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

Definitions

• the present invention concerns the production of biodegradable plastics from Brassica carinata oil with high content of erucic acid and from very long chain fatty acids, specifically fatty acids with more than twenty carbon atoms. More specifically, the invention concerns the use of very long chain fatty acids (VLCFA, having aliphatic chains with more than 20 carbon atoms) both as free fatty acids and as triglycerides (or triacyl glycerols, TAG), which are constituents of particular vegetable oils, in particular Brassica cari- nata oil with high content of erucic acid, for the production by fermentation of plastic materials of the type of bacterial polyesters, characterized by biode- gradability and biocompatibility, and having a wide range of possible co- monomers composition, thus resulting in a corresponding wide possibility of modulating the related properties.
• VLCFA very long chain fatty acids
• TAG triacyl glycerols
• PHAs are a family of aliphatic polyesters of 3-hydroxyacids generally produced in the form of intracellular granules with function of energy and carbon reserve material from a wide variety of bacteria in the presence of nutrient-limiting conditions and in the presence of excess carbon sources.
• Such polymers may be generically represented by the following general formula: wherein n represents the number of monomer units and R is a side chain or a pendant group in -3 position, which may be -CH 3 or -C 2 H 5 in short chain length PHAs or scl-PHA, represented by poly(hydroxybutyrate) - PHB or P3HB - and poly(hydroxyvalerate) - PHV o P3HV), or may range from -C 3 H 7 to -C 1 ⁇ 23 in medium chain length PHAs (mcl-PHA).
• short chain length PHAs behave as semicrystalline thermoplastic polymers, and are commonly produced by micro-organisms of the species Ralstonia eutropha and Alcaligenes latus, while medium chain length PHAs, typically produced by fluorescent pseudomonads (such as P. aeruginosa and P. oleovorans), are more amorphous compared to scl-PHAs and exhibit elastomeric properties depending on the composition of the side chain.
• fluorescent pseudomonads such as P. aeruginosa and P. oleovorans
• the substrates conventionally and commonly used as carbon sources for the PHA production by fermentation are glucose and various car- bohydrates
• the fatty acids obtainable from triacyl glycerols (TAG) have attracted the researchers' attention because they seem to be a better fermentation substrate with respect to carbohydrates from an energetic point of view. Further, it is to be taken into account that triacyl glycerols are renewable materials.
• TAG triacyl glycerols
• the first report of production of poly(3-hydroxybutyrate-co-3-hydroxy- hexanoate) (P3HB-co-3HHx) from olive oil as such dates back to the first nineties (Shima- mura, E.; Kasuya, K.; Kobayashi, G.; Shiotani, T.; Shima, Y.; Doi, Y. Macrom- lecules 1994, 27, 878-880).
• soybean oil mainly rich in triglycerides of linoleic and oleic acids
• Aeromonas caviae appeared in the literature (Kahar, P.;
• the literature of the field also includes studies on the synthesis of mcl- i PHA from pure glycerol or from the production residues of glycerol-rich biodie- sel (Ashby, R. D.; Solaiman, D. K. Y.; Foglia, T. A. J. Polym. Environ., 2004, 12, 105-112).
• the main obstacles are their variable amounts and the presence of unfermentable components. These elements unavoidably affect the productivity of the fermentation and the process line. For instance, the high cell density fermentation, which is critical to reach high yields of PHAs, is difficult to obtain with agricultural products and by-products contain- ing diluted concentrations of substrates such as sugars and glycerol.
• Brassica carinata is a plant of the Brassicaceae family, a family comprising herbaceous plants with big leaves some of which are of a vital importance for economy and human food, such as the various species of cabbage and cauliflower (Brassica oleracea), and rapeseed (Brassica napus).
• Brassica carinata or Abyssinian mustard (or Abyssinian cabbage) is a plant originating from the Ethiopian plateau and has been recently introduced in Sicily for production of oil for biodiesel starting from the seeds.
• the major component of such oil is the triglyceride of erucic acid, a monounsaturated fatty acid with 22 carbon atoms (C 22 ⁇ , a higher homologous of oleic acid).
• erucic acid a monounsaturated fatty acid with 22 carbon atoms (C 22 ⁇ , a higher homologous of oleic acid).
• C 22 ⁇ a monounsaturated fatty acid with 22 carbon atoms
• the concerned culture does not take away land to the food cultures and protects the land fertility.
• an object of the present invention is to provide a new possibility for a productive use of Brassica carinata oil which, as is the case with biodiesel production, is addressed to environmental protection, while providing at the same time products with wide and manifold possibilities of use.
