WO2023073684A1 - Method for production of pha by fermentation of archaea - Google Patents

Method for production of pha by fermentation of archaea Download PDF

Info

Publication number
WO2023073684A1
WO2023073684A1 PCT/IL2022/051061 IL2022051061W WO2023073684A1 WO 2023073684 A1 WO2023073684 A1 WO 2023073684A1 IL 2022051061 W IL2022051061 W IL 2022051061W WO 2023073684 A1 WO2023073684 A1 WO 2023073684A1
Authority
WO
WIPO (PCT)
Prior art keywords
pha
pla
culture medium
hydrolysate
weight
Prior art date
Application number
PCT/IL2022/051061
Other languages
French (fr)
Inventor
Alexander Golberg
Supratim Ghosh
Michael Gozin
Original Assignee
Ramot At Tel-Aviv University Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ramot At Tel-Aviv University Ltd. filed Critical Ramot At Tel-Aviv University Ltd.
Priority to KR1020247016094A priority Critical patent/KR20240093595A/en
Publication of WO2023073684A1 publication Critical patent/WO2023073684A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L101/00—Compositions of unspecified macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L67/00—Compositions of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Compositions of derivatives of such polymers
    • C08L67/04—Polyesters derived from hydroxycarboxylic acids, e.g. lactones
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F120/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F120/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F120/04—Acids; Metal salts or ammonium salts thereof
    • C08F120/06—Acrylic acid; Methacrylic acid; Metal salts or ammonium salts thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F120/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F120/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F120/10—Esters
    • C08F120/12—Esters of monohydric alcohols or phenols
    • C08F120/16—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms
    • C08F120/18—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms with acrylic or methacrylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F120/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F120/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F120/42—Nitriles
    • C08F120/44—Acrylonitrile
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2205/00—Polymer mixtures characterised by other features
    • C08L2205/02—Polymer mixtures characterised by other features containing two or more polymers of the same C08L -group
    • C08L2205/025—Polymer mixtures characterised by other features containing two or more polymers of the same C08L -group containing two or more polymers of the same hierarchy C08L, and differing only in parameters such as density, comonomer content, molecular weight, structure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to a process for large scale production of a polyhydroxyalkanoate (PHA) by fermentation, including outdoor fermentation, of an archaea such as Haloferax mediterranei, wherein air is constantly bubbled into the fermenter from its bottom allowing constant aeration and mixing of the culture medium, as well as removal of carbon dioxide; and further provides blends of a PHA and additional polymers, and articles made therefrom.
  • PHA polyhydroxyalkanoate
  • PHA polymers are hydrophobic, waterinsoluble, indefinitely stable in air, non-toxic, thermoplastic and/or elastomeric, and have very high purity within the cell.
  • the PHA polymer family has much potential and adaptability for the broad application of biopolymers. Therefore, the search for highly efficient microorganisms for PHA accumulation is the need of the hour.
  • Haloferax mediterranei ⁇ Hfx. mediterranei an extreme halophile
  • the archaea can accumulate high amounts of intracellular PHA utilizing a variety of substrates.
  • extremophilic nature which is characterized by high salt tolerance, enables non- sterile cultivation of this organism thereby decreasing the cost and energy required for the process.
  • PHA being an intracellular product requires high cell density cultivations, because the restricted availability of cytoplasmic space limits the maximum amount of PHA which can be accumulated in the cells.
  • Recovery of PHA begins after separating and concentrating cells from the fermentation medium. As PHA recovery from halophiles can be easily done by cell lysis using tap-water, separation of cells from the fermentation medium becomes a challenge in the overall recovery of PHA. Generally, separation of cells is performed using centrifugal force at a laboratory scale. Industrial separation of cells from the fermentation medium requires highly efficient continuous centrifuges. These methods are adequate when extracellular products are required, but when large quantities of washed bacteria are wanted, the need to scrape or wash out continuous flow centrifuge rotors makes a sterile harvest difficult to achieve.
  • the present study provides a feasible way for scaling up PHA production from inexpensive substrates, such as macroalgal hydrolysate.
  • the effect of various cultivation parameters was studied and favorable conditions for enhancement of PHA productivity were established.
  • the molecular weight analysis suggested that the PHA obtained was more homogeneous at lower aeration velocities.
  • the present invention relates to a method for production of a polyhydroxyalkanoate (PHA) by fermentation of an archaea capable of producing said PHA, said method comprising the steps of:
  • a fermenter having a cultivation, i.e., working, volume of at least about 5 or 10 liters, said fermenter containing a culture medium occupying said cultivation volume, said culture medium comprising saltwater such as seawater, supplemented with a carbon source and a nitrogen source, and suitable for culturing, i.e., promoting and supporting the growth and the survival of, said archaea;
  • the method disclosed herein may further comprise the step of purifying the PHA obtained in step (v), e.g., by repeated washing with water, an ionic liquid, or a combination thereof, to thereby obtain a purified PHA; and optionally the step of drying the purified PHA thus obtained, e.g., by a drum dryer, spray dryer, or lyophilizer.
  • the purified PHA regardeless of whether dried or not, may then be blend, i.e., mixed, with at least one polymer, e.g., a biodegradable or non-biodegradable polymer.
  • the fermentation process of the present invention may be carried out both in batches and continuously.
  • the present invention provides a blend comprising a PHA and at least two additional polymers, wherein each one of said at least two additional polymers may independently be a biodegradable or non-biodegradable polymer.
  • said at least two additional polymers comprise, or consist of, polylactic acid (PLA) and keratin, wherein the PHA:PLA ratio in said blend may range between about 1:99 to about 99:1, between about 20:80 to about 80:20, between about 30:70 to about 70:30, or between about 40:60 to about 60:40, by weight, respectively, but it is preferably between about 20:80 to about 40:60, e.g., about 25:75, about 30:70, or about 35:65, by weight, respectively (regardless of the overall amount of the other polymers, e.g., keratin, comprised within said blend).
  • one of said at least two additional polymers is keratin, which constitutes from about 1% to about 99%, e.g., from about 5% to about 90%, from about 10% to about 80%, from about 15% to about 70%, or from about 20% to about 60%, but preferably from about 10% to about 50%, by weight, of said blend.
  • the present invention provides an article comprising, or made of, a blend as disclosed herein.
  • FIGs. 1A-1D show experimental setup for optimization of aeration rates in outdoor cultivation (I L PET bottles) of Hfx. mediterranei (1A); experimental setup for cultivation of Hfx. mediterranei in 40 L sleeves (IB); schematic representation of the cultivation system in 1 L PET bottles [experimental conditions: flow rates: 0.25-2.0 Lxmin’ 1 ; fermentation volumes: 800 mL; temperature: 42+2.5°C; inoculum concentration: 50 gxL’ 1 ; initial pH: 7.2] (1C); and schematic representation of a single photobioreactor for cultivation of Hfx.
  • Figs. 2A-2F show growth and PHA production curves at different aeration rates for PHA production using Hfx.
  • mediterranei [experimental conditions: flow rates: 0.25-2.0 Lxmin’ x ; fermentation volume: 800 mL; water temperature: 42+2.5°C; air temperature: 37 ⁇ 2.5°C; inoculum concentration: 50 gxL’ 1 ; initial pH: 7.2] (2A and 2B, respectively); growth and PHA productivities under outdoor cultivation conditions for PHA production using Hfx.
  • Fig. 3 shows molecular weights (Mn, Mw, and Mz) and polydispersity index (PDI) distributions of PHA produced by Hfx. mediterranei using two standards, polystyrene (PS) and polymethyl methacrylate (PMMA).
  • PS polystyrene
  • PMMA polymethyl methacrylate
  • Figs. 4A-4C illustrate (4A) ASTM specimen dimensions of type V; (4B) the mold design; and (4C) the mold.
  • Figs. 5A-5C illustrate: (5A) ASTM specimen dimensions of type IV; (5B) the mold design; and (5C) the mold.
  • Figs. 6A-6B are graphs showing thermal properties of PLA, PHA and keratin as pure materials.
  • Figs. 7A-7B are graphs showing DSC (7A) and TGA (7B) curves of PHA and keratin blends.
  • Figs. 8A-8B are graphs showing temperature of degradation vs. keratin weight fraction (8A); and mass change vs. keratin weight fraction of PHA and keratin blends (8B).
  • Figs. 9A-9B are graphs showing DSC (9A) and TGA (9B) curves of PLA, PHA and keratin blends.
  • Figs. 10A-10B are graphs showing Temperature of degradation vs. keratin weight fraction (10A), and Mass change vs. keratin weight fraction of PLA, PHA and keratin blends (10B).
  • Figs. 11A-11G are graphs showing Stress-Strain curve of PLA specimens: (11A) of all tested PLA specimens; (11B) of PLA specimen 003-1; (11C) of Young modulus; (11D) of Poisson ratio; (HE) of PLA specimen 003-2; (HF) of Young modulus; and (11G) of Poisson ratio.
  • Figs. 12A-12G are graphs showing Stress-Strain curve of various PLA/PHA(70:30) specimens tested: (12A) of PLA/PHA(70:30) specimens; (12B) of Stress-Strain curve of PLA/PHA (70:30) specimen 102-1; (12C) of Young modulus; (12D) of Poisson ratio; (12E) of Stress-Strain curve of PLA/PHA (70:30) specimen 102-2; (12F) of Young modulus; and (12G) of Poisson ratio.
  • Figs. 13A-13J are graphs showing Stress-Strain curve of various PLA/PHA(70:30)- 70/keratin-30 specimens tested: (13A) of PLA/PHA(70:30)-70/keratin-30 specimens; (13B) of Stress-Strain curve of PLA/PHA(70:30)-70/keratin-30 specimen 402-1 ; (13C) of Young modulus; (13D) of Poisson ratio; (13E) of Stress-Strain curve of PLA/PHA(70:30)-70/keratin- 30 specimen 402-2; (13F) of Young modulus; (13G) of Poisson ratio; (13H) of Stress-Strain curve of PLA/PHA(70:30)-70/keratin-30 specimen 403-1; (131) of Young modulus; and (13J) of Poisson ratio.
  • Fig. 14 is a graph summarizing Stress-Strain curve of all the specimens.
  • PLA 003-1 and 003-2
  • PLA/PHA 102-1 and 102-2
  • PLA/PHA/keratin 402-1,402-2 and 403-1.
  • Fig. 15 presents photomicrographs showing the degradation of different composites under composting conditions.
  • Fig. 16 is a graph showing degree of disintegration of: control PLA dog-bone, PLA/PHA and PLA/PHA/keratin dog-bones under composting conditions as a function of time.
  • the present invention relates to a method for production of a PHA by fermentation of an archaea capable of producing said PHA, comprising the steps of:
  • a fermenter having a cultivation volume of at least about 5 or 10 liters, said fermenter containing a culture medium occupying said cultivation volume, said culture medium comprising saltwater such as seawater, supplemented with a carbon source and a nitrogen source, and suitable for culturing said archaea;
  • PHA polyhydroxyalkanoate
  • PHAs obtained from microbiological sources, genetically modified bacteria, and other organisms consist of monomers each of the formula [-O-C(R)H-(CH2) n -C(O)-] wherein R is H or alkyl, i.e., a linear or branched hydrocarbyl having, e.g., 1-12 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isoamyl, 2,2-dimethyl-propyl, n- hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like.
  • R is H or alkyl, i.e., a linear or branche
  • PHAs include, without being limited to, poly (3 -hydroxypropionate), poly (3 -hydroxybutyrate), poly(3-hydroxyvalerate), poly(3-hydroxyhexanoate), poly(3-hydroxyoctanoate), and polyphydroxy dodecanoate), wherein n is 1, and R is H, methyl, ethyl, propyl, pentyl, or nonyl, respectively; as well as poly(4-hydroxybutyrate) and poly (5-hydroxy valerate), wherein n is 2 or 3, and R is H, respectively.
  • archaea refers to any of a group of single-celled prokaryotic organisms that have distinct molecular characteristics separating them from bacteria and eukaryotes.
  • Non-limiting examples of archaea include species of Picrophilus methanogens, halophiles, and Pyrolobus fumarii. Particular archaea referred in this description are halophilic archaea.
  • the archaea is Hfx mediterranei, e.g., Hfx mediterranei ATCC 33500.
  • saltwater refers to water having a salinity, consisting predominantly of sodium ions and chloride ions, of at least about 2% and up to about 30, 35, or 40%.
  • said saltwater are seawater, i.e., water from a sea or an ocean, having on average a salinity of about 3.5%.
  • said saltwater has a salinity ranging between from about 4% or 5% to about 10%, or a salinity higher than 10%, e.g., of up to about 15%, 20%, 25%, or 30%.
  • oxygen-containing gaseous mixture refers to a gaseous mixture comprising at least about 15%, e.g., about 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%, but preferably about 19%, 20%, 21%, 22%, or 23%, by volume (v/v), oxygen, and additional gases, which may be selected from nitrogen, carbon dioxide, and inert gases such as helium, neon, argon, krypton, and xenon.
  • gases which may be selected from nitrogen, carbon dioxide, and inert gases such as helium, neon, argon, krypton, and xenon.
  • Particular such gaseous mixtures are air-like gaseous mixtures comprising oxygen, nitrogen, carbon dioxide, and argon, which constitute together at least 98% by volume of said gaseous mixtures.
  • the oxygen-containing gaseous mixture bubbled into the fermenter during the fermentation process disclosed is air, i.e., a gaseous mixture containing about 78% nitrogen, about 21 % oxygen, about 0.9%' argon, about 0.1 % carbon dioxide, and small amounts of other gases.
  • the culture medium used in the fermentation process disclosed herein comprises carbon and nitrogen, each independently in an amount of from about 1% to about 80% by weight, on a dry, i.e., dehydrated, mass base.
  • dry mass base refers to the dry weight of the culture medium, i.e., to the weight of overall solid ingredients constituting said culture medium.
  • the amount of carbon in said culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, by weight, on a dry (dehydrated) mass base; and/or the amount of nitrogen in said culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, by weight, on a dry (dehydrated) mass base.
  • the carbon source comprised within the culture medium used in the fermentation process disclosed comprises a sugar, more particularly a monosaccharide such as, without limiting, glucose, galactose, fructose, and xylose, or a polysaccharide such as starch; a waste product such as, without being limited to, glycerol (a major by-product in the biodiesel manufacturing process), fatty acids such as acetic acid, butyric acid and propanoic acid (produced during anaerobic degradation of organic compounds during the acidogenic fermentation, and serve as feedstock for freshwater and marine oleaginous microorganisms to produce, e.g., biodiesel), vinnase (a byproduct of the sugar or ethanol industry), stillage (a liquid waste remaining after ethanol distillation, and containing reducing sugars (fructose and glucose) and fatty acids, including acetic acid, propionic acid and butyric acid), molasses wastewater, and
  • a sugar more particularly
  • biopolymer refers to a natural polymer (consisting of covalently bonded monomeric units), produced by the cells of a living organism.
  • the major examples of such polymers classified according to the monomers used and the structure of the biopolymer formed, are polynucleotides, polypeptides and proteins, and polysaccharides.
  • Polynucleotides, such as RNA and DNA are polymers composed of 13 or more nucleotide monomers.
  • Polypeptides and proteins are polymers of amino acids and include collagen, actin and fibrin.
  • Polysaccharides are liner or branched polymeric carbohydrates and include starch, cellulose and alginate.
  • biopolymers include natural rubbers (polymers of isoprene), suberin and lignin (complex polyphenolic polymers), cutin and cutan (complex polymers of long-chain fatty acids), and melanin.
  • Polymers, as referred to hereinabove, may be glycosylated, i.e., modified by the attachment of sugar moieties.
  • amino acid refers to an organic compound comprising both amine and carboxylic acid functional groups, which may be either a natural or non-natural amino acid, and occur in both L and D isomeric forms.
  • the twenty-two amino acids naturally occurring in proteins are aspartic acid (Asp), tyrosine (Tyr), leucine (Leu), tryptophan (Trp), arginine (Arg), valine (Vai), glutamic acid (Glu), methionine (Met), phenylalanine (Phe), serine (Ser), alanine (Ala), glutamine (Gin), glycine (Gly), proline (Pro), threonine (Thr), asparagine (Asn), lysine (Lys), histidine (His), isoleucine (He), cysteine (Cys), selenocysteine (Sec), and pyrrolysine (Pyl).
  • Non-limiting examples of other amino acids include citrulline (Cit), diaminopropionic acid (Dap), diaminobutyric acid (Dab), ornithine (Om), aminoadipic acid, P- alanine, 1 -naphthylalanine, 3-(l-naphthyl)alanine, 3-(2-naphthyl)alanine, y-aminobutiric acid (GABA), 3-(aminomethyl) benzoic acid, -cthynyl-phcnylalaninc, m-ethynyl-phenylalanine, p- chlorophenylalanine (4ClPhe), -bromophcnylalaninc, -iodophcnylalaninc, p- acetylphenylalanine, p-azidophenylalanine, p-propargly-oxy-phenylalanine,
  • amino acid residue refers to a residue of an amino acid after removal of hydrogen atom from an amino group thereof, e.g., its a-amino group or side chain amino group if present, and -OH group from a carboxyl group thereof, e.g., its a-carboxyl group or side chain carboxyl group if present.
