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    • 专利摘要:
    PROCESS FOR THE PRODUCTION OF MICROALGAE, CYANOBACTERIA AND THEIR METABOLITES. The present invention relates to processes for the production of microalgae, cyanobacteria and/or their metabolics. We describe here a process involving the use of a stimulus applied to a culture of microalgae or cyanobacteria to enhance the production of one or more metabolites. Also described herein is a process for the production of microalgae and/or cyanobacteria comprising an adaptation stage in which an algae/cyanobacteria culture is grown in a process water feed load and/or under light emitting diodes (LEDs) emitting light within the spectrum of light wavelengths between about 400 nm and 700 nm, and a production stage, in which microalgae or cyanobacteria are grown under the same load of water feed and/or under the same conditions of light used in the adaptation stage. The invention also relates to specific microalgal strains.
    公开号:BR112013018377B1
    申请号:R112013018377-2
    申请日:2012-01-30
    公开日:2021-05-18
    发明作者:John Dodd；Blahsolov Marsalek；Nazir Bashir；Miroslav Vosatka
    申请人:Algaecytes Limited；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    The present invention relates to a process for the production of microalgae, cyanobacteria and/or their metabolites. Particularly, though not exclusively, the present invention relates to a process in which cultures of microalgae or cyanobacteria are exposed to a stimulus in order to intensify the production of one or more metabolites. Also described herein is a process for the production of microalgae and/or cyanobacteria comprising an adaptation stage, in which an algae/cyanobacteria culture is grown on a process water feed load and/or under light emitting diodes (LEDs ) emitting light within the spectrum of light wavelengths between about 400 nm and 700 nm, and a production stage, in which microalgae or cyanobacteria are grown in the same process water feed load and/or under them light conditions used in the adaptation stage.
    The present invention integrates methods of culturing microalgae or cyanobacteria forming lipids preferably in photobioreactor system(s), open tanks or other cultivation methods. It provides an integrated and continuous process for the production of algal biomass and conversion to high value by-products like EPA or biofuels. A specific strain for use in the invention is also described, referred to herein as ALG02.
    The present invention describes a process to enhance the exopolysaccharide (EPS) production of a unique microalgal strain (Dictyosphaerium chlorelloides ALG03) that is pre-regulated/culture-adapted for further large-scale production, using defined LED illumination spectra along with C02 and industrial wastewater, as nutrients and energy for growth in photobioreactors (PBRs). In addition, it provides an integrated and continuous process for the production of algal biomass, before/after carbohydrate extraction, which can be used for other downstream purposes, for example, to improve nutrient availability and soil adhesion in the root zones of plants irrigated with subsoil drip irrigation; as biomass for anaerobic digester units or bioethanol plants or for the production of biogas. The specific strain for use in the invention is also described, referred to herein as ALGO3. BACKGROUND OF THE INVENTION
    Algae are one of the fastest growing organisms on earth. They can reproduce (flower) in a few hours. They only need CO2 and light to grow in fresh, waste or sea water. In addition, the high levels of nutrients (mainly nitrogen and phosphates) found in industrial process waters, with other growth enhancing compounds, can increase algae biomass growth (a).
    Algae are being targeted for both future fuels and wastewater treatment solutions in ongoing research around the world. This is because algae, in the algae fuel production process, can sequester CO2 from industrial sources, and help to sequester nutrients (and some heavy metals) that are kept within the partially treated process water (b, ç).
    Algae and cyanobacteria are also valuable sources of metabolites, for example fatty acids including myristic acid, palmitic acid, palmitoleic acid, behenic acid, lauric acid, linoleic acid, alpha and gamma linolenic acid, stearic acid, arachidonic acid and eicosapentaenoic acid. Furthermore, extracellular polymeric substances from microalgae and cyanobacteria (of a polysaccharide nature) have unique biochemical properties that make them interesting from a biotechnological point of view. Cyanobacteria produce complex exopolysaccharides and their applications include food coating, emulsifying and gelling agents, flocculants, viscosifiers and moisturizing agents for the food and non-food industries. There is also a potential for its use as a source of new compounds in soft tissue adhesives in healthcare (d) . In the field of bio-decontamination, extracellular polysaccharides (EPSs) can remove toxic heavy metals from contaminated soils and water (e, f) and in the recycling of nutrients and other elements in wastewater.
    It is evident from the above that algae and cyanobacteria represent a valuable resource in several areas of technology, therefore, the present inventors sought to develop new processes for the growth and production of such Microorganisms. SUMMARY OF THE INVENTION
    According to a first aspect of the invention, there is provided a process for the intensified production of one or more metabolites in microalgae and/or cyanobacteria, said process comprising the steps of: (i) cultivating a strain of microalgae or cyanobacteria through a production phase; (ii) exposing the microalgae or cyanobacterial culture to a stimulus, where the stimulus comprises (a) a decrease in pH to a pH of no more than about pH 6, followed by an increase in pH to a pH of not less than about pH 7 and (b) an increase in light irradiance to at least 400 µmol/m2/s.
    According to a second aspect of the invention, there is provided a process for the production or growth of microalgae and/or cyanobacteria or the production of one or more metabolites derived therefrom, process comprising: (i) an adaptation stage, comprising cultivar microalgae or cyanobacteria: (a) in a process water feed load and selecting those microalgae or cyanobacteria capable of growing on the process water feed load; and/or (b) under light emitting diodes (LEDs) emitting 2 peaks of red and blue light within the spectrum of light wavelengths between about 400 and 700nm; and (ii) a production phase, comprising cultivating the microalgae or cyanobacteria selected from (i) under the same process water feed load used in the adaptation stage and/or under the same light conditions as used in the adaptation stage.
    Also provided herein is a Microorganism which is, or has the identifying characteristics of, a strain of Chlorogibba allorgei deposited with the Culture Collection of Algae and Protozoa under accession number CCAP 817/1, or a mutant strain derived therefrom. The strain of Chlorogibba allorgei deposited under accession number CCAP 817/1 is also referred to herein as ALG02.
    In addition, a microorganism is, or has the identifying characteristics of, a strain of Dictyosphaerium chlorelloides deposited with the Culture Collection of Algae and Protozoa under accession number CCAP 222/98, or a mutant strain derived therefrom. The strain of Dictyosphaerium chlorelloides deposited under accession number CCAP 222/98 is also referred to herein as ALGO3.
    The process provided herein may be for the intensified production of microalgae containing commercially valuable bioproteins, lipids and metabolites including eicosapentaenoic acid (EPA), myristic acid, palmitic acid, behenic acid, lauric acid, linoleic acid, alpha-linolenic acid and stearic acid. The process uses optimized light wavelengths for the cultivation and preferential production in photobioreactors of lipid-rich microalgal strains such as those not in the phylum Chlorophyta and selected from the Pleurochloridaceae family. Industrial by-products such as wastewater and process CO2 are used as recovered sources of nutrients and carbon to regulate/adapt the algae for intensive growth using these inputs.
    A unique two-stage process is also provided for optimizing the production of a commercially valuable exopolysaccharide in algae, in particular in Dictyosphaerium chlorelloides and can be applied to a specific strain of microalgae. The process takes place in a photobioreactor (PBR) system. The process takes advantage of the carbon dioxide (CO2)/water industrial process to grow algae and increase growth rates under similar conditions in the scaled PBR system. The process uses a pre-adaptation water culture to regulate the body with staggered process water/CO2 usage and LED lighting. This, in turn, allows the algae to produce high levels of an exopolysaccharide with commercial uses. Carbohydrate loaded algal biomass can be used in crop irrigation as a nutrient-rich, high-carbon fertilizer or, alternatively, in downstream energy production (bioethanol production or other biomass energy systems, for example , anaerobic digesters or fermentation vessels).
