METHOD OF CULTIVATING ALGAE

Abstract

The present description is related to the field of cultivating algae. It introduces a method of cultivating algae by depleting the culture of an inorganic nutrient and exposing the alga to high intensity light to obtain algal cell mass having enriched lipid content and reduced chlorophyll content.

Claims

1. A method of producing algal cells comprising steps of: a. cultivating algal cells in culture conditions and in an amount of light that support growth; b. depleting the algal cells of at least one inorganic nutrient; and c. exposing the algal cells continuously to an amount of light which is higher than in step a.; wherein step b. and step c. are started essentially at the same time.

2. The method according to claim 1, wherein the inorganic nutrient in step b. is nitrogen.

3. The method of claim 1, wherein step c. is continued for at least 3 h.

4. The method of claim 1, wherein step c. is started between one cell division before and one cell division after the inorganic nutrient is under a detection limit in a culturing medium.

5. The method of claim 1, wherein step b. is started when the algal cells have reached stationary growth phase.

6. The method of claim 1, wherein the algal cells are collected 12 h or more after induction of nutrient depletion.

7. The method of claim 1, wherein step c. is carried out by exposing the alga to an amount of light having an intensity corresponding to or exceeding a light level of E.sub.k of said alga.

8. The method of claim 7, wherein the amount of light has an intensity corresponding to or exceeding a light level of 1.5×E.sub.k, 2×E.sub.k or 3×E.sub.k of said alga.

9. An alga produced using the method of claim 1.

10. A method of obtaining a lipid extract comprising: extracting lipids from algae, the algae being produced using the method of claim 1.

11. A method of producing renewable biofuel comprising: a. culturing algal cells according to claim 1; b. isolating lipid components from the cultured algae; and c. subjecting the isolated lipid components to chemical reactions to generate hydrocarbons or alkylesters of fatty acids, whereby renewable biofuel is produced.

12. A method of producing fuel, comprising: producing an alga according to the method of claim 1 and/or producing a lipid extract from the alga for the fuel production.

13. A method of increasing a neutral lipid to chlorophyll ratio in algal cells, the method comprising: a. cultivating algal cells in culture conditions and in an amount of light that support growth; b. depleting the algal cells of at least one inorganic nutrient; and c. exposing the algal cells continuously to an amount of light which is higher than in step a.; wherein step b. and step c. are started essentially at the same time.

14. A method for reducing catalyst blocking in a biofuel conversion unit, the method comprising: producing the lipid extract of claim 10 to reduce catalyst blocking in the biofuel conversion unit.

15. A method for producing fuel, the method comprising: Producing an alga and/or producing a lipid extract from the alga for the fuel production.

16. The method of claim 2, wherein step c. is continued for at least 3 h.

17. The method of claim 16, wherein step c. is started between one cell division before and one cell division after the inorganic nutrient is under a detection limit in a culturing medium.

18. The method of claim 17, wherein step b. is started when the algal cells have reached stationary growth phase.

19. The method claim 18, wherein the algal cells are collected 12 h or more after induction of nutrient depletion.

20. The method of claim 19, wherein step c. is carried out by exposing the alga to an amount of light having an intensity corresponding to or exceeding a light level of E.sub.k of said alga.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0034] FIG. 1 discloses development of Chlorophyll and nitrate (NO.sub.3—N) concentrations during first light experiment. Daily variations are due to dilutions with fresh media. At the days 12 and 17, the culture was diluted with nitrogen free media, causing a drop in NO.sub.3 concentrations. Light-shift experimental periods when subsamples have been taken for different light treatments are shown in grey bars. Chlorophyll is either measured analytically or estimated from fluorescence. Nitrate is measured at days 11-15.

[0035] FIG. 2 discloses light doses during light-shift experimental periods and timing of sampling times. Lights were switched off for the night period.

[0036] FIG. 3 discloses Light experiment 1. Development of P. tricornutum CCAP 1055/1 cell number, Chl (chlorophyll) fluorescence (relative), Nile red fluorescence (relative), cells specific Nile Red fluorescence, photosynthetic efficiency QY (relative), dry weight and nitrate in different light levels in both exponential and stationary growth phases (nitrogen starvation). Note differences in y-scales.

[0037] FIG. 4 discloses growth rate for light experiment 1. Upper left: Growth of cell numbers in exponential phase (0-27 hours), Upper right; Growth of cell numbers in stationary phase (0-11 hours), lower left; Growth of lipids (as Nile Red) in exponential phase (0-27 hours), Lower right; Growth of lipids in stationary phase (0-11 hours). Note different y-scale.

