POLYHYDROXYALKANOATES AND METHODS THEREOF

Technical field

[0001] The present disclosure relates to polyhydroxyalkanoates (PHA) polymer and methods of obtaining thereof. Specifically, the present disclosure relates to methods of preparing PHA polymer enriched in medium-chain length (mcl) monomers.

Background

[0002] Currently, majority of the plastics are produced using non-renewable sources such as petroleum. These plastics are used in majority of the industries worldwide. The main advantage of the current forms of plastics is their cost effectiveness because they are easy to produce and dispose after use. However, they are non-biodegradable, accumulating for centuries before full degradation.

[0003] Polyhydroxyalkanoates (PHA) are polyesthers that can be synthesized by bacteria and can be completely biodegraded into CO2 and H2O. So far, over 150 different monomers have been identified; depending on the monomeric composition of the PHA, can have a very wide range of properties.

[0004] At the moment, PHA are produced using single-culture systems with very high productivities at full-scale production. A wide variety of monomers can also be produced using this current technology. However, this current process is quite expensive and an alternative using mixed microbial cultures (MMC) has arisen.

[0005] Using the typical 3-stage process for PHA production, sterilization is not necessary, thereby decreasing overall costs of production of PHA. A mixture of two monomers is often produced as a result: 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV). Owing to the fact that these monomers have a carbon chain length between 3 and 5, polymers containing them fall in the category of short-chain length PHA (scl-PHA). Medium-chain length PHA (mcl-PHA) consist of polymers containing monomers with a carbon chain length between 6 and 14 carbon atoms; longer monomers fall in the category of long-chain length PHA.

[0006] As a consequence of the length of the monomers that compose a mcl-PHA, polymers containing these monomers possess different properties than polymers containing only short-chain length monomers. For example, mcl-PHA possess improved flexibility and elastomeric properties, a high degree of elongation to break and improved biocompatibility with respect to polyhydroxybutyrate, making them suitable for a different range of applications as compared to scl-PHA.

[0007] Medium-chain length polyhydroxyalkanoates are biocompatible, biodegradable and thermoprocessable.

[0008] Back in 1986, Odham and colleagues detected 2 types of mcl-PHA (3- hydroxyhexanoate (HHx) and 3-hydroxyoctanoate) in samples of waste activated sludge collected in a wastewater treatment plant. ¹ In the study conducted by Odham and colleagues, no evidence was made about the source of these monomers.

[0009] Recently, Pisco et al reported production of HHx using MMC and sugar cane molasses as feedstock under cyclic anaerobic-aerobic conditions in a culture enriched with glycogen-accumulating organisms. ² Furthermore, Shen et al claimed to have produced a polymer containing HB and HHx using activated sludge for the first time, using synthetic lauric acid as precursor. ³ The last study achieved a maximum concentration of 0.5 gPHA L ¹ with a polymer containing 6.34% of HHx in its composition and a volumetric productivity of 0.334 g L ¹ h ¹ .

[0010] In a study from 2018, Ntaikou and colleagues reported the production of PHA containing only 2.5 % and 6 % of HHx obtained from caproic acid using mixed microbial cultures. The selection of the PHA-accumulating culture was not carried out using a caproic-rich fermentate. ⁴

[0011] Unlike fermentations using pure cultures, MMC processes can maintain a level offunctional redundancy which allowthem to be more resilientto perturbations. Hence, cheap substrates such as complex wastes become technically feasible, decreasing cost of operations beyond the non requirement for sterilization. [0012] There is a need for an alternative method of obtaining polymers enriched in medium-chain length polyhydroxyalkanoates (mcl-PHA), as current methods for mcl- PHA production utilizes only pure culture systems.

[0013] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

General Description

[0014] The present disclosure relates to polyhydroxyalkanoates (PHA) polymer and methods of obtaining thereof. Specifically, the present disclosure relates to methods of obtaining PHA polymer enriched in medium-chain length (mcl) PHA monomers.

[0015] In an embodiment, the method disclosed relates to obtaining mcl enriched PHA polymer using mixed microbial cultures and fermented liquid rich in caproate.