  • peptide refers to a short chain of amino acid monomers (residues), e.g., a chain consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid residues, linked by peptide (amide) bonds, i.e., the covalent bond formed when a carboxyl group of one amino acid reacts with an amino group of another.
  • peptide moiety refers to a moiety of a peptide as defined herein after removal of the hydrogen atom from a carboxylic group, i.e., either the terminal or a side chain carboxylic group, thereof, and/or a hydrogen atom from an amino group, i.e., either the terminal or a side chain amino group, thereof.
  • peptide bond or "amide bond”, as used herein, refers to the covalent bond -C(O)NH- formed between two molecules, e.g., two amino acids, when a carboxyl group of one of the molecules reacts with an amino group of the other molecule, causing the release of a water molecule.
  • hydrolysate refers to an aqueous product of hydrolysis, comprising proteins that are chemically or enzymatically broken down to peptides of various sizes.
  • hydrolysates that may be used as carbon and/or nitrogen sources for the culture medium include seaweed hydrolysate, olive leaves hydrolysate, agricultural waste hydrolysates, and cheese whey hydrolysate.
  • the hydrolysate referred to is seaweed hydrolysate, which may be used as either or both, but preferably both, the carbon and nitrogen source.
  • seaweed refers to any species of a macroscopic, multicellular, marine algae, and includes types of Rhodophyta (red), Phaeophyta (brown) and Chlorophyta (green) macroalgae.
  • the seaweed referred to is a green macroalgae such as an Ulva species.
  • the carbon source comprised within the culture medium used in the fermentation process disclosed comprises a seaweed hydrolysate, wherein said hydrolysate constitutes at least about 1%, preferably about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, by weight, of said carbon source; and/or the nitrogen source comprised within said culture medium comprises a seaweed hydrolysate, wherein said hydrolysate constitutes at least about 1%, preferably about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, by weight, of said nitrogen source.
  • both the carbon source and the nitrogen source consist solely of a seaweed hydrolysate, which provides all the carbon and nitrogen required for the fermentation process of said archaea.
  • the culture medium used in the fermentation process disclosed herein further comprises salts such as salts containing bromide anion, buffers, phosphorus, or a mixture thereof.
  • Phosphorus sources that may be used for the preparation of said culture medium include, without being limited to, dissolved inorganic phosphate such as potassium dihydrogen phosphate and disodium hydrogen phosphate, as well as phosphorus- containing biomolecules; and halogen sources that may be used include, without limiting, bromine.
  • the culture medium used in the fermentation process disclosed herein comprises saltwater, e.g., seawater, supplemented with said carbon source, said nitrogen source, and optionally said phosphorus, halogen, or mixture thereof, wherein the amounts of carbon and nitrogen in said culture medium each independently is from about 1% to about 80% by weight on a dry mass base; said carbon source comprises a seaweed hydrolysate, which constitutes at least about 1% by weight of said carbon source; and said nitrogen source comprises a seaweed hydrolysate, which constitutes at least about 1% by weight of said nitrogen source.
  • the amount of carbon in said culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, by weight, on a dry mass base;
  • the amount of nitrogen in said culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, by weight, on a dry mass base;
  • said carbon source comprises a seaweed hydrolysate, which constitutes at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, by weight of said carbon source;
  • said nitrogen source comprises a seaweed hydrolysate, which constitutes at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, by weight of said nitrogen source.
  • both said carbon source and said nitrogen source consist solely of a seaweed hydrolysate, which provides all the
  • the seaweed hydrolysate comprised within the culture medium used in the fermentation process disclosed, according to any one of the embodiments above, is a green macroalgae hydrolysate, such as Ulva sp. hydrolysate.
  • the pH of the culture medium prior to the inoculation step (ii), according to any one of the embodiments above, is up to 8.5.
  • the pH of said culture medium prior to the inoculation step (ii) is from about 2 to about 8.5, preferably from about 6.8 to about 7.4, e.g., 7.2.
  • the culturing step (iii), according to any one of the embodiments above, is carried out for a period of up to 14 or 21 days, e.g., for a period of at least 48, 54, 60, 66, or 72 hours.
  • an oxygen-containing gaseous mixture such as air
  • an aeration rate of at least about 0.2 vvm
  • the term “aeration rate”, measured in volume/volume/minute (vvm), represents the volumetric flow rate of the oxygen-containing gaseous mixture / fermenter working volume, i.e., refers to the ratio between the volume of the oxygen-containing gaseous mixture bubbled into the fermenter per minute and the working volume of said fermenter.
  • the aeration rate according to any one of the embodiments above is from about 0.6, 0.7, or 0.8 to about 1.2 vvm, preferably from about 0.9 to about 1.1 vvm, more preferably about 1 vvm.
  • the aeration rate of said oxygencontaining gaseous mixture may thus significantly affect the efficacy of the culturing step, e.g., the maximal archaea concentration that may be obtained in the culture medium during the fermentation process or the time required to achieve said concentration.
  • the aeration rate of said oxygen-containing gaseous mixture also affects, although not in a linear manner, the molecular weight of the PHA obtained, which varies according to the aeration rate and the archaea’s culturing period, wherein higher molecular weight PHA was clearly observed at higher aeration rates.
  • the archaea’s biomass produced is harvested from the culture medium, and the PHA is then concentrated, separated and/or extracted from the harvested biomass.
  • extraction of the PHA from the harvested biomass is carried out by physical means such as water (hydrolysis), high pressure, pulsed electric field (PEF), and centrifugation and filtration; chemical means such as a lysis buffer, deep eutectic solvent, organic solvent, biphasic solvent system, and an ionic liquid; or any combination thereof.
  • physical means such as water (hydrolysis), high pressure, pulsed electric field (PEF), and centrifugation and filtration
  • chemical means such as a lysis buffer, deep eutectic solvent, organic solvent, biphasic solvent system, and an ionic liquid; or any combination thereof.
  • Deep eutectic solvent refers to a solution of a Lewis or Bronsted acid and a base, which form a eutectic mixture, i.e., a homogeneous mixture of substances that melts or solidifies at a single temperature that is lower than the melting point of any of the constituents. Deep eutectic solvents are highly tunable through varying the structure or relative ratio of the parent components and thus have a wide variety of potential applications including separation.
  • organic solvent refers to a non-toxic, environmentally friendly, water-soluble solvent that may contain carbon, hydrogen, oxygen, sulfur, and nitrogen atoms in its molecular structure.
  • organic solvents include, without being limited to, carbonate esters, including dimethylcarbonate, and diethylcarbonate; lactate esters, including ethyl lactate; levulinate esters, including methyl levulinate and ethyl levulinate; y- valerolactone; (17?)-7,8-dioxabicyclo[3.2.1]octan-2-one (cyrene); alcohols, diols and polyols, including ethanol, isopropanol, and ethyleneglycol; and ketones, including methylethylketone.
  • carbonate esters including dimethylcarbonate, and diethylcarbonate
  • lactate esters including ethyl lactate
  • levulinate esters including methyl levulinate and ethyl levulinate
  • y- valerolactone (17?)-7,8-dioxabicyclo[3.2.1]octan-2-one (cyrene)
  • biphasic solvent system refers to a solvent system comprising two solvents separated by an interfacial layer, wherein one of said two solvents is an aqueous solvent, and the other one of said two solvents is a non-toxic, environmentally friendly, partially (or poor) water-soluble solvent that may contain carbon, hydrogen, oxygen, sulfur, and nitrogen atoms in its molecular structure, such as, but not limited to, an acetate ester, including ethyl acetate and butyl acetate; and an ether, including 2-methyltetrahydrofuran.
  • ionic liquid refers to a non-toxic, environmentally friendly, organic salt that may contain carbon, hydrogen, oxygen, sulfur, nitrogen and halogen atoms in its molecular structure.
  • Non-limiting examples of ionic liquids include salts containing dialkyl- imidazolium, A-alkyl-pyridazinium, quaternary ammonium, dialkyl-pyrrolidinium, alkyl- guanidinium, and alkyl-pyridinium cations; and a variety of envirometally friendly anions, such as acetate, formate, phosphate, hydrogen-phosphate, and chloride.
  • the method disclosed herein may further comprise the step of purifying the PHA obtained in step (v), e.g., by repeated washing with water, a solvent, an ionic liquid, or a combination thereof, to thereby obtain a purified PHA; and optionally the step of drying the purified PHA thus obtained, e.g., by a drum dryer, spray dryer, air or air/nitrogen flow drying, or lyophilizer.
  • the purified PHA may then be blend, i.e., mixed, with at least one polymer.
  • suitable polymers for blending with the PHA include biodegradable polymers, such as polylactic acid (PLA), polycaprolactone (PCL), keratin, cellulose, chitin, lignin, amylose, amylopectin, and mucin; and non-biodegradable polymers or copolymer, such as polyethylene terephthalate (PET, also known as polyester), high-density polyethylene (HDPE), polyvinyl chloride (PVC), low-density polyethylene (LDPE), polypropylene (PP), polystyrene (PS), polyethylene glycol (PEG), polyproplylene glycol (PG), polyvinyl alcohol (PVA), polyacrylonitrile (PAN), polymethacrylic acid or an ester thereof, poly(acrylonitrile-co-butadiene-co-co-
  • the preparation of blends as disclosed herein may comprise the addition of various plastisizers and/or additives, so as to modulte the properties of the PHA or of the PHA blend prepared as disclosed, e.g., in Garcia-Garcia et al., 2022; Charon et al., 2022; and Longe et al., 2022.
  • one or more plastisizers and/or additives are added to the PHA prior to mixing with the other polymer(s), to modulate the properties of said PHA before blending with the other polymer(s).
  • one or more plastisizers and/or additives are added to the PHA blend after the preparation thereof to modulate the properties of said blend.
  • the fermenter used in the method of the invention is an open fermenter, such that the culturing of said archaea is carried out in non- sterile conditions.
  • the fermenter used in the method of the invention is made of glass, metal or metallic alloy, ceramic, plastic, or cement.
  • said fermenter is an earth fermenter, also referred to as earthen-bank pond or earthen-bank construction.
  • the fermentation process of the present invention is carried out in batches following step (i)-(v) according to any one of the embodiments above.
  • said fermentation process is carried out continuously, wherien archaea’s biomass is either continuously or once in a while harvested from the culture medium (and then subjected to PHA concentration, separation and/or extraction), and fresh culture medium (in an amount equivalent to that removed for harversting) is introduced into the fermenter.
  • PLA is primarily amorphous whereas PHA and keratin are more crystalline.
  • Crystalline structures are generally very ordered, which grants them strength and rigidity. In such structures, the molecular chains are locked in place, so when a load is applied, they break rather than bend. Amorphous polymers are the opposite. Rather than being rigid, the random molecular jumble allows the chains to move across one another, when the polymer is pulled. This is what grants amorphous polymers with flexibility and elasticity.
  • the pores cause a decrease in the adhesion between the polymeric phases, therefore changing the material's properties.
  • biodegradation tests were performed as well.
  • the biopolymers synthesized were completely biodegradable under aerobic conditions in a composting environment.
  • the PLA/PHA/keratin blend was the fastest to degrade. This indicates that keratin not only helps the thermal stability of the material, but also causes it to be more biodegradable. According to these tests, these materials can be characterized as ecofriendly. In order to reinforce these findings, more tensile test repetitions should be performed.
  • the present invention thus provides a blend comprising a PHA and at least two additional polymers.
  • the polymers blended with the PHA may be selected from a biodegradable polymer, such as polylactic acid (PLA), poly caprolactone (PCL), poly(butylene succinate), poly(butylene succinate adipate), aliphatic -aromatic co-polyesters, poly(butylene adipate/terephthalate), and poly(methylene adipate/terephthalate), keratin, cellulose, chitin, lignin, amylose, amylopectin, mucin, and a PHA different from said PHA; and a non-biodegradable polymer, such as polyethylene terephthalate (PET, also known as polyester), high-density polyethylene (HDPE), polyvinyl chloride (PVC), low-density polyethylene (LDPE), polypropylene (PP), polystyrene (PS), polyethylene glycol (PEG), polyproplylene glycol (PG), poly
  • a biodegradable polymer
  • the blend disclosed herein comprises said PHA and at least two additional polymers, wherein said at least two additional polymers comprise, or consist of, PLA and keratin.
  • the PHA:PLA ratio ranges between about 1:99 to about 99:1, between about 20:80 to about 80:20, between about 30:70 to about 70:30, or between about 40:60 to about 60:40, by weight, respectively, but it is preferably between about 20:80 to about 40:60, e.g., about 25:75, about 30:70, or about 35:65, by weight, respectively (regardless of the overall amount of the other polymers, e.g., keratin, comprised within said blend).
  • one of the at least two additional polymers blended with said PHA is keratin, which constitutes from about 1% to about 99%, e.g., from about 5% to about 90%, from about 10% to about 80%, from about 15% to about 70%, or from about 20% to about 60%, but preferably from about 10% to about 50%, by weight, of said blend.
  • blend components and these components source and physical and chemical propertis (including, but not limited to molecular weight, crystallinity, chain conformation, hydrophobicity and hydrophylicity, stability to hydrolysis, thermal properties, transport properties, optical properties, toughness, elasticity, viscoelasticity, heat, electromagnetic radiation conductivity, and electrical conductivity) can affect aforemention physical and chemical properties of resulted blends and blends homogeneity and components adhesion.
  • incorporation of PHA in blends leads to a decrease in the flexural modulus with simultaneous increase in the tensile modulus.
  • the presence of keratin can influence the thermal stability and the toughness of the blends. As the keratin weight fraction increased, there was a significant increase in the degradation temperature and a reduction in the weight loss of the specimens.
  • the present invention provides an article comprising, or made of, a blend as disclosed in any one of the embodiments above.
  • Such articles may be prepared by any suitable technique known in the art, e.g., as shown in the Experimental section herein.
  • the conditions for hydrolysis were as follows: temperature of 170°C, total residence time of 20 min, salinity of 38 gxL’ 1 , and a solid load of 5%. These conditions were chosen for optimization of sugar yield.
  • the hydrolysate was separated into liquid and solid phases by centrifugation at 10,000 rpm for 3 min (Yingtai Instruments TGL- 18, China).
  • PHA production by Hfx. mediterranei. PHA production was investigated utilizing the Ulva sp. hydrolysate with the aid of Hfx. mediterranei ATCC 33500 (NCIMB 2177).
  • the Hfx. mediterranei strain were routinely grown in a rich medium (Hv-YPC).
  • the culture was grown in a shaking incubator (MRC Labs, Israel) at 42°C with a rotational speed of 180 rpm, and the media pH was adjusted to 7.2.
  • seaweed hydrolysate (aqueous phase) was supplemented to saline water (144 g NaClxL -1 ) at a working concentration of 25% v/v as observed in previous studies (Ghosh et al., 2021).
  • the experiments were performed in custom- built reactors made of 1 L PET (polyethylene teraphtalate) bottles (length 0.285 m, width 0.08 m).
  • a set of 15 PET bottles were utilized for the cultivation of Hfx. mediterranei under different aeration rates (0.25-2.0 Lxmin 1 ) in triplicates.
  • Individual reactors were connected with flowmeters to manipulate the airflow inside the reactor. The air was sparged from the bottom of the reactor for uniform flow inside the reactor.
  • the flask was maintained at 42°C in a shaking incubator at 170 rpm for approximately 48 h to late exponential/early stationary phase and removed from the shaker.
  • the optical density of the culture was 7.9 at 520 nm as measured on a benchtop spectrophotometer (ThermoFisher AquaMATE VIS, 1 cm path length), which roughly corresponds to an ash-free dry weight concentration of 3.9 gxL-1.
  • the Hfx. mediterranei culture was transferred to a closed container and stored on the benchtop for two weeks, as a test for auto-flocculation, which could improve the particle size and ultrasonic response of the culture. Auto-flocculation was not observed in the standing culture, so enhanced aggregation of Hfx.
  • mediterranei cells was attempted by Ca 2+ addition.
  • Cells were pelletized by centrifugation at l,000xg for 15 min and then twice washed in a Tris buffer (20 mM Tris-HCl and 4 M NaCl, pH 7.0).
  • the cells were then suspended at higher concentration in a salt solution (27 mM KC1, 0.24 mM NaHCO 3 , 0.49 mM NaBr, 3.7 mM NH4C1, 4.3 M NaCl, 18.5 pM FeC13-6H2O, pH 7.0).
  • a second cell suspension was washed and rinsed following the same procedure, but the final salt solution was modified to include 10 mM of CaCh.
