WO2009156950A2 - Procédés de production de polyhydroxyalcanoates à chaîne moyenne utilisant des huiles végétales comme source de carbone - Google Patents

Procédés de production de polyhydroxyalcanoates à chaîne moyenne utilisant des huiles végétales comme source de carbone Download PDF

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WO2009156950A2
WO2009156950A2 PCT/IB2009/052695 IB2009052695W WO2009156950A2 WO 2009156950 A2 WO2009156950 A2 WO 2009156950A2 IB 2009052695 W IB2009052695 W IB 2009052695W WO 2009156950 A2 WO2009156950 A2 WO 2009156950A2
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phas
monomeric units
poli
biomass
saccharide
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WO2009156950A3 (fr
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Fermín PÉREZ GUEVARA
María del Rocío LÓPEZ CUELLAR
Jorge Noel GRACIDA RODRÍGUEZ
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Centro De Investigación Y De Estudios Avanzados Del Instituto Politécnico Nacional
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    • 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
    • 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

Definitions

  • the present invention relates to biological methods for producing biodegradable plastics, specifically to fermentation methods for producing medium chain poly-3- hydroxyalkanoates (PHAs) by means of growing micro-organisms using vegetable oils as carbon source.
  • PHAs medium chain poly-3- hydroxyalkanoates
  • Plastics of chemical origin have versatile qualities, being resistant to degradation and simultaneously light, which has caused them to become an essential input for most of the industries. Nevertheless, their useful life time of a most of them is very short; many are used to produce bottling and baling materials, provoking their rejection by the environment and causing serious problems of environmental contamination such as the accumulation of non degradable plastics which, once incinerated, provoke greenhouse gas emission to the environment (methane, nitrous oxide and carbon dioxide).
  • biodegradable plastics are a viable option. In this sense, poly-3-hydroxyalkanoates (PHAs) represent the best alternative given their structural characteristics and their compatibility with the environment.
  • PHAs are polymers 100% biodegradable, thermoplastic, elastomer, insoluble in water, not poisonous and biocompatible. These kinds of polyster have characteristics similar to those of the polyethene and polyprophylen, and therefore they can be used instead of conventional plastics. Likewise they are degraded completely in aerobic and anaerobic conditions by soil, sea, lake water and residual waters microorganisms 1 ' 2 . PHAs are classified based on the number of carbon atoms in their monomehc units dividing them in two groups, short chain PHAs (PHA SC ⁇ ) with 3-5 carbon atoms and medium chain (PHA mC ⁇ ) with 6-14 carbon atoms. The chemical structure of PHAs can be observed in formula 1 :
  • n 600 to 35,000.
  • table 1 we can observe the name of the generated PHA when R is replaced by the radical indicated in its monomeric unit.
  • the PHAs are biodegradable and have thermal and mechanical properties similar to thermoplastics such as polyethylene and polyprophylene, therefore they have a big potential in medical and industrial applications 3 .
  • the PHAs family has a wide variety of mechanical properties as the incorporation of monomeric units of 6 to 14 carbons results in a major molecular weight and improves its physical characteristics since they are semicrystalline elastomers with a low melting point, a low tension force and a high extension to rupture, in addition of having a major biodegradability 4 .
  • PHAs of medium chain have a much lower percentage of crystallinity and they are more elastic, since their resistance to impact increases whereas the Young module diminishes, which gives them a wider range of applications compared to PHAs of short chain 5 (see table 2).
  • Table 2 PHAs and polyprophylene properties
  • PHAs are non toxic, biocompatible and biodegradable plastics which be produced from renewable resources. It has been reported that they have a high grade of polymerization, with a grade of crystallinity in the interval of 60 to 80%, they are active optically and isotactic (that is to say, regularly stereochemical in repeated units), piezoelectric and insoluble in water. These characteristics make them highly competitive against polyprophylene and plastic derivatives of oil 3 ' 6 .
  • PHAs initially were used in packaging film mainly in bags, containers and paper packings 6 . These films can also be used to make sheets together with other polymers such as polyvinilic alcohol 5 . Other applications of PHAs it is to use them as implements, hygienic feminine products, cosmetic containers and shampoo packing. In addition to their potential as plastic material, PHAs are also used as stereo regular components that can serve as chiral precursors for the chemical synthesis of optically active components 6 . Also they are used as biodegradable carriers for the dosage of medicines, hormones, insecticides and herbicides, and as synthetic bone materials in the growth stimulation of bones, due to its piezoelectric properties, in bone badges, surgical structures and replacement in blood vessels 3 6 .
  • PHAs are hydroxyalkanoates polyesters synthesized by at least 75 different bacteria genus including both Gram negative and Gram positive, as for example Wautersia eutropha, Bacillus megaterium, Klebsiella aerogenes, Pseudomona putida, P. oleovarans, Sphaerotilus natans, among others (see table 3). These polymers intracellular ⁇ accumulate in the bacterium as inclusions under stress conditions, such as the limitation of phosphorus, nitrogen, oxygen and a non optimum pH culture medium, to the point that PHAs can represent up to 90% of the dry weight of the dry biomass obtained in the fermentation 3 .
