Increased lipid production by metabolic activation with ionizing radiation.

Abstract

A method and system are provided for increasing the lipid, biomass, and metabolite yields of microalgal cultures of cells and other organisms with conserved metabolic pathways compared to untreated cultures or organisms when maintained under normal conditions. The method includes irradiating with electromagnetic ionizing radiation to induce rapid and reproducible hormetic metabolic activation in the organism cells. In one embodiment, irradiation can be applied during the exponential or stationary phase of microalgal growth. The hormetic effect includes upregulation of expression of lipid metabolism genes that code for enzymes involved in lipid biosynthesis, with the accumulation of energy stores in the form of lipids and / or the accumulation of other metabolites. The method can be implemented in a system that can interface with existing microalgal culture platforms, standard microalgal culture conditions, standard microalgal culture types, as well as with organisms with conserved metabolic pathways in the growth substrate.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 301,789, entitled “Increased Lipid Production In Microalgae Through Ionizing Radiation,” filed January 21, 2022, which is incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] Not applicable.

[0004] References to appendices

[0005] Not applicable. [Background technology]

[0006] FIELD OF THEINVENTION The present disclosure relates generally to novel hormesis processes for improving the performance of microalgae and organisms with conserved metabolic pathways. More specifically, the present disclosure relates to hormesis processes for improving the performance of microalgae and organisms with conserved metabolic pathways, which increase lipid yields, biomass yields, and / or metabolite yields with hormesis-beneficial doses of ionizing radiation.

[0007] 2. Description of Related Art

[0008] Microalgae and many organisms with conserved metabolic pathways are utilized for the commercial production of lipids, biomass, and metabolites that are applied to biofuels, feed, nutrition, cosmetics and pharmaceuticals, as well as a variety of other products. Growth rate, lipid yield, and metabolite yield are the parameters that most significantly impact the economic performance of microalgae farming, and are also important within organisms with conserved metabolic pathways and within the related biotechnology sector. In many cases, these parameters must be improved to reach commercial viability for bioproduct, biofuel, and biomass production. A variety of processes have been used to improve the performance of these parameters. However, in existing processes, improving one of these parameters often comes at the expense of the other. For example, existing culture processes to increase lipid productivity in microalgae often limit nutrients (usually nitrogen or phosphorus, "nutrient starvation") to stress the cells into lipid production mode, reducing the overall growth rate. Such processes may require the use of a staged production approach, or the use of different culture media for the various production stages, increasing the complexity and cost of the process. Increased lipid content may not result in higher lipid yields due to the adverse effects of stress on growth rate and biomass yield. High-dose radiation has been used to develop new organisms as genetic mutants with increased lipid productivity. However, such mutants are difficult and laborious to isolate and may have other deleterious background mutations. High-dose radiation has been used in studies to treat biomass from microalgae and other sources to increase the content of monosaccharides and other products. However, such treatments are based on the degradation of biomolecules and do not directly affect lipid or biomass yields.

[0009] A challenge remains in the art to increase the yield of lipids, biomass, and metabolites in microalgae and various organisms with conserved metabolic pathways without adversely affecting overall productivity. Summary of the Invention

[0010] The present invention provides a method for improving the performance, particularly the yield of lipids, biomass, and metabolites, of microalgae and organisms with conserved metabolic pathways, compared to untreated organisms when maintained under normal conditions. The method includes using electromagnetic ionizing radiation to induce rapid and reproducible metabolic activation through the hormetic effect of radiation applied to the organism at a specific absorbed dose and absorbed dose rate. In at least one embodiment, radiation is applied to the microalgae during the exponential or stationary growth phase. The hormetic effect of electromagnetic ionizing radiation includes upregulation of the expression of lipid metabolism genes that code for enzymes involved in lipid biosynthesis and accumulation of energy stores in the form of lipids and / or accumulation of other different metabolites and / or increase in biomass. The methods can be carried out under standard culture conditions including, but not limited to, photoautotrophic, heterotrophic, mixotrophic cultures using standard culture / harvest approaches including, but not limited to, batch, semi-continuous, continuous, using existing biotechnology culture platforms including, but not limited to, growth culture raceway ponds, photobioreactors, airlift bioreactors, bubble column bioreactors, fermentation bioreactors, biofilm reactors, and other biofilm cultures for microalgae and organisms cultured on other culture / growth substrates. Applicable organisms include classes of microalgae strains including, but not limited to, freshwater algae and marine algae such as Bacillariophyta, Chlorophyta (green algae), Cyanobacteria (blue-green algae), Haptophyta (sea algae mainly including coccoliths), Phaeophyta (brown algae), Pyrrophycophyta (dinoflagellates), Rhodophyta (red algae), and organisms with conserved metabolic pathways such as, but not limited to, Bigyra (including Thraustochytrids), Streptophyta (plants including mosses), Ascomycota (fungi including yeasts), Basidiomycota (fungi including mushrooms), bacteria, and Archaea.The present invention can interface with exemplary cells using at least one of X-rays and gamma rays to produce hormesis within the cells; and to produce metabolic activation of cells of such organisms that produces a hormetic effect in culture under defined conditions compared to untreated cultures of such cells of the same strain when maintained under the same conditions.

[0011] The present disclosure provides a method of metabolic activation of an organism, the method comprising irradiating the organism with at least one of X-rays and gamma rays that produce hormesis in the organism, and metabolically priming the organism to produce a hormesis effect under defined conditions compared to an untreated organism when maintained under the same conditions.

[0012] The present disclosure provides a method of metabolic activation of an organism by irradiation, the method comprising irradiating an organism with at least one of X-rays and gamma rays that produce hormesis in the organism, wherein the organism is at least one of a microalgae strain and an organism having a conserved metabolic pathway; and metabolically priming the organism to produce, under defined conditions, a hormesis effect of at least one of higher lipid yield, higher biomass yield, and higher metabolite yield, as compared to an untreated organism of the same species when maintained under the same conditions.

[0013] The present disclosure also provides a system for improving the performance of at least one culture of microalgae and an organism having a conserved metabolic pathway by irradiation, the system including a culture platform including at least one of a raceway pond, a photobioreactor, an airlift bioreactor, a bubble column bioreactor, a fermentation bioreactor, and a biofilm reactor; an irradiation system having at least one source of X-rays and gamma rays coupled to the culture platform; and a controller configured to control activation of the source to irradiate at least one of the microalgae and the organism having a conserved metabolic pathway at a predetermined phase of the culture for a period of time and dose rate and to metabolically prime the at least one of the microalgae and the organism having a conserved metabolic pathway to produce, under predetermined conditions, a hormesis effect of at least one of higher lipid yield, higher biomass yield, and higher metabolite yield of the culture compared to an untreated culture of the same strain of the at least one microalgae and the organism having a conserved metabolic pathway when maintained under the same conditions.

[0014] The present disclosure further provides a system for improving the performance of an organism having a conserved metabolic pathway by irradiation, the system including a growth substrate for the organism, an irradiation system having at least one X-ray or gamma ray source in operative proximity to the organism; and a controller configured to control activation of the irradiation system to irradiate the organism for a period of time and at a rate to metabolically prime the organism to produce a hormesis effect of at least one of higher lipid yield, biomass yield, and metabolite yield under specified conditions compared to an untreated organism of the same species maintained under the same conditions. [Brief description of the drawings]

[0015] A brief description of some views of the drawing [Figure 1] FIG. 1 is a flow chart of at least one example embodiment of using the methods of the present invention during stationary phase growth of microalgae and other unicellular organisms with conserved metabolic pathways related to lipid yield.

[0016] [Figure 2A] FIG. 2A is a graph showing an example of a growth curve of an organism exposed to the hormetic X-ray irradiation of the present invention in the process of FIG.

[0017] [Figure 2B] FIG. 2B is a graph showing examples of statistically significant positive effects on lipid yield of the organism of FIG. 2A for a given irradiance at various irradiance rates.

[0018] [Figure 2C] FIG. 2C is a graph showing an example where there is no statistically significant effect on the biomass yield of the organism of FIG. 2A compared to the control group when exposed to a given dose at various dose rates.

[0019] [Figure 2D] FIG. 2D is a graph showing examples of statistically significant positive effects of a given dose of radiation at various radiation rates on lipid droplets in cells of the organism of FIG. 2A.

[0020] [Figure 2E-1] FIG. 2E1 is a photomicrograph of untreated cells from the organism of FIG. 2A that had a limited number of lipid droplets.

[0021] [Figure 2E-2] FIG. 2E2 is a photomicrograph of cells from the organism of FIG. 2A treated with a given dose and rate of radiation showing increased lipid droplets compared to untreated cells of the organism of FIG. 2E1.

[0022] [Figure 2E-3] FIG. 2E3 is a photomicrograph of cells from the organism of FIG. 2A treated with a given dose and rate showing increased lipid droplets compared to untreated cells of the organism of FIG. 2E1.

[0023] [Figure 2E-4]FIG. 2E4 is a micrograph of cells from the organism of FIG. 2A treated with a given dose and rate of radiation showing increased lipid droplets compared to untreated cells of the organism of FIG. 2E1.

[0024] [Figure 2F] FIG. 2F is a graph of an example of a control group with no statistically significant effect on the fatty acid profile of cells of the organism of FIG. 2A when exposed to a given dose of radiation at various radiation rates.

