• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Disclosed herein are aquafeed, animal feed and fertilizer compositions comprising microalgae enriched with minerals and a method of enriching microalgae with minerals in non -metabolized form. Specifically, the method includes the creation of an enriched microalgae product through the assimilation, reversible chelation, and absorption of supplemental minerals required in the diet of adult fish and other aquatic animals which minimizes leaching of the supplemental minerals before ingestion by the fish. Additionally, the enriched microalgae product can be used as both a direct feed or fertilizer, or as part of an aquafeed, non-aquatic animal feed, or plant fertilizer mixture. The combination and proportion of the minerals can be adjusted to the animal or plant receiving the mineral enriched algae composition.
    公开号:AU2013221643A1
    申请号:U2013221643
    申请日:2013-02-13
    公开日:2014-08-28
    发明作者:Eneko GANUZA
    申请人:Heliae Develoopment LLC；
    IPC主号:C12N1-12
    专利说明:
    WO 2013/123032 PCT/US2013/025914 MICROALGAE ENRICHED WITH TRACE MINERALS CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/598,235, filed February 13, 2012 and U.S. Provisional Patent Application No. 61/601,970, filed February 22. 2012, and incorporates the disclosure of each application by reference. To the extent that the present disclosure conflicts with any referenced application, however, the present disclosure is to be given priority. BACKGROTUTND [0002] Aquaculture is the fastest growing alnnal-food-producing sector, with an average annual growth rate of 6.6% from 1970 to 2008 in per capita supply of food fish from aquaculture for human consumption according to the Food and Agriculture Organization of the United Nations (FAO) statistics. Due to this growth. the FAO reports that aquaculture now accounts for approximately 46% of the total food fish supply, which equates to over 50 million tons of fish. Decreasing the dependence of the aquafeed industry on fisheries may help aquaculture sustain this level of growth. Specifically, vegetable sources such as soybean, linseed, canola. etc., have been
    I
    WO 2013/123032 PCT/US2013/025914 proposed to replace a portion of the fish oil and fishmeal currently used in fish feeding formulations. [0003] Fish diets may comprise a combination of proteins, lipids, amino acids, vitamins, and trace minerals. Trace minerals for fish may comprise elements such as: Arsenic, Chroiun Cobalt, Copper. Fluorine, Iron, Iodine., Lead, Lithium, Manganese, Molybdenum, Nickel, Selenium. Silicon, Vanadium, and Zinc. The ranges for trace minerals specific for fish diets may include (in mg mineral per kg dry diet): 30-170 mg/kg Iron, 1-5 mg'kg Copper, 2-20 mg/kg Manganese 15-40 mg/kg Zinc. 0.05-1.0 img/kg Cobalt, 0.15-0.5 mg/kg Selenium, and 1-4 mg/kg Iodine. Although fish may uptake some amounts of these minerals from the water through their gills, receiving the mrinerals in their diet via the digestive system may be a more efficient method of mineral delivery. [0004] Vegetable sources may provide many of the essential lipids and amino acids present in fish meal, however one drawback with vegetable sources has been mineral deficiencies. The replacement of fish meal by vegetable sources requires an extra supplementation of minerals such as Selenium, Manganese, Zinc, Iron, Copper and Chromium three complexes. Minerals may be supplemented in an aquafeed diet as water-soluble inorganic salts, but the disadvantage of this method may be the leaching of a large portion of the minerals before being ingested by the fish. The loss of minerals in the water before ingestion by the fish may result in costs associated with wasted minerals and inefficient delivery of nutrients to the fish. [0005] A more efficient delivery of minerals to fish may occur when the minerals are in a bioavailable state. The bioavailability of the trace minerals may be subject to a munber of factors, including: the concentration of the nutrient, the form of the WO 2013/123032 PCT/US2013/025914 nutrient, the particle size of the diet, the digestibility of the diet, the nutrient interactions which may be either synergistic or antagonistic, the physiological and pathological conditions of the fish, waterborne mineral concentration, and/or the species under consideration. In an effort to ensure sufficient bioavailability of the required amount of minerals, fish feeds may be enriched with trace elements at higher concentration than needed by the fish due to the limited information on leaching and bioavailability. [0006] Adding trace elements at higher than needed concentrations may introduce a number of potential complications. One such potential complication may be that the high concentration of minerals has the potential to interact with fatty acid oxidative processes in the fish diet through the formation of hydroperoxides. In addition, minerals leaching from the aquafeed may have the potential to negatively impact the environment. The excessive leaching of minerals may stimulate phytoplankton production and increase oxygen demand. Leached minerals may also have the potential to stimulate the development of macroalgal beds and influence the benthonic ecosystem. Accordingly, a plurality of unintended consequences may be produced by leaching and high concentrations of minerals may harn the fish and aquatic envirominent. SUMMARY [0007] Disclosed herein are aquafeed, animal feed and fertilizer compositions comprising microalgae enriched with minerals and a method of enriching microalgae with minerals in non-metabolized form. Specifically, the method