Process for the production of polyhydroxyalkanoates from seaweed

Abstract

A method for producing polyhydroxyalkanoates from macroalgae, the method comprising the steps of: forming a macroalgae mixture from macroalgae and a liquid; hydrolyzing the macroalgae mixture to form a macroalgae hydrolysate; producing a growth medium comprising the macroalgae hydrolysate; fermenting the growth medium using a salt-tolerant microorganism capable of producing polyhydroxyalkanoates; and extracting polyhydroxyalkanoates from within the salt-tolerant cells using a water-based osmotic-driven lysis method.

Description

Technical Field

[0001] This invention relates to a method for producing polyhydroxyalkanoates from seaweed by hydrolysis and fermentation using halophilic microorganisms. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are natural polyesters that can be derived from the microbial fermentation of carbon from lignocellulose and other biomass feedstocks. Due to their non-fossil fuel (petrochemical-based) origin, they are often referred to as 'biopolymers' and used in the manufacture of 'bioplastics'.

[0003] During microbial fermentation, microorganisms are often deprived of nutrients such as nitrogen, oxygen, and phosphorus, but provide high levels of carbon. They produce PHA as a carbon reserve, storing it in high-refractive-index particles for later use when they have access to other nutrients needed for growth and reproduction. However, before the particles are broken down, they can be harvested from the microorganisms by lysing the cells and separating the PHA. The yield of PHA obtained from the intracellular particle contents can be as high as 80% of the organism's dry weight.

[0004] PHAs have a similar chemical structure to petrochemical-based plastics, and because they are biodegradable and do not harm living tissue, they have been used in agricultural, medical, and pharmaceutical applications. For example, PHAs have been used to produce sutures, slings, bone plates, and skin replacements. PHAs have also been used in single-use food packaging. Bioplastics formed using PHAs are generally less toxic than petrochemical-based plastics and do not contain the hormone disruptor bisphenol A (BPA).

[0005] Although research on PHA production has been conducted for over 90 years, it has recently attracted greater interest, partly due to increased public awareness of the environmental problems caused by waste plastics.

[0006] While bioplastics made from biopolymers, including PHA, may seem like a clear solution to the environmental damage we are currently experiencing from the millions of tons of petrochemical-based plastics discarded in our waterways, landfills, and microplastics in the food chain, they are not without their own pollution and sustainability issues.

[0007] Currently, the main source of PHA used in the production of bioplastics is the fermentation of starch from crops such as corn, potatoes, cassava, and sugarcane. These crops typically occupy large tracts of arable land, and if they are used for biopolymer production, they would likely replace food crops used for our growing population. Therefore, the primary issue is competition for generally limited land, and competition for fertile arable land used for growing food crops.

[0008] Further issues include the millions of liters of water required to grow crops, synthetic fertilizers and pesticides that pollute nearby waterways, and other environmental hazards associated with large-scale agricultural production. The chemical processing of harvested crops used to produce PHA also utilizes large quantities of chemicals. Furthermore, biopolymers still have a significant greenhouse gas (GHG) footprint because: (i) carbon-intensive sterilization of fermentation reactors, including steam sterilization, leads to the release of high concentrations of CO2 equivalent; and (ii) a high carbon footprint associated with crop inputs such as fertilizers.

[0009] Therefore, it would be beneficial to provide more efficient and economical methods while reducing their environmental impact, making PHA production more ideal as an alternative to petrochemical-based plastics.

[0010] The foregoing discussion of background technology is intended only to facilitate understanding of the invention. It should be understood that this discussion does not endorse or acknowledge that any material mentioned is part of common general knowledge as of the priority date of this application. Summary of the Invention

[0011] This invention provides a method for producing polyhydroxyalkanoates (PHAs) from macroalgae, comprising the following steps:

[0012] It forms a large algal mixture containing macroalgae and liquid;

[0013] Hydrolyzing a mixture of macroalgae to form macroalgae hydrolysis products;

[0014] To produce a growth medium containing hydrolysis products of large algae;

[0015] Using a halophilic microbial fermentation growth medium capable of producing PHA; and

[0016] PHA was extracted from halophilic cells using a water-based osmosis-driven lysis method.

[0017] For the purposes of describing this invention, halophilic microorganisms are those organisms that require salt in their growth medium to survive, as opposed to 'salt-tolerant' microorganisms, which are organisms that can tolerate salt in their growth medium.

[0018] The method of the present invention, as described herein, utilizes halophilic species not only due to their tolerance to the salts present in seaweed, but also because it enables very high levels of under-salt growth that suppresses most potential contamination during fermentation (thus reducing product sterilization costs and associated carbon emissions). The use of halophilic microorganisms also facilitates the extraction of PHA products from cells via osmotic shock lysis (using fresh or seawater).

[0019] In a preferred embodiment of the invention, macroalgae include seaweed. More preferably, macroalgae include cultured seaweed. Even more preferably, macroalgae include seaweed cultured in industrial quantities. In this regard, of the approximately 25,000 seaweed species currently discovered, only about 1% are currently cultured, of which about 10 are concentrated for large-scale biomass production. Therefore, the method of the present invention preferably utilizes seaweed from four families of algae, which account for approximately 95% of the current global seaweed production: Gracilariaceae, Solieriaceae, Bangiaceae, and Laminariaceae.

[0020] In one embodiment of the invention, the macroalgae include red macroalgae (Rhodophyta). More preferably, the macroalgae include macroalgae species selected from the following families: Gracilaria, Rhodophyta, Rhodophyta, Gelidiaceae, or Bonnemaisoniaceae. Even more preferably, the macroalgae include macroalgae species selected from the following genera: Gracilaria spp., Gracilariopsis spp., Kappaphycus spp., Eucheuma spp., Porphyra spp., Pyropia spp., Elidium spp., or Asparagopsis spp.

[0021] Alternatively, macroalgae may include green macroalgae (Chlorophyta), more preferably species of the genus *Ulva*. Macroalgae may also include brown macroalgae (Phaeophyta) selected from the families Laminariaceae or Lessoniaceae (kelp). More preferably, brown macroalgae species include *Saccharina japonica* (kombu), *Undaria pinnatifida* (wakame), or *Ecklonia radiata* (goldenkelp).

[0022] In one embodiment of the invention, the macroalgae mixture comprises wet macroalgae and liquid. For the purposes of describing the invention herein, 'wet macroalgae' refers to macroalgae whose internal moisture content is not (completely) dried or substantially dried. That is, in some non-limiting instances, 'wet macroalgae' may include fresh or recently cultivated, collected, and / or removed from its growth environment (e.g., seawater or saline water environment); it may also include macroalgae that have been cultivated, collected, and / or removed from its growth environment prior to use in the methods of the invention and then stored, frozen, cooled, and / or transported for a period of time; it may also include wet macroalgae that have been rotated during cleaning to remove excess water from the surface of the macroalgae; and it may also include macroalgae that have been dried to a certain extent but whose water content has not been completely removed.

[0023] In one embodiment, the wet macroalgae are collected and not completely dried prior to the step of forming the macroalgae mixture. That is, in a preferred embodiment, the method of the present invention utilizes wet macroalgae collected or harvested from water in which they once lived and grew or were even stored (e.g., marine estuaries, bays, pools, and ponds); wherein the algae grow and / or are loosely stored or attached to structures such as ropes. Macroalgae can be collected manually or by mechanical or other means, for example, in large-scale operations.

[0024] It is preferable to wash the wet macroalgae with brine or water from the collection site after collection, which may include washing with water from which macroalgae have already been collected.

[0025] Preferably, the wet macroalgae are cooled between the steps of collecting and forming the macroalgae mixture. Cooling preferably includes storing the collected and washed macroalgae on ice or refrigerated containers during storage and / or transportation prior to the step of forming the macroalgae mixture from the wet macroalgae and liquid in the method of the present invention.

[0026] Preferably, excess liquid is drained from the wet macroalgae before the step of forming the macroalgae mixture. More preferably, a swirl is used to drain excess liquid from the wet macroalgae.

[0027] Alternatively, in another embodiment of the method of the present invention, macroalgae can be dried. Macroalgae can be sun-dried or sun-dried, tunnel-dried, freeze-dried, vacuum-dried, or oven-dried. Preferably, macroalgae are sun-dried or tunnel-dried, followed by freeze-dried and / or oven-dried at a temperature between about 60°C and 80°C to remove residual moisture.

[0028] In a preferred embodiment of the method of the present invention, the step of forming a macroalgae mixture from (wet or dry) macroalgae and liquid preferably includes breaking the macroalgae into smaller fractions in the liquid and / or before mixing with the liquid to form a macroalgae slurry of broken macroalgae in the liquid. More preferably, breaking the macroalgae into smaller fractions includes the step of decomposing the macroalgae. Decomposing the macroalgae preferably includes pulverizing by grinding or mixing the macroalgae. Grinding preferably includes using a knife or a wet grinder. When broken into smaller fractions, the smaller fractions of the macroalgae contain particles preferably less than about 2 mm in diameter. It is preferable to grind the macroalgae into powder to make it easier to hydrolyze.

[0029] In one embodiment of the method of the present invention, lipids are extracted from large algae that have been broken into smaller parts before they are mixed with a liquid.

[0030] Preferably, lipids are extracted by using a mixture of macroalgae and a solution, wherein the solution preferably comprises chloroform, methanol, and water (including deionized water, distilled water, reverse osmosis water, tap water, brine, and / or seawater). Preferably, the mixture comprises 2 to 5% w / v macroalgae, and preferably, the chloroform-methanol-water solution comprises 35% to 45% chloroform, 35% to 45% methanol, and 15% to 25% water.

[0031] The mixture is preferably mixed with chloroform at room temperature for 10 to 40 minutes, more preferably 30 minutes, and the methanol phase is preferably separated from the large algal solids by centrifugation and / or filtration.

[0032] In one embodiment, the lipid contents are extracted from the chloroform phase by evaporation and / or distillation, and the remaining macroalgae solids are preferably washed with deionized water, distilled water, reverse osmosis water, tap water, salt solution and / or seawater, and the remaining solvent is removed by centrifugation and / or filtration before the remaining macroalgae are combined with the liquid to form a macroalgae mixture.

[0033] In a preferred embodiment of the method of the present invention, the liquid comprises a salt solution. For the purposes of describing the invention herein, a 'salt solution' may include: brine; salt-containing water, whether artificially produced (e.g., with distilled and / or deionized water) or derived from a natural source; it may also include seawater, whether unchanged, diluted in another liquid (e.g., water), or concentrated to increase the salt concentration or with the addition of additional salts; it may also include saline water from saltwater rivers or lakes or other bodies of water; it may also include brackish water, brine, or brine / salt water, such as desalinated brine from a desalination process, for example, using reverse osmosis and / or evaporation processes; and it may also contain a marine medium that can mimic the composition of seawater or is closely related to it.

[0034] In one embodiment of the invention, the salt solution comprises seawater. The seawater may be diluted or concentrated seawater.

[0035] In a preferred embodiment, the salinity of the salt solution is about 35 parts per thousand (ppt).

[0036] In one embodiment of the invention, the macroalgae mixture is stored at ambient temperature prior to hydrocolloid extraction, a preparatory step in preparing the fermentation growth medium. Preferably, the macroalgae mixture comprises 1:3 to 1:50 w / v macroalgae and is stored for 30 minutes to 24 hours. More preferably, the mixture comprises 1:20 w / v macroalgae and is stored at ambient temperature for 3 hours.

[0037] In a preferred embodiment of the invention, the large algae mixture is heated and stirred, and the aqueous phase containing hydrocolloids (e.g., agar) is preferably separated from the solids by filtration and / or centrifugation. Preferably, prior to separation, the mixture is heated to 80°C to 110°C, preferably for 1 to 4 hours, and stirred at 100 rpm to 1100 rpm. More preferably, prior to separation, the mixture is heated to approximately 100°C for approximately 2 hours, and preferably stirred at 200 rpm.

[0038] In one embodiment, the solid residue remaining after the above-mentioned hydrocolloid extraction is preferably washed with a liquid at a temperature between 60°C and 110°C, more preferably at about 70°C, to form another mixture, and preferably the liquid includes fresh water or a salt solution to ensure that any remaining hydrocolloids are extracted into the aqueous phase of the mixture and preferably separated from the solid residues by filtration and / or centrifugation.

[0039] In one embodiment of the invention, a solid macroalgae residue for hydrolysis is prepared by mixing with a liquid to form a further (second) macroalgae mixture. The separated liquid fraction containing the hydrocolloid extract is gelled prior to a dehydration process to form a hydrocolloid product, and / or hydrolyzed to obtain additional sugar monomers, such as galactose or glucose, which can be used in fermentation processes, for example, to produce PHA according to the invention.

[0040] In a preferred embodiment of the method of the present invention, the step of hydrolyzing the macroalgae mixture includes one or more hydrothermal, acidic, and / or enzymatic hydrolysis processes. The hydrothermal process preferably includes subcritical water extraction. The step of hydrolyzing the macroalgae mixture is preferably carried out in batches or continuously using one or more stirred reactors and / or plug flow reactors.

[0041] In a preferred embodiment, the liquid is a salt solution. Preferably, the salt solution includes a pure, concentrated, or diluted solution, such as seawater.

[0042] The hydrolysis of large algal mixtures is preferably carried out in a hydrolysis reactor or other suitable reactor.

[0043] In one embodiment, the acid hydrolysis step of the macroalgae mixture includes a strong acid (e.g., sulfuric acid, hydrochloric acid, and / or sulfamic acid) or a weak acid, preferably at a concentration between about 0.1% and 5% w / v. The acid preferably includes a weak acid, more preferably citric acid, acetic acid, formic acid, maleic acid, phosphoric acid, or oxalic acid. Even more preferably, the acid includes citric acid. The concentration of citric acid includes about 10 to 200 mM, more preferably about 25 mM.

[0044] In one embodiment, the hydrolysis of the macroalgae mixture (preferably including citric acid hydrolysis) is carried out at a temperature between about 100°C and 140°C, more preferably about 120°C. The hydrolysis of the macroalgae mixture (preferably including citric acid hydrolysis) is preferably carried out for about 10 to 120 minutes, more preferably about 30 minutes, and is preferably continuously stirred at 100 rpm to 1100 rpm, more preferably about 300 rpm. The aqueous phase is preferably separated from the macroalgae solids by centrifugation and / or filtration prior to enzyme treatment.

[0045] In one embodiment, the aqueous phase undergoes a detoxification process prior to enzyme treatment to remove fermentation inhibitors. This preferably includes excess lime treatment and / or charcoal treatment.

[0046] The use of excess lime preferably involves adjusting the pH of the aqueous phase to pH 10 to 12 with a slurry of calcium hydroxide and water (including deionized water, distilled water, reverse osmosis water, tap water or salt solution), and then separating the aqueous phase from the solids, preferably by centrifugation and / or filtration.

[0047] The carbon treatment preferably consists of the following: combining an aqueous phase with activated carbon powder to form a mixture of 0.5% to 10% w / v, preferably 2.5% w / v carbon powder, stirring the mixture (occasionally or by continuous stirring) for 10 to 120 minutes, preferably 30 minutes, then preferably separating the aqueous phase from the carbon solids by centrifugation and / or filtration, and recombining it with acid-hydrolyzed macroalgae solids to form a mixture.

[0048] In one embodiment, the enzymatic hydrolysis step of the macroalgae mixture includes adding an enzyme to the acid-hydrolyzed macroalgae mixture (including after detoxification, if applicable). The enzyme preferably comprises one or more cellulases and / or a mixture of β-glucanase, pectinase, hemicellulase, and xylanase. In a preferred embodiment, the enzyme comprises… ('Celluclast') and ('Viscozyme'). Celluclast and Viscozyme are preferably used at a ratio of about 0.01 to 2.0 ml enzyme / g macroalgae, more preferably about 0.1 ml each of the two enzymes / g macroalgae, for a total of about 0.2 ml enzyme / g macroalgae.