• the present invention proposes to employ the Brassica carinata oil, or materials related thereto, such as the free fatty acids obtainable from such starting material, as a nutrient substrate for suitably de- signed biosynthetic processes, affording the production of biodegradable and biocompatible polymeric poly(hydroxyalkanoate) materials.
• the cited works have shown that using as a carbon source saturated fatty acids with an even number of carbon atoms, such as octanoic, decanoic acids, etc., the PHA production by the microorganism grows as the number of C of the substrate increases, up to eicosanoic acid (C 20 :0), but with docosanoic acid (C 22 :0) no production is detected.
• nervonic acid may consist in its use as a substrate for the production of biodegradable and biocompatible plastics.
• the present invention specifically provides the use of starting materials based on fatty acids having aliphatic chains of more than twenty (20) carbon atoms, in the form of free acids or of the corresponding triglyce- rides, as a carbon source for the production of medium chain length poly(3- hydroxyalkanoates) (mcl-PHA) by biosynthesis from microorganisms of the genus Pseudomonas.
• starting materials based on fatty acids having aliphatic chains of more than twenty (20) carbon atoms in the form of free acids or of the corresponding triglyce- rides, as a carbon source for the production of medium chain length poly(3- hydroxyalkanoates) (mcl-PHA) by biosynthesis from microorganisms of the genus Pseudomonas.
• mcl-PHA medium chain length poly(3- hydroxyalkanoates)
• the said starting materials based on fatty acids are in the form of triglycerides and consist of Brassica ca ⁇ nata oil, preferably ⁇ . ca ⁇ nata oil with high content of erucic acid.
• the cited fatty acids- based starting materials are in the form of free fatty acids and consist of erucic and/or nervonic acid.
• the bacterium called Pseudomonas aeruginosa showed to be capable of producing PHA when it is cultured on B. ca ⁇ nata oil, on erucic acid or on nervonic acid in a nitrogen-free culture medium. More - -
• the medium chain length poly(3-hydroxyalkanoates) obtained are copolymers consisting of saturated and unsaturated aliphatic monomer units with a num- ber of carbon atoms comprised between 6 and 14.
• the different monomer units that may be identified in the mcl-PHA produced are five, namely: 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3- hydroxydecanoate, 3-hydroxydodecanoate e 3-hydroxytetradecenoate.
• the said monomer units that may be identified comprise, in addition to greater amounts of the five monomer unit mentioned above, also lesser amounts of the following: 3-hydroxyoctenoate, 3-hydroxydecenoate, 3-hydroxydodecenoate, 3-hydroxytetradecadienoate and 3-hydroxytetradecatrienoate.
• 3-hydroxyoctenoate, 3-hydroxydecenoate, 3-hydroxydodecenoate, 3-hydroxytetradecadienoate and 3-hydroxytetradecatrienoate The structures of the polymers obtained in the two cases, with the details of the various monomer units present in the polymer are shown by Formulae 1 and 2 reported further on.
• the present invention also spe- cifically concerns a process for the production of microbial medium-chain- length poly(3-hydroxyalkanoates) comprising the following fundamental steps: a) culturing a micro-organism of the genus Pseudomonas capable of pro- ducing medium-chain poly(3-hydroxyalkanoates) by biosynthesis in a culture medium deprived of nitrogen and containing, as a carbon source, starting materials based on fatty acids having aliphatic chains of more than twenty (20) carbon atoms, in the form of free acids or of the corresponding triglycerides, to obtain a culture broth containing the said medium-chain length poly(3-hydroxyalkanoates); b) separating the cells of the said micro-organism containing medium- chain length poly(3-hydroxyalkanoates) from the said culture broth and lyophilizing the same; c) recovering the said medium-chain length poly(3-hydroxyalkanoates) from the said said fundamental steps: a)
• the invention concerns the biodegradable and biocompatible polymeric materials that are obtained by applying the method and the starting materials described according to the invention.
• the said materials consist of medium-chain length poly(3- hydroxyalkanoates), obtainable by biosynthesis from microorganisms of the genus Pseudomonas, placed in a culture medium deprived of nitrogen and containing, as a carbon source, starting materials based on fatty acids having mono- or poly-unsaturated aliphatic chains of more than 20 carbon atoms, in the form of free acids or of the corresponding triglycerides.
• the fatty acids- based starting materials to be employed as the carbon source are in the form of triglycerides and consist of Brassica carinata oil.
• the PHA obtained from ⁇ . carinata oil is a transparent material having a glass transition temperature (Tg) of -47°C, totally amorphous, sticky, easily cross-linkable if left in air, exposed to light and at room temperature.
