Microalgal strain and its use for the production of lipids

Abstract

A microalgal strain of the species Nannochloropsis gaditana is characterized by a mutation of an enzyme involved in the biosynthesis of chlorophyll that changes the physiology thereof with respect to the wild-type strain of the same species. In particular, with the mutation, microalgae are formed which, with respect to the wild-type strain of the same species, have a lower chlorophyll content and a reduced capacity to absorb visible radiation (light). Further, a process for the production of lipids through the cultivation of the mutated Nannochloropsis gaditana strain and the lipids obtained can be used as synthesis intermediates, particularly in the so-called green-chemistry sector or in the production of biofuels.

Claims

1. A microalgal strain of Nannochloropsis gaditana species deposited, in accordance with Budapest Treaty, at the Culture Collection of Algae and Protozoa (CCAP), Oban, access number CCAP 849/21.

2. A process for the production of lipids, the process including the following steps: cultivating a Nannochloropsis gaditana CCAP 849/21 strain in a culture medium, under presence of visible radiation; subjecting said culture to separation, obtaining an aqueous biomass suspension of Nannochloropsis gaditana CCAP 849/21 comprising intracellular lipids and an aqueous phase; and extracting the intracellular lipids accumulated inside Nannochloropsis gaditana CCAP 849/21.

3. The process according to claim 2, wherein said process for the production of lipids is carried out: at a temperature ranging from 10 C. to 40 C.; and/or under aerobic conditions; and/or is carried out at a pH ranging from 4.5 to 9.5.

4. The process according to claim 2, wherein said process for the production of lipids is carried out starting from an inoculum in an amount ranging from 0.1% to 10% (vol/vol) of the culture medium total volume, obtained from a previous culture of said Nannochloropsis gaditana CCAP 849/21 strain, carried out in the same culture medium for a time ranging from 6 hours to 72 hours.

5. The process according to claim 2, wherein said process is carried out in a F/2 culture medium having sea salt, Tris-HCl, a Guillard (F/2) marine water enrichment solution, and nitrogen.

6. The process according to claim 2, wherein said process for the production of lipids is carried out in a discontinuous mode or in a continuous mode.

7. The process according to claim 6, wherein said process, after a time ranging from 15 hours to 175 hours after inoculation, is further carried out in the discontinuous mode or in the continuous mode, without time limit, adding at least one further source of nitrogen, in an amount such as adding to culture medium a nitrogen amount ranging from 0.02 g/l to 5 g/l.

8. The process according to claim 2, wherein the step of subjecting the culture to separation is carried out by techniques selected from: flocculation, flotation, filtration, filter pressing, microfiltration, ultrafiltration, centrifugation or combinations thereof.

9. The process according to claim 2, wherein the step of extracting the intracellular lipids is carried out by cellular lysis of Nannochloropsis gaditana CCAP 849/21 biomass.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

(1) According to the present disclosure, the chlorophyll content present inside the chloroplasts of Nannochloropsis gaditana CCAP 849/21 is reduced because of the induction of a mutation of the gene codifying uroporphyrinogen decarboxylase (UROD), which codifies for an enzyme involved in the biosynthesis of tetrapyrroles, whose final products include chlorophyll (FIG. 1 shows a schematic representation of the biosynthetic pathway of the tetrapyrroles). This mutation causes an alteration of the UROD gene expression.

(2) Advantageously, the mutation of this gene leads to a reduction of the chlorophyll content, but without causing any alterations to the composition and functionality of the photosynthetic apparatus, which comprises enzymes, proteins and molecules responsible for the sequence of stages necessary: to capture visible radiation (antenna complex), to the transformation of radiation into redox potential (reaction centre), to the translocation of redox potential (quinone and soluble cytochrome cycle), and finally to the transmembrane translocation of protons (cytochrome bcl). In fact, this mutation does not cause a reduction of the expression of the proteins of the antenna complex appointed to collect the visible radiation, which could have negative consequences on the survival of the microalga, nor does it affect the capacity of the microalga to adapt its photosynthetic apparatus to different light intensities (phenomenon known as acclimatisation).

(3) The reduction of the UROD gene expression, a key gene in the biosynthesis of chlorophyll, therefore allows the production and accumulation of chlorophyll within Nannochloropsis gaditana CCAP 849/21 to be reduced, without damaging the photosynthetic capacity of the microalga itself. As chlorophyll is the main pigment appointed to absorb visible radiation, the use inside photobioreactors of microalga provided with a reduced chlorophyll content allows improved penetration of visible radiation inside the photobioreactor, with a consequent increase in the growth rate of the microalgae, an increase in the overall production of the lipids accumulated within the microalgae and an increase in industrial production.