  • the PHAs produced by bacteria have showed a sufficiently high molecular weight as to asume characteristics similar to those of conventional plastics, such as polypropylene. It is known that the PHAs produced by microorganisms can have a variety of compositions and structures depending on the type of microorganism used for their production, as well as the composition of culture media and culture conditions.
  • Wautersia eutropha H16, ATCC no. 17699 and its mutant strains produce copolymers of 3-hydroxybutyrate acid (3HB) and 3- hydroxyvaleric acid (3HV) in diverse proportions when the sources of carbon change in the culture. It has been described 7 ' 8 , the production of binary copolymers of 3HB and 3- hidroxyhexanoic acid (3HH) by means of the culture of Aeromonas caviae with oleic acid or olive oil as carbon source.
  • Baanaado 9 describes the production of PHAs, having as monomeric units 3- hydroxyalkanoate, units from 6 to 12 carbons using as carbon source aliphatic hydrocarbur(s) and using Pseudomonas oleovorans ATCC 29347. It has been described 10 that Pseudomonas resinovorans produce a polyester with monomeric units of 3-hydroxybutyhc acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid in a ratio of 1 :15:75:9 using octanoic acid as the only carbon source.
  • Polyester also is produced with 3-hydroxybutyhc acid, 3- hydroxyhexanoic acid, acid 3-hydroxyoctanoic and 3-hydroxydecanoic acid units in a quatitative ratio of of 8:62:23:7 using hexanoic acid as the only source of carbon. It has been described 11 that Pseudomonas sp.
  • strain 613 produces a polyester of 3- hydroxyalkanoic acids, such as 3-hydroxybutyhc acid, 3-hydroxyhexanoic acid, 3- hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid, as well as of 3-hydroxyalkenoic acids, such as 3-hydroxy-5-cis-dodecanoic acid and 3-hydroxy- 5-cis-dodecanoic acid, using sodium gluconate as the only carbon source.
  • 3- hydroxyalkanoic acids such as 3-hydroxybutyhc acid, 3-hydroxyhexanoic acid, 3- hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid
  • 3-hydroxyalkenoic acids such as 3-hydroxy-5-cis-dodecanoic acid and 3-hydroxy- 5-cis-dodecanoic acid, using sodium gluconate as the only carbon source.
  • Wautersia eutropha (before Ralstonia eutropha) is a Gram negative bacteria, aerobic obligated, chemo-organotrophic, capable of accumulating from 8 to 13 granules with PHAs in the cell with a diameter that ranges from 0.2 to 0.5 ⁇ m.
  • the hydrophobic inclusions, suspended in the cell cytoplasm contain 5-10% in water weight and they are largely in an amorphous state. After a cellular destruction, when the PHA is extracted, a crystallization happens rapidly.
  • the polyhydroxybutyrate (PHB) it is the more widely studied, which has a molecular weight in an interval of 10 4 - 10 6 Da and a polydispersity of 2. It presents a vitreous transition temperature (Tg) of -4°C, whereas the melting temperature appears at 180 0 C, similar to that of the polyprophylene; nevertheless it presents an extension percentage to rupture of only 5%. Its relatively high melting point near to its thermal decomposition temperature, determines its limits of processabilitly.
  • the price of PHA depends on the cost of the substratum, yield and the efficiency of the process; this involves high levels of PHAs as percentage in dry weight and high productivity, in terms of gram of product per unit of time volume 6 , therefore improvements in the PHA yields and the substratum selection, impact the costs of the process.
  • Choice of a suitable carbon source is an important factor in the optimization of the PHAs production.
  • the nature of the carbon source not only determines the content of PHA, but also the composition, which subsequently affects the final properties of the polymer.
  • 80% corresponds to the carbon source used, therefore the price of PHAs can be decreased by using cheap substrates.
  • simple sources of carbon as glucose or fructose are used, in addition to organic volatile acids such as acetic, propionic and butyric acids.
  • valeric acid it is possible to obtain a copolymer (PHB-HV) in different proportions 12 .
  • Pradella 13 describes a production process of short chain PHAs using soybean oil or the greasy acids originated by the latter, palm, cotton, corn, sunflower or castor seed oils like carbon source.
  • the process consists of four stages, for which use the bacterial strains Wautersia eutropha IPT026 or IPT027 and the strains of Burkholderia cepacia IPT048, 064, 066, 074, 110 or 189.
  • the stage of growth of the biomass (stage II) is carried out by means of a feedback system (feed batch).
  • the weight fraction of PHAs in the cells obtained by means of this method is 90% of dry biomass with a concentration of bacterial biomass of 200 g/L of fermentation medium and a conversion of carbon mass to PHA mass of 0.6 Kg/Kg.