[0038] FIG. 5 discloses development of lipid-to-chlorophyll ratio in light experiment during exponential and stationary phases. In lower figures the values have been scaled to 1 at the start of the experiments. The scaled results for the stationary phase show the over 14-17-fold increase in the lipid:chl ratio due to high light treatment.

[0039] FIG. 6 discloses light experiment 2. Development of P. tricornutum CCAP 1055/1 cell number, Chl fluorescence, Nile Red fluorescence, cells specific Nile Red fluorescence, photosynthetic efficiency, lipid (NileRed)—to—chlorophyll concentration ratio, dry weight and nitrate under nitrogen starvation in different light levels/treatments during a time period of 34 h.

[0040] FIG. 7 discloses light experiment 3. Development of C. vulgaris cell number, Chl fluorescence, Nile red fluorescence, cells specific Nile Red fluorescence and chlorophyll absorption as indication of chlorophyll concentration, under nitrogen starvation in two different light levels during a time period of 37 h.

[0041] FIG. 8 discloses light experiment 3. Development of lipid-to-chlorophyll ratio in light experiment during transition from exponential to stationary phase.

DETAILED DESCRIPTION

[0042] The culture conditions that support growth in the present methods are culture conditions in which the algal cells grow and divide. Any growth medium typically used in algal cultivation may be used. The amount of light supporting growth is the intensity of light received by an algal cell which is sufficient to allow the cell to grow and divide in the selected conditions supporting algal cell growth and without inhibiting accumulation of algal cell mass.

[0043] Depletion of an inorganic nutrient may be accomplished by not supplementing the growth medium with the inorganic nutrient after the desired biomass concentration has been achieved, whereby the inorganic nutrient is consumed by the algal cells. Alternatively, cells can be harvested by centrifugation, or by other means suitable for separating viable cells from the growth medium, and transferred to a new growth medium depleted with at least one inorganic nutrient. However, any suitable method may be used to reach a situation where the cells are located in a medium without at least one inorganic nutrient.

[0044] In certain embodiments of the present invention the level to which the inorganic nutrient is depleted is sufficiently low to induce nutrient stress in algal cells. The depleted inorganic nutrient can be nitrogen and the level inducing nutrient stress may be DIN=0 (DIN=dissolved inorganic nitrogen), which may be determined by methods known in the art. The level may be as low as the detection level of the inorganic nitrogen, or DIN=0.

[0045] Exposing the algal cells in step c. continuously to an amount of light which is higher than used to cultivate cells in step a. can be performed using any light source which is able to provide enough light to achieve the objectives of the invention.

[0046] The step b. is started essentially at the same time with the step c. Preferably, the light exposure step c. is started at a time point that occurs during one cell division before and one cell division after the time when a dissolved inorganic nutrient is under the detection limit in the cell culturing medium. In an embodiment the light exposure is started just before the majority of the algal cells have divided for the last time before inorganic nutrient depletion. As is well known in the art, the time period for one cell division may vary depending on the culturing conditions and the time period for one cell division can be determined according to methods known in the art. Accordingly, the proper time point to start the light treatment of step c. can easily be determined by measuring the concentration of the inorganic nutrient and the time period for cell division activity. Thus, a skilled person is able to start the steps b. and c. essentially at the same time, i.e. not sooner than and not later than one cell division from the time point when the amount of inorganic nutrient falls, or is estimated to fall, below a detection limit.

[0047] The present method provides algal cell biomass having high lipid content and low chlorophyll content. Such biomass is advantageous in biofuel production, because it reduces the need to remove chlorophyll from the biomass and provides more lipid to be converted to a biofuel.

[0048] In an embodiment the above method is for increasing neutral lipid to chlorophyll ratio in an alga or in the lipid extract produced from the alga.

[0049] In an example embodiment the inorganic nutrient which is depleted in the present methods may be nitrogen, phosphorus or silica. Depletion of inorganic nitrogen is preferred because it is a commonly used and very efficient method to induce lipid accumulation in algae cells. However, other nutrients, such as phosphorus or silica, can also be used for inducing lipid accumulation.

[0050] In an example embodiment, the above step c. is continued for at least three hours. Step c may also be carried out for longer than three hours, such as 3.5 h, 4 h, 4.5 h, 6 h, 6.5 h, 7 h, 7.5 h, 8 h, 9 h, 9.5 h, 10 h, 10.5 h, 11 h, 11.5 h, 12 h, or longer. However, three hours is sufficient to increase, compared to control alga, lipid content and decrease chlorophyll content to levels that make algae suitable for biofuel production.