(4) Advantageously, the reduction of the level of expression of the UROD gene does not display any side effects on the growth and capacity of the microalga to adapt to high exposure to visible radiation because, unlike what can happen by modifying the proteins of the antenna complex, this mutation does not affect the photoprotection mechanisms. In fact, Nannochloropsis gaditana CCAP 849/21 shows that it can adapt to any conditions of excess visible radiation without suffering any damage.

(5) Preferably, the mutation characterising Nannochloropsis gaditana CCAP 849/21, according to the present disclosure, is obtained through a mutagenesis process, known to a person skilled in the art such as for example through a high voltage electroporation process, the aim of which is to induce random punctiform mutations within the genome of the microalga. Processes of this type are described in Sosnowski, Ronald G. et al., Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control, Proc. Natl. Acad. Sci. 94:1119-1123 (1997) and in Stacey Michael et al., Differential effects in cells exposed to ultra-short, high intensity electric fields: cell survival, DNA damage, and cell cycle analysis, Mutation Research 542: 65-75 (2003).

(6) Once the electroporation process has been carried out, the selection of the microalga strain containing the mutation of interest (the one affecting the UROD gene) is preferably carried out by verifying the correct formation of the membrane pores during the electroporation process. For that purpose, an exogenous DNA providing the characteristic of resistance to the Zeocin antibiotic is added to the culture broth of the microalgae. It is known that if the electroporation process takes place correctly and pores are formed on the cell membrane, the exogenous DNA can penetrate into the cell, giving the latter the characteristics dictated by the exogenous DNA, a characteristic not present in cells in which the formation of pores did not occur. This discriminant allows the strains to be selected in which the electroporation process has produced the expected results (formation of pores and inclusion of the exogenous DNA). In fact, by adding a certain amount of the antibiotic Zeocin to the culture medium of the microalgae previously subjected to electroporation, only the microalgae provided with exogenous DNA can survive, thus allowing the strains in which the electroporation took place successfully to be selected.

(7) As is known, since the formation of membrane pores only takes place when the electric discharge of the electroporation crosses the cell, it is important to carry out this type of selection in order to identify the microalgal strains that have undergone the mutation process caused by the radiation.

(8) However, as the high voltage electroporation process induces random point mutations inside the genome of the microalga, it is important to isolate and select the microalgal strains characterised by the phenotype of interest, i.e. a reduced chlorophyll content. More details on the isolation and selection process of the microalgal strains expressing a reduced chlorophyll content can be found in the article previously mentioned in the name of Perin et al., Biotechnol Biofuels (2015) 8:161.

(9) After identifying the microalgal strains expressing a reduced chlorophyll content, through gene sequencing techniques known to a person skilled in the art, it was possible to identify the mutations responsible for the phenotype. At this point the Applicant found that the mutation on the UROD gene is among the different mutations identified causing an alteration of the chlorophyll concentration level, the most advantageous one.

(10) Further subject matter of the present disclosure is a process for the production of lipids comprising: cultivating a Nannochloropsis gaditana CCAP 849/21 strain in a culture medium, under presence of visible radiation; subjecting said culture to separation, obtaining an aqueous biomass suspension of Nannochloropsis gaditana CCAP 849/21 comprising intracellular lipids and an aqueous phase; extracting the intracellular lipids accumulated inside Nannochloropsis gaditana CCAP 849/21.

(11) Said cultivation can, preferably, take place inside a photobioreactor.

(12) In accordance with a preferred embodiment of the present disclosure, said process can be carried out at a temperature ranging from 10 C. to 40 C., preferably ranging from 20 C. to 35 C.

(13) In accordance with a preferred embodiment of the present disclosure, said process can be carried out in fed-batch or continuous mode.

(14) In accordance with a preferred embodiment of the present disclosure, said process can be carried out in aerobic conditions.

(15) Said aerobic conditions can be implemented, for example, by insufflating sterile air into the culture device and through variable agitation, said agitation depending on the type of culture device used.

