Hyperhalophilic strain and use thereof for the degradation of carbon-containing substrates

Abstract

The present invention relates to a novel hyperhalophilic strain and use thereof for the degradation of carbon-containing substrates, in particular making it possible to prepare novel polymers.

Claims

1. A method for degrading a hydrocarbon substrate, the method comprising the step of contacting the hydrocarbon substrate with an isolated hyperhalophilic aerobic strain comprising a genome having at least 90% DNA-DNA hybridization with the S3S1 strain deposited on Feb. 21, 2014 at the National Collection of Microorganisms Cultures (CNCM) under accession number CNCM I-4838 under conditions which cause the hyperhalophilic aerobic strain to degrade the hydrocarbon substrate and to produce polyhydroxyalkanoate (PHA), said hydrocarbon substrate being contained in the culture medium of said hyperhalophilic aerobic strain, wherein said hydrocarbon substrate is selected from the group consisting of a product or coproduct from an oil seed plant or from liquid hydrocarbons: a hydrocarbon selected from the group consisting of a triglyceride, an alkane, an alkene, a polyene, a fatty acid and glycerol, and mixture thereof; an animal oil; and a vegetable oil.

2. The method of claim 1, wherein the isolated hyperhalophilic aerobic strain is the S3S1 hyperhalophilic aerobic strain deposited on Feb. 21, 2014 at the National Collection of Microorganisms Cultures (CNCM) under accession number CNCM I-4838.

3. The method of claim 1, wherein said produced PHA has a weight-average molecular weight above 500,000 g/mol.

4. The method of claim 1, wherein the hydrocarbon substrate is a vegetable oil selected from the group consisting of rapeseed oil, sunflower oil, corn oil, linseed oil, olive oil, castor oil, soybean oil, palm oil, palm kernel oil, coconut oil, cottonseed oil, and groundnut oil and their mixtures.

5. The method of claim 1, wherein the hydrocarbon substrate comprises a hydrocarbon selected from the group consisting of a 6 to 24 carbon alkane, a 6 to 24 carbon alkene, a 6 to 24 carbon polyene, a 6-24 carbon fatty acid, and glycerol.

6. The method of claim 1, further comprising the step of contacting a carbohydrate co-substrate with the hyperhalophilic aerobic strain.

7. The method of claim 6, wherein the carbohydrate co-substrate is glucose.

8. The method of claim 1, wherein the step of contacting the hydrocarbon substrate with the isolated hyperhalophilic aerobic strain is performed in a culture medium comprising at least one salt selected from the group consisting of NaCl, KCI, CaCl.sub.2, MgCl.sub.2, MgSO.sub.4, and hydrates thereof and their mixtures.

9. The method of claim 8, wherein the at least one salt is selected from the group consisting of a NaCl/MgCl.sub.2 mixture with a weight ratio of 15 to 100, and a NaCl/CaCl.sub.2 mixture with a weight ratio of 20 to 100.

10. The method of claim 1, wherein the step of contacting the hydrocarbon substrate with the isolated hyperhalophilic aerobic strain is performed under an atmosphere comprising less than 21% by volume of oxygen.

11. The method of claim 8, wherein the culture medium comprises nitrogen and carbon in a ratio of 1 to 300.

12. The method of claim 8, wherein the culture medium comprises phosphorus and carbon in a ratio of 80 to 800.

13. A method for preparing a PHA, the method comprising the steps of: (a) culturing an isolated hyperhalophilic aerobic strain comprising a genome having at least 90% DNA-DNA hybridization with the S3S1 hyperhalophilic aerobic strain deposited on Feb. 21, 2014 at the National Collection of Microorganisms Cultures (CNCM) under accession number CNCM I-4838 in a culture medium comprising a hydrocarbon substrate under conditions which cause the strain to produce the PHA, wherein said hydrocarbon substrate is selected from the group consisting of a product or coproduct from an oil seed plant or from liquid hydrocarbons: a hydrocarbon selected from the group consisting of a triglyceride, an alkane, an alkene, a polyene, a fatty acid and glycerol, and mixture thereofo; an animal oil; and a vegetable oil; (b) lysing the cultured hyperhalophilic aerobic strain to create a cell lysate; and (c) extracting the PHA from the cell lysate.

14. The method of claim 13, wherein the step of lysing the cultured hyperhalophilic aerobic strain to create a cell lysate is performed by a lysis method selected from the group consisting of: contacting the cultured hyperhalophilic aerobic strain with a detergent, sonicating the cultured hyperhalophilic aerobic strain, and sonicating the cultured hyperhalophilic aerobic strain in the presence of a detergent.

15. The method of claim 13, wherein the step of extracting the PHA from the cell lysate comprises adding sodium hypochlorite to the cell lysate to precipitate out the PHA and a cell fraction.

16. The method of claim 13, wherein the step of extracting the PHA from the cell lysate comprises adding sodium hypochlorite to the cell lysate to precipitate out the PHA and a cell fraction and comprising d) purifying the PHA which comprises adding an organic solvent to the PHA and to the cell fraction, to obtain a liquid fraction comprising the PHA devoid of the cell fraction.

