WO2015071908A1 - Chlorella ohadii and uses thereof - Google Patents

Abstract

The present invention relates to novel microalgae, compositions comprising same and methods of use thereof. The microalgae show high growth rate under wide conditions including extreme light intensities and is used for production of algal biomass and/or products thereof and for remediation of wastewater and contaminated soil.

Description

CHLORELLA OHADII AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to novel species of the green alga Chlorella, named Chlorella ohadii methods if use thereof and for production of same.

BACKGROUND OF THE INVENTION

Biological soil crusts (BSC) play an important role in stabilizing sandy desert areas by reducing wide erosion and by their impact on biotic compositions. Destruction of these crusts is considered an important promoter of desertification in arid and semi- arid regions.

BSCs are formed by adhesion of soil particles to extracellular polysaccharides excreted mostly by filamentous cyanobacteria, the main primary producers in desert crusts. Other microorganisms including fungi, microalgae, lichens and bacteria, are also present, particularly in humid areas that are often covered by a thick crust.

BSCs represent one of the harshest environments in nature (see, for example, Belnap et al., 2004. Oecologia 141, 306-316; Biidel and Veste, 2008. Biological crusts. In: Ecological Studies, S.W. Breckle, A. Yair, and M. Veste, eds, pp. 149-155; Garcia- Pichel et al., 2013. Science 340, 1574-1577; and references therein). Organisms inhabiting this ecosystem face frequent hydration/dehydration cycles, with hydration by early morning dew and dehydration with the rising sunlight; temperature amplitudes of from subfreezing during winter nights to 60°C in mid-summer days; limiting nutrient supplies and vast osmotic potential changes ranging from close to pure rainwater to salt crystals on the crust's upper layer. The photo synthetic organisms, in addition, must cope with extreme light intensities, much higher than required to saturate photosynthetic demand, concomitant with declining photochemical activity consequent on the desiccation (Heber, 2008. Planta 228, 641-650; Ohad et al., 2010. Plos One 5, el 1000; Ohad et al., 2011. Physiol. Plant. 142, 79-86). To cope with such harsh conditions the organisms inhabiting the BSCs must possess survival mechanisms, the nature of which remains largely unknown (Ohad et al., 2005. Photochem. Photobiol. Sci. 4, 977-982; Wright et al., 2005. J. Biol. Chem. 280, 40271-40281). Crucial for survival is the ability to sense diurnal changes in environmental conditions and rapidly activate metabolism and growth in the short periods when water and sufficient light intensity are available, but to turn metabolism off during desiccation. To cope with the limiting CO2 levels resulting from rapid increase of pH in the crust a CO2 concentrating mechanism (CCM), documented in many photosynthetic microorganisms, was described in several crust cyanobacteria.

To cope with the combination of declining water availability and rising light intensity to levels much higher than that required to saturate photosynthetic demand, photosynthetic organisms inhabiting the BSCs must possess efficient mechanisms to dissipate excess light energy (Heber, 2008). As an example, exposure of BSC- inhabiting cyanobacterium Microcoleus sp. to excess light led to a remarkable light energy quenching while retaining a maximal oxygen evolution rate (Ohad et al., 2010, ibid; Ohad et al., 2011, ibid).

In search of eukaryotic photosynthetic organisms able to withstand the harsh BSCs environment, the inventors of the present invention and co-workers isolated a new green alga that was named Chlorella ohadii (Treves et al., 2013. FEMS Microbiol. Ecol. 86, 373-380) a close relative of C. sorokiniana.

Widespread water pollution with nutrients including nitrogen and phosphate and with other contaminants is among the major environmental concerns. Although many conventional techniques and approaches are available for pollution prevention and control, these methods are usually very expensive with high energy consumption. Large quantities of sludge and/or liquid wastes generated from these systems are difficult to deal with and may also pose the risk of creating secondary contamination.

The use of renewable energy sources is becoming increasingly necessary due to today's high energy prices and impacts of climate change. Microalgae and cyanobacteria are very efficient solar energy converters when compared with land plants. In addition to fast biomass production, they can produce a great variety of metabolites. Some species are especially valuable as they can be harnessed for oil production. Other species can be used to clean polluted water, including wastewater. For example, U.S. Patent Application Publication No. 2010/0021968 discloses algal species and compositions, methods for identifying algae that produce high lipid content, possess tolerance to high CO2, and/or can grow in wastewater and methods for using such algae for lipid production, wastewater remediation, waste gas remediation, and/or biomass production.

U.S. Patent Application Publication No. 2013/0164322 discloses an extremophile green alga designated as Scenedesmus species Novo, from Jemez warm water springs, New Mexico, capable of producing high levels of microalgal biomass in wastewater under harsh ambient climatic conditions, and of yielding high levels of lipids and carotenes. The microalgae are useful in the production of biofuels, fertilizers, dietary nutrients, pharmaceuticals, polymers, as bio-filters to remove nutrients and other pollutants from wastewaters, in space technology and as laboratory research systems.

However, as of today the commercial potential of algal biomass is only scarcely exploited. One of the obstacles in the development of large scale algae farms is the requirement for lands in climate zones that can otherwise be used for agriculture. Another shortcoming is the average low growth rate observed under latge scale growth conditions.

Thus, there is a need for and would be highly advantageous to have microalgae species that can efficiently grow under optimized as well as harsh environmental conditions while maintaining their normal metabolism and high biomass production.

SUMMARY OF THE INVENTION

The present invention relates to a new species of green alga named Chlorella ohadii and to methods of use thereof. The algae were isolated from a biological sand crust (BSC) in the North- West Negev area in Israel, where its survival depends on close association with filamentous cyanobacteria or extracellular polysaccharides produced by the cynobacteria.

The present invention is based in part on the unexpected discovery that the algae are able to grow in a growth medium in an isolated form. Furthermore, the algae display an unparalleled growth rate when exposed to optimal conditions, a phenomenon contradictory to hitherto accepted dogma that organisms capable of growing under stress conditions imposed by their environments show reduced performance under optimal conditions. The productivity of the algae of the present invention is essentially unaffected by high light intensities of up to about twice the intensity of full sunlight, and exposure to such high irradiance results in significant increase in lipid and carbohydrate accumulation.

The present invention characterizes for the first time the sequences of the alga gene encoding the large subunit of the enzyme RubisCO (rbcL) and of the alga 18S ribosomal RNA gene (18S rRNA).

According to one aspect, the present invention provides isolated Chlorella ohadii composition, wherein the isolated Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l (rbcL), SEQ ID NO:2 (18S rRNA) and complements thereof.

According to some embodiments, the Chlorella ohadii genome comprises SEQ ID NO: l or a complement thereof. According to other embodiments, the Chlorella ohadii genome comprises SEQ ID NO:2 or a complement thereof. According to additional embodiments, the Chlorella ohadii genome comprises SEQ ID NO: l and SEQ ID NO:2 and/or complements thereof .

According to additional embodiments, the chlorophyll a/b ratio within the C. ohadii cells is from about 10: 1 to about 13: 1. According to certain exemplary embodiments, the ratio is 12: 1.

The isolated C. Ohadii was deposited in The Spanish Bank of Algae (BEA-Banco Espanol de Algas) on November 12, 2014 (deposit number not yet available).

According to additional aspect, the present invention provides a substantially pure culture comprising a composition of an isolated microalgae Chlorella ohadii, wherein the isolated Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l (rbcL), SEQ ID NO:2 (18S rRNA) and complements thereof and a growth medium.

According to certain embodiments, the microalgae produces a biomass of at least 10⁹ cells/ml when the culture is grown under light intensity of 100-3,500 μιηοΐε photons m^"2 s^"1. According to certain embodiments, the culture is grown under light intensity of above 500 μιηοΐε photons m^"2 s^"1, typically between 1,000-3,500 μιηοΐε photons m^"2 s^"1. According to certain exemplary embodiments, the culture is grown under light intensity of 3,000 μηιοΐε photons m^"2 s^"1. Without wishing to be bound by any specific theory or mechanism of action, this high biomass density may be due to hitherto not observed short generation time of 2.4-2.7 h.

According to further embodiments, the Chlorella ohadii composition comprises elevated content of lipids when the culture in grown under light intensity of above 1000 μιηοΐε photons m^"2 s^"1 compared to the lipid content when the culture is grown at a light intensity below 1000 μιηοΐε photons m^"2 s^"1. According to additional embodiments, the Chlorella ohadii composition comprises elevated content of carbohydrate when the culture in grown under light intensity of above 1000 μιηοΐε photons m^"2 s^"1 compared to the carbohydrate content when the culture is grown at a light intensity below 1000 μιηοΐε photons m^"2 s^"1. According to certain embodiments, the culture is grown under light intensity of between 1,000-3,500 μιηοΐε photons m^"2 s^"1. According to certain exemplary embodiments, the culture is grown under light intensity of 3,000 μιηοΐε photons m^"2 s^"1. According to some embodiments, the energy content of the microalgae is 5,200 cal/g dry weight when the culture is grown under light intensity of 3,000 μιηοΐε photons m^"2 s^"1 for at least 2 h.

According to certain embodiments, the culture is grown under photoautotrophic conditions, wherein the C0₂ source is selected from the group consisting of ambient air and ambient air supplemented with CO2 to reach 5%. According to other embodiments, the culture is grown under photomixotrophic conditions, wherein the carbon source is selected from the group consisting of a combination of ambient air and acetate and a combination of ambient air supplemented with CO2 to reach 5% and acetate.

According to certain embodiments, the culture is grown at a temperature of between 15°C-45°C. According to certain exemplary embodiments, the culture is grown at a temperature of between 25°C-37°C. According to yet additional exemplary embodiments, the culture is grown at a temperature of 35°C.

According to additional aspect, the present invention provides a method for producing algal biomass and/or products thereof, the method comprising culturing a Chlorella ohadii under conditions suitable for the Chlorella ohadii proliferation and photosynthetic activity, wherein said Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l (rbcL), SEQ ID NO:2 (18S rRNA) and complements thereof.

