Method For Increasing The Production Of Biomass and Lipids in Chlorella Vulgaris

Abstract

A method to increase the biomass production and fatty acids in the alga Chlorella vulgaris Beyerinck and obtain a lipid rich biomass type C:16 (palmitic), C18:1n-9 (oleic), C18:2n-6t (linoleidic) and C18:3n-3 (alpha-linolenic), which uses low irradiance from a dichromatic light source.

Claims

1. A method to increase the fatty acids production in Chlorella vulgaris Beyerinck and obtain a biomass from the algae with a high lipid percentage of C:16, C18:1n-9, C18:2n-6t and C18:3n-3 characterized in that it comprises the steps of: 1) Add an inoculum of the alga Chlorella vulgaris Beyerinck in exponential growth phase, to a culture medium “f”, prepared with fresh water at pH between 7.3 and 8.0, until obtaining a concentration between 0.4 to 0.5×106 cells ml−1; 2) Illuminate the solution from step 1 with a dichromatic light source with emission peaks of 450 nm and 625 nm, at irradiance of 50 μmol photon m−2 s−1, continuously in light/dark cycles of 24:0, maintaining the culture at temperature between 20° C. and 22° C. for a period between 48 and 72 hours until obtaining a cell density between 0.95 to 1.05×106 cells ml-1; 3) centrifuge the obtained biomass at between 3800 and 4200 rpm, for 5 to 10 min at temperature between 4° C. and 6° C. and; 4) lyophilize the centrifuged biomass at −50° C. and at pressure of 0.11 bar to obtain Chlorella vulgaris Beyerinck cells with a total lipid content of 18%, C20 lipids 31%, C16 lipids 17% and C18:1n9t lipid 20.63%.

2. The method according to claim 1, characterized in that the culture medium “f” comprises 150 mg of NaNO3; 10 mg of NaH2PO4.H2O; 10 mg of Fe chelate; between 30 to 60 mg of Na2SiO3.9H2O; 0.2 mg thiamine.HCl; 1.0 mg of biotin; 1.0 mg of B12; 0.0196 mg of CuSO4.5H2O; 0.044 mg of ZnSO4.7H2O; 0.020 mg of CoC12.6H2O; 0.360 mg of MnCl2.4H2O; 0.0126 mg of NaMoO4.2H2O and; 1 L cbp water.

3. The method according to claim 1, characterized in that the culture of step 2) is maintained at temperature of 21° C. and the pH between 7.3 and 8.0, with manual stirring for 1 minute every 24 hours.

4. The method according to claim 1, characterized in that the centrifugation of step 3 is carried out at 4000 rpm and temperature of 4° C.

5. The method according to claim 1, characterized in that the lyophilized Chlorella vulgaris Beyerinck cells have a lipid profile based on the total percentage of fatty acids in the exponential phase of 0.13 for C6:0; 0.49 for C8:0; 3.61 for C10:0; 0.96 for C:11; 0.13 for C12:0; 1.89 for C13:0; 0.38 for C14:0; 0.18 for C:15; 12.47 for C16:0; 0.19 for C17:0; 1.74 for C18:0; 0.21 for C:20; 0.11 for C:24; 0.43 for C14:1; 0.33 for C15:1; 9.25 for C16:1; 6.18 for C17.1; 0.06 for C24:1; 12.09 for C18:1n-9; 0.11 for C20:1n-9; 0.18 for C22:1n-9; 0.19 for C18:2n-6c; 12.04 for C18:2n-6t; 0.19 for C18:3n-6; 36.31 for C18:3n-3; 0.00 for C20:2; 0.08 for C20:3n-3; 0.06 for C20:5n-5. The lipid profile during the stationary phase corresponds to 0.15 for C6:0; from 0.36 for C8:0; 3.02 for C10:0; 1.06 for C:11; 0.00 for C12:0; 1.56 for C13:0; 0.38 for C14:0; 0.19 for C:15; 19.71 for C16:0; 0.23 for C17:0; 2.09 for C18:0; 0.30 for C:20; 0.07 for C:24; 0.33 for C14:1; 0.08 for C15:1; 8.13 for C16:1; 1.56 for C17.1; 0.09 for C24:1; 23.23 for C18:1n-9; 0.30 for C20:1n-9; 0.38 for C22:1n-9; 0.09 for C18:2n-6c; 10.42 for C18:2n-6t; 0.10 for C18:3n-6; 25.89 for C18:3n-3; 0.08 for C20:2; 0.12 for C20:3n-3; 0.08 for C20:5n-5.