(16) In accordance with a preferred embodiment of the present disclosure, said process can be carried out at a pH ranging from 4.5 to 9.5, preferably ranging from 7.0 to 8.5, even more preferably ranging from 7.5 to 8.0. For the purpose of maintaining the pH in the desired ranges, an aqueous solution can be added to the culture medium of at least one base, selected for example from: sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium dihydroxide [Ca(OH).sub.2], magnesium hydroxide [Mg(OH).sub.2], or mixtures thereof, preferably potassium hydroxide (KOH), or an aqueous solution of at least one acid selected, for example, from: phosphoric acid (H.sub.3PO.sub.4), sulphuric acid (H.sub.2SO.sub.4), hydrochloric acid (HCl), or mixtures thereof, preferably sulphuric acid (H.sub.2SO.sub.4), in an amount such as to obtain the desired pH.

(17) In accordance with a preferred embodiment of the present disclosure, said process can be carried out starting from an inoculum in an amount ranging from 0.1% to 10% (vol/vol) of the total volume of culture medium, obtained from a previous culture of said Nannochloropsis gaditana strain CCAP 849/21, according to the present disclosure, carried out in the same culture medium for a time ranging from 6 hours to 72 hours.

(18) Said previous culture can in turn be inoculated by a former culture, or can be inoculated starting from a sample of said strain CCAP 849/21, maintained at 80 C., as such or in suspension comprising 5% (vol/vol) ethylene glycol+5% (vol/vol) DMSO+10% L-Proline (vol/vol).

(19) In accordance with a preferred embodiment of the present disclosure, said process can be carried out in an F/2 culture medium preferably enriched with sea salt, Tris-HCl, Guillard (F/2) marine water enrichment solution and nitrogen.

(20) In accordance with a preferred embodiment of the present disclosure, said process, after a time ranging from 15 hours to 175 hours, after inoculation, can be further carried out in fed-batch or continuous mode, without time limits, by adding at least one further source of nitrogen, selected for example from: sodium nitrate, potassium nitrate, ammonium sulfate, ammonium carbonate or bicarbonate, ammonium chloride, urea, in an amount such as to add to the culture broth a total amount of nitrogen ranging from 0.02 g/l to 5 g/1, preferably ranging from 0.2 g/l to 5 g/1.

(21) During the culture, the cell growth rate can be evaluated through spectrophotometric methods such as, for example, through turbidity or optical density (OD) analysis, of a sample of culture broth at 750 nm (OD.sub.750), through counting the cells, or through determination of the dry weight according to methods known to a person skilled in the art.

(22) According to the process of the disclosure, the step of subjecting the culture to separation, obtaining an aqueous solution of biomass of Nannochloropsis gaditana CCAP 849/21 comprising intracellular lipids and an aqueous phase is preferably carried out through techniques that are known in the state of the art selected, for example, from: flocculation, flotation, filtration, filter pressing, microfiltration, ultrafiltration, centrifugation or combinations thereof; preferably through centrifugation. Preferably, said centrifugation can be carried out for a time ranging from 5 minutes to 30 minutes, preferably ranging from 15 minutes to 25 minutes, at a rotation speed ranging from 3000 rpm to 9000 rpm, preferably ranging from 4000 rpm to 8000 rpm. It is to be noted that the operating conditions indicated for centrifugation relate to a process carried out on laboratory scale: in the case of an industrial process, in which generally continuous centrifugation is used, a person skilled in the art will be able to adapt said operating conditions.

(23) The concentration of cellular biomass obtained can be measured in grams per litre of culture broth, determining the dry weight of the cells of microalga of a sample of culture broth of a known volume taken at pre-set intervals and at the end of said process. In particular, dry weight of cellular biomass means the weight of the cells contained in a known volume of culture broth, determined by weighing the aforesaid cells after removing all the water content through a heat treatment in a ventilated oven at 75 C. until constant weight (about 24 hours) and separating the salinity contribution of the culture medium through filtration and washing of the biomass or through determination of ash.

(24) For the purpose of extracting the intracellular lipids accumulated inside Nannochloropsis gaditana CCAP 849/21, the Nannochloropsis gaditana CCAP 849/21 biomass can be subjected to cellular lysis according to processes known in the state of the art and described, for example, in international patent application WO 2014/102254 including, for example, a heat treatment in a pressurised reactor, a heat treatment in the presence of acid, such as, for example, sulphuric acid, hydrochloric acid, phosphoric acid or mixtures thereof, a mechanical treatment with homogenizer, a treatment with microwaves or with steam (steam explosion).