    The invention provides a novel process for producing high yields of an exopolysaccharide from an adapted strain Dictyosphaerium chlorelloides ALG03 that has evolved to grow at high rates in a secondary treated wastewater source under dimmed LED lights. This treatment doubles the extracellular polysaccharide yield from 38% to 77% observed in the log phase of growth. The high percentage of polysaccharide produced can be moved to a downstream process where the polymer can be extracted for a variety of uses. The spent biomass remaining after carbohydrate extraction can be used either for animal feed (if pure water is used in the bioreactor), or for energy production, for example, in anaerobic digesters, fermentation processes or pyrolysis systems.
    Thus, the invention also relates to a specific (isolated) strain of algae belonging to the Dictyosphaeriaceae family and in particular to the Dictyosphaerium genus, more specifically a strain of Dictyosphaerium chlorelloides. The strain has been deposited with the Culture Collection of Algae and Protozoa under accession number CCAP 222/98 and accepted January 25, 2011. This strain is shown here as being useful in the production of specific metabolites.
    The present invention further provides a novel process of using algae biomass produced as a specific soil conditioner using subsoil drip irrigation systems (SDIs). Biomass grown in the decontaminated water passes to an irrigation holding tank ready to mix with normal irrigation water and other compatible chemicals. Distributing the doses of algae cells that have already absorbed phosphates and nitrates, along with other nutrients from wastewater or process water helps distribute 1 natural slow-release fertilizer to the root zone of plants. It also increases carbon in soils. and helps to aggregate soil particles around developing plant roots preventing soil erosion, which is vital in arid areas.
    These and other objects, which will become evident during the following detailed description, were achieved by the inventors' discovery that exopolysaccharide formation by pre-regulated/adapted algae strains can double the yields of the carbohydrate-rich biopolymer by manipulating its pre- - Cultivation and control during growth within PBRs. BRIEF DESCRIPTION OF THE DRAWINGS
    Figure 1 - LED Lighting Adaptation - FANE Profile FAMA HPLC profiles intensified under dimmed LED lights compared to fluorescent lighting from a species of Scenedesmus (ALG05) that underwent 6 months pre-exposure to PAR LED lighting (400-700nm). ) with 6 subculture cycles.
    HPLC profiles (FAME) obtained from algae strains grown using PAR LED illumination only on the 24 hour cycle for 6 months (6 sub-cultures). Note the appearance of higher molecular weight fatty acids (seen with arrows in right image) compared to the same strain grown with fluorescent lighting (left image) of the same irradiance. Figure 2 - Intensified growth in treated process water with pre-cultivation
    The increased growth of Trachydiscus sp. (ALG01), Chlorella sp. (ALG04) and Scenedesmus sp. (ALG05) through pre-cultivation with 6 months (6 cycles) of exposure to treated process water and regulated LED lights (PAR 27°C and 24 hours of light).
    Adapted cells were cultured for 6 months in water from a process facility (secondary treated) and re-cultured every 4 weeks in clean process water. Unadapted cells were cultured in standard 100% ZBB growth medium and re-cultured every 4 weeks in new ZBB medium. Then, both strains were cultivated for 9 days (until the stationary phase) in new process water. The results show a clear adaptation to the process water and LED lighting environment by pre-adapted cells. Figure 3 - Lipid contents of different strains
    Comparison of carbohydrate/lipid/protein production by strains in exponential growth phase and biomass calorific values. Cultures were harvested in the exponential growth phase. Figure 4 - Production of eicosapentaenoic acid (EPA) in a strain of Chlorogibba sp. (SOMETHING2)
    The percentage content of main fatty acids in the biomass sample produced under suboptimal growth conditions (no addition of CO2 or PAR irradiance) and analyzed by capillary chromatography. Other analyzes for this strain and Trachydiscus sp. ALG01 grown using PAR light sources and added CO2 showed EPA percentages in the range of 30-38% of the total fatty acid content. Significant levels of palmitic and myristic fatty acids in analyzes were detected under controlled PAR lighting. Figure 5 A and B are graphs showing the results of the experimental work carried out in relation to the present invention and illustrating the relationship between nutrient concentrations and light levels and with the growth of an algae strain Dictyosphaerium chlorelloides ALG03. 5A. Low nutrient requirements
    Growth for 4 days in Bold algae basal medium shows that the alga needs relatively low levels of nutrients for optimal growth at 27°C under optimal light irradiance. 5B. LED light irradiance levels for growth Graph shows that algae grows best in low light conditions over 7 days, on 25% BB medium at 27°C. Figure 6 is a schematic illustration of a system for producing algal biopolymers in accordance with the present invention for use in irrigation. Figure 7 is a graph showing sequential harvesting of Dictyosphaerium chlorelloides ALG03. Biomass shown as optical density readings (680nm) on a PBR showing daily harvest at 50% rates allows regrowth of the same biomass of Dictyosphaerium chlorelloides ALG03 within 24 hours when supplied with new growth medium. Figure 8 shows the adaptation of the strain for wastewater and LED lighting giving increased growth rates compared to the non-adapted strain. Increased growth of Dictyosphaerium chlorelloides by physiological adaptation with six months (6 cycles) of exposure to wastewater and regulated PAR LED lights (27°C and 24 hours of light). Figure 9 shows EPS duplication after the end of second stage cultivation (right image). Figure 10 shows comparison of carbohydrate/lipid/protein contents of cells from 3organisms and calorific values. Comparison of carbohydrate/lipid/protein production by a Dictyosphaeriiun chlorelloides ALG03 strain in the log phase of growth and biomass calorific values. Cultures were collected in the exponential growth phase. Figure 11 shows the results of using drip irrigation to grow leeks in an infertile sandy soil in a greenhouse experiment over a 12-week period. DETAILED DESCRIPTION OF THE INVENTION
    In a first aspect, the invention provides a process for the intensified production of one or more metabolites of microalgae and/or cyanobacteria, said process comprising the steps of: (i) cultivating a strain of microalgae or cyanobacteria through a production phase ; (ii) expose as microalgae or cyanobacterial culture to a stimulus, where the stimulus comprises (a) a decrease in pH to a pH of no more than about pH 6 followed by an increase in pH to a pH of not less than that about pH 7, and (b) an increase in light irradiance to at least 400 µmol/m2/s.
    In the context of the present invention, the term "cultivate" is used to mean the growth of one or more strains of microalgae or cyanobacteria in any suitable media. Such media may include standard culture media as would be known to one of skill in the art, for example Bold's basal medium or equivalent. In a preferred embodiment, strains of microalgae or cyanobacteria are grown in process water, where the term "process water" includes process water emerging from industrial systems and domestic sewage water. Process water can be treated prior to use (eg, sterilized or not) and supplemented with nutrients such that nutrient levels are within ranges found in standard growth media.
    The culture may be a batch, fed-batch, or at least partially continuous (before stationary phase) culture in certain embodiments.
    The Production Phase Growth of microalgal or cyanobacterial strains during the production phase normally occurs at an exponential rate. Therefore, in certain embodiments, the production phase corresponds to the exponential growth phase of the crop.
    The "exponential phase", also known as the "log phase" or "log phase", is a defined period during batch culture of microorganisms including microalgae and cyanobacteria, characterized by cell doubling. The number of new organisms appearing per unit of time is proportional to the existing population. If growth is not limited, doubling will proceed at a constant rate so that both the number of cells and the rate of population increase double with each consecutive time period. Actual growth rate may vary depending on the strain of Microorganism used and/or growing conditions.
    The environmental or culture conditions used during the production phase can be specifically selected to allow exponential growth of the microalgal or cyanobacterial strain being used.
