Lemna minor plant or fractions or extracts thereof, methods of producing and using same and compositions comprising same

Abstract

A method of selecting novel Lemna minor varieties. Also provided are methods of producing a Lemna minor biomass, fractions and extracts therefrom, as well as products comprising same.

Description

[0001] LEMNA MINOR PLANT OR FRACTIONS OR EXTRACTS THEREOF, METHODS OF PRODUCING AND USING SAME AND COMPOSITIONS COMPRISING SAME

[0002] RELATED APPLICATIONS:

[0003] This application claims the benefit of priority from U.S. Provisional Patent Application No. 63 / 735,923 filed on December 19, 2024, which is hereby incorporated by reference in its entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to a Lemna minor plant or fractions or extracts thereof, methods of producing and using same and compositions comprising same.

[0006] Protein components are critical for human and animal health to meet their amino acid needs. Adequate protein intake is essential for optimal building, preservation and growth processes.

[0007] Global warming accompanied by water and agricultural areas shortage are a driving force for investment in sustainable protein sources that reduce dependence on animal protein. Indeed, the alternative protein markets have been growing continuously in recent years - the alternative protein market was valued at $ 60 billion in 2021 and is expected to grow at a multi-year growth rate (CAGR) of 18.5 % to a value of $ 194 billion by 2028. The size of the global plant protein market is estimated at $ 18.49 billion in 2023. Market size is expected Grow at a CAGR of about 14.1 % from 2024 to 2029 and stand at $ 46.56 billion by 2029. The barriers to this market stem mainly from the high allergens of soy and wheat proteins, high production costs and the large areas required to grow them in the face of the constant increase in demand. Additional challenges of the alternative proteins industry is scalability, especially of plant protein powder, palatability and other organoleptic properties such as (texture, color).

[0008] In recent years, water lentils (water lens) have emerged as one of the highest quality and most worthwhile alternative sources of protein, with a number of advantages over traditional high-protein crops. Water lens grows at an extremely fast rate, doubling itself every 48 hours, and is considered the most nutritious and the smallest succulent plant in the world. The protein content of the water lens is one of the highest in the plant kingdom reaching up to 42 %. It contains other nutritional components such as flavonoids, vitamins B 12 and C, calcium, and unsaturated fatty acids.

[0009] However, commercial cultivation of water lentils is challenging, requiring a delicate balance between the growing technologies, the location of the crop, and the desired end product. Additional Related Background Art:

[0010] US 20220259259

[0011] SUMMARY OF THE INVENTION

[0012] According to an aspect of some embodiments of the present invention there is provided a method of selecting a Lemna minor variety, the method comprising: (a) providing a Lemna minor species having a protein content of about 28 % and a frond size of up to 5 mm when grown in tap water;

[0013] (b) culturing said Lemna minor species under conditions which improve frond size and protein content, wherein said conditions comprise magnesium sulfate, calcium carbonate, sodium nitrate and a fertilizer which comprises nitrogen, phosphorus and potassium;

[0014] (c) determining said protein content and frond size prior to and following (b), wherein an improvement in said protein content and frond size is indicative of a novel Lemna minor variety.

[0015] According to some embodiments of the invention, said determining is determined in the presence of said conditions and in absence of said conditions, and wherein improvement in said presence and in said absence is indicative of a stable line.

[0016] According to some embodiments of the invention, a concentration of nitrogen in said fertilizer is about 7 %.

[0017] According to some embodiments of the invention, a concentration of phosphorus in said fertilizer is about 3 %.

[0018] According to some embodiments of the invention, a concentration of potassium in said fertilizer is about 7 %.

[0019] According to some embodiments of the invention, a concentration of magnesium sulfate is about 0.5-2 mg / L.

[0020] According to some embodiments of the invention, a concentration of magnesium sulfate is about 1 mg / L.

[0021] According to some embodiments of the invention, a concentration of calcium carbonate is about 0.5-2 mg / L.

[0022] According to some embodiments of the invention, a concentration of calcium carbonate is about 1 mg / L.

[0023] According to some embodiments of the invention, a concentration sodium nitrate is 4-12 mg / L. According to some embodiments of the invention, a concentration of sodium nitrate is 10 mg / L.

[0024] According to an aspect of some embodiments of the present invention there is provided a Lemna minor variety obtainable according to the method as described herein.

[0025] According to an aspect of some embodiments of the present invention there is provided a Lemna minor variety having a frond size of at least 10 mm and an essential amino acid content of at least about 50 % of total amino acids.

[0026] According to an aspect of some embodiments of the present invention there is provided a Lemna minor variety characterized by at least one of:

[0027] (i) a frond size bigger than about 7 mm;

[0028] (i) protein content above about 34 %;

[0029] (ii) a biomass of 45 tons / hectar / year of wet biomass;

[0030] (iii) an essential amino acid content higher than that of a Lemna minor variety having a protein content of about 28-30 % (the original variety), wherein all of (i)-(iv) are at tap water conditions without additions of nutrients.

[0031] According to an aspect of some embodiments of the present invention there is provided a method of producing a Lemna minor biomass or an extract or fraction thereof, the method comprising growing the Lemna minor variety as described herein under conditions which support its growth and optionally modify its content in culture.

[0032] According to some embodiments of the invention, the method further comprises isolating the biomass from said culture.

[0033] According to some embodiments of the invention, the method further comprises fractionating a fraction of interest from said biomass.

[0034] According to some embodiments of the invention, the method further comprises extracting an extract of interest from said biomass or said fraction thereof.

[0035] According to some embodiments of the invention, said conditions comprise magnesium sulfate, calcium carbonate, sodium nitrate and a fertilizer which comprises nitrogen, phosphorus and potassium.

[0036] According to some embodiments of the invention, a concentration of nitrogen in said fertilizer is about 7 %.

[0037] According to some embodiments of the invention, a concentration of phosphorus in said fertilizer is about 3 %. According to some embodiments of the invention, a concentration of potassium in said fertilizer is about 7 %.

[0038] According to some embodiments of the invention, a concentration of magnesium sulfate is about 0.5-2 mg / L.