(25) At the end of said cell lysis, the lipids can be recovered from the suspension obtained, through extraction with polar or non-polar organic solvents, according to processes known in the state of the art and described, for example, in international patent application WO 2014/102254. Another example of prior art describing methods for extracting lipids from wet or dry cellular biomass is the document Lipid extraction methods from microalgae: a comprehensive review Ramanathan Ranjith Kumar et al., Frontiers in Energy Research 2015, Vol. 2, article 61, 1-9. Further extraction methods comprise the use of supercritical gases, ionic liquids or deep eutectic solvents (DES) as described, for example, in Deep Eutectic Solvents (DESs) and Their Applications, Emma L. Smith et al., Chem. Rev. 2014, 114, 11060-11082.

(26) The production of lipids by microalgae at the end of the process according to the present disclosure can be measured with colorimetric methods known in the state of the art, for example with Nile Red dye or with sulpho-phospho vanilline using the kit Total lipidssulpho-phospho vanilline sold by Spinreact S.A.U., Ctra. Santa Coloma, 7 E-17176 St. Esteve d'en Bas (GI), Spain; or through spectrophotometric methods such as, for example, Fourier Transform Infrared Spectroscopy (FTIR) as described in Microalgae triacylglycerols content by FTIR spectroscopy, Roberta Miglio et al., J Appl Phycol (2013) 25:1621-1631.

(27) Further, the amount of lipids produced can be determined with gravimetric methods from the fraction extracted with mixtures of organic solvents, for example with chloroform:methanol 2:1, vol/vol as described in Folch J. et al, The Journal of Biological Chemistry, vol. 226, pag. 497-509 (1957); or extracted with n-hexane:isopropanol 3:2 vol/vol as described in Hara A. et al, Analytical Biochemistry, Vol. 90, pag. 420-426 (1978), from samples of freeze-dried biomass.

(28) It is to be noted that, operating by the process according to the disclosure, the strain of the species Nannochloropsis gaditana CCAP 849/21, with respect to the wild-type strain of the same species, allows an increase in productivity of up to 25% and a corresponding increase in the lipid titer to be obtained.

(29) The lipid fraction was analysed through chromatographic techniques, for example gas chromatography or high performance liquid chromatography (HPLC) according to processes known in the prior art.

(30) Through said analytical methods it was detected that the lipids accumulated in the microalgae cells, both of the mutated strain and of the wild-type strain, are 90% represented by triglycerides, preferably esters of glycerol with fatty acids having 8 to 24 carbon atoms, such as, for example, palmitic acid, stearic acid, oleic acid and -linolenic acid.

(31) Other lipids that can be present are, for example: phospholipids, monoglycerides, diglycerides, free fatty acids, or mixtures thereof.

(32) The lipids obtained according to the process of the present disclosure can be subjected to hydrogenation/deoxygenation in the presence of hydrogen and at least one catalyst in order to produce green diesel. Hydrogenation/deoxygenation processes are known in the prior art and are described, for example, in EP1728844. Further, the aforesaid lipids can be subjected to transesterification in the presence of at least one alcohol having 1 to 4 carbon atoms, preferably methanol or ethanol, and at least one acidic or basic catalyst, in order to produce glycerol and alkyl esters, in particular methyl esters or ethyl esters. Alternatively, the aforesaid lipids can be advantageously used as synthesis intermediates, particularly in the so-called green chemistry sector.

(33) For the purpose of putting the present disclosure into practice and illustrating it more clearly, below are some non-limiting examples.

Example 1 (Obtaining the CCAP 849/21 Strain of the Nannochloropsis gaditana Species)

(34) An embodiment is described below of the method of obtaining the microalgal strain of the Nannochloropsis gaditana species deposited, in accordance with the Budapest Treaty, on 3 Oct. 2018 at the Culture Collection of Algae and Protozoa (CCAP), Oban, Argyll (Scotland, United Kingdom), access number CCAP 849/21. In this example, mutagenesis was obtained through electromagnetic radiation.