    In certain embodiments, the cell density for the microalgal or cyanobacterial strain at the point when exponential growth begins to cease, before the start of the stationary phase, the end of the exponential phase of production, should not be less than 108 cells per ml of culture medium, and preferably between 107 and 108 cells per ml. The culture conditions used to achieve optimal crop growth or exponential crop growth can be selected from the following: - continuous artificial light of a wavelength between about 400nm and 700nm; and/or - continuous artificial light between about 50 μmol/m2/s and 200 μmol/m2/s; and/or - temperature between about 20°C and 40°C; and/or - oxygen levels between about 500 mV and 800 mV; and/or - pH between about pH6 and pH9.
    It will be understood by one skilled in the art that the culture conditions can be varied depending on the strain of microalgae or cyanobacteria being cultivated. Specific conditions are discussed herein for specific strains of the invention and such conditions can be used as a guide to being applied to other strains, as would be understood by one of ordinary skill in the art.
    In preferred embodiments of the invention, the culture or growth of the strain of microalgae or cyanobacteria during the production phase takes place in a photobioreactor (PBR). As used herein, a photobioreactor is to be understood as a bioreactor that incorporates one or more light sources to provide photonic energy input into the reactor. In preferred embodiments, the microalgae or cyanobacteria are cultivated in a closed system to the (external) environment. Preferred light sources include light emitting diodes (LEDs) and, in particular, LEDs emitting PAR (photosynthetically active radiation in the 400-700 nm range) light. In certain embodiments, a PBR can be configured to include LED (highly dimmed) light sources designed to provide 360 degree angle illumination from the center of the bioreactor in order to maximize the growth of algae or cyanobacterial strains. different around the light source. In preferred embodiments, light is provided by LEDs emitting 2 peaks of red and blue light within the spectrum of PAR 400-700nm. In a preferred embodiment, light is provided by LEDs emitting a red light peak in the range between about 500-665 nm, preferably about 660 nm, and a blue light peak in the range between about 440-500 nm , preferably about 460 nm.
    When the production phase is carried out in a PBR, the density for the strain of microalgae or cyanobacteria should not be less than 10% (v/v) of the volume of the PBR at the beginning of the production phase. In the same or alternative embodiments, microalgal or cyanobacterial cultures are not exposed to natural sunlight. the stimulus
    In the second step of the process of the invention, the culture of microalgae or cyanobacteria is exposed to a stimulus, where the stimulus comprises, consists essentially of or consists of (a) a decrease in pH to a pH of no more than about pH 6 followed by an increase in pH to a pH of not less than about pH 7, and (b) an increase in light irradiance to at least about 400 µmol/m2/s.
    The pH of the culture during the production phase will typically be in the region of about pH 7, 9, and therefore the stimulus may comprise a decrease in pH from a pH of between about pH7 and pH9 to a pH of between about about pH pH3 and pH6, preferably between about pH5 and pH6. The pH of the culture can be reduced and subsequently increased by any suitable means known to the person skilled in the art, provided the viability of the culture is not compromised. In a preferred embodiment, the pH is reduced by adding carbon dioxide, CO2.
    The light irradiance delivered to the crop during the production phase will typically be in the region of 50-200 µmol/m2/s and therefore the stimulus may comprise an increase in light irradiance of between about 50-200 µmol/m2/s between about 400-2000 µmol/m2/s. The light source preferably comprises, consists essentially of or consists of one or more LEDs.
    The microalgal or cyanobacterial culture is typically exposed to the stimulus once the culture has reached the peak of exponential phase growth. This peak occurs just before the start of the stationary phase. The stationary phase is a well-defined period during batch culture of microorganisms including microalgae and cyanobacteria, in which the growth rate of the culture slows, usually as a result of nutrient depletion and accumulation of toxic products. During this phase, the microorganism's growth rate is typically equal to the microorganism's death rate. It will be understood by one of skill in the art that any culture of microalgae or cyanobacteria undergoes a transition from peak exponential growth to stationary phase. The culture can be exposed to the stimulus at any time during the peak of growth from the exponential phase or at any time during the transition from the exponential to the stationary phase.
    The growth rate of microalgae or cyanobacteria can be monitored throughout the entire production phase, for example using sampling techniques such as cell counting or by measuring chlorophyll levels in the culture. Cell numbers can be determined using spectrophotometric sampling of the culture, for example, taking readings around 680nm OD. These measurements can be used to identify the exponential growth phase, the peak of exponential phase growth, and the time when the crop begins to enter the stationary growth phase. Any proper technique can be used.
    The stimulus comprises, as a minimum, a decrease in pH to a pH of not more than about pH 6 followed by an increase in pH to a pH of not less than about pH 7, and an increase in light irradiance to at least 400 µmol/m2/s. The stimulus may further comprise the addition of a carbon source, where the carbon source is CO2 in its various forms.
    In preferred embodiments, the reduction and subsequent increase in pH precedes the increase in light irradiance. The pH can be reduced to a pH of no more than about pH 6, preferably between about pH5 and pH6, over a period of between about 30 minutes and about 2 hours. The pH can then be increased to a pH of between about pH6 and pH9, preferably between about pH7 and pH9. The pH can be raised using any suitable media as long as this does not compromise the viability of the culture. Generally, pH is restored to prestimulus levels.
    In certain embodiments, the microalgal or cyanobacterial culture is grown for an additional growth period of at least about 12, 24, 36, 48 hours etc. after exposure to the stimulus begins. In embodiments where the pH is lowered over a period of between about 30 minutes and 2 hours, and then raised, for example, to pre-stimulus levels, the culture period after exposure to the stimulus can be calculated to from the time of the initial decrease in pH, or from the time when the pH is restored and the irradiation is subsequently increased. Production of metabolites
    One of the effects of exposing a strain of microalgae or cyanobacteria to the stimulus is to induce or promote the production of particular metabolites within the microalgae or cyanobacteria. It has been observed by the present inventors that exposure of certain strains of microalgae and/or cyanobacteria to the stimulus, as defined herein, leads to the production of large amounts of particular metabolites, the particular metabolite being dependent on the strain of Microorganism used. The purpose of carrying out the claimed process described above is thus to intensify the production of one or more metabolites in microalgal and/or cyanobacterial cells. These metabolites are typically collected after the subsequent growth period, after the culture has been exposed to the stimulus. Metabolites can be harvested, purified and/or analyzed by any suitable means known to those skilled in the art, for example, as described in Bligh, E.G. and Dyer, W.J., 1959. A rapid method for total lipid extraction and purification. Can.J.Biochem.Physiol. 37:911917.
    Lipids can also be collected/analyzed according to standard industrial procedures, such as supercritical C02, variants in solvent extractions, fractional distillation and chromatography, or according to the following protocol: 25mg of the algae product sample is mixed with the internal standard if used for GC-MS measurements in culture tubes. Add 1.5 ml of 0.5N sodium hydroxide in methanol, cap the tubes and heat at 100°C for 5 minutes. Allow to cool and add 2 ml reagent BF3/methanol, cap tubes, mix and heat at 100°C for 30 minutes. Allow to cool in 2 ml of isohexane + BHT, then 5 ml of saturated sodium chloride, cap the tube and shake vigorously for 30 seconds. Cool to room temperature and allow layers to separate. The upper isohexane layer is placed through a short column of anhydrous sodium sulfate and collected. The aqueous layer is extracted with 2 ml of iso-hexane + (butylated hydroxytoluene), BHT and the upper iso-hexane layer is placed through the anhydrous sodium sulfate column and the solvent collected with the other sample. The column is washed with 2 ml of iso-hexane + BHT and also placed in the same sample tube.
    Polysaccharides can be analyzed using chromatographic techniques available to those skilled in the art. The analysis of carbohydrates and sugar alcohols can be analyzed in microalgal biomass extracts by means of high pH anion exchange chromatography (HPAE), followed by electrochemical detection (IPAD) and parallel mass spectrometry detection. In a preferred embodiment, sample extract is analyzed using an ICS-5000 modular system coupled with an MSQ Plus mass spectrometer. Analytes are separated in a CarboPac MA1 column using isocratic condition. After the analytical column, the effluent from the columns is divided so that half of it flows through the amperometric cell when integrated pulsed amperometric detection (IPAD) is performed, while the other half is continuously desalinated using a desalination device with membrane base (ASRS). The neutralized effluent is then mixed with an aqueous solution of lithium chloride in order to facilitate the detection of carbohydrates in MS as their lithium adducts.