[0016] In an embodiment, the method disclosed relates to a method of obtaining a PHA containing a fraction of HHx using a caproate-rich fermented effluent.

[0017] In an embodiment, a polymer comprising 3-hydroxyhexanoate monomers was obtained using a caproate-rich fermented feedstock. A mixed microbial culture was selected under a feast and famine regime to obtain a polymer containing HHx monomers.

[0018] In an embodiment, the typical three-stage process for obtaining PHA was operated using fruit waste as feedstock and the operating conditions of the acidogenic reactor were tailored to promote acidogenesis as well as chain elongation reactions to produce caproate.

[0019] In an embodiment, the method of the present disclosure improves the process of obtaining PHA by using mixed microbial culture. The improvement is a consequence of obtaining a third monomer - 3-hydroxyhexanoate (a mcl-PHA). Owing to the fact that it is a longer monomer and falls into the category of medium-length chain hydroxyalkanoate (as opposed to 3-hydroxybutyrate (HB), 3-hydroxyvalerate (HV), which fall into the category of short-length chain hydroxyalkanoates), a polymer

B comprising a fraction of 3-hydroxyhexanoate in its composition behaves differently, both thermally and mechanically. As a consequence, the range of applications for PHA polymers obtained using the method of the present disclosure is wider as compared to PHA polymers obtained using prior art methods. This is because the PHA polymers obtained using the method of the present disclosure comprises enriched amount of medium-length chain PHA (3-hydroxyhexanoate) in addition to short-length chain hydroxyalkanoates (3-hydroxybutyrate, 3-hydroxyvalerate). PHA polymers obtained using prior art methods which is not enriched in medium-length chain PHA (3- hydroxyhexanoate).

[0020] In an embodiment, a consortium of mixed microbial culture and fruit waste feedstock was used in the typical 3-stage process for producing PHA.

[0021] In an embodiment, caproate-rich effluent produced in the first stage of the typical 3-stage process results in a polymer comprising 3-hydroxyhexanoate (HHx), and possibly also 3-hydroxybutyrate (HB) and/or 3-hydroxyvalerate (HV), being produced. The HHx, owing to the fact that its carbon backbone contains 6 carbons, falls in the category of medium-chain length monomer, which is new in MMC processes using fermented wastes.

[0022] In an embodiment, caproate-rich effluent produced in the first stage of the typical 3-stage process results in a polymer comprising about 10% 3-hydroxyhexanoate (HHx).

[0023] The process of obtaining 3-hydroxyhexanoate (HHx), a mcl-PHA, by mixed cultures as described in the presently disclosed process is expected to be more cost effective since it does not require sterile conditions (energy savings), it requires cheaper equipment (reactors and auxiliary devices) and utilizes waste feedstocks (zero/low cost). The polymer containing 3-hydroxyhexanoate is more elastic as compared to polymers containg only scl-PHA. Hence this widens the range of applications of the polymer produced by the presently disclosed method. [0024] So far, there is no industry standard process for mcl enriched PHA polymer production using mixed microbial cultures and caproate rich feedstock. Current production of mcl-PHA is accomplished through pure cultures processes.

[0025] In an embodiment, the present disclosure relates to a method of using mixed microbial culture to obtain a polyhydroxyalkanoate (PHA) comprising at least 10% (w/w) of 3-hydroxyhexanoate, comprising the following steps: obtaining a first caproate-rich fermentate; contacting the caproate-rich fermentate with a mixed microbial culture submitted to a feast and famine regime, to obtain a PHA-accumulating culture; and contacting the PHA-accumulating culture with the first or with a second caproate- rich fermentate in order to obtain a PHA enriched in 3-hydroxyhexanoate monomer.

[0026] In an embodiment, the mixed microbial culture is an activated sludge.