6. The use of Chlorella vulgaris Beyerinck cells obtained from step 4, to produce biofuels.

7. The use of Chlorella vulgaris Beyerinck cells obtained from step 4, to prepare a food supplement.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides a novel method to increase the fatty acids production in alga Chlorella vulgaris Beijerinck, particularly to increase the lipid production of type C:16 (palmitic), C18:1n-9 (oleic), C18:2n-6t (linoleidic) and C18:3n-3 (alpha-linolenic); while the crop yield is increased, obtaining a higher biomass.

[0020] The method of the present invention allows to control the metabolism of the alga Chlorella vulgaris Beyerinck, to favor the production of C:16 type fatty acids (palmitic), C18:1n-9 (oleic), C18:2n-6t (linoleidic) and C18:3n-3 (alpha-linolenic), without it being necessary to modify in any way the conventional algae culture media basal conditions. For this, the method of the present invention uses low intensity dichromatic lighting sources with specific wavelengths applied in specific growth phases, which allow modifying the basal metabolic behavior of the algae in such a way that the production of the above-mentioned fatty acids is favored.

[0021] The method of the present invention comprises the following sequential steps:

[0022] 1.—Add an inoculum of the alga Chlorella vulgaris Beyerinck in exponential growth phase, to a culture medium “f” [(Guillard, R. R. L., Ryther, J. H. (1962). Studies of marine planktonic diatoms: I. Cyclotella nana Hustedt, and Detonula confervacea (CLEVE) Gran. Canadian Journal of Microbiology, 8 (2):229-239)], prepared with fresh water at pH between 7.3 and 8.0, until obtaining a concentration between 0.4 to 0.5×106 cells ml−1;

[0023] 2.—Illuminate the solution from step 1 with a dichromatic light source with emission peaks of 450 nm and 625 nm, at irradiance of 50 μmol photon m−2 s−1, continuously in light/dark cycles of 24:0, maintaining the culture at temperature between 20° C. and 22° C. for a period between 48 and 72 hours until obtaining a cell density between 0.95 to 1.05×106 cells ml−1;

[0024] 3.—Centrifuge the obtained biomass between 3800 and 4200 rpm, for 5 to 10 min at temperature between 4° C. and 6° C. and;

[0025] 4.—Lyophilize the centrifuged biomass at −50° C. and at pressure of 0.11 bar to obtain dry cells of Chlorella vulgaris Beyerinck with a high percentage of fatty acids.

[0026] With the above-described method, a dry biomass is obtained that has complete cells of Chlorella vulgaris Beyerinck with an average biomass production of 57.2 pg cell-1. This biomass obtained has a total lipid content of 18% in the exponential phase and 11% in the stationary phase, with the lipid content of C:16 being 12.47% in the exponential phase and 19.71% in the stationary phase, C18:1n-9 of 12.09% in the exponential phase and 23.23% in the stationary phase, C18:2n-6t of 12.04% in the exponential phase and 10.42% in the stationary phase and C18:3n-3 of 36.31% in the exponential phase and 25.89 in the stationary phase. Said lipid content, as well as its type, allow the biomass obtained to be used as raw material for conventional biofuel obtaining processes, given that the type of lipids obtained present the best profile to be used as starting material of processes for obtaining high cetane index oils. Likewise, since no type of growth accelerating hormones are used or toxic compounds are included in the culture medium, the biomass obtained can be used in the manufacture of animal feed or supplements intended for human consumption. In addition to the above, by not using strains with genetic modifications of Chlorella vulgaris Beyerinck, the environmental safety and innocuousness of the obtained biomass is guaranteed.

[0027] The biomass obtained by the methodology described above is perfectly susceptible to be used in biodiesel obtaining processes since fatty acids with a high degree of saturation (SFAs) are obtained as in the case of C16:0 with a percentage concentration in exponential phase of 12.47±0.15 and in the stationary phase of 19.71±0.73, which show high value of cetane number (CN=74.5), high values of melting point (62.9° C.), high boiling point (351.0° C.) and low density (0.85 g cm-3). The number of cetanes (CN) is one of the indicators that describe the combustion biodiesel quality during the ignition process. Particularly in biodiesel engineering, a high CN value in fuels has short ignition retardation processes compared to low CN values (Knothe, 2005). [Knothe, G. (2005) Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel processing Technology. 86(10):1059-1070]. The CN and the ignition quality are inversely related by means of a negative logarithmic function respect to the fatty acid saturation degree (Knothe et al., 2003) [Knothe. G., Matheaus, A. C., Ryan T. W. (2003) Cetane numbers of branched and straight-chain fatty esters determined in an ignition quality tester. Fuel 82(8):971-975]. The saturated fatty acids (SFAs) produced with the method above described, have high CN values, and stabilize combustion with low emissions, are resistant to degradation and consequently increase the longevity of the biodiesel obtained, they also increase oxidation resistance in places with hot climates, so the biomass obtained by the methodology above described can be used in conventional biodiesel obtaining processes.