    Spent biomass can, in turn, be used for a variety of applications, including but not limited to ruminant feed, livestock feed, aquaculture, poultry feed, carbohydrate fraction of biomass in bioethanol production and extraction of valuable amino acids from proteins.
    The process described here can be used to enhance the production of any metabolite found in microalgal and/or cyanobacterial strains. In certain embodiments of the invention, the process is used for the enhanced production of lipids, including but not limited to a fatty acid selected from myristic acid, palmitic acid, palmitoleic acid, behenic acid, lauric acid, linoleic acid, alpha acid and gamma linolenic acid, stearic acid, arachidonic acid and eicosapentaenoic acid. In a preferred embodiment, the process of the invention is for the enhanced production of eicosapentaenoic acid (EPA). The processes described herein can be used in particular to obtain desired fatty acid profiles in cultivated microalgal or cyanobacterial strains, for example, to optimize EPA production in EPA-producing strains. Fatty acids produced through the process described here can, for example, be used in the food sector for the production of liquid biofuel for cosmetic ingredients.
    In certain embodiments of the invention, the process is used for the intensified production of carbohydrates. In a preferred embodiment, the process of the invention is for the enhanced production of exopolysaccharides (EPS). Microalgae / Cyanobacteria
    Microalgae or cyanobacteria for use in conjunction with the process of the present invention may be selected from any suitable strains of algae or cyanobacteria. Appropriate microalgae strains can be selected from the following: - green microalgae; freshwater microalgae, the phylum Chlorophyta, the Pleurochloridaceae family; Trachydiscus sp; Chlorogibba sp.; and Dictyosphaerium chlorelloides. Appropriate cyanobacterial strains can be selected from the following: - the order Chroococcales; the genera Synechocystis or Synechoccocus.
    In embodiments where the process is to be used for the enhanced production of eicosapentaenoic acid, it is preferred that the microalgal strain is selected from Trachydiscus sp and Chlorogibba sp., and is in particular the strain of Chlorogibba allorgei deposited with Culture Collection of Algae and Protozoa under accession number CCAP 817/1 (also referred to herein as ALG02), or a mutant strain derived therefrom.
    In embodiments where the process is to be used for the intensified production of exopolysaccharide, it is preferred that the microalgae strain is selected from Dictyosphaerium chlorelloides, and is in particular the strain of Dictyosphaerium chlorelloides deposited with the Culture Collection of Algae and Protozoa under CCAP accession number 222/98 (also referred to herein as ALG03), or a mutant strain derived therefrom.
    As used herein, the term "mutant strain" is to be understood to mean a strain derived from the original strain of microalgae and/or cyanobacteria, which retains the characteristics of the parent strain, provided that such characteristics refer to the profile of the metabolite produced. when the strain is exposed to a stimulus according to the process of the invention, described above.
    Microalgae and/or cyanobacteria for use in conjunction with the process of the present invention may have been pre-selected, for example, on a basis of optimal growth under the same culture conditions as used during the production phase. This pre-selection or adaptation stage, which may precede the steps of the process detailed above, is described in more detail in accordance with the second aspect of the present invention described hereinafter. All embodiments described hereinafter are applicable to other aspects of the invention.
    Thus, according to a second aspect of the invention, there is provided a process for the production or growth of microalgae and/or cyanobacteria, or the production of at least one metabolite derived therefrom, which process comprises: (i) a adaptation stage, comprising culturing microalgae or cyanobacteria: (a) in a process water feed load and selecting those microalgae or cyanobacteria capable of growing on the process water feed load; and/or (b) under light emitting diodes (LEDs) emitting 2 peaks of red and blue light within the spectrum of light wavelengths between about 400 and 700nm; and (ii) the production phase, comprising cultivating the microalgae or cyanobacteria selected from (i) under the same process water feed load used in the adaptation stage and/or under the same light conditions used in the adaptation stage. Adaptation
    Process water feed load can be obtained from industrial process systems or domestic wastewater systems. Important nutrients for algae growth, such as nitrates and phosphates are typically present in said feedstock. Other algae-stimulating substances may also be present, such as vitamins and minor elements. The food, soft drinks and brewery industries produce many suitable process water streams. Other less nutrient-rich freshwater sources can be used if nutrients are added equivalent to the levels used in standard growing media.
    Microalgae or cyanobacteria are normally grown in a process water feed load during the adaptation stage under optimal culture conditions, including but not limited to the following: - continuous artificial light of a wavelength between about 400nm and 700nm; and/or continuous artificial light of between about 50 µmol/m2/s and 200 µmol/m2/s; and/or temperature between about 20°C and 29°C; and/or pH between about pH7 and pH9.
    Process water can be preconditioned, for example, in order to reduce particulates. Microalgal or cyanobacterial growth during the adaptation stage can be performed using erlenmeyer flasks (100-500 ml) or preparative photobioreactors. Light can be provided by LED sources or other unnatural light. In a preferred embodiment, light is provided by LEDs emitting 2 peaks of red and blue light within the PAR 400-700nm spectrum. In another preferred embodiment, the light is provided by LEDs emitting a red light peak in the range between about 500-665 nm, preferably about 660 nm, and a blue light peak in the range between about 440-500 nm , preferably about 460 nm.
    In certain embodiments, the goal of the adaptation stage is to identify the strains of microalgae or cyanobacteria that are capable of growing in the process water feedstock to be used in the production phase of the process.
    Alternatively, or in addition to, light can be used to adapt or "regulate" microalgae or cyanobacteria to the conditions to be used in the production phase, preferably so as to optimize growth during the production phase. In a preferred embodiment, the adaptation phase involves the growth of algae and/or cyanobacteria under LEDs emitting 2 peaks of red and blue light within the PAR 400-700nm spectrum. In another preferred embodiment, light is provided by LEDs emitting a red light peak in the range between about 500-665 nm, preferably about 660 nm, and a blue light peak in the range between about 440-500 nm, preferably about 460 nm. The adaptation stage may involve culturing the microalgal or cyanobacterial strains for a period of at least about 2, 3, 4, 5, 6 months, preferably at least about 3 months. The adaptation stage may involve culturing the microalgal or cyanobacterial strains for at least about 2, 3, 4, 5, 6, 7, 8 generations of growth, preferably at least 20 or about 6 generations of growth . In embodiments where the microalgae or cyanobacteria are cultivated for several months or for at least 2 generations of growth, the microalgae or cyanobacteria can be subcultured, where subculture is intended to mean transferring some or all of the complete culture to a new growth medium. In a preferred embodiment, the microalgae or cyanobacteria are sub-cultured once a month or once every 4 weeks.
    Once the microalgae or cyanobacteria strain was selected according to the adaptation stage described above, the second stage of the process is the production phase, which comprises cultivating the selected microalgae or cyanobacteria in the same process water feed load and /or under the same light conditions used in the adaptation stage.
    The term "the same" process water feed load is intended to mean process water from the same batch, ie with the same characteristics as used for the adaptation stage, rather than the exact same media used for cell growth during the adaptation stage.