  • the washed suspensions had an optical density of 11.8 at 520 nm, which corresponds to an ash-free dry weight concentration of 5.8 gxL-1.
  • the PHA content of the cells was measured using the Nile Red staining procedure. Briefly, the cells were washed and resuspended in 10% saline water. Further, the cell suspension was stained with Nile Red (Sigma Aldrich, USA) which had a final concentration of 3.1 pgxml 1 . The cells were incubated for 30 mins and further washing and resuspension was performed using 10% saline. The fluorescence of the suspension was measured in a 96 well plate reader (Tecan, Switzerland) at excitation and emission wavelengths of 535 and 605 nm respectively.
  • PHA commercial PHA (Sigma, USA) was utilized for preparation of standards with varying concentration (0.2-2.0 gxL 1 ). These standards were used for preparation of standard curve. A standard curve was plotted and the final concentration of PHA in the cells was determined. The unknown amount of PHA was determined from the standard curve with two repeats per point. The protocol was also verified using the crotonic acid assay for intracellular PHA content determination. Two milliliters of sulfuric acid were added to the dried cell pellet containing the polymer (PHA). The mixture was hydrolyzed by heating in a water bath at 100°C for 20 min to obtain crotonic acid.
  • a PHA — 7 ⁇ l2 C 1 wherein Bi and B2 represent the biomass concentrations (gxL 1 ) at time ti (h) and t2 (h) respectively, and Ci and C2 represent the PHA content (gxL 1 ) at time ti (h) and t2 (h) respectively.
  • the concentration was determined by PHA extraction.
  • FTIR-ATR analysis The PHA powder obtained after drying was analyzed by a Fourier-transform infrared spectroscopy (FTIR) spectrometer, equipped with attenuated total reflectance (ATR) attachment (Bruker Platinum ATR, USA). The spectrum was recorded in the range of 400 to 4000 cm 1 .
  • FTIR Fourier-transform infrared spectroscopy
  • ATR attenuated total reflectance
  • Thermogravimetric analysis I differential scanning calorimetry (TGA/DSC) analysis. 5 mg of dry PHA powder was weighed in a sealed aluminum pan. The pan was then subject to a linear temperature gradient (30 to 600°C) in a differential scanning calorimeter equipped with autoloader (Jupiter STA 449 F5, NETZSCH, Germany). The heating rate was maintained at 10 o Cxmin -1 .
  • 1 H NMR analysis The PHA produced was subjected to ’ H NMR analysis. The powdered PHA was dissolved in CDCI3 (10 mgxmr 1 ) and then analyzed in a 400 MHz spectrometer (Bruker, USA).
  • GC-MS analysis The butyl esters of PHA were analyzed using GC-MS system equipped with auto-sampler (6890/5977A, G4513A; Agilent, USA) and with HP-5MS UI column (Agilent, USA).
  • the column consisted of a stationary phase of 5% phenyl/methyl-poly- siloxane, which was 30 m in length and an i.d. of 0.25 mm.
  • Helium (99.999%) was used as a carrier gas, at a flow rate of 1.0 mLxmin 1 .
  • the samples were injected with a split ratio of 1:19 into the injector heated to a temperature of 280°C.
  • the sample injection volume was 0.2 pL.
  • the conditions for separation of analyte were as follows: initial oven temperature 70°C, which was held for 5 min. and then increased linearly to 280°C, at a rate of 15 0 Cxmin -1 . This was followed by a linear temperature increase to 320°C, at a rate of 30 o Cxmin -1 , with a holding time of 5 min at the final temperature.
  • Mass spectra analysis was performed in the El positive ion mode, using electron energy of 70 eV. Transfer line temperature and ion source temperature were maintained at 280 and 250°C, respectively. Obtained mass spectra data were collected in full-scan mode (m/z 50-400) and analyzed by using Agilent ChemStation software.
  • the maximum volumetric biomass and PH A productivity were estimated to be 64.03+0.11 mgxL ⁇ xh 1 and 34.07+0.03 mgxL ⁇ xh 1 respectively at an aeration rate of 1.0 Lxmin 1 (or 1.0 vvm).
  • a 10-15% increase in biomass productivity was observed with an increase of 6-8% in PHA productivity as compared to volumetric productivities previously observed.
  • PHB polyhydroxybutyrate
  • mediterranei might be attributed to the fact that at lower aeration rates there is a limitation of dissolved oxygen in the cultivation medium.
  • NADPH oxidase activity decreases, which further leads to an increase in overall NADPH concentration.
  • This would in turn inhibit citrate synthase and isocitrate dehydrogenase thereby increasing the acetyl-CoA concentration of the medium.
  • the excess acetyl CoA would thus be channelized to accumulation of storage products in the cells such as lipids or PHA.
  • the high NADPH/NAD ratio caused by oxygen limitation promotes synthesis of PHB, which plays the role as an alternative electron acceptor.
  • Cultivation time was supposed to be an important parameter for enhancement of PHA production using waste glycerol as a substrate. It was observed that by limiting the growth phase, a nitrogen stress is provided to the cells, which in turn enhances the PHA content of the cells. The PHA accumulation starts at the logarithmic phase, increases with the biomass and reaches a peak at the beginning of the stationary phase. PHA synthesis is delayed with respect to biomass development, reaching a maximum rate of synthesis at the end of the exponential phase. This has also been observed in previous studies (Lillo and Rodriguez- Valera, 1990). These findings suggest that PHA production in the present study was growth associated. This might be a key characteristic for conversion of the batch culture into a continuous system thereby increasing the PHA production.
  • the present study presents an alternate strategy for PHA production.
  • ADF aerobic dynamic feeding
  • the ADF process is often referred to as a feast-famine (F- F) process where the cells undergo an initial stage where they are fed with excess of external substrate followed by a later stage where there is an absence of the same (Cui et al., 2016).
  • F- F feast-famine
  • the present study utilizes a single step process where the organism can utilize external substrate in the growth phase thereby not requiring an additional step for production of PHA. This could be ascribed to the statistic that Hfx.
  • PHA is produced under controlled conditions in a bioreactor or fermenter which is operated in stirred tank mode (STR).
  • the reactor can be operated discontinuously in batch, repeated batch, or fed-batch modes, or as a continuous stirred tank reactor (CSTR) (Albuquerque et al., 2018) optionally in cascades.
  • CSTR continuous stirred tank reactor
  • the reactors are generally made of stainless steel, which produces a challenge for the cultivation of Hfx. mediterranei at higher salinities (i.e., greater than 22% salinity). This can be overcome by the use of reactors made of polymers and/or ceramics (including glass), which are non-corrosive (Hezayen et al., 2000).
  • pneumatically mixed reactors also could be utilized for the production of PHA with extreme halophiles, as their construction and design are simple.
  • the mixing is done by air bubbling in the reactor, which generates lower shear stress on the suspended cells, and in turn, utilizes lower energy for mass transfer.
  • airlift reactors ALR
  • ALR airlift reactors
  • the use of an ALR for PHB production from various carbon sources in nitrogen depleted medium by H. boliviensis has been successfully demonstrated (Ortiz- Veizan et al., 2020). Azohydromonas australica and C.
  • necator have reached about 72% PHB by weight and a biomass concentration of 10 and 32 gxL 1 , respectively (Gahlawat et al., 2012), whilst cultivation of Burkholderia sacchari in an ALR has led to 41% by weight PHB and a maximum biomass concentration of 150 g-L 1 (Pradella et al., 2010).
  • bubble column reactors could also be utilized for PHA production by extreme halophiles due to the various advantages related to pneumatically agitated bioreactors. These advantages include simple design and construction, ease of operation and lower shear stress, as compared to stirred tank reactors. There are no reports on using pneumatically agitated bioreactors for PHA production from seaweed hydrolysate using Hfx. mediterranei.
  • FTIR spectroscopy FTIR study of the PHA presented several absorption peaks. A peak was detected near 3290 cm 1 , which could be assigned to the stretching of hydroxyl group. Characteristic bond vibrations for PHA (1720-1740 cm 1 ) were observed and could be assigned to the carbonyl bond vibrations. Methyl and methylene groups stretching were detected at 2914 and 2879 cm 1 , respectively. Other peaks were observed in the range of 1450-1000 cm 1 . These peaks could be assigned to various bond vibrations, such as bending of CH3 group, wagging of CH2 group, and stretching of C-O, C-C and C-O-C.
  • P(3HB-co- 3HV) polyhydroxy-(3-hydroxybutyrate-co-3-hydroxyvalerate)
  • TGA/DSC thermal analyses The degradation temperature minima (Td) of the P(3HB- co-3HV) from Hfx. mediterranei was found to be 248°C. During the decomposition process, the polymer went through a weight loss, of 70.3%. The melting temperature was observed to be 177.1°C.
  • the molecular weight of the produced PHA was determined by using gel permeation chromatography (GPC). Table 1 shows the average molecular weight (M w ), number average molecular weight (Mn), Z-av erage molecular weight (M z ) and PDI of PHA extracted from Hfx. mediterranei at various air velocities (0.25-2.0 Lxmin 1 ).
  • the highest average M w of PHA (811 kDa using polystyrene standard; 770 kDa using PMMA standard) was obtained at air velocity of 0.25 L-min 1 with a PDI of 1.608 (polystyrene) / 1.544 (PMMA).
  • the obtained values were similar to molecular weights from previous studies of high-quality PHBV production from Hfx Mediterranei, in which a polymer with a molecular weight of 1057 kDa and a DPI of 1.5 was obtained (Koller et al., 2007).
  • the average M w ranged from 679 to 811 kDa and with a PDI in the range of 1.6-2.18 when polystyrene was used as a standard.
  • the average M w ranged from 656 to 770 kDa, with a PDI in the range of 1.544-1.959.
  • An increase in PDI was observed when the corresponding air velocities were increased (Fig. 3), suggesting that the polymer was more homogeneous at lower mixing velocities.
  • the outdoor studies yielded PHA with an average molecular weight of 716 kDa and a PDI of 1.592.
  • PHA solubility About 100 mg PHA were separately mixed with chloroform, acetone, distilled water, or acetic acid. A partial dissolution in chloroform and in acetone was observed. In contrast, only dispersions could be observed when distilled water and acetic acid were used as solvents (Table 2).
  • PLA solubility A mixture of 35 ml chloroform and 15 ml acetone was evaluated for PLA dissolution. Only a slight dissolution of PLA in this mixture of solvents was observed after stirring for 12 hours at ambient conditions. In a mixture containing 14 ml chloroform and 6 ml acetone, a partial dissolution of PLA was observed upon heating. When acetone was used as a solvent, PLA could be dissolved upon heating to 55°C for one hour (Table 2).
  • acetic acid should be used as a solvent, and the mixture should be heated.
  • PHA can partially dissolve in chloroform or in acetone.
  • PLA dissolves in acetone after heating to 55°C with continuous mixing.
  • the PLA/PHA mixture should be dissolved in excess of acetone (a ratio of 25 ml of acetone for each gram of PLA/PHA mixture) and the resulted mixture should be stirred and heated to 55 °C till homogeneity.
  • PLA/PHA solution in acetone was transferred to a tray and placed in the fumehood for evaporation. Upon acetone evaporation, a homogeneous “paper” (film) containing PLA and PHA was obtained.
  • the first mold for specimens’ preparation was made according to the ASTM D638 standard for tensile testing of plastics.
  • the selected size was type V, which is the smallest size, to allow preparation of the smallest samples, saving in the amounts of the starting materials.
  • the mold had a rectangular shape, containing four identical sockets in the form of dog-bones, as described in the ASTM (Fig. 4).
  • A1S1304 stainless steel was selected for the preparation of this mold. This stainless steel has high resistance to corrosion, and it is widely used due to its mechanical properties.
  • PLA has the lowest melting temperature, which is followed by PHA and keratin.
  • the highest decomposition temperature belongs to PLA.
  • the temperature selected for specimens’ preparation was 175°C, which is lower than the melting temperature of PHA and higher than the melting temperature of PLA, to avoid decomposition of these materials. It was preferred to heat our specimens for a longer time at a relatively low temperature, allowing the PLA to melt.
  • DSC and TGA tests were therefore performed on PHA/keratin blends.
  • the samples tested contained pure PHA, 10 wt.% of keratin, and increased keratin contents of 25, 50, 75, 90 and 100 wt.% (pure keratin) (Table 6 and Fig. 7).
  • the blend of PLA and PHA should be in a ratio of 70:30 and the keratin introduction should be between 10-50 wt.% (Table 7 and Fig. 9).
  • keratin modifies the thermal stability of the system.
  • introduction of keratin at 20 and 30 wt.% did not result in a significant change in the PLA/PHA thermal stability.
  • the PLA degradation temperatures were 323°C in both cases and the PHA degradation temperatures were 283°C (20 wt.% keratin) and 282°C (30 wt .% keratin). Based on these results, the blend that was chosen for testing was the one containing 30 wt.% of keratin.
  • the powders that were tested using FTIR were pure powders of PLA, PHA and keratin, as well as two blend powders - PLA/PHA (70:30), and PLA/PHA (70:30) 70 wt.% with 30 wt.% keratin.
  • This peak is attributed to the stretching vibrations of crystalline carbonyl groups.
  • the FTIR spectra of PLA/PHA blends showed the two major carbonyl stretching bands due to the presence of PLA and PHA. It is noticeable that the intensity ratio of these two bands changed with the composition ratio. No changes were observed in the main carbonyl peak in the FTIR spectra of the keratin-containing blends. The analysis of the FTIR spectra shows more peaks of other functional groups. Low peaks of (C-H) at range of 2933-2996 cm 1 were viewed in all spectra. In addition, a methyl group peak at 1379 cm 1 was observed for PLA and PHA.
  • the mechanical properties that can be determined from tensile tests are modulus of elasticity, stress and the strain at yield and break. At least two repetitions were performed in analyses of each specimen composition. In order to measure the strains on the specimens during the experiment, the gage length segment of the specimens was painted with black and white spray-paint in a 50:50 ratio. This was done to improve the accuracy of the pixel image processing.
  • PL A two specimens were tested: 003-1 and 003-2. Both showed similar results, as shown in Fig. 11.
  • PLA/PHA two specimens were tested: 102-1 and 102-2. Both showed similar results, except for the Poisson ratio. In addition, both specimens broke before reaching the plastic region. It appears that these specimens exhibit elastic deformation with no plastic deformation. These results are shown in Fig. 12.
  • PLA/PH A/keratin At first, only two specimens were tested: 402-1 and 402-2. Due to very different results, an additional specimen, 403-1, was also tested. Each specimen showed a different behavior. For example, unlike specimens 402-2 and 403-1 which show a curve in the graph indicating entry into the plastic region, specimen 402-1 displays a fairly linear graph which demonstrates its reamin in the elastic region. This can be seen in Fig. 13.
  • PLA specimens were semi-transparent while the specimens that contained PHA and keratin were much more opaque. This opacity is characteristic of semicrystalline polymers.
  • the semi-transparent PLA becomes opaque when crystalline PHA is inserted into its matrix.
  • the reason for the opacity of the combined PLA/PHA blend is the light scattering on the boundaries of the polymer. While amorphous materials, such as PLA, contain few boundaries, the density of these boundaries increases in crystalline materials, causing the transparency to decrease.
  • the transparency of the specimen, or lack thereof, may indicate the crystallization of the polymer.
  • SEM images of the fracture surface of the PLA specimen showed a smooth and uniform surface typical of an amorphous polymer.
  • the SEM images of the PLA/PHA specimen displayed PHA particels with a relatively small diameter in the PLA polymer matrix and with a typical sea-island morphology. The addition of keratin caused the blend to exhibit a reduction in the amount of pores. All SEM images display a homogenous distribution of PHA and keratin in the PLA matrix.
  • Fig. 15 shows the visual appearance of the dog-bones recovered at different sampling intervals.
  • the figures show that PLA degradation is enhanced when they are blended with PHA as well as PHA/keratin.
  • the fastest to degrade was the PLA/PHA/keratin composite which disintegrated in 42 days. This was followed by the PLA/PHA composite (49 days), and lastly PLA (56 days).
  • the biodegradation tests were performed under aerobic conditions in a composting environment, which was mediated by thermophilic bacteria (Iso, 2015).
  • the dog-bones of different composites were taken and were contained in a textile mesh to allow their easy removal after the composting test, but also allowing the access of moisture and microorganisms. They were buried at 4-6 cm depth in closed glass beakers of 500 mL containing a solid synthetic wet waste: 10% of compost, 30% rabbit food, 10% starch, 5% sugar, 4% corn oil, 1% urea, 40% sawdust and approximately 50 % w/w of water content and were incubated at aerobic conditions at 58°C for degradation. The aerobic conditions were guaranteed by periodical mixing of the solid synthetic wet waste.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Polymers & Plastics (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention relates to a process for large scale production of a polyhydroxyalkanoate (PHA) by fermentation of an archaea such as Haloferax mediterranei, wherein air is constantly bubbled into the fermenter from its bottom allowing constant aeration and mixing of the culture medium, as well as removal of carbon dioxide; and further provides blends of a PHA and at least two additional polymers, and articles comprising, or made of, said blends.