[0028] The biomass obtained by the method of the present invention can also be used as a food supplement, since the fatty acids type and proportion obtained make it an ideal candidate as a starting raw material for obtaining feed and supplements. Trough the methodology of the present invention, a saturated fatty acid rich biomass is obtained that are stable to oxidation because of the saturation of the carbon double bonds, so this biomass can be used to provide important characteristics in the food stability. For example, palmitic fatty acid is used in human diet and constitutes about 60% of their diet, its contribution is mainly meat and animal fats (butter, cheese, cream) as well as vegetables such as palm (44%) and seeds (8 to 20%) [Carta, G., E. Murru, S. Banni, C. Manca. 2017. Palmitic acid: physiological role metabolism and nutritional implications. Frontiers in Physiology 8, 902 https://doi.org/10.3389/fphys.2017.00902]. On the other hand, oleic fatty acid is used as a hypotensive and prevents the development of cardiovascular problems and helps control cholesterol and high-density lipoproteins (HDL) [Pérez-Rosales, R., Villanueva-Rodriguez S., Cosio-Ramirez, R. 2005. Avocado oil and its nutritional properties. E-Gnosis 3(10): 11 PP.].

[0029] To demonstrate the technical advantages of the above-mentioned method, an example of the implementation of the present invention is shown below.

Example 1. Cultivation of the Alga Chlorella vulgaris Beyerinck Under the Present Invention Methodology

[0030] A monospecific mother crop of Chlorella vulgaris Beyerinck (Beijerinick) 1890 cells obtained from the microalgae collection of the “Institute of Applied Microbiology (IAM)” of Tokyo University, Bunkyo-Ku, Tokyo, Japan was started.

[0031] Cells were taken from the mother crops in exponential growth phase and transferred to Erlenmeyer flasks containing culture medium “f” (Guillard and Ryther, 1962), prepared with fresh water, the composition of which is shown in Table 1.

TABLE-US-00001 TABLE 1 Culture medium composition “f”. Compound Concentration NaNO3 150 mg NaH2PO4•H2O 10 mg Fe Chelate 10 mg Na2SiO3•9H2O 30-60 mg Thiamine•HCl 0.2 mg Biotin 1.0 mg B12 1.0 mg CuSO4•5H2O 0.0196 mg ZnSO4•7H2O 0.044 mg CoCl2•6H2O 0.020 mg MnCl2•4H2O 0.360 mg NaMoO4•2H2O 0.0126 mg Water cbp 1 L

[0032] The cultures were started with an average cell density between 0.40 to 0.42×106 cells ml-1 and were irradiated with a dichromatic light source with emission peaks at 450 nm and 625 nm, for light and dark photoperiods of 24:0 continuously, maintaining the temperature of the cultures at 21±1° C. and pH between 7.3 and 8.0, with manual shaking for 1 minute every 24 hours.

[0033] Cell density was measured daily by direct counts with a hemocytometer and compound microscope, and cell size was assessed by randomly measuring cell diameter.

[0034] The evaluation of biomass amount produced from Chlorella vulgaris Beyerinck was carried out by collecting an aliquot of the cultures on glass fiber filters (GF/C) with a pore opening of 1 μm, the filter was rinsed with ammonium formate (3%) to eliminate salt residues. The total dry weight (TDW) was obtained by keeping the filters in an oven at 60° C. until constant weight was obtained. The ash content (AC) was obtained by incinerating the filters in a muffle at 450° C. for 12 h to subsequently obtain the sample weight. The content of the organic dry weight (ODW) was calculated by subtracting the value of the total dry weight from the ash weight ODW=TDW−AC).

[0035] The biomass production per cell was calculated by relating the sample organic weight and the cell density. The daily biomass production (DBP) was obtained by relating the organic dry weight with the cultivation time.