  • Bright 14 for example describes the production of PHAs by means of cells of plants producing vegetable oils, canola, soybean and sunflower that contain one or several genes of enzymes which are involved in the PHAs biosynthetic pathway of Wautersia eutropha, which synthesizes PHA in a natural way. It is important to notice that in order carry out this type of process it is necessary to use recombinant DNA techniques. There exist diverse and multiple works describing processes for the obtention of PHAs with very specific characteristics, by means of recombinant DNA techniques.
  • Nagaoka 15 describes the creation of a new strain of Wautersia eutropha to produce polyesters particularly containing monomeric P(3HB-co-3HH) units by means of inserting the gene of the enzyme polyhydroxyalkane acid synthetase, which participates in the PHAs synthesis, obtaining thereby a PHA with special characteristics regarding its hardness and flexibility.
  • this type of methodologies increase the price of PHAs production, while on the other hand with the manipulation of fermentation methods and the energy supply to PHAs producing microorganisms, the above mentioned polymers with the desired characteristics, can be obtained at lower costs.
  • the synthesis of medium chain PHAs requires organic acids as substrates such as octanoic, dodecanoic, lauric and mihstic acids that can incorporate monomeric units of 8 up to 14 carbons due to the different carbon content of each acid 16 . Nevertheless the cost of purification of these acids can be high, which affects the production costs of PHAs when using them as carbon source in fermentation processes.
  • Wautersia eutropha bacteria to obtain PHAs is of great use, the only way to make it grow is by using such carbon sources as glucose, propionate, valerate, methanol, sucrose and prophanol to obtain only short chain copolymers of hydroxyalkanoates as PHB-HV 17 .
  • one of the objectives of the present invention is to provide methods for the production of medium chain PHAs by means of microorganisms grown in culture media containing vegetable oils as carbon source.
  • Another objective of the invention is to provide methods for the production of medium chain PHAs by means of microorganisms grown in culture media containing vegetable oils and fructose as carbon sources.
  • Another objective of the invention is to provide methods for the production of medium chain PHAs by means of the bacteria Wautersia eutropha grown in culture media containing canola oil and fructose as carbon sources.
  • Figure 1 Shows the development of total biomass (X), residual biomass (Xr) and PHAs in cultures performed in flasks using canola oil as carbon source. A cellular growth is observed with fructose (A) and the addition of vegetable oil (B).
  • Figure 2 Shows the development of total biomass (X), residual biomass (Xr) and PHAs in cultures performed in a bioreactor with canola oil as the only carbon source. Batch culture (A), fedbatch culture (B) and PHAs production (C) are observed.
  • Figure 3. Shows the development of fructose concentration during fermentation using canola oil as the only carbon source. Batch culture (A), fedbatch culture (B) and PHAs production (C) are observed.
  • Figure 4. Shows the ammonium concentration profiles during fermentation using canola oil as the only carbon source. Batch culture (A), fedbatch culture (B) and PHAs production (C) are observed.
  • Figure 5. Shows the concentration profile of canola oil in phase 3 of the fermentation with canola oil.
  • Figure 6. Shows the concentration profiles of total biomass (X), residual biomass (Xr) and PHAs using canola oil and fructose as carbon sources. Batch culture (A), fedbatch culture (B) and PHAs production (C) are observed.
  • Figure 7. Shows the concentration profile of fructose during fermentation using oil and fructose as carbon sources. Batch culture (A), fedbatch culture (B) and PHAs production (C) are observed.
  • Figure 8 Shows the concentration profile of oil during phase 3 of fermentation with canola oil and fructose.
  • FIG. 9 Shows the concentration profile of ammonium during fermentation using oil and fructose as carbon sources. Batch culture (A), fedbatch culture (B) and PHAs production (C) are observed.
  • FIG. 10 Shows the MNR 13 C (A) and MNR 1 H (B) spectra of polyhydroxybutyrate (PHB).
  • Figure 11 Shows the MNR 13 C at 500 MHz spectrums of the obtained PHAs by the invention method, using canola oil as the only carbon source (A) or canola oil and fructose as carbon sources (B). Hydroxybutyrate (HB), hydroxyvalerate (HV), hydroxyoctanoate (HO) and hydroxydodecanoate (HDD) are clearly seen.
  • Figure 12 Shows the MNR 1 H at 125 MHz spectrums of the obtained PHAs by the method of the invention, using canola oil as the only carbon source (A) or canola oil and fructose as carbon sources (B).
  • Figure 13 Shows the FT-IR spectrum in the region 4000 to 650 cm "1 of the PHAs obtained by the method of the invention, using canola oil and fructose as carbon sources (a), using canola oil as the only carbon source (b) and
  • PHAs by the method of the invention, using canola oil and fructose as carbon sources (a), using canola oil as the only carbon source (b) and
  • FIG. 15 Shows the FT-IR spectrum in a region 1850 to 1600 cm “1 of the PHAs obtained by the method of the invention, using canola oil and fructose as carbon sources (a), using canola oil as the only carbon source (b) and PHB (C).
  • FIG 16. Shows the thermograms of the PHAs obtained by the method of the invention, using canola oil and fructose as carbon sources (A), using canola oil as the only carbon source (B) and PHB (C). Detailed description of the invention.