[0049] The enzymatic hydrolysis of the macroalgae mixture is preferably carried out at a temperature of about 30°C to 70°C and a pH of about 3.5 to 7, more preferably at a pH of about 5.0. The enzymatic hydrolysis of the macroalgae mixture is more preferably carried out at about 50°C. The enzymatic hydrolysis of the macroalgae mixture is preferably carried out under mixing conditions for about 6 to 48 hours, more preferably 20 hours, wherein mixing includes stirring at about 200 rpm.

[0050] In one embodiment, the enzymatic hydrolysis of the macroalgae mixture produces an aqueous phase ('macroalgae hydrolysis product') and a solid phase, and the macroalgae hydrolysis product is separated from the solid phase. The step of separating the macroalgae hydrolysis product preferably includes removing the solid phase and residual solids by filtration or more preferably by centrifugation.

[0051] The solid phase after hydrolysis is preferably further processed into soil treatment or protein-rich food suitable for human or animal consumption, and is preferably used as aquaculture feed product.

[0052] In a preferred embodiment of the method of the present invention, the pH of the macroalgae hydrolysis product is adjusted to approximately 7.0 by adding an acid and / or a base. The acid preferably includes HCl and the base preferably includes NaOH.

[0053] In a preferred embodiment of the method of the present invention, the step of generating a growth medium for fermentation includes adjusting the content and properties of macroalgal hydrolysates to maximize growth and / or PHA production after inoculation with halophilic microorganisms. In a preferred embodiment, the halophilic microorganism is a spp. of Halophilus or, more preferably, a Mediterranean halophilic bacterium (H. mediterranei).

[0054] In one implementation, the growth medium can be diluted with a salt solution to reduce the level of fermentation inhibitors present in the hydrolysis products of macroalgae.

[0055] The salinity and nutrient content (including carbon, nitrogen and / or micronutrient levels) of the growth medium can also be adjusted by dilution, evaporation and / or by adding components.

[0056] In one embodiment of the method, the salinity of the growth medium is adjusted to between 35 ppt and 330 ppt by adding a salt solution and / or salt (e.g., NaCl or sea salt). Preferably, when the halophilic microorganism is a Mediterranean halophilic bacterium, the salinity of the growth medium is adjusted to about 130 ppt to 200 ppt, more preferably to about 170 ppt. The salt solution and / or salt preferably contains sea salt and / or one or more of the following: NaCl, MgCl2·6H2O, Mg2O4·7H2O, CaCl2·6H2O, NaHCO3, and NaBr, more preferably in a salt ratio similar to that of ATCC medium 1176 (Halobacter broth).

[0057] In one embodiment of the method of the present invention, the nutrient content and level of the growth medium are adjusted by adding a carbon source (e.g., glucose, sucrose, and / or galactose), a nitrogen source (e.g., yeast extract, urea, ammonium chloride, and / or ammonium sulfate), and / or a micronutrient source (e.g., a micronutrient mixture) to the growth medium. Preferably, when the halophilic microorganism is a Mediterranean halophilic bacterium, the nutrient content of the growth medium is adjusted by adding a yeast extract and / or a micronutrient mixture of SL-4 or SL-6 to the growth medium.

[0058] The steps for fermentation growth medium preparation include inoculating the growth medium with a halophilic microorganism capable of producing PHA to form a fermentation culture. The inoculation volume is determined such that the optical density of the inoculated fermentation culture is 0.5 to 1 (600 nm; OD). 600 ).

[0059] In one embodiment, the growth medium is fermented in batches. In another embodiment, the growth medium is fermented in a continuous fermentation system.

[0060] In a preferred embodiment, the step of fermenting the growth medium includes culturing the fermentation culture in a continuous fermentation system using a fermentation reactor.

[0061] In one embodiment, the continuous fermentation system includes a fermentation reactor that operates continuously or semi-continuously, or two or more reactors that operate sequentially (i.e., continuously in series or cascaded).

[0062] The continuous fermentation system preferably comprises two or more reactors operating sequentially, and more preferably, has at least a first, second, and third fermentation reactor. The first fermentation reactor preferably contains a fermentation culture with a composition optimized for cell growth. The downstream fermentation reactors (e.g., the second and third fermentation reactors) preferably contain fermentation cultures with a composition optimized to increase intracellular PHA accumulation.

[0063] Preferably, the fermentation culture within the reactor is optimized by increasing its carbon-nitrogen ratio from the first fermentation reactor to the last. Preferably, each fermentation reactor also maintains its carbon-nitrogen ratio at an optimal and constant level by continuously feeding fresh growth medium into each reactor.

[0064] In one embodiment, when the halophilic microorganism is a Mediterranean halophilic bacterium, the carbon-nitrogen ratio in the fermentation culture in each reactor is preferably increased from a carbon-nitrogen ratio between 1 and 20 in the first reactor to a preferred carbon-nitrogen ratio between 21 and 40 in the last reactor.

[0065] The continuous fermentation system preferably includes three inlet feeds (one per reactor) and three outlet feeds that continuously supply fresh growth medium to the fermentation reactors: one pumps the culture from the first reactor to the second reactor, one pumps the culture from the second reactor to the third reactor, and one harvests the fermentation broth from the third reactor for further processing into extracted PHA. The flow rate of the final outlet feed is set to be equal to the sum of the flow rates of all inlet feeds to maintain a constant volume of fermentation culture in each reactor.

[0066] In one embodiment, the third fermentation reactor is fed into a fourth reactor. The fourth reactor is preferably used to collect cell biomass to separate PHA from the fermentation culture.

[0067] In one embodiment, an acid and a base are added during fermentation to maintain the pH at approximately pH 7.0. The acid preferably contains HCl and the base preferably contains NaOH. An antifoaming agent is preferably added to the first, second, and third fermentation reactors to minimize foam generation or accumulation.

[0068] In one embodiment, the temperature of the first, second, and third fermentation reactors is maintained at about 40°C and preferably at atmospheric pressure.

[0069] In a preferred embodiment, the step of extracting PHA from halophilic cells using a water-based osmosis-driven lysis method begins with the separation of cellular biomass from the fermentation culture, preferably by centrifugation and / or filtration. Separation of cellular biomass from the fermentation culture more preferably includes centrifuging the fermentation culture at a preferred rate of 10,000 to 20,000 g.

[0070] The extracted halophilic cells are then lysed by inducing hypotonic shock through immersion in an aqueous solution with a salinity lower than that of the cells. In one embodiment, the volume of the aqueous solution used for each lysis stage is approximately the same as the volume of the isolated fermentation culture (also referred to as the isolated 'fermentation broth'). After lysis, PHA and any unlysed cells are preferably extracted by filtration and / or centrifugation of an aqueous solution containing the lysed and any unlysed halophilic cells. For the purposes of this description of the invention, this lysis and extraction process is defined as the 'lysis stage'.

[0071] In one implementation, the pyrolysis stage is performed once or multiple times, preferably once. The composition of the water-based solution used in each pyrolysis stage can be adjusted according to the number of pyrolysis stages.

[0072] For the purposes of this description, the 'water-based solution' may include purchased or on-site obtained tap water, distilled water, or deionized water, including water obtained through reverse osmosis or evaporation processes applied to seawater or freshwater sources. Alternatively, the water-based solution may include a salt solution. Preferably, for each pyrolysis stage, the water-based solution comprises 50% to 100% seawater, more preferably 99.9% seawater (or almost pure seawater) and 0.05% to 0.2% surfactant, wherein the surfactant is preferably sodium dodecyl sulfate (SDS).

[0073] In one embodiment, after the lysis phase, the extracted PHA is then purified by immersing the PHA and any impurities (e.g., cell debris) in a separate aqueous solution. In one embodiment, the volume of the aqueous solution used is preferably approximately the same as the volume of the aqueous solution used for lysis. After purification, the PHA is preferably extracted by filtration and / or centrifugation of the aqueous solution containing the PHA. For the purposes of describing the invention herein, this purification and extraction process is defined as the 'purification phase'.

[0074] In one embodiment, the purification stage is performed once or more, preferably twice. The composition of the water-based solution used can be adjusted for each purification stage depending on the number of purification stages. Preferably, the water-based solution contains 50% to 100% seawater, and more preferably, for each stage except the final stage, the water-based solution contains 100% seawater. The final stage preferably contains tap water, distilled water, or deionized water, which is purchased or obtained on-site, including by reverse osmosis or evaporation processes applied to seawater or freshwater sources.

[0075] The purified PHA can be further processed to remove impurities and dried. Drying is preferably carried out between about 50°C and 80°C, more preferably at about 60°C until a constant mass is achieved, and vacuum drying is preferred.

[0076] PHA can be blended with other ingredients, granulated, and / or extruded into fibers.

[0077] The method of the present invention described herein is further different from other methods described in the art due to the following advantages:

[0078] First, the method of this invention uses cultivated or farmed macroalgae instead of terrestrial crops or waste streams as feedstock. In doing so, PHA originates from crops that do not rely on unsustainable synthetic fertilizers or scarce resources such as arable land or freshwater. Furthermore, since seaweed can be sustainably farmed over vast ocean areas, it does not suffer from the scalability issues typically associated with the distributional nature of waste feedstocks such as whey, waste lipids, waste sugar streams, and crop residues.

[0079] Given the climate and plastic pollution crises, there is a clear need to reduce the world's dependence on fossil fuel-based chemicals and fuel commodities; algal biorefineries offer an attractive solution. In this regard, seaweed has the potential to produce billions of tons of biomass annually and provide a sustainable supply of affordable health products such as food, feed, fuel, and biopolymers. Many seaweeds are rich in sugars, contain very little recalcitrant lignin, and grow rapidly, estimated to be at least three times faster than sugarcane and other terrestrial crops currently used for PHA production. Furthermore, several seaweed species are already being cultivated on a large scale for hydrocolloid production and human consumption. This is significant not only because there is an readily available supply of cultured seaweed to accelerate adoption, but also because the cultivation of these species is proven and scalable.

[0080] Secondly, since seaweed contains salt, as one embodiment, the method of the present invention employs brine hydrolysis and halophilic microorganisms to produce biopolymers via marine fermentation. Because salt generally inhibits microbial growth, the method of the present invention results in a reduced risk of contamination during the production process (from raw material storage to hydrolysis and fermentation). Therefore, the method of the present invention allows for continuous fermentation systems for PHA production with virtually no downtime for (1) sterilization and cleaning and (2) the microbial lag period between fermentation batches (which is both expensive and slow). Importantly, the process can also use seawater instead of freshwater as a water source, thereby improving sustainability.

[0081] Third, for many PHA producers today, the downstream extraction process is a key scalability barrier because it typically requires large quantities of toxic chemicals and solvents (e.g., chloroform) to extract PHA from cells. These chemicals can (1) cost more than a third of the total cost of PHA production, (2) reduce the purity of the PHA polymer, and (3) generate hazardous waste streams. Therefore, it is valuable that the method of the present invention uses a clean, water-based approach to extract PHA. In particular, since salt is present within halophilic cells (e.g., halophilic archaea and halophilic bacteria), low-salinity liquids (e.g., fresh water and seawater) can be used to lyse the cells through osmotic shock, releasing the PHA particles they contain and minimizing or eliminating the need for other chemicals or solvents.

[0082] Fourth, the method of this invention produces valuable byproducts without compromising PHA production yield. The first byproduct after hydrolysis is processed seaweed biomass with a high protein content, which can be used for human consumption and / or animal feed, including in aquaculture and fish feed due to its salt content. The second byproduct is a hydrocolloid-rich extract that can be sold to hydrocolloid underwriters for the production of alternative biomaterials and / or breakdown into additional fermentable sugars. The third and final byproduct is lipids extracted from macroalgae prior to hydrolysis. These lipids typically contain ω3 and ω6 and can be used in applications such as pharmaceuticals and nutritional supplements. Commercializing these byproducts could yield significant economic and environmental returns, such as replacing animal-derived products with vegan goods.

[0083] It is worth emphasizing that removing hydrocolloids upstream of macroalgae—for example, removing ulva-glucan from green macroalgae (Phaeophyta), carrageenan and agar from red macroalgae (Rhodophyta), and alginate from brown macroalgae (Phaeophyta)—reduces the potential chemical complexity of macroalgae biomass before hydrolysis. This results in more homogeneous macroalgae hydrolysis products. Moreover, importantly, this means that the method of the present invention (1) is more scalable than other PHA methods because it can be adapted to a variety of feedstocks, including red, green, and / or brown algae varieties, and (2) produces PHA products with more consistent molecular weight and copolymer composition (e.g., resulting in variations in PHA product composition compared to other PHA producers using variable organic waste streams).

[0084] Finally, and importantly, the method of this invention typically removes / offsets more carbon pollution than is emitted during the production process. A key reason for this is that macroalgae release carbon-containing biomass into the water in which they grow, with some of the debris being trapped in sediments beneath the farm and / or deep sea. Furthermore, brine fermentation reduces sterilization requirements, resulting in significantly lower CO2 emissions compared to the intense sterilization typically required to support freshwater fermentation. Consequently, the method of this invention not only produces carbon-friendly PHAs that can replace carbon-intensive plastics and bioplastics, but also simultaneously produces byproducts that can replace carbon-intensive commodities such as meat products and animal feed. Overall, this makes the method of this invention 'carbon-negative,' thus producing 'carbon-negative' PHAs.

[0085] In this regard, the method of the present invention includes effective tools for carbon sequestration and pollution mitigation. Environmentally friendly methods can be used to cultivate seaweed (e.g., species currently cultivated on a large scale, such as *Gracilaria*, *Kappa*, and *Euphorbia*). Furthermore, in one embodiment, wet seaweed can be used instead of dried seaweed, which reduces the energy and land requirements associated with industrial drying and solar drying, respectively. Finally, commercial seaweed production typically generates many major positive externalities, such as providing food, habitat, and refuge for marine animals, ocean oxygenation, eutrophication remediation, reduction of harmful algal bloom events, reduction of greenhouse gas emissions from feeding herbivores, and job creation. Attached Figure Description

[0086] The invention will now be described by way of example with reference to the accompanying drawings, in which:

[0087] Figure 1 Chemical analysis report of hydrolysis products of macroalgae.

[0088] Figure 2 Table showing the salt composition of ATCC medium 1176 (Halobacter broth) is provided.

[0089] Figure 3 The composition of (A)SL-6 and (B)SL-4 micrometal solutions added to growth media based on macroalgae hydrolysis products is shown.

[0090] Figure 4 The bar graph shows the sugar yield of fresh, wet macroalgae (Gracilaria) hydrolysis at different salinity, temperature, and reaction time levels.

[0091] Figure 5 Growth of Mediterranean halophytes on various sugar substrates.

[0092] Figure 6Growth of Mediterranean halophytes in diluted solutions of Gracilaria hydrolysate pretreated with (A) citric acid and (B) sulfuric acid (33%, 25%, 17% strength).

[0093] Figure 7 Growth of Mediterranean halophyte in (A) Gracilaria hydrolysate dilutions (50%, 33%, 25%, and 17% strength) with (B) yeast extract added at 2.5 g / L.

[0094] Figure 8 Changes in glucose concentration and absorbance (280 nm) during carbon treatment of Gracilaria hydrolysis products.

[0095] Figure 9 Growth of Mediterranean halophytes in diluted hydrolysates of Gracilaria hydrolysate (50% and 33% strength) with and without carbonization.

[0096] Figure 10 PHBV composition (% of dry weight) of cells in reactors 1, 2 and 3 at selected time points (121 hours) during the three-stage continuous culture period.

[0097] Figure 11 Photographs showing the color difference of agar extracted from seaweed when using fresh water (left) and seawater (right).

[0098] Figure 12 Bar graph of 5-hydroxymethylfurfural (5-HMF) levels (in g / L hydrolysis products) measured after (1) acid, (2) excess lime and (3) carbon pretreatment.