According to some embodiments, the Chlorella ohadii genome comprises SEQ ID NO: l or a complement thereof. According to other embodiments, the Chlorella ohadii genome comprises SEQ ID NO:2 or a complement thereof. According to other embodiments, the culturing conditions comprise growth under light intensity in the range of 100-3,500 μιηοΐε photons m^"2 s^"1. According to certain embodiments, the culturing is carried out under light intensity of above 500 μιηοΐε photons m^"2 s^"1. According to other embodiments, the culturing is carried out under light intensity of above 1,000 μιηοΐε photons m^"2 s^"1. According to certain exemplary embodiments culturing is carried out under light intensity of 3,000 μιηοΐε photons m^"2 s^{" l}. According to certain additional embodiments, the culture is exposed to the light intensity of above 500 or above 1,000 μιηοΐε photons m^"2 s^"1 for at least 2 hours.

According to additional embodiments, the culturing conditions comprise growth at a temperature of between 15°C-45°C. According to certain exemplary embodiments, the culturing conditions comprise growth at a temperature of between 25°C-37°C. According to yet additional exemplary embodiments, the culturing conditions comprise growth at a temperature of 35°C.

Any growth medium as is known in the art can be used for culturing the C. ohadii microalgae according to the methods of the present invention. According to some embodiments, the culturing conditions comprise photoautotrophic conditions. According to theses embodiments, the sole carbon source is ambient air or C0₂ enriched ambient air. According to other embodiment, the culturing conditions comprise photomixotrophic conditions. According to these embodiments, an additional carbon source is added to the culturing medium. According to certain exemplary embodiments, the present invention provides a method for producing algal biomass and/or products thereof, the method comprising culturing Chlorella ohadii in a growth medium comprising at least one carbon source under conditions suitable for the Chlorella ohadii proliferation, wherein said Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2 and complements thereof.

According to certain embodiments, the carbon source is acetate. According to some embodiments, the Chlorella ohadii is grown under photomixotrophic conditions and the culturing conditions comprise light source. According to other embodiments, the Chlorella ohadii is grown under heterotrophic conditions in the dark.

According to certain embodiments, the method further comprises harvesting the algal biomass from the culture. Any method known in the art for microalgae harvest can be used according to the teachings of the present invention. According to some embodiments, the step of harvesting further comprises drying the algae after harvest.

The algal biomass may be used per se as a food, particularly as a source for proteins for aquaculture animals including fish, crustaceans and mollusks; for mammals including human; or for poultry. The algal biomass can be also used in the cosmetic industry as a thickener, a water-binding agent, a coloring agent and the like. According to additional embodiments, the algal biomass can be used as fertilizer additive.

Thus, according to certain aspects the present invention provides a biomass of Chlorella ohadii produced by the methods of the present invention. According to certain embodiments, the algal biomass is formulated as a food or as a food supplement. According to certain embodiments, the food or food supplement are useful for feeding aquaculture animals, mammals or poultry.

According to yet addition aspect, the present invention provides an edible composition comprising Chlorella ohadii composition, wherein the Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2 and complements thereof, further comprising an excipient, diluent or carrier suitable for oral consumption.

According to some embodiments, the edible composition further comprises at least one additional active agent. According to certain embodiments, the active agent is selected from the group consisting of anti-oxidants, fatty acids, micro-nutrient and the like.

According to some embodiments, the edible composition is formulated as an animal food composition or as an animal food supplement. According to some exemplary embodiments, the animal food composition is for feeding aquatic animals. According to other exemplary embodiments, the animal food composition is for feeding poultry. According to yet other embodiments, the edible composition is formulated for human consumption or as human food supplement.

According to some embodiments, the algal biomass is dried before it is formulated into the edible composition.

According to certain embodiments, the composition is formulated in a form selected from the group consisting of a capsule, dragee, pill, tablet, gel, liquid, suspension, slurry, powder, pellets, cubes and flakes. Each possibility represents a separate embodiment of the present invention.

Alternatively, a product produced by the algae cells and excreted from the algae biomass is harvested. According to some embodiments, the excreted product is gaseous, particularly hydrogen.

Yet alternatively, the method further comprises isolating from the algal biomass at least one product produced by the algal cells. According to some embodiments, the product is an endogenous component naturally present in the algal cell. According to certain embodiments, the algal cell product is selected from the group consisting of lipids, carbohydrates, proteins, vitamins and pigments.

According to some exemplary embodiments, the product is a metabolite useful in controlling the growth of toxic cynobacteria. According to certain exemplary embodiments, the toxic cynobacteria is Microcystis sp.

According to other embodiments, the product is a xenogeneic product not naturally produced by the algal cells or is a product naturally produced in small amounts.

According to these embodiments, at least one cell of the Chlorella ohadii comprises at least one exogenous transcribeable polynucleotide encoding a product of interest. According to some embodiments, the product of interest is an RNAi molecule.

According to other embodiments, the product of interest is a protein or a polypeptide. Any method as is known in the art can be used for transforming Chlorella ohadii with a transcribable polynucleotide.

According to some embodiments, the protein or polypeptide has nutritional value. According to other embodiments, the protein or polypeptide has an enzymatic activity. According to some embodiments, the at least one cell comprises a plurality of transcribeable polynucleotides encoding an array of proteins or polypeptides having an enzymatic activity for producing at least one end product of interest.

According to yet additional aspect, the present invention provides a method for reducing the amount of at least one undesired substance present in waste, the method comprising culturing Chlorella ohadii in a culture medium comprising waste, wherein the Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l (rbcL), SEQ ID NO:2 (18S rRNA) and complements thereof and wherein the culturing conditions are suitable for proliferation of said Chlorella ohadii and for photosynthetic activity of said Chlorella ohadii for at least 8 h/day. According to some embodiments, the Chlorella ohadii genome comprises SEQ ID NO: l or a complement thereof. According to other embodiments, the Chlorella ohadii genome comprises SEQ ID NO:2 or a complement thereof.

According to certain embodiments, the waste is selected from the group consisting of wastewater and contaminated soil. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the method further comprises aerating the culture medium when the culturing conditions are not suitable for photosynthesis.

According to certain other embodiments, the method comprises culturing the Chlorella ohadii under continuous photosynthetic conditions.

According to certain embodiments, the method further comprises adding to the culture medium aerobic microorganisms capable of reducing the amount of the at least one undesired compound. According to certain exemplary embodiments, the aerobic microorganisms are bacteria. According to certain embodiments, the culture medium comprises at least 50% wastewater or contaminated soil. According to other embodiments, the culture medium comprises at least 60%, at least70%, at least 80% or at least 90% or more wastewater or contaminated soil. According to certain exemplary embodiments, the culture medium comprises 100% wastewater or contaminated soil. According to certain exemplary embodiments, the wastewater is partially purified. According to these embodiments, the wastewater added to the microalgae culture medium is essentially free of large debris.

According to other embodiments, the culture is grown under light intensity of 100-3,500 μιηοΐε photons m^"2 s^"1. According to certain embodiments, the culture is grown under light intensity of above 500 μιηοΐε photons m^"2 s^"1. According to certain exemplary embodiments the culture is grown under light intensity of 3,000 μιηοΐε photons m^"2 s^"1. According to certain additional embodiments, the culture is exposed to the light intensity of above 500 μιηοΐε photons m^"2 s^"1 for at least 2 hours.

According to additional embodiments, the culture is grown at a temperature of between 15°C-45°C. According to certain exemplary embodiments, the culture is grown at a temperature of between 25°C-37°C. According to yet additional exemplary embodiments, the culture is grown at a temperature of 35°C.

According to yet additional aspect, the present invention provides a composition for reducing the amount of at least one undesired substance present in wastewater or contaminated soil, the composition comprising isolated Chlorella ohadii and at least one type of aerobic microorganism capable of reducing the amount of the at least one undesired compound.

According to certain exemplary embodiments, the aerobic microorganisms are bacteria. According to some embodiments, the composition further comprises culture medium suitable for the growth of Chlorella ohadii and the at least one aerobic microorganism.

Other objects, features and advantages of the present invention will become clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows electron micrograph of a dividing C. ohadii cell isolated from a biological sand crust located near the Egypt-Israel border. The bar represents 1 μιη.

FIG. 2 demonstrates that C. ohadii growth shows ultradian rhythms. The cells were grown in TAP medium using a photo -bioreactor at 35°C and light intensities of 3000 μηιοΐε photons m^"2 s^"1 (Fig. 2A and Fig. 2B) and 100 μηιοΐε photons m^"2 s^"1 (Fig. 2C). Fig. 2A: Cell densities (presented as OD735 nm). Five biological independent experiments were performed using cells withdrawn from log phase cultures; the data were essentially identical. Fig. 2B and Fig. 2C show cell density (OD 735 nm), pH and oxygen level. The larger the deviation of 0₂ level from that expected at equilibrium with the bubbled air (100% equilibrium is about 220 μΜ 0₂) the higher the rate of 0₂ exchange between the cells and their medium. Data shown in Fig. 2B and Fig. 2C represent 4 different biologically independent experiments for each of the light intensities. FIG. 3 demonstrates the resistance of C. ohadii to photoinhibition. Fig. 3 A and Fig. 3B: Cells of C. ohadii and C. sorokiniana were withdrawn from log phase cultures and placed in TAP media (Fig. 3 A) or TAP without acetate (Fig. 3B) aerated at 100 mL air min^"1 under 30°C, 3500 μιηοΐε photons m^"2 s^"1 for 24 h. Initial cell densities corresponded to OD 730 nm = 0.06. Similar results were obtained in 3 biologically independent experiments. Fig. 3C and Fig. 3D: The levels of protein Dl in C. ohadii and C. sorokiniana during an experiment such as presented in Fig. 3 A or Fig. 3B, respectively. The cultures used in the data shown in Fig. 3D were provided with lincomycin (200 μg/mL) at time zero. Aliquots were withdrawn at TO, 6 h and 24 h and the level of Dl protein was examined by Western blot. Fig.3E shows sensitivity of photosynthetic 0₂ evolution to commonly used inhibitors of PSII, DCMU, atrazine, bromoxynil and ioxynil. Cell suspensions (corresponding to 20 μg chlorophyll/mL) were placed in the 0₂ electrode chamber, 30°C, 2 mM NaHC0₃ and light intensity of 3000 μιηοΐε photons m^"2 s^"1. The results shown here are from 3 biologically independent experiments. The 100% corresponds to 300 μιηοΐε 0₂ mgChl^"1 h^"1. FIG. 4 shows thermoluminescence (Fig. 4A) and photosynthetic 0₂ evolution and fluorescence (Fig. 4B) in C. ohadii exposed to high illumination. Fig. 4A shows thermoluminescence arising from recombination between QB^~/[Mn cluster]^"1" electron-hole pairs (Q band) and in the presence of 10 μΜ DCMU (B band) where recombination occurs between QA^~/[Mn cluster]^"1" electron-hole pairs. The signal intensity is provided in Counts per Second (CPS) detected by the photomultiplier. Three biologically independent experiments were performed yielding essentially identical data. Fig. 4B shows the dependence of 0₂ evolution and Fv/Fm on the duration of exposure of C. ohadii to excess light. The 100% on the Y axis corresponds to maximal C0₂-dependent 0₂ evolution (350+15 μιηοΐε 0₂ mg Chi^"1 h^"1). The variable fluorescence (Fv/Fm) was measured on dark adapted cells (2 min). The light intensity was then raised to 3000-3500 μιηοΐε photons m^"2 s^"1. The 100% corresponded to Fv= 0.66+0.024. Solid lines represent photosynthetic activity (0₂ evolution) and dashed lines represent fluorescence. Black lines: cells grown in TAP; grey lines: cells grown in a similar medium but without acetate.