[0051] In an example embodiment the algal cells are collected 12 h or more after induction of nutrient depletion. In a preferred example embodiment the algal cells are collected 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 h after induction of nutrient depletion.

[0052] In another example embodiment the algal cells are collected 1, 2, 3, 4, 5, 6 or 7 days after inducing nutrient depletion.

[0053] In an example embodiment the amount of light to which the algal cell is exposed in step c. corresponds to or exceeds E.sub.k, i.e. the amount of irradiance at which photosynthesis ceases to be light-limited. The light saturation parameter E.sub.k is given as E.sub.k=μ.sub.max/alpha, wherein alpha is the initial slope between growth rate and irradiance relationship for a given alga. At some irradiance level, growth rates reach a plateau. The light-saturated growth rate is denoted μ.sub.max. A skilled person is readily able to determine the E.sub.k of any algal species using methods known in the art. In an embodiment step c. is carried out by exposing the alga to an amount of light which has an intensity corresponding to or exceeding the light level of E.sub.k, 1.5×E.sub.k, 2×E.sub.k or 3×E.sub.k of said alga. Preferably the amount of light has in step c. an intensity equal to or below than needed at μ.sub.max.

[0054] In an example embodiment the amount of light in step c. may be at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 560, 600, 650, 700, 750, 800, 850, 900, 950, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950 or 2000 μmol photons m.sup.−2 s.sup.−1.

[0055] In an embodiment in the step a. the amount of light that the cell is exposed to has an intensity lower than E.sub.k of said alga.

[0056] In an example embodiment, the cultivation is done using a day-night light cycle. Examples of day-night light cycles according to the invention are 12 h light period and 12 h dark period (12-12 cycle), 11-13 cycle, 8-16 cycle, or any naturally occurring light-dark period, or a cycle mimicking the normal periodicity between day and night. The light exposure period in step c. is given and included in the light period to keep the total length of the cycle unchanged during the method.

[0057] In an example embodiment, the high light treatment is given at the start of the day period and the amount of light is kept essentially constant during the rest of the day period. Preferably, the amount of light that the cell receives is kept at an essentially constant high level during the light treatment and at an essentially constant lower level during the rest of the day period.

[0058] In an example embodiment, the above step c. is repeated at the start of each successive day period as long as the method is continued and until harvesting the algal biomass.

[0059] Algal cells are preferably started to be exposed to the light treatment when they have not yet entered the stationary growth phase. However, lipid content can be increased and chlorophyll content decreased with the present method also in the case when the cells have reached stationary phase. In either case the light treatment is not given until the nutritional depletion has begun.

EXAMPLES

[0060] The following examples are provided to illustrate various aspects of the present invention. They are not intended to limit the invention, which is defined by the accompanying claims.

[0061] Light and starvation effects in algal cells were studied both in actively growing algae cells (exponential growth) and with algae cells that had reached stationary phase. Four light levels were tested, namely 60, 200, 600 and 1700 μmol photons m.sup.−2 s.sup.−1, which represented the level of normal sun light penetrating algae cultivations to 2.2; 1.4; 0.7 and 0 cm depths (assuming algae biomass concentrations as 1 g DW L.sup.−1, Chlorophyll content of 1% of DW). Basic light level 60 μmol photons m.sup.−2 s.sup.−1 was the reference control value to which the impact of increased light was compared. Because the level 200 photons m.sup.−2 s.sup.−1 was high enough to see clear effects in algae cell physiology and behavior as lipid producers, we could identify the relation of the minimum light level that lead to strong impacts and the species-specific growth-irradiance relationship. Separate experiments were run with continuous light dosing and with pulses in second or minute scale. Best results, i.e. the fastest change to lipid accumulation phase and highest lipid:chlorophyll ratio of the algal cells, were obtained when extra light was given continuously at least for three hours.

[0062] Algae biomass was produced with maximum specific growth rate, and the nitrogen level was carefully monitored so that the photoactivation experiment was started immediately when inorganic nitrogen was under detection limit in the cultivation medium. The experimental cell density was intentionally low in order to avoid cellular shading and to provide the same light conditions to all the cells during the whole cultivation time.

[0063] The algae produced using the present method had high lipid content and lower chlorophyll content than obtained in the reference conditions. The present algae is especially useful when used for biofuel production because, in addition to high lipid content, the algae cells contain less chlorophyll which causes problems in lipid production and is difficult to remove from the algae biomass.