[0099] Figure 13: Growth curves of Mediterranean halotrophic bacteria grown in 30 mL cultures in shaker incubators (40°C, 150 rpm) with different growth media. (A) shows data using growth media prepared with undiluted seaweed hydrolysate, and (B) shows data using growth media with diluted seaweed hydrolysate (50% strength). The different curves represent different levels of yeast extract added to the flask cultures, from 2 g / L (black line) to 1 g / L (dark gray), 0.3 g / L (medium gray), and 0 g / L (light curve).

[0100] Figure 14 Bar graph showing the final PHBV (dark gray) and optical density (light gray) readings of Mediterranean halotrophic bacteria grown in different growth media. The X-axis represents whether the growth medium was prepared from diluted or undiluted seaweed hydrolysate, and the concentration (g / L) of yeast extract in this growth medium.

[0101] Figure 15: Time series curves of growth and PHBV production (A) and nutrient consumption (B) of semi-continuous fermentation using Mediterranean halophytes in a 4L bioreactor.

[0102] Figure 16 : Bar graph of biorefining byproducts with (dark gray) and without (light gray) lipid extraction steps. From right to left: grams of lipid extract, dry agar, glucose in hydrolysate, and protein in solids per 100 grams of dried seaweed biomass.

[0103] Figure 17 Total protein, carbohydrates, and lipids / fat per 100g of dried seaweed biomass. Dark gray bars show results for raw / initial seaweed biomass, while light gray bars show results for waste seaweed biomass / final solids.

[0104] Figure 18 The growth curves of halophilic bacteria in hydrolysate culture media produced under lipid extraction (dark) and lipid extraction-free (light) conditions were monitored by optical density measurement.

[0105] Figure 19 Photographs of particle samples after initial washing with solutions containing seawater of varying strengths. From left to right: 0% (100% RO water; Sample 1), 12.5% ​​(Sample 2), 25% (Sample 3), 37.5% (Sample 4), 50% (Sample 5), 62.5% (Sample 6), 75% (Sample 7), 87.5% (Sample 8), and 100% (pure seawater - 35 ppt; Sample 9). Particle color ranges from deep pink (Sample 1; 100% fresh water) to very white (Sample 9; 100% seawater). Detailed Implementation

[0106] To provide a more precise understanding of the subject matter of this invention, features of the invention will now be discussed with reference to one or more preferred embodiments.

[0107] First preferred implementation scheme

[0108] Hydrothermal hydrolysis and batch fermentation

[0109] Macroalgae collection and pretreatment. In a first preferred embodiment of the method of the invention, Gracilaria ('macroalgae') growing in the estuary of the Swan River (Western Australia) are manually collected.

[0110] Collected macroalgae biomass taken from the Swan River estuary was cleaned by using saline water from the Swan River to wash macroalgae on-site, removing debris from the surface of the macroalgae.

[0111] The cleaned macroalgae were then transported to the laboratory for processing. During transport, the macroalgae were kept at a temperature below ambient temperature in a cooler filled with ice blocks.

[0112] In the laboratory, large algae were manually drained using a 20cm diameter vegetable dehydrator made of plastic. Seawater, which served as the source of the brine solution, was then added to the drained algae, which was then mixed into small particles by mixing for 2 minutes at the highest available rpm in a mechanical food mixer (a NINJA mixer with an Auto-iQBN495UK).

[0113] The amount of water added to the drained macroalgae was calculated based on the natural water content of the macroalgae (by measuring the wet and dry weight of the collected macroalgae), resulting in a macroalgae mixture containing a mixed macroalgae slurry with a solids loading composition of approximately 10%. Using Gracilaria, approximately 200 ml of 'artificial' seawater was added per 500 g of fresh, wet (but drained) macroalgae biomass. The artificial seawater was produced by using sea salt in reverse osmosis deionized water to achieve a salinity of 35 ppt.

[0114] Refrigerate the large algae mixture (approximately 4°C) until further use.

[0115] Hydrolysis: In a subsequent step of the method of the present invention, subcritical water hydrolysis (sub-CW) is used to hydrolyze the macroalgae mixture. Sub-CW is an 'environmentally friendly' method that utilizes subcritical water to extract compounds of interest from macroalgae. Subcritical water comprises water under high pressure and at a temperature between approximately 100°C (the boiling point of water) and 374°C (the critical temperature of water).

[0116] Sub-CW hydrolysis was carried out in a 1L reactor equipped with an electric heater, magnetic stirrer, simulated barometer, temperature probe and controller.

[0117] The macroalgae mixture was hydrolyzed in batches using a sub-CW method. For each batch, 700g of macroalgae mixture was added to the reactor, and some air was forced out of the reactor for 1 minute using an industrial vacuum pump (Sparmax TC-63 Dry Piston–Single Head Vacuum Pump), after which the reactor was sealed tightly.

[0118] Throughout the hydrolysis process, a cooler using water at approximately 10°C is employed to cool the mechanical agitator and prevent overheating of the shaft bearings. Before each batch of hydrolysis, a vacuum pump is used to expel air from the reactor.

[0119] Set the reactor temperature to 175°C, stir at 280 rpm, and react for 15 minutes.

[0120] After each batch of macroalgae mixture is hydrolyzed, the hydrolyzed macroalgae mixture is removed from the reactor and centrifuged at approximately 4000g to separate the aqueous phase ('macroalgae hydrolysis product') from the residual solids.

[0121] The hydrolysis products of large algae were filtered through a glass fiber filter (GFC; pore size 0.22 μm) using a vacuum filtration unit to prepare a fermentation broth.

[0122] The residual solids were dried at approximately 60°C for 48 hours to constant weight and then stored for future analysis and potential use as biochar or fertilizer.

[0123] The hydrolysate from the macroalgae was then analyzed (by the Government of Western Australia Chem Centre), which determined that the hydrolysate contained 10.15 g / L fermentable sugars, 1.44 g / L protein nitrogen, and 2 g / L 5-hydroxymethylfurfural (5-HMF). The results are as follows: Figure 1 As shown in the report provided by the Chemistry Center.

[0124] Further analysis by the University of Western Australia determined that the monosaccharides in the hydrolysate contained 10.3% xylose, 12.7% mannose, 51.6% galactose, and 25.4% glucose.

[0125] Microorganisms used for fermentation: The microorganisms used in the fermentation step of the method of the present invention to produce PHA derived from macroalgae are the Mediterranean haloarcha from the American Type Culture Collection (ATCC33500).

[0126] Mediterranean halophilic bacteria can produce PHA from a variety of different carbon feedstock types, and it also has the ability to naturally produce PHBV (poly(3-hydroxybutyrate-co-3-hydroxyvalerate)), a type of PHA polymer with enhanced mechanical and physical properties. For example, PHBV is typically an order of magnitude more elastic than PHB (polyhydroxybutyrate).

[0127] Mediterranean halophilic bacteria thrive in very high salinity environments and have been shown to accumulate more polymers than other halophilic microorganisms.

[0128] The salinity in the Mediterranean halophilic bacteria growth medium is high enough that few other microorganisms (if they may be nearby) can grow to any significant quantity anywhere at a rate as high as or close to that achieved by the Mediterranean halophilic bacteria.

[0129] Therefore, the requirements for aseptic conditions are greatly reduced when using Mediterranean halophilic bacteria, and extremely simple production systems can be developed, as an example including open ponds similar to those used in wastewater treatment.

[0130] Fermentation: In a subsequent step of the method of the present invention, the fermentation of the hydrolysate of large algae is carried out in batches in a 6L Minifors 2 bioreactor (4L working volume).

[0131] Large-volume growth media based on macroalgae hydrolysate were prepared by mixing 1 L of macroalgae hydrolysate with 1.25 g / L of yeast extract (to increase nitrogen and other nutrient levels in the culture medium) and 3 L of water (to reduce the concentration of fermentation inhibitors (e.g., 5-HMF)) to form a fermentation culture.

[0132] Adjust the pH of the resulting fermentation culture to approximately 7.0. Increase the salinity of the fermentation culture to approximately 170 ppt by adding salt at a ratio equivalent to ATCC 1176 (Halobacter tumefaciens medium). Figure 2 As shown.

[0133] When using cheese whey as a carbon source, such as Figure 3 The SL-6 solution shown has been shown to improve galactose uptake by Mediterranean halotrophic bacteria. To utilize this finding, 0.1% SL-6 solution was added to the fermentation culture.

[0134] The bioreactor temperature was set at 40°C. The pH of the macroalgae fermentation culture was maintained at approximately 7.0 by automatically controlled addition of 5% (w / v) NaOH as alkali and 5% (w / v) H2SO4 as acid.

[0135] Dissolved oxygen in the fermentation culture was maintained at approximately 20% by automatically controlling the stirring speed between approximately 200 rpm and 650 rpm and by aeration up to approximately 6 L / min.

[0136] Defoamer A The automatic control of the addition is used to suppress foaming of the stirred fermentation culture in the bioreactor.

[0137] Batch fermentation in the bioreactor was initiated by adding approximately 2.5% v / v Mediterranean halotrophic bacteria inoculum to the fermentation culture, which is equivalent to approximately 100 ml at a culture density of 50 g / L.

[0138] Fermentation batches of the fermentation culture were carried out in a bioreactor for 96 hours.

[0139] Polymer extraction and identification. The next step after fermentation is polymer extraction of the fermentation batch, performed under hypotonic shock.

[0140] Centrifuge the fermentation culture at 4307g for 15 minutes.

[0141] Discard the supernatant and wash the remaining cell pellet with 10% w / v NaCl solution.

[0142] The cell pellet was then washed repeatedly with deionized water (at a ratio of 1 g of wet cell mass per 20 ml of water) to induce hypotonic shock until a 'white' cell pellet was obtained. During each wash, cell debris lysed during the hypotonic shock was discarded; this debris floated to the surface of the resulting supernatant. Deionized water was then obtained via reverse osmosis.

[0143] The remaining 'white' PHA granules at the end of the washing process are then dried at 70°C until a constant weight is achieved.

[0144] On a spectrometer equipped with an attenuated total internal reflection accessory, at 400 to 4000 cm⁻¹ -1 The Fourier transform infrared (FT-IR) spectra of PHA in the dried cell pellet were measured within the spectral range. Measurements confirmed that the extracted PHA contained PHBV.

[0145] Adjusting hydrolysis variables to improve sugar recovery

[0146] A key step in the conversion of seaweed biomass into PHA is the depolymerization of polysaccharides to produce monosaccharides. These sugars are the carbon source for PHA production through fermentation.

[0147] The current experiment uses subcritical water (sub-CW) hydrolysis to extract sugars from seaweed biomass, which does not involve the use of any dangerous and / or expensive chemicals, and is carried out in water heated to a relatively mild temperature (100°C-240°C).

[0148] In the first preferred embodiment of the method of the present invention described above, the parameters previously used include:

[0149] • Reaction temperature: 170℃;

[0150] • Solid loading: 10% w / v (obtained by mixing 500g of fresh, wet Gracilaria with 200ml of artificial seawater);

[0151] • Salinity of water containing macroalgae: 35 ppt;

[0152] • Reaction time: 15 minutes (plus heating and cooling time). The heating and cooling times are relatively long, approximately 70 minutes and 30 minutes respectively (for subsequent experiments, these heating and cooling times should be as short as possible, as they may impair the quality of the resulting seaweed hydrolysate).

[0153] • Sugar and nitrogen content of hydrolysate – The protein nitrogen content of the seaweed hydrolysate was determined to be 0.14% by chemical center analysis; chemical center and UWA HPLC analysis showed that the seaweed hydrolysate had a total sugar content of approximately 10 g / L.

[0154] In further experiments, different parameters were changed to improve the sugar recovery rate obtained from Gracilaria via sub-CW hydrolysis.

[0155] The constant parameters included Gracilaria species from the Swan River and a mixture of large algae with a solids load of approximately 10% w / v.

[0156] The changing parameters include:

[0157] • Reaction temperatures: 130℃, 170℃, 210℃;

[0158] • Reaction time = 0, 5, 10, 15, 20, 25, 30, 35 minutes at the target temperature; and

[0159] • The salinity of the macroalgae mixture water was 0, 35, 70, and 140 ppt. These salinities are of relevant operational significance for determining the process of the water composition, which more preferably acts as a catalyst for breaking down algal carbohydrates into fermentable sugars.

[0160] Seaweed Collection. Gracilaria specimens were manually collected at Point Resolution (Swan Estuary) and placed in a cooler containing local water and ice for transport. Upon arrival at the laboratory, the seaweed biomass was drained using a vegetable spin dryer, placed in resealable plastic bags (approximately 200g per bag), and stored at -20°C until use.

[0161] The experiment involved 12 batch reactions, each using 500g of fresh Gracilaria (see details under 'Hydrolysis' below). Therefore, 6kg of Gracilaria biomass (wet weight) was collected.

[0162] Preparation of macroalgae mixtures. To prepare hydrolyzed samples, wet seaweed biomass was thawed and divided into four 1.5 kg samples. Each of these samples was mixed with water to form a macroalgae mixture, which was then finely ground using a mechanical mixer to form a macroalgae slurry.

[0163] Water to be added to seaweed biomass was prepared using reverse osmosis deionized (RODI) water and red sea salt. Four different solutions with varying salinity were prepared and added to the seaweed biomass to prepare four types of large algae slurries:

[0164] Solution 1:0ppt

[0165] Solution 2: 35ppt

[0166] Solution 3: 70 ppt

[0167] Solution 4: 140ppt

[0168] The amount of each solution to be added to the seaweed is calculated based on the natural seaweed water content (by measuring the wet and dry weight of the collected seaweed), resulting in a slurry with a solids loading of ~10% (w / v). In the case of Gracilaria, approximately 200 ml of water is added per 500 g of wet seaweed biomass. These seaweed slurries are stored at -20°C until further use.

[0169] Hydrolysis: Sub-CW hydrolysis was carried out in a 1L reactor equipped with an electric heater, mechanical stirrer, simulated pressure gauge, temperature probe, and controller. The stirring speed was set to 70 rpm.

[0170] For each batch, add 700g of seaweed slurry to a 1L reactor, seal the reactor tightly, and then use an air pump to force some air out of the reactor. Throughout the hydrolysis process, use a cooler (10°C water) to cool the bearings of the mechanical agitator.

[0171] Twelve hydrolysis runs were conducted, each with a unique combination of temperature, salinity, and reaction time. For each run, ten samples were collected throughout the reaction to determine the optimal residence time. Sample 1 was the original seaweed slurry (used to calculate sugar yield), sample 2 was collected when the reaction temperature reached 50% of the target temperature, and samples 3 through 10 were collected at reaction times of 0, 5, 10, 15, 20, 25, 30, and 35 minutes, respectively.

[0172] Analysis. The sample was filtered through a 0.2 μm filter. The hydrolysis products of macroalgae were stored at -20 °C, and the solids were dried at 60 °C for 48 hours and then stored at ambient temperature.

[0173] The University of Western Australia analyzed the amount of total soluble sugars in the hydrolysates of macroalgae and showed that... Figure 4 In the bar chart.

[0174] When compared with results at 130°C or 210°C, the optimal temperature for producing the highest sugar content after hydrolysis is shown to be approximately 170°C.

[0175] Compared to 0 ppt, 35 ppt, and 70 ppt, the optimal salinity for producing the highest sugar content after hydrolysis is shown to be approximately 140 ppt.

[0176] Second preferred implementation scheme

[0177] Enzymatic hydrolysis and continuous fermentation

[0178] Source of macroalgae: In a second preferred embodiment of the method of the present invention, macroalgae (seaweed) of the genus Gracilaria, grown in the waters near the islands of Indonesia, are manually collected. The collected macroalgae are dried to reduce their transport weight, and then packed in vacuum-sealed bags and transported by container to Perth, Australia.

[0179] Pre-treatment: Once received at the processing facility in Perth, Australia, the large algae are stored in a dry, dark, and refrigerated environment until processing.