[0039] According to some embodiments of the invention, a concentration of magnesium sulfate is about 1 mg / L.

[0040] According to some embodiments of the invention, a concentration of calcium carbonate is about 4-12 mg / L.

[0041] According to some embodiments of the invention, a concentration of calcium carbonate is about 10 mg / L.

[0042] According to some embodiments of the invention, a concentration sodium nitrate is about 4- 12 mg / L.

[0043] According to some embodiments of the invention, a concentration of sodium nitrate is about 10 mg / L.

[0044] According to some embodiments of the invention, said extract or fraction comprises protein.

[0045] According to some embodiments of the invention, said extract or fraction comprises carbohydrates.

[0046] According to some embodiments of the invention, said extract or fraction comprises lipids.

[0047] According to some embodiments of the invention, said conditions further comprise an amount of cobalt nitrate effective at increasing B 12 content.

[0048] According to some embodiments of the invention, said amount of cobalt nitrate is about 0.5- 2 mg / L.

[0049] According to some embodiments of the invention, said conditions further comprise iron (Fe+2).

[0050] According to some embodiments of the invention, an amount of said iron (Fe+2) is about 0.005-0.015 gr / L.

[0051] According to some embodiments of the invention, an amount of said iron (Fe+2) is about 0.01 gr / L.

[0052] According to an aspect of some embodiments of the present invention there is provided an article of manufacture comprising the Lemna minor biomass of the Lemna minor variety of any one of claims 12-14 or an extract or fraction thereof.

[0053] According to some embodiments of the invention, the article of manufacture is selected from the group consisting of a human food, a protein-rich powder, Lemna-based protein isolate, Lemna oil, Lemna flour, fermented Lemna, animal feed, bioplastics, biofuels, fertilizers, nutraceuticals, and a bioremediation filter.

[0054] According to some embodiments of the invention, the article of manufacture comprises genomic DNA of the Lemna minor variety.

[0055] According to some embodiments of the invention, the article of manufacture comprises an amino acid profile as in Table 2.

[0056] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0057] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0058] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0059] In the drawings:

[0060] FIG. 1 is an embodiment of a system for the selection process for the new variety of Lemna minor.

[0061] FIG. 2 is a picture of the new variety;

[0062] FIG. 3 is an embodiment of a system for the inductive selection process for the new variety of Lemna minor characterized by improved parameters including yield and protein content;

[0063] FIG. 4 is a graph showing yield (tons dry per hectare per year) as a function of concentration of the fertilizer (NPK 7-3-7);

[0064] FIG. 5 is a graph showing protein content (% of dry weight) as a function of concentration of calcium carbonate;

[0065] FIG. 6 is a graph showing B 12 concentration (% of dry weight) as a function of concentration of calcium carbonate; FIG. 7 is a photographic presentation of the new variety versus the original variety when grown under nonselective conditions.

[0066] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0067] The present invention, in some embodiments thereof, relates to a Lemna minor plant or fractions or extracts thereof, methods of producing and using same and compositions comprising same.

[0068] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0069] The present inventors have devised a platform for the selection of improved varieties of Lemna minor. A novel variety, thus selected, is endowed with unique properties including a high growth rate, high protein content, and high essential amino acids as compared to the original variety. Conditions were found to increase its B12 content. This new variety is stable and can be grown in large scale settings, rendering it highly valuable as a protein alternative and a key player in the fiber and the medicinal industries.

[0070] This according to an aspect of the invention there is provided a method of selecting a Lemna minor variety, the method comprising:

[0071] (a) providing a Lemna minor species having a protein content of about 28 % and a frond size of up to 5 mm when grown in tap water;

[0072] (b) culturing said Lemna minor species under conditions which improve frond size and protein content, wherein the conditions comprise magnesium sulfate, calcium carbonate, sodium nitrate and a fertilizer which comprises nitrogen, phosphorus and potassium; and

[0073] (c) determining said protein content and frond size prior to and following (b), wherein an improvement in said protein content and frond size is indicative of a novel Lemna minor variety.

[0074] A “Lemna minor species” also known as “common duckweed” or “lesser duckweed”, is a species of an aquatic freshwater plant in the subfamily Lemnoideae of the arum family Araceae. Lemna minor species are typically endowed with a protein content of not more than about 28 % of dry weight (w / w) when grown in tap water (not supplemented with nutrients) and a frond size of 2-5 mm when grown in tap water, and optionally B12 concentration of less than about 12 pg / 100 gr dry weight. A “frond size” refers to an average frond size of statistically significant number of Lemna minor individuals in the culture.

[0075] Methods of determining protein content include but are not limited to Kjeldahl and Dumas nitrogen-based methods (for estimating protein content based on nitrogen levels), as well as direct colorimetric assays like the Bradford, Lowry, and BCA assays.

[0076] The sample is dried (usually in an oven at a low temperature, around 60-80 °C) to obtain its dry weight. A Lemna minor sample is considered dry at 95 % moisture.

[0077] As used herein “selecting” refers to subjecting the wild species to conditions which favor the selection of plants with at least one improved trait of interest.

[0078] According to the invention, the selection results in a stable variety which is stable throughout generations (e.g., 10-20 generations in tap water, and over 20, 50, 100, 250, 300, 500 generations in the presence of nutrients as described here) even without the conditions which favor the selection of the trait(s) of interest. A generation / passage is typically 2-3 days.

[0079] As used herein “variety” may be interchanged with “line”, “cultivar”.

[0080] As used herein “tap water” is chemically defined as primarily composed of JLO and dissolved ions such as calcium (Ca2+), magnesium (Mg2+), sodium (Na+), bicarbonate (HCCh ), chloride (CL), sulfate (SO42), fluoride (F ), and chlorine (CL), with a pH ranging from 6.5 to 8.5. Physically: it typically has a conductivity of 50 to 500 pS / cm, turbidity less than 1 NTU, and hardness varying from soft (<60 mg / L as CaCCh) to hard (>120 mg / L as CaCCh). The temperature typically ranges from 4°C to 25°C.