  • the present invention provides efficient and low cost fermentation methods to produce medium chain PHAs comprising the growth of microorganisms in culture media containing vegetal oils as substrate or vegetal oils and fructose.
  • efficient and low cost fermentation methods can be achieved using vegetal oils as carbon source which contain a large variety of fatty acids in their composition, e.g. canola oil is basically constituted by seven fatty acids in different proportions; typical composition is shown in table 4.
  • the present invention provides PHAs copolymers with monomeric units of medium chain produced by microorganisms at very low cost. Such copolymers can be useful in the industry due to the physical characteristics.
  • the PHAs of the present invention contain monomeric units with more than five carbons that those previously obtained by means of microorganisms using vegetable oils as carbon sources or with other carbon sources.
  • the PHAs of the present invention exhibit melting temperatures of 13O 0 C to 15O 0 C, more convenient than known those of PHAs obtained by present industrial processes where melting temperature is an important factor, as for example in molding processes at medium temperatures.
  • the present invention provides methods to produce medium chain PHAs comprising the culture of one or more microorganism capable of producing PHAs in such conditions that PHAs could be accumulated, for example by means of limitation of some nutrient in the culture medium, for example, nitrogen, oxygen or a source of phosphorus, which are needed for growing the microorganism but not for the accumulation of PHAs.
  • the process of the invention also comprises an earlier stage to the production of PHAs that allows the build up of biomass. This stage makes possible to increase the yields of medium chain PHAs, which can reach at least 90% of the dry weight of the biomass obtained in the fermentation.
  • the microorganism is cultured in a process comprising three stages (batch, fedbatch, and PHAs production) in a medium added with vegetable oil prior or within the PHAs production stage where the microorganism produces and accumulates polymers which comprises the monomeric units PHB, PHV, PHO and PHDD.
  • the polymers formed contain at least 5% of medium chain monomeric units, i.e. PHO and PHDD.
  • the microorganism which may be used in the method of the invention are those capable of producing short chain PHAs, which are selected from the group comprising the genera Wautersia, Klebsiella, Zoogloea, Bacillus, Aeromona, Azotobacter, Clostridium, Nocardia, Halobacterium, Burkholderia, Sphaerotilus y Pseudomonas, although bacteria within the genus Wautersia are preferred to use.
  • the essential nutrients required for the microbial growth comprise elements, which are normally present in the culture medium in an easily assimilable way, such as nitrogen, phosphorus, sulfur, potassium, sodium, magnesium, calcium and iron, together with traces of manganese, zinc and copper.
  • saccharides can be used as carbon source, which are selected of the group comprises arabinose, glucose, mannose, fructose, galactose, sorbitol, manitol and inositol, although it preferred to use fructose.
  • nitrogen sources chosen from the group comprising ammonia, ammonium salts, as for example ammonium sulphate, nitrates and/or organic nitrogen contained in such compounds as urea, casein, peptone, yeast extract and meat extract.
  • the carbon source may be used in such a quantity to generate the medium chain PHAs of the invention but without inhibit the growth of the microorganism.
  • Concentrations within the range of 2 to 50 g/L are generally used, but preferably within the range of 8 to 12 g/L are used in the culture medium.
  • the nitrogen source this can be used in concentrations within the range of 0.2 to 20 g/L, preferably of 1 to 2 g/L in the culture medium.
  • concentrations within the range of 40 to 80 g/L are generally used, but preferably within the range of 50 to 80 g/L are used in the culture medium.
  • the culture temperature can be 2O 0 C to 35 0 C, preferably 31 0 C, whereas the pH can range 6.5 to 8, preferably 6.7 to 7.5.
  • the recovery of the PHAs of the present invention can be achieved by any suitable method known in the art. Generally the cells containing PHAs can be separated from the fermentation broth by centrifugation, subsequently broken and afterwards the PHAs extracted with an organic solvent, preferably chloroform.
  • the culture medium can be varied in the quantity and sources of the different elementary nutrients, but should provide the necessary nutrients for growth of the microorganisms and the production of the PHAs of the invention.
  • methods are provided to culture such microorganisms in culture media which supply all the required essential nutrients, that is, the nutrients are present in such a concentration to allow the growth of the PHAs producing microorganism.
  • the method of the present invention aims to the production of PHAs with better thermal and mechanical properties, by means of two strategies, each of them comprising fermentations of three stages.
  • First stage Batch culture.
  • the microorganism is grown in batch culture in presence of at least one saccharide as carbon source in a concentration from 2 to 18 g/L, preferably of 8 to 12 g/L.
  • the growth takes place in presence of at least one nitrogen source, in a concentration of 0.5 to 10 g/L, preferably from 1 to 5 g/L.
  • the fermentation continues up to exhaustion of the saccharide in the culture medium until reaching a consumption of at least 80% of its initial concentration.
  • Second stage Fedbatch culture.
  • the cell biomass is increased in the broth by the controlled addition of one or more substrates to the culture (fedbatch culture).