[0180] Large algae were freeze-dried in a freeze dryer (model: BK-FD10S, Biobase Biodustry (Shandong) Co., Ltd.) that included a cooling trap operating at -65°C. Approximately 15% of the original weight was lost in this step due to the removal of moisture from the algae.

[0181] Before proceeding to the hydrolysis stage, it is necessary to reduce the size of the original large algae material, as it arrives in large chunks. First, the large algae are shredded in a shredder (model: RSH2445S, Ryobi) to obtain large algae particles of approximately 5 cm or smaller.

[0182] Then, a second size reduction is carried out in a food processor (model: Blixer 7v.v., Robot Coupe) to reduce the size of the large algae to particles equal to or less than about 1 mm, i.e., in the form of powdered large algae.

[0183] Hydrolysis: The hydrolysis of macroalgae combines acid and enzymatic methods.

[0184] A batch of hydrolysis products was prepared in a hydrolysis reactor (model: FCF-10L, Zhengzhou Keda Machinery and Instrument Equipment Co., Ltd.) using 7 liters of fresh water and 700 grams of seaweed powder (i.e., 10% w / v loading).

[0185] Add citric acid solution (25mM, ~4.8g citric acid powder / L solution) to the hydrolysis reactor.

[0186] The mixture is then preheated at 120°C and mixed for 2 hours.

[0187] After preheating, the temperature in the hydrolysis reactor is reduced to 55°C by circulating ethylene glycol through the internal coils of the hydrolysis reactor. Once the temperature is adjusted to 55°C, the pH is adjusted to 5 by adding approximately 14 ml of 2M NaOH solution per liter of solution.

[0188] A 1:1 mixture of Viscozyme and Celluclast enzyme (approximately 1.1 g / ml) was added to the hydrolysate (approximately 0.2 ml per gram of seaweed) and the reaction mixture was stirred at 200 rpm for 24 hours.

[0189] After the 24-hour reaction time is complete, the next step is to remove the solids from the hydrolysis reaction mixture. This is done by centrifuging the hydrolysis reaction mixture at 15,000 g (model: 5910-R, Eppendorf) for 30 minutes. The macroalgae hydrolysis products are then decanted from the centrifuged hydrolysis reaction mixture.

[0190] Some fermentation inhibitors are commonly generated during the hydrolysis of macroalgae, such as when sugars are degraded, and others are already present, for example, for plant defense. These inhibitors may include 5-HMF, organic acids, and polyphenolic compounds (e.g., flavonoids). To remove such inhibitors, the macroalgae hydrolysate is treated with charcoal in a hydrolysis reactor for 60 minutes, wherein 175 g of granular charcoal (2.5% w / v) is added to the macroalgae hydrolysate. The used charcoal is then removed by centrifugation at 15000 g for 30 minutes.

[0191] The completion of the hydrolysis phase involves the addition of salt to produce a suitable fermentation medium. Salt is added to the hydrolysis products to obtain the composition provided in ATCC medium 1176 (e.g., Figure 2 As shown, without yeast extract or additional glucose), then add 1 ml of SL-4 trace metal mixture per liter ( Figure 3 The salt is dissolved by mixing at 200 rpm in a hydrolysis reactor.

[0192] The final volume of the hydrolysate from the macroalgae was 7.5 L, and the glucose concentration was 20 g / L.

[0193] Fermentation: The fermentation system is a continuous fermentation system, consisting of four fermentation bioreactors (5L capacity):

[0194] Bioreactor 1: Minifors 2, Infors HT

[0195] Bioreactor 2: 5L glass fermenter, Shanghai Bailun Biotechnology Co., Ltd.

[0196] Bioreactor 3: 5L glass fermenter, Shanghai Bailun Biotechnology Co., Ltd.

[0197] Bioreactor 4: 5L glass fermenter, Shanghai Bailun Biotechnology Co., Ltd.

[0198] Four fermentation bioreactors have independent feed solutions. The feed solutions consist of a diluted solution of macroalgae hydrolysate mixed with salt, as described above. The hydrolysate is diluted with a salt-containing solution at a concentration equivalent to ATCC medium 1176 (…). Figure 2 Add in the proportion of (without yeast extract or glucose), and add 1 mL of SL-4 trace metal mixture per liter according to Table 1 (e.g., ...). Figure 3 (As shown). The purpose of the fourth bioreactor is to collect cells for harvesting and extracting PHA.

[0199] Feeding Strength of hydrolysis products (%) Bioreactor 1 50％ Bioreactor 2 75％ Bioreactor 3 100％ Bioreactor 4 No feed

[0200] Table 1. Composition of bioreactor feed.

[0201] The bioreactors were initially filled with 3.5 L of fermentation medium consisting of 50% strength hydrolysate. Each bioreactor was inoculated with approximately 6% v / v of Mediterranean halophilic bacteria and operated in batch mode until glucose was depleted. The bioreactors were then connected and operated in continuous mode, with feed flow rates and operating conditions as shown in Table 2.

[0202] Bioreactor 1 Bioreactor 2 Bioreactor 3 Bioreactor 4 Feed flow rate (L / h) 0.087 0.022 0.022 - <![CDATA[Dilution rate (h -1 )]]> 0.025 0.031 0.037 - Working volume (L) 3.5 3.5 3.5 0-4 Temperature (°C) 40 40 40 40 pH 7 7 7 7 Dissolved oxygen (%) 40 40 40 40

[0203] Table 2. Feed flow rates and operating conditions for the four bioreactors.

[0204] Harvesting: The fermentation broth was periodically removed from bioreactor 4. The biomass was then separated from the fermentation broth by centrifugation at 15000g (model: 5910-R, Eppendorf) for 15 minutes. Approximately 1% of the solution was recovered in the biomass.

[0205] After centrifugation, the biomass was processed in a cell lysis reactor (model: 5L glass fermenter, Shanghai Bailun Biotechnology Co., Ltd.), in which a 0.1% sodium dodecyl sulfate (SDS) solution was added. The amount of SDS solution added was equal to the amount of fermentation broth removed from centrifugation (i.e., 3.5L). Cell lysis was carried out for 1 hour using a magnetic stirrer at 1100 rpm.

[0206] The solution was centrifuged at 15000g (model: 5910-R, Eppendorf) for 15 minutes to recover PHA.

[0207] To remove any residual SDS, a washing step was performed by adding 3.5 L of fresh water to the PHA precipitate after removing the fermentation broth and stirring the solution with a magnetic stirrer at 1100 rpm for 1 hour. The solution was then centrifuged again at 15000 g for 15 minutes in a centrifuge (model: 5910-R, Eppendorf) to recover PHA.

[0208] Approximately 30% of the recovered biomass corresponds to collected PHA.

[0209] The harvesting stage is completed by removing the remaining moisture from the PHA particles in a vacuum oven at 60°C.

[0210] Determine the preferred conditions for the processing steps according to the second preferred embodiment.

[0211] Growth of Mediterranean halophilic bacteria on different carbon sources. Since the sugar subunits that make up macroalgal polysaccharides differ among red, green, and brown algae, experiments were conducted to determine how different sugars serve as substrates for the growth of Mediterranean halophilic bacteria, thus providing information for algae selection for PHA production.

[0212] Nine different monosaccharides (glucose, rhamnose, arabinose, galactose, xylose, glucuronic acid, mannitol, fucose, and mannose) found in macroalgae polysaccharides were selected. Growth media were prepared by adding 10 g / L of each monosaccharide to a basal medium consisting of ATCC medium 1176. Figure 2 (Preparation without glucose). Transfer 60 mL of each culture medium to a 250 mL flask and inoculate with Mediterranean halotrophic bacteria (grown overnight in ATCC 1176) until the optical density (600 nm; OD) is reached. 600 The value was approximately 0.5. The flask was incubated in a shaker at 40°C and 150 rpm for 180 hours, and the result was determined by OD. 600 Measurements are used for monitoring.

[0213] Observed growth curves ( Figure 5 This indicates that Mediterranean halophilic bacteria exhibit robust growth on glucose. Galactose was the second best substrate. Mannose, mannitol, and xylose showed similar performance, with only minor additional growth observed compared to the control culture (sugar-free substrate preparation). Arabinose, fucose, rhamnose, and glucuronic acid showed no significant differences compared to the control culture, which may indicate that Mediterranean halophilic bacteria cannot utilize these substrates.

[0214] The results showed that both green (chlorophyll) and red (rhophyll) macroalgae were the most suitable raw materials for the production of PHA using Mediterranean halophilic bacteria. The main monosaccharide produced by *Ulva* (green algae) is glucose, while *Gracilaria* (red algae) typically produces galactose primarily, followed by glucose.

[0215] Enzymatic hydrolysis—enzyme type and concentration

[0216] Experiments were conducted using different mixtures and concentrations of Viscozyme and Celluclast enzyme preparations (Novozymes) to select the enzyme treatment that provides high glucose yield while minimizing cost. Experiments were prepared by acid pretreatment (25 mM citric acid, 120°C, 2 h) of 10% (w / v) solids-loaded seaweed in an autoclave. The samples were cooled to 55°C, adjusted to pH 5 with 2 M NaOH, and then treated with the enzyme in a shaking water bath (55°C, 130 rpm) for 17 h.

[0217] The results (Table 3) showed that a high glucose concentration (19.6 g / L) was achieved with a 1:1 blend of Viscozyme and Celluclast at 0.2 mL / g biomass. Similar results (20.0 g / L) were obtained using a significantly higher enzyme loading (0.5 mL / g of a 1:3 blend of Viscozyme and Celluclast), indicating that the benefit gained from increasing the enzyme loading above 0.2 mL / g is negligible. While using only 0.2 mL / g of Viscozyme could provide results comparable to the 1:1 blend (19.2 g / L), a combination of cellulase blends with different cellulose-degrading activities is preferred because the structure and biochemical composition of seaweed can vary considerably due to taxonomy, seasonality, and location.

[0218] Enzyme treatment Glucose concentration (g / L) 0.5 mL / g Celluclast 17.8 0.2 mL / g Viscozyme 19.2 0.1 mL / g Viscozyme 15.0 0.5 mL / g 1:3 mixture (Viscozyme:Celluclast) 20.0 0.2 mL / g 1:1 mixture (Viscozyme:Celluclast) 19.6

[0219] Table 3. Glucose concentration of the final hydrolysis product of different enzyme mixtures.

[0220] Enzymatic hydrolysis—salinity

[0221] Enzymatic hydrolysis of biomass pretreated in both freshwater and seawater was used to evaluate the feasibility of using seawater to produce seaweed hydrolysates. Experiments were conducted by acid pretreatment of 10% (w / v) solid-loaded dried seaweed (Gracilaria sp.) in either freshwater (0 ppt salinity; prepared by reverse osmosis) or seawater (35 ppt salinity; obtained from Waterman's Bay, Australia) in an autoclave (25 mM citric acid, 120 °C, 2 h). The samples were then cooled to 55 °C, adjusted to pH 5 with 2 M NaOH, and treated with an enzyme (a 1:1 mixture of 0.2 mL / g Viscozyme and Celluclast) in a shaking water bath (55 °C, 130-130 rpm).

[0222] This experiment demonstrates that enzyme activity is impaired in seawater, with the glucose concentration in the seawater hydrolysate sample reaching 65±2% of that in the freshwater hydrolysate sample.

[0223] Pretreatment with strong and weak acids

[0224] Since the choice of acid type and concentration during biomass pretreatment can affect the performance of enzymatic hydrolysis (through the disruption of biomass structure) and the fermentation capacity of hydrolysates (through the generation of inhibitors), pretreatment using strong acid (sulfuric acid) and weak acid (citric acid) was compared. The performance of enzymatic hydrolysis was assessed by the concentration of glucose in the hydrolysates, and the fermentation capacity was assessed using shake-flask cultures of Mediterranean halophilic bacteria.

[0225] Experimental preparation was performed by acid pretreatment of 10% (w / v) solid-loaded seaweed in an autoclave with 25 mM citric acid or sulfuric acid (120 °C, 2 h). The samples were cooled to 55 °C, adjusted to pH 5 with 2 M NaOH, and treated with an enzyme (a 1:1 mixture of 0.2 mL / g Viscozyme and Celluclast) in a shaking water bath (55 °C, 130 rpm) for 24 h.

[0226] After enzymatic hydrolysis, the hydrolysates produced by citric acid and sulfuric acid had similar glucose concentrations, with citric acid performing slightly better at the 25 mM concentration used (citric acid and sulfuric acid were 19.5 g / L and 18.1 g / L, respectively).

[0227] To evaluate the fermentation capacity of the hydrolysate, a growth medium was prepared using the hydrolysate. The hydrolysate was adjusted to pH 7 using 2M NaOH and then combined with salts to obtain the following... Figure 2 The composition shown in ATCC medium 1176 (Halobacter broth) is provided (glucose and yeast extract omitted). All fermentation media also contain SL-4 trace metals at a final concentration of 1 mL / L. Figure 3The medium was then diluted to different strengths (33%, 25%, and 17%) with a solution containing salts as provided in ATCC Medium 1176. Figure 2 (Omit glucose and yeast extract). Transfer 60 mL of each dilution to a 250 mL flask and add 2.5 g / L yeast extract. Then inoculate the flask with Mediterranean halophyte (single colony growth in ATCC medium 1176) until the initial optical density (600 nm; OD) is reached. 600 The concentration was ~0.5. The flask was incubated at 40°C and 150 rpm, and the OD value was measured over 112 hours. 600 Measurements are used to monitor growth.

[0228] A significant difference in fermentation capacity was observed between citric acid and sulfuric acid-treated hydrolysates in a hydrolysate culture medium prepared at 33% strength, with the citric acid sample achieving a final OD of 10.0. 600 No growth was detected in sulfuric acid samples of the same dilution. Figure 6 A and Figure 6 B). This indicates that the sulfuric acid-treated hydrolysate contains high levels of inhibitory compounds (e.g., 5-HMF) compared to the citric acid sample. The growth difference between the citric acid and sulfuric acid samples was not significant when the hydrolysate was diluted to a 25% strength, and no growth difference was observed when the hydrolysate was diluted to a 17% strength, further supporting this finding and indicating that the inhibitory compounds were at sub-inhibitory concentrations at the latter dilution.

[0229] Since both types of acid produce hydrolysis products with similar glucose concentrations, citric acid pretreatment appears to result in a significantly lower production of inhibitory compounds compared to sulfuric acid, and is therefore the preferred choice.

[0230] To confirm the production of PHA from hydrolysates generated using citric acid pretreatment, PHA was isolated from cells using hypotonic shock. 50 mL of cell culture was centrifuged (4500 rpm, 25 min, 4 °C) and the supernatant was discarded. The cell pellet was then resuspended in 50 mL of a solution containing 0.1% SDS (0 ppt) and stirred to lyse the cells. PHA particles were collected by centrifugation (4500 rpm, 25 min, 4 °C) and washed twice with deionized water. The PHA was then dried overnight at 60 °C and weighed.

[0231] The PHA concentrations determined by weighing are listed in Table 4 below.

[0232] Dilution Extracted PHA (g / L) 33％ 1.48 25％ 1.52 17％ 1.20

[0233] Table 4. PHA concentration in Mediterranean halotrophic bacteria cultures grown on hydrolysates treated with different dilutions of citric acid.

[0234] Effects of dilution of yeast extract and hydrolysate

[0235] Experiments were conducted to investigate the effects of hydrolysate dilution and yeast extract supplementation on the growth of *Saccharomyces cerevisiae*. Hydrolysate dilution mitigated the effects of inhibitory compounds on cell growth but also reduced the concentrations of sugars and nitrogen in the hydrolysate, which could limit growth rate and final cell density. Yeast extract provides a rich source of nitrogen, carbon, and other nutrients but increases cost.