• Tg glass transition temperature
• this material can be used in view of its biodegradability as an ecological lacquer, but if it is derivatized by means of suitable reactions on the double bonds it can give rise to other materials potentially useful in the field of biodegradable articles.
• the PHAs produced by using erucic acid and nervonic acid as a carbon source for the fermentation are transparent as well, with a Tg of -46°C and -43°C respectively, but appear to be partially crystalline, showing a melting temperature (Tm) of 50 0 C, and therefore they show rubber-like characteristics.
• Tm melting temperature
• Their proposed use is as "scaffold" materials for tissue engineering and in the field of pharmaceutical "delivery systems".
• Figure 1 shows the 1 H-NMR spectrum at 500 MHz of poly(3-hydroxy- alkanoate) obtained according to the invention from P. aeruginosa grown on erucic acid;
• Figure 2 shows the 13 C-NMR spectrum at 125 MHz of poly(3-hydroxy- alkanoate) obtained according to the invention from P. aeruginosa grown on erucic acid;
• Figure 3 shows the 1 H-NMR spectrum at 500 MHz of poly(3-hydroxy- - -
• alkanoate obtained according to the invention from P. aeruginosa grown on Brassica carinata oil;
• Figure 4 shows the 13 C-NMR spectrum at 125 MHz of poly(3-hydroxy- alkanoate) obtained according to the invention from P. aeruginosa grown on Brassica carinata oil;
• Figure 5 shows an expansion of the 13 C-NMR spectrum of Figure 4 in the region of the olefin carbons.
• the poly(hydroxyalkanoates) have been isolated from P. aeruginosa after growth on B. carinata oil, on oleic acid, erucic acid and on nervonic acid.
• B. carinata oil has been used as a new low-cost carbon source for the production of PHAs, while as free fatty acids in the scope of the invention free erucic acid, which constitutes the major component of B. carinata oil (35-48%), and nervonic acid, which is the higher homologous of erucic acid (C 24 : 1 vs C22'1), and which is not a component of B. carinata oil, have been used.
• Oleic acid has been used as a comparison model because, as it is known, in addition to being a minor component of B. carinata oil, it is a lower homologous of erucic acid, and the structure of the correspondent PHA is known (Ballistreri et al. 2001, already cited; de Waard, P.; van der WaI, H.; Huijberts, G. N. M.; Eggink, G. J. Biol. Chem).
• the biomass of P. aeruginosa ATCC 27853 to be used for the production of PHA has been produced by inoculating the bacterium in 250 ml of Luria Bertani (LB) medium, incubating for 12-15 hours at 37°C with orbital shaking. Then, the cells were harvested by centrifugation at 8,250 x g and washed with E* medium (Volgen, H. J.; Bonner, D. M. J. Biol. Chem. 1956, 218, 97-106) deprived of the nitrogen source.
• the biomass so treated was then inoculated in 1 liter of E * medium nitrogen deprived up to a finale OD 540 of 0,8-1 , affording as carbon sources Brassica carinata oil (erucic acid content 35-48%) (kindly supplied by the Research Consortium Gian Pietro Ballatore, zona industriale Dittaino, Assoro, Enna, Italy), oleic acid, 90% erucic acid (Sigma-Aldrich, Milano, Italy) and 85% nervonic acid (Polichimica, Sondrio, Italy), at 5mM concentration.
• control cultures were set up, inoculated in complete E* medium.
• the complete E * medium was formulated as follows: (NH 4 ) 2 HPO 4 (1.1 g/l), K 2 HPO 4 (5.8 g/l), KH 2 PO 4 (3.7 g/l), MgSO 4 -7H 2 O 0,1 M (10 ml/I), MT solution (FeSO 4 -7H 2 O 10 mM, MnCI 2 -4H 2 O 10 mM, CoSO 4 -7H 2 O 10 mM,
• the fermentations were carried out at 37°c and lasted 78 hours maximum, monitored at regular time intervals, every 12 hours, to check the PHA production and build-up, by means of Nile Red fluorescent dye.
• the culture broth was divided into four 250 ml aliquots, which were centrifuged at 8,500 x g for 10 minutes. The surnatants were discarded and the pellets re-suspendend with 5-10 ml of non sterile PBS. Each re-suspended pellet was transferred in a 100 ml vacuum flask, frozen at -80°c for at least 12 hours and then underwent freeze-drying.
• the PHAs were extracted from the freeze-dried cells in chloroform by means of Soxhlet type extractor. After a period of 6 h of reflux, the chloroform solution was concentrated in a rotary evaporator. The raw polymer so obtained was solubilized in chloroform and immediately precipitated in 10 volumes of ethanol.
• the experimentation confirmed that the Brassica carinata oil and oleic, erucic and nervonic acid can support both the cell growth and the PHA build-up when the culture medium is deprived of nitrogen.