17. An isolated and lyophilized hyperhalophilic aerobic strain able to degrade a hydrocarbon substrate and produce PHA, the strain comprising a genome having at least 90% DNA-DNA hybridization with the S3S1 hyperhalophilic aerobic strain deposited on Feb. 21, 2014 at the National Collection of Microorganisms Cultures (CNCM) under accession number CNCM I-4838, in particular said strain is the S3S1 hyperhalophilic aerobic strain deposited on Feb. 21, 2014 at the National Collection of Microorganisms Cultures (CNCM) under accession number CNCM I-4838.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates an example of pigmentation with Nile red, showing the presence of PHA in strain S3S1.

(2) FIG. 2 shows the .sup.1H NMR spectrum of the PHA obtained from substrate 3 (rapeseed oil+glucose, example 1a).

(3) FIG. 3 shows the .sup.13C NMR spectrum of the PHA obtained from substrate 3 (rapeseed oil+glucose, example 1a).

DETAILED DESCRIPTION

Examples

Example 1: Degradation of a Carbon-Containing Substrate

(4) The culture media used for degradation of the substrate defined below are for example of the following composition:

(5) NH.sub.4CL, 1 g/L; KCl, 2 g/L; CaCl.sub.2.2H.sub.2O, 2 g/L; MgCl.sub.2.6H.sub.2O, 3 g/L; NaCl, 150 g/L; yeast extract, 1 g/L; substrate: (1) Glucose 2 g/L, (2) Glucose 2 g/L+10 g/L glycerol, (3) Glucose 2 g/L+10 g/L rapeseed oil, (4) Glucose 2 g/1+10 g/L of oleic acid, (5) Glycerol 10 g/L, (6) Rapeseed oil 10 g/L, or (7) Oleic acid 10 g/L.

(6) The precultures were in particular carried out in penicillin bottles containing 50 ml of culture medium.

(7) Larger volumes of culture medium (1.5 L, or even 2 L) were also used.

(8) Culture was carried out in aerobiosis, at pH 7 and 37 C.

Example 1a: Production of PHA

(9) Culture Media Used for Producing PHA: The culture media used for producing PHA are for example of the following composition: NH.sub.4CL, 1 g/L; KCl, 2 g/L; CaCl.sub.2.2H.sub.2O, 2 g/L; MgCl.sub.2.6H.sub.2O, 3 g/L; NaCl, 150 g/L; yeast extract, 1 g/L; substrate: (1) Glucose 2 g/L, (2) Glucose 2 g/L+10 g/L glycerol, (3) Glucose 2 g/L+10 g/L rapeseed oil, (4) Glucose 2 g/L+10 g/L of oleic acid, (5) Glycerol 10 g/L, (6) Rapeseed oil 10 g/L, or (7) Oleic acid 10 g/L.

(10) Higher concentrations of substrate may also be used.

(11) The precultures were in particular carried out in penicillin bottles containing 50 ml of culture medium.

(12) Larger volumes of culture medium (1.5 L, or even 2 L) were also used.

(13) Culture was carried out in aerobiosis, at pH 7 and 37 C.

(14) Detection of the Polyhydroxyalkanoates (PHAs)

(15) Detection of PHAs in the S3S1 producing strain was carried out in particular on a medium containing 15% of NaCl; NH.sub.4Cl, 1 g/l; KCl, 2 g/l; CaCl.sub.2.2H.sub.2O, 2 g/l; MgCl.sub.2.6H.sub.2O, 3 g/l; 10 g/l glycerol; 1 g/l of yeast extract, in the presence or in the absence of glucose. The pH of the medium is 7.0 and the growth temperature is 37 C.

(16) A solution of Nile red 0.5 g/ml, filtered and kept in the dark, was sprayed onto the strain with the aim of detecting the PHAs. Direct observation of the bacterial cells containing granules of PHA was carried out with a phase contrast microscope: Nikon Optiphot (Nikon, Tokyo, Japan) connected to a Nikon DS-FI 1 camera, and placed under fluorescence at 490 nm (FIG. 1).

(17) Detection of the PHAs starting from a medium comprising rapeseed oil or oleic acid as substrate, in the presence or in the absence of glucose, was carried out in a similar manner.

Example 2: Extraction of PHA

(18) Extraction of PHA was carried out starting from an isolate obtained in Example 1a, according to the following protocol: Lyophilize the bacterial culture (biomass) after recovery by centrifugation at 9000 rpm for 20 min; Add 5 ml of SDS (0.1%); Incubate for 24 h at 37 C. with stirring; Centrifuge the lysed suspension at 9000 rpm for 15 min, twice; Dissolve in sodium hypochlorite (30%); Incubate at 30 C. for 3 min; Centrifuge at 9000 rpm for 15 min; Discard the supernatant and wash the pellet with distilled water and then with an acetone/alcohol mixture (1:1); Vortex well; Dissolve in hot chloroform until evaporation occurs.