[0236] Hydrolysates were prepared as described in previous experiments (pretreated at 120°C with 25 mM citric acid at 10% solids loading for 2 h; enzymatic hydrolysis was performed with a 0.2 mL / g 1:1 mixture of Viscozyme and Celluclast; ATCC 1176 salts were added). The solutions containing salts as provided in ATCC 1176 (without yeast extract or glucose) were then diluted at 50%, 33%, 25%, and 17% strengths, and 30 mL volumes were transferred to 150 mL flasks. Two samples were prepared for each dilution, one with 2.5 g / L yeast extract and the other without. The flasks were then inoculated with Mediterranean halophytes (grown from single colonies in ATCC 1176) to the initial OD. 600 The value was approximately 0.5. The flask was incubated at 40°C and 150 rpm, and the OD value was measured over 125 hours. 600 Measure and monitor growth.

[0237] The dilution of hydrolysis products and yeast extract were observed to have a significant effect on cell growth. Figure 7 A and Figure 7 (B) At the highest hydrolysis product strength of 50%, no growth was observed with or without yeast extract, likely due to the high levels of fermentation inhibitors. At 33% strength, Mediterranean halophilic bacteria could only grow in the presence of yeast extract, suggesting that the nutrients provided by the yeast extract mitigated the effects of the inhibitory compounds. Compared to the 33% strength, cells with yeast extract grew significantly faster at 25% and 17% strengths, likely due to fewer inhibitors, but the final cell density was lower, possibly due to less glucose at these dilutions. Compared to the corresponding samples with yeast extract, the 25% and 17% strength samples without yeast extract showed significantly slower growth and lower final cell density.

[0238] The results indicate the importance of inhibitory compounds present in the hydrolysis products (whether extracted from seaweed (e.g., phenolic compounds) or generated during pretreatment (e.g., 5-HMF)) for the growth of Mediterranean halotrophic bacteria. The observation that yeast extract appears to reduce the inhibitory effect is an important finding worthy of further investigation.

[0239] Carbon treatment

[0240] Experiments were conducted to evaluate the effectiveness of activated carbon (char) in removing inhibitors such as 5-HMF and phenolic compounds from hydrolysis products. First, the hydrolysis products were treated with char, and both the absorbance of the solution at 280 nm (corresponding to phenolic groups) and the glucose concentration (to assess sugar loss during char treatment) were monitored. The effectiveness of the char treatment was then evaluated by monitoring the growth of Mediterranean halotrophic bacteria in shake-flask cultures containing growth media prepared from the hydrolysis products.

[0241] The hydrolysate was prepared as described in previous experiments (pretreated with 25 mM citric acid for 2 h at 10% solids loading at 120 °C; enzymatic hydrolysis was performed with a 0.2 mL / g 1:1 mixture of Viscozyme and Celluclast). The hydrolysate was then combined with 2.5% w / v activated charcoal granules and incubated on a rotary shaker at 150 rpm and 40 °C for 150 min. Samples were periodically removed, the charcoal was removed by centrifugation, and the absorbance at 280 nm was measured (A). 280 Glucose can be tested using glucose monitoring strips (such as the Abbott FreeStyle Optium Neo glucose monitor). Figure 8 As shown, A was observed within 60 minutes. 280 The concentration decreased by approximately 80%, then remained stable for 150 minutes, which may indicate that phenolic compounds had been removed from the hydrolysis products. Glucose concentration decreased by approximately 14% during the treatment.

[0242] Hydrolysates produced without char treatment or after char treatment (2.5% w / v) for 60 minutes were used to prepare growth media by adding ATCC medium 1176 salt as previously described. Growth media were prepared at 50% and 33% strengths and inoculated with Mediterranean halophytes as previously described.

[0243] The effect of carbon treatment is very significant in hydrolysis products with a strength of 50%. Figure 9 No growth was observed without carbon treatment, while carbon treatment allowed significant growth up to the final OD. 600 The value was 17.0. The effect of carbon treatment on 33% strength medium did not have a significant impact on the final cell density, but it may have provided a slightly faster growth rate in the early stages of culture.

[0244] The results indicate that char treatment may reduce the concentration of one or more inhibitors below critical levels, thereby allowing growth with strong hydrolysis product intensity. This allows for the use of culture media with higher levels of algal-derived sugars, providing higher final cell densities for PHBV extraction.

[0245] Large-scale production of hydrolysis products (7L)

[0246] It was demonstrated that the production of Gracilaria hydrolysate was carried out on a 7L scale and used to produce growth medium for Mediterranean halophilic bacteria bioreactor cultures.

[0247] 750g of freeze-dried, ground seaweed (Gracilaria) was placed in a 10L hydrolysis reactor (model: FCF-10L, Zhengzhou Keda Machinery Instrument Equipment Co., Ltd.) and mixed with 7.5L of 25mM citric acid solution. The reactor was sealed and heated to approximately 120°C, as determined by a gauge pressure of approximately 100kPa. The mixture was continuously stirred (200rpm) with a mechanical stirrer and maintained at this temperature for 2 hours. The reactor was then cooled to 55°C using an internal cooling coil.

[0248] Adjust the pH of the mixture to 5 using 2M NaOH. Then add 75 mL each of Viscozyme and Celluclast (0.1 mL per gram of seaweed). Maintain the reactor temperature at 55°C and continuously stir (200 rpm) for 20 hours. Then remove the mixture from the reactor and centrifuge in batches (4500 rpm, 25 minutes) to remove solid biomass.

[0249] The supernatant was then returned to the reactor and combined with 2.5% w / v activated carbon granules. The mixture was stirred for 60 minutes (200 rpm), removed from the reactor, and centrifuged in batches (4500 rpm, 25 minutes) to remove the carbon. It was then stored at 4°C until the bioreactor culture was required.

[0250] The batch produced by this method yielded 7L of liquid hydrolysate with an average glucose concentration of 18.1 ± 1.1 g / L (average of three independent batches).

[0251] Three-stage continuous fermentation system—synthetic culture medium

[0252] A three-stage continuous fermentation system for PHA production was developed and tested. The continuous fermentation system was designed to promote the growth of Mediterranean halophilic bacteria in the first bioreactor, while the second and third bioreactors were primarily used for PHA production. The fourth bioreactor served as a cell collection vessel for periodic harvesting of PHA. Bioreactors 1, 2, and 3 had independent feedstocks to allow control over the culture medium composition in each reactor.

[0253] Use synthetic culture medium (based on ATCC medium 1176) Figure 2 The three-stage continuous fermentation system was inspected. First, the three bioreactors were filled with a solution containing salts such as those provided by ATCC medium 1176 (1 mL of SL-4 metal mixture was added per liter); Figure 3 Fill to a volume of 3.5L and inoculate with a shake flask inoculum of Mediterranean halophilic bacteria to the initial OD. 600 The concentration was approximately 0.5. The bioreactors were operated in batch mode at 40°C for 43 hours, with the pH controlled at the set point of 7.0, and dissolved oxygen maintained at approximately 20% to 40% by adjusting the stirring speed and aeration rate until the glucose concentration in each bioreactor was depleted.

[0254] The system was then switched to continuous mode. Table 5 below provides the total flow rates between the sequential bioreactors and the individual feed flow rates for each reactor. The table also shows the feed composition for each bioreactor. All feed solutions consisted of solutions containing salts as provided in ATCC medium 1176 and with modified glucose and yeast extract concentrations, as described in Table 5.

[0255]

[0256] Table 5. Flow rate and feed composition of the three-stage continuous fermentation system

[0257] PHA produced by Mediterranean halotrophic bacteria in a continuous process is harvested periodically from the fourth bioreactor using the following procedure. 2 to 4 L of fermentation broth is removed from the reactor and centrifuged (10,000 rpm, 15 min) to collect cells. The supernatant is discarded, and the cells are resuspended in the same volume (0 ppt) of a solution containing 0.1% sodium dodecyl sulfate (SDS). The mixture is stirred with a magnetic stirrer (1100 rpm) for 1 hour. The solution is then centrifuged to recover solid PHA, and the supernatant is discarded. The crude PHA is then resuspended in deionized water in the same volume as in the previous extraction step. The mixture is stirred again for 1 hour, then centrifuged and the supernatant is discarded. The washing procedure is performed twice. The washed PHA is then dried in a vacuum oven at 60°C for 24 hours.

[0258] The results of PHA harvests conducted over five consecutive days of continuous cultivation are shown in Table 6 below. The results indicate that PHA yield remained consistent throughout the five days of system operation.

[0259]

[0260] Table 6. PHA harvest yield from the three-stage continuous culture system

[0261] The proportion of PHBV to cell weight was assessed using GC-MS throughout the operation. Typically, it gradually increased from the first bioreactor to subsequent bioreactors, indicating increased intracellular accumulation of PHBV. The PHBV ratio at a selected time point (121 hours) was shown as follows: Figure 10 As shown.

[0262] Three-stage continuous fermentation system—Gnaphalium hydrolysis products

[0263] The three-stage continuous bioreactor system described in previous experiments is suitable for producing PHA using hydrolysates from Gracilaria zeylanica. As mentioned above, the hydrolysates used in the experiments were produced in multiple 7L batches.

[0264] The three-stage reactor system is basically carried out as described above, with the reactor feed consisting of diluted hydrolysis products (in ATCC medium 1176 salt). Figure 2 The composition is shown in Table 7 below.

[0265]

[0266]

[0267] Table 7. Flow rate and feed composition of a three-stage continuous culture system using Gracilaria hydrolysate.

[0268] During continuous fermentation, agar dissolved in Gracilaria hydrolysates begins to precipitate and adhere to the interior of the bioreactor, causing blockage of transfer tubes between reactors. The precipitated agar also significantly interferes with biomass measurements (by optical density and dry weight). The precipitated agar is also a significant contaminant in the harvested PHA, hindering accurate quality measurements.

[0269] The results of this experiment suggest that the step of extracting hydrocolloids (agar) from seaweed (Gracilaria) biomass should be considered before the production of hydrolysis products.

[0270] Production of hydrolysis products from green seaweed (Ulva genus)

[0271] Since green algae (Chlorophyta) were identified as a potentially suitable feedstock for the production of PHBV by Mediterranean halophilic bacteria, experiments were conducted to produce hydrolysates from Ulva and use them as feedstock for a single-stage continuous fermentation system.

[0272] Ulva hydrolysis products were hydrolyzed in a 10L hydrolysis reactor via hydrothermal and enzymatic hydrolysis. Ulva biomass was received as a dry powder. 700g of the powder was combined with 7L of deionized water and heated to 150°C. It was maintained at this temperature with constant stirring (200rpm) and then cooled to 50°C. The mixture was adjusted to pH 5 using 2M NaOH, and then 700mL of Viscozyme (1.0mL / g seaweed) was added. The mixture was incubated for 24 hours and then separated by centrifugation. The liquid hydrolysis product fraction was adjusted to pH 7 using 2M NaOH and then diluted with water and salt to a 50% strength to obtain the composition for ATCC medium 1176. Figure 2 No glucose or yeast extract added). Also added is 1 mL per liter of SL-6 metal mixture (…). Figure 3 ).

[0273] Store the growth medium at 4°C until continuous fermentation experiments are required.

[0274] Single-stage continuous fermentation system—Ulva hydrolysis products

[0275] Add 3.5 L of 50% strength growth medium produced from Ulva as described above to the bioreactor (Minifors 2; Inforrs HT).

[0276] The bioreactor was heated to 40°C and then inoculated with Mediterranean halophytes to obtain an approximate initial OD of approximately 0.5. 600 The bioreactor was operated in batch mode at 40°C for 36 hours, with the pH controlled at the set value of 7.0, and the dissolved oxygen was maintained at approximately 20% to 40% by adjusting the stirring speed and aeration rate.

[0277] Then use a peristaltic pump (0.033h) -1 The 50% strength *Ulva prostrata* growth medium (dilution rate) was fed into the bioreactor at a rate of approximately 1.93 mL / min. The fermentation broth was removed from the bioreactor at the same rate using a peristaltic pump and transferred to a 5 L Erlenmeyer flask stirred with a magnetic stirrer. PHA was harvested daily from the collected fermentation broth by water extraction, as described in previous experiments.

[0278] As shown in Table 8 below, the harvest exhibited a consistent PHA yield over a six-day period.

[0279] Harvest Volume (L) PHA mass (g) PHA concentration (g / L) 1 3.0 6.25 2.08 2 2.5 8.16 3.26 3 2.5 9.20 3.68 4 2.8 8.60 3.07 5 2.6 6.40 2.46 6 4.0 7.50 1.88

[0280] Table 8. PHA harvest yield from a single-stage continuous fermentation system using Ulva hydrolysate.

[0281] Comparison of upstream process efficiency when using seawater compared to freshwater

[0282] Experiments were conducted to investigate the effects of using seawater instead of freshwater to extract agar from seaweed and produce glucose-rich hydrolysates.

[0283] Specifically, the following parameters were studied:

[0284] • Agar yield is expressed as grams of agar per 100g of dried seaweed biomass;

[0285] • Agar purity, the level of product clarity assessed through visual inspection of the sample;

[0286] • Hydrolyzed glucose yield, expressed as grams of glucose per 100g of dried seaweed biomass;

[0287] • Inhibitor formation during acid pretreatment, expressed as grams of 5-hydroxymethylfurfural (5-HMF) per liter of hydrolysis product; and

[0288] • Removal of inhibitors via detoxification (pretreatment with excess lime and charcoal), expressed as grams of 5-HMF removed.

[0289] method:

[0290] Agar extraction: 300g of dried red seaweed (Gracilaria genus, purchased from Indonesia) was processed using a food processor (Robot Coupe). 7. Particles <2mm were ground and then divided into 6 subsamples (50g each); each was soaked in 1000ml of water (1:20w / v) at ambient temperature for 3 hours. Of these 6 samples, 3 were soaked in seawater (35ppt salinity) and 3 were soaked in freshwater (0ppt salinity). Both seawater and freshwater were sourced locally in Western Australia (31.8521°S, 115.7518°E). The freshwater was tap water filtered through deionization and reverse osmosis (BOSS 031-4P system), while the seawater was obtained through the marine pumps and filtration system at the aquarium facility (Indian Ocean Marine Research Centre Watermans Bay, Western Australia).

[0291] The seaweed-water mixture was heated to 95°C in a water bath and held at that temperature for 3 hours, then centrifuged (Himac CR-3ONX, R9A2-4234 rotor; 8500 rpm, 10 min) to separate the agar-containing liquid from the solid. The solid was washed with 300 mL of hot (95°C) water (seawater or fresh water; the same as used in the first wash / agar extraction) and centrifuged again (8500 rpm, 10 min). The washed solid was set aside (for subsequent enzymatic treatment and hydrolysis product production), while the liquid fraction was combined with the first liquid fraction (containing the agar extract) and allowed to gel at room temperature overnight. The gel was frozen at -20°C, thawed at room temperature, and then dried in a 60°C oven for 48 hours. The dried agar was washed for 1 hour with 1 L of cold (4°C) fresh water on a magnetic stirrer, filtered through a muslin cloth, washed again under the same conditions (1 L of 4°C water), and dried again at 60°C for 24 hours. To remove excess water, the agar was dehydrated by soaking it in acetone (50 mL) for 1 hour and then drying it at 60°C for approximately 2.5 hours. The resulting dry agar was visually inspected to assess its transparency level (which is proportional to its purity level), and the agar yield was quantified by gravimetric analysis as the number of grams of dry agar per 100 g of dried seaweed biomass.

[0292] Acid Pretreatment: After agar extraction, the solids underwent a pretreatment process to facilitate enzymatic digestion of the cellulose. Twelve 60g wet solid samples were combined with 30mL of water (seawater or freshwater, as shown in Table 9) containing 430mg citric acid (to obtain a final concentration of 25mM). These seaweed-water mixtures were autoclaved (Biobase BKQ-B75I) at 120°C for 30 minutes, cooled to ~50°C in a water bath (ZZKD), and centrifuged (Eppendorf 5910R; 4500rpm, 20 minutes). The solids were stored until further use, while the liquid fractions were tested using an HMF test kit. Its 5-HMF level was measured, and then a detoxification process was carried out before it was remixed with the solid for enzymatic treatment (details below).