  • the same saccharide is added as carbon source to the culture in such a way that its maximum concentration does not exceed 5 g/L, preferably up to 2 g/L in the fermentation broth.
  • the total biomass increases at least 130% with regard to the total biomass obtained at the end of the first stage.
  • the nitrogen source is added in such a way that it's maximum concentration should not exceed 0.91 g/L.
  • the biomass grows quickly (see figure 2), stopping its growth when the addition of nitrogen source is stopped; likewise the saccharide is used for microbial growth at this stage; therefore the microorganism does not use the saccharide for PHAs production.
  • Medium chain PHAs are produced by adding vegetal oil as the only carbon source to the culture.
  • the concentration of the nitrogen source in the culture medium is below 0.5 g/L; therefore the microorganism encounters nitrogen limitation, such condition induce the production of PHAs from the only carbon source available in the culture (vegetable oil).
  • the accumulation of medium chain PHAs is approximately 90% of the biomass dry weight. Therefore, vegetable oil is an excellent carbon source for the production of medium chain PHAs.
  • the PHAs are obtained from the microbial cells by means of the extraction methods mentioned above.
  • the PHAs obtained with this method incorporate into their structure at least 4% of medium chain monomers, preferably at least 5% of medium chain monomers.
  • Fedbatch fermentations are commonly used to reach a high cellular density, at high productivity, while inhibition by substrate is avoided or to induce some kind of nutrient limitation to stimulate the synthesis of microbial products.
  • the reaction volume increases progressively in the fermenter.
  • the concentration of the different components in the culture medium changes continuously in the broth, as a result of volume changes, but also by the consumption of nutrients by the cells and the continuous feeding of nutrients during the fedbatch stage.
  • This stage allows the growth of the PHAs producing microorganism in the presence of at least a saccharide as carbon source and at least a nitrogen source. During this stage the microorganism consumes at least 80% of carbon and nitrogen sources initially supplied to the culture medium.
  • the saccharide is added gradually to the culture up to reach a bulk concentration of 30 to 50 g/L and the nitrogen source up to 0.2 g/L (fedbatch culture).
  • the total biomass generated in this stage is increased in at least 400% with respect to the biomass obtained in the first stage. As it can be observed, during this phase the carbon and nitrogen sources added are used solely for the generation of total biomass.
  • the PHAs are obtained from the microbial cells by means of the mentioned extraction methods previously.
  • the saccharide is preferably fructose whereas canola oil and fructose are the preferred carbon sources for the production stage of
  • the PHAs obtained with this method incorporate in their structure at least 8% of medium chain monomers, preferably at least 10% of medium chain monomers.
  • the carbon and nitrogen sources are continuously supplied to the culture medium during the second stage, approximately at
  • the methods carried out in a bioreactor generate a biomass containing up to 90% dry weight of PHAs. Consequently, allow high yields at low production costs in reasonable time.
  • medium chain PHAs that incorporate in their structure from 4 to 10% of medium chain monomers, preferably with 6 to 12 carbons in their monomeric unit.
  • PHAs having different monomers can be obtained, preferably monomers with 4 to 12 carbons in their monomeric unit, and more preferably monomers selected from the group comprising PHB, PHV, PHO and PHDD.
  • the mentioned monomers in different proportions are components of the PHAs of the present invention, particularly the medium chain monomers (PHO and PHDD) are found in proportions from 4 to 10%, while the short chain monomers (PHB and PHV) form the remaining percentage.
  • the PHAs of the invention are polymers formed by diverse monomeric units and which have the structure of formula 1 , where R is an alkyl group of 1 to 9 carbons, where preferably monomeric units form the PHAs where R is methyl, ethyl, penthyl and nonyl.
  • the PHAs of the invention contain repeated monomeric units of formula 2 which comprise 4 to 10% of monomeric units where R is an alkyl group of 3 to 9 carbons and of 90 to 95% of monomeric units where R is an alkyl group of 1 to 2 carbons.
  • the PHAs of the present invention contain 4 to 10 % of medium chain monomeric units, where they are preferably formed by monomeric units where R is penthyl and nonyl, that is, 3-hydroxyoctanoate and 3-hydroxydodecanoate; whereas the remaining percentage is formed by short chain monomeric units, where they are preferably formed by monomeric units where R is methyl and ethyl, that is, 3- hydroxybutyrate and 3-hydroxyvalerate.
  • the methods of the invention allow obtaining medium chain PHAs with high quality and better physical-chemical characteristics, as a result of the incorporation of 4 to 10% of monomeric units having 6 to 12 carbons in the polymeric structure of the PHAs.
  • the following examples illustrate the present invention in its preferred form, but these are given without the intention of limiting its scope.
  • Example 1 PHAs production using vegetable oils as substrates.
  • a flask batch culture was used for probe the production of PHAs, using fructose as carbon source for bacterial growth and the later addition of diverse vegetable oils to the culture medium.
  • An inoculum of Wautersia eutropha ATCC 17699 was prepared in a Erlenmeyer flask of 200 ml_ with 100 ml_ of modified Luha-Bertani liquid medium (LB). Inoculum development was at 200 rpm and 30 0 C. Said inoculum was later added to 250 ml_ flasks containing 50 ml_ of production medium.