[0027] In an embodiment, the present disclosure relates to method for using mixed microbial culture to obtain a polyhydroxyalkanoate (PHA) polymer enriched in medium- chain length PHA comprising at least 10% (w/w) of 3-hydroxyhexanoate, comprising the following steps: obtaining a caproate-rich fermentate; subjecting an activated sludge and the caproate-rich fermentate to feast and famine regime in a sequential batch reactor to obtain a biomass of PHA-accumulating culture; and inoculating the PHA-accumulating culture and expose it to an excess of caproate-rich fermentate in fed batch mode in order to obtain a PHA enriched in 3- hyd roxy hexa noate .

[0028] In an embodiment, the method of obtaining the polymer further comprising a previous step of subjecting a feedstock to an acidogenic anaerobic fermentation under a controlled environment in order to obtain a caproate-rich fermentate.

[0029] In an embodiment, the step of contacting the caproate-rich fermentate with a mixed microbial culture submitted to a feast and famine regime is performed, preferably in a sequential batch reactor (SBR). [0030] In an embodiment, the step of contacting the PHA-accumulating culture with a caproate-rich fermentate in order to obtain a PHA enriched in 3-hydroxyhexanoate is performed, preferably in a fed batch reactor.

[0031] In an embodiment, the acidogenic fermentation step was performed in an up- flow anaerobic sludge blanket reactor.

[0032] In an embodiment, the mixed microbial culture was inoculated and fed with the second caproate-rich fermentate in an aerated fully-mixed reactor.

[0033] The method of obtaining the polymer according to any of the previous claims, wherein the mixed microbial culture is an activated sludge.

[0034] In an embodiment, the activated sludge was inoculated and fed with the caproate-rich fermentate in an aerated fully-mixed reactor and subjected to feast and famine regime.

[0035] In an embodiment, the PHA-accumulating culture was inoculated in a mixed reactor in fed-batch mode and fed in excess of caproate-rich fermentate at regular intervals.

[0036] In an embodiment, the acidogenic fermentation is operated with a hydraulic retention time preferably ranging from 0.5 days to 2 days.

[0037] In an embodiment, the acidogenic fermentation is preferably at a temperature ranging from 25 °C to 40 °C.

[0038] In an embodiment, the acidogenic fermentation is preferably at a pH ranging from of 4.5 to 7.

[0039] In an embodiment, the acidogenic fermentation is at a recirculation speed ranging preferably from 0.5 L min ¹ to 3.5 L min ^-1 .

[0040] In an embodiment, the acidogenic fermentation is at a organic loading rate ranging preferably from 1 gCOD L ¹ d ¹ to 50 gCOD L ¹ d ¹ , preferably 10-30 gCOD L ¹ d ¹ . [0041] In an embodiment, the step of selecting a PHA-accumulating culture is operated at an organic loading rate ranging from 25 Cmmol L ¹ d ¹ to 400 Cmmol L ¹ d ¹ (1 gCOD L ¹ d ¹ to 15 gCOD L ¹ d ¹ ), preferably from 2 gCOD L ¹ d ¹ to 7 gCOD L ¹ d ¹ .

[0042] In an embodiment, the step of obtaining a biomass of PHA-accumulating culture is at a sludge retention time ranging from 0.5 days to 10 days, preferably 4 days.

[0043] In an embodiment, the step of obtaining a biomass of PHA-accumulating culture is at a hydraulic retention time ranging from 0.5 days to 10 days, preferably 1 day.

[0044] In an embodiment, the step of obtaining a biomass of PHA-accumulating culture is at a pH from 6.5 to 10, preferably 7.5 to 9.5.

[0045] In an embodiment, the step of selecting a PHA-accumulating culture is carried out with cycles between 2 hours to 24 hours, preferably 12 hours.

[0046] In an embodiment, the step of obtaining a biomass of PHA-accumulating culture is at a dissolved oxygen concentration of at least 0.5 mg L ¹ , preferably above 2 mg L ¹ .

[0047] In an embodiment, the step of obtaining a biomass of PHA-accumulating culture is at a carbon to nitrogen ratio ranging from 5 Cmol Nmol ¹ to 50 Cmol Nmol ¹ , preferably between 12.5 and 20 Cmol Nmol ¹ .

