Method for producing PHA by fermentation of archaea

Abstract

The present invention relates to a method for the large-scale production of polyhydroxyalkanoates (PHAs) by fermentation of archaea such as Haloferax mediterranei, in which air is constantly bubbled into the fermenter from the bottom, allowing constant aeration and mixing of the culture medium and removal of carbon dioxide, and further provides blends of PHA with at least two additional polymers, and articles comprising or made from said blends.

Description

[Technical field]

[0001] The present invention relates to a method for the large-scale production of polyhydroxyalkanoates (PHAs) by fermentation, including outdoor fermentation of archaea such as Haloferax mediterranei, in which air is constantly bubbled into the fermenter from the bottom, allowing for constant aeration and mixing of the culture medium and removal of carbon dioxide. Additionally, blends of PHA with additional polymers and articles made therefrom are provided. [Background technology]

[0002] The global petrochemical plastic production in 2016 was 335 million tons per year, raising urgent concerns about post-consumer waste disposal and environmental pollution by plastics. 22-43% of the polymers used worldwide end up in landfills, resulting in 10-20 million tons of plastics entering the oceans every year, causing damage to ecosystems and associated costs of 13 billion US dollars per year. Governments are currently supporting strategies to promote a bio-based circular economy by enforcing the use of bioplastics and introducing landfill bans. Among biopolymers, polyhydroxyalkanoates (PHAs) are recognized as outstanding sustainable materials due to their tunable copolymer formulations and mechanical properties, as well as their unique degradability in both marine and surrounding soil environments. In addition, unlike other biopolymers, PHA polymers are hydrophobic, water insoluble, indefinitely stable in air, non-toxic, thermoplastic, and / or elastic, and have very high purity within cells. The PHA polymer family has high potential and applicability for a wide range of biopolymer applications. Therefore, the search for microorganisms with high efficiency of PHA accumulation is an urgent need. Among wild-type strains, the extreme halophile Haloferax mediterranei (Hfx. mediterranei) is the most important strain. This archaea can utilize various substrates and accumulate large amounts of PHA intracellularly. Moreover, its extremophilic nature, characterized by high salt tolerance, allows non-sterile cultivation of this organism, thus reducing the cost and energy required for the process.

[0003] Despite decades of research, commercialization of PHA processes has been slow. This could be explained by the absence of high-value applications in the early stages of product market penetration, where the cost barrier of petroleum-based plastics is a challenge. However, the development of halophyte biorefinery processes for the production of PHA from seaweed remains challenging, and gaps exist in the potential for scale-up of these processes that would give them distinct advantages compared to current processes. For example, closed controlled fermenters currently used for bacterial PHA production are expensive and require a lot of fresh water and energy for mixing of the culture medium (Mahler et al., 2018). In addition, harvesting bacterial biomass is difficult, This is usually done in a centrifuge, which has little potential for scale-up and is not stable in the corrosive high-salt medium required for the growth of extreme halophiles. Furthermore, most of the studies on extreme halophilic archaea have been performed in shake flask cultures or bioreactors with working volumes of 1–10 L (Alsafadi and Al-Mashaqbeh, 2017;Hezayen et al., 2000). Therefore, the scale-up of PHA production from halophilic archaea using various carbon substrates is still underway. Research is needed that needs to focus on the role of oxygen in the culture medium. The high salinity of the culture medium also poses challenges for the construction materials of the reactor. Higher salinity leads to more corrosive conditions, and therefore corrosion-resistant reactors are required (Hezayen et al., 2000). Oxygen is essential for microbial growth and other metabolic activities. This becomes even more important in high salinity conditions, where the solubility of oxygen in the medium is further reduced. Oxygen limitation is required for enhanced PHA production. While oxygen-rich conditions have been studied, oxygen-rich conditions help to increase biomass concentration, thus leading to high-density cultures (Maheshwari et al., 2018). It will be important to evaluate the role of oxygen concentration on bacterial growth and therefore PHA production.

[0004] The intracellular product PHA requires high cell density culture because the limited available intracytoplasmic space limits the maximum amount of PHA that can be accumulated intracellularly. PHA recovery begins after separation and concentration of cells from the fermentation medium. PHA recovery from halophilic bacteria can be easily achieved by cell lysis using tap water, so separation of cells from the fermentation medium is the overall challenge in PHA recovery. Cell separation is generally performed using centrifugal force at laboratory scale. Industrial separation of cells from fermentation medium requires highly efficient continuous centrifuges. These methods are adequate when extracellular products are required, but when large amounts of washed bacteria are required, it becomes difficult to achieve a sterile harvest because of the need to scrape or wash the rotor of the continuous flow centrifuge. Similarly, simple filtration is time-consuming and unreliable due to filter blockage and the difficulty of washing the organisms from the filter in a sterile manner. Cell harvesting with ultrasonic standing waves offers a potential alternative to the problematic traditional separation techniques. Various studies have been carried out looking at the separation of microorganisms with very high separation efficiency using ultrasound. Recently, ultrasonic separation has been used to separate microalgal cells from the culture medium. Sonication produces highly localized cavitation and cell disruption to recover PHA from Hfx. mediterranei, a phenomenon fundamentally different from cell entrapment and concentration in ultrasonic standing waves. To determine whether bacterial cell harvesting by ultrasonic standing waves is a viable alternative to conventional dewatering techniques, its effectiveness and energy requirements need to be investigated. Summary of the Invention [Problem to be solved by the invention]

[0005] The work described herein aimed to address the challenges of halophiles in PHA production (oxygen requirement, outdoor fermentation) using Hfx. mediterranei grown in outdoor fermentation in air-agitated bioreactors and macroalgal hydrolysates. Fermentations were carried out under non-sterile conditions in 1 L PET bottles and 40 L plastic sleeves operated as bubble column reactors. Process conditions (aeration rate, fermentation time) were optimized for efficient PHA production from the hydrolysates. The structural characteristics of the resulting PHA were characterized along with the molecular weight of the final polymer.

[0006] This study provides a feasible method for scaling up PHA production from inexpensive substrates such as macroalgal hydrolysates. The effects of various culture parameters were investigated to establish favorable conditions for improving PHA productivity. Molecular weight analysis suggested that the obtained PHA was more homogeneous at lower aeration rates. [Means for solving the problem]

[0007] In one aspect, the present invention relates to a method for producing polyhydroxyalkanoates (PHA) by fermentation of archaea capable of producing said PHA, said method comprising: (i) providing a fermentor having a culture volume, i.e., working volume, of at least about 5 or 10 liters, said fermentor comprising a culture medium occupying said culture volume, said culture medium comprising salt water, such as seawater, supplemented with a carbon source and a nitrogen source, suitable for culturing, i.e., promoting and supporting the growth and survival of, said archaea; (ii) inoculating the culture medium with a seed culture of the archaea; (iii) constantly aerating the culture medium at a rate of at least about 0.2 vvm through the interior of the fermenter at the bottom thereof and at a rate of at least about 0.2 vvm so as to constantly aerate and mix the culture medium. culturing the archaea while bubbling the culture medium with ethanol until a predetermined concentration of the archaea in the culture medium is obtained; (iv) harvesting the archaeal biomass from the culture medium; and (v) concentrating, isolating and / or extracting the PHA from the harvested biomass; Includes.

[0008] The method disclosed herein may further comprise purifying the PHA obtained in step (v) by, for example, repeated washing with water, an ionic liquid, or a combination thereof to obtain a purified PHA, and optionally drying the purified PHA thus obtained, for example, by a drum dryer, a spray dryer, or a freeze dryer. The purified PHA, with or without drying, may then be blended, i.e. mixed, with at least one polymer, e.g., a biodegradable or non-biodegradable polymer.

[0009] The fermentation process of the present invention may be carried out in either batch or continuous mode.

[0010] In another aspect, the present invention provides a blend comprising a PHA and at least two additional polymers, each of which may be independently a biodegradable or non-biodegradable polymer. In certain embodiments, the at least two additional polymers comprise or consist of polylactic acid (PLA) and keratin, and the PHA:PLA ratio in the blend may be in the range of about 1:99 to about 99:1, about 20:80 to about 80:20, about 30:70 to about 70:30, or about 40:60 to about 60:40, respectively, by weight, but is preferably about 20:80 to about 40:60, e.g., about 25:75, about 30:70, or about 35:65, respectively, by weight (regardless of the total amount of other polymers, e.g., keratin, contained within the blend). In other embodiments, one of the at least two additional polymers is keratin, which comprises from about 1% to about 99% by weight of the blend, e.g., from about 5% to about 90% by weight, from about 10% to about 80% by weight, from about 15% to about 70% by weight, or from about 20% to about 60% by weight, preferably from about 10% to about 50% by weight.

[0011] In a further aspect, the present invention provides articles comprising or made from the blends disclosed herein. [Brief description of the drawings]