[0025] [Figure 2G] FIG. 2G is a graph showing an example of a statistically significant positive effect for an exemplary irradiance dose at a given dose rate on the metabolite alpha-linoleic acid (omega-3 fatty acid) content in the biomass of the organism of FIG. 2A.

[0026] [Figure 2H] FIG. 2H is a graph showing an example of a growth curve of another organism exposed to the X-ray irradiation of the present invention in the process of FIG.

[0027] [Figure 2I] FIG. 2I is a graph showing an example of a statistically significant positive effect for a given irradiance dose at various irradiation rates on the relative lipid yield of the organisms in FIG. 2H.

[0028] [Figure 2J] FIG. 2J is a graph showing an example of no statistically significant effect relative to the control for a given irradiance at various irradiation rates on biomass yield of the organisms of FIG. 2H.

[0029] [Figure 2K] FIG. 2K is a graph showing an example of the positive effect of irradiation of the present invention on upregulating expression of various classes of genes, including lipid metabolism genes, in the organism of FIG. 2A.

[0030] [Figure 2L]FIG. 2L is a graph showing an example of upregulation of expression of lipid metabolism genes for the production of fatty acids and triacylglycerol in lipid droplets in the organism of FIG. 2A.

[0031] [Figure 2M] FIG. 2M illustrates an example of the process of metabolic activation and upregulation of expression of lipid metabolism genes for the production of fatty acids and triacylglycerol in lipid droplets upon irradiation of cells of the organism of FIG. 2A.

[0032] [Figure 2N] FIG. 2N is a diagram of an example phylogenetic tree of lipid metabolism genes acetyl-CoA carboxylase, biotin carboxylase (ACCase) for the synthesis of fatty acids in lipid droplets including the green algal organisms of FIG. 2A and FIG. 2H, showing a high degree of evolutionary conservation among microalgae and classes of organisms with conserved metabolic pathways, including plants, yeast, fungi, bacteria, and archaea.

[0033] [Figure 2O] FIG. 2O is a diagram of an example phylogenetic tree of lipid metabolism genes long-chain acyl-CoA synthetase (LACS) for the synthesis of fatty acids in lipid droplets, including the green algal organisms of FIG. 2A and FIG. 2H, showing a high degree of evolutionary conservation across classes of microalgae and organisms with conserved metabolic pathways, including plants, yeast, fungi, bacteria, and archaea.

[0034] [Figure 2P] FIG. 2P is an example diagram of a phylogenetic tree of the lipid metabolism gene lysophosphatidic acid acyltransferase (LPAAT) for the synthesis of triacylglycerol in lipid droplets, including the green algal organisms of FIG. 2A and FIG. 2H, showing a high degree of evolutionary conservation across classes of microalgae and organisms with conserved metabolic pathways, including plants, yeast, fungi, bacteria, and archaea.

[0035] [Figure 2Q]FIG. 2Q is an example diagram of a phylogenetic tree of the lipid metabolism gene phosphatidic acid phosphatase (PAP) for the synthesis of triacylglycerol in lipid droplets, including the green algal organisms of FIG. 2A and FIG. 2H, showing a high degree of evolutionary conservation across classes of microalgae and organisms with conserved metabolic pathways, including plants, yeast, fungi, bacteria, and archaea.

[0036] [Figure 2R] FIG. 2R is an illustration of an example phylogenetic tree of the lipid metabolism gene diacylglycerol acyltransferase (DGAT) for the synthesis of triacylglycerol in lipid droplets including the green algal organisms of FIG. 2A and FIG. 2H, showing a high degree of evolutionary conservation across classes of microalgae and organisms with conserved metabolic pathways, including plants, yeast, fungi, bacteria, and archaea.

[0037] [Diagram 3] FIG. 3 is a flow chart of an example of at least one embodiment of using the methods of the present invention in the exponential growth phase of microalgae and other organisms with conserved metabolic pathways related to lipid yield.

[0038] [Figure 4A] FIG. 4A is an example of a growth curve for the organism of FIGS. 2A-2G, but exposed to the hormetic X-ray irradiation of the present invention in the process of FIG.

[0039] [Figure 4B] FIG. 4B is an example of the positive effect on cell density of the organism of FIG. 4A given irradiance at various irradiance rates.

[0040] [Figure 4C] FIG. 4C is an example of a statistically significant positive effect on the growth rate of the organism of FIG. 4A given irradiance at various irradiance rates.

[0041] [Figure 4D]FIG. 4D is a graph showing examples of statistically significant positive effects on biomass yield of the organisms of FIG. 4A given irradiance at various irradiation rates.

[0042] [Figure 5A] FIG. 5A is a top schematic view of an example of an illumination system installed in a raceway pond.

[0043] [Figure 5B] FIG. 5B is an enlarged schematic top view of the illumination system of FIG. 5A.

[0044] [Figure 5C] FIG. 5C is an enlarged side cross-sectional schematic diagram through a longitudinal section of the illumination system of FIG. 5A.

[0045] [Figure 5D] FIG. 5D is an enlarged side cross-sectional schematic diagram through a cross section of the illumination system of FIG. 5A.

[0046] [Figure 5E] FIG. 5E is an enlarged side cross-sectional schematic diagram of the coverage of the illumination beam through a longitudinal section of the illumination system of FIG. 5A.

[0047] [Figure 6A] FIG. 6A is a side schematic diagram of another example of an illumination system mounted on a photobioreactor.

[0048] [Figure 6B] FIG. 6B is an enlarged schematic top view of the illumination system of FIG. 6A.

[0049] [Figure 6C] FIG. 6C is an enlarged side cross-sectional schematic diagram through a longitudinal section of the illumination system of FIG. 6A.

[0050] [Figure 6D] FIG. 6D is an enlarged side cross-sectional schematic diagram through a cross section of the illumination system of FIG. 6A.

[0051] [Figure 6E] FIG. 6E is an enlarged side cross-sectional schematic diagram of the coverage of the illumination beam through a longitudinal section of the illumination system of FIG. 6A.

[0052] [Figure 7A] FIG. 7A is a side schematic view of another example of an irradiation system mounted on an airlift bioreactor.

[0053] [Figure 7B] FIG. 7B is an enlarged schematic top view of the illumination system of FIG. 7A.

[0054] [Figure 7C] FIG. 7C is a schematic diagram of an enlarged cross-sectional side view through a longitudinal section of the illumination system of FIG. 7A.

[0055] [Figure 7D] FIG. 7D is a schematic diagram of an enlarged cross-sectional side view of the coverage of the illumination beam through a longitudinal section of the illumination system of FIG. 7A.

[0056] [Figure 8A] FIG. 8A is a side schematic view of another example of an illumination system mounted on the wall of a fermentation bioreactor.

[0057] [Figure 8B] FIG. 8B is an enlarged schematic top view of the illumination system of FIG. 8A.

[0058] [Figure 8C] FIG. 8C is a schematic diagram of an enlarged cross-sectional side view through a longitudinal section of the illumination system of FIG. 8A.

[0059] [Figure 8D] FIG. 8D is a schematic diagram of an enlarged cross-sectional side view of the coverage of the illumination beam through a longitudinal section of the illumination system of FIG. 8A.

[0060] [Figure 9A]FIG. 9A is a side schematic view of another example of an irradiation system mounted on a biofilm reactor.

[0061] [Figure 9B] FIG. 9B is a schematic diagram of a cross-sectional side view of an enlarged longitudinal section of the illumination system of FIG. 9A.

[0062] [Figure 10A] FIG. 10A is a side schematic diagram of another example of a versatile, flexible irradiation system for processing organisms with conserved metabolic pathways related to lipid yield.

[0063] [Figure 10B] FIG. 10B is an enlarged schematic top view of the illumination system of FIG. 10A.

[0064] [Figure 10C] FIG. 10C is a schematic diagram of a cross-sectional side view of an enlarged longitudinal section of the illumination system of FIG. 10A.