• the yields of bio- mass and of PHAs build up are reported in the following Table.
• the PHAs produced were characterized by gel permeation chromatography (GPC) to ascertain the molecular weights, gas-chromatography (GC) for the co-monomer composition, NMR spectroscopy ( 1 H e 13 C-NMR) for the structural characterization, differential scanning calorimetry (DSC) for the thermal characterization.
• GPC gel permeation chromatography
• GC gas-chromatography
• NMR spectroscopy 1 H e 13 C-NMR
• DSC differential scanning calorimetry
• composition of the polyesters obtained was determined by gas- chromatography (GC) of the 3-hydroxyalkanoates methyl-esters prepared by total methanolysis
• GC gas- chromatography
• 2.5 mg aliquots of PHA underwent methanolysis by means of heating at 100°c for 140 min in a mixture composed of 1.00 ml CHCI 3 , 1.00 ml of CH 3 OH containing 15% of H 2 SO 4 (Gross et al. 1989, already cited).
• the reaction mixture was washed with 1.00 ml of H 2 O and the chloro- form fraction was analyzed by GC using a Perkin-Elmer 8420 Chromatographic System, equipped with a capillary column AT-50 Alltech (30 m x 0.25 mm; carrier He 1 mL/min) and a flame ionization detector (FID).
• a Perkin-Elmer 8420 Chromatographic System equipped with a capillary column AT-50 Alltech (30 m x 0.25 mm; carrier He 1 mL/min) and a flame ionization detector (FID).
• the temperature program was 80°C for 5 min, then with a gradient of
• composition so determined is reported in the following Table 2.
• Comonomers composition (mol %) of PHAs obtained from various carbon sources, detected by GC a
• the PHAs from oleic acid, erucic acid and nervonic acid consist of five monomers, with 3-hydroxyoctanoate and 3-hydroxydecanoate in greater amount, while the PHA from B. carinata oil has five monomer units more: O : i, D : i, ⁇ : - ⁇ , T :2 and T; 3 , probably originating from linoleic and linolenic acids contained as triglyce-
• Figures 1 and 2 of the enclosed drawings report the 1 H and 13 C-NMR spectra of PHA isolated from P. aeruginosa grown on erucic acid.
• the spectra of PHA from oleic and nervonic acids are essentially the same, even if the intensities of some peaks are different, and are not reported herein for brevity.
• the 13 C-NMR peaks assignments were carried out according to Gross et al. (Gross, R.A.; De MeIIo, C; Lenz, R.W.; Brandl, H.; Fuller, R.C. Macromole- cules 1989, 22, 1106-1115), according to the previous work of the present authors (Ballistreri et al.
• Figures 3 and 4 of the enclosed drawings show the 1 H and 13 C-NMR spectra of PHA isolated from P. aeruginosa grown on B. carinata oil.
• the 1 H- NMR spectrum of Fig. 3 in addition to the signals present in Fig. 1 the additional peaks between 5.2 e 5.5 ppm are noted. The latter are to be assigned to the olephinic protons of the Formula 2, while the signal at 2.7 ppm represents the protons of the methylene groups in diallyl position.
• the signal at 0.96 ppm may be assigned to the protons of the methylene groups in ⁇ -position with respect to the double bonds.
• the determination of the average molecular weights of the compounds obtained has been made by gel permeation chromatography (GPC), employing a Waters 55 pump, a set of four Styragel HR columns connected in series with each other (in the order: HR4, HR3, HR2 and HR1), and a refraction index Waters 401 detector. Chloroform at a flux of 1.00 ml/min was employed as the eluent.
• the differential thermal analysis (DSC) was performed with a Q100 calorimeter by TA Instrument, with heating rate of 20°C/min in a range from
• Table 3 shows the physical characteristics of the PHAs obtained from P. aeruginosa when grown on S. carinata oil and on oleic, eruc- ic and nervonic acids in a culture medium deprived of nitrogen.
• the average weight molecular weights determined by GPC range from about 56000 for the PHAs from B. carinata oil and from oleic acid, to about 120000 for the PHAs from erucic and nervonic acids.
• the Tgs of the four samples range from -43°C to -52°C. Only the PHAs obtained from erucic and nervonic acids also show a Tm, of 50 0 C, with a ⁇ Hm of about 16 J/g.
• the PHAs from oleic acid and B. carinata oil are totally amorphous
• the PHAs obtained by biosynthesis by P. aeruginosa from erucic and nervonic acids showing a Tm of 50 0 C and a ⁇ Hm of about 16 J/g, are partially crystalline with rubber-like characteristics.

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