[0012] [Figure 1A] Figures 1A-1D show the experimental setup for the optimization of the aeration rate in the outdoor cultivation (1 L PET bottle) of Hfx. mediterranei (1A); the experimental setup for the cultivation of Hfx. mediterranei in a 40 L sleeve (1B); a schematic diagram of the cultivation system in a 1 L PET bottle [experimental conditions: flow rate: 0.25-2.0 L x min-1; fermentation volume: 800 mL; temperature: 42 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (1C); and a schematic diagram of a single photobioreactor (40 L) for the cultivation of Hfx. mediterranei [experimental conditions: flow rate: 1.0 L min-1; fermentation volume: 10 L; temperature: 42 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (1D). [Figure 1B] Figures 1A-1D show the experimental setup for the optimization of the aeration rate in the outdoor cultivation (1 L PET bottle) of Hfx. mediterranei (1A); the experimental setup for the cultivation of Hfx. mediterranei in a 40 L sleeve (1B); a schematic diagram of the cultivation system in a 1 L PET bottle [experimental conditions: flow rate: 0.25-2.0 L x min-1; fermentation volume: 800 mL; temperature: 42 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (1C); and a schematic diagram of a single photobioreactor (40 L) for the cultivation of Hfx. mediterranei [experimental conditions: flow rate: 1.0 L min-1; fermentation volume: 10 L; temperature: 42 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (1D). [Figure 1C]Figures 1A-1D show the experimental setup for the optimization of the aeration rate in the outdoor cultivation (1 L PET bottle) of Hfx. mediterranei (1A); the experimental setup for the cultivation of Hfx. mediterranei in a 40 L sleeve (1B); a schematic diagram of the cultivation system in a 1 L PET bottle [experimental conditions: flow rate: 0.25-2.0 L x min-1; fermentation volume: 800 mL; temperature: 42 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (1C); and a schematic diagram of a single photobioreactor (40 L) for the cultivation of Hfx. mediterranei [experimental conditions: flow rate: 1.0 L min-1; fermentation volume: 10 L; temperature: 42 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (1D). [Figure 1D] Figures 1A-1D show the experimental setup for the optimization of the aeration rate in the outdoor cultivation (1 L PET bottle) of Hfx. mediterranei (1A); the experimental setup for the cultivation of Hfx. mediterranei in a 40 L sleeve (1B); a schematic diagram of the cultivation system in a 1 L PET bottle [experimental conditions: flow rate: 0.25-2.0 L x min-1; fermentation volume: 800 mL; temperature: 42 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (1C); and a schematic diagram of a single photobioreactor (40 L) for the cultivation of Hfx. mediterranei [experimental conditions: flow rate: 1.0 L min-1; fermentation volume: 10 L; temperature: 42 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (1D). [Figure 2A]Figures 2A-2F show the growth and PHA production curves at different aeration rates in PHA production using Hfx. mediterranei [experimental conditions: flow rate: 0.25-2.0 L x min-1; fermentation volume: 800 mL; water temperature: 42 ± 2.5 °C; air temperature: 37 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (2A and 2B, respectively); growth and PHA production rates under outdoor culture conditions in PHA production using Hfx. mediterranei [experimental conditions: flow rate: 1.0 L x min-1; fermentation volume: 10 L; water temperature: 42 ± 2.5 °C; air temperature: 37 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (2C and 2D, respectively); and the change in PHA% in biomass versus time in sleeve 1 and sleeve 2 (2E and 2F, respectively). [Figure 2B] Figures 2A-2F show the growth and PHA production curves at different aeration rates in PHA production using Hfx. mediterranei [experimental conditions: flow rate: 0.25-2.0 L x min-1; fermentation volume: 800 mL; water temperature: 42 ± 2.5 °C; air temperature: 37 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (2A and 2B, respectively); growth and PHA production rates under outdoor culture conditions in PHA production using Hfx. mediterranei [experimental conditions: flow rate: 1.0 L x min-1; fermentation volume: 10 L; water temperature: 42 ± 2.5 °C; air temperature: 37 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (2C and 2D, respectively); and the change in PHA% in biomass versus time in sleeve 1 and sleeve 2 (2E and 2F, respectively). [Figure 2C]Figures 2A-2F show the growth and PHA production curves at different aeration rates in PHA production using Hfx. mediterranei [experimental conditions: flow rate: 0.25-2.0 L x min-1; fermentation volume: 800 mL; water temperature: 42 ± 2.5 °C; air temperature: 37 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (2A and 2B, respectively); growth and PHA production rates under outdoor culture conditions in PHA production using Hfx. mediterranei [experimental conditions: flow rate: 1.0 L x min-1; fermentation volume: 10 L; water temperature: 42 ± 2.5 °C; air temperature: 37 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (2C and 2D, respectively); and the change in PHA% in biomass versus time in sleeve 1 and sleeve 2 (2E and 2F, respectively). [Figure 2D] Figures 2A-2F show the growth and PHA production curves at different aeration rates in PHA production using Hfx. mediterranei [experimental conditions: flow rate: 0.25-2.0 L x min-1; fermentation volume: 800 mL; water temperature: 42 ± 2.5 °C; air temperature: 37 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (2A and 2B, respectively); growth and PHA production rates under outdoor culture conditions in PHA production using Hfx. mediterranei [experimental conditions: flow rate: 1.0 L x min-1; fermentation volume: 10 L; water temperature: 42 ± 2.5 °C; air temperature: 37 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (2C and 2D, respectively); and the change in PHA% in biomass versus time in sleeve 1 and sleeve 2 (2E and 2F, respectively). [Figure 2E]Figures 2A-2F show the growth and PHA production curves at different aeration rates in PHA production using Hfx. mediterranei [experimental conditions: flow rate: 0.25-2.0 L x min-1; fermentation volume: 800 mL; water temperature: 42 ± 2.5 °C; air temperature: 37 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (2A and 2B, respectively); growth and PHA production rates under outdoor culture conditions in PHA production using Hfx. mediterranei [experimental conditions: flow rate: 1.0 L x min-1; fermentation volume: 10 L; water temperature: 42 ± 2.5 °C; air temperature: 37 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (2C and 2D, respectively); and the change in PHA% in biomass versus time in sleeve 1 and sleeve 2 (2E and 2F, respectively). [Figure 2F] Figures 2A-2F show the growth and PHA production curves at different aeration rates in PHA production using Hfx. mediterranei [experimental conditions: flow rate: 0.25-2.0 L x min-1; fermentation volume: 800 mL; water temperature: 42 ± 2.5 °C; air temperature: 37 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (2A and 2B, respectively); growth and PHA production rates under outdoor culture conditions in PHA production using Hfx. mediterranei [experimental conditions: flow rate: 1.0 L x min-1; fermentation volume: 10 L; water temperature: 42 ± 2.5 °C; air temperature: 37 ± 2.5 °C; inoculum concentration: 50 g x L-1; initial pH: 7.2] (2C and 2D, respectively); and the change in PHA% in biomass versus time in sleeve 1 and sleeve 2 (2E and 2F, respectively). [Diagram 3] FIG. 3 shows the molecular weight (Mn, Mw, and Mz) and polydispersity index (PDI) distribution of the PHA produced by Hfx. Mediterranei using two standards, polystyrene (PS) and polymethyl methacrylate (PMMA). [Figure 4A] 4A-4C show (4A) Type V ASTM specimen dimensions, (4B) mold design, and (4C) mold. [Figure 4B] 4A-4C show (4A) Type V ASTM specimen dimensions, (4B) mold design, and (4C) mold. [Figure 4C] 4A-4C show (4A) Type V ASTM specimen dimensions, (4B) mold design, and (4C) mold. [Figure 5A] 5A-5C show (5A) Type IV ASTM specimen dimensions, (5B) mold design, and (5C) mold. [Figure 5B] 5A-5C show (5A) Type IV ASTM specimen dimensions, (5B) mold design, and (5C) mold. [Figure 5C] 5A-5C show (5A) Type IV ASTM specimen dimensions, (5B) mold design, and (5C) mold. [Figure 6A] 6A-6B are graphs showing the thermal properties of PLA, PHA, and keratin as pure materials. [Figure 6B] 6A-6B are graphs showing the thermal properties of PLA, PHA, and keratin as pure materials. [Figure 7A] 7A-7B are graphs showing the DSC (7A) and TGA (7B) curves of a blend of PHA and keratin. [Figure 7B] 7A-7B are graphs showing the DSC (7A) and TGA (7B) curves of a blend of PHA and keratin. [Figure 8A] 8A-8B are graphs showing decomposition temperature versus keratin weight fraction (8A) and mass change versus keratin weight fraction (8B) of blends of PHA and keratin. [Figure 8B] 8A-8B are graphs showing decomposition temperature versus keratin weight fraction (8A) and mass change versus keratin weight fraction (8B) of blends of PHA and keratin. [Figure 9A] 9A-9B are graphs showing the DSC (9A) and TGA (9B) curves of blends of PLA, PHA, and keratin. [Figure 9B] 9A-9B are graphs showing the DSC (9A) and TGA (9B) curves of blends of PLA, PHA, and keratin. [Figure 10A]10A-10B are graphs showing decomposition temperature versus keratin weight fraction (10A) and mass change versus keratin weight fraction (10B) for blends of PLA, PHA, and keratin. [Figure 10B] 10A-10B are graphs showing decomposition temperature versus keratin weight fraction (10A) and mass change versus keratin weight fraction (10B) for blends of PLA, PHA, and keratin. [Figure 11A] 11A-11G are graphs showing the stress-strain curves of the PLA specimens, where (11A) is all the PLA specimens tested, (11B) is PLA specimen 003-1, (11C) its Young's modulus, (11D) its Poisson's ratio, (11E) is PLA specimen 003-2, (11F) its Young's modulus, and (11G) its Poisson's ratio. [Figure 11B] 11A-11G are graphs showing the stress-strain curves of the PLA specimens, where (11A) is all the PLA specimens tested, (11B) is PLA specimen 003-1, (11C) its Young's modulus, (11D) its Poisson's ratio, (11E) is PLA specimen 003-2, (11F) its Young's modulus, and (11G) its Poisson's ratio. [Figure 11C] 11A-11G are graphs showing the stress-strain curves of the PLA specimens, where (11A) is all the PLA specimens tested, (11B) is PLA specimen 003-1, (11C) its Young's modulus, (11D) its Poisson's ratio, (11E) is PLA specimen 003-2, (11F) its Young's modulus, and (11G) its Poisson's ratio. [Figure 11D] 11A-11G are graphs showing the stress-strain curves of the PLA specimens, where (11A) is all the PLA specimens tested, (11B) is PLA specimen 003-1, (11C) its Young's modulus, (11D) its Poisson's ratio, (11E) is PLA specimen 003-2, (11F) its Young's modulus, and (11G) its Poisson's ratio. [Figure 11E]11A-11G are graphs showing the stress-strain curves of the PLA specimens, where (11A) is all the PLA specimens tested, (11B) is PLA specimen 003-1, (11C) its Young's modulus, (11D) its Poisson's ratio, (11E) is PLA specimen 003-2, (11F) its Young's modulus, and (11G) its Poisson's ratio. [Figure 11F] 11A-11G are graphs showing the stress-strain curves of the PLA specimens, where (11A) is all the PLA specimens tested, (11B) is PLA specimen 003-1, (11C) its Young's modulus, (11D) its Poisson's ratio, (11E) is PLA specimen 003-2, (11F) its Young's modulus, and (11G) its Poisson's ratio. [Figure 11G] 11A-11G are graphs showing the stress-strain curves of the PLA specimens, where (11A) is all the PLA specimens tested, (11B) is PLA specimen 003-1, (11C) its Young's modulus, (11D) its Poisson's ratio, (11E) is PLA specimen 003-2, (11F) its Young's modulus, and (11G) its Poisson's ratio. [Figure 12A] 12A-12G are graphs showing the stress-strain curves of various PLA / PHA (70:30) specimens tested, where (12A) is the PLA / PHA (70:30) specimen, (12B) is the stress-strain curve of PLA / PHA (70:30) specimen 102-1, (12C) its Young's modulus, (12D) its Poisson's ratio, (12E) is the stress-strain curve of PLA / PHA (70:30) specimen 102-2, (12F) its Young's modulus, and (12G) its Poisson's ratio. [Figure 12B] 12A-12G are graphs showing the stress-strain curves of various PLA / PHA (70:30) specimens tested, where (12A) is the PLA / PHA (70:30) specimen, (12B) is the stress-strain curve of PLA / PHA (70:30) specimen 102-1, (12C) its Young's modulus, (12D) its Poisson's ratio, (12E) is the stress-strain curve of PLA / PHA (70:30) specimen 102-2, (12F) its Young's modulus, and (12G) its Poisson's ratio. [Figure 12C]12A-12G are graphs showing the stress-strain curves of various PLA / PHA (70:30) specimens tested, where (12A) is the PLA / PHA (70:30) specimen, (12B) is the stress-strain curve of PLA / PHA (70:30) specimen 102-1, (12C) its Young's modulus, (12D) its Poisson's ratio, (12E) is the stress-strain curve of PLA / PHA (70:30) specimen 102-2, (12F) its Young's modulus, and (12G) its Poisson's ratio. [Figure 12D] 12A-12G are graphs showing the stress-strain curves of various PLA / PHA (70:30) specimens tested, where (12A) is the PLA / PHA (70:30) specimen, (12B) is the stress-strain curve of PLA / PHA (70:30) specimen 102-1, (12C) its Young's modulus, (12D) its Poisson's ratio, (12E) is the stress-strain curve of PLA / PHA (70:30) specimen 102-2, (12F) its Young's modulus, and (12G) its Poisson's ratio. [Figure 12E] 12A-12G are graphs showing the stress-strain curves of various PLA / PHA (70:30) specimens tested, where (12A) is the PLA / PHA (70:30) specimen, (12B) is the stress-strain curve of PLA / PHA (70:30) specimen 102-1, (12C) its Young's modulus, (12D) its Poisson's ratio, (12E) is the stress-strain curve of PLA / PHA (70:30) specimen 102-2, (12F) its Young's modulus, and (12G) its Poisson's ratio. [Figure 12F] 12A-12G are graphs showing the stress-strain curves of various PLA / PHA (70:30) specimens tested, where (12A) is the PLA / PHA (70:30) specimen, (12B) is the stress-strain curve of PLA / PHA (70:30) specimen 102-1, (12C) its Young's modulus, (12D) its Poisson's ratio, (12E) is the stress-strain curve of PLA / PHA (70:30) specimen 102-2, (12F) its Young's modulus, and (12G) its Poisson's ratio. [Figure 12G]12A-12G are graphs showing the stress-strain curves of various PLA / PHA (70:30) specimens tested, where (12A) is the PLA / PHA (70:30) specimen, (12B) is the stress-strain curve of PLA / PHA (70:30) specimen 102-1, (12C) its Young's modulus, (12D) its Poisson's ratio, (12E) is the stress-strain curve of PLA / PHA (70:30) specimen 102-2, (12F) its Young's modulus, and (12G) its Poisson's ratio. [Figure 13A] 13A-13J are graphs showing the stress-strain curves of various PLA / PHA (70:30)-70 / keratin-30 specimens tested, (13A) is the stress-strain curve of the PLA / PHA (70:30)-70 / keratin-30 specimen, (13B) is the stress-strain curve of PLA / PHA (70:30)-70 / keratin-30 specimen 402-1, (13C) its Young's modulus, (13D) is its Poisson's ratio, (13E) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 402-2, (13F) is its Young's modulus, (13G) is its Poisson's ratio, (13H) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 403-1, (13I) is its Young's modulus, and (13J) is its Poisson's ratio. [Figure 13B] 13A-13J are graphs showing the stress-strain curves of various PLA / PHA (70:30)-70 / keratin-30 specimens tested, (13A) is the stress-strain curve of the PLA / PHA (70:30)-70 / keratin-30 specimen, (13B) is the stress-strain curve of PLA / PHA (70:30)-70 / keratin-30 specimen 402-1, (13C) its Young's modulus, (13D) is its Poisson's ratio, (13E) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 402-2, (13F) is its Young's modulus, (13G) is its Poisson's ratio, (13H) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 403-1, (13I) is its Young's modulus, and (13J) is its Poisson's ratio. [Figure 13C]13A-13J are graphs showing the stress-strain curves of various PLA / PHA (70:30)-70 / keratin-30 specimens tested, (13A) is the stress-strain curve of the PLA / PHA (70:30)-70 / keratin-30 specimen, (13B) is the stress-strain curve of PLA / PHA (70:30)-70 / keratin-30 specimen 402-1, (13C) its Young's modulus, (13D) is its Poisson's ratio, (13E) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 402-2, (13F) is its Young's modulus, (13G) is its Poisson's ratio, (13H) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 403-1, (13I) is its Young's modulus, and (13J) is its Poisson's ratio. [Figure 13D] 13A-13J are graphs showing the stress-strain curves of various PLA / PHA (70:30)-70 / keratin-30 specimens tested, (13A) is the stress-strain curve of the PLA / PHA (70:30)-70 / keratin-30 specimen, (13B) is the stress-strain curve of PLA / PHA (70:30)-70 / keratin-30 specimen 402-1, (13C) its Young's modulus, (13D) is its Poisson's ratio, (13E) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 402-2, (13F) is its Young's modulus, (13G) is its Poisson's ratio, (13H) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 403-1, (13I) is its Young's modulus, and (13J) is its Poisson's ratio. [Figure 13E]13A-13J are graphs showing the stress-strain curves of various PLA / PHA (70:30)-70 / keratin-30 specimens tested, (13A) is the stress-strain curve of the PLA / PHA (70:30)-70 / keratin-30 specimen, (13B) is the stress-strain curve of PLA / PHA (70:30)-70 / keratin-30 specimen 402-1, (13C) its Young's modulus, (13D) is its Poisson's ratio, (13E) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 402-2, (13F) is its Young's modulus, (13G) is its Poisson's ratio, (13H) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 403-1, (13I) is its Young's modulus, and (13J) is its Poisson's ratio. [Figure 13F] 13A-13J are graphs showing the stress-strain curves of various PLA / PHA (70:30)-70 / keratin-30 specimens tested, (13A) is the stress-strain curve of the PLA / PHA (70:30)-70 / keratin-30 specimen, (13B) is the stress-strain curve of PLA / PHA (70:30)-70 / keratin-30 specimen 402-1, (13C) its Young's modulus, (13D) is its Poisson's ratio, (13E) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 402-2, (13F) is its Young's modulus, (13G) is its Poisson's ratio, (13H) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 403-1, (13I) is its Young's modulus, and (13J) is its Poisson's ratio. [Figure 13G]13A-13J are graphs showing the stress-strain curves of various PLA / PHA (70:30)-70 / keratin-30 specimens tested, (13A) is the stress-strain curve of the PLA / PHA (70:30)-70 / keratin-30 specimen, (13B) is the stress-strain curve of PLA / PHA (70:30)-70 / keratin-30 specimen 402-1, (13C) its Young's modulus, (13D) is its Poisson's ratio, (13E) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 402-2, (13F) is its Young's modulus, (13G) is its Poisson's ratio, (13H) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 403-1, (13I) is its Young's modulus, and (13J) is its Poisson's ratio. [Figure 13H] 13A-13J are graphs showing the stress-strain curves of various PLA / PHA (70:30)-70 / keratin-30 specimens tested, (13A) is the stress-strain curve of the PLA / PHA (70:30)-70 / keratin-30 specimen, (13B) is the stress-strain curve of PLA / PHA (70:30)-70 / keratin-30 specimen 402-1, (13C) its Young's modulus, (13D) is its Poisson's ratio, (13E) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 402-2, (13F) is its Young's modulus, (13G) is its Poisson's ratio, (13H) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 403-1, (13I) is its Young's modulus, and (13J) is its Poisson's ratio. [Figure 13I]13A-13J are graphs showing the stress-strain curves of various PLA / PHA (70:30)-70 / keratin-30 specimens tested, (13A) is the stress-strain curve of the PLA / PHA (70:30)-70 / keratin-30 specimen, (13B) is the stress-strain curve of PLA / PHA (70:30)-70 / keratin-30 specimen 402-1, (13C) its Young's modulus, (13D) is its Poisson's ratio, (13E) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 402-2, (13F) is its Young's modulus, (13G) is its Poisson's ratio, (13H) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 403-1, (13I) is its Young's modulus, and (13J) is its Poisson's ratio. [Figure 13J] 13A-13J are graphs showing the stress-strain curves of various PLA / PHA (70:30)-70 / keratin-30 specimens tested, (13A) is the stress-strain curve of the PLA / PHA (70:30)-70 / keratin-30 specimen, (13B) is the stress-strain curve of PLA / PHA (70:30)-70 / keratin-30 specimen 402-1, (13C) its Young's modulus, (13D) is its Poisson's ratio, (13E) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 402-2, (13F) is its Young's modulus, (13G) is its Poisson's ratio, (13H) is the stress-strain curve of the PLA / PHA(70:30)-70 / keratin-30 test piece 403-1, (13I) is its Young's modulus, and (13J) is its Poisson's ratio. [Figure 14] Figure 14 is a graph summarizing the stress-strain curves of all specimens: PLA: 003-1 and 003-2, PLA / PHA: 102-1 and 102-2, PLA / PHA / keratin: 402-1, 402-2, and 403-1. [Figure 15] FIG. 15 shows optical micrographs showing the degradation of different composite materials under composting conditions. [Figure 16]FIG. 16 is a graph showing the degree of disintegration as a function of time for control PLA dog-bones, PLA / PHA dog-bones, and PLA / PHA / keratin dog-bones under composting conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In one embodiment, the present invention relates to a method for producing a PHA by fermentation of an archaea capable of producing said PHA, the method comprising the steps of: (i) providing a fermentor having a culture volume of at least about 5 or 10 liters, said fermentor comprising a culture medium occupying said culture volume, said culture medium comprising salt water, such as seawater, supplemented with a carbon source and a nitrogen source, suitable for culturing said archaea; (ii) inoculating the culture medium with a seed culture of the archaea; (iii) culturing the archaea while constantly bubbling an oxygen-containing gas mixture, such as air, inside the fermenter from the bottom thereof at an aeration rate of at least about 0.2 vvm so as to constantly both aerate and mix the culture medium, until a predetermined concentration of the archaea in the culture medium is obtained; (iv) harvesting the archaeal biomass from the culture medium; and (v) concentrating, isolating and / or extracting the PHA from the harvested biomass; Includes.