  • LB Luha-Bertani liquid medium
  • the flasks were incubated at 200 rpm and 3O 0 C.
  • a culture sample was taken every 2 h, adding 0.250 mL of oil to every flask at 15 h.
  • the concentration of the residual biomass (Xr) and of PHAs before adding the oil tested was respectively 4.9 and 1.4 g/L, whereas the resulting concentration at the end of the fermentation process is shown in table 5.
  • Table 5 shows that the PHAs concentration was approximately 2 times bigger when canola oil was used as oil source (11 g/L) compared to the PHAs production obtained with soy, olive or corn oil (5-6 g/L), whereas it was 3 times greater compared to the
  • Example 2 PHAs production at bioreactor level using canola oil as substrate.
  • canola oil was chosen to obtain PHAs in a bioreactor using two different production strategies. The first used canola oil as the only carbon source whereas the second used canola oil and fructose as carbon sources. In whichever two strategies, PHAs production was carried out in a 6 L BIOFLO 3000 (New Brunswick Scientific) bioreactor. Previously the bacteria was grown in LB culture medium and used to inoculate the reactor at 10% v/v. The medium used for production was the same as in example 1.
  • Stage 3 PHAs production with the addition of vegetal oil (previously selected at flask level), as carbon source, with nitrogen limitation, at C/N above 120.
  • Wautersia eutropha was cultured in a fermenter using a production system in three stages. The first stage was in batch culture for growing the bacteria, the second stage was a fedbatch culture in which the total biomass further increases, and the third stage was with the addition of vegetable oil to the culture medium to produce PHAs.
  • a solution of fructose and ammonium sulphate was used to feed the fermenter with concentrations of 30 g/L and 9.37 g/L respectively, with a flow of 0.9 L/h at an agitation speed of 300 rpm. There was no control over the dissolved oxygen in the medium. Once the fructose in the medium was consumed, oil was added for the production of PHAs.
  • Figure 2 shows the kinetics of biomass (X), residual biomass (Xr) and the poly- hydroxyalkanoates (PHAs).
  • the production stage was a batch culture and began with a volume of 3 L and a concentration of 10 g/L of fructose. A 5 h lag phase can be seen.
  • fructose at 30 g/L was added at 0.9 L/h during 2 h. This addition was within the range from 0.8 to 4.5 g SU bstrate /h per g re s ⁇ duai biomass- During this stage concentrations of total biomass and residual biomass of 8 and 7 g/L respectively were obtained with a fructose uptake of 3.01 g/h, which indicated that the carbon source was used for the production of residual biomass.
  • the yield achieved during the feeding process was 0.35 g xr /g s - At the end of the feeding and before adding the canola oil (begin the third stage), we confirmed that the fructose added has been consumed (see figure 3).
  • the fructose was consumed following 5 h of fermentation and concentration declined to 2 g/L, a concentration that remained during the fedbatch culture with addition of fructose. Once the feeding of fructose was finished, the reactor was operated in batch culture, consequently the residual fructose was consumed (see figure 3). Thus, W. eutropha did not use fructose to produce PHAs in the third stage.
  • the concentration of ammonium was determined during the three stages of fermentation.
  • Figure 4 shows that the ammonium declined rapidly from the initial concentration of 0.423 g/L to 0.03 g/L at the end of the first stage. The average consumption rate was 0.12 g/h.
  • FIG. 6 shows the kinetics of a 5 h adaptation phase, the biomass concentration achieved in the first stage was 3 g/L, similar to the biomass obtained in the fermentation with canola oil only. However this maximum concentration was achieved in a less time
  • the second stage began when the fructose concentration in the medium was of 2.4 g/L.
  • a fructose solution containing 117 g/L was added to the fermenter at a flow rate of
  • the percentage of consumed oil in this fermentation was 40% (see figure 8), what is somewhat greater than the percentage of oil used in the fermentation added with canola oil only as carbon source.
  • the dissolved oxygen available in the medium might explain the high consumption of oil since it was controlled by the stirring speed to support a minimum DO of 40%. Therefore, in this fermentation oxygen was not limiting for the production of PHAs.
  • the ammonium concentration was determined during the three stages of the fermentation. In figure 9 one can observe that the ammonium is rapidly consumed during the first stage, being the initial concentration of ammonium 0.43 g/L and 0.074 g/L at the end of this stage, the uptake rate was larger than the observed in the fermentation with only canola oil (0.05 g/h).
  • the larger uptake rate can be greater because the concentration of OD in the reactor was controlled well above of 40%, thus preventing limitation of oxygen during the culture.
  • ammonium sulphate was added to the reactor at a flow rate of 0.225 L/h, at concentration of 35 g/L.
  • ammonium sulphate addition to the reactor was continued, so the ammonium was maintained at concentrations above 0.4 g/L.
  • the stress to induce the synthesis of PHAs for W. eutropha was not caused by nitrogen nor by oxygen limitation in the culture medium.