[0293]

[0294]

[0295] Table 9. Types of water used for agar extraction and hydrolysis product production

[0296] The second column indicates the type of water used for agar extraction, while the third column indicates the type of water used for the hydrolysis process (i.e., acid pretreatment, detoxification step, and enzyme treatment). The samples included: (1) three samples fully treated with fresh water; (2) three samples that underwent freshwater agar extraction and seawater hydrolysis; (3) three samples that underwent seawater agar extraction and freshwater hydrolysis; and (4) three samples fully treated with seawater, resulting in a total of 12 seaweed biomass samples.

[0297] Detoxification Pretreatment: Because a very low level of glucose (<0.1 g / L) is produced by using a relatively mild acid pretreatment (120 °C, 25 mM citric acid), a detoxification process can be performed before enzymatic treatment to remove fermentation inhibitors such as phenolic compounds and 5-HMF without the risk of glucose loss. Therefore, the inventors developed a detoxification method with two treatments (details below). The first method, hereinafter referred to as excess lime treatment, involves adjusting the pH of the liquid fraction from ~3.5 to 11 using a slurry of calcium hydroxide (All Chemical, Australia) and water (1:1 w / v), stirring the solution on a rotary shaker (Thermo Scientific MaxQ 8000 shaker incubator; 40 °C, 150 rpm) for 30 minutes, and then removing the solids by centrifugation (Eppendorf 5910R; 4500 rpm, 20 minutes). The second method, hereinafter referred to as carbon treatment, consists of the following: adjusting the pH of the liquid containing excess lime to 5 with 2M HCl, then combining it with 2.5% w / v activated carbon powder (All Chemical, Australia), stirring the mixture for 30 minutes as previously described, and then centrifuging (as previously described) to remove the carbon. This is then done using an HMF test kit. The concentration of 5-HMF was measured to assess the detoxification efficiency.

[0298] Enzymatic treatment: The pretreated supernatant was recombine with its respective solids (obtained after acid pretreatment) and then subjected to an enzymatic hydrolysis process aimed at breaking down cellulose into glucose. Two cellulase preparations—Celluclast and Viscozyme (0.1 mL each per gram of dry seaweed biomass)—were added, and the mixture was incubated in a water bath at 50°C and 130 rpm for 20 hours. Finally, the mixture was separated by centrifugation (4500 rpm, 20 min) to separate the waste seaweed biomass (solid) from the hydrolysis products (liquid). The salinity (in parts per thousand (ppt)) and glucose concentration (in g / L) of the hydrolysis products were measured using an EcoSense EC300 salinity meter. ).

[0299] Results and Discussion:

[0300] Agar yield and quality. When using freshwater, the average agar extraction yield was 22.7 g ± 1.7 standard deviations (std) per 100 g of dried seaweed biomass, and 16.5 g (±0.5 standard deviations) per 100 g of dried seaweed biomass when using seawater. Although using seawater resulted in a 6% reduction in agar yield, the seawater-based product was lighter in color, likely due to its higher purity level. Figure 11 This means that marine products may have higher market value and / or require less downstream processing (such as bleaching or further purification) to produce market-ready and more sustainable products.

[0301] Glucose yield. The average glucose yield obtained by seawater hydrolysis and freshwater hydrolysis was comparable, but negatively correlated with the salinity of the culture medium (Table 10). This may be due to the impaired performance of cellulase at higher salt concentrations.

[0302]

[0303] Table 10. Average glucose percentage (g glucose per 100g of dried seaweed biomass), glucose concentration (g glucose per liter of hydrolysis product), and culture medium salinity (ppt) under different schemes using seawater and freshwater.

[0304] Formation and removal of fermentation inhibitors. The level of the fermentation inhibitor 5-HMF increased proportionally to the salinity level in the culture medium after acid pretreatment. Figure 12 This indicates that higher salt concentrations lead to faster polysaccharide degradation, meaning that shorter pretreatment times, milder temperatures, and / or lower acid concentrations may reduce the generation of 5-HMF in the presence of seawater.

[0305] Excess lime and carbon treatment both contribute to 5-HMF removal; the combination of these two steps reduced inhibitor levels by 50%, regardless of salinity levels (see [link to article]). Figure 12 The inventors noted that different salinity levels (formed by using seawater and / or freshwater; see Table 9) affect the level of 5-HMF formed during acid pretreatment, but do not affect the detoxification efficiency of excess lime or char treatment.

[0306] Test the seawater-based method of the present invention on an experimental scale.

[0307] The purpose of conducting the experiment is:

[0308] • Test the efficiency of upstream processes based on seawater at a scale of ~20L.

[0309] • Evaluate the effects of hydrolysis product dilution (100%, 50% intensity) and yeast extract concentration (0, 0.3, 1, 2 g / L) on microbial growth and PHA yield.

[0310] • Demonstrates semi-continuous production of PHA using a 4L capacity bioreactor.

[0311] method:

[0312] Agar extraction. An upstream seawater-based method developed in previous laboratory-scale experiments, consisting of agar extraction, acid pretreatment, detoxification, and enzymatic treatment. Comparison of upstream process efficiency when using seawater compared to freshwater The production has been expanded to ~20L of hydrolysate for the production of PHA via brine fermentation (see the next section, 'Marine Fermentation,' for details).

[0313] Using a food processing machine (Robot Coupe) 7. Grind 2 kg of dried red seaweed (Gracilaria genus, purchased from Indonesia) with a particle size <2 mm, and then soak it overnight in 40 L of seawater at ambient temperature.

[0314] The seaweed-seawater mixture was heated to 100°C for 2 hours in a 50L double-walled glass reactor (model: S-50L, Zhengzhou Keda Machinery Instrument Equipment Co., Ltd.) equipped with a mechanical stirrer, and stirred at a constant speed of 200 rpm using a heat-conducting fluid (Duratherm 450, DurathermFluids, USA) in a circulating heater (model: GDX-50L-30C, Zhengzhou Keda Machinery Instrument Equipment Co., Ltd.). The mixture was then cooled to approximately 80°C, and the agar was separated by filtration through a cotton cloth. The removed solids were washed with approximately 8L of hot (80°C) seawater to further remove the agar.

[0315] Acid pretreatment. Divide the wet solid material weighing approximately 10 kg into two batches of 5 kg each for acid pretreatment.

[0316] Each batch was combined with 2.7 L of seawater and 34 g of citric acid in a 10 L capacity stainless steel hydrolysis reactor (model: FCF-10L, Zhengzhou Keda Machinery Instrument Equipment Co., Ltd.) equipped with an electric heating element, mechanical stirrer, and cooling coil (ZZKD, China). The reactor was heated to 120 °C with constant stirring (300 rpm) until the gauge pressure of the reactor reached 1 bar. The mixture was held at this pressure and stirred for 20 minutes, then cooled through an internal cooling coil containing a 30% v / v ethylene glycol / water mixture recirculated via a cooling device (Kegland IceMaster G40). The two pretreated batches were combined and filtered through a cotton cloth to separate the solids. The solids were washed with approximately 4 L of hot (80 °C) seawater and combined with the previously obtained liquid fraction.

[0317] Detoxification pretreatment. The pH of the combined liquid was adjusted from ~3.5 to 11 using a slurry of calcium hydroxide and water, and incubated at room temperature for 30 minutes with occasional manual stirring. The mixture was then filtered through a perlite bed to remove precipitated solids.

[0318] The pH of the recovered liquid was adjusted to 5 using a 2M HCl solution and mixed with approximately 2.5% w / v of powdered char. The mixture was then incubated again at room temperature for 30 minutes with occasional manual stirring. The char mixture was then centrifuged in batches (Himac CR-3ONX, R9A2-4234 rotor; 8500 rpm, 15 min) to separate the char solids.

[0319] Enzyme treatment. The liquid fraction was recombine with seaweed biomass and transferred to a 50 L glass reactor (the same one used for agar extraction) and heated to 50 °C. 200 mL of Celluclast and 200 mL of Viscozyme (Novozymes) were added, and the mixture was incubated at 50 °C with continuous stirring (200 rpm) for 18 h. The enzyme-treated mixture was removed from the reactor and centrifuged in batches (Himac CR-3ONX, R9A2-4234 rotor; 8500 rpm, 15 min) to remove solids. The glucose concentration in the final liquid hydrolysate was measured (in g / L using the D-glucose HK assay kit). ) and 5-HMF levels (in g / L, using an HMF assay kit; ).

[0320] Fermentation medium preparation. The liquid hydrolysis product fraction (35 ppt salinity) was adjusted to pH 7 with 2 M NaOH and combined with the following salts per liter (adapted from ATCC medium 1176 formulation to achieve a final salinity of 170 ppt): NaCl, 124 g; MgCl2·6H2O, 16 g; CaCl2·2H2O, 0.8 g; KCl, 3 g; NaHCO3, 0.16 g; NaBr, 0.4 g. Trace metals were supplied by adding 1 mL of SL-6 trace metal per liter. Figure 3 The culture medium was then cooled overnight to 4°C and centrifuged in batches (8500 rpm, 15 minutes, 4°C) to remove any remaining precipitate.

[0321] Fermentation-shake flask experiment. The fermentation capacity and PHA production of the prepared seaweed-based growth medium (170 ppt salinity, pH 7) were evaluated by shake flask experiment. This medium contained different dilutions of hydrolysate (50% and 100% strength) and different levels of supplemental yeast extract (0, 0.3, 1, and 2 g yeast per liter of growth medium). For the diluted medium, a solution containing 1 mL of SL-6 micrometal mixture per liter, along with salts as provided in ATCC medium 1176, was used. Figure 2 (Does not contain glucose or yeast extract) Dilute ( Figure 3 ).

[0322] A total of 16 shake-flask cultures were prepared, each measured in 30 mL volumes in 150 mL Erlenmeyer flasks. The yeast extracts at all four levels were tested in 50% and 100% strength hydrolysates, with each condition repeated twice.

[0323] The inoculum used in this fermentation experiment was prepared by growing a single colony of Mediterranean halophilic bacteria (ATCC 33500) for 24 hours in a 50 mL Falcon tube at 40°C and 150 rpm (Thermo Scientific MaxQ8000). Each tube contained 5 mL of a solution containing salts as provided in ATCC medium 1176. Figure 3 The 5 mL culture was then transferred to a 250 mL flask containing 45 mL of the same medium and cultured for another 72 hours under the same fermentation conditions. 50 mL of the culture was centrifuged (Eppendorf 5910R; 4500 rpm, 20 min, 4°C) and the cells were resuspended in salt solution (based on ATCC 1176 medium without yeast extract or glucose). Sixteen algae growth medium cultures were inoculated with the cell suspension to the initial optical density (OD) at 600 nm. 600 ); using Eppendorf The value (measured by BioSpectrometer) is approximately 0.5.

[0324] The culture was incubated under the same conditions as the inoculum (40°C, 150 rpm), and the OD was measured every 12-24 hours. 600 Growth was assessed. Cells were harvested by centrifugation (Eppendorf 5910R; 10,100 rpm, 20 min, 4 °C) after growth had ceased or all glucose had been consumed (assessed using an Abbott FreeStyle Optium Neo glucose monitor), and the final polyhydroxyalkanoate (PHA) concentration was determined by GC-MS (using the same method described below for monitoring the semi-continuous fermentation process in the bioreactor).

[0325] Fermentation—Semi-continuous production in a 4L bioreactor. The inventors demonstrate a semi-continuous marine continuous fermentation system operating in a 4L bioreactor for the production of PHA (Infrared Minifors). The operating volume was 3.5 L. Mediterranean halotrophic bacteria cultures were grown in the optimal seaweed-based growth medium determined by the above flask experiments, namely, a 50% dilution of hydrolysate (diluted with modified ATCC medium 1176 as described above) with the addition of 2 g / L yeast extract.

[0326] Semi-continuous fermentation was carried out by removing 80% (2.8 L) of the working volume from the bioreactor every ~24 hours and replacing it with fresh algae-based growth medium. A total of five such harvests were performed over 138 hours. The collected fermentation broth was used for PHA extraction as described below.

[0327] Inoculation and Operating Conditions. The bioreactor was autoclaved (121°C, 20 min; Biobase BKQ-B75I) and 3.4 L of growth medium (a 50% dilution of hydrolysate medium containing 2 g / L yeast extract) was added. The bioreactor was heated to 40°C before inoculation. For inoculation, 50 mL of Mediterranean halotrophic bacteria culture was prepared as described in the shake-flask experiment above. This was then added to 180 mL of fresh solution in a 1 L Erlenmeyer flask containing the salts provided in ATCC medium 1176, and incubated (40°C, 150 rpm) for 72 h. 140 mL of this culture was centrifuged (4500 rpm, 20 min) and the cells were resuspended in 100 mL of salt solution as previously described. The resuspended cells were added to the culture medium in the bioreactor to obtain an initial OD of ~0.5. 600 Throughout the fermentation process, the temperature was maintained at 40°C, and the pH was maintained at 7 by automatically adding 2M HCl and 2M NaOH. Dissolved oxygen was maintained at 40% through cascaded control of stirrer speed (450 to 800 rpm) and gas flow rate (0 to 8 L / min). Foaming was controlled by automatically adding antifoam (Anti-foam 2010; ChemSupply, Australia). Cell growth was monitored using an online turbidimeter (CGQ Sensor BIOR, Aquila Biolabs). Samples were taken every 4 to 12 hours for analysis of biomass, glucose, PHA, and nitrogen as described below. A peristaltic pump (Longer G100-1J) was used to remove and replace the fermentation broth at harvest.

[0328] Biomass monitoring. Throughout the fermentation process, the cell biomass concentration in the culture medium (in g / L) was measured every 12 hours as ash-free dry weight. 10 mL of fermentation broth was centrifuged and the supernatant was discarded. The cell pellet was frozen at -80°C for at least 1 hour, then transferred to a freeze dryer (Biobase BK-FD10S) and freeze-dried for 48 hours. The freeze-dried biomass was transferred to a crucible, weighed, and then incinerated in an oven (ThermoFisher M104) at 400°C for 3 hours. The incinerated biomass was cooled and weighed again to determine the ash-free dry weight.

[0329] PHA monitoring. Throughout the fermentation process, the PHBV concentration in the culture medium (in g / L) was measured by GC-MS every ~12 hours. 7 mL of fermentation broth was centrifuged (Eppendorf 5910R; 10100 rpm, 20 min, 4 °C), and the cell pellet was lyophilized as described above. The lyophilized cell pellet was weighed and then methanol-decomposed in 2 mL of 15% sulfuric acid in methanol and 2 mL of chloroform at 100 °C for 2 h 20 min. The chloroform phase containing methyl hydroxyalkanoate was then removed for analysis by GC-MS using benzoic acid as an internal standard. Butyric acid and methyl valerate were evaluated using a ZB-Wax capillary column (30 m long, 0.25 mm diameter, and 0.25 μm thickness) via gas chromatography-coupled mass spectrometry (QP2010; Shimadzu). 1 μL of sample was injected at 250 °C using a 20 split and helium as the gas carrier. The temperature was increased from 60°C to 250°C at a rate of 20°C per minute and held at 250°C for 5 minutes. Quantification was performed by comparison with a standard curve prepared from PHBV of known purity and composition.

[0330] Glucose and nitrogen monitoring. Throughout the fermentation process, measure glucose and total nitrogen concentrations in the culture medium (in g / L) every ~12 hours. Use the D-Glucose (HK) kit according to the manufacturer's instructions. Glucose concentration was determined. Total nitrogen concentration was determined by persulfate digestion using a Hach DRB200 digester, a Hach DR3900 spectrophotometer, and the accompanying Hach proprietary reagents.