[0048] In an embodiment, the PHA accumulation step is at a pH ranging from 6.5 to 10, preferably between 7.5 and 9.5.

[0049] In an embodiment, the PHA accumulation step is at a dissolved oxygen concentration of at least 0.5 mg L ¹ .

[0050] In an embodiment, the caproate-rich fermentate is a fermented fruit waste or a fermented cheese whey.

[0051] In an embodiment, the acidogenic fermentation process is performed using an upflow anaerobic sludge blanket reactor.

[0052] Another aspect of the present disclosure relates a polyhydroxyalkanoate (PHA) polymer enriched in medium-chain length PHA monomer comprising at least 10% (w/w) 3-hydroxyhexanoate; this polymer is obtained by the method described in the present disclosure. Preferably, the polymer may further comprise at least one of the following monomers: 3-hydroxybutyrate, 3-hydroxyvalerate. Preferably the polymer may further comprise 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxyhexanoate.

[0053] In an embodiment, the polymer may comprise 10% (w/w) - 60% (w/w) of of 3- hydroxyhexanoate; preferably 40% (w/w) - 60% (w/w) of 3-hydroxyhexanoate.

[0054] In an embodiment, the polymer may comprise 25% (w/w) to 90% (w/w) of 3- hydroxybutyrate, from 0% (w/w) to 50% (w/w) of 3-hydroxyvalerate, from 10% (w/w) to 60% (w/w) of 3-hydroxyhexanoate.

[0055] In an embodiment, the polymer may comprise 46% (w/w) of 3-hydroxybutyrate, 14% (w/w) of 3-hydroxyvalerate and 40% (w/w) of 3-hydroxyhexanoate.

Brief Description of the Drawings

[0056] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.

[0057] Figure 1 shows the general structure of PHA. R = CH ₃ (3-hydroxybutyrate); R = CH2CH ₃ (3-hydroxyvalerate); R= CH2CH2CH ₃ (3-hydroxyhexanoate).

[0058] Figure 2 shows the experimental set-up for obtaining the disclosed medium- chain length (mcl) enriched PHA polymer.

Detailed Description

[0059] The present disclosure relates to polyhydroxyalkanoates (PHA) polymer and methods of obtaining thereof. Specifically, the present disclosure relates to methods of obtaining PHA polymer enriched in medium-chain length (mcl) PHA monomers.

[0060] In an embodiment, the method disclosed relates to obtaining mcl enriched PHA polymer using mixed microbial cultures and fermented liquid rich in caproate.

[0061] In an embodiment, the method disclosed relates to a method of obtaining a PHA containing a fraction of HHx using a caproate-rich fermented effluent. [0062] In an embodiment, a polymer comprising 3-hydroxyhexanoate monomers was obtained using a caproate-rich feedstock for a feast and famine process using mixed microbial culture. The use of a caproate-rich stream to select a PHA-accumulating culture under a feast and famine regime and accumulate PHA in a separate step has not been reported previously.

[0063] In an embodiment, the typical three-stage process for obtaining PHA was operated with fruit waste as feedstock and the operating conditions of the acidogenic reactor were tailored to promote acidogenesis as well as chain elongation reactions to produce caproate.

[0064] In an embodiment, the fruit waste feedstock preferably has the characteristics as shown in example Table 1 below.

Table 1: Characterization of the fruit waste used in fermentation reactor.

[0065] In an embodiment, as illustrated in example Table 1 above, the waste is characterized by a high solids content and high COD.

[0066] In an embodiment, caproate-rich effluent produced in the first stage of the typical 3-stage process results in a mcl-PHA containing 3-hydroxybutyrate (HB), 3- hydroxyvalerate (HV) and 3-hydroxyhexanoate (HHx) being produced. The HHx, owing to the fact that its carbon backbone contains 6 carbons, falls in the category of medium- chain length monomer, which is new in MMC processes using fermented wastes.

[0067] Typically, the process for the production of PHA comprises three steps: an acidogenic fermentation of the waste into suitable products for PHA production (step 1); the selection of an undefined mixed microbial culture capable of accumulating PHA based on a feast and famine regime (step 2); accumulation of PHA using the precursors produced in step 1 and the culture selected in step 2 (step 3).