[0081] According to a specific embodiment, the Lemna minor is obtained from Hushnia (also known as “Hawaja”) spring in the Golan Heights in Israel.

[0082] As mentioned, the Lemna minor species is cultured under conditions which improve protein content and frond size.

[0083] These conditions are typically regulated involving a controlled lighting regimen and temperatures (e.g., 10,000-25,000 LUX, 12-30 °C).

[0084] According to a specific embodiment, the conditions used for selection comprise tap water supplemented with magnesium sulfate, calcium carbonate, sodium nitrate and a fertilizer which comprises nitrogen, phosphorus and potassium, also collectively referred to as “nutrients”). Nutrients can also be added to water at the stage of production (biomass) of the novel variety. Generally, these are the same conditions though calcium carbonate and fertilizer may change. These conditions are also referred to as enriched water. According to a specific embodiment, the concentration of nitrogen in said fertilizer is about 7 %.

[0085] According to a specific embodiment, the concentration of phosphorus in said fertilizer is about J O zo.

[0086] According to a specific embodiment, the concentration of potassium in said fertilizer is about 7 %.

[0087] According to a specific embodiment, the fertilizer composition is present in the growth solution at a percentage of about 0.05 % -0.175 % e.g., about 0.15 % (v / v of the nitrogen, phosphorus and potassium of the fertilizer).

[0088] According to a specific embodiment the fertilizer is NPK.

[0089] According to a specific embodiment, the fertilizer is NPK 7-3-7.

[0090] As used herein “NPK 7- -7” also referred to as FERTICELL NPK 7-3-7 is a soluble powdered fertilizer, having the composition percentage (w / w) Total Nitrogen (N) 7.00% Total Phosphor (P205) 3.00% Total Potassium (K20) 7.00% Ammoniacal Nitrogen 0.49 Organic Nitrogen 7.13 Total Amino acids 33.45% Total Organic Matter 69.30% Total Organic Carbon (C) 40.20% C / N relation 5,74 %.

[0091] Vendors of NPK fertilizers include but are not limited to, Yara International, Nutrien, CF Industries, The Mosaic Company, K+S Group, PhosAgro, and ICL Fertilizers.

[0092] NPK 7-3-7 alternatives include, but are not limited to, NPK 8-3-8, NPK 10-3-10, NPK 6-3- 6, and NPK 7-2-7, each offering a similar balance of nitrogen, phosphorus, and potassium, with slight variations in the concentrations of nitrogen and potassium. These fertilizers maintain a comparable phosphorus content of around 3%. Non-NPK alternatives include custom blends such as a mixture of ammonium nitrate, superphosphate, and potassium chloride, which can be tailored to approximate the 7-3-7 ratio. Organic options, like combining blood meal, bone meal, and potassium sulfate, also provide a balanced nutrient profile. Other possible mixes include urea, triple superphosphate, and potassium sulfate, which can be adjusted to match the desired nutrient balance. A blend of fish emulsion, rock phosphate, and greensand can offer a similar nutrient composition. A combination of calcium nitrate, monoammonium phosphate, and potassium nitrate can be used.

[0093] According to a specific embodiment, the concentration of magnesium sulfate is about 0.5-2 mg / L (e.g., 0.75-1.25. 0.8-1.2, 0.9-1.1 mg / L).

[0094] According to a specific embodiment, the concentration of magnesium sulfate is about 1 mg / L.

[0095] According to a specific embodiment, the concentration of calcium carbonate is about 0.5-2 mg / L (e.g., 0.75-1.25. 0.8-1.2, 0.9-1.1 mg / L). According to a specific embodiment, the concentration of calcium carbonate is about 1 mg / L. According to a specific embodiment, the concentration sodium nitrate is about 4-12 mg / L. According to a specific embodiment, the concentration of sodium nitrate is about 10 mg / L. It will be appreciated that sodium nitrate can be replaced with potassium nitrate at the same concentration.

[0096] As used herein “culturing” refers to growing the Lemna species under controlled culture conditions. Culturing can be for a number of generations, at least, 10, 50, 100, 200, 300, 500 or 1000 generation. Figure 1 shows an embodiment of a system for culturing and selection of varieties of interest.

[0097] As mentioned, the method further comprises determining protein content and frond size prior to and following (b), wherein an improvement in said protein content and frond size is indicative of a novel Lemna minor variety.

[0098] As used herein “improvement” or “increase” is a statistically significant increase.

[0099] According to a specific embodiment, the increase in protein content is at least about 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, or 2 fold.

[0100] According to a specific embodiment, the increase in frond size is at least about, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold or 20 fold.

[0101] According to a specific embodiment, determining is done in the presence of the aforementioned conditions (i.e., fertilizer, calcium carbonate, sodium nitrate and magnesium sulfate) and in absence of said conditions, and wherein improvement in the presence and in the absence of the conditions is indicative of a stable line.

[0102] This was done as shown in the Examples section which follows, specifically Example 2 in the presence of NPK 7-3-7, calcium carbonate, sodium nitrate and magnesium sulfate and Example 6 in their absence (solely tap water). In both instances an improvement in size and protein content was indicated, substantiating the formation of a stable variety

[0103] Whilst employing these teachings, the present inventors were able to generate a stable variety of Lemna minor.

[0104] Thus, according to an aspect of the invention there is provided a Lemna minor variety obtainable according to the embodiments of the method as described herein.

[0105] According to an alternative or an additional aspect of the invention, there is provided a Lemna minor variety having a frond size of at least 10 mm and an essential amino acid content of at least about 50 % of total amino acids. According to an alternative or an additional aspect there is provided a Lemna minor variety characterized by at least one of:

[0106] (i) a frond size bigger than about 7 mm;

[0107] (i) protein content at least about 34 %;

[0108] (ii) a biomass of 45 tons / hectar / year of wet biomass;

[0109] (iii) an essential amino acid content higher than that of a Lemna minor variety having a protein content of about 28-30 % (the original variety), wherein all of (i)-(iv) are at tap water conditions without additions of nutrients.