[0014] As used herein, the term "polyhydroxyalkanoates" (PHAs) refers to polyesters produced naturally by microorganisms, including by bacterial / archaeal fermentation of sugars or lipids. Within this family, over 150 different monomers can be combined to yield materials with different properties. PHAs are biodegradable thermoplastic or elastomeric materials with melting points in the range of 40°C to 180°C. Specific PHAs obtained from microbial sources, genetically modified bacteria, and other organisms each have the formula [-OC(R)H-(CH2) n-C(O)-, where R is H or alkyl, i.e., linear or branched hydrocarbyl having, for example, 1 to 12 carbon atoms, and is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, Examples of PHAs include, but are not limited to, poly(3-hydroxypropionate), poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), poly(3-hydroxyhexanoate), poly(3-hydroxyoctanoate), and poly(3-hydroxydodecanoate), where n is 1 and R is H, methyl, ethyl, propyl, pentyl, or nonyl, respectively, as well as poly(4-hydroxybutyrate) and poly(5-hydroxyvalerate), where n is 2 or 3 and R is H, respectively.

[0015] As used herein, the term "archaebacteria" refers to any group of unicellular prokaryotes that have distinct molecular characteristics that distinguish them from bacteria and eukaryotes. Non-limiting examples of archaea include species of Picrophilus, methanogens, halophiles, and Pyrolobus fumarii. Certain archaea referred to herein are halophilic archaea. In certain embodiments, the archaea is Hfx. mediterranei, e.g., Hfx. mediterranei ATCC 33500.

[0016] As used herein, the term "salt water" refers to water having a salinity of at least about 2% and up to about 30, 35, or 40%, consisting primarily of sodium and chloride ions. In certain embodiments, the salt water is seawater, i.e., water from a sea or ocean, and has an average salinity of about 3.5%. In other embodiments, the salt water has a salinity in the range of about 4% or 5% to about 10%, or a salinity of more than 10%, for example, up to about 15%, 20%, 25%, or 30%.

[0017] As used herein, the term "oxygen-containing gas mixture" refers to a gas mixture comprising at least about 15% oxygen on a volumetric basis (vol / vol), e.g., about 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%, but preferably about 19%, 20%, 21%, 22%, or 23%, and an additional gas that may be selected from nitrogen, carbon dioxide, and inert gases such as helium, neon, argon, krypton, and xenon. Particular such gas mixtures are air-like gas mixtures that include oxygen, nitrogen, carbon dioxide, and argon that together constitute at least 98% by volume of the gas mixture. In a more specific embodiment, the oxygen-containing gas mixture bubbled into the fermenter during the disclosed fermentation process is air, i.e., a gas mixture containing about 78% nitrogen, about 21% oxygen, about 0.9% argon, about 0.1% carbon dioxide, and small amounts of other gases.

[0018] In certain embodiments, the culture medium used in the fermentation process disclosed herein comprises carbon and nitrogen, each independently, in an amount of about 1% to about 80% by weight, based on dry weight, i.e., dehydrated weight. The term "dry weight basis" refers to the dry weight of the culture medium, i.e., the weight of all the solid components that make up the culture medium. In certain embodiments, the amount of carbon in the culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% by weight, based on dry (dehydrated) weight, and / or the amount of nitrogen in the culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by weight, based on dry (dehydrated) weight.

[0019] In certain embodiments, the carbon dioxide contained in the culture medium used in the disclosed fermentation process The sources can be sugars, more specifically monosaccharides such as, but not limited to, glucose, galactose, fructose, and xylose, or polysaccharides such as starch; glycerol (the major by-product in the biodiesel production process); fatty acids such as acetic acid, butyric acid, and propanoic acid (produced during the anaerobic decomposition of organic compounds during acidogenic fermentation and used as feedstock for freshwater and marine oil-producing microorganisms, e.g., for biodiesel production); vinase (a by-product of the sugar or ethanol industry); stillage; ) (wastewater remaining after ethanol distillation, containing reducing sugars (fructose and glucose) and fatty acids including acetic acid, propionic acid, and butyric acid), molasses wastewater, and olive mill wastewater (side stream from olive oil production); polyols, i.e., organic compounds containing multiple hydroxyl groups, hydrolysates, such as, but not limited to, seaweed hydrolysates, olive leaf hydrolysates, agricultural waste hydrolysates, and cheese whey hydrolysates; or mixtures thereof; and / or the nitrogen source contained in the culture medium comprises organic or inorganic nitrogen, such as proteins, glycosylated proteins, biopolymers including DNA fragments, RNA fragments, and nitrogen-containing oligosaccharides, peptides, amino acids, and nitrogen salts, hydrolysates, such as seaweed hydrolysates, olive leaf hydrolysates, paper waste, agricultural waste hydrolysates, and cheese whey hydrolysates, or mixtures thereof. In certain embodiments, both the carbon source and the nitrogen source consist exclusively of hydrolysates, such as, for example, seaweed hydrolysates, which provide all the carbon and nitrogen required for the archaeal culture process.

[0020] As used herein, the term "biopolymer" refers to a natural polymer (consisting of covalently linked monomeric units) produced by cells of an organism. Primary examples of such polymers, classified according to the monomers used and the structure of the biopolymer formed, are polynucleotides, polypeptides and proteins, and polysaccharides. Polynucleotides, such as RNA and DNA, are polymers composed of 13 or more nucleotide monomers. Polypeptides and proteins are polymers of amino acids and include collagen, actin, and fibrin. Polysaccharides are linear or branched polymeric carbohydrates and include starch, cellulose, and alginates. Further examples of biopolymers include natural rubber (polymers of isoprene), suberin and lignin (complex polyphenolic polymers), cutin and cutan (complex polymers of long chain fatty acids), and melanin. The polymers described herein above may be modified by glycosylation, i.e., by the attachment of sugar moieties.

[0021] As used herein, the term "amino acid" refers to an organic compound that contains both amine and carboxylic acid functional groups, may be either a natural or unnatural amino acid, and exists in both L- and D-isomers. The 22 amino acids that naturally occur in proteins are aspartic acid (Asp), tyrosine (Tyr), leucine (Leu), tryptophan (Trp), arginine (Arg), valine (Val), glutamic acid (Glu), methionine (Met), phenylalanine (Phe), serine (Ser), alanine (Ala), glutamine (Gln), glycine (Gly), proline (Pro), threonine (Thr), asparagine (Asn), lysine (Lys), histidine (His), isoleucine (Ile), cysteine ​​(Cys), selenocysteine ​​(Sec), and pyrrolysine (Pyl). Non-limiting examples of other amino acids include citrulline (Cit), diaminopropionic acid (Dap), diaminobutyric acid (Dab), ornithine (Orn), aminoadipic acid, β-alanine, 1-naphthylalanine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, γ-aminobutyric acid (GABA), 3-(aminomethyl)benzoic acid, p-ethynyl-phenylalanine, m-ethynyl-phenylalanine, p-chlorophenylalanine (4ClPhe), p-bromophenylalanine, p-iodophenylalanine, p-acetylphenylalanine, p-azidophenylalanine, p-propargly-oxy-phenylalanine, indanylglycine (Igl), (benzyl)cysteine, Indolyl, norleucine (Nle), azidonorleucine, 6-ethynyl-tryptophan, 5-ethynyl-tryptophan, 3-(6-chloroindolyl)alanine, 3-(6-bromoindolyl)alanine, 3-(5-bromoindolyl)alanine, azidohomoalanine, α-aminocaprylic acid, O-methyl-L-tyrosine, N-acetylgalactosamine-α-threonine, and N-acetylgalactosamine-α-serine.

[0022] As used herein, the term "amino acid residue" means the residue of an amino acid following removal of a hydrogen atom from the amino group of the amino acid, e.g., its α-amino group or a side chain amino group, if present, and removal of an -OH group from the carboxyl group of the amino acid, e.g., its α-carboxyl group or a side chain carboxyl group, if present.

[0023] The term "peptide" refers to a short chain of amino acid monomers (residues), e.g., a chain consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid residues, linked by peptide (amide) bonds, i.e., by covalent bonds formed when the carboxyl group of one amino acid reacts with the amino group of another. As used herein, the term "peptide moiety" refers to that portion of a peptide as defined herein after removal of a hydrogen atom from its carboxyl group, i.e., from either its terminal or side chain carboxyl group, and / or from its amino group, i.e., from its terminal or side chain amino group.

[0024] As used herein, the term "peptide bond" or "amide bond" refers to the covalent bond, -C(O)NH-, that forms between two molecules, e.g., two amino acids, when the carboxyl group of one of the molecules reacts with the amino group of the other molecule, resulting in the release of a water molecule.