(19) Results of the extraction of PHA, from cultures carried out in a large volume (1.5 L), comprising from 2.1 to 2.5 g of biomass, are shown in Table 1 below.

(20) TABLE-US-00001 TABLE 1 Substrate used Quantity of PHA extracted (mg) Rapeseed oil (6) 90 Rapeseed oil + Glucose (3) 140 Glycerol + Glucose (2) 40 Glucose (1) 120

Example 3: Production of PHA without Sterilization of the Culture Medium

(21) Growth of strain S3S1 could be obtained in a media comprising 150 or 200 g/L of NaCl, without autoclaving said media.

Example 4: Structure of the PHAs

(22) The PHA obtained from substrate 3 (rapeseed oil+glucose, example 1a) was analysed by NMR, DSC and GPC.

(23) Material and Methods

(24) Analysis by NMR

(25) The .sup.1H and .sup.13C NMR spectra are recorded in CDCl.sub.3 (Bruker 400 MHz instrument).

(26) Analysis by DSC

(27) The thermal properties are determined by DSC (Diamond DSCPerkin Elmer). The melting point is determined at the 1st passage and the glass transition temperature at the 2nd passage.

(28) The temperature programme used is as follows:

(29) 1) from 20 C. to 50 C. at 40 C./min;

(30) 2) 3 min at 50 C.;

(31) 3) 1st passage: from 50 C. to 200 C. at 20 C./min;

(32) 4) from 180 C. to 50 C. at 200 C./min;

(33) 5) 3 min at 50 C.;

(34) 6) 2nd passage: 50 C. to 200 C. at 20 C./min.

(35) Analysis by GPC

(36) The samples are dissolved in chloroform (5 mg/mL) before analysis. Two methods of detection were used: the differential refractometer (RI) and light scattering (LS).

(37) The molecular weights determined by RI are given in polystyrene equivalents.

(38) The equipment used is as follows: the Shimadzu LC-20AD pump is equipped with 2 columns PL Gel Polymer Laboratories 5 m mixed C and two detectors: a Wyatt Optilab Rex differential refractometer and a Wyatt Dawn Heleos 8 light scattering detector.

(39) The polymers are analysed by light scattering using a do/dc of 0.034, a value that is characteristic of short side chain PHAs in chloroform.

(40) Results

(41) Analysis by DSC

(42) The melting points (Mp) and the fusion enthalpies are determined at the 1st passage, and the glass transition temperatures (Tg) at the 2nd passage. The Tg is measured at the point of inflexion, and the Mp is measured at the top of the peak.

(43) The exothermic peak is due to the phenomenon of crystallization and it appears during the 2nd passage.

(44) The results are shown in Table 2.

(45) TABLE-US-00002 TABLE 2 Measurements carried out Results Tg ( C.) +5 Mp ( C.) 157 HF (J/g) 70
Determination of the Molecular Weights by SEC
The results for the number-average molecular weights Mn, the weight-average molecular weights Mw and the polymolecularity index PMI (sometimes called polydispersity index) are shown in Table 3.

(46) TABLE-US-00003 TABLE 3 Measurements carried out Mn(RI) Mw(RI) PDI(RI) Mn(LS) Mw(LS) PMI(LS) Results 642 000 1 097 000 1.7 597 400 732 900 1.2
Determination of Chemical Structure by NMR
The .sup.1H and .sup.13C NMR spectra as well as the attributions are shown in FIGS. 2 and 3 respectively.

(47) Conclusions

(48) In proton NMR, the peaks at 1.2, 2.5 and 5.2 ppm are characteristic of the CH.sub.3, CH.sub.2 and CH groups, respectively, of poly(3-hydroxybutyrate), PHB (FIG. 2). These groups are also clearly identified by .sup.13C NMR (19.6, 40.9 and 67.7 ppm). The peak at 169 ppm is also characteristic of the ester function (FIG. 3).

(49) The sample consists predominantly of PHB. However, NMR indicates the presence of other groups in smaller quantities.

(50) In .sup.1H NMR, the peaks at 0.8, 1.5 and 5.05 are characteristic of the presence of hydroxyvalerate units (HV) (CH.sub.3, CH.sub.2 of the side chain and CH of the macromolecular backbone).

(51) In .sup.13C NMR, these groups can also be seen at 9.5, 27 and 72 ppm.

(52) By finding the ratios of the integrations of the peaks located at 0.8 ppm (PHV) and at 1.2 ppm (PHB), it can be deduced that the proportion of HV units is 4%.

(53) It is therefore a P(HB-co-HV) polymer in proportions 96/4.

(54) This chemical structure is consistent with the results obtained in DSC, as the glass transition temperature Tg is 5 C. and the melting point Mp is 157 C., which is in agreement with the known results for the scl PHAs.

(55) It should be noted, in connection with the behaviour of said polymer in solution, that said solution is perfectly clear but very viscous. The weight-average molecular weight determined by LS is particularly high (732 900 g/mol) and the polymolecularity index is 1.7 (Table 3).