Description

METHOD FOR PRODUCTION OF PHA BY FERMENTATION OF ARCHAEA
TECHNICAL FIELD
[0001] The present invention relates to a process for large scale production of a polyhydroxyalkanoate (PHA) by fermentation, including outdoor fermentation, of an archaea such as Haloferax mediterranei, wherein air is constantly bubbled into the fermenter from its bottom allowing constant aeration and mixing of the culture medium, as well as removal of carbon dioxide; and further provides blends of a PHA and additional polymers, and articles made therefrom.
BACKGROUND
[0002] Global petrochemical plastics production of 335 million tons per year in 2016 is raising urgent concerns regarding end-of-life disposal and plastic contamination of the environment. Between 22-43% of polymers used worldwide are disposed of in landfills, and 10-20 million tons of plastics end up in oceans each year, causing damage to our ecosystem and an associated cost burden of 13 billion USD per year. Governments are now supporting strategies to promote a bio-based and circular economy by enforcing the use of bioplastics and introducing bans on landfills. Amongst biopolymers, polyhydroxyalkanoates (PHAs) are recognized as outstanding sustainable materials due to their tunable co-polymer formulation and mechanical properties and unique degradability in both marine and ambient soil environments. In addition, unlike other biopolymers, PHA polymers are hydrophobic, waterinsoluble, indefinitely stable in air, non-toxic, thermoplastic and/or elastomeric, and have very high purity within the cell. The PHA polymer family has much potential and adaptability for the broad application of biopolymers. Therefore, the search for highly efficient microorganisms for PHA accumulation is the need of the hour. Among wild-type strains, Haloferax mediterranei {Hfx. mediterranei), an extreme halophile, is of utmost significance. The archaea can accumulate high amounts of intracellular PHA utilizing a variety of substrates. Moreover, its extremophilic nature, which is characterized by high salt tolerance, enables non- sterile cultivation of this organism thereby decreasing the cost and energy required for the process.
[0003] Despite decades of research, the commercialization of PHA processes is slow. This could be explained by the lack of high-value applications for the initial stages of product penetration to the market, where the cost barriers of petroleum-based plastics are challenging. However, the development of the halophyte biorefinery processes for PHA production from seaweeds is still challenging and there are gaps in the scalability of these processes that will give them a clear advantage over current processes. For example, closed controlled fermenters that are used for PHA production with bacteria today are expensive and require a lot of freshwater and energy for mixing of culture media (Mahler et al., 2018). In addition, bacterial biomass harvesting is usually done with centrifuges, which are barely scalable and are not stable for the corrosive high salt media required for extreme halophiles growth. Moreover, most of the studies regarding extreme halophilic archaea have been performed in shake flask cultures or bioreactors of working volumes of 1-10 L (Alsafadi and Al-Mashaqbeh, 2017; Hezayen et al., 2000). Therefore, studies are required which need to be focused on the scale up of PHA production from halophilic archaea using various carbon substrates. The high salinity of the cultivation medium also poses challenges with regard to the material of construction of reactors. Higher salinities provide more corrosive conditions thereby requiring corrosion resistant reactors (Hezayen et al., 2000). Oxygen is a requisite for the growth of microorganisms and other metabolic activities. This becomes more important in highly saline conditions where solubility of oxygen in the media decreases further. Oxygen limiting conditions have been studied for enhancement of PHA production whereas oxygen replete conditions help in increasing the biomass concentrations thereby leading to high density cultivations (Maheshwari et al., 2018). Therefore, it becomes important to assess the role of oxygen concentration on growth as well as the PHA production in extreme halophilic archaea.
[0004] PHA being an intracellular product requires high cell density cultivations, because the restricted availability of cytoplasmic space limits the maximum amount of PHA which can be accumulated in the cells. Recovery of PHA begins after separating and concentrating cells from the fermentation medium. As PHA recovery from halophiles can be easily done by cell lysis using tap-water, separation of cells from the fermentation medium becomes a challenge in the overall recovery of PHA. Generally, separation of cells is performed using centrifugal force at a laboratory scale. Industrial separation of cells from the fermentation medium requires highly efficient continuous centrifuges. These methods are adequate when extracellular products are required, but when large quantities of washed bacteria are wanted, the need to scrape or wash out continuous flow centrifuge rotors makes a sterile harvest difficult to achieve. Similarly, filter blockage and difficulty in washing organisms off filters aseptically make simple filtration time-consuming and unreliable. Harvesting cells in an ultrasonic standing wave provides a potential alternative to problematic conventional separation technologies. There have been various studies that discuss the separation of microorganisms using ultrasound with very high separation efficiencies. Recently, ultrasonic separation has been utilized for separation of microalgal cells from the cultivation medium. Sonication produces highly localized cavitation and cell disruption for recovery of PHA from Hfx. mediterranei and is a fundamentally different phenomena from the trapping and concentration of cells in an ultrasonic standing wave. The effectiveness and energetic requirements of harvesting bacteria cells by ultrasonic standing waves need to be explored to determine whether it presents a viable alternative to conventional dewatering technologies.
SUMMARY OF THE INVENTION
[0005] The study described herein was aimed at addressing the challenges of halophiles (oxygen requirement, outdoor fermentation) for PHA production, using Hfx. mediterranei grown in pneumatically agitated bioreactors in outdoor fermentation, using macroalgal hydrolysates. The fermentation was performed in 1 L PET bottles, and 40 L plastic sleeves, under non-sterile conditions, which were operated as bubble column reactors. The process conditions (aeration rate, time of fermentation) were optimized for efficient PHA production from the hydrolysate. The structural characteristics of the PHA obtained were determined along with the molecular weight of the final polymer.
[0006] The present study provides a feasible way for scaling up PHA production from inexpensive substrates, such as macroalgal hydrolysate. The effect of various cultivation parameters was studied and favorable conditions for enhancement of PHA productivity were established. The molecular weight analysis suggested that the PHA obtained was more homogeneous at lower aeration velocities.
[0007] In one aspect, the present invention relates to a method for production of a polyhydroxyalkanoate (PHA) by fermentation of an archaea capable of producing said PHA, said method comprising the steps of:
(i) providing a fermenter having a cultivation, i.e., working, volume of at least about 5 or 10 liters, said fermenter containing a culture medium occupying said cultivation volume, said culture medium comprising saltwater such as seawater, supplemented with a carbon source and a nitrogen source, and suitable for culturing, i.e., promoting and supporting the growth and the survival of, said archaea;
(ii) inoculating said culture medium with a seed culture of said archaea; (iii) culturing said archaea while constantly bubbling an oxygen-containing gaseous mixture, such as air, inside the fermenter from its bottom, at an aeration rate of at least about 0.2 vvm, thereby constantly both aerating and mixing said culture medium, until a pre-defined concentration of said archaea in said culture medium is obtained;
(iv) harvesting said archaea’s biomass from said culture medium; and
(v) concentrating, separating and/or extracting said PHA from the harvested biomass.
[0008] The method disclosed herein may further comprise the step of purifying the PHA obtained in step (v), e.g., by repeated washing with water, an ionic liquid, or a combination thereof, to thereby obtain a purified PHA; and optionally the step of drying the purified PHA thus obtained, e.g., by a drum dryer, spray dryer, or lyophilizer. The purified PHA, regardeless of whether dried or not, may then be blend, i.e., mixed, with at least one polymer, e.g., a biodegradable or non-biodegradable polymer.
[0009] The fermentation process of the present invention may be carried out both in batches and continuously.
[0010] In another aspect, the present invention provides a blend comprising a PHA and at least two additional polymers, wherein each one of said at least two additional polymers may independently be a biodegradable or non-biodegradable polymer. In certain embodiments, said at least two additional polymers comprise, or consist of, polylactic acid (PLA) and keratin, wherein the PHA:PLA ratio in said blend may range between about 1:99 to about 99:1, between about 20:80 to about 80:20, between about 30:70 to about 70:30, or between about 40:60 to about 60:40, by weight, respectively, but it is preferably between about 20:80 to about 40:60, e.g., about 25:75, about 30:70, or about 35:65, by weight, respectively (regardless of the overall amount of the other polymers, e.g., keratin, comprised within said blend). In other embodiments, one of said at least two additional polymers is keratin, which constitutes from about 1% to about 99%, e.g., from about 5% to about 90%, from about 10% to about 80%, from about 15% to about 70%, or from about 20% to about 60%, but preferably from about 10% to about 50%, by weight, of said blend.
[0011] In a further aspect, the present invention provides an article comprising, or made of, a blend as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figs. 1A-1D show experimental setup for optimization of aeration rates in outdoor cultivation (I L PET bottles) of Hfx. mediterranei (1A); experimental setup for cultivation of Hfx. mediterranei in 40 L sleeves (IB); schematic representation of the cultivation system in 1 L PET bottles [experimental conditions: flow rates: 0.25-2.0 Lxmin’1; fermentation volumes: 800 mL; temperature: 42+2.5°C; inoculum concentration: 50 gxL’1; initial pH: 7.2] (1C); and schematic representation of a single photobioreactor for cultivation of Hfx. mediterranei (40 L) [experimental conditions: flow rate: 1.0 L min 1; fermentation volume: 10 L; temperature: 42±2.5°C; inoculum concentration: 50 gxL’1; initial pH: 7.2] (ID).
[0013] Figs. 2A-2F show growth and PHA production curves at different aeration rates for PHA production using Hfx. mediterranei [experimental conditions: flow rates: 0.25-2.0 Lxmin’ x; fermentation volume: 800 mL; water temperature: 42+2.5°C; air temperature: 37±2.5°C; inoculum concentration: 50 gxL’1; initial pH: 7.2] (2A and 2B, respectively); growth and PHA productivities under outdoor cultivation conditions for PHA production using Hfx. mediterranei [experimental conditions: flow rate: 1.0 Lxmin’1; fermentation volume: 10 L; water temperature: 42±2.5°C; air temperature: 37±2.5°C; inoculum concentration: 50 gxL’1; initial pH: 7.2] (2C and 2D, respectively); and change in PHA% in biomass with respect to time in Sleeve 1 and Sleeve 2 (2E and 2F, respectively).
[0014] Fig. 3 shows molecular weights (Mn, Mw, and Mz) and polydispersity index (PDI) distributions of PHA produced by Hfx. mediterranei using two standards, polystyrene (PS) and polymethyl methacrylate (PMMA).
[0015] Figs. 4A-4C illustrate (4A) ASTM specimen dimensions of type V; (4B) the mold design; and (4C) the mold.
[0016] Figs. 5A-5C illustrate: (5A) ASTM specimen dimensions of type IV; (5B) the mold design; and (5C) the mold.
[0017] Figs. 6A-6B are graphs showing thermal properties of PLA, PHA and keratin as pure materials.
[0018] Figs. 7A-7B are graphs showing DSC (7A) and TGA (7B) curves of PHA and keratin blends.
[0019] Figs. 8A-8B are graphs showing temperature of degradation vs. keratin weight fraction (8A); and mass change vs. keratin weight fraction of PHA and keratin blends (8B).
[0020] Figs. 9A-9B are graphs showing DSC (9A) and TGA (9B) curves of PLA, PHA and keratin blends. [0021] Figs. 10A-10B are graphs showing Temperature of degradation vs. keratin weight fraction (10A), and Mass change vs. keratin weight fraction of PLA, PHA and keratin blends (10B).
[0022] Figs. 11A-11G are graphs showing Stress-Strain curve of PLA specimens: (11A) of all tested PLA specimens; (11B) of PLA specimen 003-1; (11C) of Young modulus; (11D) of Poisson ratio; (HE) of PLA specimen 003-2; (HF) of Young modulus; and (11G) of Poisson ratio.
[0023] Figs. 12A-12G are graphs showing Stress-Strain curve of various PLA/PHA(70:30) specimens tested: (12A) of PLA/PHA(70:30) specimens; (12B) of Stress-Strain curve of PLA/PHA (70:30) specimen 102-1; (12C) of Young modulus; (12D) of Poisson ratio; (12E) of Stress-Strain curve of PLA/PHA (70:30) specimen 102-2; (12F) of Young modulus; and (12G) of Poisson ratio.
[0024] Figs. 13A-13J are graphs showing Stress-Strain curve of various PLA/PHA(70:30)- 70/keratin-30 specimens tested: (13A) of PLA/PHA(70:30)-70/keratin-30 specimens; (13B) of Stress-Strain curve of PLA/PHA(70:30)-70/keratin-30 specimen 402-1 ; (13C) of Young modulus; (13D) of Poisson ratio; (13E) of Stress-Strain curve of PLA/PHA(70:30)-70/keratin- 30 specimen 402-2; (13F) of Young modulus; (13G) of Poisson ratio; (13H) of Stress-Strain curve of PLA/PHA(70:30)-70/keratin-30 specimen 403-1; (131) of Young modulus; and (13J) of Poisson ratio.
[0025] Fig. 14 is a graph summarizing Stress-Strain curve of all the specimens. PLA: 003-1 and 003-2, PLA/PHA: 102-1 and 102-2, PLA/PHA/keratin: 402-1,402-2 and 403-1.
[0026] Fig. 15 presents photomicrographs showing the degradation of different composites under composting conditions.
[0027] Fig. 16 is a graph showing degree of disintegration of: control PLA dog-bone, PLA/PHA and PLA/PHA/keratin dog-bones under composting conditions as a function of time.
DETAILED DESCRIPTION
[0028] In one aspect, the present invention relates to a method for production of a PHA by fermentation of an archaea capable of producing said PHA, comprising the steps of:
(i) providing a fermenter having a cultivation volume of at least about 5 or 10 liters, said fermenter containing a culture medium occupying said cultivation volume, said culture medium comprising saltwater such as seawater, supplemented with a carbon source and a nitrogen source, and suitable for culturing said archaea;
(ii) inoculating said culture medium with a seed culture of said archaea; (iii) culturing said archaea while constantly bubbling an oxygen-containing gaseous mixture such as air inside the fermenter from its bottom, at an aeration rate of at least about 0.2 vvm, thereby constantly both aerating and mixing said culture medium, until a pre-defined concentration of said archaea in said culture medium is obtained;
(iv) harvesting said archaea’s biomass from said culture medium; and
(v) concentrating, separating and/or extracting said PHA from the harvested biomass.
[0029] The term “polyhydroxyalkanoate” (PHA), as used herein, refers to a polyester produced in nature by a microorganism, including through bacterial/archaea fermentation of sugars or lipids. More than 150 different monomers can be combined within this family to give materials with different properties. PHAs are biodegradable thermoplastic or elastomeric materials, with melting points ranging from 40°C to 180°C. Particular PHAs obtained from microbiological sources, genetically modified bacteria, and other organisms consist of monomers each of the formula [-O-C(R)H-(CH2)n-C(O)-] wherein R is H or alkyl, i.e., a linear or branched hydrocarbyl having, e.g., 1-12 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isoamyl, 2,2-dimethyl-propyl, n- hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like. Examples of such PHAs include, without being limited to, poly (3 -hydroxypropionate), poly (3 -hydroxybutyrate), poly(3-hydroxyvalerate), poly(3-hydroxyhexanoate), poly(3-hydroxyoctanoate), and polyphydroxy dodecanoate), wherein n is 1, and R is H, methyl, ethyl, propyl, pentyl, or nonyl, respectively; as well as poly(4-hydroxybutyrate) and poly (5-hydroxy valerate), wherein n is 2 or 3, and R is H, respectively.
[0030] The term “archaea”, as used herein, refers to any of a group of single-celled prokaryotic organisms that have distinct molecular characteristics separating them from bacteria and eukaryotes. Non-limiting examples of archaea include species of Picrophilus methanogens, halophiles, and Pyrolobus fumarii. Particular archaea referred in this description are halophilic archaea. In certain embodiments, the archaea is Hfx mediterranei, e.g., Hfx mediterranei ATCC 33500.
[0031] The term “saltwater”, as used herein, refers to water having a salinity, consisting predominantly of sodium ions and chloride ions, of at least about 2% and up to about 30, 35, or 40%. In certain embodiments, said saltwater are seawater, i.e., water from a sea or an ocean, having on average a salinity of about 3.5%. In other embodiments, said saltwater has a salinity ranging between from about 4% or 5% to about 10%, or a salinity higher than 10%, e.g., of up to about 15%, 20%, 25%, or 30%.
[0032] The term “oxygen-containing gaseous mixture”, as used herein, refers to a gaseous mixture comprising at least about 15%, e.g., about 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%, but preferably about 19%, 20%, 21%, 22%, or 23%, by volume (v/v), oxygen, and additional gases, which may be selected from nitrogen, carbon dioxide, and inert gases such as helium, neon, argon, krypton, and xenon. Particular such gaseous mixtures are air-like gaseous mixtures comprising oxygen, nitrogen, carbon dioxide, and argon, which constitute together at least 98% by volume of said gaseous mixtures. In more particular embodiments, the oxygen-containing gaseous mixture bubbled into the fermenter during the fermentation process disclosed is air, i.e., a gaseous mixture containing about 78% nitrogen, about 21 % oxygen, about 0.9%' argon, about 0.1 % carbon dioxide, and small amounts of other gases.
[0033] In certain embodiments, the culture medium used in the fermentation process disclosed herein comprises carbon and nitrogen, each independently in an amount of from about 1% to about 80% by weight, on a dry, i.e., dehydrated, mass base. The term “dry mass base” refers to the dry weight of the culture medium, i.e., to the weight of overall solid ingredients constituting said culture medium. In particular embodiments, the amount of carbon in said culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, by weight, on a dry (dehydrated) mass base; and/or the amount of nitrogen in said culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, by weight, on a dry (dehydrated) mass base.
[0034] In certain embodiments, the carbon source comprised within the culture medium used in the fermentation process disclosed comprises a sugar, more particularly a monosaccharide such as, without limiting, glucose, galactose, fructose, and xylose, or a polysaccharide such as starch; a waste product such as, without being limited to, glycerol (a major by-product in the biodiesel manufacturing process), fatty acids such as acetic acid, butyric acid and propanoic acid (produced during anaerobic degradation of organic compounds during the acidogenic fermentation, and serve as feedstock for freshwater and marine oleaginous microorganisms to produce, e.g., biodiesel), vinnase (a byproduct of the sugar or ethanol industry), stillage (a liquid waste remaining after ethanol distillation, and containing reducing sugars (fructose and glucose) and fatty acids, including acetic acid, propionic acid and butyric acid), molasses wastewater, and olive mill wastewater (the side-stream of olive oil production); a polyol, i.e., an organic compound containing multiple hydroxyl groups, a hydrolysate such as, without limiting, a seaweed hydrolysate, olive leaves hydrolysate, agricultural waste hydrolysates, and cheese whey hydrolysate; or a mixture thereof; and/or the nitrogen source comprised within said culture medium comprises an organic or inorganic nitrogen, such as biopolymers including proteins, glycosylated proteins, DNA fragments, RNA fragments, and nitrogen-containing oligosaccharides, peptides, amino acids, and nitrogen salts, a hydrolysate such as a seaweed hydrolysate, olive leaves hydrolysate, paper production waste, agricultural waste hydrolysates, and cheese whey hydrolysate, or a mixture thereof. In particular embodiments, both the carbon source and the nitrogen source consist solely of a hydrolysate, e.g., a seaweed hydrolysate, which provides all the carbon and nitrogen required for the cultivation process of the archaea.
[0035] The term “biopolymer”, as used herein, refers to a natural polymer (consisting of covalently bonded monomeric units), produced by the cells of a living organism. The major examples of such polymers, classified according to the monomers used and the structure of the biopolymer formed, are polynucleotides, polypeptides and proteins, and polysaccharides. Polynucleotides, such as RNA and DNA, are polymers composed of 13 or more nucleotide monomers. Polypeptides and proteins are polymers of amino acids and include collagen, actin and fibrin. Polysaccharides are liner or branched polymeric carbohydrates and include starch, cellulose and alginate. Additional examples of biopolymers include natural rubbers (polymers of isoprene), suberin and lignin (complex polyphenolic polymers), cutin and cutan (complex polymers of long-chain fatty acids), and melanin. Polymers, as referred to hereinabove, may be glycosylated, i.e., modified by the attachment of sugar moieties.
[0036] The term "amino acid", as used herein, refers to an organic compound comprising both amine and carboxylic acid functional groups, which may be either a natural or non-natural amino acid, and occur in both L and D isomeric forms. The twenty-two amino acids naturally occurring in proteins are aspartic acid (Asp), tyrosine (Tyr), leucine (Leu), tryptophan (Trp), arginine (Arg), valine (Vai), glutamic acid (Glu), methionine (Met), phenylalanine (Phe), serine (Ser), alanine (Ala), glutamine (Gin), glycine (Gly), proline (Pro), threonine (Thr), asparagine (Asn), lysine (Lys), histidine (His), isoleucine (He), cysteine (Cys), selenocysteine (Sec), and pyrrolysine (Pyl). Non-limiting examples of other amino acids include citrulline (Cit), diaminopropionic acid (Dap), diaminobutyric acid (Dab), ornithine (Om), aminoadipic acid, P- alanine, 1 -naphthylalanine, 3-(l-naphthyl)alanine, 3-(2-naphthyl)alanine, y-aminobutiric acid (GABA), 3-(aminomethyl) benzoic acid, -cthynyl-phcnylalaninc, m-ethynyl-phenylalanine, p- chlorophenylalanine (4ClPhe), -bromophcnylalaninc, -iodophcnylalaninc, p- acetylphenylalanine, p-azidophenylalanine, p-propargly-oxy-phenylalanine, indanylglycine (Igl), (benzyl)cysteine, norleucine (Nle), azidonorleucine, 6-ethynyl-tryptophan, 5-ethynyl- tryptophan, 3-(6-chloroindolyl)alanine, 3-(6-bromoindolyl)alanine, 3-(5-bromoindolyl) alanine, azidohomoalanine, a-aminocaprylic acid, O-methyl-L-tyrosine, N-acetyl- galactosamine-a-threonine, and N-acetylgalactosamine-a-serine.
[0037] The term "amino acid residue", as used herein, refers to a residue of an amino acid after removal of hydrogen atom from an amino group thereof, e.g., its a-amino group or side chain amino group if present, and -OH group from a carboxyl group thereof, e.g., its a-carboxyl group or side chain carboxyl group if present.