  • the stress to stimulate the synthesis of PHAs was caused by the absence of phosphates, this because during the culture potassium phosphate is used to regulate the pH of the medium, nevertheless this one was not added afterwards to the medium, which indicates that W. eutropha only used the initial phosphate, consuming it up to reaching a limitation of phosphates.
  • Wautersia eutropha is capable of using vegetable oils as carbon source to produce PHAs. Because it has been reported that the structure of the carbon skeletons of the hydroxyalkanoic acids that incorporate to the PHAs during fermentation is related to the substrata used as precursors during the synthesis of PHAs 1 , the composition and thermal characteristics of the PHAs obtained by means of the method of the invention were determined by different techniques.
  • the cells of Wautersia eutropha previously dried were submitted to boiling in chloroform during 10 min.
  • the chloroformic extract containing PHAs dissolved was filtered to remove the cell biomass. The latter step was repeated twice. Subsequently the PHA was precipitated with cold hexane, filtered and the residual solvent eliminated by evaporation.
  • MNR Magnetic nuclear resonance
  • the MNR was carried out in a Broker equipment (DM X 500 MHz) using deuterated chloroform as solvent.
  • the 13 C and 1 H spectra were obtained at 500 and 125 MHz, respectively.
  • the spectrums corresponding to 1 H were analyzed using the Spinkworks program version 2.5.5.
  • the 13 C and 1 H spectrums of pure PHB showed the expected signals in accordance with the reported in literature 19 20 , whereas the signals of the primary chain of the PHA produced by means of the method of the invention (see figure 11 ) correspond to the expected structures, coinciding with the reported structures 17 .
  • Figure 11 shows the chemical displacements inside the intervals earlier mentioned corresponding to the carbons of the principal chain of the produced PHA.
  • Matsusaki 21 reports the resonance of a methyl group with a chemical displacement of 14 ppm corresponding to a terminal methyl of hydroxyoctanoate (C8) and hydroxydodecanoate (C12). They also report the hydroxyoctanoate and hydroxydodecanoate methylenes with a chemical displacement between 22.66 - 34 ppm.
  • the 1 H spectrum (figure 12) of the PHAs produced by the method of the invention shows the chemical displacements expected in an interval of 2.45 - 2.6 ppm, that correspond to the methylenes protons of the principal chain.
  • the chemical displacement at 0.87 ppm indicated the presence of terminal methyl protons of medium chain, a signal that has been reported as a methyl group joined to a chain of 4 methylenes for the case of hydroxyoctanoate and 8 methylenes for hydroxydodekanoate.
  • Besides a chemical displacement can be identified at 0.97 ppm that identifies as a methyl group corresponding to CH 3 of a monomeric chain of PHV (polihydroxyvalerate) 22 ' 23 .
  • the PHAs obtained by means of the method of the invention is a polymer formed of hydroxybutyrate (HB), hydroxyvalerate (HV), hydroxyoctanoate (HO) and hydroxydodekanoate (HDD).
  • FT-IR - Infrared spectroscopy
  • the band at 2935 cm “1 corresponds to the stretching of anti symmetrical chains of CH 2 , showing that in the PHA obtained with canola oil and fructose presents a more intense band than the PHA obtained with only canola oil and the PHB, these functional groups correspond to the lateral chains of the monomeric units.
  • this signal indicates a major quantity of methyl asymmetric groups, contained in the monomeric chains of the PHA.
  • the band that corresponds to asymmetric methyls also is observed in the spectrum at 2978 cm "1 . This band is more intense for the PHB, and different authors indicate that this signal increases gradually with the presence of crystalline structures in material 25 .
  • DSC Differentiated sweep calorimetrv
  • the DSC studies were done in a differential scanning calorimeter (Mettler Toledo DSC823). In order to erase the thermal history, the samples (4 mg approx.) were cooled at -2O 0 C to be warmed later at 18O 0 C. They were subsequently cooled down rapidly up to -2O 0 C being maintained 5 minutes at this temperature, to later carry out the thermal analysis warming them slowly up to 18O 0 C. The warming ramp was of 5 0 C min "1 .
  • the percentage of crystallinity was estimated from the melting enthalpies determined in the calorimeter. The melting enthalpies were used in equation 1 26 , taking as basis the melting enthalpy of the polymer 100% crystalline, presuming an enthalpy of 146 J/g for PHB 27 .
  • Wp proportion of the polymer when dealing with a polymeric mixture
  • ⁇ H° m melting enthalpy of the 100% crystalline polymer.
  • the DSC results show the signals that comprise the melting temperatures of the obtained PHAs.
  • the obtained thermograms indicated a PHB melting temperature (Tm) of 17O 0 C with a ⁇ H m of 84.74 Jg "1 (see figure 16), very similar to the reported in literature 21 .
  • the melting temperature (Tm) which present the samples of the obtained PHAs with the method of the invention from canola oil and fructose and only canola oil, were 132 0 C and 15O 0 C respectively with a ⁇ H m 44.24 for both materials.