    The production step of the second aspect of the invention may be carried out in accordance with any of the embodiments of the production step as described above for the process of the first aspect. For example, the growth of the adapted microalgal or cyanobacterial strain can be carried out under conditions that allow for exponential growth. Furthermore, growth during the production phase is preferably carried out in a photobioreactor in accordance with the above definitions. The condition used in the production phase may mirror the conditions used in the adaptation stage. Thus, in addition to using process water as the feed load, adaptation can extend to other environmental conditions as described above (wavelength of light, irradiance levels, temperature, pH etc.). More specifically, the adaptation may be to use LED lighting as described herein. In a preferred embodiment, adaptation is for LED illumination provided in 2 peaks of red and blue light within the PAR 400-700nm spectrum. In another preferred embodiment, the light is provided by LEDs emitting a red light peak in the range between about 500-665 nm, preferably about 660 nm, and a blue light peak in the range between about 440-500 nm , preferably about 460 nm.
    In certain embodiments, the production phase may be followed by a step comprising exposing the microalgal or cyanobacterial culture to a stimulus to enhance metabolite production. All embodiments of the stimulus described above in the context of the first aspect of the invention apply mutatis mutandis to the process of the second aspect of the invention. In addition, microalgal and/or cyanobacterial strains for use in accordance with the process of the second aspect of the invention may be selected from any of the phyla, orders, families and/or species of microalgae and/or cyanobacteria already described above. .
    The process of the second aspect of the invention can be used to enhance the production of microalgal and/or cyanobacterial biomass. Such biomass can be used in applications such as the production of biofuels, biodiesel, gasoline, kerosene or for use as a soil conditioner or biofertilizer, including underground irrigation systems as described herein below. Alternatively, or in addition to, the process can be used for the production of metabolites, including, but not limited to, enhanced production of lipids and/or carbohydrates. In preferred embodiments, the process is for the enhanced production of eicosapentaenoic acid. In another preferred embodiment, the process is for the enhanced production of exopolysaccharide.
    In a further aspect of the invention, a Microorganism is provided which is, or has the identifying characteristics of, a strain of Chlorogibba allorgei deposited with the Culture Collection of Algae and Protozoa under accession number CCAP 817/1, or a mutant strain derived from it.
    In yet another aspect, a Microorganism is provided which is, or has the identifying characteristics of, a strain of Dictyosphaerium chlorelloides deposited with the Culture Collection of Algae and Protozoa under accession number CCAP 222/98, or a mutant strain derived from same.
    The present invention describes a process for the production of lipids and metabolites of eicosapentaenoic acid, myristic acid, palmitic acid, behenic acid, lauric acid, linoleic acid, alpha-linolenic acid and stearic acid and the like from algae belonging to the Pleurochloridaceae family ( for example, Trachydiscus sp. and Chlorogibba sp.). Thus, the invention provides a process for priming initiation and selection of adapted cells from said algae cultures ready for the bioreactor and/or other culture conditions used to produce algal biomass products containing lipids and metabolites described herein. More particularly, the present invention provides a process for producing and/or increasing the yields of certain lipids containing fatty acids. The method may comprise a method of preparing algae belonging to the Pleurochloridaceae family and containing the aforementioned lipids and fatty acids by increasing the photosynthetic efficiency, adjustment and metabolic activity of nutrients for the downstream production of biodiesel, gasoline, kerosene and other chemicals of high value. The present invention also provides a process for obtaining desired fatty acid profiles in algae strains forming eicosapentaenoic acid. This process allows optimization for or production of eicosapentaenoic acid for the food sector or saturated fatty acids or fatty acids - myristic acid, palmitic acid, behenic acid, or lauric acid, linoleic acid, alpha-linolenic acid and stearic acid and the like , for the production of liquid biofuel(s) or for cosmetic ingredients by adjusting certain nutrient levels (including nitrogen and sulfur) in the growth medium, along with altered light irradiance levels. The present invention also provides a method for preparing species of the order Chroococcales cyanobacteria in the genera Synechocystis and Synechoccocus containing some or all of the above mentioned lipids and fatty acids increasing photosynthetic efficiency and metabolic activity. The present invention further provides a process for obtaining the specific production of fatty acids in lipids by regulating the photosynthetic efficiency in the presence of selected bands within the photosynthetically active radiation (PAR) wavelengths using continuous light.
    The invention also relates to a specific (isolated) strain of algae belonging to the Pleurochloridaceae family and in particular to the genus Chlorogibba, more specifically a strain of Chlorogibba allorgei. The strain has been deposited with the Culture Collection of Algae and Protozoa under accession number CCAP 817/1 accepted January 25, 2011. This strain is shown here as being usable in the production of specific metabolites.
    The present invention further provides a process for producing an algal biomass product containing eicosapentaenoic acid and the aforementioned lipids, fatty acids and metabolites thereof; suitable for the downstream production of bio-diesel, gasoline, kerosene and other such liquid biofuels and high value industrial chemicals comprising the steps of: a) an upstream culture stage, which is used to isolate sub-cultivate and prepare regulated algae belonging to species within the Chlorophyta specifically including the EPA-rich Pleurochloridaceae family and the cyanobacteria order Chroococcales in the genera Synechocystis and Synechoccocus using regulated continuous light PAR wavelengths and process water stress. Algae belonging to the Pleurochloridaceae family and the cyanobacteria order Chroococcales in the genera Synechocystis and Synechoccocus are now ready for use in bioreactors using similar water sources and light supply. The results show that up to a two-fold increase in growth rates compared to the same crops grown in normal water with nutrients and daylight or fluorescent lighting can be expected in different strains. b) a stage in a photobioreactor in which algae belonging to Chlorophyta, the Pleurochloridaceae family or the order of Chroococcales cyanobacteria in the genera Synechocystis and Synechoccocus are grown in industrial process water, +/- nutrients (100% standard growth medium comprising o NaNO3 (0.25 g/L); CaCl2 .2H2O (0.025 g/L); MgSO4 .7H2O (0.075 g/L); K2HPO4 (0.075 g/L); NaCl (0.025 g/L); KH2PO4 (0.175 g) g/L); FeSO4. 7H20 (4.98 mg/L); H2SO4 (0.01 μl/L); H3BO3 (0.1142 g/L); ZnSO4. 7H2O (0.00882 g/L); MnCl2 . 4H2O (0.00144 g/L); Mo03 (0.00071 g/L); CuSO4 .5H2O (0.00157 g/L); Co(N03)2 .6H2O (0.00049 g/L); EDTA (0.005 g/L); KOH (0.031 g/L)), PAR light wavelengths and specific irradiance levels in a continuous flow system, is kept in exponential growth phase to allow daily harvesting at a fixed ratio of the biomass product. The biomass product contains lipids, fatty acids and their metabolites and bio-proteins mentioned above belonging to the Pleurochloridaceae family and the order of the cyanobacteria Chroococcales in the genera Synechocystis and Synechoccocus. The bioreactor is replenished with process water or nutrient altered process water to allow an algae cell density to recover the exponential growth phase before the next harvest.
    Algae generally contain about 7-60% by weight of the lipid content relative to the total weight of dried algae. In the case of algae belonging to the Pleurochloridaceae family, eicosapentaenoic acid comprises 25 to 40% of the total fatty acids in the lipid and the remaining fatty acids are mainly said saturated or unsaturated fatty acids. Thus, algae belonging to the Pleurochloridaceae family have a higher amount of EPA than traditional sources derived from fish oils.
    According to the embodiment of the present invention, lipids and metabolites of eicosapentaenoic acid, myristic acid, palmitic acid, behenic acid, lauric acid, linoleic acid, alpha-linolenic acid and stearic acid and the like belonging to the Pleurochloridaceae family can be verified as being present as free fatty acids and/or in situ in the growth medium and/or obtained from the wet and/or homogenized algal biomass product and/or the algae after lyophilization or air drying and extracted in the presence of a appropriate organic solvent and/or sonicated or using supercritical CO2.