Example 1

[0064] The first example was done using P. tricornutum CCAP 1055/1, as it has a high growth rate, it is easy to cultivate and the chlorophyll analysis are reliable. Cells were cultivated in N-replete media using salinity of 6 PSU (8.12 mg NO.sub.3—N). Algae was cultivated at 60 μmol q m.sup.−2 s.sup.−1 and was kept in the exponential phase by diluting with nutrient replete culture media. Algae concentration was kept low to avoid self-shading. The daily dilutions kept the chlorophyll levels relatively low (around 100-200 μg L.sup.−1) and thus light conditions inside the cultivation units remained similar throughout the experiment (FIG. 1). With these chlorophyll levels roughly 80-90% of nitrogen is in an inorganic form (NO.sub.3) and only 10-20% is taken up in cells. The cultivation volume was gradually increased during the experiment, from 1.8 L in the beginning to 15-17 L in the experimental days. This allowed us to have several units (each 1.5 L) for light shift experiments. Light shift experiments were started at days 12 (exponential growth phase) and at day 17 (stationary growth phase) (FIG. 1). In the experiment, algae culture was divided into several subsamples, which were incubated in different light treatments. Light treatment with duration of 27 hours was conducted using four light levels (60, 200, 600 & 1700 μmol q m.sup.−2 s.sup.−1, LED Light Source SL 3500, Photon Systems Instruments), lowest and highest light levels had replicate cultures. In all light experiments lights were switched off for the night (off after the 12 h measurements and switched on sampling for 24 h measurements) (FIG. 2). Cultures were kept in 2 L polycarbonate bottles with continuous aeration (air pump capacity 550 ml/h), which is enough to keep pH and inorganic carbon levels constant when such low biomass is used. High ventilation was used and most of the dissolved oxygen was removed and enough carbon dioxide was incorporated to guarantee that CO.sub.2 could not be a growth limiting factor in our trials. Inorganic phosphorus was measured during light-shift experiments, and it was always above 0.4 mg L.sup.−1, thus in excess for algae growth. The first measuring period was conducted at the exponential phase. The second measuring period took place at the transition to stationary phase, at the onset of nutrient limitation. For this purpose part of the original culture was cultivated at 60 μmol q m.sup.−2 s.sup.−1 until NO.sub.3 was depleted (FIG. 1).

[0065] In the experiment, the amount of cells (flowCAM), lipid accumulation (Nile Red) and photophysiology of the cells (AquaPen) were measured. Photophysiological measurements included light absorption, chl content and photochemical activity using variable fluorescence techniques. The latter was measured using fluorescence-irradiance curve technique (rapid light curves, Suggett et al 2003). For this analysis, subsamples were taken from each culture, as for other analyses, and the fluorescence induction curves were measured using various light levels. From fluorescence response vs. light level curves typical production-photosynthesis parameters were calculated (Maclntyre et al 2002).

[0066] In the exponential phase light dose affected the growth rate, but clearly the growth was already saturated at 200 μmol q m.sup.−2 s.sup.−1 (FIGS. 3 & 4). In lowest light the cell number increased 2.5-fold, while in higher irradiances the increase was 3-fold. Nutrient analyses showed the average nitrate and phosphate concentrations at 7600 and 590 μg L.sup.−1. These values are very high and during the experimental period hardly any decrease was observed (FIG. 3). Based on variable fluorescence measurements, the cells growing at the highest light level were somewhat stressed by excess light. In that light level Fv/Fm (or QY) values were around 0.50, while other cultures showed values around 0.60, which is typical for unstressed cells (Seppälä 2009). As an acclimation to high light, chlorophyll concentration or fluorescence did not change as much as cell numbers (FIGS. 3 & 5). While cells were dividing faster in high light than in low light, they did not make any new Chlorophyll in high light. This is clearly visible in results of cellular Chlorophyll content, which shows 50% decrease of Chlorophyll content within one day (FIG. 5). Chlorophyll to dry weight ratio (FIG. 5) did not show similar decrease in chlorophyll content, but it must be noted that dry weight measurements were not very reliable, especially for the samples taken at first hours of exponential phase. Part of the excess light energy was used in making up lipids and the lipid content of cells in higher light was 2 times higher than those at 60 μmol q rn.sup.−2 s.sup.−1 (FIG. 3). When this is coupled with decrease of Chlorophyll content, lipid-to-Chlorophyll ratio increased rapidly and was 2.5-3.5 times higher in saturated irradiance levels, compared to growth irradiance at 60 μmol q m.sup.−2 s.sup.−1 (FIG. 3). When looking at the rate of lipid production, much of the differences between cultures seem to be due to decreased production rate of lipids in lowest light (FIG. 4). In other cultures the lipid production rate is slightly higher than the cell growth rate, as seen also in roughly 20-40% increase of their lipid per cell values.