[0038] The term "peptide" refers to a short chain of amino acid monomers (residues), e.g., a chain consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid residues, linked by peptide (amide) bonds, i.e., the covalent bond formed when a carboxyl group of one amino acid reacts with an amino group of another. The term "peptide moiety", as used herein, refers to a moiety of a peptide as defined herein after removal of the hydrogen atom from a carboxylic group, i.e., either the terminal or a side chain carboxylic group, thereof, and/or a hydrogen atom from an amino group, i.e., either the terminal or a side chain amino group, thereof.
[0039] The term "peptide bond" or "amide bond", as used herein, refers to the covalent bond -C(O)NH- formed between two molecules, e.g., two amino acids, when a carboxyl group of one of the molecules reacts with an amino group of the other molecule, causing the release of a water molecule.
[0040] The term “hydrolysate”, as used herein, refers to an aqueous product of hydrolysis, comprising proteins that are chemically or enzymatically broken down to peptides of various sizes. Examples of hydrolysates that may be used as carbon and/or nitrogen sources for the culture medium include seaweed hydrolysate, olive leaves hydrolysate, agricultural waste hydrolysates, and cheese whey hydrolysate. In particular embodiments, the hydrolysate referred to is seaweed hydrolysate, which may be used as either or both, but preferably both, the carbon and nitrogen source.
[0041] The term “seaweed”, as used herein, refers to any species of a macroscopic, multicellular, marine algae, and includes types of Rhodophyta (red), Phaeophyta (brown) and Chlorophyta (green) macroalgae. In particular embodiment, the seaweed referred to is a green macroalgae such as an Ulva species.
[0042] In certain embodiments, the carbon source comprised within the culture medium used in the fermentation process disclosed comprises a seaweed hydrolysate, wherein said hydrolysate constitutes at least about 1%, preferably about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, by weight, of said carbon source; and/or the nitrogen source comprised within said culture medium comprises a seaweed hydrolysate, wherein said hydrolysate constitutes at least about 1%, preferably about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, by weight, of said nitrogen source. In particular embodiments, as exemplified herein, both the carbon source and the nitrogen source consist solely of a seaweed hydrolysate, which provides all the carbon and nitrogen required for the fermentation process of said archaea.
[0043] In certain embodiments, the culture medium used in the fermentation process disclosed herein further comprises salts such as salts containing bromide anion, buffers, phosphorus, or a mixture thereof. Phosphorus sources that may be used for the preparation of said culture medium include, without being limited to, dissolved inorganic phosphate such as potassium dihydrogen phosphate and disodium hydrogen phosphate, as well as phosphorus- containing biomolecules; and halogen sources that may be used include, without limiting, bromine.
[0044] In certain embodiments, the culture medium used in the fermentation process disclosed herein, according to any one of the embodiments above, comprises saltwater, e.g., seawater, supplemented with said carbon source, said nitrogen source, and optionally said phosphorus, halogen, or mixture thereof, wherein the amounts of carbon and nitrogen in said culture medium each independently is from about 1% to about 80% by weight on a dry mass base; said carbon source comprises a seaweed hydrolysate, which constitutes at least about 1% by weight of said carbon source; and said nitrogen source comprises a seaweed hydrolysate, which constitutes at least about 1% by weight of said nitrogen source.
[0045] In particular such embodiments, the amount of carbon in said culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, by weight, on a dry mass base; the amount of nitrogen in said culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, by weight, on a dry mass base; said carbon source comprises a seaweed hydrolysate, which constitutes at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, by weight of said carbon source; and said nitrogen source comprises a seaweed hydrolysate, which constitutes at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, by weight of said nitrogen source. In more particular such embodiments, both said carbon source and said nitrogen source consist solely of a seaweed hydrolysate, which provides all the carbon and nitrogen required for the fermentation process of said archaea.
[0046] In certain embodiments, the seaweed hydrolysate comprised within the culture medium used in the fermentation process disclosed, according to any one of the embodiments above, is a green macroalgae hydrolysate, such as Ulva sp. hydrolysate.
[0047] In certain embodiments, the pH of the culture medium prior to the inoculation step (ii), according to any one of the embodiments above, is up to 8.5. In particular embodiments, the pH of said culture medium prior to the inoculation step (ii) is from about 2 to about 8.5, preferably from about 6.8 to about 7.4, e.g., 7.2.
[0048] In certain embodiments, the culturing step (iii), according to any one of the embodiments above, is carried out for a period of up to 14 or 21 days, e.g., for a period of at least 48, 54, 60, 66, or 72 hours.
[0049] According to the present invention, an oxygen-containing gaseous mixture, such as air, is constantly bubbled during the culturing step (iii) into the fermenter from its bottom, at an aeration rate of at least about 0.2 vvm, to thereby constantly both aerate and mix the culture medium, and consequently also constantly remove carbon dioxide, produced by the archaea.
[0050] The term “aeration rate”, measured in volume/volume/minute (vvm), represents the volumetric flow rate of the oxygen-containing gaseous mixture / fermenter working volume, i.e., refers to the ratio between the volume of the oxygen-containing gaseous mixture bubbled into the fermenter per minute and the working volume of said fermenter. In certain embodiments, the aeration rate according to any one of the embodiments above is from about 0.6, 0.7, or 0.8 to about 1.2 vvm, preferably from about 0.9 to about 1.1 vvm, more preferably about 1 vvm.
[0051] As stated above, the oxygen-containing gaseous mixture constantly bubbled into the fermenter during the culturing step (iii) both aerates and mixes the culture medium, and consequently removes carbon dioxide from said medium. The aeration rate of said oxygencontaining gaseous mixture may thus significantly affect the efficacy of the culturing step, e.g., the maximal archaea concentration that may be obtained in the culture medium during the fermentation process or the time required to achieve said concentration.
[0052] As shown herein, the aeration rate of said oxygen-containing gaseous mixture also affects, although not in a linear manner, the molecular weight of the PHA obtained, which varies according to the aeration rate and the archaea’s culturing period, wherein higher molecular weight PHA was clearly observed at higher aeration rates. [0053] According to the present invention, upon completion of the culturing step (iii), the archaea’s biomass produced is harvested from the culture medium, and the PHA is then concentrated, separated and/or extracted from the harvested biomass. In certain embodiments, extraction of the PHA from the harvested biomass, according to any one of the embodiments above, is carried out by physical means such as water (hydrolysis), high pressure, pulsed electric field (PEF), and centrifugation and filtration; chemical means such as a lysis buffer, deep eutectic solvent, organic solvent, biphasic solvent system, and an ionic liquid; or any combination thereof.
[0054] The term “deep eutectic solvent”, as used herein, refers to a solution of a Lewis or Bronsted acid and a base, which form a eutectic mixture, i.e., a homogeneous mixture of substances that melts or solidifies at a single temperature that is lower than the melting point of any of the constituents. Deep eutectic solvents are highly tunable through varying the structure or relative ratio of the parent components and thus have a wide variety of potential applications including separation.
[0055] The term “organic solvent”, as used herein with respect to extraction of the PHA from the harvested biomass at various temperatures, refers to a non-toxic, environmentally friendly, water-soluble solvent that may contain carbon, hydrogen, oxygen, sulfur, and nitrogen atoms in its molecular structure. Examples of such organic solvents include, without being limited to, carbonate esters, including dimethylcarbonate, and diethylcarbonate; lactate esters, including ethyl lactate; levulinate esters, including methyl levulinate and ethyl levulinate; y- valerolactone; (17?)-7,8-dioxabicyclo[3.2.1]octan-2-one (cyrene); alcohols, diols and polyols, including ethanol, isopropanol, and ethyleneglycol; and ketones, including methylethylketone. [0056] The term “biphasic solvent system”, as used herein with respect to extraction of the PHA from the harvested biomass at various temperatures, refers to a solvent system comprising two solvents separated by an interfacial layer, wherein one of said two solvents is an aqueous solvent, and the other one of said two solvents is a non-toxic, environmentally friendly, partially (or poor) water-soluble solvent that may contain carbon, hydrogen, oxygen, sulfur, and nitrogen atoms in its molecular structure, such as, but not limited to, an acetate ester, including ethyl acetate and butyl acetate; and an ether, including 2-methyltetrahydrofuran.
[0057] The term “ionic liquid”, as used herein with respect to extraction of the PHA from the harvested biomass at various temperatures, refers to a non-toxic, environmentally friendly, organic salt that may contain carbon, hydrogen, oxygen, sulfur, nitrogen and halogen atoms in its molecular structure. Non-limiting examples of ionic liquids include salts containing dialkyl- imidazolium, A-alkyl-pyridazinium, quaternary ammonium, dialkyl-pyrrolidinium, alkyl- guanidinium, and alkyl-pyridinium cations; and a variety of envirometally friendly anions, such as acetate, formate, phosphate, hydrogen-phosphate, and chloride.
[0058] The method disclosed herein, according to any one of the embodiments above, may further comprise the step of purifying the PHA obtained in step (v), e.g., by repeated washing with water, a solvent, an ionic liquid, or a combination thereof, to thereby obtain a purified PHA; and optionally the step of drying the purified PHA thus obtained, e.g., by a drum dryer, spray dryer, air or air/nitrogen flow drying, or lyophilizer.
[0059] The purified PHA, regardeless of whether dried or not, may then be blend, i.e., mixed, with at least one polymer. Examples of suitable polymers for blending with the PHA include biodegradable polymers, such as polylactic acid (PLA), polycaprolactone (PCL), keratin, cellulose, chitin, lignin, amylose, amylopectin, and mucin; and non-biodegradable polymers or copolymer, such as polyethylene terephthalate (PET, also known as polyester), high-density polyethylene (HDPE), polyvinyl chloride (PVC), low-density polyethylene (LDPE), polypropylene (PP), polystyrene (PS), polyethylene glycol (PEG), polyproplylene glycol (PG), polyvinyl alcohol (PVA), polyacrylonitrile (PAN), polymethacrylic acid or an ester thereof, poly(acrylonitrile-co-butadiene-co-styrene) (ABS), polyamide, polyacrylamide (PAM), polysiloxanes, grafted polymers, and dendrimers such as polyamidoamine (PAMAM, made of repetitively branched subunits of amide and amine functionality).
[0060] The preparation of blends as disclosed herein may comprise the addition of various plastisizers and/or additives, so as to modulte the properties of the PHA or of the PHA blend prepared as disclosed, e.g., in Garcia-Garcia et al., 2022; Charon et al., 2022; and Longe et al., 2022. In certain embodiments, one or more plastisizers and/or additives are added to the PHA prior to mixing with the other polymer(s), to modulate the properties of said PHA before blending with the other polymer(s). In other embodiments, one or more plastisizers and/or additives are added to the PHA blend after the preparation thereof to modulate the properties of said blend.
[0061] In certain embodiments, the fermenter used in the method of the invention, according to any one of the embodiments above, is an open fermenter, such that the culturing of said archaea is carried out in non- sterile conditions.
[0062] In certain embodiments, the fermenter used in the method of the invention, according to any one of the embodiments above, is made of glass, metal or metallic alloy, ceramic, plastic, or cement. In other embodiments, said fermenter is an earth fermenter, also referred to as earthen-bank pond or earthen-bank construction.
[0063] In certain embodiments, the fermentation process of the present invention is carried out in batches following step (i)-(v) according to any one of the embodiments above. In other embodiments, said fermentation process is carried out continuously, wherien archaea’s biomass is either continuously or once in a while harvested from the culture medium (and then subjected to PHA concentration, separation and/or extraction), and fresh culture medium (in an amount equivalent to that removed for harversting) is introduced into the fermenter.
[0064] Many studies have been conducted to examine the combination of PLA with either PHA or keratin. The present study was done to examine the combination of all three materials together, in order to create bioplastic from these biodegradable materials. As described in the Experimental section herein, blends of PLA, PHA and keratin were prepared by taking the powders of the three materials and combining them to form a homogeneous powder. The blended powders were then melted in the oven. The morphology and the thermal and mechanical properties of the blends were investigated by SEM and digital microscopy, DSC, TGA, FTIR and tensile experiments. The thermal analysis showed that adding keratin affected the thermal stability of the blend. As the keratin weight fraction increased, there was a significant increase in the degradation temperature and a reduction in the weight loss of the specimens. The differences between the amorphous materials and those that are more crystalline could be seen in FTIR. PLA is primarily amorphous whereas PHA and keratin are more crystalline. Crystalline structures are generally very ordered, which grants them strength and rigidity. In such structures, the molecular chains are locked in place, so when a load is applied, they break rather than bend. Amorphous polymers are the opposite. Rather than being rigid, the random molecular jumble allows the chains to move across one another, when the polymer is pulled. This is what grants amorphous polymers with flexibility and elasticity. These characteristics were reflected in the results of the tensile tests; the specimens of PLA with PHA and keratin were broken in the elastic region without being deformed at all. In addition, the morphology tests showed a typical brittle fracture with no evidence of plasticity. It should be noted that, in contrary to other studies that found that adding PHA to PLA will make the specimens more ductile and less brittle, the addition of PHA to PLA in these experiments caused the opposite. This could most likely be explained by the type of PHA that was used; different monomers of PHA can provide materials with different properties. Another explanation for the results may be the existence of pores that were probably formed during the production process. A good adhesion between both phases of the blends is very important. The pores cause a decrease in the adhesion between the polymeric phases, therefore changing the material's properties. To avoid the creation of pores in the specimens, it will be better to store the powders of the materials in the desiccators until use in order to reduce moisture absorption. In addition to the characterization of the blends, biodegradation tests were performed as well. The biopolymers synthesized were completely biodegradable under aerobic conditions in a composting environment. The PLA/PHA/keratin blend was the fastest to degrade. This indicates that keratin not only helps the thermal stability of the material, but also causes it to be more biodegradable. According to these tests, these materials can be characterized as ecofriendly. In order to reinforce these findings, more tensile test repetitions should be performed.
[0065] In another aspect, the present invention thus provides a blend comprising a PHA and at least two additional polymers.
[0066] The polymers blended with the PHA, according to the present invention, may be selected from a biodegradable polymer, such as polylactic acid (PLA), poly caprolactone (PCL), poly(butylene succinate), poly(butylene succinate adipate), aliphatic -aromatic co-polyesters, poly(butylene adipate/terephthalate), and poly(methylene adipate/terephthalate), keratin, cellulose, chitin, lignin, amylose, amylopectin, mucin, and a PHA different from said PHA; and a non-biodegradable polymer, such as polyethylene terephthalate (PET, also known as polyester), high-density polyethylene (HDPE), polyvinyl chloride (PVC), low-density polyethylene (LDPE), polypropylene (PP), polystyrene (PS), polyethylene glycol (PEG), polyproplylene glycol (PG), polyvinyl alcohol (PVA), polyacrylonitrile (PAN), polymethacrylic acid or an ester or amide thereof, poly(acrylonitrile-co-butadiene-co-styrene) (ABS), polyamide, polyacrylamide (PAM), polysiloxanes, grafted polymers, and dendrimers such as PAMAM.
[0067] In certain embodiments, the blend disclosed herein comprises said PHA and at least two additional polymers, wherein said at least two additional polymers comprise, or consist of, PLA and keratin. In certain paticular such blends, the PHA:PLA ratio ranges between about 1:99 to about 99:1, between about 20:80 to about 80:20, between about 30:70 to about 70:30, or between about 40:60 to about 60:40, by weight, respectively, but it is preferably between about 20:80 to about 40:60, e.g., about 25:75, about 30:70, or about 35:65, by weight, respectively (regardless of the overall amount of the other polymers, e.g., keratin, comprised within said blend). [0068] In other embodiments, one of the at least two additional polymers blended with said PHA is keratin, which constitutes from about 1% to about 99%, e.g., from about 5% to about 90%, from about 10% to about 80%, from about 15% to about 70%, or from about 20% to about 60%, but preferably from about 10% to about 50%, by weight, of said blend.
[0069] The variation in blend components and these components source and physical and chemical propertis (including, but not limited to molecular weight, crystallinity, chain conformation, hydrophobicity and hydrophylicity, stability to hydrolysis, thermal properties, transport properties, optical properties, toughness, elasticity, viscoelasticity, heat, electromagnetic radiation conductivity, and electrical conductivity) can affect aforemention physical and chemical properties of resulted blends and blends homogeneity and components adhesion. For example, incorporation of PHA in blends leads to a decrease in the flexural modulus with simultaneous increase in the tensile modulus. The presence of keratin can influence the thermal stability and the toughness of the blends. As the keratin weight fraction increased, there was a significant increase in the degradation temperature and a reduction in the weight loss of the specimens.
[0070] In a further aspect, the present invention provides an article comprising, or made of, a blend as disclosed in any one of the embodiments above. Such articles may be prepared by any suitable technique known in the art, e.g., as shown in the Experimental section herein.
[0071] Unless otherwise indicated, all numbers referring, e.g., to aeration rate, amounts of carbon and/or nitrogen in a culture medium, pH of said culture medium, and ratios between polymers in a blend, used in the present specification are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this description and claims are approximations that may vary by up to plus or minus 10% depending upon the desired properties sought to be obtained by the invention.
[0072] The invention will now be illustrated by the following non limiting Examples.
EXAMPLES
Materials and Methods
[0073] Macroalgae biomass production. Ulva sp. was cultivated in [custom made] macroalgae photo-bioreactors (MPBR) (Polytiv, Israel, length 100 cm, thickness 200 mm, width 40 cm), under controlled conditions and natural irradiance, from September 15 to November 3, 2016. The conditions utilized for growing the seaweeds were those previously found to be the optimal. Elemental analysis of the Ulva sp. biomass was done using a CHNS analyzer (Flash 2000, Thermo Scientific, USA). The ash and moisture content were determined by burning the biomass at 550°C in a muffle furnace (M.G. Furnaces, Faridabad, India), and the starch content was analyzed using total starch assay.
[0074] Subcritical hydrolysis of macroalgal biomass. The hydrolysis of seaweed biomass was performed in a batch reactor (0.25 E working volume) equipped with electric heating system (CJF-0.25, Keda Machinery, China). The temperature and pressure inside the reactor were monitored using a digital temperature meter (MRC Ltd., Israel) and a pressure gauge (MRC Ltd., Israel) respectively. In order to mix the hot slurry inside the reactor, it was equipped with a stirrer which was cooled using chilled water from a chiller (Guangzhou Teyu Electromechanical Co. Ltd., China). To eliminate residual air from the reactor, a vacuum pump (MRC Ltd., Israel) was utilized. The conditions for hydrolysis were as follows: temperature of 170°C, total residence time of 20 min, salinity of 38 gxL’1, and a solid load of 5%. These conditions were chosen for optimization of sugar yield. The hydrolysate was separated into liquid and solid phases by centrifugation at 10,000 rpm for 3 min (Yingtai Instruments TGL- 18, China).
[0075] PHA production by Hfx. mediterranei. PHA production was investigated utilizing the Ulva sp. hydrolysate with the aid of Hfx. mediterranei ATCC 33500 (NCIMB 2177). The Hfx. mediterranei strain were routinely grown in a rich medium (Hv-YPC). The culture was grown in a shaking incubator (MRC Labs, Israel) at 42°C with a rotational speed of 180 rpm, and the media pH was adjusted to 7.2. For the experiment, seaweed hydrolysate (aqueous phase) was supplemented to saline water (144 g NaClxL-1) at a working concentration of 25% v/v as observed in previous studies (Ghosh et al., 2021). The experiments were performed in custom- built reactors made of 1 L PET (polyethylene teraphtalate) bottles (length 0.285 m, width 0.08 m). A set of 15 PET bottles were utilized for the cultivation of Hfx. mediterranei under different aeration rates (0.25-2.0 Lxmin 1) in triplicates. Individual reactors were connected with flowmeters to manipulate the airflow inside the reactor. The air was sparged from the bottom of the reactor for uniform flow inside the reactor. On the top of the reactor, an outlet was provided for gases. The cultivation was further scaled up to 10 L, using cylindrical, sleeve-like MPBRs (Polytiv, Israel, length 1.55 m, thickness 0.02 m, width 0.4 m) which were used for the cultivation of Ulva sp. The reactor configurations used for the cultivation of Hfx. mediterranei and their schematic representations are shown in Fig. 1. [0076] For the ultrasonic separation tests, cells were grown in 600 mL of rich Hv-YPC medium (pH 7.2) in a 1 L baffled Erlenmeyer flask. The flask was maintained at 42°C in a shaking incubator at 170 rpm for approximately 48 h to late exponential/early stationary phase and removed from the shaker. The optical density of the culture was 7.9 at 520 nm as measured on a benchtop spectrophotometer (ThermoFisher AquaMATE VIS, 1 cm path length), which roughly corresponds to an ash-free dry weight concentration of 3.9 gxL-1. The Hfx. mediterranei culture was transferred to a closed container and stored on the benchtop for two weeks, as a test for auto-flocculation, which could improve the particle size and ultrasonic response of the culture. Auto-flocculation was not observed in the standing culture, so enhanced aggregation of Hfx. mediterranei cells was attempted by Ca2+ addition. Cells were pelletized by centrifugation at l,000xg for 15 min and then twice washed in a Tris buffer (20 mM Tris-HCl and 4 M NaCl, pH 7.0). The cells were then suspended at higher concentration in a salt solution (27 mM KC1, 0.24 mM NaHCO3, 0.49 mM NaBr, 3.7 mM NH4C1, 4.3 M NaCl, 18.5 pM FeC13-6H2O, pH 7.0). A second cell suspension was washed and rinsed following the same procedure, but the final salt solution was modified to include 10 mM of CaCh. The washed suspensions had an optical density of 11.8 at 520 nm, which corresponds to an ash-free dry weight concentration of 5.8 gxL-1.