FIG. 5 demonstrates the acclimation of C. ohadii to excess light. Fig. 5A: Effect of excess light on light-dependent photosynthetic 0₂ evolution. Three biological independent experiments were performed; data depicted here are the average values. The variability was lower than + 5% of the average. Fig. 5B: Effect of excess light on cellular content of lipids, carbohydrates and proteins. Lipids and carbohydrates levels were examined in C. ohadii cells exposed to 3000 μιηοΐε photons m^"2 s^"1 for 2 h. The relative abundance of each pool in control C. ohadii was estimated by Fourier transform infra-red (FTIR) spectroscopy.

(C) The Nile red signal as affected by the duration of exposure of C. ohadii cells to excess light. Nile red (500 μg of 9-diethylamino-5Hbenzo[a]phenoxazine-5-one per 1 ml acetone was used as a fluorescent probe of intracellular lipids and hydrophobic domains of proteins. To estimate lipid content, 10 μΐ of Nile red was added to 200 μΐ of C. ohadii cells withdrawn before and after exposure to 3500 μιηοΐε photons m^"2 s^"1. Fluorometric analyses were performed 10 min after staining using a plate fluorometer with a 485 nm narrow band excitation filter and a 590 nm narrow band emission filter.

FIG. 6 shows the effect of excess light on the cell structure and C0₂-dependent photosynthetic 0₂ evolution. Fig.6A-Fig. 6C: Electron micrographs showing C. ohadii cells grown in medium TAP, 100 μιηοΐε photons m^"2 s^"1 and 30°C (Fig. 6A) and after exposure for 2 h to either 3000 μιηοΐε photons m^"2 s^"1 (Fig. 6B) or to TAP lacking acetate media bubbled with air (Fig. 6C). Pyrenoids are marked P. The bars represent 500 nm. Fig. 6D: Number of packed thylakoids in the control and after 2 h of exposure to the high illumination. Thylakoids were counted in over 60 randomly selected cross sections from 8 fields for each treatment. Fig. 6E: The rate of photosynthetic 0₂ evolution as affected by inorganic carbon concentration provided to C. ohadii cells treated as in Fig. 6A-C above. Cell suspensions (corresponding to 20 μg chlorophyll/mL) were placed in an 0₂ electrode chamber, 30°C, with light intensity 1300 μηιοΐε photons m^"2 s^"1. LC: low C0₂. HL: High light.

FIG. 7 shows the correlation between C. ohadii cell count and optical density at 735 nm. Samples were withdrawn during the growth of C. ohadii in batch cultures as described in Methods. Three biologically independent experiments were performed. FIG. 8 shows Coomassie blue stained gels used for the Western blot analyses shown in Fig. 3C (Fig. 8A) and Fig. 3D (Fig. 8B).

FIG. 9 shows electron micrograph (EM) picture of C. ohadii cells 2 h after transfer from growth medium (TAP, 30°C, 100 μιηοΐε photons m^"2 s^"1) to medium of TAP lacking acetate bubbled with air, under otherwise identical conditions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel Chlorella species, named Chlorella ohadii, which is advantageous over hitherto known Chlorella species in its capability to rapidly grow and produce biomass under intense irradiation as high as twice the sunlight irradiation, while its photosynthetic activity is not negatively affected and furthermore, while producing high amounts of lipids and carbohydrates. These characteristic make the microalgae highly suitable for outdoor growth in areas not suitable for agriculture and for production of algal biomass to be used per se or as a factory for the synthesis of biological substances. Definitions

The terms "microalga" or "alga" in its single or plural forms are used herein interchangeably and refer to unicellular alga species which exist individually, or in chains or groups. According to certain embodiments, the terms refer to the novel Chlorella of the present invention, Chlorella ohadii. The term "Chlorella ohadii" as used herein refers to a microlaga comprising in its genome at least one of SEQ ID NO: l, SEQ ID NO:2 or a sequence complemented thereto. Cells of C. ohadii are about 2 μιη in diameter.

The isolated C. Ohadii was deposited in The Spanish Bank of Algae (BEA-Banco Espanol de Algas) on November 12, 2014 (deposit number not yet available). The term "isolated" as used herein refers to at least 90% of the algae present in the composition being of the recited algal type of Chlorella ohadii. According to certain embodiments, at least 95%, 96%, 97%, 98%, 99% or more of the algae present in the composition are of the species Chlorella ohadii. The isolated Chlorella ohadii. - composition can be cultured or stored in solution, frozen, dried, or on solid agar plates. The term "substantially pure" when referring to a culture comprising the microalgae Chlorella ohadii refers to a culture comprising at least 90% of isolated Chlorella ohadii composition disclosed herein out of the total microorganisms present in the culture. According to certain embodiments, at least 95%, 96%, 97%, 98%, 99% or more of the culture microorganisms are the Chlorella ohadii composition. According to a first aspect, the present invention provides isolated Chlorella ohadii composition, wherein the isolated Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l (rbcL), SEQ ID NO:2 (18S rRNA) and complements thereof.

It is to be expectedly understood that each of SEQ ID NO: l and SEQ ID NO:2 disclosed herein for the first time or sequences complementary thereto can serve as a marker for the novel Chlorella of the present invention, that distinguishes Chlorella ohadii from any hitherto known Chlorella species.

The algae of the invention were isolated from biological sand crust from the desert in Israel. Unexpectedly, the present invention now shows that contrary to its natural growth characteristics requiring the presence of filamentous cyanobacteria or extracellular polysaccharides produced therefrom, the algae grow well in an axenic culture.

In addition, the ability to acclimate to extreme environments is usually accompanied by reduced performance under optimal conditions (Brock, 1978. Thermophilic microorganisms and life at high temperatures; New York: Springer- Verlag; Kushner, 1978. Microbial life in extreme environment; London: Academic press). Unexpectedly, this is not the case for C. ohadii that exhibits unparalleled growth rate and withstands an array of stressing conditions in its natural habitat. To the best ability of the inventors to ascertain, the division times observed for C. ohadii (Table 1 below) are the fastest ever reported for a photosynthetic organism and close to the maximal attained by unicellular heterotrophic eukaryotes (Bowler et al., 2010. Curr. Opin. Plant Biol. 13, 623-630; Flynn et al., 2010. J. Phycol. 46, 1-12; Raven et al., 2013. Phil. Trans. R. Soc. B. 368, 2012.026).

The isolated Chlorella ohadii composition of the present invention is characterized in that (i) it is able to grow as an axenic culture as well as non-axenic culture; (ii) it can grow under various trophic conditions, including photoautotrophic conditions, photomixotrophic conditions and heterotrophic conditions; (iii) it shows an extremely high proliferation rate (iv); it is essentially non sensitive to photoinhibition; and (v) it shows ultradian growth rhythms with elevated photosynthesis rate under high light intensities, resulting in elevation of oxygen production and accumulation of lipids and carbohydrates.

According to additional aspect, the present invention provides a substantially pure culture comprising a composition of the isolated microalgae Chlorella ohadii and a growth medium.

According to certain embodiments, the microalgae produces a biomass of at least 10⁹ cells/ml when the culture is grown under light intensity of 100-3,500 μιηοΐε photons m -2 s -1.

Ultradian rhythms were observed in various, mostly multicellular, organisms and models describing this phenomenon were proposed. However, in most cases the mechanisms involved and their interaction with the circadian rhythms are largely unknown. Ultradian rhythms were reported in several unicellular organisms such as yeast (Robertson et al., 2008. Proc. Natl. Acad. Sci. U. S. A. 105, 17988-17993), algae (Jenkins et al., 1990. J. Interdiscip. Cycle Res. 21, 75-80), cyanobacteria (Cerveny et al., 2013. Proc. Natl. Acad. Sci. U. S. A. 110, 13210-13215) and photo synthetic bacteria (Min et al., 2005. FEBS Lett. 579, 808-812). In the latter case a shift from a circadian to an ultradian rhythm is strongly affected by the ambient 0₂ level that alters the redox state of the cells. The mere fact that an ultradian rhythm was observed in unicellular organisms clearly indicates that the population is synchronized but the nature of the signal harmonizing the cells is not known.