[0065] Detailed Description The figures above and the following specific structural and functional descriptions are not presented to limit the scope of the applicant's invention or the scope of the appended claims. Rather, the figures and descriptions are provided to teach one of ordinary skill in the art how to make and use the invention for which patent protection is sought. One of ordinary skill in the art will appreciate that not all features of a commercial embodiment of the present invention are described or shown for clarity and understanding. One of ordinary skill in the art will also appreciate that the development of an actual commercial embodiment incorporating aspects of the present disclosure will require numerous implementation-specific decisions to achieve the developer's end goal for the commercial embodiment. Such implementation-specific decisions may include, but are not limited to, compliance with system-related, business-related, government-related, and other constraints, which may vary depending on the particular implementation, location, or time. While the developer's efforts may be complex and time-consuming in absolute terms, such efforts are routine for one of ordinary skill in the art having the benefit of this disclosure. It should be understood that the invention disclosed and taught herein is susceptible to numerous and various modifications and alternative forms. The use of singular terms such as "a" is not intended to limit the number of items. Furthermore, the various methods and embodiments of the system may be included in combination with one another to create variations of the disclosed methods and embodiments. A description of a singular element may include a plurality of elements and vice versa. A reference to at least one item may include one or more items. Also, various aspects of the embodiments may be used in combination with each other to achieve the realized goals of the present disclosure. Unless the context requires otherwise, the term "comprise" or variations such as "comprises" or "comprising" should be understood to mean the inclusion of at least the recited element or step, group of elements or steps, or equivalents thereof, and not to imply the exclusion of a greater number or other elements or steps, group of elements or steps, or equivalents thereof. A device or system may be used in various orientations and directions.Directional terms such as "top", "up", "upper", "lower", "lower", "below" and the like are used to indicate directions relative to the figures and their illustrated orientations, and are not absolute relative to a fixed reference such as the earth used commercially. Terms such as "inner", "inward", "internal" and the like refer to directions toward a central portion of an assembly or component, e.g., a longitudinal centerline of the assembly or component, and terms such as "outer", "outward", "external" and the like refer to directions away from the central portion of an assembly or component. Terms such as "coupled", "coupled", "coupler", and the like are used broadly herein and can include any method or device for, for example, mechanically, magnetically, electrically, chemically, manipulatively, directly or indirectly, fixing, joining, adhering, fastening, attaching, joining, inserting, forming, communicating, or otherwise associating an intermediate element, one or more pieces or members, and further including, but not limited to, integrally forming one functional member with another functional member. Coupling can occur in any direction, including rotational direction. Unless otherwise limited, the sequence of steps can occur in various orders. Various steps described herein may be combined with other steps, inserted into the steps described, or split into multiple steps. Similarly, elements are described functionally and may be embodied as separate components or combined into components having multiple functions. Some elements are designated by device names for brevity and are understood to include systems of related components that are known to those skilled in the art and may not be specifically described. Some elements are designated by device names for brevity and are understood to include systems of related components that are known to those skilled in the art and may not be specifically described. Various examples are provided in the description and figures that perform various functions and are not limited in shape, size, or description, but serve as exemplary structures that may be modified as known to those skilled in the art given the teachings contained herein. Thus, the use of the term "exemplary" is the adjective form of the noun "example," and similarly refers to exemplary structures and not necessarily to preferred embodiments.Element numbers with letter suffixes such as "A", "B", or numbers with primes, double primes, such as 1, 1', 1'', etc., designate different elements within a group of similar elements having similar structure or function, while corresponding element numbers without letters generally refer to one or more similar elements. Element numbers in the claims that correspond to elements disclosed in the application are exemplary and not exclusive, since several embodiments are disclosed that use various element numbers for similar elements. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods, materials, and / or systems similar or equivalent to those described herein can be used in the practice of the present disclosure, suitable methods, materials, and / or systems are described below. Furthermore, unless otherwise stated, the methods, materials, and / or systems are exemplary and not intended to be limiting. Publications, patents, and other references mentioned herein are incorporated by reference in their entirety, but in the event of a conflict, the present specification, including definitions, will control.

[0066] In general, the present invention provides a method for improving the performance, particularly the yield of lipids, biomass, and metabolites, of microalgae and organisms with conserved metabolic pathways, compared to untreated organisms when maintained under normal conditions. The method includes using electromagnetic ionizing radiation to induce rapid and reproducible metabolic activation through the hormetic effect of radiation applied to the organism at a specific absorbed dose and absorbed dose rate. In at least one embodiment, radiation is applied to the microalgae during the exponential or stationary growth phase. The hormetic effect of electromagnetic ionizing radiation includes upregulation of the expression of lipid metabolism genes that code for enzymes involved in lipid biosynthesis and accumulation of energy stores in the form of lipids, and / or accumulation of other different metabolites, and / or increased biomass. The methods can be performed using existing biotechnology culture platforms including, but not limited to, raceway ponds, photobioreactors, airlift bioreactors, bubble column bioreactors, fermentation bioreactors, biofilm reactors, and suspension or biofilm cultures for microalgae and organisms cultured on other culture / growth substrates; standard culture conditions including, but not limited to, photoautotrophic, heterotrophic and mixotrophic cultures; standard culture types including, but not limited to, batch, semi-continuous and continuous cultures, and other platforms. Applicable organisms include, but are not limited to, classes of microalgae strains including freshwater algae and marine algae, such as, but not limited to, diatoms, green algae, cyanobacteria, blue-green algae, haptophytes (marine algae mainly including coccoliths), phaeophytes (brown algae), dinoflagellates, rhodophytes (red algae), and organisms with conserved metabolic pathways, such as, but not limited to, bigila (including thraustochytrids), streptophytes (plants including mosses), ascomycota (fungi including yeasts), basidiomycota (fungi including mushrooms), bacteria, and archaea. The present invention can interface with a typical cell using at least one of X-rays and gamma rays to produce hormesis in the cells, and the metabolic activation of the cells of such organisms produces a hormesis effect in culture under given conditions compared to untreated cultures of the same strain of such cells when maintained under the same conditions.Generally, the illumination system is placed in operational proximity to the organism to produce the hormesis effect described herein.

[0067] As used herein, the following terms and their derivatives apply:

[0068] "Biomass yield" refers to the dry mass of cell mass harvested per volume of microalgal culture. Harvesting the biomass means separating the cells from the aqueous medium. An increase in biomass yield is generally the result of an increase in biomass productivity, where productivity means the rate at which biomass is produced.

[0069] "Drying" means substantially removing moisture from harvested biomass.

[0070] "Electromagnetic ionizing radiation" refers to electromagnetic radiation of sufficient energy to cause the dissolution of water molecules, such as X-rays and gamma rays. Irradiation with electromagnetic ionizing radiation involves uniformly exposing a microalgae culture or other organism to a specific dose of radiation at a specific rate. The dose can be delivered continuously or in fractions. One source of radiation is an X-ray tube, which has a selected voltage, current, and filters to deliver one or more specific doses and rates.

[0071] "Exponential phase" refers to the early stage of microalgal culture growth exhibiting an exponential increase in cell density or biomass, and for purposes of the present invention includes lag phase, early exponential phase, and late exponential phase as known to those of skill in the art.

[0072] "Growth rate" refers to the rate of change in cell density or biomass per day. Cell density is measured using the OD 750 The optical density at 750 nm (OD 750 ) and shows a correlation coefficient close to 1.

[0073] "Hormesis" refers to the biological phenomenon in which exposure to low doses of agents that are toxic or lethal at high doses results in beneficial effects such as improved growth, stress resistance, or other priming-related metabolic activation (such as accumulation of energy stores or accumulation of specific metabolites). These beneficial effects of hormesis are due to "metabolic activation" that may involve modulation of mRNA transcription, post-transcriptional modifications, and metabolic pathways, and are not due to genetic mutations.

[0074] "Lipid metabolism genes" refer to genes that encode enzymes involved in lipid metabolism, including fatty acid and triacylglycerol synthesis.

[0075] "Lipid yield" refers to the total mass of lipids in the biomass harvested per volume of culture. Common lipids include triglycerides, diglycerides, free fatty acids, and phospholipids. Lipids can be extracted by standard methods known and available to those skilled in the art, including mechanical grinding or sonication, extraction with organic solvents and heating, or extraction with supercritical carbon dioxide. The increase in lipid yield is the result of increased lipid productivity. Lipid yield differs from lipid content, which refers to the percentage of lipids in the biomass, and does not take into account the harvested biomass yield, which can be significantly reduced with conventional irradiation methods, leading to a net loss of lipid yield from the reduced biomass.

[0076] "Metabolite yield" refers to the total mass of metabolites in biomass harvested per volume of culture. Common metabolites include, but are not limited to, pigments, antioxidants, nutrients, drug precursors, omega-3 fatty acids, proteins, carbohydrates, and other products of primary and secondary metabolism. Metabolites can be extracted by standard methods known to those skilled in the art, including, but not limited to, mechanical grinding and extraction with selected solvents and heat.

[0077] "Microalgae" refers to unicellular eukaryotic and prokaryotic microorganisms that undergo photosynthesis or are phylogenetically derived from photosynthetic genes, including, but not limited to, green algae, dinoflagellates, diatoms, brown algae, marine algae (including coccoliths), cyanobacteria, and red algae.

[0078] "Microalgae culture" refers to microalgae cultured in aqueous or solid media under conventional conditions for commercial, research, or other uses.

[0079] "Normal conditions" refers to conditions commonly known to those of skill in the art, including, but not limited to, conditions recommended for photoautotrophic, heterotrophic or mixotrophic cultivation of a particular microalgae strain, or cultivation of organisms with conserved metabolic pathways for a particular cultivation platform and cultivation type. Normal conditions include commonly used media or growth substrates that are not intentionally nutrient limiting, and recommended temperature ranges, nutrient concentrations, light intensities, and regimes known to those of skill in the art.

[0080] "Organisms with conserved metabolic pathways" refers to the presence of functionally equivalent genes that control metabolism similar to that in microalgae, including, but not limited to, plants, fungi, yeasts, thraustochytrids, bacteria, and archaea.

[0081] "Stationary phase" of growth refers to a later stage of microalgal culture growth that generally exhibits a growth rate close to zero or day-to-day fluctuations around a zero value. The stationary phase follows the exponential phase, and for purposes of the present invention includes pre-stationary, early stationary, late stationary, and death (or lysis) phases known to those skilled in the art.

[0082] "Transcriptional upregulation" refers to a significantly increased number of copies of a messenger RNA in response to a particular stimulus.

[0083] In at least one embodiment of the present invention, a method is provided for inducing metabolic activation in an organism to increase lipid yield without substantial changes (i.e., statistically insignificant changes) in biomass yield compared to untreated organisms when maintained under the same normal conditions by irradiating the organism in a hormetic manner. Hormetic irradiation results in upregulation of expression of lipid metabolism genes, including genes of enzymes that synthesize lipids. Organisms include microalgae and organisms with conserved metabolic pathways related to lipid yield. Lipid yield includes omega-3 fatty acid yield. The method includes irradiation during a particular growth period of the organism, such as exposing microalgae in a stationary growth phase.