[0077] Determination of intracellular PHA content. The PHA content of the cells was measured using the Nile Red staining procedure. Briefly, the cells were washed and resuspended in 10% saline water. Further, the cell suspension was stained with Nile Red (Sigma Aldrich, USA) which had a final concentration of 3.1 pgxml 1. The cells were incubated for 30 mins and further washing and resuspension was performed using 10% saline. The fluorescence of the suspension was measured in a 96 well plate reader (Tecan, Switzerland) at excitation and emission wavelengths of 535 and 605 nm respectively. Commercial PHA (Sigma, USA) was utilized for preparation of standards with varying concentration (0.2-2.0 gxL 1). These standards were used for preparation of standard curve. A standard curve was plotted and the final concentration of PHA in the cells was determined. The unknown amount of PHA was determined from the standard curve with two repeats per point. The protocol was also verified using the crotonic acid assay for intracellular PHA content determination. Two milliliters of sulfuric acid were added to the dried cell pellet containing the polymer (PHA). The mixture was hydrolyzed by heating in a water bath at 100°C for 20 min to obtain crotonic acid. The amount of accumulated polymer was quantified by recording the UV absorbance at 235 nm using concentrated sulfuric acid as blank with multiplate reader (Tecan, Switzerland). [0078] Calculation of biomass and PHA productivities . To determine the volumetric biomass productivity (Xsiomass, gxL-1xh-1) and volumetric PHA productivity (XPHA, gxL-1xh-1), the following equations were used:
_ B2 - B1 (1)
A Biomass l2 C1
_ C2 - C1 (2)
APHA — 7 ~ l2 C1 wherein Bi and B2 represent the biomass concentrations (gxL 1) at time ti (h) and t2 (h) respectively, and Ci and C2 represent the PHA content (gxL 1) at time ti (h) and t2 (h) respectively. For determination of PHA productivity, the concentration was determined by PHA extraction.
[0079] Extraction of PHA from cells. After fermentation, the broth was centrifuged at 10,000 rpm for 10 mins (Yingtai Instruments TGL-18, China). The cell pellet thus formed was dried for 12 h at a temperature of 60°C in a convection oven (MRC Laboratories, Israel). In order to extract the PHA from the cells, the cell pellet was treated with 0.1% SDS (sodium dodecyl sulfate) in distilled water and incubated at 32°C for 24 h. This induced lysis of the cells thereby releasing the intracellular PHA into the water. The suspension was further centrifuged at 9000 rpm for 15 min to obtain the PHA as pellet. The pellet was washed with distilled water repeatedly, until it became white and was then dried for further analysis. The PHA obtained from individual fermentations was analyzed separately.
Characterization of PHA
[0080] FTIR-ATR analysis. The PHA powder obtained after drying was analyzed by a Fourier-transform infrared spectroscopy (FTIR) spectrometer, equipped with attenuated total reflectance (ATR) attachment (Bruker Platinum ATR, USA). The spectrum was recorded in the range of 400 to 4000 cm 1.
[0081] Thermogravimetric analysis I differential scanning calorimetry (TGA/DSC) analysis. 5 mg of dry PHA powder was weighed in a sealed aluminum pan. The pan was then subject to a linear temperature gradient (30 to 600°C) in a differential scanning calorimeter equipped with autoloader (Jupiter STA 449 F5, NETZSCH, Germany). The heating rate was maintained at 10oCxmin-1. [0082] 1H NMR analysis. The PHA produced was subjected to ’ H NMR analysis. The powdered PHA was dissolved in CDCI3 (10 mgxmr1) and then analyzed in a 400 MHz spectrometer (Bruker, USA).
[0083] GC-MS analysis. The butyl esters of PHA were analyzed using GC-MS system equipped with auto-sampler (6890/5977A, G4513A; Agilent, USA) and with HP-5MS UI column (Agilent, USA). The column consisted of a stationary phase of 5% phenyl/methyl-poly- siloxane, which was 30 m in length and an i.d. of 0.25 mm. Helium (99.999%) was used as a carrier gas, at a flow rate of 1.0 mLxmin 1. The samples were injected with a split ratio of 1:19 into the injector heated to a temperature of 280°C. The sample injection volume was 0.2 pL. The conditions for separation of analyte were as follows: initial oven temperature 70°C, which was held for 5 min. and then increased linearly to 280°C, at a rate of 150Cxmin-1. This was followed by a linear temperature increase to 320°C, at a rate of 30oCxmin-1, with a holding time of 5 min at the final temperature. Mass spectra analysis was performed in the El positive ion mode, using electron energy of 70 eV. Transfer line temperature and ion source temperature were maintained at 280 and 250°C, respectively. Obtained mass spectra data were collected in full-scan mode (m/z 50-400) and analyzed by using Agilent ChemStation software.
[0084] Molecular weight analysis. Gel permeation chromatography (GPC) analyses were performed to determine the weighted average molecular weight (Mw), number average molecular weight (Mn), z-average molecular weight (Mz), and polydispersity index (PDI) of the PHA biopolymer samples, using a high-performance liquid chromatography (HPLC) system fitted with a refractive index (RI) detector (Agilent 1260, Agilent, USA) and two Phenomenex columns (Phenomenex Inc., USA), and operated at column temperature of 40°C. The mobile phase used was tetrahydrofuran (THF) at a flow rate of 1 mLxmin 1. The samples were injected in an amount of 10 pL. Calibration of the GPC system was done with linear polystyrene and poly(methylmethacrylate), as the internal standards.
Example 1. Effect of different aeration rates on biomass and PHA productivity
[0085] The aeration rates required for mixing in outdoor cultivation of Hfx. mediterranei were optimized in the 1 L PET bottle experiments. The PET bottles were used as bubble column reactors for the sequential scale up from 100 mL cultivation volume to 1 L of reactor volume, as well as PHA production studies under outdoor cultivation conditions. The profiles for biomass and PHA concentration over time are shown in Figs. 2A-2B, respectively. After a cultivation time of 72 h, the volumetric productivity was calculated. This was also the maximum time for PH A production. The maximum volumetric biomass and PH A productivity were estimated to be 64.03+0.11 mgxL^xh 1 and 34.07+0.03 mgxL^xh 1 respectively at an aeration rate of 1.0 Lxmin 1 (or 1.0 vvm). A 10-15% increase in biomass productivity was observed with an increase of 6-8% in PHA productivity as compared to volumetric productivities previously observed.
[0086] A previous study was performed in 100 mL bottles, which were used for archaea growth in media supplemented with 25% v/v seaweed hydrolysate. The archaea were cultivated aerobically in a shaking incubator, with uniform mixing at 120 rpm and at temperature of 42°C. Lower aeration rates (up to 1.0 Lxmin 1) were suitable for biomass and PHA production whereas higher aeration rates (above 1.0 Lxmin 1) were suitable for biomass production rather than PHA accumulation. These studies showed that 1 .0 Lxmin 1 was more suitable aeration condition for PHA accumulation. Our studies also demonstrated that PHA production could be obtained along with growth of the organism, suggesting that it was a growth-related product formation. Studies on polyhydroxybutyrate (PHB) production using glucose and glycerol, as substrates, provided an insight on the effect of various aeration rates, and observed that at lower aeration rates, a higher PHB concentration was obtained. A higher aeration rate led to higher biomass production using various substrates, but lower aeration rates provided higher PHB accumulation (De Almeida et al., 2010). Aeration rate of 1 vvm was observed to be optimal for maximum biomass and PHA production using Cupriavidus necator. Because oxygen is only partially soluble in aqueous culture broths, even a short interruption of aeration results in the available oxygen becoming quickly exhausted, causing irreversible damage to the culture. The accumulation of PHA in the cells of Hfx. mediterranei might be attributed to the fact that at lower aeration rates there is a limitation of dissolved oxygen in the cultivation medium. Under limited oxygen conditions with an excess of carbon in the medium, NADPH oxidase activity decreases, which further leads to an increase in overall NADPH concentration. This would in turn inhibit citrate synthase and isocitrate dehydrogenase thereby increasing the acetyl-CoA concentration of the medium. The excess acetyl CoA would thus be channelized to accumulation of storage products in the cells such as lipids or PHA. The high NADPH/NAD ratio caused by oxygen limitation promotes synthesis of PHB, which plays the role as an alternative electron acceptor.
Example 2. Effect of different cultivation time on biomass and PHA productivity
[0087] The effect of different cultivation time was studied in 1 L PET bottles at an aeration rate of 1 vvm. The experiments were conducted for up to 196 hrs. The productivity increased with an increase in time, and the maximum biomass and PHA productivities of 68.01+0.11 mgxL^xh 1 and 28.02+0.03 mgxL^xh 1 respectively were observed at a cultivation time of 72 h (Figs. 2C-2D). PHA productivity decreased significantly after 72 hrs. This might be due to the consumption of PHA by the organism during the starvation phase. Hfx. mediterranei produces PHA as a storage energy product and has intracellular PHA depolymerases, which allows the PHA to be utilized for survival under stress conditions. Cultivation time was supposed to be an important parameter for enhancement of PHA production using waste glycerol as a substrate. It was observed that by limiting the growth phase, a nitrogen stress is provided to the cells, which in turn enhances the PHA content of the cells. The PHA accumulation starts at the logarithmic phase, increases with the biomass and reaches a peak at the beginning of the stationary phase. PHA synthesis is delayed with respect to biomass development, reaching a maximum rate of synthesis at the end of the exponential phase. This has also been observed in previous studies (Lillo and Rodriguez- Valera, 1990). These findings suggest that PHA production in the present study was growth associated. This might be a key characteristic for conversion of the batch culture into a continuous system thereby increasing the PHA production.
[0088] The present study presents an alternate strategy for PHA production. Usually, for inexpensive PHA production from mixed microbial consortia (MMC), an aerobic dynamic feeding (ADF) strategy is utilized. The ADF process is often referred to as a feast-famine (F- F) process where the cells undergo an initial stage where they are fed with excess of external substrate followed by a later stage where there is an absence of the same (Cui et al., 2016). The present study utilizes a single step process where the organism can utilize external substrate in the growth phase thereby not requiring an additional step for production of PHA. This could be ascribed to the statistic that Hfx. mediterranei cultivates quicker than other known members of the Halobacteriaceae family, and also has an excessive salt tolerance. An additional surprising detail is that it is metabolically adaptable: it cultivates both on complex media and on simple defined media, using the major variety of single carbon sources of any haloarchaeon, and it secretes exo-enzymes that hydrolyze proteins, polysaccharides and lipids. Moreover, the very high salinities (>25%) provides the process with lower levels of contaminants thereby eliminating the requirement for an enrichment step. The PHA content reported for ADF process is in the range of 25-70% dry cell weight (DCW), which is comparable to the yields observed in the present study. Therefore, the present strategy is suitable for efficient and cost effective PHA production from wastes resources. Example 3. Scale up of PHA production by Hfx. mediterranei in 40 L sleeves
[0089] We further scaled up the PHA production process by cultivation in 40 L plastic sleeves. Initial cultivation volumes attempted were 10 L which were sequentially scaled up to 40 L. The experimental conditions were determined after optimization - initial culture density of 50 gxL 1 (Ghosh et al., 2021), aeration rate of 1 vvm, and cultivation time of 72 h. The maximum mass fraction of PHA achieved in biomass was calculated to be 56% w/w. The maximum biomass productivity observed was 50.1+0.11 mgxL^xh 1 with a PHA productivity of 27.03+0.01 mgxL^xh 1. The conversion yield was calculated to be 0.107 g PHAxg Ulva w~ x. The carbon balance was also calculated from the present study. Out of the input carbon content of Ulva sp. biomass (33.4% C, as calculated by CHNS analysis), 32.03% was utilized for PHA production by Hfx. mediterranei, and the remaining content was either utilized for volatile fatty acids (VFAs) production during fermentation, or as a residual carbon, which remains unutilized in the medium. This was confirmed by further analysis of the spent medium. A slight decrease in overall productivities (biomass and PHA) was observed as compared to 1 L PET bottle cultivation, possibly due to inefficient air circulation within the reactor (a batch fermentation of 10 L in a 40 L bioreactor). Inefficient air circulation leads to unavailability of media components within the reactor, which might lead to decrease in accumulation of cell biomass thereby effectively decreasing the volumetric biomass and PHA productivities. This effect was observed in our studies and might explain the slight decrease in productivity within sequential steps of scale up for the process. Sequential scale up to 40 L cultivation could further increase the biomass and PHA productivities in the reactor. The biomass and PHA productivities showed a bell-shaped curve with maximum productivities at 72 h cultivation time. The PHA production also showed similarity with biomass productivity, suggesting that the production was a growth-dependent process which decreased with cultivation time. The decrease in PHA content with time could be explained by the fact that PHA is produced in archaeal cells as an energy storage product. With the increase in cultivation time, the nutritional stress in the cells increases leading to utilization of PHA granules for energy generation. Various wastes have been utilized for production of PHA. Batch cultivation experiments yielded a PHA productivity of 27 mgxL^xh 1 where the fermentation experiment utilized tuna condensate, as a substrate.
[0090] Technologically, PHA is produced under controlled conditions in a bioreactor or fermenter which is operated in stirred tank mode (STR). The reactor can be operated discontinuously in batch, repeated batch, or fed-batch modes, or as a continuous stirred tank reactor (CSTR) (Albuquerque et al., 2018) optionally in cascades. The reactors are generally made of stainless steel, which produces a challenge for the cultivation of Hfx. mediterranei at higher salinities (i.e., greater than 22% salinity). This can be overcome by the use of reactors made of polymers and/or ceramics (including glass), which are non-corrosive (Hezayen et al., 2000). Moreover, pneumatically mixed reactors also could be utilized for the production of PHA with extreme halophiles, as their construction and design are simple. The mixing is done by air bubbling in the reactor, which generates lower shear stress on the suspended cells, and in turn, utilizes lower energy for mass transfer. Recently, airlift reactors (ALR) have been utilized to produce PHB by H. boliviensis from starch hydrolysate in a batch system. The use of an ALR for PHB production from various carbon sources in nitrogen depleted medium by H. boliviensis has been successfully demonstrated (Ortiz- Veizan et al., 2020). Azohydromonas australica and C. necator have reached about 72% PHB by weight and a biomass concentration of 10 and 32 gxL 1, respectively (Gahlawat et al., 2012), whilst cultivation of Burkholderia sacchari in an ALR has led to 41% by weight PHB and a maximum biomass concentration of 150 g-L 1 (Pradella et al., 2010). Like airlift reactors, bubble column reactors could also be utilized for PHA production by extreme halophiles due to the various advantages related to pneumatically agitated bioreactors. These advantages include simple design and construction, ease of operation and lower shear stress, as compared to stirred tank reactors. There are no reports on using pneumatically agitated bioreactors for PHA production from seaweed hydrolysate using Hfx. mediterranei.
[0091] We utilized PET bottles (1 L), as well as sleeve-like macroalgae photo-bioreactors (40 L), which are not undergoing corrosion and can significantly reduce maintenance costs. The high salinity of the medium inhibits the growth of contaminants, thereby increasing the possibility of outdoor cultivation using extreme halophilic archaea. Such a process is feasible and could generate higher revenues for seaweed farmers. Analyzing the greenhouse gas emissions to the current feedstocks for bioplastic production, we observed that our emissions were comparable. An efficient design of the cultivation system (both offshore and onshore) could be a determining factor in the feasibility of the process. Another way by which we can increase the sustainability of the process is by producing PHA and biochar simultaneously in a biorefinery concept. When paired with biochar production, the economic feasibility and sustainability improves to a great extent (Ghosh et al., 2021). We wanted to provide sustainable solutions to the onshore cultivation of seaweed in pneumatically agitated reactors, which could then be utilized for PHA production in an economically feasible manner. There are challenges related to further scale up of the process. The high salinity of the medium could prove to be an impediment in the whole bioprocess. A further unit for concentration of brine could be utilized where the outlet from the system can be recycled to the cultivation system. In addition, and as mentioned above, the process can be operated in a biorefinery concept where various products can be achieved from a single biomass substrate thereby increasing the cost efficiency of the process.
Example 4. PHA Structural analysis (FTIR, TGA/DSC, 'H NMR, GC-MS)
[0092] FTIR spectroscopy . FTIR study of the PHA presented several absorption peaks. A peak was detected near 3290 cm 1, which could be assigned to the stretching of hydroxyl group. Characteristic bond vibrations for PHA (1720-1740 cm 1) were observed and could be assigned to the carbonyl bond vibrations. Methyl and methylene groups stretching were detected at 2914 and 2879 cm 1, respectively. Other peaks were observed in the range of 1450-1000 cm 1. These peaks could be assigned to various bond vibrations, such as bending of CH3 group, wagging of CH2 group, and stretching of C-O, C-C and C-O-C. These observations indicated that the analyzed polymer is polyhydroxy-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co- 3HV)). P(3HB-co-3HV) co-polyesters production without supplementation of 3HV- structurally related precursor compounds like, e.g., valerate, is a scarce feature and typically found in some haloarchaea like Hfx. mediterranei.
[0093] TGA/DSC thermal analyses . The degradation temperature minima (Td) of the P(3HB- co-3HV) from Hfx. mediterranei was found to be 248°C. During the decomposition process, the polymer went through a weight loss, of 70.3%. The melting temperature was observed to be 177.1°C.
[0094] GC-MS and 1H NMR analyses. The GC-MS analysis of hydrolyzed biopolymer showed two peaks in the chromatogram, which had retention times of 9.46 and 10.654 min. After mass spectrum analysis, it was suggested that these peaks were butyl esters of 3 -hydroxybutyrate (3HB) and 3 -hydroxy valerate (3HV). The GC-MS analysis indicated that the polymer produced by Hfx. mediterranei consisted of the monomers 3HB and 3HV. According to the 1 H NMR spectrum of the biopolymer, peaks at 0.83 and 1.26 ppm suggested the presence of two different CH3 groups, which could be related to the presence of 3HB and 3HV groups in the polymer, respectively. Peaks at 2.58 and 2.48 ppm were assigned to CH2 groups of 3HV and 3HB, respectively. Peaks at 5.25 ppm were assigned to CH group. The monomers composition of the PHA was calculated according to the 1 H NMR spectrum. These calculations indicated a composition of 90.4% of 3HB and 10.6% of 3HV.
[0095] The molecular weight of the produced PHA. The molecular weights for PHA produced at different air velocities were determined by using gel permeation chromatography (GPC). Table 1 shows the average molecular weight (Mw), number average molecular weight (Mn), Z-av erage molecular weight (Mz) and PDI of PHA extracted from Hfx. mediterranei at various air velocities (0.25-2.0 Lxmin 1). The highest average Mw of PHA (811 kDa using polystyrene standard; 770 kDa using PMMA standard) was obtained at air velocity of 0.25 L-min 1 with a PDI of 1.608 (polystyrene) / 1.544 (PMMA). The obtained values were similar to molecular weights from previous studies of high-quality PHBV production from Hfx Mediterranei, in which a polymer with a molecular weight of 1057 kDa and a DPI of 1.5 was obtained (Koller et al., 2007). In our study, the average Mw ranged from 679 to 811 kDa and with a PDI in the range of 1.6-2.18 when polystyrene was used as a standard. Using PMMA as a standard, the average Mw ranged from 656 to 770 kDa, with a PDI in the range of 1.544-1.959. An increase in PDI was observed when the corresponding air velocities were increased (Fig. 3), suggesting that the polymer was more homogeneous at lower mixing velocities. The outdoor studies yielded PHA with an average molecular weight of 716 kDa and a PDI of 1.592.
Table 1. MW distribution and PDI vs. various standards (polystyrene and PMMA) for PHA produced from Hfx. mediterranei at different aeration rates
Figure imgf000028_0001
[0096] Various studies have been performed to determine the impact of various parameters on the molecular weight of PHA, and to study the dependence of PHA synthase activity on the molecular weight distribution and PDI of PHA. Another study claimed that the molecular weight was a function of the type of substrate used (Quagliano et al., 2001). The dependence of the molecular weight of PHA in different mixing velocities was observed by Kshirsagar et al., 2013. Our study demonstrates that by controlling the agitation and aeration velocity, the molecular weight and PDI of the produced PHA could be controlled. The ability to control the molecular weight of the PHA is an advantage in polymer manufacturing and processing. Example 5. Blends preparation
Solubility tests of PLA, PH A and keratin
[0097] To prepare blends containing the three materials, solubility tests were first performed. [0098] Keratin solubility. About 100 mg keratin were mixed in either acetic acid or distilled water (Ruzgar el al., 2020), and the mixtures were stirred for 12 hours. As found, the keratin did not dissolve at all in either of these solvents under ambient conditions. A colloid dispersion of keratin in acetic acid could be obtained upon heating (Table 2).
[0099] PHA solubility. About 100 mg PHA were separately mixed with chloroform, acetone, distilled water, or acetic acid. A partial dissolution in chloroform and in acetone was observed. In contrast, only dispersions could be observed when distilled water and acetic acid were used as solvents (Table 2).