[0110] These parameters are improved when grown in the presence of the nutrients as mentioned above, fertilizer, magnesium sulphate, calcium carbonate and sodium nitrate.

[0111] Thus, according to a specific embodiment, there is provided a Lemna minor variety characterized by at least one of:

[0112] (i) a frond size bigger than about 10 mm (e.g., 10-15 mm);

[0113] (i) protein content at least about 38 % of dry weight (e.g., 38-42 %);

[0114] (ii) a biomass of 60 tons / hectar / year of wet biomass;

[0115] (iii) an essential amino acid content higher than that of a Lemna minor variety having a protein content of about 28-30 % (the original variety in tap water) and reaching at least about 50 % of the total amino acids, wherein all of (i)-(iv) are at water enriched conditions.

[0116] According to a specific embodiment, the variety is stable in tap water (e.g., having conductivity of the above mentioned microsiemens, e.g., 450).

[0117] Once the variety is obtained it can be produced in small to large scale settings.

[0118] Thus, according to an aspect of the invention there is provided a method of producing a Lemna minor biomass or an extract or fraction thereof, the method comprising growing the Lemna minor variety (e.g., also referred to herein “the novel variety”) under conditions which support its growth and optionally modify its content in culture.

[0119] As used herein “biomass” refers to the whole plant.

[0120] As used herein “fraction” refers to a part of the plant.

[0121] As used herein “extract” refers to a specific component or group of components obtained from a source material (the plant or fraction thereof) typically through a solvent process.

[0122] According to some embodiments, the extract or fraction comprises protein. The primary proteins in Lemna minor include those involved in photosynthesis, such as Rubisco and lightharvesting complex proteins; Structural proteins include, but are not limited to actin and tubulin; Storage proteins, including albumins and globulins, which act as reservoirs of amino acids and nitrogen, especially during nutrient stress; Enzymes like amylase and nitrate reductase; Transport proteins, such as aquaporins. The identity of the protein as Lemna proteins can be determined based on their sequence.

[0123] According to some embodiments, the extract or fraction comprises carbohydrates. Lemna minor contains cellulose and hemicellulose in its cell walls for structural support, starch as a primary energy storage form, and soluble sugars like glucose, fructose, and sucrose for metabolic processes. Additionally, fructans may also be present in certain conditions, serving as another carbohydrate storage form

[0124] According to some embodiments, the extract or fraction comprises lipids. Lemna minor contains a variety of lipids, including phospholipids, glycolipids, neutral lipids (mainly triglycerides), sterols, and free fatty acids, which contribute to its metabolic functions and its potential as a source of biofuels.

[0125] Thus, the fraction or extract can be at least 50 %, 60 %, 70 %, 80 %, 90 %, 95 % pure.

[0126] Typical growth conditions comprise a temperature of 12-30 °C, e.g., 20-25 °C and an uncovered container (within the indicated temp range). Covering can be done using a shadow net e.g., 50 % sun-light filtering.

[0127] Systems for growing Lemna minor are well known in the art. These systems typically use a variety of technologies, such as vertical farming, photobioreactors, closed-loop systems, and floating platforms, to cultivate Lemna at a large scale

[0128] Examples of large scale growth settings are provided infra.

[0129] GreenTech Agro (LemnaTec), uses automated monitoring and precise environmental control systems to optimize the growth conditions of Lemna, ensuring efficient production in high-density cultivation environments. Fertile Earth has large-scale Lemna cultivation systems that incorporate vertical farming racks, hydroponic systems, and controlled-environment greenhouses to efficiently grow Lemna for various applications. Phycobloom employs vertical farming techniques and automated nutrient management to ensure consistent production of high-protein duckweed biomass in large-scale operations. Renewable Energy Solutions (RES) utilizes floating platforms, photobioreactors, and closed-loop systems to create optimal growing conditions for Lemna, enhancing efficiency and scalability. Advanced BioEnergy incorporates automated harvesting systems, nutrient management tools, and bioreactor designs to maximize Lemna yield for bioethanol production and other bio-based products. Lemna Solutions uses scalable floating pond-based systems and aerated bioreactors, which optimize water use and minimize energy consumption for large-scale Lemna cultivation. Algae. Tec integrates closed-loop photobioreactors and vertical farming towers to cultivate Lemna and other plants in controlled environments, aiming to reduce environmental impacts while maintaining high productivity. Seawater Greenhouse adapts its solar-powered desalination and closed-loop greenhouse systems to enable the cultivation of Lemna in water-scarce regions with high salinity.

[0130] The volume range for large-scale Lemna cultivation systems typically falls between 100 cubic meters to 10,000 cubic meters of water, depending on the technology and application. Smaller systems may be around 100-500 cubic meters, while larger industrial-scale operations, particularly those focused on biofuels, aquaculture, or wastewater treatment, can scale up to 5,000-10,000 cubic meters or more. These volumes can vary significantly depending on the design, modularity, and specific goals of the cultivation system.

[0131] Exemplary alternative or additive embodiments for growth are provided below:

[0132] According to some embodiments, the conditions comprise magnesium sulfate, calcium carbonate, sodium nitrate and a fertilizer which comprises nitrogen, phosphorus and potassium.

[0133] According to some embodiments, the concentration of nitrogen in said fertilizer is about 7 %.

[0134] According to some embodiments, the concentration of phosphorus in said fertilizer is about O zo.

[0135] According to some embodiments, the concentration of potassium in said fertilizer is about 7 %.

[0136] According to a specific embodiment, the fertilizer composition is present in the growth solution at a percentage of about 0.05 %-0.175 %, e.g., about 0.15 % (of the nitrogen, phosphorus and potassium of the fertilizer).

[0137] According to some embodiments, the concentration of calcium carbonate is about 4-12 mg / L (e.g., about 6-12, 7-12, 8-12, 9-11, 5-12, 6-10, 6-8 mg / L).

[0138] According to some embodiments, the concentration of calcium carbonate is about 10 mg / L.