[0025] As used herein, the term "hydrolysate" refers to the aqueous product of hydrolysis, which contains proteins that have been chemically or enzymatically degraded into peptides of various sizes. Examples of hydrolysates that can be used as carbon and / or nitrogen sources in culture medium include seaweed hydrolysates, olive leaf hydrolysates, agricultural waste hydrolysates, and cheese whey hydrolysates. In a particular embodiment, the hydrolysate referred to is seaweed hydrolysates, which can be used as either or both carbon and nitrogen sources, but preferably both.

[0026] As used herein, the term "seaweed" refers to any species of large, multicellular marine algae, including the Rhodophyta (red algae), Phaeophyta (brown algae), and Chlorophyta (green algae) classes of macroalgae. In certain embodiments, the seaweed referred to is a green macroalgae, such as an Ulva species.

[0027] In certain embodiments, the carbon source included in the culture medium used in the disclosed fermentation processes comprises a seaweed hydrolysate, said hydrolysate constituting at least about 1% by weight of said carbon source, preferably about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or more by weight, and / or the nitrogen source included in the culture medium comprises a seaweed hydrolysate, said hydrolysate constituting at least about 1% by weight of said nitrogen source, preferably about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or more by weight. In certain embodiments, as exemplified herein, both the carbon and nitrogen sources consist solely of seaweed hydrolysate, which provides all the carbon and nitrogen required for the archaeal fermentation process.

[0028] In certain embodiments, the culture medium used in the fermentation process disclosed herein further comprises a salt, such as a salt containing a bromide anion, a buffer, phosphorus, or a mixture thereof. Phosphorus sources that may be used in the preparation of the culture medium include, but are not limited to, dissolved inorganic phosphates, such as potassium dihydrogen phosphate and disodium hydrogen phosphate, as well as phosphorus-containing biomolecules, and halogen sources that may be used include, but are not limited to, bromine.

[0029] In certain embodiments, the culture medium used in the fermentation process disclosed herein according to any one of the preceding embodiments comprises the carbon source, the nitrogen source, and salt water, e.g., seawater, optionally supplemented with the phosphorus, halogen, or mixture thereof, wherein the amount of carbon and nitrogen in the culture medium is each independently from about 1% to about 80% by weight on a dry weight basis, the carbon source comprises seaweed hydrolysate constituting at least about 1% by weight of the carbon source, and the nitrogen source comprises seaweed hydrolysate constituting at least about 1% by weight of the nitrogen source.

[0030] In certain such embodiments, the amount of carbon in the culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% by weight on a dry weight basis; the amount of nitrogen in the culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by weight on a dry weight basis; and the carbon source is In some embodiments, the nitrogen source comprises seaweed hydrolysate constituting at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or more by weight of the source, and the nitrogen source comprises seaweed hydrolysate constituting at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or more by weight of the source. In more particular such embodiments, both the carbon source and the nitrogen source consist solely of seaweed hydrolysate, which provides all the carbon and nitrogen required for the archaeal fermentation process.

[0031] In certain embodiments, the seaweed hydrolysate included in the culture medium used in the disclosed fermentation process according to any one of the previous embodiments is a green macroalgae hydrolysate, such as an Ulva sp. hydrolysate.

[0032] In certain embodiments, the pH of the culture medium prior to seeding step (ii) according to any one of the previous embodiments is up to 8.5. In certain embodiments, the pH of said culture medium prior to seeding step (ii) is from about 2 to about 8.5, preferably from about 6.8 to about 7.4, e.g. 7.2.

[0033] In certain embodiments, the culturing step (iii) according to any one of the preceding embodiments is carried out for a period of up to 14 or 21 days, such as at least 48 hours, 54 hours, 60 hours, 66 hours, or 72 hours.

[0034] According to the invention, during the cultivation step (iii), an oxygen-containing gas mixture, such as air, is constantly bubbled into the fermenter from the bottom thereof at an aeration rate of at least about 0.2 vvm, so that the culture medium is both constantly aerated and constantly mixed, and, as a result, the carbon dioxide produced by the archaea is also constantly removed.

[0035] The term "aeration rate" is measured in volumes per volume per minute (vvm) and refers to the rate at which an oxygen-containing gas mixture Volumetric flow rate / fermenter working volume, i.e., the volume of fluid bubbled into the fermenter per minute The aeration rate refers to the ratio between the volume of the oxygen-containing gas mixture being fed to the fermentor and the working volume of the fermentor. In certain embodiments, the aeration rate according to any one of the above embodiments is about 0.6, 0.7, or 0.8 to about 1.2 vvm, preferably about 0.9 to about 1.1 vvm, and more preferably about 1 vvm.

[0036] As mentioned above, the oxygen-containing gas mixture that is constantly bubbled into the fermenter during the culture step (iii) both aerates and mixes the culture medium, and as a consequence removes carbon dioxide from said medium. The aeration rate of said oxygen-containing gas mixture can therefore have a significant effect on the effectiveness of the culture step, e.g. the maximum archaeal concentration that can be obtained in the culture medium during the fermentation process or the time required to achieve said concentration.

[0037] As shown herein, the aeration rate of the oxygen-containing gas mixture also has a non-linear effect on the molecular weight of the resulting PHA, which varies depending on the aeration rate and the cultivation period of the archaea, with higher molecular weight PHAs clearly being observed at higher aeration rates.

[0038] According to the present invention, after completion of the culturing step (iii), the produced archaeal biomass is harvested from the culture medium, and then the PHA is concentrated, separated and / or extracted from the harvested biomass. In certain embodiments, the extraction of PHA from the harvested biomass according to any one of the above embodiments is performed by physical means such as water (hydrolysis), high pressure, pulsed electric fields (PEF), and centrifugation and filtration; chemical means such as cell lysis buffers, deep eutectic solvents, organic solvents, biphasic solvent systems, and ionic liquids; or any combination thereof.

[0039] As used herein, the term "deep eutectic solvent" refers to a solution of a Lewis or Bronsted acid and a base that forms a eutectic mixture, i.e., a homogeneous mixture of substances that melts or solidifies at a single temperature below the melting point of either component. Deep eutectic solvents are highly tunable by varying the structure or relative ratios of the parent components and therefore have a wide variety of potential applications, including separations.

[0040] As used herein with respect to extracting PHA from harvested biomass at various temperatures, the term "organic solvent" refers to a non-toxic, environmentally friendly, water-soluble solvent that may contain carbon, hydrogen, oxygen, sulfur, and nitrogen atoms in its molecular structure. Examples of such organic solvents include, but are not limited to, carbonate esters, including dimethyl carbonate and diethyl carbonate; lactate esters, including ethyl lactate; levulinic acid esters, including methyl levulinate and ethyl levulinate; gamma-valerolactone; (1R)-7,8-dioxabicyclo[3.2.1]octan-2-one (cyrene); alcohols, diols, and polyols, including ethanol, isopropanol, and ethylene glycol; and ketones, including methyl ethyl ketone.

[0041] The term "two-phase solvent system," as used herein with respect to extracting PHAs from harvested biomass at various temperatures, refers to a solvent system comprising two solvents separated by an interfacial layer, one of the two solvents being an aqueous solvent and the other of the two solvents being a non-toxic, environmentally friendly, partially (or poorly) water-soluble solvent that may contain carbon, hydrogen, oxygen, sulfur, and nitrogen atoms in its molecular structure, such as, but not limited to, acetates, including ethyl acetate and butyl acetate, and ethers, including 2-methyltetrahydrofuran.

[0042] As used herein with respect to the extraction of PHAs from harvested biomass at various temperatures, the term "ionic liquid" refers to an ionic liquid that does not contain carbon, hydrogen, oxygen atoms in its molecular structure. By ionic liquids we mean non-toxic, environmentally friendly organic salts that may contain cations such as cations of dialkyl-imidazolium, N-alkyl-pyridazinium, quaternary ammonium, dialkyl-pyrrolidinium, alkyl-guanidinium, and alkyl-pyridinium, and various environmentally friendly anions such as acetate, formate, phosphate, hydrogen phosphate, and chloride.

[0043] The method disclosed herein according to any one of the above embodiments may further comprise purifying the PHA obtained in step (v), e.g., by repeated washing with water, a solvent, an ionic liquid, or a combination thereof, to obtain a purified PHA, and, if desired, drying the purified PHA thus obtained, e.g., by drum dryer, spray dryer, air or air / nitrogen stream drying, or freeze dryer.

[0044] The purified PHA, with or without drying, may then be blended or mixed with at least one polymer. Examples of polymers suitable for blending with PHA include biodegradable polymers such as polylactic acid (PLA), polycaprolactone (PCL), keratin, cellulose, chitin, lignin, amylose, amylopectin, and mucin, as well as non-biodegradable polymers or copolymers such as polyethylene terephthalate (PET, also known as polyester), high density polyethylene (HDPE), polyvinyl chloride (PVC), low density polyethylene (LDPE), polypropylene (PP), polystyrene (PS), polyethylene glycol (PEG), polypropylene glycol (PG), polyvinyl alcohol (PVA), polyacrylonitrile (PAN), polymethacrylic acid or its esters, poly(acrylonitrile-co-butadiene-co-styrene) (ABS), polyamides, polyacrylamide (PAM), polysiloxanes, graft polymers, and dendrimers such as polyamidoamines (PAMAM, composed of repeating branched subunits with amide and amine functionality).

[0045] The preparation of the blends disclosed herein can be carried out in a variety of ways, for example as described in Garcia-Garcia et al., 2022; Charon et al., 2022; and Longe et al., 2022, PHA or prepared The preparation of the PHA blend may include the addition of various plasticizers and / or additives to adjust the properties of the resulting PHA blend. In certain embodiments, one or more plasticizers and / or additives are added to the PHA prior to mixing with other polymers to adjust the properties of the PHA prior to blending with other polymers. In other embodiments, one or more plasticizers and / or additives are added to the PHA blend after its preparation to adjust the properties of the blend.

[0046] In one particular embodiment, the fermentor used in the method of the invention according to any one of the previous embodiments is an open fermentor, such that the cultivation of said archaea is carried out under non-sterile conditions.

[0047] In certain embodiments, the fermentor used in the method of the invention according to any one of the above embodiments is made from glass, metal or metal alloy, ceramic, plastic, or cement, hi other embodiments, the fermentor is an earthen fermentor, also called an earthen-bank pond or earthen-bank construction.

[0048] In certain embodiments, the fermentation process of the invention is carried out in batches according to steps (i) to (v) according to any one of the previous embodiments. In other embodiments, the fermentation process is carried out continuously, in which archaeal biomass is continuously or at intervals harvested from the culture medium (and then subjected to PHA concentration, separation and / or extraction) and fresh culture medium (in an amount equal to the amount removed for harvesting) is introduced into the fermenter.

[0049] Many studies have been done to explore the combination of PLA with either PHA or keratin. This study was done to explore the combination of all three materials together with the goal of creating bioplastics from these biodegradable materials. As described in the experimental section herein, blends of PLA, PHA, and keratin were prepared by using powders of the three materials and combining them to form a homogenous powder. The blend powder was then melted in an oven. The morphology, as well as the thermal and mechanical properties of the blends were investigated by SEM and digital microscopy, DSC, TGA, FTIR, and tensile experiments. Thermal analysis showed that the addition of keratin affected the thermal stability of the blends. There was a significant increase in the decomposition temperature and a decrease in the weight loss of the specimens as the weight fraction of keratin increased. The difference between the amorphous and more crystalline materials can be seen in the FTIR. PLA is primarily amorphous, whereas PHA and keratin are more crystalline. The crystalline structures are generally very ordered, which imparts strength and rigidity to the structures. In such structures, the molecular chains are fixed in place, so that when a load is applied, they break rather than bend. Amorphous polymers are the opposite. Instead of being rigid, the random intermixing of the molecules allows the chains to move across each other when the polymer is pulled. This gives amorphous polymers flexibility and elasticity. These properties are also reflected in the tensile test results, where the PHA and PLA with keratin specimens broke in the elastic region without any deformation. In addition, morphological tests showed a typical brittle fracture, with no signs of plasticity. It is noteworthy that in contrast to other studies that found that the addition of PHA to PLA increased the ductility and decreased the brittleness of the specimens, in these experiments the addition of PHA to PLA caused the opposite. This can most likely be explained by the type of PHA used, where different PHA monomers can result in materials with different properties. Another explanation for this result could be the presence of pores, which were probably formed during the manufacturing process. Good adhesion between both phases of the blend is very important.Pores cause a decrease in the adhesion between the polymer phases and therefore change the properties of the material. To avoid the creation of pores in the specimens, the powder of the material is stored in a desiccator until use in order to reduce moisture absorption. In addition to the characterization of the blends, biodegradation tests were also performed. The synthesized biopolymers were completely biodegradable under aerobic conditions in a composting environment. The PLA / PHA / keratin blend was degraded the fastest. This indicates that keratin not only aids the thermal stability of the material but also makes it more biodegradable. According to these tests, these materials can be characterized as environmentally friendly. Further repeated tensile tests should be performed to corroborate these findings.

[0050] In another aspect, the invention therefore provides a blend comprising a PHA and at least two additional polymers.