[0036] The lipid content of the Chlorella vulgaris Beyerinck cultures was obtained by collecting an aliquot of a volume of the cultures on glass fiber filters (GF/C) with a pore opening of 1 μm. The lipids were extracted by macerating the sample and using chloroform, methanol, and water (2:2:1). The quantification was carried out following the sulfuric acid method. The lipid content was expressed as a percentage based on the organic dry weight of the sample. Tripalmitin (99%) was used as standard for the calibration curve. To obtain the fatty acid profile, the biomass of Chlorella vulgaris Beyerinck cells was harvested by centrifugation at 4000 rpm, for 5 to 10 min and at 4° C. The wet biomass of the Chlorella vulgaris Beyerinck cells was lyophilized at −50° C. and pressure of 0.110 bar until obtaining dry biomass. The extraction of lipids from Chlorella vulgaris Beyerinck cells was carried out by cold gravimetric methods using chloroform for extraction. Methylation was performed and the fatty acid profile was analyzed by gas chromatography. For identification of the fatty acids, the fatty acids retention time of a commercial standard was taken into consideration.

[0037] All the titrations were carried out in the exponential growth phase (E) day 2 and stationary (S) day 8. The results of the culture titrations are shown below:

TABLE-US-00002 TABLE 2 Growth rate (μ: divisions day-1), maximum cell concentration (MCC: × 106 cells ml-1), total dry weight (TDW: pg cell-1), organic dry weight (ODW: pg cell-1), cell size (CS: μm) and daily biomass production values (DBP: g L-1 day-1) for Chlorella vulgaris Beyerinck in growth phase: exponential (E) and stationary (S). TDW ODW CS DBP μ MCC E S E S E S E S 0.47 ± 0.04b 2.29 ± 0.15 80.90 ± 3.37 68.51 ± 5.99 57.20 ± 0.82 68.51 ± 5.99 14.95 ± 2.94 14.86 ± 2.40 0.065 ± 0.001 0.078 ± 0.001

TABLE-US-00003 TABLE 3 Lipid content (%) of Chlorella vulgaris Beyerinck in two growth phases: exponential (E) and stationary (S). Lipids E S 18.35 ± 0.25 11.27 ± 0.70

TABLE-US-00004 TABLE 4 Fatty acid composition (based on the percentage of total fatty acids) of Chlorella vulgaris Beyerinck grown in two growth phases: exponential and stationary. Growth phase Lipid Type Exponential Stationary Saturated Fatty Acids (SFA) C6:0 0.13 ± 0.01 0.15 ± 0.01 C8:0 0.49 ± 0.03 0.36 ± 0.02 C10:0 3.61 ± 0.10 3.02 ± 0.12 C11:0 0.96 ± 0.03 1.06 ± 0.03 C12:0 0.13 ± 0.01 C13:0 1.89 ± 0.08 1.56 ± 0.05 C14:0 0.38 ± 0.01 0.38 ± 0.01 C15:0 0.18 ± 0.00 0.19 ± 0.01 C16:0 12.47 ± 0.15  19.71 ± 0.73  C17:0 0.19 ± 0.01 0.23 ± 0.02 C18:0 1.74 ± 0.10 2.09 ± 0.03 C20:0 0.21 ± 0.01 0.30 ± 0.03 C21:0 C24:0 0.11 ± 0.01 0.07 ± 0.00 Monounsaturated fatty acids C14:1 0.43 ± 0.02 0.33 ± 0.01 C15:1 0.33 ± 0.02 0.08 ± 0.01 C16:1 9.25 ± 0.48 8.13 ± 0.05 C17:1 6.18 ± 0.22 1.56 ± 0.09 C24:1 0.06 ± 0.00 0.09 ± 0.00 C18:1n-9 12.09 ± 0.13  23.23 ± 0.41  C20:1n-9 0.11 ± 0.01 0.30 ± 0.00 C22:1n-9 0.18 ± 0.01 0.38 ± 0.01 Polyunsaturated fatty acids (PUFA) C18:2n-6c 0.19 ± 0.01 0.09 ± 0.00 C18:2n-6t 12.04 ± 0.11  10.42 ± 0.09  C18:3n-6 0.19 ± 0.00 0.10 ± 0.01 C18:3n-3 36.31 ± 0.73  25.89 ± 0.81  C20:2 0.08 ± 0.01 C20:3n-3 0.08 ± 0.00 0.12 ± 0.00 C20:4n-6 C20:5n-3 0.06 ± 0.00 0.08 ± 0.00 TOTAL 100.00 100.00

[0038] The previous results unequivocally show that, with the methodology of the present invention, the proportion of fatty acids produced by Chlorella vulgaris Beyerinck can be increased, obtaining a lipid profile suitable for use as a starting material in biodiesel obtaining processes and with high cetane index values.

[0039] The present invention has been described in accordance with one of its preferred embodiments; however, it will be apparent to a person skilled in the art that modifications can be made to the invention without departing from its spirit and scope.