[0067] In the onset of stationary phase the cell growth was rather similar in lowest light level than in exponential phase. In higher light levels the growth was even higher than during first part of the experiment. Nutrient results indicated that nitrogen was consumed at the onset of the second part of the experiment and was thus not yet limiting the growth to large extent (FIG. 3). As a first sign of nitrogen limitation, Chlorophyll content was not increasing with the same rate as cell numbers, causing more rapid decrease of cellular Chlorophyll content, even in the lowest light level. Variable fluorescence levels were rather high for the first light period, except for highest light treatment which indicated higher light stress than during exponential growth phase. Highest values are close to the theoretical maximum of 0.65-0.70 which can be obtained in healthy cells (Seppälä 2009). Also Chlorophyll fluorescence dropped in those samples, indicating that fluorescence is decreased by non-photochemical quenching. In highest light, compared to lowest light level, chlorophyll concentration decreased 35% during 24 hours. As the number of cells was rather similar in lowest and highest light levels, in the end of stationary phase experiment, the cell chlorophyll quota also decreased roughly 30% due to high light treatment (FIG. 3).

[0068] Lipids started to increase rapidly after nitrogen was depleted. Within 11 hours Nile Red fluorescence was 4-7 fold compared to start of the period (FIG. 3), resulting in much higher production rates than the cell growth rate was (FIG. 4). Together with the decrease of Chlorophyll content, the change in lipid-to-Chlorophyll ratio was even more dramatic. The increase lasted throughout the experiment, and in the end up to 17-fold increase in the ratio was observed (FIG. 5 and Table 1).

[0069] In the stationary phase it seems that nutrient stress combined with high light stress decreased the growth, caused large decrease of Chl fluorescence (non-photochemical quenching or photodamage) and decrease of photochemical activity. However, in the lipid increase was equal at all high light cultures. Relative decrease of chlorophyll in cell when grown in high light did not compromise lipid production.

TABLE-US-00001 TABLE 1 Relative increase of Nile Red fluorescence and ratio between Nile Red fluorescence and Chlorophyll concentration during the Light experiment 1. Time Irradiance 0 1 4 8 12 24 27 Exponential Increase of Lipids [Nile red fl] 60 1.00 1.03 0.97 0.40 0.54 1.32 1.40 60 1.00 1.06 1.05 0.41 0.46 1.31 1.34 200 1.00 1.10 1.40 1.37 2.27 2.46 3.06 600 1.00 1.17 1.58 1.52 2.38 2.57 3.75 1700 1.00 1.07 1.75 1.89 2.64 3.09 3.49 1700 1.00 1.10 1.66 1.67 2.80 2.82 3.28 Increase of Lipids vs. Chl [Nile red fl./Chl μgL−1] 60 1.00 1.06 0.89 0.33 0.39 0.81 0.82 60 1.00 1.02 0.93 0.31 0.32 0.78 0.76 200 1.00 1.06 1.34 1.32 2.06 1.77 2.11 600 1.00 1.18 1.54 1.42 2.18 1.92 2.66 1700 1.00 1.05 1.73 1.86 2.71 2.56 2.81 1700 1.00 1.09 1.58 1.67 2.88 2.24 2.48 Stationary Increase of Lipids [Nile red fl] 60 1.00 11.02 1.63 2.67 3.80 5.61 6.96 60 1.00 1.06 1.26 2.26 3.76 5.68 7.73 200 1.00 0.93 1.79 4.10 6.65 10.93 12.82 600 1.00 1.11 2.29 5.20 7.02 11.98 13.56 1700 1.00 1.02 1.68 4.14 6.05 10.77 11.42 1700 1.00 0.89 1.83 4.30 6.05 10.61 11.98 Increase of Lipids vs. Chl [Nile red fl./Chl pgL−1] 60 1.00 1.02 1.54 2.34 3.30 5.20 6.42 60 1.00 1.07 1.18 2.01 3.28 5.47 7.38 200 1.00 0.94 1.74 3.96 6.41 12.23 14.38 600 1.00 1.08 2.22 5.17 7.12 14.18 16.51 1700 1.00 0.99 1.66 4.36 6.53 15.57 17.28 1700 1.00 0.89 1.77 4.58 6.79 15.40 16.86