[0051] According to the present invention, the polymers blended with the PHAs include biodegradable polymers such as polylactic acid (PLA), polycaprolactone (PCL), poly(butylene succinate), poly(butylene succinate adipate), aliphatic-aromatic copolyesters, poly(butylene adipate / terephthalate) and poly(methylene adipate / terephthalate), keratin, cellulose, chitin, lignin, amylose, amylopectin, mucin, and PHAs different from the above PHAs, as well as polyethylene terephthalate (PET, also called polyester), high density polyethylene (HPE), and the like. The polymer may be selected from non-biodegradable polymers such as polyvinyl chloride (PVC), low density polyethylene (LDPE), polypropylene (PP), polystyrene (PS), polyethylene glycol (PEG), polypropylene glycol (PG), polyvinyl alcohol (PVA), polyacrylonitrile (PAN), polymethacrylic acid or its esters or amides, poly(acrylonitrile-co-butadiene-co-styrene) (ABS), polyamides, polyacrylamide (PAM), polysiloxanes, graft polymers, and dendrimers such as PAMAM.

[0052] In certain embodiments, the blends disclosed herein comprise the PHA and at least two additional polymers, the at least two additional polymers comprising or consisting of PLA and keratin. In certain such blends, the ratio of PHA:PLA ranges from about 1:99 to about 99:1, about 20:80 to about 80:20, about 30:70 to about 70:30, or about 40:60 to about 60:40, respectively, by weight, but preferably is about 20:80 to about 40:60, e.g., about 25:75, about 30:70, or about 35:65, respectively, by weight (regardless of the total amount of other polymers, e.g., keratin, contained within the blend).

[0053] In other embodiments, one of the at least two additional polymers blended with the PHA is keratin, which constitutes from about 1% to about 99% of the blend by weight, e.g., from about 5% to about 90%, from about 10% to about 80%, from about 15% to about 70%, or from about 20% to about 60%, but preferably from about 10% to about 50%.

[0054] Variations in the blend components, the sources of these components, and the physical and chemical properties (including but not limited to molecular weight, crystallinity, chain conformation, hydrophobicity and hydrophilicity, hydrolytic stability, thermal properties, transport properties, optical properties, toughness, elasticity, viscoelasticity, thermal, electromagnetic radiation conductivity, and electrical conductivity) can affect the aforementioned physical and chemical properties of the resulting blend, the homogeneity of the blend, and the adhesive properties of the components. For example, the incorporation of PHA into the blend results in a decrease in the flexural modulus and a concomitant increase in the tensile modulus. The presence of keratin can affect the thermal stability and toughness of the blend. As the weight fraction of keratin increases, the decomposition temperature increases significantly and the weight loss of the test specimens decreases.

[0055] In a further aspect, the present invention provides an article comprising or made from the blend disclosed in any one of the preceding embodiments. Such an article may be made by any suitable technique known in the art, for example as shown in the experimental section herein.

[0056] Unless otherwise indicated, all numerical values ​​used herein, e.g., referring to aeration rates, amounts of carbon and / or nitrogen in the culture medium, pH of said culture medium, and ratios between polymers in a blend, are to be understood as being modified in all instances by the term "about." Thus, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary by plus or minus 10% depending upon the desired properties sought to be obtained by the present invention.

[0057] The invention will now be illustrated by the following non-limiting examples. EXAMPLES

[0058] Materials and Methods Macroalgae biomass production. Ulva sp. was cultivated in a [custom-made] macroalgae photobioreactor (MPBR) (Polytiv, Israel, 100 cm long, 200 mm thick, 40 cm wide) under controlled conditions and natural light from September 15 to November 3, 2016. The conditions used for the growth of this seaweed were those found to be optimal so far. Elemental analysis of the Ulva sp. biomass was performed using a CHNS analyzer (Flash The biomass was analyzed using a 500 sieve centrifuge (Thermo Scientific, USA) at 550°C for 20 min. The ash and moisture content were determined by burning the biomass in a muffle furnace (MGFurnaces, Faridabad, India) at 550°C and the starch content was analyzed using a total starch assay.

[0059] Subcritical hydrolysis of macroalgal biomass. Hydrolysis of seaweed biomass was carried out in a batch reactor (0.25 L working volume) (CJF-0.25, Keda Machinery, China) equipped with an electric heating system. The temperature and pressure inside the reactor were monitored using a digital thermometer (MRC Ltd., Israel) and a pressure gauge (MRC Ltd., Israel), respectively. A chiller (Guangzhou Teyu Electromechanical Co., Ltd., Israel) was used to mix the hot slurry inside the reactor. An agitator cooled with cold water (MRC Ltd., China) was installed. A vacuum pump (MRC Ltd., Israel) was used to remove residual air from the reactor. The hydrolysis conditions were as follows: temperature 170°C, total residence time 20 min, salt concentration 38 g × L -1 , and solid loading 5%. These conditions were selected for optimizing sugar yield. The hydrolysate was separated into liquid and solid phases by centrifugation (Yingtai Instruments TGL-18, China) at 10,000 rpm for 3 min.

[0060] PHA production by Hfx. mediterranei. PHA production was investigated using hydrolysates of Ulva sp. supplemented with Hfx. mediterranei ATCC 33500 (NCIMB 2177). Hfx. mediterranei strains were grown in a nutrient rich medium (Hv-YPC) using routine procedures. Cultures were grown at 42 °C in a shaking incubator (MRC Labs, Israel) with a rotation speed of 180 rpm and the pH of the medium was adjusted to 7.2. In the experiment, the seaweed hydrolysate (aqueous phase) was mixed with brine (144 g NaCl × L) at a working concentration of 25% v / v as seen in a previous study (Ghosh et al., 2021). -1 The experiments were carried out in a custom-made reactor constructed from 1 L PET (polyethylene teraphthalate) bottles (length 0.285 m, width 0.08 m). A set of 15 PET bottles was used to test the aeration rate at different rates (0.25–2.0 L × min -1Cultivation of Hfx. mediterranei in a 1000L MPBR was carried out in triplicate. Flow meters were connected to each reactor to manipulate the airflow inside the reactor. Air was sparged from the bottom of the reactor to ensure uniform flow inside the reactor. An outlet for gas was provided at the top of the reactor. The cultivation was further scaled up to 10 L using a cylindrical sleeve MPBR (Polytiv, Israel, 1.55 m long, 0.02 m thick, 0.4 m wide) which was used for the cultivation of Ulva sp. The reactor configuration used for the cultivation of Hfx. mediterranei and its schematic diagram are shown in Figure 1.

[0061] For ultrasonic dissociation studies, cells were grown in 600 mL of nutrient-rich Hv-YPC medium (pH 7.2) in 1 L baffled Erlenmeyer flasks. Flasks were maintained in a shaking incubator at 170 rpm at 42°C for approximately 48 hours until late exponential / early stationary phase and removed from the shaker. The optical density of the culture was 7.9 at 520 nm as measured with a benchtop spectrophotometer (ThermoFisher AquaMATE VIS, 1 cm path length), which corresponds to an ash-free dry weight concentration of 3. This is roughly equivalent to 9 g × L-1. Hfx. mediterranei cultures were transferred to sealed containers and kept on the benchtop for 2 weeks as a test for autoaggregation, which may improve the particle size and ultrasonic response of the cultures. Since no autoaggregation was observed in the static cultures, Ca 2+We attempted to promote aggregation of Hfx. mediterranei cells by adding 10 mM CaCl2. The cells were pelleted by centrifugation at 1,000 × g for 15 min and subsequently washed twice with Tris buffer (20 mM Tris-HCl and 4 M NaCl, pH 7.0). The cells were then suspended at a higher concentration in salt solution (27 mM KCl, 0.24 mM NaHCO3, 0.49 mM NaBr, 3.7 mM NH4Cl, 4.3 M NaCl, 18.5 μM FeCl3·6H2O, pH 7.0). The second cell suspension was washed and rinsed following the same procedure, but the final salt solution was modified to contain 10 mM CaCl2. The optical density of the suspension after washing was 11.8 at 520 nm, which corresponds to an ash-free dry weight concentration of 5.8 g × L-1.

[0062] Identification of intracellular PHA content. The cellular PHA content was measured using the Nile Red staining method. Briefly, cells were washed and suspended in 10% saline. The cell suspension was then diluted with 10% saline at a final concentration of 3.1 μg × ml -1 The cells were stained with 10% Nile Red (Sigma Aldrich, USA). The cells were incubated for 30 min, washed and resuspended with 10% saline. The fluorescence of the suspension was measured at excitation and emission wavelengths of 535 and 605 nm, respectively, using a 96-well plate reader (Tecan, Switzerland). Standards of various concentrations were prepared using commercially available PHA (Sigma, USA) (0.2–2.0 g × L). -1). These standards were used to construct a standard curve. The standard curve was plotted to determine the final concentration of PHA in the cells. Unknown amounts of PHA were determined from the standard curve with two replicates per point. This protocol was also validated using a crotonic acid assay to determine the intracellular PHA content. Two milliliters of sulfuric acid were added to the dried cell pellet containing the polymer (PHA). The mixture was hydrolyzed by heating in a water bath at 100 °C for 20 min to obtain crotonic acid. The amount of accumulated polymer was quantified by recording the UV absorbance at 235 nm on a multiplate reader (Tecan, Switzerland) using concentrated sulfuric acid as a blank.

[0063] Calculation of biomass and PHA production rates. Volumetric biomass production rate (X Biomass , g×L -1 ×time -1 ) and volumetric PHA production rate (X PHA , g×L -1 ×time -1 ) using the following formula:

number

[0064] Extraction of PHA from cells. After fermentation, the broth was centrifuged at 10,000 rpm for 10 min (Yingtai Instruments TGL-18, China). The cell pellet thus formed was dried in a convection oven (MRC Laboratories, Israel) at a temperature of 60° C. for 12 h. To extract PHA from cells, the cell pellet was treated with 0.1% SDS (sodium dodecyl sulfate) in distilled water and incubated at 32° C. for 24 h. This induced cell lysis and released intracellular PHA into the water. The suspension was further centrifuged at 9000 rpm for 15 min to obtain PHA as a pellet. The pellet was repeatedly washed with distilled water until it was white, and then dried for further analysis. The PHA obtained from each fermentation was analyzed separately.

[0065] Characterization of PHAs FTIR-ATR analysis. The PHA powder obtained after drying was analyzed by a Fourier transform infrared spectroscopy (FTIR) spectrometer (Bruker Platinum ATR, USA) equipped with an attenuated total reflectance (ATR) attachment. The spectra were recorded from 400 to 4000 cm -1 was recorded in the range of

[0066] Thermogravimetric analysis / differential scanning calorimetry (TGA / DSC) analysis. 5 mg of dried PHA powder was weighed into a sealed aluminum pan. The pan was then subjected to a linear temperature gradient (from 30°C to 600°C) in a differential scanning calorimeter (Jupiter STA 449 F5, NETZSCH, Germany) equipped with an autoloader. The heating rate was 10°C x min. -1 was maintained.

[0067] 1 H NMR analysis. The produced PHA was 1 The powdered PHA was dissolved in CDCl3 (10 mg × ml -1 ), followed by analysis with a 400 MHz spectrometer (Bruker, USA).

[0068] GC-MS analysis. Butyl esters of PHA were analyzed using a GC-MS system equipped with an autosampler (6890 / 5977A, G4513A; Agilent, USA) and an HP-5MS UI column (Agilent, USA). The column consisted of a stationary phase of 5% phenyl / methylpolysiloxane, had a length of 30 m, and an inner diameter of 0.25 mm. Helium (99.999%) was used as the carrier gas, and the flow rate was 1.0 mL × min. -1 The sample was injected into an injector heated to a temperature of 280°C with a split ratio of 1:19. The sample injection volume was 0.2 μL. The conditions for separation of the analytes were as follows: initial oven temperature 70°C held for 5 min, then heated to 280°C for 15°C x min. -1 The temperature was then increased linearly at a rate of 30°C x min. -1 The temperature was linearly increased to 320 °C at a rate of 100 Hz and held at this final temperature for 5 min. Mass spectral analysis was performed in EI positive ion mode with an electron energy of 70 eV. The transfer line and ion source temperatures were maintained at 280 and 250 °C, respectively. The mass spectral data obtained were collected in full scan mode (m / z 50-400) and analyzed using Agilent ChemStation software.

[0069] Molecular weight analysis. Gel permeation chromatography (GPC) analysis was performed to determine the weight-average molecular weight (M) of the PHA biopolymer samples using a high-performance liquid chromatography (HPLC) system equipped with a refractive index (RI) detector (Agilent 1260, Agilent, USA) and two Phenomenex columns (Phenomenex Inc., USA) operated at a column temperature of 40 °C. w ), number average molecular weight (M n ), z-average molecular weight (M z The mobile phase used was tetrahydrofuran (THF) at a flow rate of 1 mL × min. -1 The sample was injected in a volume of 10 μL. The GPC system was calibrated using linear polystyrene and poly(methyl methacrylate) as internal standards.

[0070] Example 1. Effect of different aeration rates on biomass and PHA production rates The aeration rate required for mixing in outdoor cultivation of Hfx. mediterranei was optimized in a 1 L PET bottle experiment. The PET bottle was used as a bubble column reactor for stepwise scale-up from 100 mL culture volume to 1 L reactor volume and further PHA production tests under outdoor cultivation conditions. The biomass and PHA concentration profiles over time are shown in Figure 2A-B, respectively. The volumetric production rate was calculated after 72 h cultivation time, which was also the time of maximum PHA production. Aeration rate 1.0 L x min -1 The maximum volumetric biomass productivity and PHA productivity at 1.0 vvm were 64.03 ± 0.11 mg × L, respectively. -1 ×time -1 and 34.07±0.03mg×L -1 ×time -1 Compared with the volumetric productivity rates observed so far, the observed increases in biomass productivity were 10-15% and in PHA productivity were 6-8%.