[0084] In at least another aspect of the invention, a method is provided for inducing metabolic activation in organisms, such as microalgae and organisms with conserved metabolic pathways, resulting in increased growth rates and biomass yields compared to untreated cultures when maintained under the same normal conditions, comprising hormetic irradiation of the organisms during a particular growth phase, such as a microalgae culture in the exponential growth phase.

[0085] The invention also features increased lipid and biomass yields in microalgae cultures grown and / or maintained under abnormal conditions by any of the methods described herein.

[0086] The invention also features upregulating expression of lipid metabolism genes that encode enzymes that synthesize lipids and increase lipid yields in organisms having a conserved metabolic pathway by any of the methods described herein.

[0087] The present invention also features the regulation of genes involved in other metabolic pathways for the ability to produce other metabolites and products by any of the methods described herein.

[0088] For illustrative purposes, the specification and embodiments focus on microalgae, however, it is understood that the methods and principles described herein can be applied to other organisms that have conserved metabolic pathways.

[0089] FIG. 1 is a flow chart of at least one example embodiment of using the methods of the present invention during stationary phase growth of microalgae and other organisms with conserved metabolic pathways related to lipid yield.

[0090] Generally, in at least one embodiment of the present invention, a microalgae culture can be cultured under normal conditions until it reaches a stationary phase of growth after initial inoculation of the medium. The culture can be briefly exposed to electromagnetic ionizing radiation, such as X-rays, while in the stationary phase. The culture can be maintained under normal conditions until biomass harvesting, where the microalgae cells are collected. In biomass processing, lipids can be extracted from the biomass. Using the teachings of the present invention, irradiated microalgae cultures exhibit higher lipid yields compared to untreated cultures of the same microalgae strains cultured under the same conditions.

[0091] More specifically, the method 10 can include an inoculation step 12, in which a microalgal culture can be formed by inoculating the cells into a medium recommended for the selected microalgal strain. The culture can be maintained under normal conditions necessary to undergo a series of growth phases. The culture enters an exponential phase 14, followed by a stationary phase 16. Biomass is highest during the stationary phase and does not change significantly during this phase. In an irradiation step 18, the microalgal culture can be irradiated with an applied dose of electromagnetic ionizing radiation during the stationary phase. The irradiation can be applied continuously or in portions. By way of example, but not by way of limitation, the microalgal culture can be exposed to X-rays at an exposure rate between 0.05 Gy and 2 Gy per minute of absorbed dose, to a total absorbed dose of 1 to 15 Gy. After irradiation, the microalgae can be maintained in a maintenance step 20 for an optimal period of time from irradiation, for example, 6 hours to 10 days. After maintenance, the microalgae can be subjected to a biomass harvesting step 22, which includes harvesting the microalgal cells. After harvesting, biomass processing step 24 can include mechanical disruption of the microalgae cells. Lipid extraction step 26 and / or metabolite extraction step 28 can be performed, such as by using a mixture of organic solvents and high temperatures, or by using supercritical carbon dioxide extraction protocols specific to the selected microalgae culture, and can be optimized by one of skill in the art.

[0092] Thus, in at least one embodiment, the invention provides a method for increasing lipid yield and / or metabolite yield in a culture compared to an untreated culture of the same strain when maintained under the same conditions.

[0093] 2A-2L are exemplary graphs, photographs, and schematic diagrams of actual test data based on application of electromagnetic ionizing radiation of an exemplary method of the present invention to stationary phase growth of microalgae under otherwise normal conditions.

[0094] Stationary Phase Experiment 1

[0095] Figure 2A is a graph showing an example of a growth curve for an organism exposed to the hormetic X-rays of the present invention in the process of Figure 1. Figure 2B is a graph showing an example of a statistically significant positive effect on lipid yield for the organism of Figure 2A for a given irradiance at various irradiation rates. Figure 2C is a graph showing an example of a no significant negative effect on biomass yield for the organism of Figure 2A for a given irradiance at various irradiation rates.

[0096] Cultures of the microalga Chlorella sorokiniana (CCAP 211 / 8K, also known as UTEX 1230), an example of a green algae strain, were grown in 3N-BBM+V medium (recipe available at https: / / www.ccap.ac.uk / media / documents / 3N_BBM_V.pdf ) at 22°C on an orbital shaker (120 rpm) and at 120 μmol m -2 s -1The microalgae cultures were cultured under normal conditions with a continuous photon flux of 1000 x 1000 cells / mL. The microalgae cultures were inoculated into fresh medium at 0.5 x 106 cells / mL and grew through an exponential growth phase before reaching a stationary phase on day 20 (Figure 2A). The microalgae cultures were irradiated with X-rays on day 20 in irradiation step 18 using a CellRad system (FaxitronBioptics LLC, tube power: 750 W, filtration: 1.6 mm Be and 0.5 mm Al, energy 120 kV, dose and rate adjusted by changing the current). The absorbed dose was 1, 2, or 5 Gy. The absorption rate was 0.25 or 0.5 Gy / min. The irradiated cultures were further maintained under the same normal conditions for 24 h. The biomass was harvested on day 21 in biomass harvesting step 22 by centrifugation at 5000 g for 5 min and drying the pellet at 50 °C for 24 h. (The increased lipids were stored for a significant period after irradiation.) The biomass was mechanically pulverized and lipids were extracted using a 2 / 1 (v / v) mixture of chloroform and methanol and a Soxhlet extractor operated at 80 °C for 4 h. The extracted lipids were dried at 50 °C for 12 h. The irradiated samples showed a significant increase in lipid yield of 22%-29% in just 24 h compared to untreated cultures grown under the same normal conditions (Figure 2B). Irradiation did not cause a decrease in biomass yield (Figure 2C).

[0097] FIG. 2D is a graph of an example of a statistically significant positive effect on lipid droplets in cells of the organism of FIG. 2A when a given dose of radiation is applied at various radiation rates. FIG. 2E1 is a micrograph of untreated cells of the organism of FIG. 2A, which have a limited number of lipid droplets. FIG. 2E2 is a micrograph of cells of the organism of FIG. 2A, which have been treated with a given dose and rate of radiation, showing an increase in lipid droplets compared to untreated cells of the organism of FIG. 2E1. FIG. 2E3 is a micrograph of cells of the organism of FIG. 2A, which have been treated with a given dose and rate of radiation, showing an increase in lipid droplets compared to untreated cells of the organism of FIG. 2E1. FIG. 2E4 is a micrograph of cells of the organism of FIG. 2A, which have been treated with a given dose and rate of radiation, showing an increase in lipid droplets compared to untreated cells of the organism of FIG. 2E1.

[0098] Cells were collected from the same samples immediately prior to biomass harvest and analyzed using transmission electron microscopy. Irradiated cells contained more lipid droplets27 than untreated cells grown under the same normal conditions (Figure 2D). The accumulation of lipid droplets27 as a response to irradiation dose shown in Figure 2C was further confirmed by transmission electron microscopy by comparing untreated cells (Figure 2E1) with cells treated with a dose of 1 Gy at a dose rate of 0.25 Gy / min (Figure 2E2), cells treated with 2 Gy at 0.25 / min (Figure 2E3), and cells treated with 5 Gy at 0.5 Gy / min (Figure 2E4).

[0099] Figure 2F is a graph of an example of no substantial effect on the fatty acid profile of cells of the organism of Figure 2A for a given irradiance at various dose rates, and Figure 2G is a graph of an example of a statistically significant positive effect on the metabolite alpha-linoleic acid (omega-3 fatty acid) content in the biomass of the organism of Figure 2A for an exemplary irradiance at a given dose rate.

[0100] The fatty acid profile of the extracted lipids was measured by gas chromatography. The fatty acids in the extracted lipids were transesterified by a methanol-sulfuric acid protocol, collected in hexane, and dissolved in dichloromethane for analysis. Irradiation did not alter the fatty acid profile (saturated, monounsaturated, and polyunsaturated fatty acids) and the degree of saturation (Figure 2F), both of which are relevant parameters for biodiesel production and other applications. Cells irradiated with 5 Gy at 0.5 Gy / min contained increased amounts of alpha-linoleic acid than untreated cells cultured under the same normal conditions (Figure 2G). Alpha-linoleic acid is an essential omega-3 fatty acid in human nutrition and a precursor of other omega-3 fatty acids.

[0101] Stationary phase experiment 2

[0102] Figure 2H is a graph showing an example of a growth curve for another organism exposed to the X-ray irradiation of the present invention in the process of Figure 1. Figure 2I is a graph showing an example where there was a statistically significant positive effect on lipid yield for the organism of Figure 2H when given a given dose at various dose rates. Figure 2J is a graph showing an example where there was no significant negative effect on biomass yield for the organism of Figure 2H when given a given dose at various dose rates.

