USE OF NITRIC OXIDE OR NITRIC OXIDE DONOR FOR INDUCING THE PRODUCTION OF TRIACYLGLYCEROLS IN MICROALGAE

Abstract

The invention relates to a method for triggering triacylglycerols (TAG) accumulation in microalgae comprising the step of contacting a source of exogenous nitroxide (NO) with said microalgae in their growth medium.

Claims

1. A method for triggering triacylglycerols (TAG) accumulation in microalgae comprising the step (i) of contacting a source of exogenous nitroxide (NO) with said microalgae in their growth medium.

2. The method according to claim 1, wherein the source of exogenous nitroxide is a nitroxide donor (NO donor), notably an organic NO donor.

3. The method according to claim 1, wherein the growth medium contains nitrates (NO.sub.3.sup.−) and/or nitrites (NO.sub.2.sup.−)

4. The method according to claim 1, wherein the microalgae are selected from microalgae of the Diatom phylum, the Chromalveolata phylum, and the Archaeplastidae phylum.

5. The method according to claim 4, wherein the microalgae are selected from the Diatom micro-algae species Phaeodactylum tricornutum and Thalassiosira pseudonana, and the Archaeplastidae micro-algae species Chlamydomonas, Ostreococcus, Chlorella.

6. The method according to claim 2 wherein the organic NO donor is selected from Diazeniumdiolates (NONOates), and S-Nitrosothiols.

7. The method according to claim 6, wherein the organic NO donor is a S-Nitrosothiol.

8. The method according to claim 7, wherein the S-Nitrosothiol is selected from S-nitroso-N-acetylpenicillamine (SNAP), le S-nitroso-N-valerylpenicillamine (SNVP), S-nitroso-glutathione (GSNO).

9. The method according to claim 1, wherein the concentration of exogenous NO in the growth medium is from 0.25 mM to 5 mM.

10. The method according to claim 1, further comprising a step (ii) of recovering the accumulated triacylglycerols in microalgae.

11. The method according to claim 1, for further producing fatty acids, biofuels, pharmaceutical or cosmetic compositions or food supplements.

12. A method for producing biofuels comprising: steps (i) and (ii) as defined in claim 1; a step (iii) of transesterifying the triacylglycerols recovered in step (ii); and optionally a step (iv) of recovering the obtained transesterified triacylglycerols.

13. A method for triggering triacylglycerols (TAG) accumulation in microalgae comprising the use of an exogenous nitroxide.

14. The method of claim 13, wherein the exogenous nitroxide is released from a nitroxide donor.

15. The method of claim 14, wherein the nitroxide is an organic nitroxide donor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In addition to the above provisions, the invention also comprises other provisions which will emerge from the remainder of the description which follows, and also to the appended drawings in which:

[0041] FIGS. 1 to 4: Effect of increasing concentrations of SNAP on the production of TAG in Phaedocatylum tricornutum. The medium used was 10×ESAW; incubation was performed in 500 μl, inoculated at 1E+06 cells/ml, with immediate addition of chemicals. Measurements were performed after 2 days of incubation. Data of FIGS. 1 and 2 and 3 are the results of 3 biological replicates, FIG. 4 was a time course without replicates. NAP was used as a non-active analogue of SNAP.

[0042] FIG. 1: Effect of increasing concentrations of SNAP on growth. Growth is measured in cell/mL.

[0043] FIG. 2: Effect of increasing concentrations of SNAP on TAG level per cell. TAG level per cell is given in relative fluorescence units/10.sup.6 cells.

[0044] FIG. 3: Effect of increasing concentrations of SNAP on TAG productivity. TAG productivity is given in relative fluorescence unit (Rfu) corresponding to the fluorescence of Nile Red per mL and per day.

[0045] FIG. 4: NO release following treatment with 1 mM or 3 mM SNAP compared to the untreated control.

DETAILED DESCRIPTION

Examples

[0046] 1) Materials & Methods

[0047] Cell Cultivation.

[0048] Phaeodactylum tricornutum (Pt1) Bohlin Strain 8.6 CCMP2561 (Culture Collection of Marine Phytoplankton, now known as NCMA: National Center for Marine Algae and Microbiota) was used in all experiments. Pt1 was grown at 20° C. in 250 mL flask in artificial seawater (ESAW) medium (composition of the medium see table 1) using ten times enriched nitrogen and phosphate sources (5.49.Math.10.sup.−3 M NaNO.sub.3 and 2.24.Math.10.sup.−4 NaH.sub.3PO.sub.4) called “10×ESAW”, or nitrogen-depleted medium. Cells were grown on a 12:12 light (30 μE m.sup.−2.Math.sec.sup.−1)/dark cycle. Cells were sub-cultured twice a week by inoculating 1.Math.10.sup.6 cells/ml with fresh media. Growth was evaluated by cell counting using a Malassez counting chamber, or by the absorption at 750 nm using a plate reader.