  • Table 8 and figure 16 it is possible to observe that with the incorporation of small percentages of monomeric units of medium chain, the melting temperature of the PHAs is notably affected. Table 8. Comparison between melting and enthalpy temperatures for different

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Abstract

La présente invention concerne des procédés de fermentation efficaces et économiques pour la production efficace de poly-3-hydroxyalcanoates (PHA) à chaîne moyenne, comprenant la croissance de micro-organismes de production de PHA en utilisant des huiles végétales comme source de carbone, en présence ou en l’absence de saccharide. Les procédés selon l’invention comprennent la production de biomasse en culture discontinue, la production ultérieure de biomasse en culture à écoulement discontinu par l’ajout de saccharide et d’une source d’azote au milieu de culture et enfin la production de PHA par l’ajout d’huile végétale comme source de carbone avec limitation d’azote ou de phosphore. Grâce au procédé selon l’invention, au moins 90% en poids sec des PHA à chaîne moyenne par rapport à la biomasse sèche est obtenu à partir du milieu de culture, présentant entre 5 et 10% d’unités monomères à chaîne moyenne dans leur structure.
PCT/IB2009/052695 2008-06-23 2009-06-23 Procédés de production de polyhydroxyalcanoates à chaîne moyenne utilisant des huiles végétales comme source de carbone WO2009156950A2 (fr)

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MX2008008292A MX2008008292A (es) 2008-06-23 2008-06-23 Metodos para la produccion de polihidroxialcanoatos (pha) de cadena media usando aceite vegetal como fuente de carbono.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014032633A1 (fr) 2012-08-27 2014-03-06 Vysoke Uceni Technicke V Brne Procédé de production de polyhydroxyalcanoates (pha) sur la base d'un substrat huileux
WO2014096276A1 (fr) * 2012-12-21 2014-06-26 Carbios Préparation de poly(hydroxyacides gras) à longue chaîne
WO2015176940A1 (fr) * 2014-05-02 2015-11-26 Institut De Recherche Pour Le Developpement (I.R.D.) Nouvelle souche hyperhalophile et son utilisation pour la degradation des substrats carbones
CN108374026A (zh) * 2018-01-18 2018-08-07 同济大学 利用酵母菌发酵合成油脂的方法
AU2019200943B2 (en) * 2018-02-12 2020-11-12 Zoetis Services Llc Vaccine against Bovine Viral Diarrhea Virus
CN114769296A (zh) * 2022-05-20 2022-07-22 中国环境科学研究院 一种利用有机废物发酵液培养产pha颗粒污泥的方法及系统

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0533144A2 (fr) * 1991-09-17 1993-03-24 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Copolymère et son procédé de production
WO1997007229A1 (fr) * 1995-08-21 1997-02-27 The Procter & Gamble Company Extraction par solvant de polyhydroxyalcanoates (pha) de la biomasse, facilitee par l'utilisation d'un non-solvant marginal pour pha
EP1557460A1 (fr) * 2002-10-10 2005-07-27 Kaneka Corporation Methode de culture permettant de reguler la composition d'un polyester copolymere

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0533144A2 (fr) * 1991-09-17 1993-03-24 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Copolymère et son procédé de production
WO1997007229A1 (fr) * 1995-08-21 1997-02-27 The Procter & Gamble Company Extraction par solvant de polyhydroxyalcanoates (pha) de la biomasse, facilitee par l'utilisation d'un non-solvant marginal pour pha
EP1557460A1 (fr) * 2002-10-10 2005-07-27 Kaneka Corporation Methode de culture permettant de reguler la composition d'un polyester copolymere

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014032633A1 (fr) 2012-08-27 2014-03-06 Vysoke Uceni Technicke V Brne Procédé de production de polyhydroxyalcanoates (pha) sur la base d'un substrat huileux
WO2014096276A1 (fr) * 2012-12-21 2014-06-26 Carbios Préparation de poly(hydroxyacides gras) à longue chaîne
WO2015176940A1 (fr) * 2014-05-02 2015-11-26 Institut De Recherche Pour Le Developpement (I.R.D.) Nouvelle souche hyperhalophile et son utilisation pour la degradation des substrats carbones
US10144977B2 (en) 2014-05-02 2018-12-04 Institut De Recherche Pour Le Developpement (I.R.D.) Hyperhalophilic strain and use thereof for the degradation of carbon-containing substrates
CN108374026A (zh) * 2018-01-18 2018-08-07 同济大学 利用酵母菌发酵合成油脂的方法
AU2019200943B2 (en) * 2018-02-12 2020-11-12 Zoetis Services Llc Vaccine against Bovine Viral Diarrhea Virus
CN114769296A (zh) * 2022-05-20 2022-07-22 中国环境科学研究院 一种利用有机废物发酵液培养产pha颗粒污泥的方法及系统
CN114769296B (zh) * 2022-05-20 2024-02-09 中国环境科学研究院 一种利用有机废物发酵液培养产pha颗粒污泥的方法及系统

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