[0331] PHA extraction. PHA was extracted from harvested Mediterranean halotrophic bacteria biomass by rupturing / lysing cells under hypotonic shock. Approximately 2.8 L of fermentation medium was removed from the bioreactor and the cells were separated from the fermentation broth by centrifugation (Himac CR-3ONX, R9A2-4234 rotor; 8500 rpm, 25 min, 4 °C). The cell pellet was then resuspended in reverse osmosis water containing 0.1% w / v sodium dodecyl sulfate (SDS) (0 ppt salinity, 0 total dissolved solids; BOSS 031-4P system). The precipitated biomass was resuspended in 0.1% SDS solution so that the final mixture contained approximately 1.1% w / v Mediterranean halotrophic bacteria biomass to ensure proper lysis. Proper dispersion of the precipitated cells in the SDS-water mixture was achieved by vigorous agitation at approximately 1000 rpm using a magnetic stirrer unit. Agitation was continued for 1 hour until complete cell lysis. Subsequently, the mixture of lysed cells and SDS solution was centrifuged (8500 rpm, 25 min, 4 °C) to precipitate coarse PHA particles. The PHA precipitate was resuspended in the same volume of SDS solution from the lysis phase to wash away any residual cell debris. This washing was performed using a magnetic stirrer at 1000 rpm for 30 min. After washing, the PHA particles were precipitated from the mixture by centrifugation as described above. A final wash was then performed by resuspending the precipitate in SDS-free fresh water (the same volume as the previous wash) with a magnetic stirrer at 1000 rpm for 30 min. A final precipitation step was then performed by centrifugation (8500 rpm, 25 min) to obtain the PHA particles. The extracted PHA particles were dried in a vacuum oven at 60 °C for 24 h.

[0332] Results and discussion:

[0333] Hydrolysis product yield. 2 kg of dried seaweed successfully produced 21 liters of seaweed hydrolysis products.

[0334] The hydrolysate contains 12.0 g of glucose per liter, equivalent to 12.6 g of glucose per 100 g of dried seaweed. This glucose yield is similar to previous experiments ( Comparison of upstream process efficiency when using seawater compared to freshwater The glucose yield obtained on a small scale during the period (mean ± standard = 12.4 ± 0.7 g / 100 g) was comparable, indicating that our hydrolysis product production process has good scalability. However, the glucose concentration (12.0 g / L) was lower than that of previous experiments ( Compared to fresh water Upstream process efficiency when using seawater The concentration obtained on a small scale was 16.7 ± 1 g / L. This was due to the higher dilution caused by the process differences in solid / liquid separation, as some separation was carried out by filtration in a 20L process and additional water was required to displace the liquid in the filter bed.

[0335] Fermentation-shake flask experiment. Cell growth was observed in both diluted and undiluted media, but significantly stronger growth was observed in the diluted medium (50% v / v) (Figure 13). This is likely due to the presence of inhibitory compounds in the hydrolysis products. Given the relatively low concentration of 5-HMF in the hydrolysis products (0.27 g / L), the inhibition is likely due to the presence of other inhibitory compounds present in the algae (rather than those generated during hydrolysis). Potential inhibitors include plant metabolites with antibacterial activity (e.g., flavonoids and other phenolic compounds) or heavy metal ions.

[0336] The addition of yeast extract enhanced growth rates and final cell densities in both diluted and undiluted media (see Figure 13). Yeast extract is a complex substrate that provides carbon, nitrogen, vitamins, and other growth factors to fermentation media. Given the higher nitrogen and glucose content in undiluted media compared to diluted media, it is unclear whether the growth enhancement observed with yeast extract is primarily due to improved nitrogen or carbon supply, or whether it is more related to specific compounds (e.g., amino acids, cofactors, nucleotides) that can improve cell health and resilience in the presence of growth-inhibiting compounds in hydrolysates.

[0337] The final PHBV concentration in the culture medium was proportional to the cell density, varying between 0.4 and 1.2 g PHBV per liter of growth medium. Figure 14 As mentioned above, due to poor cell growth, the PHBV concentration in the undiluted medium (0.27 ± 0.10 g / L) was significantly lower than that in the diluted medium (0.93 ± 0.21 g / L).

[0338] Fermentation—4L bioreactor, continuous fermentation system. Biomass growth and PHBV yield were consistent in five replicate cultures of the semi-continuous process (Figure 15). The bioreactor was harvested approximately every 24 hours after the average PHBV concentration reached 0.94 ± 0.20 g / L (as determined by GC-MS). PHBV accumulation was observed to lag behind biomass growth, increasing with carbon and nitrogen limitation (see Figure 15).

[0339] The yield of PHBV extracted from the harvest by hypotonic shock (0.49 ± 0.14 g / L) was significantly lower than the PHBV concentration in the culture medium as determined by GC-MS (0.94 ± 0.20 g / L), indicating significant loss of PHBV during extraction. This is likely due to the PHBV being released from the precipitate after centrifugation and lost during the removal of the supernatant.

[0340] The nitrogen concentration at each harvest was consistently 0.17 ± 0.01 g / L. It is noteworthy that this is equivalent to the nitrogen concentration in the hydrolysate medium diluted before the addition of the yeast extract. This may indicate that most of the nitrogen present in the hydrolysate exists in forms that are not readily accessible to Mediterranean halophilic bacteria (e.g., indigestible proteins).

[0341] Adding a lipid extraction step to the seaweed-based method of this invention

[0342] This study investigates the effectiveness of integrating the upstream lipid extraction step into the seaweed-based production process of this invention. More specifically, the experiment aims to:

[0343] • To test the effect of adding upstream lipid extraction process on the yield of our following biorefining byproducts: grams of (1) dry agar, (2) glucose in hydrolysate and (3) protein in residual biomass per 100g of dried seaweed biomass;

[0344] • Determine the composition of lipids extracted from seaweed;

[0345] • Evaluate the efficiency of our lipid extraction process, expressed as a percentage of total lipids successfully extracted.

[0346] • Compare the growth curves and PHA productivity of Mediterranean halotrophic bacteria in a seaweed-based growth medium prepared after lipid extraction with those in a seaweed-based growth medium without upstream lipid extraction.

[0347] method:

[0348] Lipid extraction: 200g of dried red seaweed (Gracilaria genus, purchased from Indonesia) was processed using a food processor (Robot Coupe). 7. The biomass (particle size <2 mm) was ground and then divided into four subsamples (50 g each); two of these were used for lipid extraction. The latter was performed by combining each 50 g sample with 2075 mL of chloroform:methanol:water solution (containing 830 mL chloroform, 830 mL methanol, and 415 mL water). These mixtures were shaken on a rotary shaker at room temperature for 30 minutes and then centrifuged (Eppendorf 5910R; 3000 rpm, 10 minutes, 4 °C). The chloroform and methanol phases were removed separately, and the biomass was washed with 1 L of deionized water and centrifuged again under the same conditions to remove the solvent. The chloroform phase was stored at -80 °C for lipid and fatty acid determination, while the particulate biomass phase was dried at 60 °C overnight for the next biorefining step (agar extraction).

[0349] Two mL of a chloroform phase containing lipids was dried under nitrogen and the total lipid mass was quantified by gravimetric analysis. The lipids were methylated to fatty acid methyl esters (FAME) by suspending dried aliquots of the lipids in BF3 (7% methanol) and toluene. The mixture was heated to 100 °C for 45 min, and after cooling, water was added and FAME was extracted with hexane. FAME was evaluated by gas chromatography-coupled mass spectrometry (QP2010; Shimadzu) using a ZB-Wax capillary column (30 m long, 0.25 mm diameter, and 0.25 μm thickness). A 10 split and helium flow rate of 1 mL / min was used. -1 1 μL of sample was injected at a flow rate of 220 °C. The temperature was increased from 60 °C to 240 °C at a rate of 5 °C per minute and held at 240 °C for 10 minutes. Supelco37 Component FAME Mix (Merck) was used as a standard to identify and quantify the FAMEs based on their retention times and linear equations.

[0350] Agar Extraction. Four 50g biomass samples (two with upstream lipid extraction and two without) were soaked in 1 liter of seawater at our laboratory in Western Australia (31.8521°S, 115.7518°E). This seawater was obtained using marine pumps and filtration systems at our aquarium facility (Watermans Bay Indian Ocean Marine Research Centre, Western Australia). The seaweed-water mixture was heated to 95°C in a water bath (ZZKD) and maintained at this temperature for 3 hours, followed by centrifugation (Eppendorf 5910R; 4500 rpm, 15 min) to separate the agar-containing liquid from the solids used for hydrolysis product production. The solid fraction was washed again with 1 L of hot seawater (70°C) and centrifuged again (4500 rpm, 15 min). The washed solids were set aside (for subsequent enzymatic treatment and hydrolysis product production), while the liquid fraction was combined with the first liquid fraction (containing agar extract) and allowed to gel at room temperature overnight. The gel was frozen at -20°C, thawed at room temperature, and then dried in an oven at 60°C for 48 hours. The dried agar was washed for 1 hour with 1 L of cold (4°C) fresh water on a magnetic stirrer, filtered through a cotton cloth, washed again under the same conditions (1 L of 4°C water), and dried at 60°C for 24 hours. To remove residual water, the agar was dehydrated by soaking in acetone (50 mL) for 1 hour, followed by drying at 60°C for approximately 2.5 hours. The agar yield was quantified by gravimetric analysis as grams of dry agar per 100 g of dried seaweed biomass.

[0351] Acid pretreatment. After agar extraction, the solids underwent a pretreatment process to make the cellulose more readily digestible by enzymes. Four samples were mixed with 500 mL of seawater containing citric acid (25 mM). These seaweed-water mixtures were heated to 120 °C for 30 minutes in an autoclave (Biobase BKQ-B75I), cooled to ~50 °C in a water bath, and centrifuged (Eppendorf 5910R; 4500 rpm, 20 minutes). The solids were stored until further use, while the liquid fractions underwent a detoxification process before being remixed with the solids for enzymatic treatment (see details below).

[0352] Detoxification Pretreatment. Since a relatively mild acid pretreatment (120°C, 25 mM citric acid) produces very low levels of glucose (<0.1 g / L; data not shown), a detoxification process can be performed prior to enzymatic treatment to remove fermentation inhibitors such as phenolic compounds and 5-HMF without the risk of glucose loss. Therefore, the inventors describe a detoxification process with two treatments (see details below). The first method, hereinafter referred to as excess lime treatment, involves adjusting the pH of the liquid fraction from ~3.5 to 11 using a slurry of calcium hydroxide (All Chemical, Australia) and water (1:1 w / v), stirring the solution on a rotary shaker (Thermo Scientific MaxQ 8000 shaker incubator; 40°C, 150 rpm) for 30 minutes, and then precipitating the solids by centrifugation (Eppendorf 5910R; 4500 rpm, 20 minutes). The second method, hereinafter referred to as carbon treatment, consists of the following: adjusting the pH of the liquid after adding excess lime to 5 with 2M HCl, then combining it with 2.5% w / v activated carbon powder (All Chemical, Australia), stirring the mixture for 30 minutes as described above, and then centrifuging (as described above) to remove the carbon.

[0353] Enzymatic hydrolysis. The pretreated supernatant was recombine with its respective solids (obtained after acid pretreatment) and then subjected to an enzymatic hydrolysis process aimed at breaking down cellulose into glucose. Two cellulase preparations—Celluclast and Viscozyme (0.1 mL each per gram of dry seaweed biomass)—were added, and the mixture was incubated in a water bath at 50°C and 130 rpm for 20 hours. Finally, the mixture was separated by centrifugation (4500 rpm, 20 min) to separate the waste seaweed biomass (solid) from the hydrolysis product (liquid). The latter was analyzed using a D-glucose HK assay kit. Glucose concentration was measured (in g / L). The solids were washed with distilled water (1 L), dried overnight in an oven (60°C), and then sent to Agrifood Technology (Perth, Australia) for determination of total protein, lipids, and carbohydrates. For comparative purposes, the same analyses were also performed on the original seaweed biomass (Gracilaria).

[0354] Fermentation medium preparation. The pH of the four liquid hydrolysates was adjusted to 7 with 2M NaOH and mixed with the following amounts of salt per liter (adapted from ATCC medium 1176 formulation to achieve a final salinity of 170 ppt): NaCl, 124 g; MgCl2·6H2O, 16 g; CaCl2·2H2O, 0.8 g; KCl, 3 g; NaHCO3, 0.16 g; NaBr, 0.4 g. Trace metals were supplied by adding 1 mL of SL-6 trace metal per liter. Figure 3 The medium was then cooled overnight to 4°C and centrifuged in batches (8500 rpm, 15 minutes, 4°C) to remove any remaining precipitate. Then, it was mixed with ATCC medium 1176 containing 1 mL of SL-6 metal mixture per liter. Figure 2 The four growth media were diluted (50% strength) with the saline solution provided in the preparation (without glucose or yeast extract). Finally, a 2 g / L concentration of yeast extract was added to all four growth media.

[0355] Fermentation-shake flask experiments. To test whether the addition of our lipid extraction step had any effect on the fermentation capacity and PHA production of our process, we conducted shake flask experiments. A total of 12 250 mL shake flask cultures were prepared, 6 of which contained 60 mL of algae-derived medium for lipid extraction and 6 of algae-derived medium without lipid extraction.

[0356] The inoculum used in this fermentation experiment was prepared by growing a single colony of Mediterranean halotrophic bacteria (ATCC 33500) in a 50 mL Falcon tube (Thermo Scientific MaxQ8000) at 40 °C and 150 rpm for 24 hours. The tube contained 5 mL of a solution containing salts as provided in ATCC medium 1176. This 5 mL culture was then transferred to a 250 mL flask containing 45 mL of the same medium and cultured for another 72 hours under the same fermentation conditions. The 50 mL culture was centrifuged (Eppendorf 5910R; 4500 rpm, 20 min, 4 °C) and the cells were resuspended in the aforementioned salt solution. The cell suspension was then inoculated with 12 different seaweed growth media to the initial optical density (OD) at 600 nm. 600 ); using Eppendorf The value (measured by BioSpectrometer) is ~0.8.

[0357] The culture was incubated under the same conditions as the inoculum (40°C, 150 rpm), and the optical density OD was measured every 12 to 24 hours. 600 Growth was assessed. Cells were harvested by centrifugation (Eppendorf 5910R; 10,100 rpm, 20 min, 4 °C) after growth had ceased or all glucose had been consumed (assessed using an Abbott FreeStyle OptiumNeo glucose monitor), and the final polyhydroxyalkanoate (PHA) concentration was determined by GC-MS and direct gravimetric analysis.

[0358] The final PHBV concentration (in g / L) of 7 mL of fermentation broth sample was measured by GC-MS. The sample was centrifuged (Eppendorf 5910R; 10100 rpm, 20 min, 4 °C), and the cell pellet was lyophilized. The lyophilized cell pellet was weighed and then methanol-decomposed in 2 mL of 15% sulfuric acid in methanol and 2 mL of chloroform at 100 °C for 2 h 20 min. The chloroform phase containing methyl hydroxyalkanoate was then removed for analysis by GC-MS using benzoic acid as an internal standard. Butyric acid and methyl valerate were evaluated using a ZB-Wax capillary column (30 m long, 0.25 mm diameter, and 0.25 μm thickness) via gas chromatography-mass spectrometry (QP2010; Shimadzu). 1 μL of sample was injected at 250 °C using a 20 split and helium as the gas carrier. The temperature was increased from 60 °C to 250 °C at a rate of 20 °C per minute and held at 250 °C for 5 min. Quantification was performed by comparing the results with a standard curve prepared from PHBV of known purity and composition.

[0359] The final PHBV concentration (in g / L) was also measured directly by extracting PHA from a 40 mL volume of each culture. The extract was centrifuged (Eppendorf 5910R; 10100 rpm, 20 min, 4 °C) and the supernatant was discarded. The cell pellet was frozen overnight at -20 °C. The pellet was then thawed and combined with 40 mL of reverse osmosis deionized water containing 0.1% (w / v) sodium dodecyl sulfate (SDS). The cell pellet was resuspended and stirred at 150 rpm for 1 h at 40 °C. The suspension was centrifuged again, the supernatant was discarded, and the pellet was washed in another 40 mL of 0.1% SDS. The centrifugation and washing process was then repeated twice with SDS-free reverse osmosis deionized water. The pellet was then transferred to a pre-weighed tray and dried in an oven at 60 °C for 24 h, then weighed to determine the dry weight.