[0071] Previous studies were carried out in 100 mL bottles, which were used to grow archaea in media supplemented with 25% v / v seaweed hydrolysate. Archaea were cultured aerobically in a shaking incubator at a temperature of 42 °C with uniform mixing at 120 rpm. Lower aeration rate (1.0L x min -1 Aeration rates up to 1.0 L x min 2 were suitable for biomass and PHA production, but higher aeration rates (up to 1.0 L x min 2 -1 From these studies, the 1.0 L × min -1It was shown that aeration at high aeration rates was more suitable for PHA accumulation. Our study also demonstrated that PHA production could be obtained along with the growth of the organism, suggesting that it is a growth-associated product formation. A study on polyhydroxybutyrate (PHB) production using glucose and glycerol as substrates provided insight into the effect of different aeration rates, and it was observed that higher PHB concentrations were obtained at lower aeration rates. Higher aeration rates resulted in higher biomass production with different substrates, but lower aeration rates resulted in higher PHB accumulation (De Almeida et al., 2010). Caprifolius capitis. Towards maximum biomass and PHA production using necator (Cupriavidus necator) observed that an aeration rate of 1 vvm was optimal. Since oxygen is only partially soluble in the aqueous culture broth, even a short interruption in aeration results in a rapid depletion of available oxygen, causing irreversible damage to the culture. The accumulation of PHA in Hfx. mediterranei cells may be due to the limited dissolved oxygen in the culture medium at low aeration rates. Under oxygen-limited conditions with an excess of carbon in the medium, NADPH oxidase activity is reduced, which further leads to an increase in the overall NADPH concentration. This, in turn, inhibits citrate synthase and isocitrate dehydrogenase, thereby increasing the acetyl-CoA concentration in the medium. The excess acetyl-CoA is therefore directed towards the accumulation of intracellular storage products such as lipids or PHA. The high NADPH / NAD ratio resulting from oxygen limitation promotes the synthesis of PHB, which serves as an alternative electron acceptor.

[0072] Example 2. Effect of different incubation times on biomass and PHA production rates The effect of different incubation times was tested in 1 L PET bottles at an aeration rate of 1 vvm. The experiments were carried out for up to 196 hours. The productivity increased with increasing time and reached 68.01 ± 0.11 mg × L at 72 hours incubation time, respectively. -1 ×time -1 and 28.02±0.03mg×L -1 ×time -1 The maximum biomass and PHA production rates were observed (Figures 2C-D). The PHA production rate significantly decreased after 72 h. This may be due to the consumption of PHA by the organism during the starvation phase. Hfx. mediterranei produces PHA as a stored energy product and has an intracellular PHA depolymerase, which allows it to utilize PHA for survival under stress conditions. The incubation time was estimated to be an important parameter for promoting PHA production using waste glycerol as a substrate. It was observed that limiting the growth phase provides nitrogen stress to the cells, resulting in an increased PHA content in the cells. PHA accumulation begins in the logarithmic phase, increases with biomass, and peaks at the beginning of the stationary phase. PHA synthesis lags behind biomass growth and reaches its maximum synthesis rate at the end of the exponential phase. This has also been observed in previous studies (Lillo and Rodriguez-Valera, 1990). These findings suggest that PHA production in this study is related to growth. This may be a key property for converting batch cultures to continuous systems, thereby increasing PHA production.

[0073] In this study, we present an alternative strategy for PHA production. Typically, the aerobic dynamic feeding (ADF) strategy is utilized to produce PHA from mixed microbial consortia (MMC) at low cost. The ADF process is often referred to as the feast-famine (FF) process, in which cells are fed with external substrates. It undergoes an early stage where the substrate is supplied in excess, followed by a later stage where the substrate is absent (Cui et al., 2016). In this study, we used a single-step process that does not require additional steps for PHA production, due to the organism's ability to utilize external substrates during the growth phase. This is because Hfx. mediterranei is a member of the Halobacteriaceae family, unlike other known members of the family. This may be based on the statistics that it is faster to cultivate than other halophilic archaea and also has extreme salt tolerance. Further surprising details are its high metabolic adaptability, i.e., it can be cultivated in both complex and simple defined media, uses a single carbon source with great diversity among halophilic archaea, and secretes extracellular enzymes that hydrolyze proteins, polysaccharides, and lipids. Furthermore, the very high salinity (>25%) reduces the level of contaminants in the process, thus making a concentration step unnecessary. The PHA contents reported for the ADF process are in the range of 25-70% by dry cell weight (DCW), which is comparable to the yields observed in this study. Thus, the present strategy is suitable for efficient and cost-effective PHA production from waste resources.

[0074] Example 3. Scale-up of PHA production by Hfx. mediterranei in a 40 L sleeve The present inventors further scaled up the PHA production process by culturing in a 40 L plastic sleeve. The initial culture volume was 10 L, which was scaled up stepwise to 40 L. The experimental conditions were determined after optimization, with an initial culture density of 50 g x L. -1 (Ghosh et al., 2021), aeration rate of 1 vvm, and incubation time of 72 h were used. The maximum mass fraction of PHA achieved in the biomass was calculated to be 56 wt / wt%. The maximum biomass productivity observed was 50.1 ± 0.11 mg × L -1 ×time -1 The PHA production rate was 27.03±0.01mg×L -1 ×time -1 The conversion yield was 0.107 g PHA x g UlvaDW -1 The carbon balance from this study was also calculated. Of the input carbon content of the Ulva sp. biomass (33.4% C as calculated by CHNS analysis), 32.03% was utilized for PHA production by Hfx. mediterranei, and the remaining content was utilized for volatile fatty acid (VFA) production during fermentation or was residual carbon remaining unused in the medium. This was confirmed by further analysis of the spent medium. A slight decrease in the overall production rate (biomass and PHA) was observed compared to the 1 L PET bottle culture, which may be due to inefficient air circulation in the reactor (10 L batch fermentation in a 40 L bioreactor). Inefficient air circulation leads to unavailability of medium components in the reactor, which may result in a decrease in the accumulation of cell biomass, substantially reducing the volumetric biomass and PHA production rate. This effect was also observed in our study and may explain the slight decrease in production rate at each stage of the stepwise scale-up of the process. Stepwise scale-up to 40 L culture could further improve the biomass and PHA production rates in the reactor. The biomass and PHA production rates showed a bell-shaped curve with the maximum production rate at 72 h of culture time. PHA production also showed similarity to the biomass production rate, suggesting that the production was a growth-dependent process that decreased with culture time. The decrease in PHA content over time can be explained by the fact that PHA is produced as an energy storage product in the archaeal cells. With increasing culture time, the nutritional stress in the cells increases, and the PHA granules become available for energy generation. Various waste materials have been used for PHA production. In batch culture experiments, 27 mg × L -1 ×time -1 In this fermentation experiment, tuna condensate was used as the substrate. there was.

[0075] Technically, PHAs are produced under controlled conditions in bioreactors or fermenters operated in stirred tank mode (STR). Reactors may be operated discontinuously in batch, repeated batch, fed-batch mode, or as continuous stirred tank reactors (CSTR) (Albuquerque et al., 2018), and may be cascaded if desired. Reactors are generally made of stainless steel, which creates challenges in the cultivation of Hfx. mediterranei at higher salinities (i.e., salinities above 22%). This can be overcome by using reactors made of polymers and / or ceramics (including glass), which are non-corrosive (Hezayen et al., 2000). Furthermore, air-mixed reactors have been used to culture Hfx. mediterranei at higher salinities (i.e., salinities above 22%). The pneumatically mixed reactor is also extremely useful due to its simple structure and design. It can be used for PHA production by extremely halophilic bacteria. Mixing is achieved by air bubbling in the reactor, which reduces the shear stress on the suspended cells and decreases the energy used for mass transfer. Recently, airlift reactors (ALR) have been used to produce H. boliviensis from starch hydrolysates in a batch system. Successful use of ALR has been demonstrated for PHB production from various carbon sources in nitrogen-deficient medium by H. boliviensis (Ortiz-Veizan et al., 2020). Azohydromonas australica and C. necator had approximately 72% PHB by weight and biomass concentrations of 10 and 32 g × L, respectively. -1 (Gahlawat et al., 2012), whereas Burkholderia sacchari Cultivation of Burkholderia sacchari in ALR produced 41% PHB by weight and a maximum biomass of 1000 mg / kg. Mass concentration 150g L -1 (Pradella et al., 2010). Similar to the air-agitated bioreactor, a bubble column reactor can also be utilized for PHA production by extreme halophiles due to the various advantages associated with air-agitated bioreactors. These advantages include simple design and construction, ease of operation, and lower shear stress compared to stirred tank reactors. There have been no reports on the use of air-agitated bioreactors for PHA production from seaweed hydrolysates using Hfx. mediterranei.

[0076] We used PET bottles (1 L) and even sleeve-shaped macroalgae photobioreactors (40 L), which are not subject to corrosion and can significantly reduce maintenance costs. The high salinity of the medium inhibits the growth of pollutants, thereby enhancing the possibility of outdoor cultivation with extreme halophilic archaea. Such a process is feasible and could potentially generate higher revenues for seaweed farmers. We analyzed the greenhouse gas emissions against current feedstocks for bioplastic production and found that our emissions are comparable. The efficient design of the cultivation system (both offshore and onshore) could be a factor in determining the feasibility of this process. Another way in which the sustainability of the process can be increased is by simultaneously producing PHA and biochar in a biorefinery concept. When combined with biochar production, the economic feasibility and sustainability are significantly improved (Ghosh et al., 2021). The inventors wanted to provide a sustainable solution for land-based cultivation of seaweed in air-agitated reactors that could be subsequently used for PHA production in an economically feasible way. There are challenges associated with further scale-up of the process. The high salinity of the medium can be an obstacle to the entire bioprocess. An additional unit for concentrating the brine could be used, in which case the discharge from the system could be recycled to the cultivation system. In addition, and as mentioned above, the process could be operated in a biorefinery concept, in which case various products could be obtained from a single biomass substrate, thereby increasing the cost-effectiveness of the process.

[0077] Example 4. PHA structural analysis (FTIR, TGA / DSC, 1 H NMR, GC-MS) FTIR spectroscopy. FTIR examination of PHA showed several absorption peaks. -1 A peak was detected around 1720-1740 cm, which can be attributed to the stretching of hydroxyl groups. -1) was observed, which can be attributed to the vibration of the carbonyl bond. The stretches of the methyl and methylene groups are at 2914 and 2879 cm, respectively. -1 Other peaks were detected at 1450–1000 cm -1 These peaks can be assigned to various bond vibrations, such as bending of the CH3 group, wagging of the CH2 group, and stretching of C-O, C-C, and C-O-C. These observations indicate that the analyzed polymer is polyhydroxy-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)). The production of P(3HB-co-3HV) copolyesters without the addition of 3HV-structure related precursor compounds, such as valerate, is a rare feature and is typically achieved by the addition of Hfx. It is found in some halophilic archaea such as Nei.

[0078] TGA / DSC thermal analysis. Hfx. The minimum decomposition temperature (T d ) was found to be 248°C. During the degradation process, the weight of the polymer decreased by 70.3%. The melting temperature was observed to be 177.1°C.

[0079] GC-MS and 1 H NMR analysis. GC-MS analysis of the hydrolyzed biopolymer showed two peaks in the chromatogram with retention times of 9.46 and 10.654 min. After mass spectrum analysis, these peaks were suggested to be the butyl esters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). GC-MS analysis showed that the polymer produced by Hfx.mediterranei was composed of 3HB and 3HV monomers. 1From the H NMR spectrum, the peaks at 0.83 ppm and 1.26 ppm indicate the presence of two different CH3 groups, which may be associated with the presence of 3HB and 3HV groups in the polymer, respectively. The peaks at 2.58 ppm and 2.48 ppm were assigned to the CH2 groups of 3HV and 3HB, respectively. The peak at 5.25 ppm was assigned to the CH group. 1 The monomer composition of the PHA was calculated according to the H NMR spectrum. These calculations indicated a composition of 90.4% 3HB and 10.6% 3HV.

[0080] Molecular weight of PHA produced. The molecular weight of PHA produced at different aeration rates was determined using gel permeation chromatography (GPC). Table 1 shows the molecular weight of PHA produced at various aeration rates (0.25-2.0 L × min -1 The average molecular weight (M w ), number average molecular weight (M n ), Z average molecular weight (M z ), and PDI. w (811 kDa using polystyrene standard, 770 kDa using PMMA standard) at an aeration rate of 0.25 L min -1 The molecular weights obtained were similar to those from a previous study of high-quality PHBV production from Hfx. mediterranei, where a polymer with a molecular weight of 1057 kDa and a PDI of 1.5 was obtained (Koller et al., 2007). In our research, when polystyrene was used as the standard, the average M w The molecular masses were in the range of 679–811 kDa, and the PDIs were in the range of 1.6–2.18. Using PMMA as a standard, the average M wThe molecular weights ranged from 656 to 770 kDa and the PDIs ranged from 1.544 to 1.959. A corresponding increase in PDI was observed with increasing aeration rate (Figure 3), suggesting that the polymer was more homogeneous at lower mixing speeds. In a field study, a PHA with an average molecular weight of 716 kDa and a PDI of 1.592 was obtained.