(35) For that purpose, 7*10.sup.6 cells/ml of a wild-type strain of microalga of the Nannochloropsis gaditana species CCAP 849/5 were grown for 5 days in Drechsel bottles having a diameter of 5 cm, working volume of 250 ml and subjected to bubbling with air enriched with 5% (v/v) of CO.sub.2. The culture medium used was F/2 with sea salt (32 g/1, Sigma-Aldrich), 40 mM Tris-HCl (pH 8) and enriched Guillard sea water solution (F/2) (Sigma-Aldrich) additioned with nitrogen (final concentration 12 mM NaNO.sub.3). In the growth chamber, the intensity of the visible radiation (fundamental for the growth of the microalgae) was set to illumination of 120 mol photons m.sup.2 s.sup.1 at 221 C. To induce the mutation, 5*10.sup.8 cells of N gaditana were washed four times with sorbitol 375 mM at 4 C. and re-suspended in 100 l of cold sorbitol 375 mM. Subsequently, the cells were subjected to an electroporation process in 2 mm cuvettes using the set for electroporation Electro Cell Manipulator ECM630 BTX (500 , 50 F and 2400 V). Subsequently, the cells were re-suspended in 10 ml of F/2 medium and left for 48 hours at 221 C. under stirring and 20 mol photons m.sup.2 s.sup.1 before being plated. The mutation of the microalga genome was induced through electroporation of the cells using a high voltage in order to induce random punctiform mutations. In order to verify the correctness of the electroporation process, exogenous DNA was added, as a selection marker, for resistance to the antibiotic Zeocin. In this way, by selecting the colonies resistant to the antibiotic Zeocin, it was possible to guarantee that these colonies had correctly undergone the electroporation process and had therefore developed some random punctiform mutations within their endogenous DNA.

Example 2 (Analysis of the CCAP 849/21 Strain of the Nannochloropsis Gaditana Species)

(36) Mutation of the UROD Gene

(37) Nannochloropsis gaditana CCAP 849/21 is characterised by a mutation on the UROD gene. This mutation is a punctiform mutation, which led to the formation of a premature stop codon and therefore to the suppression of the expression of the mutant allele of the gene (FIG. 2).

(38) FIG. 2A represents a schematic structure of part of the UROD gene in which the punctiform mutation is indicated: the insertion of a cytosine, upstream of a thymine, within the first exon of the gene. FIG. 2B instead represents the comparison by the nucleotide sequence, with related translation codons, of the wild-type gene UROD (WT) with respect to the mutated gene (I 29), the sequence of which are reported in SEQ ID NO:1 and SEQ ID NO:2. In this figure it is possible to see how the mutation, the insertion of a cytosine upstream of the thymine in position 86 determines, further downstream, the formation of a stop codon.

(39) Effects of the Mutation of the UROD Gene

(40) As can be seen from the graphs in FIG. 3, Nannochloropsis gaditana CCAP 849/21 has shown a 20% reduction of the chlorophyll content with respect to the wild-type strain of the same species, which accompanies greater photosynthetic electron transport, an indicator of a greater ability to use light energy for photosynthesis.

(41) In fact, FIG. 3A shows a graph representing the chlorophyll content of microalgae of the wild-type strain (left column) and the chlorophyll content of Nannochloropsis gaditana CCAP 849/21 (right column). The statistically significant difference between the two strains is identified with an asterisk (one-way ANOVA, p-value <0.05). Instead, FIG. 3B shows a graph representing the saturation kinetics of the ETR (Electron Transfer Rate), determined through PAM (Pulse Amplitude Modulation) fluorimeter at increasing light intensities over time. The dark grey shows the wild-type strain whereas the light grey shows Nannochloropsis gaditana CCAP 849/21.

(42) Productivity Evaluation

(43) The productivity of Nannochloropsis gaditana CCAP 849/21 was also evaluated when grown inside an experimental photobioreactor. The aim of this study was that of verifying the effect of the mutation of the UROD gene with reference to the production of biomass over time, when the culture reaches a high optical density level and the penetration of the visible radiation becomes limiting. To simulate industrial conditions a fed-batch reactor was used in which, every two days, the culture was diluted until an initial pre-established concentration was reached. The productivity was evaluated in terms of biomass (FIG. 4).

(44) FIG. 4A schematically represents the experimental photobioreactor used: it comprises a Drechsel bottle having a diameter of 5 cm, a tube for insufflating a mixture of air and 5% CO.sub.2 and a set of LED lamps for providing the visible radiation. FIG. 4B shows a graph representing the concentration of the biomass, contained in the photobioreactor, over time. The initial concentration of the biomass was set to 15010.sup.6 cells/ml. By simulating the industrial conditions of a fed-batch reactor, the culture was brought back to the initial concentration every two days through dilution with fresh culture medium.

(45) In order to evaluate the productivity of Nannochloropsis gaditana CCAP 849/21 with respect to the wild-type strain, expressed in terms of concentration of biomass inside the photobioreactor, two culture conditions were considered:

(46) 1) initial concentration of 15010.sup.6 cells/ml; light intensity 400 mol photons m.sup.2s.sup.1;

(47) 2) initial concentration of 25010.sup.6 cells/ml; light intensity 1200 mol photons m.sup.2s.sup.1.