The ultradian rhythm is highly pronounced in C. ohadii under both high and low light intensities (3000 and 100 μιηοΐε photons m^"2 s^"1, Figure 2B and Figure 2C, respectively) although the magnitude is affected by the illumination. The large alterations in 0₂ exchange and H, characteristic of the transitions between the various growth phases reflects major redox/metabolic changes such as observed during shifts in light intensity or carbon supply (Figure 5 and Figure 6). The rising maximal O2 evolution rate after exposure of cells grown in TAP (Tris, Acetate, Phosphate buffer) to high light or photoautotrophic conditions (Figure 6E) reflects a metabolic shift from acetate utilization to CO2 reduction. Metabolic shifts such as transfer from photomixotrophic to photoautotrophic metabolism during growth under constant conditions were also reflected by changes in media pH, O2 level (Figure 2) and CO2 exchange. The fastest photosynthetic rate (where the highest O2 level was observed) occurred when the growth rate was relatively slow, as if the cells were accumulating reserve resources for the next growth burst. A rise in respiratory activity during the fast growth phase (phase III) resulted in net O2 consumption and acidification due to CO2 release (Figure 2B and Figure 2C), however it should be noted that protons and O2 fluxes were not coupled throughout the cycle.

According to certain embodiments, the culture is grown at a temperature of between 15°C-45°C. According to certain exemplary embodiments, the culture is grown at a temperature of between 25°C-37°C. According to yet additional exemplary embodiments, the culture is grown at a temperature of 35°C. The growth of Chlorella ohadii is hardly affected by light intensity, and the alga is resistant to very high light intensities. According to additional embodiments, the culture is grown under light intensity of 100-3,500 μιηοΐε photons m^"2 s^"1. According to certain embodiments, the culture is grown under light intensity of above 500 μιηοΐε photons m^"2 s i. According to certain exemplary embodiments, the culture is grown under light intensity of y between 1,000-3,500 μιηοΐε photons m^"2 s^"1. According to certain further exemplary embodiments, the culture is grown under light intensity of 3,000 μιηοΐε photons m^"2 s^"1.

Modulation of the light-harvesting cross sections and efficiency of energy transfer from the antenna to the core pigments is one of the strategies used by photosynthetic organisms to acclimate to changing ambient conditions in their habitat, such as light intensity. Attempts are being made to reduce the antenna size in algae. This is expected to lower the photon flux to the reaction centre under excess light intensity and thus improve light penetration in the water column of algal growth facilities. In a recent study Melis and colleagues were able to raise a mutant of C. reinhardtii, tla3, with both a lower chlorophyll content per cell and a higher Chi a/b ratio (13: 1) than in the corresponding wild-type (3: 1) and with improved solar energy conversion efficiency and photosynthetic productivity (Min et al., 2005, ibid). The chlorophyll a/b ratio in C. ohadii is naturally very high, 12: 1, about 4-fold higher than in other green algae such as C. reinhardtii. Chlorophyll b is solely located in the light-harvesting complexes but its low abundance suggests a relatively reduced size of the light-harvesting cross section. Without wishing to be bound by any theory or mechanism of action, a relatively small light-harvesting antenna in C. ohadii may reduce the photon flux to the reaction center, potentially minimizing the damaging excess light.

Taken together, the present invention now discloses unique functional properties of the photosynthetic machinery in C. ohadii, particularly of PSII lending support to the suggestion that it differs from that observed in other photosynthetic organisms, leading to essentially complete stability even under very high light intensity. The Western blot analyses (Figure 3C-D and Figure 8) showed a slow decline in Dl level even in the presence of lincomycin, suggesting a slow degradation of Dl protein unlike the case in other photosynthetic organisms such as C. reinhardtii (Zer and Ohad, 1995. Eur. J. Biochem. 231, 448-453) and C. sorokiniana (Figure 3). A smaller gap in TL peak temperatures (in the presence or absence of DCMU) following exposure to high light was observed in several organisms such as the cyanobacterium Microcoleus vaginatus (Ohad et al., 2010, ibid; Ohad et al., 2011, ibid) and the diatom Phaeodactylum tricornutum (Eisenstadt et al., 2008. Environm. Microbiol. 10, 1997-2007) but not in others such as C. reinhardtii (Ohad et al., 2010, ibid). Apparently, C. ohadii is "locked" in the low energy gap phase already under normal growth light. Raising the light intensity reduced the signal intensity but hardly affected the temperature of maximal signal.

0₂ evolution and fluorescence are widely used to assess PSII activity. The contradicting behavior of these two parameters in response to high irradiance (Figure 4B) provided another support to the notion that C. ohadii' s PSII functions differently than in model photosynthetic organisms such as C. reinhardtii. The "uncoupling" between these two parameters was also observed to some extent in some other photosynthetic organisms (Maenpaa et al., 1995. Plant Physiol. 107, 187-19; Eisenstadt et al., 2008; Ohad et al., 2010, ibid; Ohad et al., 2011, ibid). Based on results obtained with Microcoleus sp. isolated from the same BSC, it is proposed that activation of a non-radiative electron route in PS II may explain the decline of fluorescence whereas PSII activity (0₂ evolution) increased. It has been previously suggested that enhancement of non-radiative reaction centre processes that ensue following the change in light intensity may reduce harmful radiative recombination events, thereby lowering ^l02 generation and oxidative photodamage under excess illumination (Ohad et al., 2011, ibid).

Any suitable medium for cultivating algae of the present invention can be used according to the teachings of the present invention. As disclosed herein, the algae are capable of growing under various trophic conditions. Thus, according to certain embodiments, the algae of the invention grow photosynthetically on CO2 and sunlight, plus the necessary amounts of trace nutrients. According to certain exemplary embodiments, the growth medium contains TAP buffer without acetate. According to other embodiments, the algae of the invention grow photosynthetically on CO2 and sunlight plus trace nutrients and additional carbon source. According to certain exemplary embodiments, the growth medium contains TAP buffer and ambient air enriched to 5% CO2 bubbled into the medium.

Nutrients that can be used in the systems described herein include, for example, nitrogen (in the form of NO3^" or NH₄, phosphorus, and trace metals (Fe, Mg, K, Ca, Co, Cu, Mn, Mo, Zn, V, and B). The nutrients can come, for example, in a solid form or in a liquid form. If the nutrients are in a solid form they can be mixed with water prior to being delivered to the liquid containing the microalgae, or prior to being delivered to a photobioreactor.

The volume of growth medium can be any volume suitable for cultivation of the algae for any purpose, whether for standard laboratory cultivation, to large scale cultivation for use in, for example, algal biomass production, bioremediation, production of the algae natural cell components and/or production of foreign compounds expressed within the algal cell. The volume of the growth medium further depends on the system in which the algae are grown. Any system as is known in the art may be used, typically depending on the purpose of the algae growth (biomass production, bioremediation etc.)

Typically, the algae are grown under conditions that enable photosynthesis. Since photosynthesis requires sunlight and C0₂ and the microalgae further require water (either fresh or wastewater) optionally mixed with the appropriate fertilizers to grow, microalgae can be cultivated in, for example, open ponds and lakes. However, the open systems are more vulnerable to contamination than a closed system. In addition, in open systems there is less control over water temperature, CO2 concentration, and lighting conditions. The growing season is largely dependent on location and, aside from tropical areas, is limited to the warmer months of the year. An open system, however, is cheaper to set up and/or maintain than a closed system.

Another approach to growing the microalgae is thus to use a semi-closed system, such as covering the pond or pool with a structure, for example, a "greenhouse -type" structure. While this can result in a smaller system, it addresses many of the problems associated with an open system. The advantages of a semi-closed system are that it can allow for the C. ohadii being dominant over an invading organism by allowing the microalgae to out-compete the invading organism for nutrients required for its growth, and it can extend the growing season. For example, if the system is heated or cooled, the microalgae can grow year round.

Alternatively, the microalgae can be grown in closed structures such as photobioreactors, where the environment is under stricter control than in open systems or semiclosed systems. A photobioreactor is a bioreactor which incorporates some type of light source to provide photonic energy input into the reactor. The term photobioreactor can refer to a system closed to the environment and having no direct exchange of gases and contaminants with the environment. A photobioreactor can be described as an enclosed, illuminated culture vessel designed for controlled biomass production of phototrophic or mixotrophic liquid cell suspension cultures. Examples of photobioreactors include, for example, glass containers, plastic/glass tubes, tanks, plastic sleeves, and bags. Examples of light sources that can be used to provide the energy required to sustain photosynthesis include, for example, fluorescent bulbs, LEDs, and natural sunlight. Because these systems are closed everything that the organism needs to grow (for example, carbon dioxide, nutrients, water, and light) must be introduced into the bioreactor. Photobioreactors, despite the costs to set up and maintain them, have several advantages over open systems. They can, for example, prevent or minimize contamination, offer better control over the culture conditions (for example, pH, light, C0₂ and temperature), prevent water evaporation, lower CO2 losses due to degassing, and permit higher cell concentrations. On the other hand, certain requirements of photobioreactors, such as cooling, mixing, control of oxygen accumulation and bio-fouling, make these systems more expensive to build and operate than open systems or semi-closed systems. Photobioreactors can be set up to be continually harvested (as is with the majority of the larger volume cultivation systems), or harvested one batch at a time (for example, as with polyethlyene bag cultivation). A batch photobioreactor is set up with, for example, nutrients, microalgae, and water, and the microalgae is allowed to grow until the batch is harvested. A continuous photobioreactor can be harvested, for example, either continually, daily, or at fixed time intervals.

According to additional aspect, the present invention provides a method for producing algal biomass or products thereof, the method comprising culturing Chlorella ohadii under conditions suitable for the Chlorella ohadii proliferation and photosynthetic activity, wherein said Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l (rbcL), SEQ ID NO:2 (18S rRNA) and complements thereof.