[0103] In a second experiment, a culture of the microalga Chlamydomonas reinhardtii (strain CCAP11 / 32C) was grown in TAP medium (recipe available from Harris, EH (1989) The Chlamydomonas Sourcebook. Academic Press, San Diego) at 22°C on an orbital shaker (120 rpm) at 120 μmol m -2 s -1 The microalgae cultures were grown under standard conditions with a continuous photon flux of 2x10 6 cells / mL were inoculated and with growth, they went through an exponential phase and reached a stationary phase on day 40 (Figure 2H). Microalgal cultures were irradiated with X-rays at day 40 in irradiation step 18 using a CellRad system. The absorbed dose was 5 or 12 Gy with an absorption rate of 0.25 or 0.5 Gy / min, respectively. The irradiated samples were cultured for an additional 24 h under the same normal conditions. The biomass was harvested (step 22). Relative lipid yields were measured in microalgal cultures using a Nile Red fluorescent dye assay. The fluorescence intensity of this dye, which excites at 530 nm and emits at 570 nm in microalgal cultures, is proportional to the amount of lipid per volume. The fluorescence of irradiated culture samples was normalized to the fluorescence of non-irradiated cultures to measure the relative lipid yield. X-ray irradiation of microalgal cultures caused a significant increase in lipid yield of about 21% to 26% compared to untreated cultures cultured under the same normal conditions (Figure 2I). Irradiation had no significant adverse effect on biomass yield (Figure 2J). Biomass yield was harvested by centrifugation at 5000 g for 5 min and drying the pellet at 50 °C for 24 h.

[0104] Gene Expression Experiment 3

[0105] FIG. 2K is a graph showing an example of the positive effect of irradiation of the present invention on the upregulation of expression of various classes of genes, including lipid metabolism genes, in the organism of FIG. 2A. FIG. 2L is a graph showing an example of upregulation of expression of lipid metabolism genes for the production of fatty acids and triacylglycerols in lipid droplets in the organism of FIG. 2A. FIG. 2M is a diagram illustrating the process of metabolic activation by irradiation of cells of the organism of FIG. 2A and an example of upregulation of expression of lipid metabolism genes for the production of fatty acids and triacylglycerols in lipid droplets. FIG. 2N is a diagram showing an example of a phylogenetic tree of lipid metabolism genes acetyl-CoA carboxylase, biotin carboxylase (ACCase) for fatty acid synthesis in lipid droplets including green algae of FIG. 2A and FIG. 2H, showing high evolutionary conservation among microalgae and classes of organisms with conserved metabolic pathways, including plants, yeasts, fungi, bacteria, and archaea. FIG. 2O shows an example of a phylogenetic tree of the lipid metabolism gene long-chain acyl-CoA synthetase (LACS) for fatty acid synthesis in lipid droplets including green algae of FIG. 2A, showing high evolutionary conservation among microalgae and classes of organisms with conserved metabolic pathways, including plants, yeast, fungi, bacteria, and archaea. FIG. 2P shows an example of a phylogenetic tree of the lipid metabolism gene lysophosphatidic acid acyltransferase (LPAAT) for synthesis of triacylglycerol in lipid droplets including green algae of FIG. 2A, showing high evolutionary conservation among microalgae and classes of organisms with conserved metabolic pathways, including plants, yeast, fungi, bacteria, and archaea. FIG. 2Q shows an example of a phylogenetic tree of the lipid metabolism gene phosphatidic acid phosphatase (PAP) for synthesis of triacylglycerol in lipid droplets including green algae of FIG. 2A, showing high evolutionary conservation among microalgae and classes of organisms with conserved metabolic pathways, including plants, yeast, fungi, bacteria, and archaea. Figure 2R is an illustration of an example phylogenetic tree of the lipid metabolism gene diacylglycerol acyltransferase (DGAT) for the synthesis of triacylglycerol in lipid droplets including the green algal organism in Figure 2A, showing a high degree of evolutionary conservation across microalgae and classes of organisms in which the metabolic pathway is conserved including plants, yeast, fungi, bacteria, and archaea.

[0106] For example, gene expression profiles and identification of transcriptionally up- and down-regulated metabolic genes in response to 5 Gy of radiation at 0.5 Gy / min in C. sorokiniana were determined by sequencing of extracted messenger RNA (mRNA). RNA extraction was performed by incubation in TRIzol reagent and chloroform, centrifugation at 12,000 g and 4 °C for 15 min to collect the upper layer, and RNA precipitation with isopropanol. The RNA pellet was washed twice, centrifuged at 12,000 g / 5 min at 4 °C, and resuspended in sterile deionized water. RNA libraries were generated using the TruSeq® Stranded mRNA assay according to the manufacturer's protocol, Illumina, Inc. In irradiated cells, expression of over 30 lipid metabolism genes was elevated, including genes encoding enzymes important for the synthesis of triacylglycerol, which accumulates in lipid droplets, as well as numerous genes related to other important metabolic and cellular functions (Figures 2K-2M). The evolutionary conservation of lipid metabolism genes whose expression is upregulated in response to irradiation in this green algal strain was determined by using the Basic Local Alignment Search tool to perform a gene sequence search of related genes in other microalgal organisms such as red algae, brown algae, marine algae (including coccoliths), diatoms, dinoflagellates, and cyanobacteria, as well as other organisms such as thraustochytrids, plants, yeast, fungi, bacteria, and archaea with conserved metabolic pathways. Amino acid sequence alignments and maximum likelihood phylogenetic analysis were performed to create a set of phylogenetic trees for each gene. The upregulated genes encoding enzymes important for the synthesis and regulation of fatty acids and triacylglycerols (Figure 2L) have been shown to be highly evolutionarily conserved in microalgae and organisms with conserved metabolic pathways, including but not limited to macroalgae and plants (Figure 2N-2R).

[0107] When the acetyl-CoA carboxylase (ACCase) gene from a representative green algae strain Chlorella sorokiniana was compared to representative genes from other organisms tested, there was an average sequence similarity of 46%, with many organisms showing high sequence similarity of 43-72% (Figure 2N). For example, the ACCase genes from the red algae Porphyra umbilicalis and the cyanobacterium Nostoc punctiforme showed 58% and 68% sequence similarity with C. sorokiniana ACCase, respectively. When the long-chain acyl-CoA synthetase (LACS) gene from C. sorokiniana (green algae) was compared to LACS genes from other organisms, it showed high sequence similarity of 36-64% (Figure 2O). For example, the LACS genes from the thraustochytrid Schizochytrium aggregatum and the plant Arabidopsis thaliana showed 52% and 64% sequence similarity with C. sorokiniana LACS, respectively. Comparison of the lysophosphatidic acid acyltransferase (LPAAT) gene from C. sorokiniana (green algae) with LPAAT genes from other organisms showed high sequence similarity of 35-43% (Figure 2P). For example, the LPAAT genes from the yeast Saccharomyces cerevisiae and the dinoflagellate Symbiodinium microadriaticum showed 40% and 43% sequence similarity with C. sorokiniana LPAAT, respectively. Comparison of the phosphatidic acid phosphatase (PAP) gene from C. sorokiniana (green algae) with PAP genes from other organisms showed high sequence similarity values ​​of 33-46% (Figure 2Q). For example, the PAP genes from the brown alga Ectocarpus siliculosus and the plant Beta vulgaris showed 40% and 46% sequence similarity with C. sorokiniana PAP, respectively. When the diacylglycerol acyltransferase (DGAT) gene from C. sorokiniana (a green alga) was compared with DGAT genes from other organisms, the average sequence similarity was 42%, with high sequence similarity values ​​ranging from 35 to 54% ( Figure 2R ).For example, the DGAT genes of the diatom Phaeodactylum tricornutum and the fungus Rhizoctonia solani showed 43% and 45% sequence similarity, respectively, with the C. sorokiniana DGAT. All these high sequence similarity values ​​(>40%) indicate a high degree of evolutionary conservation across the various organism classes examined.

[0108] Furthermore, it has been demonstrated that these lipid metabolism genes in other microalgae and organisms with conserved metabolic pathways are also upregulated in response to stress conditions and are involved in the accumulation of triacylglycerol, which accumulates in lipid droplets under stress. For example, in salinity-stressed diatoms, genes such as ACCase, LACS, and DGAT are upregulated (Cheng, RL, et al., 2014, Transcriptome and gene expression analysis of an oleaginous diatom under different salinity conditions. BioEnergy Research, vol. 7, pp. 195-205). For example, in red algae, genes such as PAP and DGAT are upregulated under nitrogen starvation (Imamura, S., et al., 2015, Target of rapamycin (TOR) plays a critical role in triacylglycerol acceleration in microalgae. Plant Molecular Biology, vol. 89, pp. 309-318). For example, in dinoflagellates in response to nitrogen starvation, genes such as DGAT are upregulated (Shi, X., et al., 2021, Transcriptome response of the dinoflagellate Karenia mikimotoi driven by nitrogen deficiency. Harmful Algae, vol. 103, article 101977). For example, in filamentous fungi, genes required for TAG metabolism such as LPAAT and DGAT are upregulated in response to nitrogen starvation (Chen, Y., et al., 2018, Nitrogen-starvation triggers cellular accumulation of triacylglycerol in Metarhizium robertsii. Fungal Biology vol. 122, pp. 410-419).For example, in mycobacteria, the DGAT gene required for accumulation of triacylglycerol is induced by stresses such as acid stress and hypoxic stress (Sirakova, TD, et al., 2006, Identification of a diacylglycerol acyltransferase gene involved in accumulation of triacylglycerol in Mycobacterium tuberculosis under stress. Microbiology, vol. 152, pp. 2717-2725). The mechanism of metabolic activation regulated by ionizing radiation (Figure 2M) involves the generation of reactive oxygen species that act as signaling molecules and can control metabolic pathways by activating transcription factors. Reactive oxygen species-dependent signaling pathways in response to stress have been documented in many organisms, including plants and algae (for example, as discussed in Foyer, CH et al., 2018, Redox regulation of cell proliferation: bioinformatics and redox proteomics approach to identify redox-sensitive cell cycle generators. Free Radical Biology and Medicine, vol. 122, pp. 137-149; and Pokora, W., et al., 2022, Cross talk between hydroperoxide and nitric oxygen in the unicellular green algae cell cycle: how does it work? Cells, vol. 11, article 2425). At least 10 transcription factor genes were significantly upregulated in irradiated microalgae (grouped within “DNA binding and transcription”, Figure 2K), consistent with the upregulation of other metabolic and cellular function genes. These Chlorella sorokiniana transcription factors belong to a family of transcription factor genes that are conserved across life spans.Collectively, this provides evidence that the mechanisms by which metabolic genes are upregulated in response to ionizing radiation and the regulation of metabolic activation are highly conserved across microalgae and organisms with conserved metabolic pathways.