[0081] [Table 1]

[0082] Various studies have been carried out to determine the effect of different parameters on the molecular weight of PHA and to investigate the dependence of PHA synthase activity on the molecular weight distribution and PDI of PHA. Another study claimed that the molecular weight depends on the type of substrate used (Quagliano et al., 2001). Kshirsagar et al., 2013, reported that the effect of different mixing speeds on the PHA synthase activity was not observed in the control group. A molecular weight dependence of HA was observed. Our study demonstrates that by controlling the agitation and aeration rates, the molecular weight and PDI of the PHA produced can be controlled. Being able to control the molecular weight of PHA is advantageous in the manufacture and processing of polymers.

[0083] Example 5. Preparation of blends Solubility Testing of PLA, PHA, and Keratin To prepare blends containing these three materials, a solubility test was first performed.

[0084] Keratin solubility. Approximately 100 mg of keratin was mixed with either acetic acid or distilled water (Ruzgar et al., 2020), and the mixture was stirred for 12 hours. As found, keratin solubility was significantly increased in acetic acid and distilled water (Ruzgar et al., 2020). Keratin did not dissolve at all in any of these solvents under ambient conditions. Colloidal dispersions of keratin in acetic acid could be obtained by heating (Table 2).

[0085] Solubility of PHA. Approximately 100 mg of PHA was mixed with chloroform, acetone, distilled water, or acetic acid, separately. Partial dissolution was observed in chloroform and acetone. In contrast, only dispersion was observed when distilled water and acetic acid were used as solvents (Table 2).

[0086] Solubility of PLA. A mixture of 35 ml chloroform and 15 ml acetone was evaluated for the solubility of PLA. In this solvent mixture, only slight dissolution of PLA was observed after stirring at ambient conditions for 12 hours. Partial dissolution of PLA was observed in a mixture containing 14 ml chloroform and 6 ml acetone upon heating. When acetone was used as the solvent, PLA was able to dissolve upon heating to 55°C for 1 hour (Table 2).

[0087] [Table 2]

[0088] In summary, to prepare a keratin colloidal dispersion, acetic acid should be used as the solvent and the mixture should be heated. PHA can be partially dissolved in chloroform or acetone. PLA dissolves in acetone when heated to 55°C with continuous mixing.

[0089] Material Blend Preparation of PLA and PHA solutions: 100 mg of a binary blend consisting of 70 wt% PLA and 30 wt% PHA was made and the solubility of this blend in acetone was tested in two different vials to determine if the order of material addition to the solvent could affect the formation of a homogenous mixture. In the first vial, both solids, PLA and PHA, were added simultaneously to 10 ml of acetone, while in the second vial, PLA was added to 10 ml of acetone and PHA was added after dissolution. As observed, the dissolution rate of PLA was the same in both vials, i.e., with or without PHA. However, in the first vial, a homogenous solution was obtained, whereas in the second vial, a dark precipitate formed after the addition of PHA. In conclusion, these materials should be added simultaneously to acetone before heating.

[0090] Another test was performed to determine the rate of dissolution in the presence of excess acetone: 100 mg of a PLA / PHA (70:30) mixture was dissolved in 50 ml of acetone, and as was found, the rate of dissolution was increased.

[0091] To see if this mixture could be used as a solid, the material was added directly to a dog-bone mold and mixed every few minutes, this method was not convenient as the material was difficult to mix in the hot oven, which resulted in inhomogeneous test pieces.

[0092] In conclusion, the PLA / PHA mixture should be dissolved in an excess of acetone (25 ml of acetone for 1 g of PLA / PHA mixture) and the resulting mixture should be stirred and heated to 55° C. until homogenous.

[0093] Example 6. Preparation of "Paper" After preparing the PLA / PHA solution in acetone, the solvent could be removed. When the solution was placed in the dog-bone mold, the evaporation rate of acetone was very slow. Therefore, the evaporation of acetone should be done before placing the mixture in the dog-bone mold. The PLA / PHA solution in acetone was transferred to a tray and placed in a fume hood to evaporate. Once the acetone had evaporated, a homogeneous "paper" (film) containing PLA and PHA was obtained.

[0094] [Table 3]

[0095] Subsequently, blends of PLA / PHA and keratin were prepared, maintaining the same ratio of 70% PLA and 30% PHA as in the previous experiments, with the only difference between the various blends being their keratin content (Table 3).

[0096] Example 7. Preparation of tensile test specimens Dog-bone molded V-type test specimen The first mold for making the test specimens was made according to the ASTM D638 standard for tensile testing of plastics. The size chosen was the smallest size, Type V, to save on the amount of starting material and to make the smallest samples.

[0097] The mold was rectangular and had four identical sockets in the shape of dog bones, as described in ASTM (Figure 4). AlSl304 stainless steel was selected for the fabrication of the mold. This stainless steel has high corrosion resistance and is widely used for its mechanical properties.

[0098] To prepare Type V specimens, a 2 gram PLA / PHA "paper" strip (cut with scissors) can first be added to the mold, and the mold is placed in an oven heated to 220° C. Once the first portion of the strip was melted, more "paper" strip was added to the mold until the entire volume of the mold was filled.

[0099] Example 8. Redesign of the mold used for dog-bone specimen type IV Tensile tests of the dogbone specimens were performed on a universal mechanical testing machine with a load capacity of 2 kN. It was also decided to fabricate new specimens according to ASTM D638 Type IV that were twice the size of the previous specimens (Figure 5). The newly designed mold allowed all the material to be added at once and had a top section that allowed the material in the mold to be compressed, resulting in more uniform specimens. These specimens contained 4 gram A blend containing 70% PLA and 30% PHA was used, but the keratin content was varied as specified in Table 4.

[0100] [Table 4]

[0101] Keratin was ground into a fine powder before being added to a solution of 70% PLA and 30% PHA in acetone. The resulting PLA / PHA / keratin mixture was stirred for 15 minutes for homogenization, followed by transfer to a tray for evaporation and "paper" formation. The formed PLA / PHA / keratin "paper" was shredded into a fine powder in a grinder, and the powder was added to a Type IV mold. The mold with the powder was heated in an oven at 175°C for 60 minutes, then cooled to room temperature over 2 hours.

[0102] The resulting PLA / PHA / keratin Type IV specimens were then removed from the mold and painted with 50% white paint and 50% black paint for better imaging prior to tensile testing. The coating was sprayed using

[0103] Example 9. Powder characterization thermal analysis DSC and TGA studies of various formulations were carried out to determine the melting temperature of the pure material and various blends, as well as the effect of keratin incorporation on the decomposition temperature and mass change of PHA and PLA / PHA blends (Table 5 and Figure 6).

[0104] [Table 5]

[0105] PLA has the lowest melting temperature, followed by PHA and keratin. PLA has the highest decomposition temperature. The temperature chosen for preparing the test specimens was 175°C, lower than the melting temperature of PHA and higher than that of PLA, in order to avoid decomposition of these materials. For our test specimens, it was preferable to melt the PLA by heating at a relatively low temperature for a longer time. At the start of the experiments, the possibility of preparing blends without PLA was investigated. Therefore, DSC and TGA tests were performed on PHA / keratin blends. The samples tested included pure PHA, 10 wt% keratin, and 25, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, 180, 190, 200, 220, 240, 260, 280, 300, 320, 360, 380, 400, 420, 460, 480, 500, 520, 540, 560, 640, 760, 780, 800, 900, 1000, 1100, 1200, 1400, 1600, 1800, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 2800, 3200, 3600, 3800, 42 ... Increasing contents of keratin were included: 75, 90, and 100% by weight (pure keratin) (Table 6 and Figure 7).

[0106] As the weight fraction of keratin in the blend increased, the decomposition temperature increased and the weight loss decreased (Figure 8). As found, the blend could not be based only on PHA and keratin. As found, the blend of PLA and PHA should be in a ratio of 70:30 and the incorporation of keratin should be between 10 and 50 wt% (Table 7 and Figure 9).

[0107] [Table 6]

[0108] [Table 7]

[0109] With increasing weight fraction of keratin, the decomposition temperature of PHA increased significantly, whereas that of PLA did not change significantly. In addition, the rate of weight loss decreased with increasing weight fraction of keratin (Figure 10).

[0110] The addition of keratin significantly increased the decomposition temperature and reduced the weight loss, indicating that the addition of keratin improves the thermal stability of the blends.

[0111] PLA alone decomposed in a one-step process with the maximum decomposition peak at 365°C. When PHA and keratin were added to PLA, a two-step decomposition behavior was observed. The first peak (about 280°C) was due to the thermal decomposition of PHA, while the second peak (about 320°C) was due to the thermal decomposition of PLA. Furthermore, the addition of PHA to PLA reduced the main decomposition peak (344°C) compared to PLA alone (365°C). The addition of keratin to the blends reduced the main decomposition peak (344°C). The addition of keratin increased the temperature of the first decomposition peak, demonstrating that keratin alters the thermal stability of the system.

[0112] It should be noted that the addition of 20% and 30% by weight keratin did not result in any significant change in the thermal stability of the PLA / PHA. The decomposition temperature of PLA was 323°C in both cases, and the decomposition temperatures of PHA were 283°C (20% by weight keratin) and 282°C (30% by weight keratin). Based on these results, the blends selected for testing were those containing 30% by weight keratin.

[0113] FTIR The powders examined using FTIR were pure powders of PLA, PHA, and keratin, as well as two blends of PLA / PHA (70:30) and 70% PLA / PHA (70:30) with 30% keratin by weight.

[0114] The FTIR spectrum showed typical absorption bands of PLA, as well as several absorption bands specific to PHA and keratin. PLA is primarily amorphous, whereas PHA is more crystalline. The difference in the initial crystallinity of the polymers causes differences in the (C=O) band width and transmittance. The (C=O) FTIR band characteristics were significantly different between PLA and PHA. The FTIR spectrum showed a band around 1750 cm -1 The spectrum of the PHA blend showed a strong peak at 1720 cm, which is due to the amorphous carbonyl stretching vibration of PLA. This peak remained constant for all PLA / PHA blends. In addition, the spectrum of the PHA blend showed a strong peak at 1720 cm -1 A sharp peak was observed at 2933-2996 cm. This peak is due to the stretching vibration of crystalline carbonyl groups. The FTIR spectra of the PLA / PHA blends showed two main carbonyl stretching bands due to the presence of PLA and PHA. It is noteworthy that the intensity ratio of these two bands changed with the composition ratio. The FTIR spectra of the keratin-containing blends showed no change in the main carbonyl peak. When the FTIR spectra were analyzed, further peaks of other functional groups were seen. In all the spectra, the peaks at 2933-2996 cm were observed. -1 In addition, a low peak of (CH) was observed in the range of 1379 cm for PLA and PHA. -1 The methyl group peaks were observed at 1633, 1538, and 1233 cm. The amide I, II, and III vibrations derived from the peptide bonds of keratin were observed at 1633, 1538, and 1233 cm, respectively. -1 These peaks indicate that the extracted keratin has a beta-sheet secondary structure similar to that of feathers. -1 The peak is due to the NH stretching vibration of the peptide bond (-CO-NH-).

[0115] Example 10. Characterization of test specimens Mechanical Property Tests - Tensile Tests Mechanical properties that can be determined from tensile testing are modulus, stress, strain at yield, and strain at break. Each specimen composition was analyzed in at least two replicates. To measure the strain of the specimens during the course of the experiment, gage length segments of the specimens were painted 50:50 with black and white spray paint. This was done to improve the accuracy of pixel image processing.

[0116] The specimen was photographed during the experiment, and the images were then processed to calculate the strain on the specimen.

[0117] [Table 8]

[0118] Two specimens were tested, PLA: 003-1 and 003-2, both of which showed similar results, as shown in FIG.

[0119] Two specimens, PLA / PHA: 102-1 and 102-2, were tested. Both showed similar results except for the Poisson's ratio. In addition, both specimens broke before reaching the plastic region. These specimens appear to exhibit elastic deformation without plastic deformation. The results are shown in Figure 12.

[0120] [Table 9]

[0121] PLA / PHA / Keratin: Initially, only two specimens, 402-1 and 402-2, were tested. As the results were very different, an additional specimen, 403-1, was also tested. Each specimen exhibited different behavior. For example, unlike specimens 402-2 and 403-1, which show a curve in the graph indicating entry into the plastic region, specimen 402-1 shows a fairly linear graph indicating it remains in the elastic region. This can be seen in Figure 13.

[0122] Mechanical Analysis of PLA, PLA / PHA, and PLA / PHA / Keratin Specimens The results are summarized in Table 9 and FIG.

[0123] The results of the different specimens were averaged for each blend (PLA, PLA / PHA, PLA / PHA / Keratin). The three specimens showed very variable results, so an average result was not calculated. The results of specimen 402-2 showed the best results, so it was chosen to represent the PLA / PHA / Keratin blends (Table 10).