According to certain embodiments, the algal biomass is used for at least one of animal food (for fish, mammals and poultry), fertilizer additive, cosmetics and feedstock for biodiesel production. According to other embodiments, the algal biomass is used as a source for the production of at least one of proteins, lipids (including fatty acids), carbohydrate, pigments and secondary metabolites either naturally present in the algae cells or heterogeneously expressed by the alga cells. Each possibility represents a separate embodiment of the present invention. As described hereinabove, the growth of C. ohadii is not adversely affected by intense light; moreover, exposure of C. ohadii to a high irradiance led to significant structural, biochemical and physiological changes rather than to photodamages like other photosynthetic organisms. The abundance of condensed thylakoid bundles observed in transmission electron microscope (TEM) photographs increased from 2.91+0.54 in TAP grown cells to 14.1+2.8 within 2 h after transfer to photoautotrophic growth under air level of C0₂, in the absence of acetate (Figure 6D). Nevertheless the chlorophyll content per cell was hardly affected, maintained at 4.83xl0^"7+7.36e^"8 and 4.09xl0^"7+7.15e^"8 μg Chi cell^"1, respectively, suggesting dynamic architectural alterations in the photosynthetic apparatus within the thylakoids.

Appearance of pyrenoids is clearly observed in cells grown in TAP medium exposed to high light or transferred to photoautotrophic conditions (Figure 6B and Figure 6C). The pyrenoids play an essential role in CO2 fixation in alga and the CCM that enables efficient photosynthetic CO2 utilization. In C. reinhardtii, where this aspect has been intensively investigated (Rawat et al., 1996. Planta 198, 263-270), most of the ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), the primary carboxylating enzyme, is located in these bodies. The thylakoids traversing the pyrenoids bear carbonic anhydrase, which facilitates the formation of CO2 from bicarbonate in close proximity to RubisCO and thereby raises the apparent photosynthetic affinity for extracellular CO2 (Raven et al., 2002. Funct. Plant Biol. 29, 355-378). Indeed, the apparent photosynthetic affinity for inorganic carbon (Ci) increased in cells exposed for 2 h to excess light or bubbled with air in the absence of acetate. The maximal rate of O2 evolution also increased after the transfer to the new conditions, presumably reflecting the metabolic shift from acetate to CO2 reduction and thereby contributing to the dissipation of excess redox (Figure 6E). The large fluxes of inorganic carbon and protons associated with CCM activity affects the pH homeostasis of the cells (Kaplan and Reinhold, 1999. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 539-570) and may help dissipate excess light energy (Fukuzawa et al., 2012. In Photosynthesis: Plastid Biology, Energy Conversion and Carbon Assimilation, J.J. Eaton Rye, B.C. Tripathy, and T.D. Sharkey, eds, pp. 625-650.).

For animal food or fertilizer additive, the algae may be directly harvested from the growth culture or algae residuals after extraction of the algae cell products may be used.

According to certain embodiments, the method further comprises harvesting the algal biomass. As is known to a person skilled in the art, various methods can be used for separating the algae cells from the growth medium. Non-limiting examples include screening, centrifugation, rotary vacuum filtration, pressure filtration, hydrocycloning, flotation and gravity settling. Other techniques, such as addition of precipitating agents, flocculating agents, or coagulating agents, etc., can also be used in conjunction with these techniques. Two or more stages of separation can also be used. When multiple stages are used, they can be based on the same or a different technique. Non-limiting examples include screening of the bulk of the algal culture contents, followed by filtration or centrifugation of the effluent from the first stage. The harvested cell mass can optionally be dried, using any method known in the art, including, but not limited to, freeze-drying, spray-drying and heat-drying, including drying under the sunlight.

The algal biomass may be used per se as a food or as a food supplement to human beings, other mammal, poultry or aquacultures. According to certain aspects, the present invention provides an edible composition comprising Chlorella ohadii composition, wherein the Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2 and complements thereof, further comprising an excipient, diluent or carrier suitable for oral consumption.

According to some embodiments the edible composition is formulated in a form selected from the group consisting of solutions, suspensions, dry soluble lyophilized powders, emulsions, microemulsions, dispersions, liposomal dosage forms, lipid complexes such as with cholesterol derivatives and phospholipids, capsules, soft gel capsules, cubes, flakes and pellet. The skilled Artisan can select the formulation according to the animal to be feed (e.g. aquatic animals, land mammals, humans and poultry).

According to some embodiments the solutions and vehicles are selected from aqueous and non-aqueous solutions. Optionally, at least one additional ingredient selected from the group consisting of preservatives, antioxidants and tonicity controlling agents may be added to the formulation. According to some exemplary embodiment the preservatives are selected from the group consisting of benzyl alcohol, methyl paraben, propyl paraben, and sodium salts of methyl paraben. According to other exemplary embodiment the tonicity controlling agents are selected from the group comprising of sodium chloride, mannitol, dextrose, glucose, lactose and sucrose.

According to certain embodiments the edible composition of the present invention is a solid composition selected from the group consisting of tablets, capsules, sachets, granules, lozenges, powders cubes and pellets.

In certain embodiments the solid edible composition contain in addition to the algal biomass suitable excipients including, but not limited to, starches, gum arabic, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulations can additionally include lubricating agents such as, for example, talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propyl hydroxybenzoates; sweetening agents; or flavoring agents. Polyols, buffers, and inert fillers may also be used. Examples of polyols include, but are not limited to: mannitol, sorbitol, xylitol, sucrose, maltose, glucose, lactose, dextrose, and the like. Suitable buffers encompass, but are not limited to, phosphate, citrate, tartarate, succinate, and the like. Other inert fillers, which may be used, encompass those which are known in the art and are useful in the manufacture of various dosage forms. If desired, the solid compositions may include other components such as bulking agents and/or granulating agents, and the like.

According to some embodiments, the C. ohadii biomass is produced for the isolation of foreign substances. According to these embodiments, at least one cell of the Chlorella ohadii is transformed with at least one exogenous transcribeable polynucleotide encoding a product of interest. Any method for transforming microalgae as is known in the art can be used according to the teachings of the present invention. Transformation methods include particle bombardment, electroporation, microporation, vortexing cells in the presence of exogenous DNA, acid washed beads and polyethylene glycol-mediated transformation.

Typically, to prepare vectors for making the transgenic algae, the polynucleotide encoding the exogenous protein is first cloned into an expression vector, a plasmid that can integrate into the algal genome. In such an expression vector, the DNA sequence which encodes the exogenous protein is operatively linked to an expression control sequence, i.e., a promoter, which directs mRNA synthesis. The promoter can be an endogenous promoter, i.e., a promoter that directs transcription of genes that are normally present in the algae. According to certain embodiments, the vector further comprises a polynucleotide encoding a resistance gene to enable selection of transformed algae.

According to yet additional aspect, the present invention provides a method for reducing the amount of at least one undesired substance present in waste comprising culturing a Chlorella ohadii in a culture medium comprising wastewater, wherein the Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:l (rbcL), SEQ ID NO:2 (18S rRNA) and complements thereof and wherein the culturing conditions are suitable for proliferation and photosynthetic activity for at least 8 h/day of said Chlorella ohadii. According to some embodiments, the Chlorella ohadii genome comprises SEQ ID NO: l or a complement thereof. According to other embodiments, the Chlorella ohadii genome comprises SEQ ID NO: 2 or a complement thereof.

As used herein, the term "reducing the amount of at least one undesired substance present in waste" refers to lowering the amount of the undesired substance, which may be organic or inorganic, from its amount in the waste before culturing C. ohadii. According to certain embodiments, the waste is selected from the group consisting of wastewater and contaminated soil. Each possibility represents a separate embodiment of the present invention.

The terms "wastewater" and "contaminated soil" are used herein in its broadest meaning and refers to water or soil that has been adversely affected in quality by any human activity, including, but not limited to, municipal sewage, industrial sewage and water or soil contaminated with toxic spills.

According to certain embodiments, the culture medium comprises at least 50% waste. According to other embodiments, the culture medium comprises at least 60%, at least70%, at least 80% or at least 90% or more waste. According to certain exemplary embodiments, the culture medium comprises 100% waste.

As exemplified herein below, oxygen evolution is increased during exposure to excess illumination above that required to saturate CO2 fixation (Figure 4B). Together with the finding that C. ohadii is capable of growing in swage water (following a first treatment phase), this phenomenon enables the use of C. ohadii compositions for the cleaning of wastewater or contaminated soil, by providing optimal conditions for the bioremediation activity of aerobic microorganisms already present or added to the wastewater or soil.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

MATERIALS AND METHODS Growth in batch and photobioreactor cultures

C. ohadii was isolated as a contaminant of a cyanobacterial isolation culture from a desert sand crust.A BSC sample taken near Nizzana sand dune field station of the Herbrew University Arid Ecossystem Reaeracg Center (AERC)m situated close to the Isreali-Egyption border (30°56'N, 34°23'E, elevation 190 m m.a.s.l.; annual average rainfall about 100 mm). ((Treves et al., 2013). Crust samples (Fig. la) were placed in medium BGl l (Stanier et al., 1971. Bacteriol. Rev 35, 171-205), typically used to grow cyanobacteria, in an attempt to raise axenic cultures of the filamentous cyanobacteria inhabiting the crust. Within the developing cultures, mostly consisting of Microcoleus sp. and Leptolyngbia sp., a small green alga we observed, which was isolated and grown in a TAP (Tris, acetate, phosphate buffer) medium (Zito et al., 1997. Plant Mol Biol 31,79-86) at 30°C under continuous fluorescent light (200 μιηοΐ pho-tons m^"2 s^"1). Following several dilutionVinoculation cycles and resuspension on agar plates containing TAP, an axenic culture of a small, about 2 μιη in diameter, green alga was isolated. (Treves H et al., FEMS Microbial Ecol 2013. 86(3):373-380). Growth experiments on various inorganic carbon concentrations were carried out using TAP medium without acetate with bubbling of air, or C02-enriched air (5% CO2 in air), or with no forced aeration.