[00100] PLA solubility. A mixture of 35 ml chloroform and 15 ml acetone was evaluated for PLA dissolution. Only a slight dissolution of PLA in this mixture of solvents was observed after stirring for 12 hours at ambient conditions. In a mixture containing 14 ml chloroform and 6 ml acetone, a partial dissolution of PLA was observed upon heating. When acetone was used as a solvent, PLA could be dissolved upon heating to 55°C for one hour (Table 2).
Table 2. Solubility tests of keratin, PHA and PLA in various solvents
Figure imgf000029_0001
[00101] In summary, in order to prepare a keratin colloid dispersion, acetic acid should be used as a solvent, and the mixture should be heated. PHA can partially dissolve in chloroform or in acetone. PLA dissolves in acetone after heating to 55°C with continuous mixing.
Blending the materials together
[00102] Preparation of a PLA and PHA solution: 100 mg of a binary blend composed of 70 wt.% of PLA and 30 wt.% of PHA was made, and the solubility of the blend in acetone was tested in two different vials to understand whether the order of materials addition to the solvent may affect the formation of a homogeneous mixture. In the first vial, both PLA and PHA solids were simultaneously added to 10 ml of acetone; and in the second vial, PLA was added to 10 ml of acetone, and upon dissolution, PHA was added. As observed, the dissolution rate of the PLA was the same in both vials, i.e., with or without the PHA. Yet, while in the first vial a homogeneous solution was obtained, in the second vial, a dark-colored precipitate was formed after PHA was added. In conclusion, the materials should be added to acetone simultaneously before heating.
[00103] Another test was performed to examine the dissolution rate with excess of acetone. 100 mg of PLA/PHA (70:30) mixture was dissolved in 50 ml of acetone, and as found, the dissolution rate was higher.
[00104] In order to examine whether the mixture can be used as a solid, the materials were added directly to a dog-bone mold and mixed every few minutes. This method was not convenient due to difficulty in mixing of the materials in a hot oven, leading to non- homogeneous specimens.
[00105] In conclusion, the PLA/PHA mixture should be dissolved in excess of acetone (a ratio of 25 ml of acetone for each gram of PLA/PHA mixture) and the resulted mixture should be stirred and heated to 55 °C till homogeneity.
Example 6. Preparation of "Papers"
[00106] After preparing the PLA/PHA solution in acetone, the solvent could be removed. When the solution was placed in the dog-bone mold, the rate of acetone evaporation was very slow. Therefore, the acetone evaporation should be done before the mixture is placed in the dog-bone mold. PLA/PHA solution in acetone was transferred to a tray and placed in the fumehood for evaporation. Upon acetone evaporation, a homogeneous “paper” (film) containing PLA and PHA was obtained.
Table 3. Amounts of materials in type V specimens
Figure imgf000030_0001
[00107] Subsequently, blends of PLA/PHA with keratin were prepared. In these blends the ratio of 70% PLA and 30% PHA was maintained as in the previous experiments, in which the only difference between the various blends was in their keratin content (Table 3).
Example 7. Production of tensile test specimens
Specimens of dog-bone mold type V
[00108] The first mold for specimens’ preparation was made according to the ASTM D638 standard for tensile testing of plastics. The selected size was type V, which is the smallest size, to allow preparation of the smallest samples, saving in the amounts of the starting materials.
[00109] The mold had a rectangular shape, containing four identical sockets in the form of dog-bones, as described in the ASTM (Fig. 4). A1S1304 stainless steel was selected for the preparation of this mold. This stainless steel has high resistance to corrosion, and it is widely used due to its mechanical properties.
[00110] For creating type V specimens, initially, 2 grams of PLA/PHA “paper” shreds (cut by scissors) could be added to the mold and the mold was placed in an oven heated to 220°C. Upon melting of the first portion of shreds, more “paper” shreds were added to the mold in order to fill the entire volume of the mold.
Example 8. Redesign of used mold for dog-bone specimens type IV
[00111] The tensile tests of the prepared dog-bone specimens were performed on the universal mechanical testing machine using a 2 kN of force capacity. It was also decided to prepare new specimens according to type IV of the ASTM D638, with twice the size of the previous specimens (Fig. 5). The newly designed mold allowed to add all the material in one portion and had an upper part, capable of pressing the material in the mold and thus creating more uniform specimens. For these specimens, an amount of 4 grams of material was required. Blends containing 70% PLA and 30% PHA were used, where the keratin content was varied as specified in Table 4.
Table 4. Amounts of materials in type IV specimens
Figure imgf000031_0001
[00112] Before adding keratin to the solution of 70% PLA and 30% PHA in acetone, it was grinded into a fine powder. The resulting PLA/PHA/keratin mixture was stirred for 15 minutes for homogenization and was then transferred to a tray for evaporation and “paper” formation. The PLA/PHA/keratin “paper” formed was shredded by a grinder to a fine powder and the powder was added to the type IV mold. The mold with the powder was heated at 175°C in an over for 60 minutes and then cooled to room temperature for 2 hours.
[00113] The resulting PLA/PHA/keratin type IV specimens were then removed from the mold and, prior to the tensile testing, were spray -painted using 50% white paint and 50% black paint for better image processing.
Example 9. Powder characterization
Thermal analysis
[00114] DSC and TGA tests of different formulations were conducted to determine the melting temperatures of the pure materials and the various blends, and the effect of the keratin introduction on the degradation temperature and mass change of PHA and PLA/PHA blends (Table 5 and Fig. 6).
Table 5. Thermal properties of pure PLA, PHA and keratin
Figure imgf000032_0001
[00115] PLA has the lowest melting temperature, which is followed by PHA and keratin. The highest decomposition temperature belongs to PLA. The temperature selected for specimens’ preparation was 175°C, which is lower than the melting temperature of PHA and higher than the melting temperature of PLA, to avoid decomposition of these materials. It was preferred to heat our specimens for a longer time at a relatively low temperature, allowing the PLA to melt. At the beginning of the experiment, the possibility of preparing blends without PLA was investigated. DSC and TGA tests were therefore performed on PHA/keratin blends. The samples tested contained pure PHA, 10 wt.% of keratin, and increased keratin contents of 25, 50, 75, 90 and 100 wt.% (pure keratin) (Table 6 and Fig. 7).
[00116] As the weight fraction of keratin in the blend increased, the decomposition temperature increased, and the weight loss decreased (Fig. 8). As found, the blend could not be based only on PHA and keratin. As discovered, the blend of PLA and PHA should be in a ratio of 70:30 and the keratin introduction should be between 10-50 wt.% (Table 7 and Fig. 9).
Table 6. Thermal properties of PHA/keratin blends
Figure imgf000033_0001
Table 7. Thermal properties of PLA/PHA/keratin blends
Figure imgf000033_0002
[00117] As the weight fraction of keratin increased, the decomposition temperature of the PHA increased significantly while that of the PLA did not change significantly. In addition, increasing the keratin weight fraction caused the percentage of weight loss to decrease (Fig. 10).
[00118] The addition of keratin produced a significant increase in the degradation temperature and a reduction in the weight loss, indicating that it increases the thermal stability of the blend. [00119] PLA alone decomposed in a single-step process with a maximum degradation peak at 365 °C. When PHA and keratin were added to the PLA, a two-step degradation behavior was observed. The first peak (~280°C) was attributed to the PHA thermal degradation, while the second peak (~320°C) was attributed to the PLA thermal degradation. Moreover, the addition of PHA to PLA caused the main degradation peak to decrease (344°C), when compared to only PLA (365 °C). When keratin was added to the blends, there was an increase in the temperature of the first degradation peak. This proves that keratin modifies the thermal stability of the system. [00120] It should be noted that the introduction of keratin at 20 and 30 wt.% did not result in a significant change in the PLA/PHA thermal stability. The PLA degradation temperatures were 323°C in both cases and the PHA degradation temperatures were 283°C (20 wt.% keratin) and 282°C (30 wt .% keratin). Based on these results, the blend that was chosen for testing was the one containing 30 wt.% of keratin.
FTIR
[00121] The powders that were tested using FTIR were pure powders of PLA, PHA and keratin, as well as two blend powders - PLA/PHA (70:30), and PLA/PHA (70:30) 70 wt.% with 30 wt.% keratin.
[00122] FTIR spectra showed typical absorption bands of PLA, and several absorption bands specific for PHA and keratin. PLA is primarily amorphous, whereas PHA is more crystalline. Differences in the initial crystallinity of polymers cause a difference in the band width and transmittance of (C=O). The (C=O) FTIR band characteristics differed significantly between PLA and PHA. FTIR spectra showed a strong peak at 1750 cm 1, which is attributed to the amorphous carbonyl stretching vibration of PLA. This peak remains constant in all PLA/PHA blends. In addition, a sharp peak at 1720 cm 1 was observed in the spectra of PHA blends. This peak is attributed to the stretching vibrations of crystalline carbonyl groups. The FTIR spectra of PLA/PHA blends showed the two major carbonyl stretching bands due to the presence of PLA and PHA. It is noticeable that the intensity ratio of these two bands changed with the composition ratio. No changes were observed in the main carbonyl peak in the FTIR spectra of the keratin-containing blends. The analysis of the FTIR spectra shows more peaks of other functional groups. Low peaks of (C-H) at range of 2933-2996 cm 1 were viewed in all spectra. In addition, a methyl group peak at 1379 cm 1 was observed for PLA and PHA. The vibration of amides I, II and III from peptide bonds in keratin caused peaks at 1633, 1538 and 1233 cm' l, respectively. These peaks indicate that extracted keratin has a P-sheet secondary structure, similar to that of a feather. The peak at 3272 cm'1 is attributed to the N-H stretching vibration of peptide bonds (-CO-NH-).
Example 10. Specimen characterization
Mechanical property testing - tensile tests
[00123] The mechanical properties that can be determined from tensile tests are modulus of elasticity, stress and the strain at yield and break. At least two repetitions were performed in analyses of each specimen composition. In order to measure the strains on the specimens during the experiment, the gage length segment of the specimens was painted with black and white spray-paint in a 50:50 ratio. This was done to improve the accuracy of the pixel image processing.
[00124] During the experiment, the specimens were filmed. Later, image processing was performed to calculate the strains on the specimens.
Table 8. Calculation of the cross-sectional area in the gage length segment of the specimens
Figure imgf000035_0001
[00125] PL A: two specimens were tested: 003-1 and 003-2. Both showed similar results, as shown in Fig. 11.
[00126] PLA/PHA: two specimens were tested: 102-1 and 102-2. Both showed similar results, except for the Poisson ratio. In addition, both specimens broke before reaching the plastic region. It appears that these specimens exhibit elastic deformation with no plastic deformation. These results are shown in Fig. 12.
Table 9. Tensile properties
Figure imgf000035_0002
[00127] PLA/PH A/keratin : At first, only two specimens were tested: 402-1 and 402-2. Due to very different results, an additional specimen, 403-1, was also tested. Each specimen showed a different behavior. For example, unlike specimens 402-2 and 403-1 which show a curve in the graph indicating entry into the plastic region, specimen 402-1 displays a fairly linear graph which demonstrates its reamin in the elastic region. This can be seen in Fig. 13.
[00128] The results of the mechanical analysis for the PLA, PLA/PHA and PLA/PHA/keratin specimens are summarized in Table 9 and Fig.14.
[00129] For each type of blend (PLA, PLA/PHA, PLA/PHA/keratin), the results of the different specimens were averaged. Since the three specimens exhibited such variable results, the average results were not calculated. The results for specimen 402-2 were chosen to represent the PLA/PHA/keratin blend as it displayed the best results (Table 10).
Table 10. Average of tensile properties
Figure imgf000036_0001
[00130] In summary, the addition of PHA caused a substantial reduction in the PLA strength, as evident from a decrease in the break stress. The greatest elastic modulus was obtained for PLA/PHA blends. Following keratin addition, a reduction in the elastic modulus was observed,, confirming that keratin addition causes a decrease in the resistance to elastic deformation. The elongation at break of the specimens strongly decreased when PHA and keratin were added to PLA, exhibiting a reduction in the blends' ductility. In contrast to expectations that the addition of PHA to PLA would casue the specimens to be more ductile, they became more rigid. The specimens were broken in the elastic region without being deformed at all. This might have occurred due to the type of PHA used, PHB poly(3-hydroxybutyrate). While pure PHB is rigid, due to its fairly crystalline structure, other PHA compositions, such as P3HB-co-P4HB, are more rubbery and flexible. Upon adding P4HB (poly(4-hydroxybutyrate)) to PHA, the percentage of crysallinity decreases, while the percentage of elongation and break increases. The melting temperature (DSC) of PHA used in our experiments was 176°C, indicating that it was PHB that has a typical melting temperature of 178°C (Table 11). [00131] Another reason for the decrease in mechnical properties could be the pores created in the specimen during the production process. Such voids can cause a decrease in adhesion between the materials.
[00132] The Poisson ratio results are not significantly different between the various blends. PLA seems to have the highest ratio, followd by PLA/PHA/keratin and PLA/PHA, respectively. High Poisson ratio indicates strong resistance to deformation.
Table 11. Physical and Thermal properties of P3HB-co-4HB copolymers
Figure imgf000037_0001
Example 11. Morphology, micro-structure and transparency of the fracture surface
[00133] Following the tensile test experiments, three specimens - one of each blend type (PLA, PLA/PHA and PLA/PHA/keratin) were tested for morphology, micro-structure and transparency of the fracture surface using SEM and a digital microscope (data not shown). The images obtained from these tests provide evidence to the source of changes in the mechanical properties of the blends. As found in those images, there was no evidence of plasticity in all specimens. This is very typical for pure PLA, but not for PLA/PHA, wherein it was expected that the specimens will undergo a substantial ductile plastic deformation and elongation before breaking. Looking at the 200 micron resolution SEM images of the specimens of the different blends, it was easy to notice many pores That were most likely formed during the production process due to air imprisoned in the specimens from the moisture in the original powders of PLA, PHA and keratin used. PLA specimens were semi-transparent while the specimens that contained PHA and keratin were much more opaque. This opacity is characteristic of semicrystalline polymers. The semi-transparent PLA becomes opaque when crystalline PHA is inserted into its matrix. The reason for the opacity of the combined PLA/PHA blend is the light scattering on the boundaries of the polymer. While amorphous materials, such as PLA, contain few boundaries, the density of these boundaries increases in crystalline materials, causing the transparency to decrease. Therefore, the transparency of the specimen, or lack thereof, may indicate the crystallization of the polymer. SEM images of the fracture surface of the PLA specimen showed a smooth and uniform surface typical of an amorphous polymer. The SEM images of the PLA/PHA specimen displayed PHA particels with a relatively small diameter in the PLA polymer matrix and with a typical sea-island morphology. The addition of keratin caused the blend to exhibit a reduction in the amount of pores. All SEM images display a homogenous distribution of PHA and keratin in the PLA matrix.
Example 12. Biodegradation tests
[00134] Fig. 15 shows the visual appearance of the dog-bones recovered at different sampling intervals. The figures show that PLA degradation is enhanced when they are blended with PHA as well as PHA/keratin. The fastest to degrade was the PLA/PHA/keratin composite which disintegrated in 42 days. This was followed by the PLA/PHA composite (49 days), and lastly PLA (56 days).
[00135] The visual disintegration was also supported by calculating the weight loss during composting conditions, as shown in Fig. 16. The goal of sample disintegrability was considered to be 90% (Arrieta et al., 2014), as indicated in the current legislation for biodegradable materials (Iso, 2015). Degradation of PLA composites started on the 14th day of the composting experiment, but no noticeable disintegration of PLA was observed until day 21. Previous studies on PLA/PHB blends showed similar trends of degradation. Arrieta, Lopez, Rayon and Jimenez (2014) studied the degradation of PLA/PHB blends with and without plasticizers. They showed similar degradation profiles of PLA along with blends of PLA and plasticizers (Arrieta et al., 2014). Similar observations were made in studies on PHB degradation as well as PLA/keratin degradation (Pooma et al., 2019; Ahn et al., 2011).
Example 13. Biodegradation tests
[00136] The biodegradation tests were performed under aerobic conditions in a composting environment, which was mediated by thermophilic bacteria (Iso, 2015). The dog-bones of different composites were taken and were contained in a textile mesh to allow their easy removal after the composting test, but also allowing the access of moisture and microorganisms. They were buried at 4-6 cm depth in closed glass beakers of 500 mL containing a solid synthetic wet waste: 10% of compost, 30% rabbit food, 10% starch, 5% sugar, 4% corn oil, 1% urea, 40% sawdust and approximately 50 % w/w of water content and were incubated at aerobic conditions at 58°C for degradation. The aerobic conditions were guaranteed by periodical mixing of the solid synthetic wet waste. Samples of the composites were taken in 7-day intervals, until reaching complete degradation. At each sampling point, the dog-bones were cleaned with distilled water, dried in an oven for 24 h, and re-weighed. Photographs of all samples were taken each time they were extracted from the composting medium. REFERENCES
Ahn, H.K.; Huda, M.S.; Smith, M.C.; Mulbry, W.; Schmidt, W.F.; Reeves III, J.B., Biodegradability of injection molded bioplastic pots containing polylactic acid and poultry feather fiber. Bioresource Technology, 2011, 102(7), 4930-4933
Alsafadi, D.; Al-Mashaqbeh, O., A one-stage cultivation process for the production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) from olive mill wastewater by Haloferax mediterranei. N. Biotechnol., 2017, 34, 47-53
Arrieta M.P.; Lopez, j.; Rayon, e.; Jimenez, A., Disintegrability under composting conditions of plasticized PLA-PHB blends, Polymer Degradation and Stability, 2014, 108, 307 Charon, G.; Peixinho, J.; Michely, L.; Guinault, A.; Langlois, V., Rosin natural terpenes as processing aid for poly hydroxy alkanoate: Thermal, mechanical, and viscoelastic properties. J Appl Polym Sci., 2022, e53052
Cui, Y.-W .; Zhang, H.-Y.; Lu, P.-F.; Peng, Y.-Z., Effects of carbon sources on the enrichment of halophilic polyhydroxyalkanoate-storing mixed microbial culture in an aerobic dynamic feeding process. Sci. Rep., 2016, 6(1), 30766 de Almeida, A.; Giordano, A.M.; Nikel, P.I.; Pettinari, M.J., Effects of aeration on the synthesis of poly (3 -hydroxybutyrate) from glycerol and glucose in recombinant Escherichia coli. Appl. Environ. Microbiol., 2010, 76(6), 2036-2040
Gahlawat, G.; Sengupta, B.; Srivastava, A.K., Enhanced production of poly(3- hydroxybutyrate) in a novel airlift reactor with in situ cell retention using Azohydromonas australica. J. Ind. Microbiol. Biotechnol., 2012, 39(9), 1377-1384
Garcia-Garcia, D.; Quiles-Carillo, L.; Balart, R.; Torres-Giner, S.; Arrieta, M.P., Innovative solutions and challenges to increase the use of poly (3 -hydroxybutyrate) in food packaging and disposables. European Polymer Journal, 2022, 178, 111505
Ghosh, S.; Greiserman, S.; Chemodanov, A.; Siegers, P.M.; Belgorodsky, B.; Epstein, M.; Kribus, A.; Gozin, M.; Chen, G.-Q.; Golberg, A., Polyhydroxyalkanoates and biochar from green macroalgal Ulva sp. biomass subcritical hydrolysates: Process optimization and a priori economic and greenhouse emissions break-even analysis. Sci. Total Environ., 2021, 770, 145281
Hezayen, F.F.; Rehm, B.H.; Eberhardt, R.; Steinbiichel, A., Polymer production by two newly isolated extremely halophilic archaea: Application of a novel corrosion-resistant bioreactor. Appl. Microbiol. Biotechnol., 2000, 54(3), 319-325
Iso, U., Determination of the degree of disintegration of plastic materials under simulated composting conditions in a laboratory- scale test. UNE-EN ISO-202002015
Koller, M., Hesse, P., Bona, R., Kutschera, C., Atlic, A., Braunegg, G., Biosynthesis of high quality polyhydroxyalkanoate Co- and terpolyesters for potential medical application by the archaeon Haloferax mediterranei. Macromol. Symp., 2007, 253(1), 33-39
Kshirsagar, P., Suttar, R., Nilegaonkar, S., Kulkarni, S., Scale up production of poly hydroxy alkanoate (PHA) at different aeration, agitation and controlled dissolved oxygen levels in fermenter using Halomonas campisalis MCM B-1027. J. Biochem. Technol., 2013, 4, 512-517
Lillo, J.G., Rodriguez- Valera, F., 1990. Effects of culture conditions on poly(beta- hydroxybutyric acid) production by Elaloferax mediterranei. Appl. Environ. Microbiol., 1990, 56(8), 2517-2521
Longe, L.F.; Michely, L.; Gallos. A.; De Anda, A.R.; Vahabi, H.; Renard, E.; Latroche, M.; Allais, F.; Langlois, V., Improved processability and antioxidant behavior of poly(3- hydroxybutyrate) in presence of ferulic acid-based additives. Bioengineering, 2022, 9(3), 100
Maheshwari, N., Kumar, M., Thakur, I.S., Srivastava, S., Production, process optimization and molecular characterization of polyhydroxyalkanoate (PHA) by CO2 sequestering B. cereus SS105. Bioresour. Technol., 2018, 254, 75-82
Mahler, N., Tschirren, S., Pfliigl, S., Herwig, C., Optimized bioreactor setup for scale- up studies of extreme halophilic cultures. Biochem. Eng. J., 2018, 130, 39-46
Ortiz-Veizan, N., Daga-Quisbert, J., Perez-Zabaleta, M., Guevara-Martinez, M., Larsson, G., Quillaguaman, J., Improvements of poly (3 -hydroxybutyrate) production in an airlift reactor using simple production media. Bioresour. Bioprocess., 2020, 7 (22)
Quagliano, J.C., Amarilla, F., Fernandes, E.G., Mata, D., Miyazaki, S.S., Effect of simple and complex carbon sources, low temperature culture and complex carbon feeding policies on poly-3-hydroxybutyric acid (PHB) content and molecular weight (Mw) from Azotobacter chroococcum 6B. World J. Microbiol. Biotechnol., 2001, 17, 9-14
Ruzgar, D.G.; Kurtoglu, S.A.; Bhullar, S.K., A Study on extraction and characterization of keratin films and nanofibers from waste wool fiber. Journal of Natural Fibers, 2020, 17(3)
Sabapathya, P.C., Devaraja, S., Parthipanb, A., Kathirvela, P., Polyhydroxyalkanoate production from statistically optimized media using rice mill effluent as sustainable substrate with an analysis on the biopolymer’s degradation potential. International Journal of Biological Macromolecules, 2019, 126, 977-986

