) content) was determined by GC-MS, while, as before, the molecular weight was assessed by GPC . The native polymer was extracted with chloroform as mentioned in Example 3. Table 5 summarises the results and clearly shows that within the polymeric composition ranges tested the monomeric composition of the polymer was not affected by the extraction process, with any differences being well within the analysis error.

Table 5 - Effect of the extraction on hydroxybutyrate (HB) and hydroxyvalerate (HV)

The parameters related to the concentration and ranges of the reagents as well the duration of the digestion/ reaction are interrelated . The skilled person taking into account the teachings of the present invention is able to choose the proper parameters in order to control the purity and the molecular weight of the extracted polymer . Further, the temperature of the reaction is also selected upon the digestion of speci fic microbial biomasses and targeting speci fic polymer speci fications .

When the product obtained by the process of the invention is used in the production of plastic products , it was confirmed that there is an overall improvement o f the melting properties of the puri fied material when compared to the previous known processes and that no chlorine smell was detected in the final product .

Claims

CLAIMS Process for extraction and purification of polyhydroxyalkanoates (PHA) characterized by reacting PHA-containing material with H2O2 and adding an alkali up to a pH value equal to 9 or higher. Process according to claim 1 characterized by adding the alkali up to a pH value above 11. Process according to claim 2 characterized by adding the alkali up to a pH value above 11,5. Process according to any of claims 1-3 characterized by the ratio of H2O2 to PHA-containing material being within a range of 20 mL H2O2 30vol/g of biomass (dry weight) to 0.1 mL H2O2 30vol/g of biomass (dry weight) . Process according to any of claims 1-4 characterized by adding the alkali simultaneously with H2O2. Process according to any of claims 1-4 characterized by adding the alkali sequentially to H2O2. Process according to any of claims 1-6 characterized by the alkali being selected from the group consisting of NaOH, KOH and Ca(OH)₂. Process according to claim 7 characterized by the alkali being NaOH. Process according to claim 1 characterized by the PHA-containing material being a mixed microbial culture or a pure culture containing intracellular PHAs .

10. Process according to any of claims 1-9 characterized by further comprising a pre-treatment step. 11. Process according to claim 10 characterized by the pretreatment being selected from a chemical, mechanical, electric and enzymatic treatment.