The algae growth rate was assessed from cell counting (using a light microscope, Eclipse E200, Nikon, Melville, NY, USA and a hemocytometer; for each data point counting 10 squares containing up to 30 cells); the optical density at 735 nm (spectrophotometer, Genesys 5, Spectronic Instruments, Leeds, UK); and from the level of chlorophyll after extraction (see below). Figure 7 shows the correlation between cell count and OD at 735 nm for samples withdrawn from cultures maintained under the range of conditions used herein. Batch cultures were grown in Erlenmeyer flasks, on a shaker, in medium TAP at 35°C, 100 RPM, 100 μιηοΐ photons m^"2 s^"1. The data presented in Table 1 comparing the growth rates of C. ohadii and C. sorokiniana were obtained from such experiments. The data presented in Figure 2A and Figure 2B were derived from experiments where the cells were grown in flat panel photobioreactors (FMT-150, PSI, Drasov, Czech Republic) as described previously (Nedbal et al., 2008. Biotechnol. Bioeng. 100, 902-91; Cerveny et al., 2013, ibid). Unless specified otherwise, the temperature was 35.0 + 0.3°C and irradiance was 100 or 3000 μιηοΐ photons m^~2 s^-1. Air bubbling was supplied using an air-pump at a rate of -100 ml min^" \ pH, dissolved oxygen concentration, OD 680nm and OD 735 nm were monitored in situ every 5 min as described in (Nedbal et al., 2008 ibid; Cerveny et al., 2013, ibid).

DNA sequencing

As part of a genomic project of C. ohadii, in progress, DNA was extracted, purified, and sequenced using 454 and Illumina technologies. The sequences of the 18S rRNA gene and of rbcL (a single copy on the chloroplast genome) encoding the large subunit of RubisCO were first compared with known sequences through the NCBI- BLAST alignment tool with a standard nucleotide collection (nr/nr) MEGABLAST query that identified it as a close relative to Chlorella species. Relevant gene sequences of the genus Chlorella were obtained from the Silva comprehensive ribosomal RNA database (Quast C et al., 2013. Nuc Acids Res 41, D590-D596). 18S rRNA gene sequences were aligned using the SINA aligner (Pruesse E et al., 2012. Bioinformatics 28, 1823-1829) rejecting sequences below 70% identity. The rbcL (encod-ing the large subunit of RubisCO) sequences were aligned using T-Coffee (http://www.tcoffee.org/) with default parameters. Sensitivity to high light (photoinhibition)

C. ohadii and C. sorokiniana cells were grown to mid log phase cultures in medium TAP or TAP without acetate at 30°C and under 100 μιηοΐ photons m^~2- s^_1. Aliquots were then transferred to the growth cylinders shown in Figure 3A and Figure 3B to reach an initial cell density corresponding to OD 735 nm=0.06, and grown at 30°C. The cylinders were bubbled with air and the light intensity was raised to 3,500 μιηοΐε photons m^"2 s^"1 for 24 h.

Western blot analyses

For SDS PAGE and Western blot, an equal amount of protein samples, 15μg/lane, were loaded on 14% polyacrylamide gels. The gels were than stained with Coomassie blue or electro-transferred to a PVDF membrane. Dl level was determined using commercially available polyclonal antibodies (Agrisera) and a CDP star chemiluminescent protein detection system (Perkin-Elmer). The Coomassie blue stained gels are shown in Figure 8. CO2- and light intensity- dependent O2 exchange

The rate of C -dependent O2 evolution as a function of Ci concentrations was determined using a Clark type O2 electrode (PS2108, Passport dissolved O2 sensor Roseville, CA, USA) essentially as described in Treves et al. (2013, ibid). The cells were harvested by centrifugation for 10 min at 4000 rpm in a swinging-bucket rotor and resuspended in CC -free medium containing 20 mM Hepes. pH was then adjusted to 7.5 with saturated NaOH. 200 μΐ of CC -free cells were then diluted in 4 ml of the same media and incubated in the O2 electrode chamber, a temperature-controlled perspex holder (optical path 0.7 cm, 30°C). Light was provided using a LED array (warm white, 3000°K) surrounding the cell suspension held in temperature-controlled glass columns (optical path 1.9 cm, 30°C). CO2 curves were established using ascending concentrations of sodium bicarbonate. For light intensity-dependent O2 evolution measurements, cells were incubated in darkness for at least 5 min followed by exposure to increasing light of known intensities. A saturating NaHC0₃ (2 mM) concentration was added to ensure that the cells were not Ci limited. Fourier transform infra-red (FTIR) spectroscopy

Samples were prepared as described in Palmucci et al., (2011. J. Phycol. 47, 313- 323.). FTIR spectra were recorded with a Tensor 27 FTIR spectrometer (Bruker Optics, Ettlingen, Germany). The ratios of the FTIR absorbance of the carbohydrate, lipid and protein pools were calculated from the integrals of the absorption bands of the relevant functional groups as assigned in (Giordano et al., 2001. J. Phycol. 37, 271-279).

Microscopy TEM

Samples were centrifuged at 6,000g for 5 min in Eppendorf tubes. Pellets were fixed in 0.5 ml fixative (2% formaldehyde and 2.5% glutaraldehyde in cacodylate buffer 0.1 M pH=7.4) overnight. Pellets were then centrifuged at 10,000 g for 2 min and the fixative was discarded, followed by incubation of the pellets in 0.5 ml fixative (1% Os0₄ in 0.1 M cacodylate buffer + 1.5% potassium ferricyanide) for 1 h. Samples were then incubated in ascending concentrations of ethanol for dehydration, embedded in resin molds and baked at 60°C for 48 h. Blocks were cut using LKB 8800 Ultrotome 3. Between 70-90 nm sections were collected on 200 nm thin bar copper grids and then stained with 5% aqueous uranyl acetate solution and lead citrate. Thin sections were observed with a Tecnai 12 (FEI Phillips, Eindhoven, The Netherlands) electron microscope equipped with MegaView 2 CCD camera and AnalySIS version 3.0 software (Soft Imaging System GmbH, Munster, Germany). Chlorophyll a and b levels

Pellets of 1 ml samples were re-suspended in 100% acetone and centrifuged at 4,000g for 2 min to pellet cell debris. Supernatant was collected for separation and analysis. Pigments were separated by High Performance Liquid Chromatography (HPLC) using a Waters system consisting of Waters 600 pump, Waters 996 photodiode array detector and Waters 717 plus Autosampler (Waters, Milford, MA). The ODS2 C18 reversed-phase columns were applied using a gradient of solvents, acetonitrile:water (9: 1; designated A) and ethylacetate (B), at a constant flow rate of 1 ml/min or 1.6 ml/min for the Phenomenex column or the Waters column, respectively. Gradients were: 100% to 80% A during 8 min; 80% to 65% A during 4 min, followed by 65% to 45% A during 14 min and a final segment at 100% B. The absorption of the eluting HPLC solvent at 250-800 nm was recorded. Chlorophylls were identified by their absorption spectra and retention time. Quantification was performed by integrating the peak areas using the Millennium chromatography software (Waters).

Chlorophyll fluorescence and Thermoluminescence

Light-induced fluorescence parameters were neasured using an FL3000 fluorimeter (PSI, Brno, Czech Republic) as described in Ohad et al., (2010, ibid). The light excitation intensity was 1800 μιηοΐ photons m^"2 s^"1 for 30 s.

C. ohadii cells (corresponding to OD 730 nm=0.8) were placed in a chamber where 0₂ concentration and fluorescence could be measured simultaneously. The cells, in medium TAP, were provided with a saturating level of inorganic carbon (2 mM) and the light intensity gradually increased until the maximal rate of CC -dependent O2 evolution was attained, at 700-800 μιηοΐε photons m^"2 s^"1. Light excitation intensity was 1800 μιηοΐ photons m^"2 s^"1 for 30 s.

Thermoluminescence was measured as described in Ohad et al. (2010, ibid). Samples (0.4 ml, 4-5 μg chlorophyll) were dark-adapted at 25°C for 2 min, rapidly frozen to -22°C and excited by saturating light flashes (3 μ8, xenon arc discharge). The samples were then heated at a rate of 0.6°C s^"1 to 50°C while counting photon emissions (B band). For detection of the Q band, the herbicide DCMU (Sigma, Aldrich, Germany), which binds to the QB site, was added before dark- adaptation at concentrations completely inhibiting oxygen evolution (20 μΜ). Estimating Lipid content using Nile red

To estimate lipid content, 200 μΐ of cells that were exposed to high light for various durations were loaded in triplicates on a 96-well plate and the OD 750 nm measured before staining. Ten μΐ of Nile red (500 μg of 9-diethylamino- 5Hbenzo[a]phenoxazine-5-one per 1 ml acetone, Sigma), a fluorescent probe of intracellular lipids and hydrophobic domains of proteins, were added to the cells. Fluorometric analyses were performed 10 min after staining using a fluorescence plate reader with narrow bands 485 nm excitation and 590 nm emission filters. With this technique, cellular storage or neutral lipids show yellow-golden fluorescence (Greenspan et al., 1985. J. Cell Biol. 100, 965-973; McGinnis et al., 1997. J. Appl. Phycol. 9, 19-24). Example 1; Growth and ultradian rhythm

An axenic culture of Chlorella ohadii (a dividing cell is shown in Figure 1) was isolated from a biological soil crust (BSC) samples collected near the Egypt - Israel border(Treves et al., 2013, ibid). Growth rate was examined under a wide range of ambient conditions including 15-45°C (optimal growth was reached at 35-37°C), light intensities from darkness to 3000 μιηοΐε photons m^"2 s^"1, photoautotrophic and photomixotrophic (in the presence of acetate) conditions where the C0₂ source was either air or 5% CO2 in air, and heterotrophic growth with acetate as the sole carbon source in darkness. Growth was assessed by several means including cell counting under the microscope, optical density (OD, the correlation between cell count and OD735 nm is shown in Figure 7) and chlorophyll content. In batch cultures maintained under all the trophic conditions examined here (Table 1) the maximal growth rate of C. ohadii was considerably faster than observed in other algae (Bowler et al., 2010. Curr. Opin. Plant Biol. 13, 623-630; Raven et al., 2013, ibid) including its close relative, C. sorokiniana which is widely used in algal growth facilities due to its relatively fast growth rate and resistance to high illumination (Vigeolas et al., 2012. J. Biotechnol. 162, 3-12). Much longer generation times were obtained using other model algal species, examined under their respective optimal growth conditions, such as 6-7 h in Chlamydomonas reinhardtii 137C and 17 h in Chlorella variabilis. Table 1: Generation time (in hours) of C. ohadii and C. sorokiniana in batch cultures

Data from the first 6 h of growth is provided to minimize the impact of self -shading and bleaching of C. sorokiniana during growth under high light. The cells were grown in medium TAP (Tris, Acetate, Phosphate buffer) or in TAP without acetate (TAP-A) where CO₂ in air was the sole carbon source. Growth temperature was 30°C. The light intensity in μπιοΐε photons m ² s ¹ is provided. Cell counting every hour was used to assess the growth as explained in Methods. When the cells were aerated with 5% CO₂ in air the photoautotrophic growth rate was 15-18% faster than under air.