    In certain embodiments of the invention when the growth medium (using water for decontamination or water altered with nutrients) flows through the bioreactor with the algae, it is exposed to regulated sets of LED lights dimmable internally or externally to the bioreactor comprising unique patterns of red and blue light-emitting diodes (LEDs), which are configured to operate at different wavelengths depending on the algae strain. The angulation of the LEDs is optimized for maximum irradiance feed to algae cultures growing inside PBR to promote and maintain the exponential growth phase of the algae. Algal biomass is monitored by cell density, but also by lipid/fatty acid content to see if they have reached target levels. Biomass water is then removed and it is passed to the appropriate downstream process for biofuels or high value fatty acids are extracted. The remaining biomass can be used either for animal feed (if pure water is used in the bioreactor), or for energy production in anaerobic digesters (AD) or pyrolysis systems. Bioreactor
    The present invention provides an integrated process of using any combination of standard or renewable energies such as solar, wind, hydro, geothermal and thermal and/or other renewables to power a photobioreactor and produce continuous high yields of algal biomass. Using effective and cost-effective methods algal biomass is further refined to produce biodiesel, biokerosene, gasoline, ethanol and other valuable co-products.
    The system encompasses highly highly regulated LED light sources, which are designed to maximize the growth of different algae strains. The system includes, but is not limited to, inclusion of highly regulated LED light sources that are designed to provide 360 degree angle illumination from the center of a bioreactor to maximize the growth of different strains of algae (growth in around the light source). Energy sources The system can be self-energized with solar, wind, hydraulic, geothermal and thermal and/or renewable energies.
    These different energy sources are managed through a commercial energy management system that uses a scalable storage facility (such as a battery bank) to store and supply energy.
    Process temperature is controlled in two ways: water heating through the supply of process water naturally emerging from industrial systems or domestic wastewater and regulated by thermostats or heat exchangers or managed through an automated process control unit. Low energy light sources
    The process employs a wide range of low voltage lights (such as LEDs as well as other unique light emitting sources) that are powered by a combination of standard and/or renewable energy sources, where the lights are dimmed to optimize growth of algae, without compromising other lipid or other by-products of commercial value.
    In preferred embodiments, an array of lights, emitting light covering wavelengths with a precise angle of beam emissions within photosynthetically active radiation (PAR) wavelengths of 400700 nm, is equipped internally or externally to the PBR. This helps to encourage the algae to maximize biomass growth at a cost effective production rate.
    The algae were selected from previous adaptive conditioning to the conditioned environment, using specific management of light sources and wavelengths, process water, temperature and pH, to provide strains capable of optimal growth under reduced energy inputs.
    In one embodiment, the present invention provides a new process for enhancing the exopolysaccharide (EPS) of the alga Dictyosphaerium chlorelloides ALG03 within any photobioreactor system. This comprises (a) a pre-cultivation to adapt the strain for LED lighting regulated within photosynthetically active radiation (PAR) wavelengths and industrial process water conditions (eg, secondary treated process water), (b) growing the strain forming EPS within a PBR supplemented by regulated PAR LED illumination (400-700nm photosynthetically active radiation) to provide continuous illumination over any period between 1 and 24 hours of the algae each day, (c) after the phase peak growth exponential move a portion so that approximately 50% of the yield to a static light tank system with gentle aeration (room air with no added CO2) to enhance EPS formation for 3-4 days (d) remove the water from the algae slurry for biopolymer extraction or for other uses described above or for energy production through ethanol fermentation or other processes (eg anaerobic digestion or biomass gasification).
    Examples of gelatinous coated microalgal strains suitable for use in the invention may reside in Chlorophyta and selected from the Dictyosphaeriaceae family. Adapted algae strains
    The microalgae strain used for EPS production is maintained in heterogeneous industrial process water supplies in standard bottle cultures and PAR lighting provided via low energy LED fixtures with regulated light patterns. Light can be provided internally within the PBR or externally. Cultures are sub-cultured each month to select cells adapted to wastewater constituents and LED lighting. This phenotypic selection process includes an evaluation step to verify EPS production in each subculture. The mother cultures are maintained under these growing conditions to continue the adaptation process.
    Algae cultivation in photobioreactors ("PBRs") A great deal of work has been done to develop small scale PBRs for the production of microalgae (g) . Commercial scale PBRs (>100,000 L) must have large volume capacities and have a small footprint in terms of floor space. Furthermore, they must have transparent surfaces, high mass transfer rates, and must be able to produce large yields of biomass. Furthermore, any LIC project must take into account the specific needs of individual microalgae strains and be robust and low maintenance.
    It has been suggested that PBRs can also act as culture vessels for outdoor tank growth systems. Since outdoor PBRs are generally naturally lit with sunlight, biomass productivity will depend on the prevailing year-round environmental conditions in a given location. Seasonal variations in temperatures and sunlight occur throughout the year in most regions that were tested (often desert environments), so it is difficult to carry out year-round outdoor mass cultivation of algae in such regions. There are a number of projects for PBRs in the public domain being sold commercially and projects in academic articles, but there is no definitive standard "best practice" model.
    In a first set of embodiments, any PBR apparatus (tubular design with air lift or flat tank) can be employed, which has a device to drain or collect the cells on a daily basis and can be illuminated for part or a day of 24-hour continuous, with PAR wavelengths of LED illumination (internally or externally). Algae growth is monitored by an integrated process control device to measure 680nm OD, which is correlated with cell numbers per previous computation and allows determination of peak exponential growth from PBR authorization. The system includes, but is not limited to, inclusion of highly regulated LED light sources that are designed to provide 360 degree angle illumination from the center of a bioreactor to maximize the growth of different strains of algae (growth around of the light source).
    Upon reaching peak exponential growth, 50% of the cells are harvested daily into a separate batch holding tank that has built-in aeration (ambient air mixture) and 24/7 LED lighting. Algae strains are kept under prescribed conditions for 3-4 days and tank samples monitored daily visually under a microscope for EPS production.
    These cells are finally extracted through water removal for either carbohydrate extraction or for the uses described above. Lighting Sources
    Algae farming systems can be lit by artificial light, sunlight, or both. One of the most important factors to control the production of high biomass microalgae in closed PBR is the light irradiance and the quality of the light spectrum provided. If the PBR is positioned outdoors, it may be limited to the high light levels observed during the day and during the summer months, but this may vary geographically. Ultraviolet ("UV") light levels in natural sunlight can also cause algae photoinhibition at certain times of the day and thus lack the potential control of PBR grown using artificial light.
    A major light source that can provide specific wavelengths of light is the range of light-emitting diodes ("LED"), which are now being developed by several major manufacturers. LEDs have the ability to save energy and have a long life expectancy. Its electrical efficiency helps to minimize heat generation. This high efficiency and sharp spectrum have eliminated the need for a cooling and filter system, thus significantly reducing energy consumption requirements. These factors open up the potential to optimize light delivery for specific strains of microalgae (h).
    It is known that parts of the photosynthetically active radiation (PAR) spectrum (400-700nm, which covers the red and blue ends of the spectrum) can help maximize the efficient photosynthesis of algae in general, but there is little data (in the public domain) on specific interactions of such light irradiance on the growth and physiological state of biofuel precursors (lipids/carbohydrates) for different strains of algae cultivated under artificial light in PBRs. Applicants found the precise red/blue LED requirements and distributed irradiance levels for a range of microalgae and for the two-stage process of EPS production here.
    "Sustainable" Energy Inputs for PBR Systems The potential use of "waste" streams, where scrubbed flue gases supply CO2 with modified process water used as a nutrient medium for algae growth has been debated at length in the literature. There are some significant, or successful, demonstration projects that have been established to date.
    It has been estimated that using hybrid systems, algae can be used to recycle 20% of C02 emissions from industrial power generation. If new and inexpensive sources of C02 capture can be developed, this will reduce the energy requirements and costs of PBRs.
    The process of growing algae in industrial process waters can reduce nutrient levels to potentially required levels with the evolution of strict targets in legislation in different parts of the world for the discharge of water into the environment and particular bodies of water.
