NOVEL METHOD FOR CULTURE OF ALGAE, IN PARTICULAR MICROALGAE

Abstract

This relates to the field of culture of algae, in particular microalgae. Disclosed is a method for culture of algae, advantageously of microalgae. The particular culture conditions in terms of lighting and nutrients enables a biomass to be obtained which is constituted of microalgae having a quantity of chlorophyll that is at least low, advantageously very low, or even zero and a high quantity of antioxidant agents.

Claims

1-27. (canceled)

28. Method for producing a biomass of microalgae consisting of microalgae having a lower quantity of chlorophyll and an increased quantity of antioxidant agents, comprising the following steps: a) culturing the microalgae in a culture medium comprising at least one carbon source under conditions of illumination with radiation having a narrow wavelength spectrum between 460 and 490 nm and centered at 475 nm, and b) harvesting the biomass of microalgae produced.

29. Method according to claim 28, wherein the illumination is produced by one or more lamp(s), one or more tube(s), one or more light-emitting diode(s) (LEDs).

30. Method according to claims 28 wherein the illumination is provided in the form of flashes with the number of flashes between 2 and 3.6 10.sup.4 per hour.

31. Method according to claim 28 wherein the illumination is provided in the form of flashes with the number of flashes between 3.6×10.sup.4 and 5.4×10.sup.9 per hour.

32. Method according to claim 30, wherein the flash duration is between 1/150000 of second and 1799 seconds (29 minutes and 59 sec).

33. Method according to claim 30, wherein the intensity of the light provided in the form of flashes is between 5 and 5000 μmol. m.sup.−2. s.sup.−1.

34. Method according to claim 28 wherein the illumination is not interrupted by phases of darkness and that the light intensity varies over time.

35. Method according to claim 34, wherein the variations of light intensity have an amplitude between 5 μmol. m.sup.−2. s .sup.−1 and 2000 μmol. m.sup.−2. s.sup.−1.

36. Method according to claim 28 wherein the culture is carried out at a temperature between 15° C. and 38° C.

37. Method according to claim 28 wherein the microalgae is selected from the group consisting of microalgae of the genera Chlorella, Scenedesmus, Desmodesmus and Monoraphidium.

38. Method according to claim 28 wherein the carbon source is selected from the group consisting of glucose, cellulose derivatives, lactate, starch, lactose, sucrose, acetate and glycerol.

39. Method according to claim 28 wherein the carbon source is at a concentration between 5 mM and 1.5 M.

40. Method according to claim 28, wherein the biomass harvested has a chlorophyll content lower than 500 ppm.

41. Method according to claim 28, wherein the biomass harvested has a quantity of carotenoids between 50 and 10000 ppm.

42. Method according to claim 28 wherein the microalgae culture is subjected to a nitrogen deficiency.

43. Method according to claim 42, the nitrogen concentration in the culture medium is between 0 and 0.5 mM.

44. Method according to claim 31, wherein the flash duration is between 1/150000 of second and 1799 seconds (29 minutes and 59 sec).

45. Method according to claim 31, wherein the intensity of the light provided in the form of flashes is between 5 and 5000 μmol. m.sup.−2. s.sup.−1.

Description

EXAMPLE 1: PRODUCTION OF CHLOROPHYLL PIGMENTS AND CAROTENOIDS BY THE MICROALGAE SCENEDESMUS AND DESMODESMUS

[0097] Scenedesmus abundans and Desmodesmus pannonicus cultures are prepared in 250 mL flasks with 50 mL of BG11 culture medium enriched in nitrogen (Rippka et al., 1979). The carbon substrate used is 10 g/L glucose. The culture temperature is set to 26° C.

[0098] The illumination conditions are as follows:

[0099] For heterotrophy: the cultures are maintained in darkness.

[0100] For mixotrophy: the cultures are illuminated in flashes (0.5 hertz, 1800 flashes per hour) with an intensity of 200 μmol.m.sup.−2.s.sup.−1. The light provision is obtained by LED lamps the wavelength of which extends from 450 nm to 500 nm, the peak being centered at 475 nm, for mixotrophy limited to blue light.

[0101] For “white” mixotrophy, the LEDs used cover a light spectrum from 370 to 700 nm, with 3 peaks centered at 445, 550 nm and 630 nm.

[0102] After 7 days of culture, the cells are centrifuged then lyophilized. Methods for extracting pigments are known to the person skilled in the art. The table below presents the results of 3 biological replicates.