When C. ohadii cells were grown in photo -bioreactors (PSI, Drasov, Czech Republic) under optimal conditions and continuous illumination, initiated at low density corresponding to OD735 nm = 0.02, division times shorter than 1.5 h were initially recorded during the first 3 h (Figure 2A). Ultradian growth rhythms were observed with the development of the cultures (Figure 2B and Figure 2C) indicating that the cells became synchronized although they were maintained in continuous light. Cells were grown in TAP medium contained 10 mM Tris buffer whose pK is about 8.06.

The cells grew rapidly for about 16-17 h (phase I) but then, abruptly, the growth rate declined for about 6-8 h (phase II) but then resumed their fast growth (Phase III) for an additional 7-9 h. In this open system, flushed by bubbling of air, rates of oxygen evolution were high enough to reach super-saturation. The highest levels of 0₂, an indicator of the fastest rate of photosynthetic O2 evolution, were observed when the cells entered the slower growing phases (II and IV). In cultures exposed to 3000 μιηοΐε photons m^"2 s^"1 (Figure 2B) the O2 level reached 125% air saturation despite bubbling with air at 100 mL/min into the 1L cell suspension. When the cultures resumed fast growth, in phase III, they lowered O2 production and, at some point, even started to consume (net) O2 at a very fast rate and acidified the medium presumably due to respiratory CO2 release. This was followed by another growth arrest during which the cells shifted again to net O2 evolution and alkalization of the media (phase IV). Similar behavior was observed in cells exposed either to 3000 or 100 μιηοΐε photons m^"2 s^"1 (Figure 2B and Figure 2C, respectively) although the growth rates, cell densities reached, the pH and O2 concentration amplitudes rose with increasing irradiance.

Example 2: Resistance to photodamage

In its natural environment C. ohadii encounters a combination of rising light intensity with declining photochemical activity due to desiccation. Photosynthetic organisms undergo photodamage, a process that significantly lowers global photosynthetic productivity, particularly when exposed to light intensities higher than required to saturate CO2 fixation. This process involves the destruction of photosystem II (PSII) which is the more sensitive of the two photosynthetic reaction centers to excess light (Barber J and Andersso B., 1992. Trends Biochem. Sci. 17, 61-66; Keren N et al., 2000. Photosynth. Res. 63, 209-216; Vinyard D J et al., 2013. Annu. Rev. Biochem. 82, 577-606; Kangasjarvi S et al., 2014. Plant. Physiol. Biochem. 81, 128-134). To examine C. ohadii susceptibility to photodamage, its growth under intense light was followed. C. ohadii cells grown in medium TAP, 30°C, 100 μιηοΐε photons m^"2 s^"1 were exposed at TO to 3000 μιηοΐε photons m^"2 s^"1 for various durations followed by analysis of the rate of photo synthetic 0₂ evolution as affected by light intensity. Cell suspensions (corresponding to 20 μg chlorophyll/mL) were placed in the O2 electrode chamber, 30°C, 2 mM NaHCOs.

This light intensity is almost twice the maximal sunlight and considerably higher than required to saturate CC -dependent O2 evolution (500 μιηοΐε photons m^"2 s^"1, see To in Figure 5A). The generation time of C. ohadii under this illumination was about 2.4 h in the presence and absence of acetate, i.e. similar to that observed during prolonged photomixotrophic growth under a much lower illumination (Table 1 hereinabove). These data indicated that photoautotrophic and photomixotrophic growth of C. ohadii was hardly affected by the excess light. In contrast, under both trophic conditions, the cultures of C. sorokiniana bleached completely (Figure 3A and Figure 3B) even though it is considered highly resistant to photoinhibition (De-Bashan L E et al., 2008. Bioresource Technol. 99, 4980-4989). A faster destruction of protein Dl in the photo synthetic reaction center II (PS II) followed by its replacement is the main cause of the decline in photosynthetic activity (photoinhibition) in photosynthetic organisms exposed to excess light (Aro E-M et al., 1993. Biochim. Biophys. Acta 1143, 113-134.; Ohad I et al., 2011, ibid; Krupnik T et al., 2013 J. Biol. Chem. 288, 23529-23542 and references therein). Degradation of Dl is commonly accepted as a hallmark of the extent of photodamage (Barber and Andersson, 1992, ibid; Keren et al., 2000, ibid). Western blots analysis of Dl levels in experiments such as presented in Figure 3A and Figure 3B showed a very fast decline in C. sorokiniana but much slower in C. ohadii even in the presence of lincomycin (Figure 3C and Figure 3D; the respective Coomassie blue gels are shown in Figure 8Aand Figure 8B) This antibiotic blocks protein biosynthesis in the chloroplast and hence blocks the replacement of damaged Dl during photoinhibition. Treatment of C. ohadii with lincomycin completely arrested growth within 24 h, indicating its effectiveness, and was expected to cause a fast decline in Dl level of cells exposed to excess light. This was the case in C. sorokiniana but clearly not in C. ohadii (Figure 3D) indicating a much slower damaging of Dl in the latter.

Example 3: Function of PSII in Chlorella ohadii

The data presented in Figure 3 suggested a modified function of PSII in C. ohadii as compared with other model photosynthetic organisms. This notion is supported by several additional observations:

1) Lower sensitivity of C. ohadii to inhibitors commonly used to block PSII activity. The inhibitors used in the present study (Figure 3E) are known to compete with Q_B for the quinone-binding pocket in protein Dl and thereby inhibit electron transport (Ohad et al., 2010, ibid). C. ohadii is not inhibited at all by Bromoxynil or Ioxynil at concentrations as high as ΙΟΟμΜ and is far less sensitive to 3-(3,4-dichlorophenyl)-l,l- dimethyl urea (DCMU) than other organisms. As an example, the DCMU concentration required for 50% inhibition of 0₂ evolution was approximately 10 times higher than the concentration required to reach the same inhibition in Chlamydomonas reinhrdtii (Giordano et al., 2005. Eur J. Phycol. 40, 345-352), often used as a model system to study photosynthesis in algae (Rochaix, 1995; Gutman and Niyogi, 2004; Finazzi et al., 2006; Merchant et al., 2007; Gonzalez-Ballester et al., 2011). 2) Thermoluminescence (TL) emission is often used as a reporter of the functionality of PSII electron transport (see, for example, Vass, 2003. Photosynth. Res. 76, 303-318). The emission results from charge recombination of S₂,3/QB^~ or S₂/QA^~ in the presence of DCMU (which blocks electron transfer from QA to Q_B), designated B and Q bands, respectively. TL measurements for C. ohadii (Figure 4A) showed a maximal B band signal at about 25°C and a Q band maximum at 21°C, i.e. only about 4°C lower than in the absence of DCMU. In other photosynthetic organisms including another Chlorella sp. (Yewalkar et al., 2013. Photosynthetica 51, 565-57) the difference in maximal B and Q emissions is 15-20°C. These data suggested that the redox potential gap between QA and Q_B is significantly smaller in C. ohadii than in other organisms, thus supporting the notion of an altered structural organization in C. ohadii. Example 4: Oxygen evolution and cell products

Exposure to high illumination led to enhanced 0₂ evolution that was less pronounced in cells grown in the absence of acetate (Figure 4B); possibly since these cells are already acclimated to limiting carbon supply reflected in the lower fluorescence at time zero. The rising O2 evolution during exposure to excess illumination above that required to saturate CO2 fixation (Treves et al., 2013, ibid; Figure 4B and Figure 5A) supported the suggestion that PSII was not damaged and indicated the activation of a process whereby relatively reduced end product(s) such as lipids are formed. Indeed, Fourier Transform Infra-Red (FTIR) spectroscopy and Nile red analyses (Figure 5B and Figure 5C, respectively) showed a large increase in the lipid and carbohydrate contents within 2 h of exposure to 3000 μιηοΐε photons m^"2 s^"1. Although it is not absolutely specific, Nile red is frequently used to examine changes in lipid levels (see, e.g. Haimovich-Dayan et al., 2013. Environm. Microbiol. 13, 1767- 1777). The Nile red signal kept rising during the entire 2 h high light treatment and reached a value 4-fold higher than the control thus supporting a rise in lipid content. This was further confirmed by the elevated abundance of lipid bodies (Figure 6B) and a 20% increase in the "energy content" (from 4300 to 5200 cal/gr DW, assessed by calorimetric analyses), during the 2 h exposure to high light.

Fluorescence parameters are frequently used to assess PSII activity and its quantum yield (Jursinic and Dennenberg, 1993. Biochim. Biophys. Acta 1183, 281- 291). However, while O2 evolution due to water cleavage in PSII increased by 40% within 20-40 min of high irradiance the fluorescence (Fv/Fm) declined by 60-80% (Figure 4B). This indicates that fluorescence can't be used to assess the photosynthetic electron transport, which was not damaged by the high light treatment in C. ohadii. In addition to the rise in O2 evolution, massive structural changes were observed within less than 2 h of transfer of photomixotrophic grown cells (Figure 6 A) to excess light (Figure 6B) or photoautotrophic conditions where CO2 in air was the sole carbon source (Figure 6C). These included the development of a pyrenoid (marked P in Figures 6B and 6C) with traversing thylakoid membranes and surrounded with starch grains, and an over 4-fold rise in the abundance of the thylakoid membranes (Figure 6D and Figure 9). The pyrenoid plays an essential role in CO2 concentrating mechanism (CCM) and fixation in eukaryotic phytoplankton (Kaplan and Reinhold, 1999, ibid; Giordano et al., 2005. Ann. Rev. Plant Biol. 56, 99-131). Their development led to a large rise in the photosynthetic Vmax and apparent affinity to external inorganic carbon, from 160 μΜ Ci in TAP grown cells to 10 or 16 μΜ Ci in cells exposed for 2 h to excess light or bubbled with air, respectively (Figure 6E), consequent on the activation of the CCM.