TABLE-US-00003 TABLE 3 Ingredients of solutions for ESAW 1 x cultivation medium.. ESAW-Medium Composition Reagents Per Liter NaCl 21.19 g Na.sub.2SO.sub.4 3.55 g KCl 0.599 g Na.sub.2HCO.sub.3 0.174 g KBr 0.0863 g H.sub.3BO.sub.3 0.023 g NaF 0.0028 g MgCl.sub.2*6H.sub.2O 9.592 g CaCl.sub.2*2H.sub.2O 1.344 g SrCl.sub.2 0.0218 mg NaNO.sub.3 46.7 mg NaH.sub.2PO.sub.4*H.sub.2O 3.09 mg Na.sub.2SiO.sub.3*9H.sub.2O 30 mg Metal Stock I 1 ml Metal Stock II 1 ml Vitamin Solution 1 ml Metal Stock I Na.sub.2EDTA*2H.sub.2O 3.09 g FeCl.sub.3*6H.sub.2O 1.77 g Metal Stock II Na.sub.2EDTA*2H.sub.2O 2.44 g ZnSO.sub.4*7H.sub.2O 0.073 g CaSO.sub.4*7H.sub.2O 0.016 g MnSO.sub.4*4H.sub.2O 0.54 g Na.sub.2MoO.sub.4*2H.sub.2O 1.48 mg Na.sub.2SeO.sub.3 0.173 mg NiCl2*6H.sub.2O 1.49 mg Vitamin Solution Biotin (Vitamin H) 1 mg Thiamine HCl (Vitamin B.sub.i) 100 mg Cyanocobalamin (Vitamin B.sub.i2) 2 mg pH = 8.2
Incubation with a Nitric Oxide (NO) Donor Agent.

[0049] S-Nitroso-N-acetylpenicillamine (SNAP) is a compound that spontaneously releases NO, when dissolved. Nitroso-acetylpenicillamine (NAP) is used as a non-active compound, which does not release NO and can therefore be used for control experiments.

Measure of Nitric Oxide Using a Fluorescent Reporter

[0050] The fluorophore 4-amino-5-methylamino-2′,7′-difluororescein diacetate (DAF-FM) allows the sensitive detection of low levels of nitric peroxide (ONOO.sup.−), which is in equilibrium with NO and thus indicates NO levels (St Laurent C D, Moon T C, Befus A D. 2015. Measurement of nitric oxide in mast cells with the fluorescent indicator DAF-FM diacetate. Methods Mol Biol. 1220:339-45) and was previously used to detect NO levels in P. tricornutum cells (Vardi et al., 2008). 10 ml culture were diluted to 10.sup.6 cells/ml and cells were incubated with 20 μl 5 mM DAF-FM (1.5 h, room temperature, darkness, shaking). Cells were washed and resuspended in 10 ml 10×ESAW media and aliquoted to 500 μl cultures on a 48 well culture plate to which the SNAP was added. For the examination of DAF-FM-dependent detection of nitric peroxide, 150 μl of the culture were transferred into a 96 well plate and fluorescence was measured with a TECAN infinite M1000Pro plate reader (excitation wavelength at 488 nm, emission at 529 nm).

Measure of TAG Accumulation by Nile Red Staining

[0051] Accumulation of TAG droplets was monitored by Nile Red (Sigma Aldrich) fluorescent staining (Excitation wavelength at 485 nm; emission at 525 nm) following the principles previously described (Abida et al., 2015). In brief, cells were diluted and adjusted to a cell density that was linearly correlated with Nile Red fluorescence. Nile Red solution (40 μl of 2.5 μg/mL stock concentration, in 100% DMSO) was added to 160 μl cell suspension. Oil bodies stained with Nile Red were then visualized using a Zeiss AxioScope.A1 microscope (FITC filter; Excitation wavelength at 488 nm; emission at 519 nm).The productivity, corresponding to the accumulation of TAG per volume and per time unit was calculated based on the staining by Nile Red, and expressed in relative fluorescence unit (Rfu) of Nile Red per mL and per day of incubation. Alternatively, Nile red fluorescence values were normalized to the cell concentration.

[0052] 2) Results

[0053] A 2-day incubation of P. tricornutum with 1 mM SNAP, in a 500 μL volume, induces a reduction of cell growth (FIG. 1), but triggers a 2.2 fold increase of TAG per cell (FIG. 2) and also a >2 fold increase of productivity, corresponding to the level of TAG per volume of culture and per day (FIG. 3).