Example 2

[0070] The second experiment replicated the first experiment partly, but only light levels 60 and 200 μmol q m.sup.−2 s.sup.−1 were used and the measuring period was in the transition to stationary phase only. In addition we tested if the mode of supplying light in different pulses has an effect on lipid accumulation and photophysiology. This was done by applying pulsed light during measuring periods. Pulses were done at sec-min scale (45 sec dim light, 15 sec of high light) or min-hour scales (45 min dim light, 15 min high light). The light levels were dim light=60 μmol q m.sup.−2 s.sup.−1 and high light=600 μmol q m.sup.−2 s.sup.−1. The accumulated light dose for both pulsed light treatments equal the one with continuous level of 200 μmol q m.sup.−2 s.sup.−1.

[0071] The cell growth was highest at constant light at 200 μmol q m.sup.−2 s.sup.−1 (FIG. 6). Similarly, highest lipid yield (absolute, per cell or per chlorophyll) was obtained with this treatment. From the growth curves it seems evident that cells were not able to use the extra energy provided by light pulses. The number of cells was even slightly lower in pulsed light than in low light. Pulsed or especially higher light level decreased photosynthetic efficiency during the experiment.

[0072] Chlorophyll content of cells (FIG. 7) decreased as in first experiment, but again, pulsed treatments seemed not to influence cell chlorophyll content. The effect of light treatment on spectral absorption was less than in the first experiment. As well, in light-fluorescence relationship the parameters had quite similar ranges in low and high light, and in pulsed light conditions. This indicates very low dynamic light acclimation capacity of cells during this experiment, as during the latter part of the first experiment. The slight differences in pigmentation and lipid content between first and second experiment may be explained by slight shifts in nutrient status of cells. While looking at nitrogen concentrations, similar values observed at the initial stage of second experiment (approx. 30 μg NO.sub.3—N L.sup.−1) are found at 2-3 hours is in the first experiment. As the NO.sub.3 uptake may have been low during dark period, the actual difference between developments of nitrogen limitation may be somewhat longer than this time difference. The results indicate that the values at the beginning of second experiment are quite similar to values at 4-11 hours in the first experiment (stationary phase). We may therefore consider that the second experiment shows a case were nutrient limitation has been developed a bit further than in first experiment. This may partly explain why in the second experiment the responses were weaker than in the first experiment; cells were already more nitrogen limited in the second one.

[0073] It is likely, from the experimental data collected here, that the time-scale for changes in pigmentation and lipid accumulation is from hours to days, as the shorter minute or second scale light pulses did not cause such strong acclimation.

Example 3

[0074] The experiment was carried out using the green algae Chlorella vulgaris. Based on the previous experiment, we used only two light treatments, and samples were taken at the beginning and at the end of light periods, for two days. Between experimental hours 11 to 24, the cells were in darkness, while at other times at given irradiances. Like for the previous experiments, cells were first cultivated at lowest irradiance (here, 80 μmol q m.sup.−2 s.sup.−1). At the onset from exponential to stationary phase, which is due to nitrogen depletion, the culture was divided into four bottles,

[0075] C. vulgaris showed high growth, in both cell numbers and in lipids, at high light (FIG. 8). The time of lipid increase took place after cell growth had stopped, after first light period during nitrogen limited conditions. The halt in cell growth was very rapid and the increase in cellular lipids was two-fold. The higher light dosing resulted in somewhat higher lipid-to-chlorofyll ratio. The pigmentation did not respond to the increase of light level, though a slight increase of blue-to-red absorption ratio was noted in the last sample in high light.

[0076] The foregoing description has provided, by way of non-limiting examples of particular implementations and embodiments of the invention, a full and informative description of the best mode presently contemplated by the inventors for carrying out the invention. It is however clear to a person skilled in the art that the invention is not restricted to details of the embodiments presented in the foregoing, but that it can be implemented in other embodiments using equivalent means or in different combinations of embodiments without deviating from the characteristics of the invention.

[0077] Furthermore, some of the features of the afore-disclosed embodiments of this invention may be used to advantage without the corresponding use of other features. As such, the foregoing description shall be considered as merely illustrative of the principles of the present invention, and not in limitation thereof. Hence, the scope of the invention is only restricted by the appended patent claims.