[0109] Experiment 4 on the exponential phase

[0110] FIG. 3 is a flow chart of at least one embodiment of the present invention in the exponential phase growth of microalgae and other organisms with conserved metabolic pathways related to lipid yield. In general, in at least one embodiment of the present invention, the microalgae culture can be cultured under normal conditions after the initial inoculation of the medium to reach exponential phase growth. The culture can be briefly exposed to electromagnetic ionizing radiation, e.g., X-rays, while in the exponential phase. The culture can be further cultured under normal conditions until biomass harvesting of the entire culture is performed, such as in a batch culture type, or until biomass harvesting of a portion of the culture is performed, such as in a semi-continuous culture type, while the remainder of the culture is left to grow and then irradiate again. The biomass can be treated according to the requirements of further use. According to the teachings of the present invention, irradiated microalgae cultures show higher growth rates and biomass yields compared to untreated cultures of the same microalgae cultured under the same conditions.

[0111] More specifically, the method 10' can include an inoculation step 12, where a microalgal culture can be formed by inoculating cells into a medium recommended for the selected microalgal strain. The culture can be maintained under normal conditions necessary to go through a series of growth phases. The culture can enter an exponential phase 14, in which case an irradiation step 18 is generally applied at an early stage of the exponential phase. The microalgal culture can be irradiated with an applied dose of electromagnetic ionizing radiation. The irradiation can be applied continuously or in portions. By way of example, but not limited to, the microalgal culture can be exposed to X-rays at a dose of about 1-15 Gy at a rate of about 0.05-2 Gy / min. The microalgal culture can be maintained under normal conditions for an additional 1 to 50 days, including transition to stationary phase 16, in a maintenance step 20. After the maintenance period, a biomass harvest 22 is performed. After harvesting, a biomass processing 24 is performed, which can include any processing depending on the requirements for further use.

[0112] 4A-4D are exemplary graphs of actual test data of the application of electromagnetic ionizing radiation of the exemplary method of the present invention during the exponential phase of growth of microalgae. FIG. 4A is an example of a growth curve of the organism of FIGS. 2A-2G, but exposed to the hormetic X-rays of the present invention in the process of FIG. 3. FIG. 4B is a graph showing an example of a positive effect on cell density of the organism of FIG. 4A when exposed to a given radiation dose at various dose rates. FIG. 4C is a graph showing a statistically significant positive effect on growth rate of the organism of FIG. 4A when exposed to a given dose at various dose rates. FIG. 4D is a graph showing an example of a statistically significant positive effect on biomass yield of the organism of FIG. 4A when exposed to a given dose at various dose rates.

[0113] The microalga Chlorella sorokiniana (CCAP211 / 8K, also known as UTEX1230) was cultured in 3N-BBM+V medium at 22°C on an orbital shaker (120 rpm) with 120 μmol m -2 s -1The experiments were carried out under normal conditions with a continuous photon flux of 1000 x 1000 x 1000 cells / mL. The microalgal cultures were inoculated with 0.5 x 106 cells / mL. The microalgal cultures were irradiated with X-rays using a CellRad system at day 3 of the exponential phase in irradiation step 18. The absorbed dose was 1 or 2 Gy. The absorption rate was 0.25 or 0.05 Gy / min. The irradiated samples were cultured for another 24 days under the same normal conditions. The cell density was analyzed daily (Figure 4A). Irradiation of the exponential phase microalgal cultures had a positive effect on the growth rate (Figure 4B-4C). At the stationary phase, 25 days after irradiation, in biomass harvesting step 22, the biomass was harvested by centrifugation at 5000 g for 5 min and the pellet dried at 50 °C for 24 h. When compared to untreated cultures of the same microalgal strains cultured under the same conditions, the irradiated microalgal cultures had a 25% increase in biomass yield (Figure 4D).

[0114] 5A-10C show various embodiments of systems employing aspects of the invention described herein for microalgae and organisms with conserved metabolic pathways. Growth substrates for microalgae and organisms with conserved metabolic pathways include inorganic and organic materials, nutrients, and the like.

[0115] 5A-5E are exemplary schematic diagrams of at least one embodiment of an illumination system of the present invention configured to apply an exemplary method to an existing microalgae cultivation platform in a raceway pond under otherwise normal conditions. FIG. 5A is a top view schematic diagram of an example of an illumination system installed in a raceway pond. FIG. 5B is an enlarged top view schematic diagram of the illumination system of FIG. 5A. FIG. 5C is an enlarged side cross-sectional view schematic diagram through a longitudinal section of the illumination system of FIG. 5A. FIG. 5D is an enlarged side cross-sectional view schematic diagram through a transverse section of the illumination system of FIG. 5A. FIG. 5E is an enlarged side cross-sectional view schematic diagram of the coverage of the illumination beam through a longitudinal section of the illumination system of FIG. 5A.

[0116] Standard raceway pond platforms are known to those skilled in the art and generally include a vessel 32 with a growth substrate 56 of a volume of water divided into channels (manufactured or formed underground) by a central baffle 34 to form a flow circuit 35 around the vessel, a motive force 36 such as a paddle wheel for moving the microalgae culture 37 in the vessel around the flow circuit, and a sensor 38 for sensing the culture conditions (such as phase and other parameters to be monitored). In at least one embodiment, an illumination raceway pond system 50 can include an embodiment of the invention for carrying out the disclosed method by coupling an illumination system 40A with a raceway pond platform to illuminate the microalgae culture (FIG. 5A). The illumination system 40A with associated control system 50 includes a housing 46 with electrical equipment containing an illumination device 42, such as an X-ray tube, which can be located, for example, but not limited to, a cooling system and an illumination case 44, such as a lead box with oil for heat dissipation (FIGS. 5B-5D). The illumination system 40A can contain a controller 50 that can be connected to the sensors and used to control the activation of the illumination system. The sensor can be, but is not limited to, an optical density probe or a turbidity sensor. The sensor can provide a signal to the controller 50 to activate the irradiator 42 at a predefined value of optical density or other selected parameter. The housing 46 can be placed on a framework having supports 48 for mounting and stability. Additional protection from radiation can be provided by a radiation shield 52 ("skirt"), such as a 0.5 mm lead layer bonded to the housing (FIGS. 5B-5D). Radiation, such as X-rays, can be irradiated through an irradiation window 54, such as a Be and Al window, onto the culture 37 flowing past the irradiation system 40A. The beam angle θ and distance from the source can be optimized along with the spacing S between the beams to cover a large surface and maximize the penetration of the X-rays into the microalgae culture, which is typically, but not limited to, a depth D of 20-40 cm in raceway ponds (FIG. 5E). In at least one embodiment, the irradiation system 40A is movable to irradiate other raceway ponds.In at least another embodiment, the illumination system 40A can be fixedly mounted to the raceway pond. The system requires a power source. Other embodiments of the illumination raceway pond system are possible and contemplated.

[0117] 6A-6E are exemplary schematic diagrams of at least one embodiment of an illumination system of the present invention, configured to apply the exemplary method to an existing microalgae culture platform in a photobioreactor under otherwise normal conditions. FIG. 6A is a side schematic diagram of another example of an illumination system installed in a photobioreactor. FIG. 6B is an enlarged top schematic diagram of the illumination system of FIG. 6A. FIG. 6C is an enlarged side cross-sectional schematic diagram through a longitudinal section of the illumination system of FIG. 6A. FIG. 6D is an enlarged side cross-sectional schematic diagram through a transverse section of the illumination system of FIG. 6A. FIG. 6E is an enlarged side cross-sectional schematic diagram of the coverage of the illumination beam through a longitudinal section of the illumination system of FIG. 6A.