(48) The results of the cultures carried out with these two conditions are shown in Table 2.

(49) TABLE-US-00002 TABLE 2 Dry weight of the biomass (g/l/day) Culture condition 1 Culture condition 2 Wild-type strain (n = 37) 0.33 0.05 0.48 0.11 Nannochloropsis 0.39 0.05** 0.47 0.12 gaditana CCAP 849/21 (n = 10) **statistically significant difference between the wild-type strain and Nannochloropsis gaditana CCAP 849/21 (one-way ANOVA, p-value < 0.05).

(50) As can be seen in Table 2, Nannochloropsis gaditana CCAP 849/21 demonstrated higher productivity with respect to the wild-type strain in the condition of culture 1, where it manages to accumulate over 14% more biomass with respect to the wild-type strain whereas, in culture condition 2, it does not demonstrate any significant differences with respect to the wild-type strain.

(51) As the condition of culture 2 envisages a high level of light intensity (1200 mol photons m.sup.2s.sup.1), this latter datum demonstrates that the mutation of the UROD gene of Nannochloropsis gaditana CCAP 849/21 does not make the strain photosensitive. Also in high light intensity conditions Nannochloropsis gaditana CCAP 849/21 in fact demonstrates equal productivity of biomass with respect to the wild-type strain.

(52) For both strains, under the same culture conditions, the chlorophyll content was also evaluated (FIG. 5). In fact, FIG. 5 shows a graph expressing the concentration of chlorophyll, per microalga cell (pg Chl/cell), contained both in the wild-type strain (dark grey), and in Nannochloropsis gaditana CCAP 849/21 (light grey), when grown in the aforesaid culture conditions (culture condition 1, indicated in the graph as 150_400 and culture condition 2, indicated in the graph as 250_1200; the statistically significant differences were highlighted with an asterisk, one-way ANOVA, p-value <0.05). From FIG. 5 it is clear that, in culture condition 1, Nannochloropsis gaditana CCAP 849/21 demonstrates having a 43% lower chlorophyll concentration with respect to the wild-type strain; whereas in culture condition 2 the concentration of chlorophyll between the two strains is not significantly different.

(53) The data that demonstrate the reduction of the chlorophyll concentration and the increased productivity of biomass of Nannochloropsis gaditana CCAP 849/21 when grown in the condition of culture 1, confirm the hypothesis that a reduced chlorophyll content is more advantageous in limited light conditions (such as in the condition of culture 1), as this reduced chlorophyll content guarantees greater diffusion of the limited visible radiation within the entire photobioreactor, also in high cell density conditions, thus guaranteeing a high level of productivity of biomass over time.

Example 3 [Non Photochemical Quenching (NPQ) and Productivity]

(54) In order to evaluate the Non Photochemical Quenching (NPQ) of Nannochloropsis gaditana CCAP 849/21 with respect to the wild-type strain and to the mutant of Nannochloropsis gaditana E2 obtained as disclosed in the article of Perin et al., Generation of random mutants to improve light-use efficiency of Nannochloropsis gaditana cultures for biofuels production, Biotechnol Biofuels (2015) 8:161, above reported, the same were subjected to the following cultures conditions: initial concentration of 15010.sup.6 cells/ml; light intensity up to 2000 mol photons m.sup.2s.sup.1 (FIG. 6). FIG. 6 clearly shows that the Nannochloropsis gaditana CCAP 849/21 (G14) reaches a NPQ activation comparable to the wild-type strain, while the mutant of Nannochloropsis gaditana E2 reaches a NPQ activation lower than both Nannochloropsis gaditana CCAP 849/21 (G14) and wild-type strain.

(55) Moreover, the productivity of Nannochloropsis gaditana CCAP 849/21 with respect to the wild-type strain and to the mutant of Nannochloropsis gaditana E2 was evaluated under these culture conditions: initial concentration of 15010.sup.6 cells/ml; light intensity 500 mol photons m.sup.2s.sup.1 (FIG. 7). FIG. 7 clearly shows that the Nannochloropsis gaditana CCAP 849/21 (G14) has a growth rate (expressed in d.sup.1) higher than the wild-type strain, while the mutant of Nannochloropsis gaditana E2 has a growth rate lower than both Nannochloropsis gaditana CCAP 849/21 (G14) and wild-type strain.