[0068] In an embodiment, as an example illustrated in Figure 2, the experimental setup for obtaining the mcl enriched PHA polymer described in the present disclosure comprises 3 reactors with volumes in the range from 60 L to 100 L that are inoculated with a biomass from wastewater treatment plants.

[0069] In an embodiment, a fermentation reactor was assembled to convert fruit waste into suitable precursors for obtaining PHA. The fermented effluent of the acidogenic fermentation step was filtered and used in the subsequent steps.

[0070] In am embodiment, in order to select a PHA-accumulating culture, activated sludge was inoculated in a sequential batch reactor (SBR) and operated in a feast and famine regime to stimulate storage response and select a undefined mixed microbial culture capable of producing HHx-rich PHA.

[0071] In an embodiment, a third reactor was assembled and inoculated with excess biomass from the SBR and fed discontinuously with fermentation products (FP) from the acidogenic reactor in fed-batch mode with the goal of producing PHA. 2 reactors were inoculated similarly to the latter reactor to carry out batch assays under different operating conditions, mimicking the last step of the process.

[0072] In an embodiment, in order to achieve the desired product of the present disclosure, step 1 (acidogenic fermentation) of the process must result in a caproate- rich effluent, possibly containing other fermentation products such as formate, acetate, propionate, acetone, isopropanol, butyrate, iso-butyrate, succinate, butanol, 2,3- butanediol, valerate, iso-valerate, iso-caproate, heptanoate, octanoate, lactate and ethanol. [007B] In an embodiment, the method of producing mcl enriched PHA polymer comprises the following steps:

Acidogenic fermentation of fruit waste; selection of a PHA-accumulating culture; and PHA accumulation.

[0074] In an embodiment, the process of acidogenic fermentation comprises an upflow anaerobic sludge blanket (UASB) reactor with a total volume of 100 L and a working volume of 60 L; the upflow anaerobic sludge blanket (UASB) reactor was assembled and inoculated with granularsludge from a full scale methanogenic reactorfrom a treatment plant of the brewer industry with up to 20 L of granules. It was operated with a variable organic loading rate (OLR), starting at around 5 gCOD L ^-1 d ^-1 and it was gradually increased up to 27.9 gCOD L ^-1 d ^-1 .

[0075] In an embodiment, the pH of the upflow anaerobic sludge blanket (UASB) reactor was kept at 5.07 ± 0.19, a hydraulic retention time (HRT) of 1 d was applied, the temperature of the reactor was set to 30 °C using an external jacket and a mixture of fruit waste and nutrients (MgSC>4: 120 mg L ^-1 , CaCh: 480 mg L ^-1 and FeCU^^O: 80 mg d ^-1 ) was fed constantly with a C/N/P ratio of 100/0.5/0.1 (gCOD/gN/gP). Sodium bicarbonate was added in order to buffer the pH of the reactor. The fermented effluent was filtered and used as carbon source in the subsequent steps of the process.

[0076] In an embodiment, as an alternative, the step of acidogenic fermentation of the feedstock may be skipped if the feedstock is already rich in caproate such as for example, a caproate-rich effluent.

[0077] In an embodiment, the process of acidogenic fermentation of waste is alternatively replaced with the use of pure caproate or a chemically defined media containing caproate.

[0078] In an embodiment, a sequential batch reactor (SBR), preferably a 100 L sequential batch reactor (SBR) was inoculated with activated sludge from the wastewater treatment and operated with a feast and famine regime. The reactor was fed with fermented fruit waste obtained from the acidogenic step at an OLR of 3.0 gCOD L ^-1 d ^-1 . A sludge retention time (SRT) of 4 d along with a HRT of 1 d were applied.