[0360] Results and discussion:

[0361] Biorefining: The aforementioned "biorefining" processes, including hydrocolloid, protein feed, and PHA production, have been successfully completed with or without lipid extraction. Adding an upstream lipid extraction step has no significant impact on the final yield of our biorefined products. Figure 16 This solvent extraction process appears to leave no impurities that would affect the efficiency of the hydrolysis process, nor does it alter the biochemistry of the seaweed by removing and / or degrading its hydrocolloids (agar) and cellulose (which breaks down into glucose during the enzymatic process).

[0362] Byproducts obtained from our complete seaweed biorefining process include lipid extracts (yield = 1.8 ± 0.3 g lipids per 100 g dry seaweed biomass), agar (15.3 ± 0.4 g dry agar per 100 g dry seaweed biomass), glucose-rich hydrolysates (11.6 ± 0.4 g glucose per 100 g dry seaweed biomass), and protein-rich solids (7.7 g protein per 100 g dry seaweed biomass). The latter contains a higher protein content (36.8 g protein per 100 g dry weight) than the original seaweed biomass (7.2 g protein per 100 g dry weight), making it an attractive food / feed source, particularly suitable for aquaculture (due to the presence of salt). The increased protein content is due to the removal of ~97% of carbohydrates (30.6 g carbohydrates per 100 g dry seaweed biomass) from the original seaweed biomass through agar removal and cellulose hydrolysis. Figure 17 ).

[0363] Regarding the fatty acid composition of the extracted lipids, nearly 99% were saturated fatty acids (palmitic acid, stearic acid, and myristic acid), with only one unsaturated fatty acid (oleic acid) detected (Table 11). Given the low yield and market value of lipids extracted from this specific seaweed, omitting the lipid extraction step may be more advantageous, allowing the lipids to remain in the final solid and enhancing their nutritional value as a potential aquaculture feed. It is also worth noting that, as the seaweed lipid content was successfully extracted at 63.5 ± 9.4%, there is room for improvement in this lipid extraction process. This could be improved by repeating the solvent extraction once or multiple times.

[0364]

[0365] Table 11. Fatty acid composition of lipid extracts (n=2) from the red algae Gracilaria.

[0366] Fermentation—Cell Growth and PHA Production. The cell growth modes of the two fermentation media used in this study ( Figure 18Similar to: (1) seaweed hydrolysate (50% strength, 2 g / L yeast extract) produced after lipid extraction, and (2) seaweed hydrolysate (50% strength, 2 g / L yeast extract) produced without any prior lipid extraction. It is noteworthy that, compared with previous studies ( The seawater-based method of the present invention was tested on a pilot scale. Compared to those described in [previous study], the cultures in this experiment grew significantly faster and reached higher cell densities (see Figure 13). While this difference may be partly due to the different glucose concentrations in the culture medium (7.2 ± 0.8 g / L vs. 5.9 ± 0.5 g / L at 50% intensity in the previous experiment), it is more likely due to the use of a more active growth inoculum and a higher initial cell density (OD). 600 0.8 compared to 0.5).

[0367] The PHA productivity obtained for both growth media (i.e., with and without lipid extraction; Table 12) was also similar. Notably, the concentration of PHBV as analyzed by GC-MS was lower than that of the extracted PHBV. This is due to impurities in the extracted material, with the mean (± standard deviation) purity of the extracted PHBV being ~76±5%. Potential impurities include unlysed cells and cell debris such as proteins and membrane lipids.

[0368] Overall, the average PHBV yield (based on extraction mass) per 100g of dried seaweed was 3.8 ± 0.3g. Potential strategies to improve this metric include:

[0369] (i) Research other seaweed species that can produce higher yields of fermentable sugars;

[0370] (ii) The hydrolysis product production process includes a protease digestion step to increase carbon and nitrogen concentrations;

[0371] (iii) Better removal of fermentation inhibitors.

[0372]

[0373] Table 12. PHBV yield (in g / L medium) and cellular percentage of PHBV estimated by direct PHA extraction and GC-MS

[0374] Seawater lysis of Mediterranean halophilic bacteria for PHBV extraction

[0375] The inventor's PHA extraction method is based on lysing halophilic cells (approximately 170 ppt) through hypotonic shock when exposing saline cells to a solution with a lower salt content.

[0376] In previous implementations, Mediterranean halophilic bacteria cells were lysed by exposing them to a solution containing reverse osmosis (RO) water, followed by hypotonic shock. However, large-scale use of freshwater (0 ppt) can be costly and unsustainable, making seawater (35 ppt) an attractive alternative.

[0377] The study aimed to evaluate the efficiency of lysing Mediterranean halotrophic bacteria cells (~170 ppt) and effectively extracting intracellular PHA using solutions containing different proportions of RO water and seawater (salinity range = 0 to 35 ppt).

[0378] method:

[0379] Fresh Mediterranean halotrophic bacteria cells were harvested from a 9×40 mL fermentation broth sample (poured into a 50 mL FAlcon tube) by centrifugation (15000 g, 10 min) and discarding the supernatant.

[0380] The resulting nine cell pellets were resuspended in 40 mL solutions containing 0.1% SDS and different levels of seawater: 0% (100% RO water; 0 ppt), 12.5%, 25%, 37.5%, 50%, 62.5%, 75%, 87.5%, and 100% (pure seawater; 35 ppt). These solutions were prepared by mixing RO water with filtered seawater from the Watermans Bay Indian Ocean Marine Research Centre in Western Australia. The suspensions were then incubated at 25°C with stirring (250 rpm) for 1 hour, centrifuged (15000 g, 10 min), and the supernatant was discarded. The color of the resulting pellets (representing the level of cell lysis) was examined and recorded by photograph. Since whole Mediterranean halophilic bacteria cells are pink and PHA particles are white, fewer pink particles indicate better cell lysis.

[0381] Results and Discussion:

[0382] Different degrees of cell lysis were observed, with better lysis levels occurring when halophilic cells were immersed in seawater rather than freshwater. This was clearly visible by observing the color of the resulting particles. Figure 19 The particles exhibit the strongest pink color in freshwater treatment and the whitest appearance in seawater treatment, with the pink color gradually disappearing from solutions with a higher proportion of freshwater to solutions with a higher proportion of seawater.

[0383] Our hypothesis is that the better lysis in seawater is due to salt ions preventing free DNA strands from interacting with and binding with proteins, resulting in salt-containing cells (~170 ppt) being better exposed to the solution (<35 ppt). The higher viscosity at lower salinity is clearly visible, with many cells trapped in some of the higher viscosity portions of the culture medium.

[0384] other

[0385] References to material or information cited in the text should not be construed as an admission that the material or information is part of common general knowledge or is known in Australia or any other country.

[0386] Every document, reference, patent application, or patent cited in this article is explicitly incorporated in its entirety by reference, meaning that the reader should read and consider it as part of this article. References, patent applications, or patents cited in this article are not repeated solely for the sake of brevity.

[0387] The alternative embodiments of the invention may also be widely present in the parts, elements and features individually or jointly mentioned or indicated herein, as well as any or all combinations of two or more parts, elements or features, and wherein the specific integers mentioned herein have equivalents known in the field of the invention, which are considered to be incorporated herein as if set forth separately.

[0388] It should be understood that references to "an example" or "example" of the invention are not exclusive. Thus, one example may exemplify certain aspects of the invention, while other aspects are exemplified in different examples. These examples are intended to assist those skilled in the art in practicing the invention and are not intended to limit the overall scope of the invention in any way, unless the context clearly indicates otherwise.

[0389] It should be understood that the terminology used above is for descriptive purposes and should not be considered limiting. The described embodiments are intended to illustrate the invention and not to limit its scope. The invention can be implemented with various modifications and additions readily apparent to those skilled in the art.

[0390] Other definitions of the selected terms used herein can be found in the detailed description of the invention and throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0391] This document describes, in textual and / or graphical form, various substantially practical and useful exemplary embodiments of the claimed subject matter, including the best mode known to the inventors for performing the claimed subject matter (if any).

[0392] Those skilled in the art will understand that the invention described herein is readily adaptable to variations and modifications beyond those specifically described. It should be understood that the invention encompasses all such variations and modifications. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in the specification, as well as any and all combinations or any two or more steps or features.

[0393] The inventor expects those skilled in the art to appropriately adopt such changes, and the inventor intends to practice the claimed subject matter in a manner different from that specifically described herein. Therefore, as permitted by law, the claimed subject matter includes and covers all equivalents of the claimed subject matter and all modifications thereof. Furthermore, every combination of the foregoing elements, activities, and all possible variations thereof is included within the claimed subject matter, unless otherwise clearly indicated, explicitly and specifically waived, or otherwise manifestly contradictory to the context.

[0394] The scope of this invention is not limited to the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the invention described herein.

[0395] The use of any and all instances or exemplary language (e.g., "such as" or "for example") provided herein is intended only to better illustrate one or more embodiments and does not constitute a limitation on the scope of any claimed subject matter unless otherwise stated. No language in this specification should be construed as indicating that any unclaimed subject matter is necessary for the practice of the claimed subject matter.

[0396] The use of words indicating direction or orientation should not be considered restrictive. Therefore, words such as “front,” “rear,” “rear,” “side,” “up,” “down,” “above,” “below,” “top,” “bottom,” “forward,” “backward,” “towards,” “far,” “near,” “inside,” and “outside,” as well as their synonyms, antonyms, and derivatives, are chosen solely for convenience, unless the context otherwise requires. The inventors envision various exemplary embodiments of the claimed subject matter that can be provided in any particular direction, and the claimed subject matter is intended to include such directions.

[0397] In the context of describing various embodiments (especially in the context of the claimed subject matter), the use of the terms “a,” “an,” “the,” “the,” and / or similar indicators shall be construed as covering both the singular and plural, unless otherwise stated herein or clearly contradicted by the context. Unless otherwise stated, the terms “comprising,” “having,” “including,” and “containing” shall be construed as open-ended terms (i.e., meaning “including but not limited to”).

[0398] Throughout the specification and claims, unless the context otherwise requires, the word “comprising” or variations such as “including” or “containing” shall be understood to imply inclusion of the specified integer or group of integers, but not to exclude any other integer or group of integers.

[0399] Furthermore, when any number or range is described herein, it is approximate unless otherwise explicitly stated. Unless otherwise stated herein, the enumeration of numerical ranges herein is intended only as a shorthand method for individually referring to each individual value falling within that range, and each individual value and each individual subrange defined by such individual value is incorporated into the specification as if it were individually enumerated herein. For example, if a range of 1 to 10 is described, the range includes all values ​​therein, such as 1.1, 2.5, 3.335, 5, 6.179, 8.9999, etc., and includes all subranges therein, such as 1 to 3.65, 2.8 to 8.14, 1.93 to 9, etc.

[0400] Therefore, apart from the claims themselves, each part of this application (e.g., title, technical field, background art, summary of the invention, detailed embodiments, abstract, drawings, etc.) should be regarded in nature as illustrative rather than restrictive; the scope of the subject matter protected by any patent filed based on this application is defined only by the claims of that patent.

[0401] In the accompanying drawings, which are incorporated to illustrate features of a non-limiting exemplary embodiment, the same reference numerals are used to identify the same parts throughout the drawings.

Claims

1. A method for producing polyhydroxyalkanoates from macroalgae, comprising the following steps: A mixture of large algae and brine; The macroalgae mixture is enzymatically hydrolyzed to form macroalgae hydrolysis products, wherein the enzymatic hydrolysis of the macroalgae mixture produces an aqueous phase and a solid phase of the macroalgae hydrolysis products, and the aqueous phase and the solid phase of the macroalgae hydrolysis products are separated. A growth medium containing the hydrolysis products of the macroalgae is produced; The growth medium was fermented using halophilic microorganisms capable of producing polyhydroxyalkanoates; and The polyhydroxyalkanoate was extracted from halophilic cells using a saline-based lysis method.

2. The method according to claim 1, wherein, The brine includes seawater and / or water with added salt.

3. The method according to claim 1, wherein, The macroalgae mentioned include cultured Gracilariaceae, Rhodophytaceae, Rhodophytaceae, Ulvaceae, Pterygaceae, Megaphytaceae, or Laminariaceae.

4. The method according to claim 3, wherein, The macroalgae include the group consisting of macroalgae species selected from the following genera: Gracilaria, Gracilaria, Kappaella, Euphorbia, Porphyra, Boragina, Gracilaria, Asparagus, Ulva, Undaria, Laminaria, or Laminaria.

5. The method according to claim 1, wherein, The halophilic microorganisms are halotrophic bacteria or Mediterranean halotrophic bacteria.

6. The method according to any one of the preceding claims, wherein, The macroalgae mixture comprises wet macroalgae and brine.

7. The method according to claim 1, wherein, The large algae are broken into smaller pieces before they are formed into the mixture.

8. The method according to claim 7, wherein, The smaller portions of the large algae contain particles with a diameter of less than 2 mm.

9. The method according to claim 7, wherein, Hydrocolloids are removed from the macroalgae before the macroalgae mixture is formed.

10. The method according to claim 9, wherein, The large algae mixture was treated with a weak acid.

11. The method according to claim 10, wherein, The weak acid includes citric acid.

12. The method according to claim 1, wherein, The enzymes include one or more of cellulase, β-glucanase, pectinase, hemicellulase, and xylanase.

13. The method according to claim 1, wherein, The hydrolysate of the large algae is detoxified.

14. The method according to claim 1, wherein, The step of fermenting the growth medium using halophilic microorganisms includes: inoculating the growth medium with halophilic microorganisms capable of producing polyhydroxyalkanoates to form a fermentation culture.

15. The method according to claim 14, wherein, The fermentation culture is grown in a continuous fermentation system.

16. The method according to claim 15, wherein, The continuous fermentation system includes a semi-continuous fermentation system.

17. The method according to claim 15, wherein, The continuous fermentation system comprises two or more fermentation reactors that operate sequentially, in series, or in cascade.

18. The method according to claim 14, wherein, Additional growth medium is added to the fermentation culture during fermentation.

19. The method of claim 17, wherein, The carbon-nitrogen ratio was increased from the first fermentation reactor to the last to optimize the fermentation culture within the reactor.

20. The method of claim 17, wherein, The carbon-nitrogen ratio of the fermentation culture in each reactor is kept constant by feeding additional growth medium into each reactor.

21. The method according to claim 17, wherein, The continuous fermentation system includes an inlet feed for providing growth medium to the fermentation reactor and an outlet feed for pumping the fermentation culture from a first reactor through each subsequent reactor to the two or more reactors.

22. The method according to claim 21, wherein, The flow rate of the feed from the outlet of the last reactor is set to be equal to the sum of the inlet feed flow rates of all the continuous fermentation systems.

23. The method of claim 17, wherein, During fermentation, acid and alkali are added to maintain the fermentation culture at pH 7.0, and the temperature of the fermentation reactor is maintained at 40°C.

24. The method according to claim 1, wherein, The step of extracting polyhydroxyalkanoates from the halophilic cells using a saline-based lysis method includes harvesting the cell biomass by separating the cell biomass from the fermentation culture by centrifugation or filtration.

25. The method according to claim 24, wherein, The isolated halophilic cells were immersed in a saline-based solution to lyse them.

26. The method of claim 25, wherein, The salt-based solution contains seawater or water with added salt.

27. The method according to claim 26, wherein, The salt-based solution contains 0.05 to 0.2% surfactant.

28. The method according to claim 27, wherein, The surfactant includes sodium dodecyl sulfate.

29. The method according to claim 24, wherein, Polyhydroxyalkanoates are recovered from halophilic cells by immersing them in a saline-based solution and then centrifuging or filtering them from the saline-based solution once or more to remove the aqueous phase.

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