TABLE-US-00001 Trophic Chlorophyll a Chlorophyll b Lutein Strain mode (ppm) (ppm) (ppm) Scenedesmus Heterotrophy  99 (±40)  58 (±29)  68 (±27) abundans Scenedesmus “Blue” 210 (±31) 304 (±58) 3189 (±245) abundans mixotrophy (450-500 nm) Scenedesmus “White” 7705 (±354) 2101 (±287) 3568 (±332) abundans mixotrophy Desmodesmus Heterotrophy 270 (±36)  540 (±134)  54 (±11) pannonicus Desmodesmus “Blue” 235 (±76) 688 (±66) 1857 (±163) pannonicus mixotrophy (450-500 nm) Desmodesmus “White” 13886 (±204)  4217 (±157) 2289 (±141) pannonicus mixotrophy

[0103] The test carried out here shows the advantages of the method according to the invention. It is noted indeed that the “Blue” mixotrophy culture makes it is possible to obtain a biomass having a low chlorophyll a or b content relative to a “White” mixotrophy culture with a virtually identical but largely improved lutein level compared to a heterotrophic culture.

[0104] “Blue” mixotrophy combines the advantages of heterotrophy in terms of the quantity of chlorophyll a or b and of “White” mixotrophy in terms of lutein.

[0105] EXAMPLE 2: COMPARISON OF THE EFFECT OF ILLUMINATION (WAVELENGTH AND FLASHES) ON PIGMENT PRODUCTION BY CHLORELLA PROTOTHECOIDES UTEX B25

[0106] Composition of the medium:

TABLE-US-00002 Yeast extract 4 g/L MgSO.sub.4, 7H.sub.20 500 mg/L KH.sub.2PO.sub.4 1 g/L CaCl.sub.2 44 mg/L Stock solution Fe—EDTA (6.9 g/L FeSO.sub.4 and 9.3 g/L 3 mL/L EDTA—Na.sub.2) Trace metal solution (3.09 g/L EDTA—Na.sub.2; 0.080 g/L 4 mL/L CuSO.sub.4, 5H.sub.2O; 2.860 g/L H.sub.3BO.sub.3; 0.040 g/L NaVO.sub.3, 4H.sub.2O; 1.820 g/L MnCl.sub.2; 0.040 g/L CoCl.sub.2, 6H.sub.2O; 0.220 g/L; ZnSO.sub.4, 7H.sub.2O, 0.017 g/L Na.sub.2SeO.sub.3; 0.030 g/L (NH.sub.4).sub.6Mo.sub.7O.sub.24, 4H.sub.2O) Glucose 30 g/L

[0107] Culture conditions:

[0108] In each Erlenmeyer flask 100 mL of 1% medium is inoculated with a 7-day-old Chlorella protothecoides preculture.

[0109] To test the effect of the light, the Erlenmeyer flasks are illuminated independently with a system of white LEDs or blue LEDs at 455 nm or at 475 nm. The light intensity for each condition is 0 μmol m.sup.−2 s.sup.−1 (μE) in heterotrophic conditions, 100 μmol m.sup.−2 s.sup.−1 with a frequency of 1 second of illumination every 10 seconds, and 5000 μmol m.sup.−2 s.sup.−1 with a frequency of 1 second of illumination every 60 seconds in mixotrophic conditions. The cells are cultivated at a temperature of 26° C. with moderate shaking (200 rpm). Cell growth is monitored every 24 hours by measuring absorbance at 800 nm. When the stationary phase is reached (7 days), 50 mL of cell suspension is taken to carry out the analysis of the quantities of pigments, chlorophyll and carotenoids contained in the biomass.

TABLE-US-00003 Carotenoids (ppm) White Blue light Blue light Hetero- Light intensity and flash light (455 nm) (475 nm) trophy 0 μE — — — 58  100 μE/1 sec every 10 s 120 109 101 — 5000 μE/1 sec every 60 s 180 134 162 —

TABLE-US-00004 Chlorophylls (ppm) White Blue light Blue light Hetero- Light intensity and flash light (455 nm) (475 nm) trophy 0 μE — — — 0  100 μE/1 sec every 10 s 81 0 0 — 5000 μE/1 sec every 60 s 200 106 20 —

[0110] The results show that in the absence of light (0 μE) there is an absence of chlorophyll and a presence of carotenoids. In the presence of low-intensity flashes (100 μE) the quantity of carotenoids in the biomass is higher than in heterotrophy irrespective of the wavelengths used. However, it can be noted that in blue light (455 nm or 475 nm) there is no chlorophyll production unlike that which is observed in white light. Flashes of very high intensity and low frequency seem to have an increased effect on carotenoid production compared to flashes of low intensity and high frequency. It should be noted that in the presence of white light and blue light at 455 nm the quantity of chlorophyll increases substantially by 200 and 106 ppm, respectively, whereas at 475 nm the quantity of chlorophyll is 10 to 5 times smaller.