[0079] In an embodiment, the 12h-cycle configuration comprises a period of 11 hours of aeration (0.7 vvm) and 1 h of settling with no aeration or stirring. The aeration phase started with a 10-minute feeding of fermented fruit waste along with mineral solution and after about 2 h, a solution rich in nutrients was fed to the reactor. Every two cycles, 10.5 h after the beginning of the cycle, the reactor was purged. About 15 minutes before the end of the settling period, up to 50 L of liquid phase of the reactor was withdrawn. The phases of the cycle were controlled by timers and a software programmed in Labview. The composition of the mineral solution was as follows (mg L ^-1 ): FeCl _3» 6H ₂ 0: 3.00, H ₃ BO ₃ : 0.30, CoCI _2» 6H ₂ 0: 0.30, MnCI _2» 4H ₂ 0: 0.24, ZnS0 _4« 7H ₂ 0: 0.24, Na ₂ Mo0 _4« 2H ₂ 0: 0.12, CuS0 _4» 5H ₂ 0: 0.06, Kl: 0.06.

[0080] In an embodiment, the nutrients solution comprises NH ₄ CI and KH ₂ P0 ₄ in a proportion to the carbon source of 100/7/1 (Cmol/Nmol/Pmol) along with thiourea with a concentration of 10 mg L ^-1 . Temperature and pH were monitored and uncontrolled.

[0081] In an embodiment, the PHA accumulation process comprises using a fed batch reactor, preferably a 60 L fed batch reactor for the PHA accumulation. The reactor was inoculated with biomass selected in the SBR (25 L) and fed with the FP-rich effluent obtained from the first step in DO-based pulse-wise mode. The volume of each pulse was preferably 1/4 the volume fed to each cycle of the SBR, to maintain the F/M ratio of the previous step. The reactor was fully aerated (at least 1 vvm) during operation to maintain the DO above 2 mg L ^-1 and pH and temperature were monitored but not controlled.

[0082] In an embodiment, the fermented fruit waste composition comprises (COD basis): lactate (0.09), acetate (1.03), butyrate (1.61), valerate (0.03) and caproate (11.5).

[0083] In an embodiment, batch tests were carried out. Two reactors of 1 L were assembled to carry out the batch tests (BioFlo/CelliGen 115 Fermentor and Bioreactor, Eppendorf). The pH of the reactor was uncontrolled and no nutrients were added. The experiments differed by the number of feed pulses given, and the composition of feedstock used and Table 2 summarizes the compositions used in each experiment.

Table 2: Composition of the feedstock used in each batch experiment (COD basis).

Lactate (Lac), acetate (Ace), propionate (Pro), ethanol (EtOH), butyrate (But), iso valerate (i-Val), valerate (Val) and caproate (Cap)

*This assay was carried out separately from the others so the fermented fruit waste had a different composition; hence, FP profile was adjusted with pure reagents to be closer to the one used in assay 1.

[0084] In an embodiment, cell dry weight was determined by total suspended solids (TSS) measurements as described in standard methods and the COD was assessed using LCK 914 Hach Lange kits (Hach-Lange, Germany). Fermentation products (acetate, ethanol, propionate, lactate, butyrate, isobutyrate, valerate, iso-valerate and caproate) were quantified by high performance liquid chromatography (HPLC) using a VWR Hitachi Chromaster equipped with a Biorad 125-0129 pre-column (30 x 4.6 mm), Aminex HPX- 87H column (300 x 7.8 mm, Biorad) and Rl and UV (l = 210 nm) detectors. Samples (99 m) were eluted with 0.01 N H2SO4 at a flow rate of 0.6 mL min ¹ with an operational temperature of 60 °C for 65 min. Samples were centrifuged at 10000 rpm for 3 min and filtered through 0.2 mm membranes before injection.