[0139] According to a specific embodiment, the concentration of magnesium sulfate is about 0.5-2 mg / L (e.g., 0.75-1.25. 0.8-1.2, 0.9-1.1 mg / L).

[0140] According to a specific embodiment, the concentration of magnesium sulfate is about 1 mg / L.

[0141] According to some embodiments, a concentration sodium nitrate is 4-12 mg / L.

[0142] According to some embodiments, a concentration of sodium nitrate is 10 mg / L.

[0143] It will be appreciated that potassium nitrate can be used instead of sodium nitrate at the indicated concentrations. According to some embodiments, the conditions further comprise an amount of cobalt nitrate effective at increasing B 12 content.

[0144] According to some embodiments, the amount of cobalt nitrate is about 0.5-2 mg / L (e.g., about 0.6-1.8, 1.1-1.9, 1.3-1.8 mg / L).

[0145] According to some embodiments, the amount of cobalt nitrate is about 1.5 mg / L.

[0146] The present inventors have shown that the addition of microbes to the culture can be used to increase the growth rate of the new variety.

[0147] According to a specific embodiment, the microbe is bacteria.

[0148] Microbes that enhance the growth of Lemna minor include nitrogen-fixing bacteria (such as Azospirillum and Rhizobium), phosphorus-solubilizing bacteria (like Bacillus and Pseudomonas), plant growth-promoting rhizobacteria (such as Pseudomonas putida and Bacillus subtilis), and certain algae and endophytic organisms. These microbes can improve nutrient availability, promote stress resistance, and enhance overall plant health, which can lead to faster growth and higher biomass production of Lemna minor.

[0149] It will be appreciated that the type of microbe can be selected based on the intended use of the Lemna minor variety. Thus, for instance, microbes, which are generally considered as safe can be used for products that are intended for human consumption, while others can be used in wastewater treatment, bioremediation and the like. According to a specific embodiment, the microbe is bacteria e.g., Aquitalea spp., Pseudomonas spp. or combination of same.

[0150] According to a specific embodiment, the bacteria in culture include not more than 10, 8, 7, 6, 5, 4, 3, 2, or a single bacterial strains or species.

[0151] Once a sufficient biomass is provided the method further comprises isolating the biomass from the culture.

[0152] According to a specific embodiment, the isolation is done using a harvesting machine. Such are described in WO2024099863 which is hereby incorporated by reference in its entirety.

[0153] According to a specific embodiment, the method further comprises fractionating a fraction of interest from said biomass.

[0154] According to a specific embodiment, the method further comprises extracting an extract of interest from said biomass or said fraction thereof.

[0155] Methods of extracting Lemna are well known in the art. See for example WO / 2022 / 151689 which is hereby incorporated by reference in its entirety.

[0156] Using such teachings the present inventors were able to produce more than 60 tons per hectar per year (300 days) of dry novel variety. As research into its potential expands, Lemna minor is proving to be a promising candidate for several innovative applications, including as a food product, animal feed, bioplastics, biofuels, fertilizers, pharmaceuticals, as well as a bioremediation filter for water purification.

[0157] Thus according to an aspect of the invention there is provided an article of manufacture comprising the Lemna minor biomass of the Lemna minor variety as described herein or an extract or fraction thereof.

[0158] According to a specific embodiment, the article of manufacture is selected from the group consisting of a human food, a protein-rich powder, Lemna-based protein isolate, Lemna oil, Lemna flour, fermented Lemna, animal feed, bioplastics, biofuels, fertilizers, nutraceuticals, and a bioremediation filter.

[0159] According to a specific embodiment, the article of manufacture comprises genomic DNA of the Lemna minor variety.

[0160] According to a specific embodiment, the article of manufacture comprises an amino acid profile as in Table 2.

[0161] Thus, one of the most exciting applications of Lemna minor is its use as a protein source in the food industry. Lemna minor can be processed into protein-rich powder, which can be incorporated into various food products such as protein bars, dairy products, smoothies, and plant-based meat alternatives. This protein powder is highly digestible and contains high levels of all nine essential amino acids, making it a complete protein source. The powder can be used to fortify food products, especially in regions with limited access to traditional sources of protein. Furthermore, the protein isolate derived from Lemna minor offers a sustainable, low-cost alternative to animal-based proteins, which are resource-intensive to produce.

[0162] Beyond protein powder, Lemna minor can also be processed into Lemna-based protein isolate, which has a higher concentration of protein and can be used in specialized dietary supplements or functional foods. This protein isolate is particularly valuable in the market for plant-based protein alternatives. T he unique nutritional profile of Lemna minor and the novel variety in particular, which includes high levels of essential amino acids, vitamins (e.g., B12), and minerals, makes it an attractive ingredient for developing nutritionally balanced products. Lemna protein isolate can be a key component in the formulation of various food and beverage products, such as meal replacements, protein shakes, and vegetarian or vegan foods.

[0163] In addition to protein, the Lemna minor can also be processed into Lemna oil. Lemna minor contains a significant amount of lipids, particularly unsaturated fatty acids like oleic and linoleic acids, which are beneficial for human health. This oil can be extracted and used as an ingredient in food products, cosmetics, and health supplements. The high levels of polyunsaturated fatty acids make Lemna oil a potential source of heart-healthy fats. It can also be of use in the formulation of functional foods aimed at improving cardiovascular health. The oil’s potential to be used in the cosmetics industry is also notable, as it can be incorporated into creams, lotions, and other skincare products due to its moisturizing and anti-inflammatory properties.

[0164] Another important application of Lemna minor is its use in the production of Lemna flour. The plant’s biomass is rich in carbohydrates, and when dried and processed, it can be ground into a fine flour that can be used as an alternative to traditional grains. Lemna flour can be incorporated into a variety of baked goods, such as bread, cookies, and pancakes, providing a gluten-free and highly nutritious alternative. The flour is also a good source of dietary fiber, which is beneficial for digestive health.