[0118] Standard photobioreactors are known to those skilled in the art and generally include conduits 62, such as glass or plastic tubing, to provide a flow circuit 66 for the aqueous growth substrate 56 flowing through a solar collection array 64, a motive force 65, such as a pump, to move the microalgae culture 63 in the conduits around the flow circuit, feed and degassing vessels 67, and sensors 68 to sense the culture conditions, such as monitoring phase and other parameters. In at least one embodiment, an illumination photobioreactor system 60 can include an embodiment of the invention for carrying out a method, for example, by coupling an illumination system 40B to the photobioreactor near the conduits 62 (FIG. 6A). The illumination system 40B with associated controller 50 includes, but is not limited to, a housing 46 with electrical equipment containing an illumination device 42, such as an X-ray tube, which can be located in an illumination case 44, such as a lead box with a cooling system and oil for heat dissipation (FIGS. 6B-6D). The illumination system 40B can contain a controller 50 that can be connected to sensors and used to control the activation of the illumination system. The sensor can be, but is not limited to, an optical density probe or a turbidity sensor. The sensor can provide a signal to the controller 50 to activate the irradiation device 42 at a predefined value of optical density or other selected parameter. The housing 46 can be placed on a framework having a support 48 for mounting and stability. Additional protection from radiation can be provided by a radiation shield 52 ("skirt"), such as a 0.5 mm lead layer bonded to the housing (FIGS. 6C-6D). Radiation, e.g., X-rays, can be irradiated through an irradiation window 54, such as a Be and Al window, onto the culture 37 flowing past the irradiation system 40B. The beam angle θ and distance from the source can be optimized to cover the cross section of the tube (FIG. 6E). In at least one embodiment, the irradiation system 40B is movable to irradiate other photobioreactors. In at least another embodiment, the irradiation system 40B can be stationary and installed on the photobioreactor. A power source is required for the system. Other embodiments of the irradiation photobioreactor system are possible and contemplated.

[0119] 7A-7D are exemplary schematic diagrams of at least one embodiment of an illumination system of the present invention configured to apply the exemplary method to an existing microalgae cultivation platform of an airlift or bubble column bioreactor under normal conditions. FIG. 7A is a side schematic diagram of another example of an illumination system mounted on an airlift bioreactor. FIG. 7B is an enlarged top schematic diagram of the illumination system of FIG. 7A. FIG. 7C is an enlarged side cross-sectional schematic diagram through a longitudinal section of the illumination system of FIG. 7A. FIG. 7D is an enlarged side cross-sectional schematic diagram of the illumination range of the illumination beam through a longitudinal section of the illumination system of FIG. 7A.

[0120] Standard airlift or bubble column bioreactors are known to those skilled in the art and generally include a vessel 72 forming a bioreactor column, a power source 74, such as an air pump, for injecting gas into an aqueous growth substrate 56 for a microalgal culture 76 in the bioreactor column to form bubbles and agitate and mix the microalgal culture in the column or channel, and sensors 78 for sensing culture conditions, such as phase and other parameters to be monitored. In at least one embodiment, the irradiation airlift or bubble column bioreactor 70 can include an embodiment of the invention for carrying out the disclosed method by coupling an irradiation system 40C for irradiating the culture through a transparent glass or plastic surface in the vessel 72 (FIG. 7A). The irradiation system 40C with associated control system 50 includes a system housing 46 with, for example, a cooling system and electrical equipment, which contains an irradiation device 42, such as an X-ray tube, which can be placed in an irradiation case 44, such as a lead box with oil for heat dissipation (FIGS. 7B-7C). The irradiation system 40C can contain a controller 50 that can be connected to the sensors and used to control the start-up of the irradiation system. The sensor can be, but is not limited to, an optical density probe or a turbidity sensor. The sensor can provide a signal to the controller 50 to activate the irradiator 42 at a predefined value of optical density or other selected parameter. The housing 46 can be placed on a framework having a support 48 for mounting and stability. Radiation, such as X-rays, can be emitted to the culture 76 in the airlift bioreactor column through an irradiation window 54, such as a Be and Al window. The beam angle θ and distance from the source can be optimized to cover a large surface and allow maximum penetration of the X-rays into the microalgae culture (FIG. 7D). A radiation shield 52, such as a 0.5 mm lead layer, can provide protection from the X-rays and can be placed on the opposite side of the airlift or bubble column bioreactor compared to the irradiation system. In at least one embodiment, the irradiation system 40C is movable to irradiate other airlift bioreactors.In at least another embodiment, the irradiation system 40C can be fixedly mounted to the airlift bioreactor. The system requires a power source. Other embodiments of the irradiation airlift or bubble column bioreactor system are possible and contemplated.

[0121] 8A-8D are exemplary schematic diagrams of at least one embodiment of an illumination system of the present invention, configured to apply the exemplary method to an existing microalgae cultivation platform of a fermentation bioreactor under otherwise normal conditions. FIG. 8A is a side schematic diagram of another example of an illumination system mounted on a wall of a fermentation bioreactor. FIG. 8B is an enlarged top schematic diagram of the illumination system of FIG. 8A. FIG. 8C is an enlarged side cross-sectional schematic diagram through a longitudinal section of the illumination system of FIG. 8A. FIG. 8D is an enlarged side cross-sectional schematic diagram of the coverage of the illumination beam through a longitudinal section of the illumination system of FIG. 8A.

[0122] Standard fermentation bioreactors are known to those skilled in the art and generally include a vessel 82 with an aqueous growth substrate 56 with an agitation system 84, a power source 86 such as a pump for injecting a gas such as air into the microalgae culture 88 in the vessel, an air shaft 90 for ventilation, an exhaust valve 92, and sensors 94 for sensing the culture conditions (such as phase and other parameters to be monitored). In at least one embodiment, the irradiation fermentation bioreactor 80 can include an embodiment for carrying out the method of the present invention, for example, by connecting an irradiation system 40D to the vessel 82. The irradiation system 40D with associated control system 50 can include a housing 46 with cooling system and electrical equipment containing an irradiation device 42 such as an X-ray tube that can be placed in an irradiation case 44, such as, for example, but not limited to, a lead box with oil for heat dissipation (FIGS. 8B-8C). The irradiation system can be positioned to irradiate through a window 54, such as a Be and Al window, which allows radiation such as X-rays to reach the microalgae culture (FIGS. 8A and 8D). The X-rays can irradiate only a portion of the volume of the fermentation bioreactor tank. Agitating the culture can provide more uniform irradiation of the microalgae culture (FIGS. 8A and 8D). A radiation shield 52, such as a 0.5 mm lead layer, can provide protection from X-rays that can be placed over the vessel. The irradiation system 40D can include a controller 50 that can be connected to a sensor and used to control activation of the irradiation system. The sensor can be, but is not limited to, an optical density probe or a turbidity sensor. The sensor can provide a signal to the controller 50 to activate the irradiator 42 at a predefined value of optical density or other selected parameter. The housing 46 can be bonded to the vessel or placed on a framework with supports for mounting and stability. The beam angle θ and distance from the source can be optimized to cover a large surface and maximize the penetration of the X-rays into the microalgae culture (FIG. 8E). In at least one embodiment, the irradiation system 40D is portable to irradiate other fermentation bioreactors.In at least another embodiment, the irradiation system 40D can be fixedly mounted on the container. The system requires a power source. Other embodiments of the irradiation fermentation bioreactor system are possible and contemplated.

[0123] 9A-9B are exemplary schematic diagrams of at least one embodiment of an illumination system of the present invention configured to apply an exemplary method to an existing microalgae culture platform in a biofilm reactor under normal conditions. FIG. 9A is a side schematic diagram of another example of an illumination system attached to a biofilm reactor. FIG. 9B is an enlarged side cross-sectional schematic diagram of a longitudinal section of the illumination system of FIG. 9A.

[0124] Standard biofilm reactors are known to those skilled in the art and generally include a source layer as a support surface 102 on which a water and gas permeable / porous surface 106 is attached, through which water and gas flow to provide a growth substrate 56, and microalgae 107 is held on one side of the surface 106. The water and gas can be circulated by a motive force 104, such as a pump or injector, and a sensor 108 for sensing the state of the organism suitable for controlling the pump and other equipment. In at least one embodiment, the irradiation biofilm reactor system 100 can include an embodiment for carrying out the method of the present invention by coupling an irradiation system 40E to the biofilm reactor in the vicinity of the support surface 102 containing the microalgae (FIG. 9A). The irradiation system 40E with associated controller 50 includes a housing 46 with, for example, a cooling system and electrical equipment, including an irradiation device 42, such as an X-ray tube, which can be placed in an irradiation case 44, such as a lead box with oil for heat dissipation (FIG. 9B). A support 48', such as a handle coupled to the housing, aids in moving the irradiation system. The irradiation system 40E includes a controller 50 that can be used to connect to sensors and control activation of the irradiation device 42 at a predetermined value. The housing 46 can be placed on the support 48 for mounting and stability. Additional protection from radiation can be provided by a radiation shield 52, such as a 0.5 mm lead layer attached to the support surface 102 distal from the irradiation system 40E (FIG. 9A). Radiation, such as X-rays, can be irradiated to the microalgae 107 through an irradiation window 54, such as a Be and Al window. The beam angle θ and distance from the source can be optimized to irradiate the microalgae (FIG. 9A). In at least one embodiment, the irradiation system 40E is movable to irradiate other biofilm reactors. In at least another embodiment, the irradiation system 40E can be fixedly installed in the biofilm reactor. The system requires a power source. Other types of biofilm reactors, such as membrane aerated biofilm reactors, moving bed biofilm reactors, etc., can be adapted using the principles described herein, and other embodiments of biofilm reactors are possible and contemplated.

[0125] 10A-10C are exemplary schematic diagrams of at least one embodiment of the illumination system of the present invention configured to apply the exemplary method to the cultivation of plants or other organisms with conserved metabolic pathways. FIG. 10A is a side schematic diagram of another example of a multi-purpose mobile illumination system for the processing of organisms with conserved metabolic pathways related to lipid yield. FIG. 10B is an enlarged top schematic diagram of the illumination system of FIG. 10A. FIG. 10C is an enlarged side cross-sectional schematic diagram through a longitudinal section of the illumination system of FIG. 10A.