[0124] [Table 10]

[0125] In summary, the addition of PHA significantly reduced the strength of PLA as evidenced by the reduction in breaking stress. The highest modulus was obtained for the PLA / PHA blend. A decrease in modulus was observed with the addition of keratin, confirming that the addition of keratin causes a decrease in resistance to elastic deformation. The addition of PHA and keratin to PLA significantly reduced the elongation at break of the specimens, indicating a decrease in the ductility of the blend. In contrast to the expectation that the addition of PHA to PLA would make the specimens more ductile, the specimens became stiffer. The specimens broke in the elastic region without any deformation. This may have occurred due to the type of PHA used, PHB poly(3-hydroxybutyrate). Pure PHB is stiff due to its rather crystalline structure, while other PHA compositions such as P3HB-co-P4HB are more rubbery and flexible. Addition of P4HB (poly(4-hydroxybutyrate)) to PHA reduces the percentage of crystallinity while increasing the percent of elongation and breakage. The melting temperature (DSC) of the PHA used in our experiments was 176°C, indicating that it was a PHB with a typical melting temperature of 178°C (Table 11).

[0126] Another reason for the deterioration of mechanical properties may be pores that occur in the specimens during the manufacturing process. Such voids may cause a decrease in the adhesion between materials.

[0127] The Poisson's ratio results are not significantly different among the various blends. PLA appears to have the highest Poisson's ratio, followed by PLA / PHA / keratin and PLA / PHA, respectively. A high Poisson's ratio indicates a strong resistance to deformation.

[0128] [Table 11]

[0129] Example 11. Fracture surface morphology, microstructure, and transparency After the tensile testing experiments, three specimens, one each from the various blends (PLA, PLA / PHA, PLA / PHA / keratin), were examined for fracture surface morphology, microstructure, and transparency using SEM and digital microscopy (data not shown). The images obtained from these tests provide evidence of the cause of the changes in the mechanical properties of the blends. As can be seen in these images, there was no evidence of plasticity in all specimens. This is quite typical for pure PLA, but not for PLA / PHA, where the specimens would be expected to undergo substantial ductile plastic deformation and elongation before breaking. Looking at the 200 micron resolution SEM images of the specimens of the different blends, one can easily notice many pores, which were most likely formed during the manufacturing process by air trapped within the specimens originating from the moisture in the raw powders of PLA, PHA, and keratin used. The PLA specimens were translucent, while the specimens containing PHA and keratin were much more opaque. This opacity is characteristic of semi-crystalline polymers. Translucent PLA becomes opaque when crystalline PHA is inserted into its matrix. The reason for the opacity of combined PLA / PHA blends is the scattering of light at the polymer boundaries. Amorphous materials such as PLA contain few boundaries, whereas crystalline materials have a high density of such boundaries, causing a decrease in transparency. Thus, the transparency, or lack of transparency, of the specimens may indicate the crystallization of the polymer. SEM images of the fracture surfaces of the PLA specimens showed a smooth and uniform surface, typical of amorphous polymers. SEM images of the PLA / PHA specimens showed PHA particles with relatively small diameters and a typical sea-island morphology in the PLA polymer matrix. With the addition of keratin, the blends exhibited a reduced amount of pores. All SEM images show a homogeneous distribution of PHA and keratin in the PLA matrix.

[0130] Example 12. Biodegradation test Figure 15 shows the visual appearance of dog bones collected at different sampling intervals. The figure shows that the degradation of PLA is accelerated when blended with PHA as well as with PHA / keratin. The fastest degraded was the PLA / PHA / keratin composite, which disintegrated in 42 days, followed by the PLA / PHA composite (49 days) and lastly the PLA (56 days).

[0131] The visual disintegration was also supported by calculating the weight loss under composting conditions, as shown in Figure 16. The target for sample disintegration was considered to be 90% (Arrieta et al., 2014), as indicated by the current legislation for biodegradable materials (Iso, 2015). Degradation of PLA composites The degradation of PLA started on the 14th day of the composting experiment, but no significant degradation of PLA was observed until the 21st day. Previous studies on PLA / PHB blends showed similar degradation trends. Arrieta, Lopez, Rayon and Jimenez (2014) investigated the degradation of PLA with and without plasticizers. The degradation of A / PHB blends was studied, which showed a similar degradation profile of PLA along with blends of PLA with plasticizers (Arrieta et al., 2014). Similar observations were made in studies on the degradation of PHB and also PLA / keratin (Poorna et al., 2019;Ahn et al., 2011).

[0132] Example 13. Biodegradation test Biodegradation tests were carried out under aerobic conditions in a thermophilic composting environment (Iso, 2015). Different composite dog bones were used and they were easily degraded after the composting test. The dog bones were placed in a textile mesh to allow access to the soil, but still allow access for moisture and microorganisms. They were buried at a depth of 4-6 cm in 500 mL sealed glass beakers containing 10% compost, 30% fresh vegetables, 10% starch, 5% sugar, 4% corn oil, 1% urea, 40% sawdust, and solid synthetic wet waste with a water content of about 50% (w / w) and incubated at 58 °C in aerobic conditions for degradation. Aerobic conditions were ensured by mixing the solid synthetic wet waste periodically. Samples of the composite were taken at intervals of 7 days until complete degradation was reached. At each sampling time point, the dog bones were washed with distilled water, dried in an oven for 24 h, and then weighed again. Photographs were taken of all samples after removal from the composting medium.

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Claims

1. 1. A method for producing polyhydroxyalkanoates (PHAs) by fermentation of archaea capable of producing said PHAs, said method comprising: (i) providing a fermenter having a culture volume of at least about 5 liters, the fermenter comprising a culture medium occupying the culture volume, the culture medium comprising saltwater supplemented with a carbon source and a nitrogen source suitable for culturing the archaea, the carbon source comprising a green macroalgae hydrolysate constituting at least about 1% by weight of the carbon source, and / or the nitrogen source comprising a green macroalgae hydrolysate constituting at least about 1% by weight of the nitrogen source; (ii) inoculating the culture medium with a seed culture of the archaea; (iii) culturing the archaea while constantly bubbling an oxygen-containing gas mixture into the fermentor from the bottom thereof at an aeration rate of at least about 0.2 vvm so as to constantly aerate and mix the culture medium, until a predetermined concentration of the archaea in the culture medium is obtained; (iv) harvesting the archaeal biomass from the culture medium; and (v) concentrating, separating, and / or extracting the PHA from the harvested biomass; A method comprising:

2. The method of claim 1, wherein the archaea is Haloferax mediterranei.

3. 3. The method of claim 2, wherein the Haloferax mediterranei is Haloferax mediterranei ATCC 33500.

4. The method of claim 1, wherein the green macroalgae hydrolysate constitutes at least about 5% by weight, 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight, 35% by weight, 40% by weight, 50% by weight, 60% by weight, 70% by weight, 80% by weight, 90% by weight, or more of the carbon source and / or constitutes at least about 5% by weight, 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight, 35% by weight, 40% by weight, 50% by weight, 60% by weight, 70% by weight, 80% by weight, 90% by weight, or more of the nitrogen source.

5. 10. The method of claim 1, wherein the culture medium comprises carbon and nitrogen, each independently, in an amount of from about 1% to about 80% by weight on a dry weight basis.

6. The amount of carbon in the culture medium is at least about 10% by weight, 20% by weight, on a dry weight basis.

6. The method of claim 5, wherein the amount of nitrogen in the culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by weight on a dry weight basis.

7. The method of claim 1, wherein the carbon source further comprises sugars, waste products, polyols, hydrolysates, or mixtures thereof, and / or the nitrogen source further comprises organic or inorganic nitrogen, hydrolysates, or mixtures thereof.

8. 8. The method of claim 7, wherein the sugars are monosaccharides or polysaccharides; the waste is glycerol and fatty acids, vinasse, stillage, molasses wastewater, or olive mill wastewater; the hydrolysate is olive leaf hydrolysate, paper waste, agricultural waste hydrolysate, or cheese whey hydrolysate; the organic or inorganic nitrogen is proteins, glycosylated proteins, DNA fragments, RNA fragments, and biopolymers including nitrogen-containing oligosaccharides, peptides, amino acids, and nitrogen salts; and the hydrolysate is olive leaf hydrolysate, agricultural waste hydrolysate, or cheese whey hydrolysate.

9. The method of claim 1, wherein the oxygen-containing gas mixture is air.

10. The method described in claim 1, wherein the ventilation rate is up to approximately 1.2 vvm.

11. 10. The method of claim 1, wherein the culture medium further comprises a salt, a buffer, phosphorus, or a mixture thereof.

12. the culture medium comprises salt water supplemented with the carbon source, the nitrogen source, and optionally the phosphorus, halogen, or mixture thereof; 10. The method of claim 1, wherein the amount of carbon and nitrogen in the culture medium is each independently from about 1% to about 80% by weight on a dry weight basis.

13. 13. The method of claim 12, wherein the amount of carbon in the culture medium is at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70% by weight on a dry weight basis; the amount of nitrogen in the culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by weight on a dry weight basis; and the green macroalgae hydrolysate independently constitutes at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or more by weight of each of the carbon source and the nitrogen source.

14. (a) the green macroalgae hydrolysate is an Ulva sp. hydrolysate; (b) the pH of the culture medium prior to the inoculation step (ii) is at most 8.5; (c) the culturing step (iii) is carried out for at least 48 hours, 60 hours, or 72 hours; (d) the airflow rate is at most about 1.2 vvm; and / or (e) step (v) comprises extracting the PHA by physical means, chemical means, or a combination thereof; The method of claim 1.

15. (a) the pH of the culture medium prior to the seeding step (ii) is within the range of about 6.8 to about 7.4; (b) the airflow rate is about 1 vvm; and / or (c) the physical means is selected from water (hydrolysis), high pressure, pulsed electric fields, and centrifugation and filtration; and the chemical means is selected from cell lysis buffer, deep eutectic solvent, organic solvent, two-phase solvent system, and ionic liquid.

15. The method of claim 14.

16. The method according to any one of claims 1 to 15, wherein the molecular weight of the obtained PHA varies depending on the aeration rate and the culture period of the archaea.

17. The method of claim 1, further comprising purifying the PHA obtained in step (v) to obtain a purified PHA.

18. 18. The method of claim 17, wherein the purification step is carried out by repeatedly washing the obtained PHA with water, an ionic liquid, or a combination thereof.

19. The method of claim 17, further comprising drying the purified PHA.

20. The method of claim 19, wherein the drying is performed by a drum dryer, a spray dryer, air or air / nitrogen stream drying, or a freeze dryer.

21. 20. The method of any one of claims 17 to 19, further comprising blending the purified PHA with at least one polymer.

22. 22. The method of claim 21, wherein the at least one polymer is a biodegradable polymer; or a non-biodegradable polymer or copolymer.

23. The method of claim 22, wherein the biodegradable polymer is selected from polylactic acid (PLA), polycaprolactone (PCL), keratin, cellulose, chitin, lignin, amylose, amylopectin, and mucin; or the non-biodegradable polymer or copolymer is selected from polyethylene terephthalate (PET, also known as polyester), high density polyethylene (HDPE), polyvinyl chloride (PVC), low density polyethylene (LDPE), polypropylene (PP), polystyrene (PS), polyethylene glycol (PEG), polypropylene glycol (PG), polyvinyl alcohol (PVA), polyacrylonitrile (PAN), polymethacrylic acid or its ester, poly(acrylonitrile-co-butadiene-co-styrene) (ABS), polyamide, polyacrylamide (PAM), polysiloxane, graft polymer, and dendrimer.

24. 2. The method of claim 1, wherein the fermentor is an open fermentor and the culturing of the archaea is carried out under non-sterile conditions.

25. 10. The method of claim 1, wherein the fermentor is made of glass, ceramic, plastic, cement, or the fermentor is an earthen fermentor (banked pond).

26. 10. The method of claim 1, which is carried out continuously.

27. A blend comprising a polyhydroxyalkanoate (PHA) and at least two additional polymers, one of which is keratin. and said keratin comprises from about 1% to about 99% by weight of said blend.

28. The blend described in claim 27, wherein the keratin constitutes from about 10% to about 50% by weight of the blend.

29. The blend described in claim 27, wherein the other of the at least two additional polymers is each independently a biodegradable polymer; or a non-biodegradable polymer.

30. The blend of claim 29, wherein the biodegradable polymer is selected from polylactic acid (PLA), polycaprolactone (PCL), keratin, cellulose, chitin, lignin, amylose, amylopectin, mucin, and a PHA different from the PHA; or the non-biodegradable polymer is selected from polyethylene terephthalate, high density polyethylene (HDPE), polyvinyl chloride (PVC), low density polyethylene (LDPE), polypropylene (PP), polystyrene (PS), polyethylene glycol (PEG), polypropylene glycol (PG), polyvinyl alcohol (PVA), polyacrylonitrile (PAN), polymethacrylic acid or its ester, poly(acrylonitrile-co-butadiene-co-styrene) (ABS), polyamide, polyacrylamide (PAM), polysiloxane, graft polymer, and dendrimer.

31. 30. The blend of claim 29, wherein the at least two additional polymers comprise or consist of PLA and keratin.

32. 32. The blend of claim 31, wherein the ratio of PHA:PLA is from about 1:99 to about 99:1 by weight, respectively.

33. 33. The blend of claim 32, wherein the ratio of PHA:PLA is from about 20:80 to about 40:60, about 25:75, about 30:70, or about 35:65, respectively, by weight.

34. An article comprising or made from the blend of any one of claims 27 to 33.

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