[0165] In addition to human consumption, fermented Lemna can be used as a potential feed ingredient for animals. Thus the novel variety can be subjected to fermentation processes that enhance the digestibility of Lemna and improve its nutritional profile by increasing its bioavailability. This makes it an attractive option for use in animal feed, especially for livestock, poultry, and aquaculture. Duckweed-based animal feed is rich in proteins, amino acids, and essential micronutrients, making it a highly efficient and cost-effective alternative to traditional animal feed ingredients such as soybeans and fishmeal. Furthermore, because the variety is endowed with high growth rate it can be cultivated in aquatic environments, it has the potential to reduce the pressure on land-based agricultural systems, providing a more sustainable source of feed.

[0166] Lemna minor can be used in the production of bioplastics. Duckweed-derived bioplastics are biodegradable and can be used in a variety of applications, including packaging materials, disposable cutlery, and agricultural films. The rapid growth rate and high biomass yield of Lemna minor make it an ideal candidate for bioplastic production, offering a renewable source of raw material for the industry. Furthermore, the use of Lemna in bioplastics could help reduce reliance on food crops like corn or sugarcane, which are typically used for bioplastic production.

[0167] Another application of Lemna minor is in the field of biofuels. Lemna’ s high lipid content makes it a promising candidate for biodiesel production. The plant’s ability to grow quickly in nutrient-rich water makes it a highly efficient feedstock for biofuel production. Lemna minor can be cultivated in a variety of aquatic environments, including wastewater, making it an ideal candidate for waste-to- energy solutions. By growing Lemna on wastewater or other nutrient-rich sources, it is possible to produce biofuels while simultaneously treating polluted water, creating a sustainable and environmentally friendly system. In addition to biodiesel, Lemna biomass can be used for biogas production through anaerobic digestion, providing another renewable energy source.

[0168] In the agricultural sector, Lemna minor has great potential as a fertilizer. As a nitrogen-fixing plant, Lemna can improve the nutrient content of soil and water, making it a valuable addition to organic farming practices. The biomass of Lemna minor can be used as a natural fertilizer to enrich the soil with essential nutrients like nitrogen, phosphorus, and potassium. In addition, the plant's rapid growth and ability to absorb excess nutrients from the water make it an effective tool in nutrient management systems, helping to reduce nutrient runoff and improve the quality of soil and water. Duckweed can also be used in aquaponics systems, where it helps to purify water and provide nutrients for fish and other plants.

[0169] Nutraceuticals is another area where Lemna minor is being used. Due to its rich nutrient content, including vitamins, minerals, and antioxidants, Lemna minor has been explored as a source of natural health supplements. The plant’s high content of flavonoids, phenolic compounds, and carotenoids makes it an attractive option for the development of functional foods and dietary supplements aimed at improving health and preventing disease. Lemna’ s potential as a natural antioxidant and anti-inflammatory agent is of particular interest, as these properties are associated with various health benefits, including improved immune function and reduced risk of chronic diseases such as heart disease and cancer.

[0170] In addition, one of the most notable environmental applications of Lemna minor is its role as a bioremediation filter. Lemna has the ability to absorb and remove various pollutants from water, including heavy metals, organic compounds, and excess nutrients such as nitrogen and phosphorus. This makes it an effective tool for cleaning up polluted water bodies, such as wastewater treatment ponds, stormwater runoff, and contaminated rivers and lakes. Lemna’ s fast growth and high biomass production allow it to rapidly absorb pollutants, and its use in bioremediation can help restore the ecological health of water ecosystems while also providing valuable biomass for other applications, such as biofuels or animal feed.

[0171] It is expected that during the life of a patent maturing from this application many relevant products which contain biomass, extract or fraction of the novel variety will be developed and the scope of the term “article of manufacture” or “product” is intended to include all such new technologies a priori.

[0172] As used herein the term “about” refers to ± 10 %.

[0173] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to". The term “consisting of’ means “including and limited to”.

[0174] The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0175] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0176] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0177] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. [

[0178] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0179] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0180] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0181] EXAMPLES

[0182] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0183] Example 1

[0184] Selection of a novel variety of Lemna Minor

[0185] Lemna minor plants naturally growing in a spring in the Golan Heights, Israel have a growth habit of growth almost all year round, when at the beginning of the winter growth rate decreases and returns to a full growth rate around March. These plants are termed as “the original variety”.

[0186] The selection process was carried out in 5 main stages:

[0187] 1. Selection of the largest individuals in the population and their continued growth separately.

[0188] 2. Identifying conditions for maximizing the output per area for the selected large individuals.

[0189] 3. Based on the maximum yield conditions, consider additives that increase the percentage of protein in the plant.

[0190] 4. Increasing the concentration of vitamin B12 in the plant.

[0191] The selection and separation process were done is a facility as in Figure 1.

[0192] The selection conditions consisted of tap water supplemented with NPK7-3-7 0.25 ml per liter (0.025 % v / v), Magnesium Sulfate - 1 mg / L (0.0001%), Calcium Carbonate - 1 mg / L (0.0001%), Sodium Nitrate - 1 mg / L and (0.0001%).

[0193] After a few months of selecting the large plants, a population of "new" plants was created that looked visibly different from the original population.

[0194] As a result of this operation, the present inventors were able to reach a plant size of about 15 mm. It is significantly larger than the original plant from nature. The original variety reaches about 5 mm maximum (see Figure 2). Example 2

[0195] Conditions for maximizing the yield per area for the selected large variety

[0196] Experimental Procedures

[0197] The experimental system was based on 8 pools of the Dolev company (see Figure 3). For each pool, a different concentration of nutrients was introduced. This allows determining the relative effect of the tested nutrients on the examined parameter.

[0198] To do this, a base solution was prepared, which includes the following components:

[0199] 1.NPK 7-3-7 Solution

[0200] 2. Magnesium Sulfate

[0201] 3. Calcium Carbonate

[0202] 4. Sodium Nitrate

[0203] In the first stage, the solution was identical to the solution in which the plants were selected [tap water supplemented with NPK7-3-7 (abbreviated as NPK) 0.25 ml per liter (0.025 % v / v) Magnesium Sulfate - 1 mg / L (0.0001%), Calcium Carbonate - 1 mg / L (0.0001%), Sodium Nitrate - 1 mg / L and (0.0001%)] and then the effect of the change in concentrations on the various parameters was examined according to the following parameters:

[0204] A. Quantity of dry plant that can be harvested per year per unit area;

[0205] B. Protein concentration in dry matter;

[0206] C. B12 concentration;

[0207] Concentrations of NPK were tested at levels of 0.25 to 2.0 ml per liter (in increments of 0.25 ml per liter).