Claims

1. A method for production of a polyhydroxyalkanoate (PHA) by fermentation of an archaea capable of producing said PHA, said method comprising the steps of:
(i) providing a fermenter having a cultivation volume of at least about 5 liters, said fermenter containing a culture medium occupying said cultivation volume, said culture medium comprising saltwater (e.g., seawater) supplemented with a carbon source and a nitrogen source, and suitable for culturing said archaea;
(ii) inoculating said culture medium with a seed culture of said archaea;
(iii) culturing said archaea while constantly bubbling an oxygen-containing gaseous mixture, such as air, inside the fermenter from its bottom, at an aeration rate of at least about 0.2 vvm, thereby constantly both aerating and mixing said culture medium, until a pre-defined concentration of said archaea in said culture medium is obtained;
(iv) harvesting said archaea’s biomass from said culture medium; and
(v) concentrating, separating and/or extracting said PHA from the harvested biomass.
2. The method of claim 1, wherein said archaea is Haloferax mediterranei.
3. The method of claim 2, wherein said Haloferax mediterranei is Haloferax mediterranei ATCC 33500.
4. The method of claim 1, wherein said culture medium comprises carbon and nitrogen, each independently in an amount of from about 1% to about 80% by weight on a dry mass base.
5. The method of claim 4, wherein the amount of carbon in said culture medium is at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70%, by weight, on a dry mass base; and the amount of nitrogen in said culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, by weight, on a dry mass base.
6. The method of claim 1, wherein said carbon source comprises a sugar such as a monosaccharide or polysaccharide, a waste product such as glycerol and fatty acids, vinnase, stillage, molasses wastewater, and olive mill wastewater, a polyol, a hydrolysate such as a seaweed hydrolysate, olive leaves hydrolysate, paper production waste, agricultural waste hydrolysates, and cheese whey hydrolysate, or a mixture thereof; and/or said nitrogen source comprises organic or inorganic nitrogen such as biopolymers including proteins, glycosylated proteins, DNA fragments, RNA fragments, and nitrogen-containing oligosaccharides, peptides, amino acids, and nitrogen salts, a hydrolysate such as a seaweed hydrolysate, olive leaves hydrolysate, agricultural waste hydrolysates, and cheese whey hydrolysate, or a mixture thereof.
7. The method of claim 1, wherein said carbon source comprises a seaweed hydrolysate, which constitutes at least about 1%, preferably about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, by weight, of said carbon source; and/or said nitrogen source comprises a seaweed hydrolysate, which constitutes at least about 1%, preferably about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, by weight, of said nitrogen source.
8. The method of claim 1, wherein said culture medium further comprises salts such as salts containing bromide anion, buffers, phosphorus, or a mixture thereof.
9. The method of any one of claims 1-8, wherein said culture medium comprises saltwater, e.g., seawater, supplemented with said carbon source, said nitrogen source, and optionally said phosphorus, halogen, or mixture thereof, wherein the amounts of carbon and nitrogen in said culture medium each independently is from about 1% to about 80% by weight on a dry mass base; said carbon source comprises a seaweed hydrolysate, which constitutes at least about 1% by weight of said carbon source; and said nitrogen source comprises a seaweed hydrolysate, which constitutes at least about 1% by weight of said nitrogen source.
10. The method of claim 9, wherein the amount of carbon in said culture medium is at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70%, by weight, on a dry mass base; the amount of nitrogen in said culture medium is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, by weight, on a dry mass base; said carbon source comprises a seaweed hydrolysate, which constitutes at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, by weight of said carbon source; and said nitrogen source comprises a seaweed hydrolysate, which constitutes at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more, by weight of said nitrogen source.
11. The method of any one of claims 6-10, wherein said seaweed hydrolysate is a green macroalgae hydrolysate, such as Ulva sp. hydrolysate.
12. The method of any one of claims 1-11, wherein the pH of said culture medium prior to the inoculation step (ii) is up to 8.5.
13. The method of claim 12, wherein the pH of said culture medium prior to the inoculation step (ii) ranges from about 2 to about 8.5, preferably from about 6.8 to about 7.4, e.g., 7.2.
14. The method of any one of claims 1-13, wherein the culturing step (iii) is carried out for a period of at least 48, 60, or 72 hours.
15. The method of any one of claims 1-14, wherein said aeration rate is from about 0.8 to about 1.2 vvm, preferably about 1 vvm.
16. The method of any one of claims 1-15, wherein step (v) comprises extracting said PHA by physical means, such as water (hydrolysis), high pressure, pulsed electric field, and centrifugation and filtration; chemical means, such as a lysis buffer, deep eutectic solvent, organic solvent, biphasic solvent system, and an ionic liquid; or a combination thereof.
17. The method of any one of claims 1-16, wherein the molecular weight of the PHA obtained varies according to the aeration rate and the archaea’s culturing period.
18. The method of claim 1, further comprising the step of purifying the PHA obtained in step (v) to thereby obtain a purified PHA.
19. The method of claim 18, wherein the purification step is carried out by repeated washing the PHA obtained with water, an ionic liquid, or a combination thereof.
20. The method of claim 18 or 19, further comprising the step of drying the purify ed PHA, e.g., by a drum dryer, spray dryer, air or air/nitrogen flow drying, or lyophilizer.
21. The method of any one of claims 18-20, further comprising the step of blending the purifyed PHA with at least one polymer.
22. The method of claim 21, wherein said at least one polymer is a biodegradable polymer such as polylactic acid (PLA), polycaprolactone (PCL), keratin, cellulose, chitin, lignin, amylose, amylopectin, and mucin; or a non-biodegradable polymer or copolymer such as polyethylene terephthalate (PET, also known as polyester), high-density polyethylene (HDPE), polyvinyl chloride (PVC), low-density polyethylene (LDPE), polypropylene (PP), polystyrene (PS), polyethylene glycol (PEG), polyproplylene glycol (PG), polyvinyl alcohol (PVA), polyacrylonitrile (PAN), polymethacrylic acid or an ester thereof, poly (aery lonitrile-co- butadiene-co- styrene) (ABS), polyamide, polyacrylamide (PAM), polysiloxanes, grafted polymers, and dendrimers such as polyamidoamine (PAMAM).
23. The method of claim 1, wherein said fermenter is an open fermenter and the culturing of said archaea is carried out in non-sterile conditions.
24. The method of claim 1, wherein said fermenter is made of glass, ceramic, plastic, cement, or said fermenter is an earth fermenter (earthen-bank pond).
25. The method of claim 1, carried out continuously.
26. A blend comprising a poly hydroxy alkanoate (PHA) and at least two additional polymers.
27. The blend of claim 26, wherein said at least two additional polymers each independently is a biodegradable polymer such as polylactic acid (PLA), polycaprolactone (PCL), keratin, cellulose, chitin, lignin, amylose, amylopectin, mucin, or a PHA different from said PHA; or a non-biodegradable polymer such as polyethylene terephthalate (PET, also known as polyester), high-density polyethylene (HDPE), polyvinyl chloride (PVC), low-density polyethylene (LDPE), polypropylene (PP), polystyrene (PS), polyethylene glycol (PEG), polyproplylene glycol (PG), polyvinyl alcohol (PVA), polyacrylonitrile (PAN), polymethacrylic acid or an ester thereof, poly(acrylonitrile-co-butadiene-co-styrene) (ABS), polyamide, polyacrylamide (PAM), polysiloxanes, grafted polymers, and dendrimers such as PAMAM.
28. The blend of claim 27, wherein said at least two additional polymers comprise or consist of PLA and keratin.
29. The blend of claim 28, wherein the PHA:PLA ratio is between about 1 :99 to about 99:1, by weight, respectively.
30. The blend of claim 29, wherein the PHA:PLA ratio is between about 20:80 to about 40:60, e.g., about 25:75, about 30:70, or about 35:65, by weight, respectively.
31. The blend of claim 27, wherein one of said at least two polymers is keratin, and said keratin constitutes from about 1% to about 99%, preferably from about 10% to about 50%, by weight, of said blend.
32. An article comprising, or made of, a blend according to any one of claims 26-31.
PCT/IL2022/051061 2021-10-20 2022-10-06 Method for production of pha by fermentation of archaea WO2023073684A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020247016094A KR20240093595A (en) 2021-10-20 2022-10-06 Method for producing PHA by fermentation of archaea

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163257655P 2021-10-20 2021-10-20
US63/257,655 2021-10-20

Publications (1)

Publication Number Publication Date
WO2023073684A1 true WO2023073684A1 (en) 2023-05-04

Family

ID=86157487

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2022/051061 WO2023073684A1 (en) 2021-10-20 2022-10-06 Method for production of pha by fermentation of archaea

Country Status (2)

Country Link
KR (1) KR20240093595A (en)
WO (1) WO2023073684A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5536419A (en) * 1993-04-29 1996-07-16 Repsol Quimica S.A. Procedure for extraction of polyhydroxyalkanoates from halophilic bacteria which contain them

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5536419A (en) * 1993-04-29 1996-07-16 Repsol Quimica S.A. Procedure for extraction of polyhydroxyalkanoates from halophilic bacteria which contain them

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BUTT FATIMA IMRAN; MUHAMMAD NAWSHAD; HAMID ALMAS; MONIRUZZAMAN MUHAMMAD; SHARIF FAIZA: "Recent progress in the utilization of biosynthesized polyhydroxyalkanoates for biomedical applications – Review", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 120, 4 September 2018 (2018-09-04), NL , pages 1294 - 1305, XP085493164, ISSN: 0141-8130, DOI: 10.1016/j.ijbiomac.2018.09.002 *
CUI YOU-WEI, SHI YUN-PENG, GONG XIAO-YU: "Effects of C/N in the substrate on the simultaneous production of polyhydroxyalkanoates and extracellular polymeric substances by Haloferax mediterranei via kinetic model analysis", RSC ADVANCES, vol. 7, no. 31, 1 January 2017 (2017-01-01), pages 18953 - 18961, XP093061351, DOI: 10.1039/C7RA02131C *
LU QIUHE, HAN JING, ZHOU LIGANG, ZHOU JIAN, XIANG HUA: "Genetic and Biochemical Characterization of the Poly(3-Hydroxybutyrate- co -3-Hydroxyvalerate) Synthase in Haloferax mediterranei", JOURNAL OF BACTERIOLOGY, vol. 190, no. 12, 15 June 2008 (2008-06-15), US , pages 4173 - 4180, XP093061349, ISSN: 0021-9193, DOI: 10.1128/JB.00134-08 *
PARDO-IBÁÑEZ PABLO, LOPEZ-RUBIO AMPARO, MARTÍNEZ-SANZ MARTA, CABEDO LUIS, LAGARON JOSÉ MARÍA: "Keratin-polyhydroxyalkanoate melt-compounded composites with improved barrier properties of interest in food packaging applications", JOURNAL OF APPLIED POLYMER SCIENCE, vol. 131, no. 4, 15 February 2014 (2014-02-15), US , pages 39947, XP093061353, ISSN: 0021-8995, DOI: 10.1002/app.39947 *
ZHAO DAHE, CAI LEI, WU JINHUA, LI MING, LIU HAILONG, HAN JING, ZHOU JIAN, XIANG HUA: "Improving polyhydroxyalkanoate production by knocking out the genes involved in exopolysaccharide biosynthesis in Haloferax mediterranei", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 97, no. 7, 1 April 2013 (2013-04-01), Berlin/Heidelberg, pages 3027 - 3036, XP093061348, ISSN: 0175-7598, DOI: 10.1007/s00253-012-4415-3 *

Also Published As

Publication number Publication date
KR20240093595A (en) 2024-06-24

Similar Documents

Publication Publication Date Title
Muneer et al. Microbial polyhydroxyalkanoates (PHAs): efficient replacement of synthetic polymers
Turco et al. In vivo and post-synthesis strategies to enhance the properties of PHB-based materials: A review
Muthuraj et al. Recent developments in short-and medium-chain-length Polyhydroxyalkanoates: Production, properties, and applications
Muhammadi et al. Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: production, biocompatibility, biodegradation, physical properties and applications
Kovalcik et al. Characterization of polyhydroxyalkanoates produced by Synechocystis salina from digestate supernatant
Kurian et al. Comparative analysis of various extraction processes based on economy, eco-friendly, purity and recovery of polyhydroxyalkanoate: A review
Eraslan et al. Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)(PHBH): Synthesis, properties, and applications-A review
Yu et al. Kinetics and mechanism of the monomeric products from abiotic hydrolysis of poly [(R)-3-hydroxybutyrate] under acidic and alkaline conditions
Tokiwa et al. Biotechnological production of (R)-3-hydroxybutyric acid monomer
Södergård et al. Properties of lactic acid based polymers and their correlation with composition
Lenz et al. Bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology
Sharma et al. A handbook of applied biopolymer technology: synthesis, degradation and applications
Zhao et al. Biodegradation behavior of polycaprolactone/rice husk ecocomposites in simulated soil medium
Silva et al. Microbial production of medium-chain length polyhydroxyalkanoates
JP7123909B2 (en) Polyhydroxyalkanoate particles and aqueous dispersion thereof
Joyyi et al. Composite properties and biodegradation of biologically recovered P (3HB-co-3HHx) reinforced with short kenaf fibers
JP6860489B2 (en) A microorganism having a gene encoding a PHA synthase, and a method for producing PHA using the microorganism.
Kushwah et al. RETRACTED ARTICLE: Towards understanding polyhydroxyalkanoates and their use
Liu et al. Biodegradable polyhydroxyalkanoates production from wheat straw by recombinant Halomonas elongata A1
Wu Polylactide-based renewable composites from natural products residues by encapsulated film bag: characterization and biodegradability
Sain et al. Effect of modified cellulose fibres on the biodegradation behaviour of in-situ formed PMMA/cellulose composites in soil environment: Isolation and identification of the composite degrading fungus
Sinaei et al. Production and characterization of poly 3-hydroxybutyrate-co-3-hydroxyvalerate in wheat starch wastewater and its potential for nanoparticle synthesis
BR102014015395A2 (en) microorganism of the genus pseudomonas, process for producing long or medium chain length phas, phb, use of a microorganism and use of a pha synthase
Sharma et al. Polyhydroxybutyrate as an Eco‐Friendly Alternative of Synthetic Plastics
Mojaveryazdia et al. Production of biodegradable polymers (PHA) through low cost carbon sources: Green Chemistry

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22886302

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2024524003

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE