WO2011069244A1 - Polymère de polyhydroxyalcanoate à longueur de chaîne moyenne et procédé de fabrication de celui-ci - Google Patents
Polymère de polyhydroxyalcanoate à longueur de chaîne moyenne et procédé de fabrication de celui-ci Download PDFInfo
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- WO2011069244A1 WO2011069244A1 PCT/CA2010/001930 CA2010001930W WO2011069244A1 WO 2011069244 A1 WO2011069244 A1 WO 2011069244A1 CA 2010001930 W CA2010001930 W CA 2010001930W WO 2011069244 A1 WO2011069244 A1 WO 2011069244A1
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- 239000010802 sludge Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
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- BYKRNSHANADUFY-UHFFFAOYSA-M sodium octanoate Chemical compound [Na+].CCCCCCCC([O-])=O BYKRNSHANADUFY-UHFFFAOYSA-M 0.000 description 1
- FIWQZURFGYXCEO-UHFFFAOYSA-M sodium;decanoate Chemical compound [Na+].CCCCCCCCCC([O-])=O FIWQZURFGYXCEO-UHFFFAOYSA-M 0.000 description 1
- NMTDPTPUELYEPL-UHFFFAOYSA-M sodium;heptanoate Chemical compound [Na+].CCCCCCC([O-])=O NMTDPTPUELYEPL-UHFFFAOYSA-M 0.000 description 1
- LTOCMXUTASYUOC-UHFFFAOYSA-M sodium;nonanoate Chemical compound [Na+].CCCCCCCCC([O-])=O LTOCMXUTASYUOC-UHFFFAOYSA-M 0.000 description 1
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- ISXSCDLOGDJUNJ-UHFFFAOYSA-N tert-butyl prop-2-enoate Chemical compound CC(C)(C)OC(=O)C=C ISXSCDLOGDJUNJ-UHFFFAOYSA-N 0.000 description 1
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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
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
- C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
Definitions
- the field of the invention is biodegradable plastic. More specifically, the field of the invention is a method of using microorganisms to prepare biodegradable polymeric materials such as medium-chain-length poly(3-hydroxyalkanoate).
- Biodegradable plastics are plastics that decompose in the natural environment. That is, upon exposure to microorganisms in the natural environment (e.g., soil), biodegradable plastics are metabolized and their molecular structure is broken down. Biodegradability in plastics is desirable because such plastics may be diverted from landfill sites. When disposed in microbially rich environments such as active compost and activated sludge, biodegradable plastics rapidly convert to nitrogen-, phosphorus-, and carbon-containing components that can be taken up by organisms such as plants.
- biodegradable plastics In contrast to non-biodegradable plastics, which are mostly produced from non-renewable resources such as petroleum and hydrocarbon raw materials, most biodegradable plastics are produced from renewable natural sources and provide sustainable technology. Markets for biodegradable plastics include, for example, products that end up in sewage systems and marine environments.
- a particular type of biodegradable plastic is a family of polyesters called “polyhydroxyalkanoate” or simply "PHA”.
- PHAs also offer unique advantages such as resistance to water hydrolysis and low gas permeability when compared to other types of biodegradable plastics such as polylactides and starch-derived plastics.
- PHAs Polyhydroxyalkanoates
- PHAs are polyesters synthesized and accumulated by many microorganisms. They are considered a major class of biopolymers and have attracted extensive research and industry interests in recently years. Their properties resemble those of many petroleum-based plastics, while offering inherent biodegradability and biocompatibility.
- PHAs are produced from renewable resources such as sugars and plant oils.
- SCL-PHA short- chain-length poly(3-hydroxyalkanoate)
- MCL-PHA medium-chain-length poly(3- hydroxyalkanoate)
- SCL-PHAs are classified as having 3 to 5 carbons in their repeating units.
- MCL-PHAs are classified as having more than 5 carbons in the repeating units.
- Characteristics such as crystallinity and melting points differ between SCL-PHAs and MCL-PHAs. Accordingly, SCL-PHAs are better suited for certain applications while for others, MCL-PHAs are better suited.
- SCL- PHAs there are numerous microorganisms that are able to synthesize SCL- PHAs such as poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV).
- PHB poly(3-hydroxybutyrate)
- PHBV poly(3-hydroxybutyrate-co-3- hydroxyvalerate)
- Cupriavidus necator formerly known as Ralstonia eutropha
- SCL-PHA production by SCL- PHA-synthesizing bacteria has been well studied and it has been determined that nitrogen (N) or phosphate (P) limitation often stimulates rapid SCL-PHA synthesis.
- MCL- PHAs have only been found in pseudomonads belonging to the rRNA homology group I, such as Pseudomonas fluorescens and Pseudomonas putida (Diard, S., et al. (2002) Syst. Appl. Microbiol. 25: 183-188).
- MCL-PHAs are generally thermo-elastomeric materials with low melting temperature and low degree of crystallinity. Their use as biodegradable adhesives, rubbers, coatings, tissue engineering scaffolds, controlled drug delivery carriers, and toner agents has been proposed. However, due to the lack of an efficient production method for a variety of MCL-PHAs, these polyesters are not yet well studied. In particular, since under previously known technology there is no method to reliably produce MCL-PHAs with certain selected ratios of monomers, there is a lack of research as to the applications of such polymers. Thus there are unexplored avenues of new polymers.
- a first aspect of the invention provides a method of controlling microbial synthesis of MCL-PHA copolymer, comprising providing MCL-PHA-producing microbial cells in a medium suitable for microbial growth, the medium comprising a substrate that is structurally related to MCL-PHA; a carbon source that is not structurally related to MCL-PHA; and a ⁇ -oxidation pathway inhibitor; and allowing microbial cell growth to occur in the medium wherein the microbial cells produce MCL- PHA copolymer; wherein the MCL-PHA copolymer has a selected ratio of
- the first aspect of the invention provides a method of controlling microbial synthesis of MCL-PHA copolymer, comprising providing MCL- PHA-producing microbial cells in a medium suitable for microbial growth, the medium comprising a substrate that is structurally related to MCL-PHA; a carbon source that is not structurally related to MCL-PHA; and a ⁇ -oxidation pathway inhibitor; allowing microbial cell growth to occur in the medium wherein the microbial cells produce MCL- PHA copolymer; collecting the MCL-PHA copolymer; wherein the MCL-PHA copolymer has a selected ratio of comonomers that differs from the ratio of comonomers obtained in the absence of the ⁇ -oxidation pathway inhibitor.
- the structurally-related substrate is a fatty acid. In certain embodiments of the first aspect, the structurally- related substrate is a C 6 -C 18 fatty acid. In some embodiments of the first aspect, the structurally-related substrate is hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, or dodecanoic acid.
- the carbon source is not susceptible to ⁇ -oxidation.
- the MCL-PHA- producing microbial cells are of the genus Pseudomonas.
- the structurally-related substrate is a functionalized fatty acid. In certain embodiments of the first aspect, the structurally-related substrate is a functionalized fatty acid.
- functionalized fatty acid is a functionalized hexanoic acid, functionalized heptanoic acid, functionalized octanoic acid, functionalized nonanoic acid, functionalized decanoic acid, functionalized undecanoic acid, or functionalized dodecanoic acid.
- the ⁇ -oxidation pathway inhibitor is acrylic acid, 2-butynoic acid, 2-octynoic acid, phenylpropionic acid, propionic acid, trans-cinnamic acid, salicylic acid, methacrylic acid, 4-pentenoic acid, or 3- mercaptopropionic acid.
- the carbon source is a
- the method further comprises purifiying the collected MCL-PHA.
- the carbon source is added in a fed-batch manner or a continuous manner.
- a second aspect of the invention provides a method of making MCL- PHA polymer, comprising: providing MCL-PHA-producing microbial cells in a medium suitable for microbial growth, the medium comprising: a substrate that is structurally related to MCL-PHA; a carbon source that is not structurally related to MCL-PHA; and a ⁇ -oxidation pathway inhibitor; and allowing microbial cell growth to occur in the medium wherein the microbial cells synthesize MCL-PHA polymer; wherein the MCL- PHA polymer is a homopolymer or a copolymer that has a selected ratio of
- Another embodiment of the second aspect provides a method of making MCL-PHA polymer, comprising providing MCL-PHA-producing microbial cells in a medium suitable for microbial growth, the medium comprising a substrate that is structurally related to MCL-PHA; a carbon source that is not structurally related to MCL-PHA; and a ⁇ -oxidation pathway inhibitor; allowing microbial cell growth to occur in the medium wherein the microbial cells synthesize MCL-PHA polymer; and collecting the MCL-PHA polymer, wherein the MCL-PHA polymer is a homopolymer or a copolymer that has a selected ratio of comonomers that differs from that obtained in the absence of the ⁇ -oxidation pathway inhibitor.
- Embodiments of the second aspect provide a method of making MCL-
- PHA polymer wherein MCL-PHA-producing microbial cells are genetically modified microorganisms.
- the genetic modification increases production of PHA, increases oxygen uptake capacity, increases solvent toxicity resistance, decreases autolysis, modifies the ratio of PHA comonomers, or any combination thereof. In some such embodiments modifying the ratio of PHA
- embodiments of the second aspect the MCL-PHA-producing microbial cells are naturally occurring.
- the structurally-related substrate is a fatty acid. In some embodiments of the second aspect, the structurally- related substrate is a C 6 -Ci 8 fatty acid. In certain embodiments of the second aspect, the structurally-related substrate is nonanoic acid or octanoic acid. In certain embodiments of the second aspect, the carbon source is not susceptible to ⁇ - oxidation. In some embodiments of the second aspect, the MCL-PHA-producing microbial cells are of the genus Pseudomonas.
- the structurally-related substrate is a functionalized fatty acid.
- the functionalized fatty acid is a functionalized hexanoic acid, functionalized heptanoic acid, functionalized octanoic acid, a functionalized nonanoic acid, a functionalized decanoic acid, a functionalized undecanoic acid, or a functionalized dodecanoic acid.
- the ⁇ -oxidation pathway inhibitor is acrylic acid, 2-butynoic acid, 2-octynoic acid, phenylpropionic acid, propionic acid, trans- cinnamic acid, salicylic acid, methacrylic acid, 4-pentenoic acid, or 3- mercaptopropionic acid.
- the carbon source is a carbohydrate such as glucose or sucrose.
- the method further comprises purifiying the collected MCL-PHA.
- the carbon source is added in a fed-batch manner or a continuous manner.
- a third aspect of the invention provides a polyhydroxyalkanoate polymer produced by the method of the second aspect, that is at least about 89 mol % or higher of a selected monomer.
- the at least about 89 mol % or higher is 89 mol %.
- the at least about 89 mol % or higher is 90 mol %.
- the at least about 89 mol % or higher is 95 mol %.
- the at least about 89 mol % or higher is 99 mol %.
- the structurally-related substrate is hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid; decanoic acid, undecanoic acid, or dodecanoic acid.
- the ⁇ -oxidation pathway inhibitor is acrylic acid, 2-butynoic acid, 2-octynoic acid, phenylpropionic acid, propionic acid, trans-cinnamic acid, salicylic acid, methacrylic acid, 4-pentenoic acid, or 3-mercaptopropionic acid.
- a fourth aspect of the invention provides poly(3-OH-nonanoate-co-3- OH-heptanoate) (PHN) comprising about 71 mol % or higher 3-OH-nonanoate.
- the PHN comprises about 75, about 81 , about 83, about 86, about 88, about 90, about 92, about 94, about 95, or about 96 mol % 3-OH- nonanoate.
- a fifth aspect of the invention provides poly(3-OH-octanoate-co-3-OH- hexanoate) (PHO) comprising about 89 mol % or higher 3-OH-octanoate.
- PHO poly(3-OH-octanoate-co-3-OH- hexanoate)
- the PHO comprises about 93, about 94, about 96, about 97, or about 98 mol % 3-OH-octanoate.
- a sixth aspect of the invention provides polyhydroxyalkanoate polymer comprising a C n monomer that has n carbons and a C n . 2 monomer that has n-2 carbons, where n is 8 to 12, and where the polymer is at least 70 mol % C Pain monomer.
- a seventh aspect of the invention provides polyhydroxyalkanoate polymer comprising a C fatigue monomer that has n carbons, where n is 6 to 18, and where the polymer is 70 mol % or higher C Compute monomer.
- An eighth aspect of the invention provides polyhydroxyalkanoate polymer comprising a C fatigue monomer that has n carbons, where n is 6 to 12, and where the polymer is 70 mol % or higher C n monomer.
- a ninth aspect of the invention provides polyhydroxyalkanoate polymer comprising a C fatigue monomer that has n carbons, where n is 8 to 18, and
- the C n monomers is: 3-
- the at least 70 mol % is 80 mol %. In some embodiments, the at least 70 mol % is 85 mol %. In some embodiments, the at least 70 mol % is 90 mol %. In some embodiments, the at least 70 mol % is 95 mol %. In some embodiments, the at least 70 mol % is 99 mol %. [0030] In some embodiments of the sixth to ninth aspects, the 70 mol % or higher is 100 mol %.
- Another aspect of the invention provides a polyhydroxyalkanoate polymer produced by the method of the first aspect, that is at least about 89 mol % or higher of a selected monomer.
- the at least about 89 mol % is 89 mol %.
- the at least about 89 mol % is 90 mol %.
- the at least about 89 mol % is 95 mol %.
- the at least about 89 mol % is 99 mol %.
- the at least about 89 mol % is 100 mol %.
- Embodiments of this aspect provide a polyhydroxyalkanoate polymer, wherein the structurally-related substrate is octanoic acid, nonanoic acid; decanoic acid, undecanoic acid, or dodecanoic acid.
- Yet another embodiment of this aspect provides polyhydroxyalkanoate polymer, wherein the ⁇ -oxidation pathway inhibitor is acrylic acid, 2-butynoic acid, 2- octynoic acid, phenylpropionic acid, propionic acid, trans-cinnamic acid, salicylic acid, methacrylic acid, 4-pentenoic acid, or 3-mercaptopropionic acid.
- the ⁇ -oxidation pathway inhibitor is acrylic acid, 2-butynoic acid, 2- octynoic acid, phenylpropionic acid, propionic acid, trans-cinnamic acid, salicylic acid, methacrylic acid, 4-pentenoic acid, or 3-mercaptopropionic acid.
- Another aspect of the invention provides a polyhydroxyalkanoate polymer comprising a C Intel monomer that has n carbons and a C Compute. 2 monomer that has n- 2 carbons, where n is 6 to 18, and where the polymer is at least about 89 mol % or higher C Intel monomer.
- Yet another aspect of the invention provides a polyhydroxyalkanoate polymer comprising a Cn monomer that has n carbons, where n is 6 to 18, and where the polymer is 89 mol % or higher Cn monomer.
- An aspect of the invention provides a polyhydroxyalkanoate polymer comprising a C n monomer that has n carbons, wherein when n is 6, 7, 9, 10, or 1 1 , the polymer is at least about 71 mol % or higher C n monomer, wherein when n is 8, the polymer is at least about 89 mol % or higher C fatigue monomer, and wherein when n is 12 to 18, the polymer is at least about 21 mol % or higher C Pain monomer.
- Yet another aspect of the invention provides a polyhydroxyalkanoate polymer prepared in the presence of a ⁇ -oxidation pathway inhibitor, comprising a ratio of comonomers that differs from the ratio of comonomers obtained in the absence of ⁇ - oxidation pathway inhibition.
- An embodiment of this aspect provides polyhydroxyalkanoate polymer, wherein the C n monomer is: 3-OH-hexanoate, 3-OH-heptanoate, 3-OH-octanoate, 3- OH-nonanoate, 3-OH-decanoate, 3-OH-undecanoate, or 3-OH-dodecanoate.
- Another aspect of the invention provides a polyhydroxyalkanoate polymer comprising about 21 mol % or higher 3-OH-dodecanoate.
- Another aspect of the invention provides a polyhydroxyalkanoate polymer comprising at least about 35 mol % or higher 3-OH-dodecanoate.
- Yet another aspect of the invention provides a polyhydroxyalkanoate polymer comprising at least about 60 mol % or higher 3-OH-dodecanoate.
- the invention provides polyhydroxyalkanoate polymer of any of the preceeding aspects, wherein the MCL-PHA-producing microbial cells are genetically modified microorganisms.
- the genetic modification increases production of PHA, increases oxygen uptake capacity, increases solvent toxicity resistance, decreases autolysis, modifies the ratio of PHA comonomers, or any combination thereof.
- modifying the ratio of PHA comonomers is increasing the amount of predominant monomer.
- the MCL-PHA-producing microbial cells are naturally occurring.
- Figure 1 is a plot of Absorbance at a wavelength ( ⁇ ) of 650 nm for shake flask cultures of Pseudomonas putida KT2440 grown at various initial concentrations of acrylic acid; data is presented for culturing times of 12 h ( ⁇ ) and 24 h
- Figures 2A and 2B graphically show the effect of a ⁇ -oxidation pathway inhibitor on cell growth, PHA content (which in this case is PHN), and PHA
- FIG. 2A is a plot of biomass concentration (see ⁇ symbols and left y-axis), PHA content (see ⁇ symbols and right y- axis), and residual acrylic acid concentration (AA, see A symbols and right secondary y-axis) versus inflow acrylic acid concentration (x-axis).
- Figure 2B is a plot of monomeric composition (C 9 and C 7 ) of collected MCL-PHA versus inflow acrylic acid concentration.
- Figures 3A and 3B graphically show the effect of a ⁇ -oxidation pathway inhibitor on cell growth, PHA content (which in this case is PHN), and PHA
- FIG. 3A is a plot of biomass concentration (see ⁇ symbols and left y-axis), PHA content (see ⁇ symbols and right y- axis) and residual acrylic acid concentration (AA, see A symbols and right secondary y-axis) versus inflow acrylic acid concentration (x-axis).
- Figure 3B is a plot of monomeric composition of isolated MCL-PHA (C 9 and C 7 ) versus inflow acrylic acid concentration.
- Figures 4A and 4B graphically show the effect of a ⁇ -oxidation pathway inhibitor on cell growth, PHA content (which in this case is PHO), and PHA
- FIG. 4A is a plot of biomass concentration (see ⁇ symbols and left y-axis), PHA content (see ⁇ symbols and right y- axis) and residual acrylic acid concentration (AA, see A symbols and right secondary y-axis) versus inflow acrylic acid concentration (x-axis).
- Figure 4B is a plot of monomeric composition of isolated MCL-PHA (C 8 and C 6 ) versus inflow acrylic acid concentration.
- Figures 5A and 5B graphically show the time profile of biomass concentration, and PHA content (which in this case is PHN) (see Figure 5A) and monomeric composition (C 9 and C 7 ) (see Figure 5B) of isolated MCL-PHA during a fed-batch fermentation with co-feeding of nonanoic acid and glucose with a mass ratio of 1 :1 , and in the absence of acrylic acid.
- Figures 6A and 6B graphically show the time profile of biomass concentration, PHA content (which in this case is PHN) (see Figure 6A) and monomeric composition of isolated MCL-PHA (C 9 and C 7 ) (see Figure 6B) during a fed-batch fermentation with co-feeding of nonanoic acid, glucose, and acrylic acid at a mass ratio of 1.25:1 :0.03.
- Figure 7 is an overlay of the Differential Scanning Calorimetry (DSC) up-heating curves of various poly(3-OH-nonanoate-co-3-OH-heptanoate)
- PHN-70 is the abbreviation of poly(3-OH-nonanoate-co-3-OH- heptanoate) with 70 mol % nonanoate and 30 mol % heptanoate.
- PHN-81 is the abbreviation of poly(3-OH-nonanoate-co-3-OH-heptanoate) with 81 mol % nonanoate and 19 mol % heptanoate.
- PHN-86 is the abbreviation of poly(3-OH-nonanoate-co-3- OH-heptanoate) with 86 mol % nonanoate and 14 mol % heptanoate.
- PHN-95 is the abbreviation of poly(3-OH-nonanoate-co-3-OH-heptanoate) with 95 mol % nonanoate and 5 mol % heptanoate.
- Figure 8 is an overlay of the Differential Scanning Calorimetry (DSC) up-heating curves of various poly(3-OH-octanoate-co-3-OH-hexanoate) compositions.
- DSC Differential Scanning Calorimetry
- PHO-88 is the abbreviation of poly(3-OH-octanoate-co-3-OH-hexanoate) with 88 mol % octanoate and 12 mol % hexanoate.
- PHO-94 is the abbreviation of poly(3-OH- octanoate-co-3-OH-hexanoate) with 94 mol % octanoate and 6 mol % hexanoate.
- PHO-96 is the abbreviation of poly(3-OH-octanoate-co-3-OH-hexanoate) with 96 mol % octanoate and 4 mol % hexanoate.
- PHO-98 is the abbreviation of poly(3-OH- octanoate-co-3-OH-hexanoate) with 98 mol % octanoate and 2 mol % hexanoate.
- Figure 9 is a schematic of major pathways involved in MCL-PHA synthesis by pseudomonads (Adapted from Vanderleij FR, Witholt B (1995) "Strategies for the sustainable production of new biodegradable polyesters in plants - a review.” Canadian Journal of Microbiology 41:222-238).
- alkane hydroxylase (1) alkane hydroxylase (1); alcohol dehydrogenase (2); aldehyde dehydrogenase (3); thiokinase (4); acyl-CoA dehydrogenase (5); enoyl- CoA hydratase or crotonase, (6); (S)-3-OH-acyl-CoA dehydrogenase (7); 3-ketoacyl- CoA thiolase (8); enoyl-CoA hydratase (9); 3-OH-acyl-CoA epimerase (10); ketoacyl- CoA reductase (1 ); PHA polymerase (12); (R)-3-OH-acyl (ACP-CoA) transferase (13); 3-OH-acyl-ACP dehydratase (14); enoyl-ACP reductase (15); fatty acid thioesterase (16); 3-ketoacyl synthe
- Figures 10A and 10B graphically show the effect of a ⁇ -oxidation pathway inhibitor on cell growth, PHA content, and PHA composition for cultures grown at various inflow concentrations of acrylic acid with cofeeding of dodecanoic acid and glucose in a continuous fermentation. Samples were taken once systems had reached steady state.
- Figure 10A is a plot of biomass concentration (see ⁇ symbols and left y-axis), PHA content (see ⁇ symbols and right y-axis) versus inflow acrylic acid concentration (x-axis).
- Figure 10B is a plot of monomeric composition of isolated MCL-PHA (C 2 , C 10 , C 8 and C 6 ) versus inflow acrylic acid concentration.
- the term "monomer” as used herein means a repeating unit of a polymer.
- the term “comonomer” as used herein means one of at least two monomers that are present in a polymer.
- the term “polymer” as used herein means a molecular chain of subunits that may be linear or branched, and includes homopolymers and copolymers.
- the term “homopolymer” as used herein means a polymer with a single repeating unit.
- copolymer as used herein means a polymer with at least two different repeating units.
- aliphatic refers to hydrocarbon moieties that are straight chain, branched or cyclic, may be alkyl, alkenyl or alkynyl, and may be substituted or unsubstituted.
- PHA polyhydroxyalkanoate
- MCL-PHA medium-chain-length poly(3-hydroxyalkanoate) which are poly(3-hydroxyalkanoates classified as having more than 5 carbons in their repeating units.
- SCL-PHA short-chain-length poly(3- hydroxyalkanoate) which are poly(3-hydroxyalkanoates classified as having 3 to 5 carbons in their repeating units.
- SCL-co-MCL PHA as used herein means a copolymer of SCL-PHA and MCL-PHA.
- PHA as used herein means a PHA comprising at least 50 mole % of 3-OH-octanoic acid monomer (i.e., predominant monomer is 3-OH-octanoic acid).
- PPN as used herein means a PHA comprising at least 50 mole % of
- 3-OH-nonanoic acid monomer i.e., predominant monomer is 3-OH-nonanoic acid.
- PHDD as used herein means a PHA comprising at least 50 mole % of 3-
- OH-dodecanoic acid monomer i.e., predominant monomer is 3-OH-dodecanoic acid.
- the term "functionalized” is intended to encompass compounds bearing a substituent(s) selected from the group consisting of halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphate ester, phosphonato, phosphinato, cyano, amino, acylamino, amide, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, dithiocarboxylate, sulfate, sulfonato, sulfamoyl, sulfonamido, nitro,
- structurally-related substrate means a compound that can be metabolized via a ⁇ -oxidation pathway (see Figure 9) by MCL-PHA-synthesizing- microbial cells to generate a MCL-PHA precursor.
- structurally-related carbon sources include: aliphatic acids such as octanoic acid, nonanoic acid, decanoic acid; or compounds that can be oxidized to form aliphatic acids such as octane, nonane, and decane.
- structurally-unrelated and “not structurally related” mean a compound that is metabolized by MCL-PHA-synthesizing-microbial cells by metabolic routes other than the ⁇ -oxidation pathway (i.e., not susceptible to ⁇ -oxidation).
- carbon source refers to a nutrient (such as sugar) that provides carbon needed for cellular respiration, cellular combustion, and/or synthesis of new organic molecules.
- ⁇ -oxidation inhibitor and " ⁇ -oxidation pathway inhibitor” refer to a chemical compound that inhibits an enzyme(s) of the fatty acid ⁇ -oxidation pathway.
- C 6 is used for convenience to represent a
- C 7 is used for convenience to represent a polyhydroxyalkanoate monomer that has seven carbons (three of the carbons in its backbone and four carbons in its sidechain).
- C 8 is used for convenience to represent a polyhydroxyalkanoate monomer that has eight carbons (three of the carbons in its backbone and five carbons in its sidechain).
- C 9 is used for convenience to represent a polyhydroxyalkanoate monomer that has nine carbons (three of the carbons in its backbone and six carbons in its sidechain).
- C 10 is used for convenience to represent a polyhydroxyalkanoate monomer that has ten carbons (three of the carbons in its backbone and seven carbons in its sidechain).
- Cn is used for convenience to represent a
- C 12 is used for convenience to represent a polyhydroxyalkanoate monomer that has twelve carbons (three of the carbons in its backbone and nine carbons in its sidechain). See Table 1 for structural formulae of examples of polyhydroxyalkanoates discussed herein.
- PHA has attracted commercial interest since it is renewable, biodegradable and biocompatible.
- PHA has properties similar to polyethylene, polypropylene, synthetic polyesters and acrylics, but it can be produced from renewable resources.
- Suggested uses for currently produced PHAs include packaging such as fast food containers, disposable syringes and bottles, and coatings.
- MCL-PHAs are a distinct group of polymers in the PHA family that have more than five carbons in their monomer units.
- MCL-PHAs with 6 to 14 carbons in their repeating units show promise in latex form, and as thermoelastomers for biomedical applications such as drug delivery and tissue engineering.
- aspects of the invention provide methods of controlling the monomeric ratio of MCL-PHA produced by microbial cells; optionally, the microbial cells are wild type.
- MCL-PHA monomeric ratio control
- Such monomeric ratio control is obtained by modulating a metabolic pathway that is involved both in MCL-PHA synthesis and in energy generation.
- MCL-PHAs were produced that differ significantly from MCL-PHAs that were produced by previously known methods using the same microorganism. Accordingly, the invention provides a method of making a variety of copolymers with a broad range of properties.
- aspects of the invention provide polyhydroxyalkanoates with a broad range of monomeric ratios.
- previously known methods used mutant or recombinant microorganisms and provided PHAs of limited monomeric compositions.
- the efficiency of such methods was typically low.
- MCL-PHAs produced by methods of the invention significantly expand the range of compositions and properties of MCL-PHAs as a class of biopolyester.
- methods of the invention to produce MCL-PHAs possess such efficiency that they are capable of being easily scaled up for industrial production.
- MCL-PHAs are produced by naturally occurring microorganisms such as Pseudomonas species of the rRNA homology group I (Diard, S., et al. (2002) Syst. Appl. Microbiol. 25:183-188), for example, Pseudonomas putidta KT2440 (ATCC (American Type Culture Collection) No. 47054).
- Pseudomonas species of the rRNA homology group I Diard, S., et al. (2002) Syst. Appl. Microbiol. 25:183-188
- Pseudonomas putidta KT2440 ATCC (American Type Culture Collection) No. 47054
- microorganisms are fermented under appropriate conditions. Conditions to be maintained during fermentation include appropriate levels of nutrients, dissolved oxygen, temperature and pH. Required nutrients include carbon-, nitrogen-, phosphorus-, and sulfur-containing sources as well as mineral salts.
- Methods of the invention may be applied to any microorganism capable of producing MCL-PHA from structurally-related carbon sources via the fatty acid ⁇ - oxidation route of MCL-PHA synthesis (see Figure 9).
- microorganisms include, but are not limited to, rRNA homology group I Pseudomonas (Diard, S., et al. (2002) Syst. Appl. Microbiol.
- Pseudomonas putida e.g., strains KT2440 and GPo1
- Pseudomonas aeruginosa e.g., strain PAO
- Pseudomonas resinovorans Pseudomonas citronellolis
- Pseudomonas fluorescens for example, Pseudomonas putida (e.g., strains KT2440 and GPo1), Pseudomonas aeruginosa (e.g., strain PAO), Pseudomonas resinovorans, Pseudomonas citronellolis and Pseudomonas fluorescens.
- the microorganism may also be a recombinant strain if a similar ⁇ -oxidation MCL-PHA synthesis route is present, for example, recombinant Escherichia coli.
- the microorganisms may be cultured in any conventional way of bacterial cultivation, such as, for example, in a batch mode shake flask, in a batch mode bioreactor fermentation, in a fed-batch mode bioreactor fermentation, and in a continuous bioreactor fermentation.
- culture medium contains (i) a structurally- related carbon source, (ii) a structurally-unrelated carbon source, (iii) a ⁇ -oxidation inhibitor, and other nutrients generally necessary for microbial growth.
- nutrients are typically added to the culture medium in water soluble salt forms, and include nitrogen, phosphorus, sulfur, magnesium, sodium, potassium, iron, and other trace metal elements.
- Other culture conditions such as temperature, pH, and dissolved oxygen (DO) also need to be maintained at appropriate levels for the culture.
- non-limiting examples of (i) structurally-related carbon sources include: fatty acids such as C 6 -C 18 fatty acids which include nonanoic acid, octanoic acid, decanoic acid, undecanoic acid, dodecanoic acid; and functionalized C 6 -C 18 fatty acids which include functionalized nonanoic acid, functionalized octanoic acid, functionalized decanoic acid and functionalized
- non-limiting examples of (ii) structurally-unrelated carbon sources include carbohydrate such as glucose or sucrose, or any other energy source that is not susceptible to ⁇ -oxidation, such as glycerol, citric acid and acetic acid.
- ⁇ - oxidation inhibitors include acrylic acid, 2-butynoic acid, 2-octynoic acid,
- phenylpropionic acid propionic acid, trans-cinnamic acid, salicylic acid, methacrylic acid, 4-pentenoic acid, and 3-mercaptopropionic acid.
- the ⁇ -oxidation pathway ulitizes substrates that are structurally-related to PHA, such as fatty acids, aliphatic alkanes, alkenes, and intermediates thereof, in the synthesis of MCL-PHA.
- substrates that are structurally-related to PHA, such as fatty acids, aliphatic alkanes, alkenes, and intermediates thereof.
- fatty acids are oxidized sequentially into enoyl-CoA, acyl-CoA, and ketoacyl-CoA (steps 4, 5, and 6) before two carbons are removed from the ketoacyl-CoA in the form of acetyl- CoA (step 8).
- the acyl-CoA with two carbons less than the previous acyl-CoA enters the ⁇ -oxidation cycle again.
- any of the three intermediates of one ⁇ -oxidation cycle can be transformed into (R)-3-hydroxy-acyl-CoA (steps 9, 10, 11), which is the only stereoisomeric precursor accepted by the MCL- PHA synthase, and polymerized into MCL-PHA (step 2).
- the PHA that is produced has at least two comonomers that differ from one another by one C 2 unit (e.g., C 9 and C 7 ) (Lee, S.Y., et al. (2000) Biotechnol.
- nonanoic acid for example, is the fatty acid carbon source on the left side of Figure 9
- a C 9 comonomer is present in the resulting MCL-PHA product by following one of three enzymatic paths: (4), (5) and (9); (4), (5), (6) and (10); or (4), (5)
- MCL-PHA synthesis route is much less efficient energetically than the direct fatty acid ⁇ -oxidation route, as considerable energy is required to produce MCL-fatty acids from structurally-unrelated carbon compounds.
- MCL-PHAs synthesized by conventional methods have limited variation in monomeric composition and physical properties. Such MCL- PHAs have low melting temperatures and a low degree of crystallinity. In order to expand the applicability of MCL-PHA, it is important to obtain a wider variety of monomeric compositions than naturally occurs with a given combination of culture and carbon source.
- MCL-PHA monomeric composition of MCL-PHA. It was reported by Park et a/, in Enzyme and Microbial Technology 33 (2003) 62-70 that, by amplifying the fadO and fadE genes in recombinant Escherichia coli, MCL-PHA with enriched 3-OH-decanoate molar content was obtained when sodium decanoate was used as the carbon source. It was also reported by Ouyang ef al.
- MCL-PHA with enriched monomers having the same number of carbons as the employed alkanoate substrates could be obtained.
- MCL-PHA with very high relative amounts of one monomer over the other, ultimately approaching a substantially homopolymeric PHA was also attained.
- aspects of the invention provide inhibiting of the ⁇ -oxidation pathway to control the ratio of comonomers in the MCL-PHA product.
- Studies described herein have been conducted to determine the effects of inhibiting the ⁇ -oxidation pathway on cell growth, PHA content, and PHA monomer ratios. Such studies are described in the working examples, figures, and tables.
- acrylic acid was used as a ⁇ -oxidation pathway inhibitor. The concentration of acrylic acid was varied to quantify its effects. Other ⁇ -oxidation pathway inhibitors are expected to have similar results. Without wishing to be bound by theory, the inventors suggest that the mechanism of action of acrylic acid as a ⁇ -oxidation inhibitor involves interaction with ⁇ -ketoacyl-CoA thiolase.
- ⁇ -ketoacyl-CoA thiolase acts at conversion step (8) of Figure 9, before chain scission of 3-ketoacyl-CoA to form Acyl-CoA with one C 2 unit less that the Acyl- CoA that is obtained through conversion step (4).
- ⁇ -oxidation pathway By inhibiting the ⁇ -oxidation pathway at this site, generation of energy required for cell growth and maintenance is substantially decreased, and many accumulated intermediates are diverted to PHA synthesis. Unless efficient cell growth and maintenance are achieved in another way, inhibition of the ⁇ -oxidation pathway results in low cell concentrations and low PHA accumulation. Accordingly, when the ⁇ -oxidation pathway was inhibited, cell growth was maintained by providing a carbon-source that was structurally-unrelated to PHA.
- This structurally-unrelated carbon source was used for energy production and cell growth while the C-source that was structurally-related to PHA was used for PHA production. Therefore, with the combination of a structurally-related carbon source, a structurally-unrelated carbon source, and a ⁇ -oxidation inhibitor, not only was the final MCL-PHA composition successfully modulated, but good cell growth and MCL-PHA content were also attained.
- an aspect of the invention provides a method of controlling the ratio of monomers in MCL-PHA by growing bacteria in the presence of representatives from three groups of compounds. These three groups are: (i) a carbon source that is structurally-related to PHA; (ii) a carbon source that is structurally-unrelated to PHA but that supports cell growth; and (iii) a ⁇ -oxidation pathway inhibitor.
- the first group (i) a substrate that is structurally-related to PHA, comprises carbon sources that can be converted to MCL-PHA synthesis precursors substantially directly via the fatty acid ⁇ -oxidation pathway, which is shown in Figure 9. Their structure is similar to the structure of the repeating units in the resulting MCL- PHA.
- Structurally-related compounds include, but are not limited to: alkanes such as heptane, octane, nonane, decane, undecane, dodecane; alkenes such as heptene, octene, nonene, decene, undecene, dodecene; alcohols such as hexanol, heptanol, octanol, nonanol, decanol; saturated and unsaturated aliphatic acids and their salt form such as hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, octanoic acid, nonenoic acid, undecenoic acid, sodium heptanoate, sodium octanoate, sodium nonanoate; and long chain fatty acids such as lauric acid, oleic acid.
- medium-chain-length fatty acids or their salt forms are used, such as octanoate, nonanoate, and undecenoate.
- Octanoic acid may be conveniently derived from coconut and palm kernel oils.
- Nonanoic acid may be produced from oleic, linoleic, erucic and other carboxylic acids derived from temperate zone plants such as, for example, canola.
- the structurally-related carbon source may be used in an amount that is sufficient for MCL-PHA production but does not inhibit the growth of the culture.
- the feeding of the carbon source is carried out such that the concentration of the carbon source in the bioreactor does not exceed 20 g L "1 , preferably does not exceed 10 g L '1 , and more preferably does not exceed 3 g L "1 .
- the carbon source concentration in the inflow medium can be relatively high, as long as the concentration in the bioreactor does not exceed the concentrations that are acceptable in cultivation.
- the second group (ii) a carbon-source that is not structurally-related to PHA but that supports cell growth, does so through Entner-Doudoroff pathway and Krebs cycle, or Embden-Meyerhof pathway (glycolysis) and Krebs cycle.
- Structurally- unrelated carbon sources include, but are not limited to: saccharides such as glucose, fructose, sucrose, and mannose; glycerol; organic acids such as acetic acid; and complex carbon sources such as nutrient broth, tryptone, and yeast extract.
- the structurally-unrelated carbon source is a saccharide such as glucose.
- the structurally-unrelated carbon source may be used in a concentration that is sufficient for biomass and energy generation but does not inhibit the growth of the culture.
- up to 40 g L "1 of structurally-unrelated carbon source can be used.
- up to 10 g L "1 of structurally-unrelated carbon source is used.
- up to 5 g L " of structurally-unrelated carbon source is used.
- the feeding of the carbon source is carried out such that the concentration of the carbon source in the bioreactor does not exceed 20 g L " , preferably does not exceed 10 g L '1 , and more preferably does not exceed 5 g L "1 .
- the structurally-related carbon source concentration in the inflow medium can be relatively high, as long as the concentration in the bioreactor does not exceed the concentrations that are acceptable in cultivation.
- the third group is (iii) a ⁇ -oxidation pathway inhibitor. By inhibiting the ⁇ -oxidation pathway, the amount of group (i) substrate that completes the ⁇ -oxidation pathway is controlled.
- This pathway includes conversion of 3-ketoacyl-CoA to acyl- CoA through enzyme (8) 3-ketoacyl-CoA thiolase, such that the acyl-CoA is modified to have one less C 2 unit than (i).
- fatty acid ⁇ -oxidation inhibitors include, but are not limited to, acrylic acid, 2-butynoic acid, 2-octynoic acid, phenylpropionic acid, propionic acid, trans-cinnamic acid, salicylic acid, methacrylic acid, 4-pentenoic acid, and 3-mercaptopropionic acid.
- the inhibitors may be an acid form or a corresponding salt form, such as, for example, sodium acrylate.
- acrylic acid or acrylate is the preferred ⁇ -oxidation inhibitor.
- the inhibitor may be used in an amount that will be sufficient to modulate the ⁇ -oxidation pathway and the final MCL-PHA compositions, but at a level such that the growth of the culture is not substantially inhibited.
- the inhibitor concentration in the inflow medium may be 0 to 10 mM depending on the desired final MCL-PHA composition.
- the initial inhibitor concentration may be up to 10 mM depending on the desired final MCL-PHA composition.
- the feeding of the inhibitor is carried out such that the concentration of the inhibitor in the reactor does not exceed 0 mM, and preferably not exceed 4 mM, while the feeding amount depends on the desired final MCL-PHA composition.
- Another aspect of the invention provides a method to produce from microbial cells, MCL-PHA having a selected specific monomer content.
- the microbial cells may be wild type, i.e., not genetically engineered.
- Embodiments of the invention that are described herein were tested and produced high cell concentration, high PHA content, and high yield. Monomer content was controlled by the amount of (iii) ⁇ -oxidation pathway inhibitor that was added, the choice of and the ratio of (i) and (ii).
- Pseudomonas species of the rRNA homology group I microorganisms were grown and fed in a manner well established in the art and detailed in the Working Examples.
- the growth medium comprised a compound from each of the three groups in a controlled manner, (i) nonanoic acid; (ii) glucose, and (iii) acrylic acid.
- PHA is produced with a chosen ratio of comonomers.
- such ratios include: 100:0; 99:1 ; 98:2; 97:3; 96:4; 95:5; 94:6; 93:7; 92:8; 91 :9; 90:10; 89:11 ; 88:12; 87:13; 86:14; 85:15; 84:16; 83:17; 82:18; 81 :19; 80:20; 79:21 ; 78:22; 77:23; 76:24; 75:25; 74:26; 73:27; 72:28; 71 :29; 70:30; 69:31 ; 68:32; 67:33; 66:34; and 65:35.
- MCL-PHAs with a variety of relative amounts of monomer (e.g., 80:20, 85:15, 90:10; 95:5) can be reliably produced.
- substrate (i) is a fatty acid, which may be a functionalized fatty acid.
- the MCL-PHAs obtained by these methods are polymers whose monomeric constituents are determined by the nature of the structurally-related carbon source, and whose monomeric compositions are determined according to the selected concentrations of the structurally-related carbon source, the ⁇ -oxidation inhibitor, and the structurally-unrelated carbon source.
- the melting point, degree of crystallinity, and rate of crystallinity, as well as other related physical and mechanical properties of the MCL-PHAs, are affected by the molar fraction of the longer chain comonomers.
- MCL-PHAs were obtained that were polymers of poly(3-OH-octanoate-co-3-OH-hexanoate) with a 3-OH-octanoate content of 88 mol % to 98 mol % and with a 3-OH-hexanoate content of 12 mol % to 2 mol %.
- MCL-PHAs were obtained that were polymers of poly(3- OH-nonanoate-co-3-OH-heptanoate) with a 3-OH-nonanoate content of 65 mol % to 96 mol % and with a 3-OH-heptanoate content of 35 mol % to 4 mole %.
- the average molecular weight was between 50,000 and 200,000 g moL '1 , typically between 80,000 and 150,000 g moL "1 .
- Glass transition temperatures ranged from -50 °C to -30°C. Melting temperatures (Tm) were 40°C to 75°C. Young's modulus was up to 12 MPa. Tensile stress at maximum load was up to 16 MPa. Elongation at break was up to 1400%.
- FIGS. 2A and 2B plots are shown that graphically present the effect of a ⁇ -oxidation pathway inhibitor on cell growth, PHA (which in this case is PHN) content, and PHA composition for cultures grown at various inflow concentrations of acrylic acid with a single feeding of nonanoic acid in a continuous fermentation. Samples were taken after systems had reached steady state as judged by attainment of stable rates of carbon dioxide production.
- Figure 2A is a plot of biomass concentration, PHA content, and residual acylic acid concentration versus inflow acrylic acid concentration.
- Figure 2B is a plot of monomeric composition of isolated MCL-PHA (C g and C 7 ) versus inflow acrylic acid concentration.
- FIGS 3A and 3B plots are shown to demonstrate the effect of ⁇ -oxidation pathway inhibitor on cell growth, PHN content, and PHN composition for cultures grown at various concentrations of acrylic acid with cofeeding of nonanoic acid and glucose in a continuous fermentation. The sample point was taken once the system had reached steady state.
- Figure 3A is a plot of biomass concentration, PHA (which in this case is PHN) content, and residual acrylic acid concentration versus inflow acrylic acid concentration.
- Figure 3B is a plot of monomeric composition of isolated MCL-PHA (C 9 and C 7 ) versus inflow acrylic acid concentration.
- Figure 3B shows results of polymer syntheses with a 3-OH-nonanoate mol percent content of 68, 75, 83, 90, 92, 94, 95, and 96 mol % and corresponding 3-OH- heptanoate mole percentages.
- FIGS. 4A and 4B plots are shown to demonstrate the effect of ⁇ -oxidation pathway inhibitor on cell growth, PHO content, and PHO composition for cultures grown at various concentrations of acrylic acid with cofeeding of octanoic acid and glucose in a continuous fermentation. Samples were taken once steady state had been reached.
- Figure 4A is a plot of biomass concentration, PHA (which in this case is PHO) content, and residual acrylic acid concentration versus inflow acrylic acid concentration.
- Figure 4B is a plot of monomeric composition of isolated PHO (C 8 and C 6 ) versus inflow acrylic acid concentration.
- FIGS. 5A and 5B plots are shown of the time profile of biomass concentration, PHA (which in this case is PHN) content, and monomeric composition of isolated MCL-PHA (C 9 and C 7 ) during a fed-batch fermentation with cofeeding of nonanoic acid and glucose with a mass ratio of 1 :1 , and in the absence of acrylic acid.
- Fed-batch is the most commonly used bacterial fermentation technique (compared to batch mode and continuous mode fermentation) at industrial scale.
- an experiment was conducted to determine the feasibility of the cofeeding of structurally-related carbon source (nonanoic acid) and structurally-unrelated carbon source (glucose) method in such fermentation.
- the monomeric composition of MCL-PHA produced was consistent at 69.5 ⁇ 0.4 mol % C 9 (3-OH-nonanoate) and the remaining comonomer was C 7 (3-OH- heptanoate) throughout the entire fermentation.
- Such MCL-PHA composition was also consistent with the composition obtained under similar cofeeding conditions during continuous fermentation, when ⁇ -oxidation pathway inhibitor (acrylic acid) was absent.
- FIGS. 6A and 6B plots are shown of the time profile of biomass concentration, PHN content, and monomeric composition of isolated MCL- PHA (C 9 and C 7 ) during a fed-batch fermentation with cofeeding of nonanoic acid, glucose and acrylic acid with a mass ratio of 1.25:1 :0.03.
- an experiment was conducted to determine the feasibility of the cofeeding of structurally related carbon source (nonanoic acid), structurally-unrelated carbon source (glucose), and ⁇ -oxidation pathway inhibitor (acrylic acid) method in such fermentation.
- alkane hydroxylase (1) alkane hydroxylase (1); alcohol dehydrogenase (2); aldehyde dehydrogenase (3); thiokinase (4); acyl-CoA dehydrogenase (5); enoyl-CoA hydratase or crotonase, (6); (S)-3-OH-acyl-CoA dehydrogenase (7); 3-ketoacyl-CoA thiolase (8); enoyl-CoA hydratase (9); 3-OH-acyl- CoA epimerase (10); ketoacyl-CoA reductase (11); PHA polymerase (12); (R)-3-OH- acyl (ACP-CoA) transferase (13); 3-OH-acyl-ACP dehydratase (14); enoyl-ACP reductase (15); fatty acid thioesterase (16); 3-ketoacyl
- FIGS. 10A and B plots are shown to demonstrate the effect of ⁇ -oxidation pathway inhibitor on cell growth, monomer content, and polymer composition for cultures grown at various concentrations of acrylic acid with cofeeding of dodecanoic acid and glucose in a continuous fermentation. Samples were taken once steady state had been reached.
- Figure 10A is a plot of biomass concentration and PHA content versus inflow acrylic acid concentration.
- Figure 10B is a plot of monomeric composition of isolated PHA (C 12 , Ci 0 , C 8 and C 6 ) versus inflow acrylic acid concentration.
- experiments were conducted to examine the effects of acrylic acid on cell growth and PHA production in carbon-limited chemostat culture with dodecanoic acid and glucose cofeeding.
- a basic glucose-only shake flask medium was a 100 mL liquid medium in a 500 mL Erlenmeyer flask.
- the medium comprised (g L ⁇ 1 ): (NH 4 ) 2 S0 (4.70), MgSCv7H 2 0 (0.80), Na 2 HP0 4 « 7H 2 0 (12.0), KH 2 P0 4 (2.70), glucose (9.0), nutrient broth (1.0).
- Continous fermentations were carried out in a 1.5 L stirred tank bioreactor (Bioflo lie, New Brunswick Scientific, New Jersey, USA) with a working volume of 1.1 L.
- the fermentor was operated at 28.5 ⁇ 1 °C and the pH was automatically controlled at 6.85 ⁇ 0.05 using 2 N potassium hydroxide solution.
- the dissolved oxygen was monitored with an Ingold polarographic probe (available from Mettler-Toledo Ingold Inc., Bedford, MA, USA) and maintained no lower than 30% air saturation with an aeration rate of 0.8 vvm (void volumes per minute) and an agitation speed up to 900 rpm.
- Nonanoic acid was fed separately at controlled speed by a peristaltic nutrient pump on the bioreactor. All other aqueous medium was mixed and fed together.
- the dilution rate of all continuous fermentations was controlled at 0.25 h ' by fixing the working volume and a constant aqueous medium feeding rate.
- microbial cells grown according to methods of the present invention as a source of polymer wherein cellular material is diluted and/or destroyed in subsequent process steps.
- Known amount of dried biomass, or purified MCL-PHA, or MCL-PHA standard with known composition and elution time of each of its components were reacted in 2 mL chloroform plus 1 mL methanol containing 15% (v/v) concentrated sulfuric acid as acidifying agent and 0.2% (w/v) benzoic acid as internal standard, at 100°C for 4 hours. After the reaction, 1 mL distilled water was added and the sample was mixed vigorously by vortex mixer for 1 min and phase separation was allowed to take place.
- DSC Differential scanning calorimetry
- T g glass transition temperature
- T m melting temperature
- ⁇ relative crystallinity expressed as heat of fusion
- TA Instruments DSC Q100 available from TA Instruments, New Castle, DE, USA.
- Purified MCL-PHA samples were melted at 70°C and allowed to crystallize at 25°C for 5 days before being analyzed.
- a sample of known mass was placed in a special aluminum pan and sealed. The sample was first heated from -70°C up to 80°C at a rate of 10°C/min. During this heating, an endothermic peak was observed and the temperature value at the peak point was taken as the T m .
- Pseudomonas putida KT2440 (ATCC 47054). Pseudonomas putida KT2440 (ATCC 47054) was maintained on nutrient agar plates at 4°C before inoculation. The toxicity of a ⁇ -oxidation inhibitor, acrylic acid, to the growth of Pseudomonas putida KT2440 was determined by shake flask experiment. The cultivation conditions were 28°C in a New Brunswick Shaker-Incubator at 200 rpm. The medium used was the basic gluose-only shake flask medium described above, plus appropriate amount of acrylic acid.
- MCL-PHAs with controlled composition were produced in continuous fermentation of Pseudomonas putida KT2440 by increasing the concentration of acrylic acid.
- the inoculum was grown in the basic glucose medium described above. After 18 h of cultivation, 200 mL of such culture was inoculated into a 1.5 L bioreactor with 1.1 L working volume (Bioflo II, New Brunswick Scientific, New Jersey, USA). The bioreactor was then operated in continuous fermentation mode with a medium dilution rate of 0.25 h "1 .
- the inflow medium was composed of the following components (per litre): carbon source, nonanoic acid (3.80 g ⁇ 0.1 g for each steady state); ⁇ -oxidation inhibitor, acrylic acid (0, 0.02, 0.05, 0.10, 0.20 g for each steady state); nutrients (NH 4 ) 2 S0 4 (5.02 g); MgS0 4 « 7H 2 0 (1.03 g); Na 2 HP0 4 -7H 2 0 (2.24 g); KH 2 P0 4 (0.50 g); and trace element solution (1.40 mL).
- the trace element solution was composed of the following components (per litre): FeS0 « 7H 2 0 (10.00 g); CaCI 2 -2H 2 0 (3.00 g); ZnSCv7H 2 0 (2.20 g); MnSCv4H 2 0 (0.50 g); H 3 BO 3 (0.30 g); CoCI 2 « 6H 2 0 (0.20 g); Na 2 MoCv2H 2 0 (0.15 g); NiCI 2 « 6H 2 0 (0.02 g); and CuSCv5H 2 0 (1.00 g).
- Five different bacterial physiological steady states were established at each acrylic acid increment by allowing at least 5 reactor working volumes of medium flow through the bioreactor and by monitoring the carbon dioxide production rate to reach constant value. Sample was then taken at such steady state and analyzed. See the data from these studies in Figures 2A and 2B.
- MCL-PHAs obtained from nonanoic acid as the only carbon source were always composed of C 9 (3-OH-nonanoate) and C 7 (3-OH-heptanoate) monomers but their relative fraction could be controlled by increasing inhibitor concentration.
- Example 3 Preparation of MCL-PHAs with controlled composition prepared in the presence of a structurally-related carbon source (nonanoic acid), a structurally-unrelated carbon source (glucose), and increasing concentration of ⁇ -oxidation inhibitor (acrylic acid) using continuous fermentation
- MCL-PHAs with controlled composition were produced in continuous fermentation of Pseudomonas putida KT2440 by providing both nonanoic acid and glucose as carbon sources, and by increasing the concentration of acrylic acid.
- the operation of the continuous fermentations was similar to that described in Example 2.
- the inflow medium was composed of the following components (per litre): carbon source, nonanoic acid (2.90 ⁇ 0.15 g for each steady state); glucose (3.90 g for each steady state); ⁇ -oxidation inhibitor acrylic acid (0, 0.02, 0.05, 0.10, 0.20 g for each steady state); nutrients; and trace element solution (1.40 ml_).
- MCL-PHAs with controlled composition were produced in continuous fermentation of Pseudomonas putida T2440 by providing both octanoic acid and glucose as carbon source, and by increasing the concentration of acrylic acid.
- the operation of continuous fermentations was similar to that described in Example 2.
- the inflow medium was composed of the following components (per litre): carbon source, octanoic acid (3.60 g ⁇ 0.16 g for each steady state); glucose (3.90 g for each steady state); ⁇ -oxidation inhibitor acrylic acid (0, 0.02, 0.05, 0.10, 0.20 g for each steady state); nutrients; and trace element solution (1.40 ml_).
- Example 5 Preparation of MCL-PHAs with controlled composition prepared in the presence of a structurally-related carbon source (nonanoic acid) and a structurally-unrelated carbon source (glucose), and in the absence of ⁇ - oxidation inhibitor (acrylic acid) using fed-batch fermentation
- MCL-PHA was produced in fed-batch fermentation by providing nonanoic acid as MCL-PHA-generating carbon source and glucose as energy- providing carbon source, without providing acrylic acid as ⁇ -oxidation inhibitor.
- the inoculum was grown in the basic glucose medium described above. After 18 h of cultivation, 300 mL of such culture was inoculated into a 5 L bioreactor with 3 L working volume (Minifors, INFORS HT, Bottmingen, Switzerland). The fermentation was then operated at fed-batch mode with programmed nonanoic acid and glucose feeding rate that controlled the culture specific growth rate at 0.25 h "1 .
- the medium was composed of the following components (per litre): nonanoic acid and glucose (1 : 1 (w/w) fed at a programmed rate); nutrients; and trace element solution (10.00 mL).
- Nutrients included (NH 4 ) 2 S0 4 (4.70 g); MgSCv7H 2 0 (0.80 g); Na 2 HPCv7H 2 0 (12.00 g); and KH 2 P0 (2.70 g).
- the fermentation was operated at 28 °C and the pH was controlled by feeding ammonium water. Results that were obtained after 28 hours fermentation are presented in Figures 5A and 5B.
- Example 6 Preparation of MCL-PHAs with controlled composition prepared in a fed-batch fermentation in the presence of a structurally-related carbon source (nonanoic acid), a structurally-unrelated carbon source (glucose), and ⁇ - oxidation inhibitor (acrylic acid) using fed-batch fermentation
- a structurally-related carbon source nonanoic acid
- a structurally-unrelated carbon source glucose
- ⁇ - oxidation inhibitor acrylic acid
- MCL-PHA with controlled composition was produced in fed-batch fermentation by providing nonanoic acid as MCL-PHA-generating carbon source and glucose as energy-providing carbon source, and by providing acrylic acid as ⁇ - oxidation inhibitor.
- the setup of the fermentation was similar to that of Example 5.
- the weight ratio of nonanoic acid, glucose and acrylic acid in the feeding was 1.25:1 :0.03.
- the bioreactor was operated in fed-batch fermentation mode with programmed carbon source feeding rate such that the culture specific growth rate was 0.15 h "1 .
- the medium was composed of the following components (per litre): nonanoic acid, glucose and ⁇ -oxidation inhibitor acrylic acid were all fed at
- Poly(3-OH-nonanoate-co-3-OH-heptanoate) (PHN) of varied monomeric compositions were produced by methods described in Example 3. After the MCL-PHA samples were purified and annealed for certain period of time, the samples' melting behavior was recorded using Differential Scanning Calorimetry (DSC) with a heating profile from -70°C to 80°C at 5°C per min. Results are presented as up-heating curves, which are overlaid in Figure 7.
- PHN-70 is the abbreviation of poly(3-OH- nonanoate-co-3-OH-heptanoate) with 70 mol % nonanoate and 30 mol % heptanoate.
- Tm, °C melting temperature
- HN 3-hydroxy-nonanoate
- Poly(3-OH-octanoate-co-3-OH-hexanoate) (PHO) of varied monomeric compositions were produced by methods described in Example 4. After the MCL-PHA samples were purified and annealed for certain period of time, their melting behavior was recorded using Differential Scanning Calorimetry (DSC). The heating profile was from -70 °C to 80 °C at 5 °C per min. The up-heating curves are overlaid in Figure 8.
- PHO-88 is the abbreviation of poly(3-OH-octanoate-co-3-OH-hexanoate) with 88 mol % octanoate and 12 mol % hexanoate.
- Tm, °C melting temperature
- HO 3-hydroxy-octanoate
- Example 9 Stress-strain measurements of various PHN and PHO samples
- dumbbell-shaped specimens were then used to produce dumbbell-shaped specimens according to ASTM D638 Type V dimension (American Society for Testing and Materials International (ASTM) Standard D638, 2004, “Standard Test Method for Tensile Properties of Plastics," ASTM International, West Conshohocken, PA, 2004, DOI: 10.1520/C0033-03, www.astm.org.).
- the width of the narrow part of the sample was about 3.3 mm, while the thickness depended on the thickness of the original sample sheet, which was between 1.3 mm and 2.7 mm. Stress-strain measurements of these dumbbell-shaped specimens were obtained using a tensile testing instrument (model Instron 3369, available from Instron, Norwood, MA, USA).
- the Instron tensile test used a crosshead speed of 100 mm per min. Properties such as annealing time (h), tensile stress at maximum load (MPa), Young's modulus (MPa), and extension at break (%) of various samples are summarized in Table 2.
- Example 10 Preparation of MCL-PHAs with controlled composition prepared in the presence of a structurally-related carbon source (dodecanoic acid), a structurally-unrelated carbon source (glucose), and increasing concentration of ⁇ -oxidation inhibitor (acrylic acid) using continuous fermentation
- MCL-PHAs with controlled composition were produced in continuous fermentation of Pseudomonas putida KT2440 by providing both dodecanoic acid and glucose as carbon source, and by increasing the concentration of acrylic acid.
- the operation of continuous fermentations was similar to that described in Example 2.
- the inflow medium had the following components (per litre): carbon source, dodecanoic acid (1.14 g for each steady state); glucose (3.00 g for each steady state); ⁇ -oxidation inhibitor acrylic acid (0, 0.10, 0.20 g for each steady state); nutrients; and trace element solution (1.40 ml_).
- the nutrients included (NH 4 ) 2 S0 (5.02 g); MgS0 4 » 7H 2 0 (1.03 g); Na 2 HP0 4 « 7H 2 0 (2.24 g); and KH 2 P0 4 (0.50 g).
- results demonstrated that, by providing both a structurally-related carbon source such as dodecanoic acid and a structurally-unrelated carbon source such as glucose, and by providing acrylic acid as ⁇ -oxidation inhibitor, MCL-PHA monomeric composition could be controlled.
- a structurally-related carbon source such as dodecanoic acid
- a structurally-unrelated carbon source such as glucose
- acrylic acid as ⁇ -oxidation inhibitor, MCL-PHA monomeric composition could be controlled.
- the inlet concentration of acrylic acid was increased from 0 to 0.1 to 0.2 g L "1
- molar C12 content increased from 20.4 % to 34.5 % to 59%.
- Example 11 Preparation of MCL-PHAs with controlled composition prepared in the presence of a structurally-related carbon source (nonanoic acid), a structurally-unrelated carbon source (glucose), and increasing concentration of ⁇ -oxidation inhibitor (acrylic acid) using continuous fermentation
- MCL-PHAs with controlled composition were produced in continuous fermentation of Pseudomonas citronellolis DSM 50332 (ATCC 13674) by providing both nonanoic acid and glucose as carbon source, and by increasing the concentration of acrylic acid.
- the operation of continuous fermentations was similar to that described in Example 2.
- the inflow concentration of ⁇ -oxidation inhibitor acrylic acid was varied to be 0, 0.01 , 0.02, 0.025, 0.030, 0.035, 0.040 g for each steady state.
- the MCL-PHAs synthesized at any steady state comprised 3-OH-nonanoate and 3-OH-heptanoate (C 9 and C 7 ).
- the amount of 3-OH-nonanoate in the MCL-PHA product was successfully controlled.
- Compositions comprising from 65 mole % to 76 mole % 3-OH- nonanoate were obtained for the range of acrylic acid tested, with the remaining mole % being 3-OH-heptanoate.
- R 2 are side chains, and where x and y
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Abstract
La présente invention concerne un procédé pour produire un poly(3-hydroxyalcanoate) à longueur de chaîne moyenne (MCL-PHA) avec un rapport choisi de monomères. La présente invention concerne en outre un procédé de contrôle du rapport de monomères dans des MCL-PHA, qui consiste à assurer la fermentation de micro-organismes d'origine naturelle avec un substrat d'acide gras, une source d'aliment et un additif. Les procédés décrits ne sacrifient pas la croissance et la maintenance cellulaire et présentent des rendements élevés de MCL-PHA. La présente invention concerne en outre des MCL-PHA qui sont des copolymères de monomères en Cn et Cn-2, où (n est 6 à 18).
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EP10835329.3A EP2510037A4 (fr) | 2009-12-07 | 2010-12-03 | Polymère de polyhydroxyalcanoate à longueur de chaîne moyenne et procédé de fabrication de celui-ci |
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KR101112590B1 (ko) | 2009-07-27 | 2012-03-13 | 경상대학교산학협력단 | 폴리하이드록시 알칸산의 제조방법 |
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Also Published As
Publication number | Publication date |
---|---|
US20110166318A1 (en) | 2011-07-07 |
EP2510037A4 (fr) | 2014-12-17 |
EP2510037A1 (fr) | 2012-10-17 |
CA2781699C (fr) | 2015-01-27 |
US8273852B2 (en) | 2012-09-25 |
CA2781699A1 (fr) | 2011-06-16 |
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