[0208] Magnesium sulfate and calcium carbonate concentrations remained constant at the level of 1 mg / L per substance.

[0209] At all measurements, water temperature was identical (20-25 °C), the level of light was uniform 16 / 8 hours summer 10 / 14-12 / 12 hours winter). and all the pools were pH corrected to a level of 6.5, which is the optimal level for the utilization of fertilizer by the plant. pH was titrated with sulfuric acid (10 %).

[0210] Results

[0211] Yield - Figure 4 shows the yield of the novel variety as a function of the concentration of NPK.

[0212] The amount of dry matter / year was calculated according to the following assumptions:

[0213] 95% moisture (oven dry matter); Operation 300 days a year.

[0214] Protein - After determining the optimal fertilizer concentrations for maximizing yield, the effect of nitrogen supplementation in the form of sodium nitrate on the protein concentration in the final product was examined by the Dumas-nitrogen-based method.

[0215] The nitrate concentrations tested were 2-14 mg / L of growing water.

[0216] Based on the results of the experiment that are shown in Figure 5, a concentration of 10 mg / L was determined as the optimal concentration.

[0217] Example 3

[0218] Amino acid profile

[0219] The protein composition was examined in order to elucidate the amino acids profile of the novel variety, and in particular the essential amino acids that the human body cannot produce.

[0220] Experimental Procedures

[0221] Amino acid profiles were determined by HPLC.

[0222] Results

[0223] Below is Table 1 showing the composition of the essential amino acids in our Lemna protein and a comparison versus egg white and whey protein.

[0224] Table 1

[0225] The value of BCAA expresses the sum of 3 amino acids (isoleucine, leucine, and valine) which are essential amino acids for building muscle.

[0226] As can be seen, in the selected variety about 50 % (49.88) of all the amino acids are essential amino acids. The novel variety also showed a higher amino acid content as compared to the original variety. Results are shown in Table 2 below (original variety grown in spring water vs the new variety grown in the presence of nutrients). Table 2

[0227] About the same results were obtained from the variety on 2023(data not shown).

[0228] Table 3

[0229] New variety (lemna++) vs original variety comparison with respect to amino acid profile in unsupplemented tap water.

[0230] Example 4

[0231] Improving the concentration of B12 in the plant

[0232] One of the most outstanding features of the Lemna minor species is its unique ability, in the plant world, to naturally produce a vitamin B 12

[0233] The possibility of increasing the amount of the vitamin with cobalt nitrate was examined.

[0234] Experimental Procedures

[0235] The concentration of cobalt tested was in the range of 0.5-2 mg to 10 liters, specifically 0.1 mg / L.

[0236] Results

[0237] Figure 6 shows B 12 concentration in dry weight of the novel variety.

[0238] Example 5

[0239] Comparison between the novel variety and the parent under similar non-selective growth conditions

[0240] After a growing period of one year, the present inventors determined if the observed differences between the original variety and the improved variety are stable even if both are grown under the same conditions, without the addition of fertilizers.

[0241] Protein concentration in the plant: The concentrations of the protein in the original plants and in plants of the improved species were examined.

[0242] It was found that the concentration of protein in plants selected and grown under special conditions was about 25% higher than in natural plants.

[0243] The concentration of protein in natural plants is about 30%, the concentration of protein in improved plants is about 40 % and sometimes even exceeds this value. These results are in the presence of nutrients i.e., .25 ml per liter (0.025 % v / v) Magnesium Sulfate - 1 mg / L (0.0001%), Calcium Carbonate - 1 mg / L (0.0001%), Sodium Nitrate - 10 mg / L and (0.0001%), NPK 7-3-7 1.5 ML / LITER (0.000015 %).

[0244] Hence, the purpose of this protocol is to present a test program that can support the fact that the improved "large" novel plants constitute a novel variety of Lamena Minor.

[0245] The basic assumption is that if a new variety of Lamena Minor is created, the properties that distinguish it from the original variety will be stable even in comparative cultivation in water without the addition of chemicals.

[0246] Hence, the following parameters were validated with respect to the new variety: a higher protein content that is higher than the original plant; size of the plants that is larger than the original plant; rate of coverage of the area of the experimental tank of the plant that is higher than the rate of coverage in the original plant.

[0247] Experimental Procedure

[0248] Comparative cultivation was performed over a period of 4 weeks, in a system of 4 plastic growing containers. As mentioned, the growing water was tap water with a conductivity of 450 microsiemens.

[0249] All four tanks were equipped with circulation pumps. The pumps created a movement of water according to the surface and prevented the growth of greenery that could have interfered with the process.

[0250] In 2 containers the original Lemna minor plant was sown and in 2 others the new variety was sown. The rest of the environmental conditions - temperature and day light hours were identical.

[0251] Protein level was determined by Dumas.

[0252] Results

[0253] Figure 7 shows photographs of the 2 populations after 2 weeks of growth. The left panel is the original variety and the right panel is the new variety, in tap water, same magnification. The lower panel shows one item of each population in the same field. The size difference is clear. Clearly, the new variety is larger even in the absence of the nutrients.

[0254] Comparing the level of protein in the plant after 3 weeks of growth was done next. Four samples were taken - 2 from the new variety and 2 from the original variety.

[0255] The new variety:

[0256] Sample 1 - 35 % dry -based protein

[0257] Sample 2 - 34 % dry-based protein

[0258] The original variety:

[0259] Sample 1 - 28 % dry-based protein

[0260] Example 2 - 28 % dry -based protein

[0261] The results show that the new variety filled the growing tank in 48 hours less than the original variety.

[0262] In order to verify the growth rate, the entire population was weighed at the end of the cycle: 21 days of growth.