Example 5: Oxygen evolution and wastewater remediation

Industrial or municipal wastewater is typically treated with three defined steps: (a) removal of large debris. This stage is typically performed by mechanical means for separating large solids present in the wastewater from the aqueous solution; (b) aerobic bacteria treatment in which decomposition of the organic materials occurs. The activity of the bacteria is highly depended on 0₂ supply; and (c) removal of minerals.

As described in Example 4 hereinabove, exposure to high illumination led to enhanced O2 evolution by Chlorella ohadii of the present invention. Chlorella ohadii is added to wastewater after the first step of debris removal. The microalgae is added directly to the wastewater, or, alternatively, to wastewater diluted with a culture medium (TAP or TAP without acetate). The microalgae have the ability to thrive in wastewater of various sources, including industrial, municipal and agricultural. The culture comprising the microalgae and wastewater is exposed to light intensity of between 100 μιηοΐε photons m^"2 s^"1 to 3,500 μιηοΐε photons m^"2 s^"1. When the culture is placed outdoors, it is exposed to the natural sunlight during the day. Optionally, the culture is aerated with ambient air during the night. When the culture is placed indoors, it is illuminated with artificial light, typically at an intensity of above 1,000 μιηοΐε photons m^"2 s^"1. Optionally, aerobic bacteria are added to the culture together with the addition of

C. ohadii. The microalgae and the bacteria can be added as separate compositions or can be first combined in a single composition to be added to the culture.

Chemical oxygen demand (COD) test is used to measure the amount of organic pollutants found in water and is expressed in milligrams per liter (mg/L) or PPM. Reduction is the COD indicates reduction of organic pollutant within the wastewater. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. An isolated Chlorella ohadii composition, wherein the isolated Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2 and complements thereof.

2. The isolated Chlorella ohadii composition of claim 1, wherein the isolated Chlorella ohadii genome comprises the nucleic acid sequence set forth in SEQ ID NO: l or a complement thereof.

3. The isolated Chlorella ohadii composition of claim 1, wherein the isolated Chlorella ohadii genome comprises the nucleic acid sequence set forth in SEQ ID NO: 2 or a complement thereof.

4. The isolated Chlorella ohadii composition of claim 1, wherein chlorophyll a/b ratio within cells of the isolated Chlorella ohadii is from 10: 1 to 13: 1.

5. A substantially pure culture comprising a composition of an isolated microalgae Chlorella ohadii, wherein the isolated Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2 and complements thereof and a growth medium.

6. The substantially pure culture of claim 5, wherein the isolated Chlorella ohadii genome comprises the nucleic acid sequence set forth in SEQ ID NO: l or a complement thereof.

7. The substantially pure culture of claim 5, wherein the isolated Chlorella ohadii genome comprises the nucleic acid sequence set fort in SEQ ID NO:2 or a complement thereof.

8. The substantially pure culture of claim 5, wherein chlorophyll a/b ratio within cells of the isolated Chlorella ohadii is from 10: 1 to 13: 1.

9. The substantially pure culture of claim 5, wherein the microalgae produces a biomass of at least 10⁹ cells/ml when the culture is grown under light intensity of 100-3,500 μιηοΐε photons m^"2 s^"1.

10. The substantially pure culture of claim 9, wherein the culture is grown under light intensity of above 500 μιηοΐε photons m^"2 s^"1.

11. The substantially pure culture of claim 10, wherein the culture is grown under light intensity of 3,000 μιηοΐε photons m^"2 s^"1.

12. The substantially pure culture of claim 5, wherein the microalgae produces elevated content of lipids when the culture in grown under light intensity of above 1,000 μιηοΐε photons m^"2 s^"1 compared to the lipid content when the culture is grown at a light intensity below 1,000 μιηοΐε photons m^"2 s^"1.

13. The substantially pure culture of claim 5, wherein the microalgae produces elevated content of carbohydrates when the culture in grown under light intensity of above 1,000 μιηοΐε photons m^"2 s^"1 compared to the lipid content when the culture is grown at a light intensity below 1,000 μιηοΐε photons m^"2 s-¹.

14. Τΐιε substantially pure culture of claim 5, said culture is grown und8r trophic conditions selected from the group consisting of photoautotrophic conditions, photomixotrophic conditions and heterotrophic conditions.

15. A method for producing algal biomass and/or products thereof, the method comprising culturing Chlorella ohadii microalgae under conditions suitable for the Chlorella ohadii proliferation and photosynthetic activity, wherein said Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2 and complements thereof.

16. The method of claim 15, wherein the Chlorella ohadii genome comprises the nucleic acid sequence set forth in SEQ ID NO: l or a complement thereof.

17. The method of claim 15, wherein the Chlorella ohadii genome comprises the nucleic acid sequence set forth in SEQ ID NO:2 or a complement thereof.

18. The method of claim 15, wherein chlorophyll a/b ratio within cells of the

Chlorella ohadii is from 10: 1 to 13: 1.

19. The method of claim 15, wherein the culturing conditions comprise light intensity of 100-3,500 μιηοΐε photons m^"2 s^"1.

20. Τΐιε nrethod of claim 19, wherein the culturing conditions comprise light intensity of above 1,000 μιηοΐε photons m^"2 s^"1.

21. The method of claim 20, wherein the culturing conditions comprise light intensity of 3,000 μιηοΐε photons m^"2 s^"1.

22. The method of claim 20, wherein the culturing conditions comprise light intensity of above 1,000 μιηοΐε photons m^"2 s^"1 for at least 2h.

23. The method of claim 15, wherein the culturing conditions comprise growth medium comprising at least one carbon source.

24. The method of claim 15, said method further comprises harvesting the algal biomass from the culture.

25. The method of claim 24, wherein the algal biomass is used as a food or a food supplement for an aquaculture animal, mammal or poultry.

26. The method of claim 15, said method further comprises harvesting a product produced by the microalgae cells and excreted from the algal biomass.

27. The method of claim 15, said method further comprises extracting at least one product produced by the microalgae cells.

28. The method of any one of claim 27, wherein the product is selected from the group consisting of a product naturally present in the microalgae cells and a product xenogeneic to the microalgae cells.

29. The method of claim 28, wherein the product produced by the microalgae cells is selected from the group consisting of lipids, carbohydrates, proteins, vitamins and pigments.

30. The method of claim 28, wherein the product is a metabolite useful in controlling the growth of toxic cynobacteria naturally present in the microalgae cells.

31. The method of claim 28, wherein at least one cell of the Chlorella ohadii comprises at least one exogenous transcribeable polynucleotide encoding a product of interest.

32. The method of claim 31, wherein the product of interest is selected from the group consisting of RNAi molecule, a protein or a polypeptide.

33. The method of claim 31, wherein the at least one cell comprises a plurality of transcribeable polynucleotides encoding an array of proteins or polypeptides having an enzymatic activity for producing at least one end product of interest.

34. A biomass of Chlorella ohadii produced by the method of any one of claims 15-24.

35. The biomass of claim 34, said biomass is formulated as a food for at least one of aquaculture animals, poultry and mammals.

36. The biomass of claim 34, said biomass is formulated as a food supplement for at least one of aquaculture animals, poultry and mammals.

37. An edible composition comprising Chlorella ohadii composition, wherein the Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2 and complements thereof, further comprising an excipient, diluent or carrier suitable for oral consumption.

38. The edible composition of claim 37, said composition is formulated as a food for at least one of aquaculture animals, poultry and mammals.

39. The edible composition of claim 38, formulated in a form selected from the group consisting of a capsule, dragee, pill, tablet, gel, liquid, suspension, slurry, powder, pellets, cubes and flakes

40. The edible composition of claim 37, said composition is formulated as a food supplement for at least one of aquaculture animals, poultry and mammals.

41. A method for reducing the amount of at least one undesired substance present in waste, the method comprising culturing Chlorella ohadii microalgae in a culture medium comprising waste, wherein the Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2 and complements thereof and wherein the culturing conditions are suitable for the Chlorella ohadii proliferation and for photosynthetic activity for at least 8 h/day.

42. The method of claim 41, wherein the Chlorella ohadii genome comprises the nucleic acid sequence set forth in SEQ ID NO: l or a complement thereof.

43. The method of claim 41, wherein the Chlorella ohadii genome comprises the nucleic acid sequence set forth in SEQ ID NO:2 or a complement thereof.

44. The method of claim 41, wherein chlorophyll a/b ratio within cells of the Chlorella ohadii is from 10: 1 to 13: 1.

45. The method of claim 41, said method further comprises adding to the culture medium aerobic microorganisms capable of reducing the amount of the at least one undesired compound.

46. The method of claim 41, wherein the aerobic microorganisms are bacteria.

47. The method of claim 41, wherein the waste is selected from the group consisting of wastewater and contaminated soil

48. The method of claim 41, wherein the culture medium comprises at least 50% waste.

49. The method of claim 48, wherein the waste is partially purified wastewater.

50. The method of claim 41, wherein the culturing conditions suitable for photosynthetic activity comprise light intensity of 100-3,500 μιηοΐε photons m^"

V¹.

51. The method of claim 41, said method further comprises aerating the culture medium when the culturing conditions are not suitable for photosynthesis.

52. The method of claim 41, wherein the culturing conditions comprise growth temperature of between 15°C-45°C .

53. A composition for reducing the amount of at least one undesired substance present in wastewater or contaminated soil, the composition comprising isolated Chlorella ohadii and at least one type of aerobic microorganism capable of reducing the amount of the at least one undesired compound.

54. The composition of claim 53, wherein the Chlorella ohadii genome comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2 and complements thereof.

55. The composition of claim 53, wherein the aerobic microorganisms are bacteria.

56. The composition of any one of claims 53-55, said composition further comprises culture medium suitable for the growth of Chlorella ohadii and the at least one aerobic microorganism.