    Applicants have discovered the optimal nutrient levels required under controlled temperature and light irradiance levels for cost-effective growth of the algae described in this document. Algae biomass and biopolymers
    Algal polysaccharides are currently used commercially (d) . Soil algae are known to excrete a variety of extracellular polymeric substances (EPS) mainly polysaccharides which may play an important role in their vital function (e). The algae strain forming exopolysaccharide (EPS) has the advantage of also being used in water to decontaminate by sequestering potentially toxic elements within the EPS formed in the batch stage and before. There is clear evidence that elements such as copper can be linked by these mucilaginous compounds (i) . This has clear value where cleaning water for discharge or downstream use is the primary objective. Carbohydrate analysis performed on the EPS composition showed 94-95% neutral monosaccharides and about 5-6% uronic acid. The latter is particularly important for binding heavy metals.
    Soil algae are known to intensify soil formation and water retention, soil stabilization, increase nutrient availability from nearby plants and reduce soil erosion. They have been introduced as soil conditioners in many countries and have also been suggested for use as biofertilizers (j) •
    Decreased "fresh" water supply will increase the need to use recycled water and secondary treated water has been used for fresh vegetable and fruit production in Israel using irrigation systems. However, this actually needs regulation in many countries in order to be modified for its widespread use even with SDI, where the treated water does not reach the edible parts of the crop (above the land).
    Traditional drip irrigation is found on the above part, but recently numerous companies manufacturing drip irrigation systems have invented and implemented underground drip irrigation (SDI) systems. Lines are buried below ground for longer service life. Subsoil irrigation allows for the precise application of water, nutrients and other agro-chemicals directly to the root zone of plants. This allows the farmer to optimize the growing environment and lead to higher quality crop results.
    When properly managed SDI is one of the most efficient methods of irrigating plants with an efficiency >90%. The savings in applied water can be as high as 50% compared to other methods. The amount of water available for irrigation is expected to decrease in the coming decades and water conservation is more important. This is where SDI is crucial when extending the life of aquifers. Its use for biofuel crops has great potential and it has been shown in poor soils that the use of SDI can double sugarcane production yields and increase sugar content while conserving 70% of the water requirement (k).
    The exopolysaccharide from an adapted strain of Dictyosphaerium chlorelloides ALG03 can also be used for the downstream production of bioethanol using the extracted carbohydrates. The remaining carbohydrates and proteins can be used for other by-products such as food additives and animal feed.
    The invention will be further understood with reference to the following non-limiting examples. EXAMPLES Example 1 Algae belonging to the Pleurochloridaceae family or cyanobacteria order Chroococcales in the genera Synechocystis and Synechoccocus were pre-incubated in nutrient-modified water and continuous PAR lighting conditions (within wavelengths 400-700nm) for a period of 6 months and sub-cultured at regular intervals. Isolated cultures were further sub-cultured to bring them to the exponential growth phase and used in a series of 50L bioreactors. The starting cell density of the algal inoculum was 106 cells per milliliter taken from the subcultured algae which were transferred to a 50 L bioreactor containing nutrient modified water and allowed to reach the exponential growth phase in the presence of PAR lighting conditions continuous (wavelengths between 400-700nm). The process was controlled by several parameters, including: pH (maintained between 6-9), temperature (maintained between 20-40°C); aeration (0.02-1.0 v/v/m - volume of air per volume of liquid per minute), and/or C02, (0% at startup ending at least 0.7% in first harvest, but can reach 5% within the overtime system); 02 kept between 500 and 800mV); maximum light irradiance in first cells in PBR (600 mmol m-2 s-1) aeration, C02 (starting at 0% CO2 added and increasing to 0.7% per harvest), cell density, temperature (27°C on average ) and light supply (24 hours a day). Upon reaching the maximum exponential growth close to 500g of wet algal biomass were removed from the reactors. A solvent mixture containing methanol/acetyl chloride/hexane was added producing methyl esters of native lipid acids. The methyl esters were separated by chromatography using hexane and an acetone solution and then evaporated to obtain 1.6 g of eicosapentaenoic acid. Chromatography also provided pure samples of myristic acid, palmitic acid, behenic acid, lauric acid, linoleic acid, alpha-linolenic acid and stearic acid, and the like. Example 2 50.08g of biomass was dried for 6 hours at 105°C. After cooling in a desiccator, the sample was placed for 1 hour in an oven at 105°C and weighed again. In a Soxhlet extraction device, dry biomass was extracted in 260 ml of petroleum ether for 8 hours in an extraction capsule. After evaporation of the solvent in a vacuum rotation evaporator, the sample was further dried by passing a fine flow of nitrogen over the sample and weighed again after cooling. To obtain the lipids, the sample was dissolved in 50 ml of hexane and divided in half. Ten grams of activated carbon were added to the first sample and the solution was filtered and again evaporated and put in a stream with nitrogen and weighed. There was still some color present, so for the second sample 20g of activated carbon was used.
    The samples were analyzed using an Agilent Technologies 6890N and an HP 88 column, cyanopropyl - length: 100m - diameter: 0.25mm, layer width: 0.2 Oμm. Table 1 - The results of the analysis of the percentages of 3 fatty acids bound by their metabolic pathway from 3 different batch PBR cycles.


    The results clearly indicate switching from GLA to EPA via ARA as the algal strain is growing and then induced to enter the stationary phase after stimulation as described above. This is evidence 5 that the process described here is directly controlling the perceived metabolic pathways that allow interconversion between y-linolenic acid and eicosapentaenoic acid via arachidonic acid in freshwater microalgae (1). Example 3
    Dictyosphaerium chlorelloides (ALG03) was cultivated for 4 days in Bold basal medium. Figure 5a shows that algae need relatively low levels of nutrients for optimal growth at 27°C under optimal light irradiance. Figure 5b shows that algae grows best in low light conditions for 7 days on 25% BB medium at 27°C.
    During a continuous production process, Dictyosphaerium chlorelloides (ALG03) were harvested daily. Figure 7, with biomass shown as optical density readings (68 0nm) in PBR, shows that daily harvesting at 50% rates allows re-growth of the same biomass of Dictyosphaerium chlorelloides (ALG03) within 24 hours, when supplemented with new growth medium.
    Physiologically adapted cells were grown for 6 months in wastewater from municipal facilities (treated secondary) and re-cultured every 4 weeks in fresh wastewater. Unadapted cells were grown in standard ZBB growth medium and re-grown every 4 weeks in fresh ZBB medium. Then, both strains were grown for 9 days (until stationary phase) in fresh wastewater. The results (Figure 8) show a clear adaptation to the wastewater environment and LED lighting by pre-adapted cells. Figure 9 shows the doubling of EPS production after the 2-stage PBR process.
    Subsequent steps of the method included purification, EPS isolation and sample hydrolysis using methanolic HCI followed by trifluoroacetic acid. neutral monosaccharides, with 5-6% uronic acid. Some neutral monosaccharides are partially methylated. The predominant sugars were: galactose (~20-21%) 5 glucose (~20-21%) unidentified hexose (~12-13%) rhamnose (~12%) unidentified methylated hexose 9-10%) unidentified methylated hexose identified (~ 7-8%) 10 mannose (~ 6-7%) xylose (~ 3-4%) arabinose (~ 1-2%) unknown monosaccharide (~ up to 0.5%) unknown monosaccharide (~ 0.5 -1.0%) uronic acids 5-6%).
    The molar ratio of sugars in dry gel and lyophilized samples of hydrolyzed EPS is shown in Table 2. Table 2

    Example 3
    The results of using drip irrigation to grow leeks in an infertile sandy soil in a greenhouse experiment over a 12-week period can be seen in the image in Figure 11. Left to right: control without algae, fungal symbionts mycorrhizals (Glomus spp. mixture) used in planting; seaweed used weekly; algae and mycorrhizal fungal symbionts. The results clearly show a growth-promoting effect of Dictyosphaerium chlorelloides ALG03 cells added twice weekly on the growth and nutrition of leeks. A synergistic effect of using algae cells with a mixture of adapted mycorrhizal fungal symbionts was observed.