[0263] Container 1 (New Variety) Container Area 0.2016 m2

[0264] Weight at the end of the cycle - 1350 g

[0265] Container 2 (New Variety) Container Area 0.124 m2

[0266] Weight at the end of the cycle - 780 g

[0267] Container 3 (Origin Variety) Container Area 0.124 m2

[0268] Weight at the end of the cycle - 600 g

[0269] Weight 4 (Origin Variety) Container Area 0.124 m2

[0270] Weight at the end of the cycle - 620 grams

[0271] The results reflect a growth rate of about 3.5 tons per year per dunam (300 days) of dry matter of the original variety and about 4.5 tons per year per dunam (300 days) of the new variety( 10,000- 20,000 lux , water temperature 25-28 °C).

[0272] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0273] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of selecting a Lemna minor variety, the method comprising:(a) providing a Lemna minor species having a protein content of about 28 % and a frond size of up to 5 mm when grown in tap water;(b) culturing said Lemna minor species under conditions which improve frond size and protein content, wherein said conditions comprise magnesium sulfate, calcium carbonate, sodium nitrate and a fertilizer which comprises nitrogen, phosphorus and potassium;(c) determining said protein content and frond size prior to and following (b), wherein an improvement in said protein content and frond size is indicative of a novel Lemna minor variety.

2. The method of claim 1, wherein said determining is determined in the presence of said conditions and in absence of said conditions, and wherein improvement in said presence and in said absence is indicative of a stable line.

3. The method of any one of claims 1-2, wherein a concentration of nitrogen in said fertilizer is about 7 %.

4. The method of any one of claims 1-3, wherein a concentration of phosphorus in said fertilizer is about 3 %.

5. The method of any one of claims 1-3, wherein a concentration of potassium in said fertilizer is about 7 %.

6. The method of any one of claims 1-5, wherein a concentration of magnesium sulfate is about 0.5-2 mg / L.

7. The method of any one of claims 1-6, wherein a concentration of magnesium sulfate is about 1 mg / L.

8. The method of any one of claims 1-7, wherein a concentration of calcium carbonate is about 0.5-2 mg / L.

9. The method of any one of claims 1-8, wherein a concentration of calcium carbonate is about 1 mg / L.

10. The method of any one of claims 1-9, wherein a concentration sodium nitrate is 4-12 mg / L.

11. The method of any one of claims 1-10, wherein a concentration of sodium nitrate is 10 mg / L.

12. A Lemna minor variety obtainable according to the method of any one of claims 1-11.

13. A Lemna minor variety having a frond size of at least 10 mm and an essential amino acid content of at least about 50 % of total amino acids.

14. A Lemna minor variety characterized by at least one of:(i) a frond size bigger than about 7 mm;(i) protein content above about 34 %;(ii) a biomass of 45 tons / hectar / year of wet biomass;(iii) an essential amino acid content higher than that of a Lemna minor variety having a protein content of about 28-30 % (the original variety), wherein all of (i)-(iv) are at tap water conditions without additions of nutrients.

15. A method of producing a Lemna minor biomass or an extract or fraction thereof, the method comprising growing the Lemna minor variety of any one of claims 12-14 under conditions which support its growth and optionally modify its content in culture.

16. The method of claim 15, further comprising isolating the biomass from said culture.

17. The method of any one of claims 15-16 further comprising fractionating a fraction of interest from said biomass.

18. The method of any one of claims 15-17 further comprising extracting an extract of interest from said biomass or said fraction thereof.

19. The method of any one of claims 15-18, wherein said conditions comprise magnesium sulfate, calcium carbonate, sodium nitrate and a fertilizer which comprises nitrogen, phosphorus and potassium.

20. The method of any one of claims 15-19, wherein a concentration of nitrogen in said fertilizer is about 7 %.

21. The method of any one of claims 15-20, wherein a concentration of phosphorus in said fertilizer is about 3 %.

22. The method of any one of claims 15-21, wherein a concentration of potassium in said fertilizer is about 7 %.

23. The method of any one of claims 19-22, wherein a concentration of magnesium sulfate is about 0.5-2 mg / L.

24. The method of any one of claims 19-23, wherein a concentration of magnesium sulfate is about 1 mg / L.

25. The method of any one of claims 19-24, wherein a concentration of calcium carbonate is about 4-12 mg / L.

26. The method of any one of claims 19-25, wherein a concentration of calcium carbonate is about 10 mg / L.

27. The method of any one of claims 19-26, wherein a concentration sodium nitrate is about 4-12 mg / L.

28. The method of any one of claims 19-27, wherein a concentration of sodium nitrate is about 10 mg / L.

29. The method of any one of claims 15-28, wherein said extract or fraction comprises protein.

30. The method of any one of claims 15-28, wherein said extract or fraction comprises carbohydrates.

31. The method of any one of claims 15-28, wherein said extract or fraction comprises lipids.

32. The method of any one of claims 15-31, wherein said conditions further comprise an amount of cobalt nitrate effective at increasing B12 content.

33. The method of claim 32, wherein said amount of cobalt nitrate is about 0.5-2 mg / L.

34. The method of any one of claims 15-33, wherein said conditions further comprise iron (Fe+2).

35. The method of claim 34, wherein an amount of said iron (Fe+2) is about 0.005-0.015 gr / L.

36. The method of claim 34, wherein an amount of said iron (Fe+2) is about 0.01 gr / L.

37. An article of manufacture comprising the Lemna minor biomass of the Lemna minor variety of any one of claims 12-14 or an extract or fraction thereof.

38. The article of manufacture of claim 37 being selected from the group consisting of a human food, a protein-rich powder, Lemna-based protein isolate, Lemna oil, Lemna flour, fermented Lemna, animal feed, bioplastics, biofuels, fertilizers, nutraceuticals, and a bioremediation filter.

39. The article of manufacture of claim 37 or 38 comprising genomic DNA of the Lemna minor variety.

40. The article of manufacture of any one of claims 37-39, comprising an amino acid profile as in Table 2.

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