[0126] Cultivation of microalgae and other organisms with conserved metabolic pathways associated with the production of lipids and other metabolites is known to those skilled in the art. In at least one embodiment, the method can be carried out using a mobile platform with an irradiation system. For example, a mobile irradiation system 110 includes the use of an irradiation system 40F that can be coupled to a lifting device 112 using a mobile platform 114, such as a truck or other vehicle (FIG. 10A). In at least one embodiment, the lifting device 112 includes a crane or rail system for moving the irradiation system. The irradiation system 40F can be moved near an organism 116 supported by a growth substrate 56 for irradiation. The irradiation system 40F with an associated control system 50 includes a housing 46 that includes, for example, a cooling system and electrical equipment. The housing 46 includes an irradiation device 42, such as an X-ray tube, that can be placed in an irradiation case 44, such as a lead box, with oil for heat dissipation (FIGS. 10B-10C). The irradiation system 40F includes a controller 50 for operating the irradiation device. In at least one embodiment, the irradiation system can be coupled to a sensor 118 that is used to control the activation of the irradiation system. The sensor is adapted to sense one or more parameters of the particular organism that are relevant for the activation of irradiation. The sensor can provide an appropriate signal (wired or wireless) to the controller 50 to activate the irradiation device 42. The housing 46 can be coupled to a support 48', such as a swivel, that can be coupled to the lifting device 112. Additional protection from radiation can be provided by a radiation shield 52 ("skirt"), such as a 0.5 mm lead layer coupled to the housing (FIG. 10C). Radiation, such as X-rays, can be emitted by the irradiation system 40F to the organism 116 through an irradiation window 54, such as a Be and Al window. The beam angle θ and distance from the source can be optimized to cover a large surface (FIG. 10A). The system can be easily transported to irradiate a variety of growth cultures and systems using a mobile platform. A power source is required for the system. Other embodiments of the mobile irradiation system are possible and contemplated.

[0127] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although suitable methods and materials are described herein, methods and materials similar or equivalent to those described herein can be used to practice or test the present invention. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including the definitions herein, will control. Furthermore, the materials, methods, and examples are illustrative and not intended to be limiting.

[0128] Other and further embodiments utilizing one or more aspects of the invention described above can be devised without departing from the spirit of the applicant's invention. For example, the use of gamma rays instead of X-rays in the irradiation system, irradiation of organisms with conserved metabolic pathways, irradiation of organisms cultured in solid media instead of liquid media, multiple irradiations, irradiation systems for alternative microalgae culture platforms or other plant and microbial culture systems, irradiation systems that are not mobile but integrated into bioreactors and other platforms, the use of electromagnetic ionizing radiation to increase metabolite yields, various growth substrates, growth of microalgae and organisms with conserved metabolic pathways under conditions that are not normal according to the definition provided herein, microalgae cultured on other culture platforms, culture conditions, culture types, other doses, rates and durations between irradiation and harvest, other related and unrelated microorganisms, and other variations may occur within the scope of the claims.

[0129] The present invention is described in the context of preferred and other embodiments, and not all embodiments of the present invention are described. Obvious modifications of the method and system include variations that would occur to one skilled in the art given the teachings of this specification. The disclosed and undisclosed embodiments are not intended to limit or restrict the scope or applicability of the inventions devised by the applicant, but rather are governed by the patent laws. The applicant intends to fully protect all such modifications and improvements that fall within the scope of the following claims.

Claims

1. A method for metabolic activation of an organism by irradiation, comprising the steps of: irradiating the organism with at least one of X-rays and gamma rays to induce hormesis, wherein the organism is at least one of a microalgae strain and an organism having a conserved metabolic pathway; and metabolically priming the organism to produce a hormesis effect under defined conditions, at least one of a higher lipid yield, a higher biomass yield, and a higher metabolite yield, as compared to an untreated organism of the same species when maintained under the same conditions. A method comprising:

2. The method of claim 1 , wherein the hormesis effect occurs in cultured microalgae cells.

3. 2. The method of claim 1, wherein the hormesis effect occurs without a significant decrease in biomass yield compared to a control on biomass yield.

4. 3. The method of claim 2, wherein the culture is exposed to normal conditions for the cultivation of the particular strain of microalgal cells at the time of irradiation, and the culture is maintained under said normal conditions for a period of time after irradiation until harvesting.

5. The method of claim 2, wherein the culture is in a stationary phase of growth at the time of irradiation.

6. 6. The method of claim 5, wherein irradiation has a hormetic effect of higher lipid yield from the organism.

7. 6. The method of claim 5, wherein the culture is exposed to normal conditions for the cultivation of the particular microalgae strain at the time of irradiation, and the culture is maintained under said normal conditions for a period of time after irradiation until harvesting.

8. Living things, Microalgal strains, including freshwater algae and marine algae, including diatoms (diatoms), chlorophytes (green algae), cyanobacteria (blue-green algae), haptophytes (seaweeds, mainly including coccoliths), phaeophytes (brown algae), pyrophytes (dinoflagellates), and rhodophytes (red algae); and Organisms with conserved metabolic pathways such as microalgae strains related to lipid yield, including Bigilla (including Thraustochytrids), Streptophytes (plants including mosses), Ascomycota (fungi including yeasts), Basidiomycota (fungi including mushrooms), bacteria, and Archaea The method of claim 1 , comprising at least one of:

9. The organism is exposed to the usual conditions recommended for culture at the time of irradiation; and The organism is maintained under said normal conditions for a period of time after irradiation until harvest; The system of claim 8.

10. 2. The method of claim 1, wherein the hormesis effect comprises upregulation of expression of lipid metabolism genes encoding enzymes involved in the biosynthesis of lipids in microalgae and organisms having conserved metabolic pathways related to lipid yield.

11. The method of claim 10, wherein the lipid metabolism genes include lipid metabolism genes for the production of fatty acids and triacylglycerol in lipid droplets.

12. Living things, Microalgal strains, including freshwater algae and marine algae, including diatoms (diatoms), chlorophytes (green algae), cyanobacteria (blue-green algae), haptophytes (seaweeds, mainly including coccoliths), phaeophytes (brown algae), dinoflagellates (dinoflagellates), and rhodophytes (red algae); and Organisms with conserved metabolic pathways such as microalgae strains related to lipid yield, including Bigilla (including Thraustochytrids), Streptophytes (plants including mosses), Ascomycota (fungi including yeasts), Basidiomycota (fungi including mushrooms), bacteria, and Archaea The method of claim 10, comprising at least one of:

13. The organism is exposed to the usual conditions recommended for culture at the time of irradiation; and The organism is maintained under said normal conditions for a period of time after irradiation until harvest; The system of claim 12.

14. 3. The method of claim 2, wherein the organism is in the exponential growth phase at the time of irradiation.

15. 15. The method of claim 14, wherein the culture is exposed to normal conditions recommended for the cultivation of the particular microalgal cell strain at the time of irradiation, and the culture is maintained under said normal conditions for a period of time after irradiation until harvesting.

16. 15. The method of claim 14, wherein the irradiation has a hormetic effect of higher biomass yield.

17. The method of claim 1 , wherein the metabolite yield comprises omega-3 fatty acids.

18. 18. The method of claim 17, wherein the omega-3 fatty acid comprises alpha-linoleic acid.

19. 1. A system for improving the performance of at least one culture of a microalgae and an organism having a conserved metabolic pathway by irradiation, comprising: a cultivation platform comprising at least one of a raceway pond, a photobioreactor, an airlift bioreactor, a bubble column bioreactor, a fermentation bioreactor, and a biofilm reactor; an irradiation system coupled to the culture platform, the irradiation system having a source of at least one of X-rays and gamma rays; and and a controller configured to control activation of the radiation source to irradiate at least one of the microalgae and the organism having the conserved metabolic pathway at a predetermined phase of the culture for a period of time and at a dose rate, and to metabolically prime at least one of the microalgae and the organism having the conserved metabolic pathway to produce, under the predetermined conditions, a hormesis effect of at least one of a higher lipid yield, a higher biomass yield, and a higher metabolite yield of the culture compared to an untreated culture of the same strain of the at least one of the microalgae and the organism having the conserved metabolic pathway when maintained under the same conditions. Including, the system.

20. 20. The system of claim 19, further comprising a sensor configured to determine at least one parameter of the microalgae cells and the microalgae cultivation platform and provide a signal to the controller to control activation of the illumination system.

21. The culture is exposed to the usual conditions recommended for the cultivation of the particular microalgal cell strain at the time of irradiation; and The cultures are maintained under normal conditions for a period of time after irradiation until harvest.

20. The system of claim 19.

22. 1. A system for enhancing the performance of an organism having a conserved metabolic pathway by irradiation, comprising: a growth substrate for said organism; an irradiation system having a source of at least one of X-rays or gamma rays in operative proximity to the organism; and a controller configured to control activation of the illumination system to illuminate the organism for a period of time and dose rate and to metabolically prime the organism to produce a hormesis effect under predetermined conditions, at least one of a higher lipid yield, a higher biomass yield, and a higher metabolite yield, as compared to an untreated organism of the same species when maintained under the same conditions. Including, the system.

23. The culture is exposed to the usual conditions recommended for the culture of the organism at the time of irradiation; and The organism is maintained under said normal conditions for a period of time after irradiation until harvest; 23. The system of claim 22.

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