[0085] In an embodiment, gas chromatography with flame ionization detector (GC-FID) was used to determine the concentration of polyhydroxyalkanoates (PHA) as described by Pereira et at, with a few modifications. Sample (2-5 mg) were liophilized and digested at 100 °C for 4 hours with 2 mL of acidic methanol (20% (v/v)) and 2 mL of heptadecane in chloroform (1 g L ¹ ) as internal standard. After digestion, samples were cooled to 20- 27 °C, 2 mL of water were added and the samples stirred. Finally, the chloroform phase was extracted and injected. Resulting methyl esthers were analysed using a 430-GC Bruker equipped with flame ionization detector and Restek column of 60 m, 0.53 mm ID, 1 m df, Crossbond, Stabilwax. The detector heating ramp consisted in the following program: 20 C min ¹ until 100 °C; 3 °C min ¹ until 155 °C; and 20 C min ¹ until 220 °C. 2 m of sample was injected in splitless mode with a running time of 32 min at constant pressure of 14.5 psi, using helium as carrier gas. Copolymer PHBV (88:12 molar ratio) from Sigma-Aldrich was used as standards in the range of 0.03-3 g L ¹ . Additionally, methyl 3-hydroxyhexanoate (SigmaAldrich) was added in the same range of concentrations to quantify HHx.

[0086] In an embodiment, ammonia and phosphate concentrations were measured by a segmented flow analyser through the Skalar San++ system.

[0087] In an embodiment, the acidification yield (%) of the acidogenic reactor was calculated by the ratio between the sum of all FP in COD and inlet COD concentration (after subtracting any residual FP); the productivity (gCOD L ^-1 d ^_1 ) was determined as the ratio between the outlet FP concentration and the HRT.

[0088] In an embodiment, PHA content was determined as 100*PHA/TSS in gPHA gTSS ^-1 . FP concentration was determined as the sum of all FP quantified by HPLC: lactate, acetate, propionate, ethanol, isobutyrate, butyrate, iso-valerate, valerate, caproate, heptanoate and octanoate on a COD basis. Volumetric rates (G ^RHA MAC, G ^RR MAC) were calculated by the slope of PHA or FP as function of the corresponding time period. The yields of PHA production per substrate consumption (YPHA/FP) were determined by the ratio of the maximum PHA production rates per maximum substrate uptake rates; the yields of HHx production per caproate consumed (Y _HHx /c _ap ) were determined by the ratio of the maximum HHx production rates per maximum caproate uptake rates.

[0089] Typically, scl-PHA are composed of 3-hydroxybutyrate (HB) or a mixture of 3- hydroxybutyrate (HB) and 3-hydroxyvalerate (HV). In the case of the homopolymer of PHB, this polymer is highly crystalline, brittle and stiff with a high melting point, high young's modulus and high tensile strength. The addition of HV monomers, results in a copolymer with improved properties: lower crystallinity, lower melting point, young's modulus as well as tensile strength.

[0090] In an embodiment, the polymer obtained using the presently disclosed method comprises 46% of 3-hydroxybutyrate, 14% of 3-hydroxyvalerate and 40% of 3- hydroxyhexanoate. This results in a completely different product with a molecular weight of 7.90 x 10 ⁵ Da, melting point of 152 °C, young's modulus of 0.78 MPa, tension at break of 5.15 MPa and deformation at break of 269 %. As a consequence of its mechanical properties, this polymer has improved flexibility and elastomeric properties. Typically, these polymers are produced used single-culture processes.

[0091] Table 2 shows a comparison of the mechanical properties of example polymers based on PHA and polypropylene (PP) as compared to the mechanical properties of the polymers obtained by the method of the present disclosure. When compared to other typical materials produced by MMC, such as PHB and P(HBV), the polymer of present disclosure is far more elastic. This is represented by the high elongation to break, low Young's modulus and tensile strength. In fact, the elongation to break of the polymer herein disclosed is far more comparable to polymers produced by pure cultures systems such as P(PHHxHO). Besides, this polymer possesses the lowest Young's Modulus from the polymers herein reported, which means that it is the one that requires the least amount of stress to be stretched.

Table 2 - Summary of mechanical properties of some polymers based on PHA and polypropylene (PP). (P(HBV), poly(hydroxybutyrate-co-hydroxyvalerate); P(HHxHO), poly(hydroxyhexa noate-co-hyd roxyocta noate)

[0092] The above described embodiments are combinable.

[0093] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0094] The following claims further set out particular embodiments of the disclosure.

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