    The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the above description and the accompanying figures. Such modifications are intended to be within the scope of the appended claims. Furthermore, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the present invention (including, in insulation) as appropriate.
    Various publications are cited herein, the descriptions of which are incorporated by reference in their entirety. References a. Chisti Y - 2007 - Biodiesel from microalgae. Biotechnol. Adv. 25(3):294-306. B. Olaizola M - 2005 Microalgal removal of CO2 from flue gases: Changes in medium pH and flue gas composition do not appear to affect the photochemical yield of microalgal cultures. The Korean Society for Biotechnology and Bioengineering 8: 60-367. ç. Braun AR - 1996 Reuse and Fixation of CO2 in Chemistry, Algal Biomass and Fuel Substitutions in the Traffic Sector. Energy Convers Manage., 37:1229-1234. d. Bitton R & Bianco-Peled H - 2008 Novel Biomimetic Adhesives Based on Algae Glue. Macromolecular Bioscience 8:393-400. and. Otero A & Vincenzini M - 2003 Extracellular polysaccharide synthesis by Nostoc strains as affected by N source and light intensity. J. Biotechnol. 102:143-152. f. Gonzalez-Chavez C., D'haen J, Vangronsveld J. & Dodd JC - 2002 Copper sorption and accumulation by the extra radical mycelium of different Glomus spp. of arbuscular mycorrhizal fungi isolated from the same polluted soil. Plant and Soil 240: 287-297. g. Ugwu CU, Aoyagi H and Uchiyama H - 2 008 Photobioreactors for mass cultivation of algae. Bioresource Technology, 99, 4021-4028. H. Choul-Gyun Lee - 1999 Calculation of Light Penetration Depth in Photobioreactors Biotechnol. Bioprocess Eng., 4, 78-81. i. Garcia-Meza, JV, Barrangue C & Admiraal W - 2005 Biofilm formation by algae as a mechanism for surviving on mine tailings. Environ. Toxic. & Chemistry. 24:573-581. j. Painter TJ - 1993 Carbohydrate polymers in desert complaint-the potential of microalgal biofertilizers. Carbohydrate Polymers, 20, 77-86. k. http://www.netafim.com/article/sugarcane-- Philippines. (1) Khozin-Goldberg, I., Didi-Cohen, S., Cohen, Z., 2002. Biosynthesis of eicosapentaenoic acid (EPA) in the freshwater eustigmatophyte Monodus subterraneus. J. Phycol. 60 38, 745-756.
    权利要求:
    Claims (17)
    [0001]
    1. Process for the intensified production of (a) eicosapentaenoic acid in selected microalgae of Chlorogibba sp.; Trachydiscus sp. from the Pleurochloridaceae family or (b) exopolysaccharide in microalgae of the genus Dictyosphaerium, the referred process characterized by the fact that it comprises the steps of: (i) cultivating a strain of microalgae through a production phase; (ii) exposing the microalgal culture to a stimulus, where the stimulus comprises (a) a decrease in pH to a pH of not more than pH 6, followed by an increase in pH to a pH of not less than pH 7 and (b) an increase in light irradiance from between 50 and 200 µmol/m2/s to up to between 400 and 2000 µmol/m2/s.
    [0002]
    2. Process according to claim 1, characterized in that the stimulus comprises a decrease in pH from a pH between pH 7 and pH 9 to a pH between pH 5 and pH 6.
    [0003]
    3. Process according to claim 1 or 2, characterized in that the production phase corresponds to the exponential growth phase.
    [0004]
    4. Process according to any one of claims 1 to 3, characterized in that the production phase involves the growth of the microalgae strain under conditions that allow exponential growth.
    [0005]
    5. Process according to any one of claims 1 to 4, characterized in that the production phase involves the growth of the microalgae strain in a photobioreactor.
    [0006]
    6. Process according to any one of claims 1 to 5, characterized in that the production phase involves the growth of microalgae under LEDs emitting 2 peaks of red and blue light within the 400-700 nm photosynthetically active radiation spectrum .
    [0007]
    7. Process according to any one of claims 1 to 7, characterized in that the crops are not exposed to natural sunlight.
    [0008]
    8. Process according to any one of claims 1 to 7, characterized in that the microalgae culture is exposed to the stimulus at the peak of the exponential growth phase and/or at the beginning of the stationary growth phase.
    [0009]
    9. Process according to any one of claims 1 to 8, characterized in that the stimulus additionally comprises the addition of a carbon source.
    [0010]
    10. Process according to any one of claims 1 to 9, characterized in that the decrease in pH is initiated by the addition of CO2.
    [0011]
    11. Process according to any one of claims 1 to 10, characterized in that the pH is reduced to a pH of between pH 5 and pH 6 for a period of between 30 minutes and 2 hours, and said period precedes the increase in light irradiance.
    [0012]
    12. Process according to claim 11, characterized in that after reducing the pH, the pH is increased to a pH of between pH 7 and pH 9.
    [0013]
    13. Process according to any one of claims 1 to 12, characterized by the fact that after exposure of the microalgae to the stimulus, the microalgae are cultivated for an additional period of 48 hours before harvesting the metabolite.
    [0014]
    14. Process according to any one of claims 1 to 13, characterized in that the microalgae strain is selected from the Dictyosphaerium chlorelloides species.
    [0015]
    15. Process according to any one of claims 1 to 13, characterized in that the microalgae strain is a Chlorogibba allorgei strain deposited with the Culture Collection of Algae and Protozoa under accession number CCAP 817/1.
    [0016]
    16. Process according to any one of claims 1 to 13, characterized in that the microalgae strain is a strain of Dictyosphaerium chlorelloides deposited with the Culture Collection of Algae and Protozoa under accession number CCAP 222/98.
    [0017]
    17. Process according to any one of claims 1 to 16, characterized in that the process comprises the additional step of harvesting one or more metabolites after an additional period of growth after the culture has been exposed to the stimulus.
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    同族专利:
    公开号 | 公开日
    EP2668259A2|2013-12-04|
    ES2622628T3|2017-07-06|
    CA2825856C|2019-08-27|
    PL2668259T3|2017-09-29|
    JP2017060478A|2017-03-30|
    PT2668259T|2017-05-22|
    ZA201306422B|2018-12-19|
    EP2668259B1|2017-03-29|
    NZ614107A|2015-08-28|
    JP2014509188A|2014-04-17|
    US9499784B2|2016-11-22|
    AU2012210354A1|2013-08-29|
    AU2012210354C1|2017-01-19|
    AU2012210354B2|2016-08-25|
    US20140051131A1|2014-02-20|
    WO2012101459A2|2012-08-02|
    DK2668259T3|2017-05-01|
    CN103459585B|2017-05-24|
    JP6414904B2|2018-10-31|
    CN103459585A|2013-12-18|
    WO2012101459A3|2012-11-08|
    BR112013018377A2|2016-09-20|
    CA2825856A1|2012-08-02|
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    法律状态:
    2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-06-11| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|
    2019-10-29| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|
    2021-03-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-05-18| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 30/01/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    GB1101487.5|2011-01-28|
    GBGB1101489.1A|GB201101489D0|2011-01-28|2011-01-28|Two stage photobloreactor process for production of exopolysaccharide|
    GBGB1101487.5A|GB201101487D0|2011-01-28|2011-01-28|Management of metabolite profiles of micro-algae|
    GB1101489.1|2011-01-28|
    PCT/GB2012/050194|WO2012101459A2|2011-01-28|2012-01-30|Process for production of microalgae, cyanobacteria and metabolites thereof|
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