includes the creation of an enriched microalgae product through the assimilation, reversible chelation, and absorption of supplemental minerals required in the diet of adult fish and other aquatic -3 WO 2013/123032 PCT/US2013/025914 annals which minimizes leaching of the supplemental minerals before ingestion by the fish. Additionally, the enriched microalgae product can be used as both a direct feed or fertilizer, or as part of an aquafeed, non-aquatic animal feed, or plant fertilizer mixture. The combination and proportion of the minerals can be adjusted to the anmial or plant receiving the mineral enriched algae composition DETAILED DESCRIPTION [0008] The present invention may be described in terms of functional block components and various processing steps, Such functional blocks may be realized by any number of components configured to perform the specified functions and achieve the various results. For example, the present invention may employ various process steps, apparatus, systems, methods, etc. In addition, the present invention may be practiced in conjunction with any number of systems and methods for providing microalgae as a vegetable source for aquafeed, and the system described is merely one exemplary application for the invention. Various representative implementations of the present invention may be applied to any type of live aquaculture. Certain representative implementations may include, for example, providing the microalgae preparation to the aquaculture to at least partially meet the nutritional needs of the aquaculture. [0009] The particular implementations described are illustrative of the invention and its best mode and are not intended to otherwise limit the scope of the present invention in any way. For the sake of brevity, conventional manufacturing, connection, preparation, and other functional aspects of the system may not be described in detail. Many altermnative or additional functional relationships or physical connections may be present in a practical system. 4 WO 2013/123032 PCT/US2013/025914 [0010] Various embodiments of the invention may provide methods, apparatus. and systems for providing an aquafeed vegetable source comprising microalgae that may be grown quickly and/or year round to ensure a readily available supply. In some embodiments, mcroalgae may provide an alternative vegetable source for aquafeed which may possess a beneficial amino acid profile and/or a high unsaturated fatty acid profile. Microalgae may also provide a bio-absorption capacity. For example, microalgae may absorb, chelate and/or assimilate trace minerals from a medium, even at very low concentrations. The ability to absorb, chelate, and assimilate minerals from a medium may be due to several characteristics of mnicroalgae such as, but not limited to, a large surface to volume ratio, the presence of high-affinity metal binding groups on the microalgae cell surface, and/or efficient metal uptake and storage systems. These mineral uptake characteristics of microalgae may provide a potential advantage over other vegetable sources for fish feed. [0011] Microalgae, such as Nannochloropsis, Chlorela or Scenecdesmus; may be a rich source of minerals. fatty acids of chain length ClO-C24, and proteins, which may provide a nutritious and natural source for feeding fish, For example, every 100g of Nannochloropsis contains 972 ing Calcium (Ca), 533 mg Potassium (K), 659 mug Sodium (Na), 316 mg Magnesium (Mg), 103 nmg Zinc (Zn), 136 mg Iron (Fe) 3A mg Manganese (Mn 35.0 mg Copper (Cu). 0.22 mug Nickel (Ni), and <0.1 mug Cobalt (Co). Additionally, Nannocidoropsis may possess an essential fatty acid and amino acid profile having nutritional value. Together, the growth characteristics and nutritional composition may make microalgae a leading vegetable source alternative for aquafeed.
    WO 2013/123032 PCT/US2013/025914 [0012] In addition to the growtli characteristics and nutritional composition, microalgae may have other characteristics associated with their nanoparticle properties that provide unique benefits as an aquafeed over other vegetable sources. The large surface to volume ratio and the presence of high affinity, metal binding groups confers the microalgae the ability to adsorb trace minerals from a inedium. The microalgae cells may sequestrate soluble ions from water and concentrate them at their specific requirements. For example, Chlorella and Scenedesnms may absorb Zn2+ and Cr6+ and concentrate them above 02 % of dry weight. This ability of microalgae to bind metals and minerals may be subject to multiple variables such as, but not limited to. the pH of the water medium, the temperature of the water medium, the concentration of the minerals, the mass of the microalgae, and the time allowed for the metal to bind to the surface of the microalgae. In some embodiments, these variables may be adjusted as parameters in a method of making an aquafeed to produce an aquafeed of a desired composition, for a desired purpose, or at a desired cost. [0013] Also, in some embodiments, the microalgae cells may be able to prevent toxicity at high concentrations by preventing the indiscriminate entry of the minerals into the microalgae cell. Minerals may reversibly chelate to the microalgae cell wall or the extra-celllar polymers before they interact with the cellular metabolism. Mineral chelation may refer to a mineral that is bound to amino acids or proteins. Mineral chelation may comprise the metabolization of the mineral by the microalgae into its organic configuration. Unlike the reversible chelation that occurs in the cell wall of the microalgae, the chelated minerals vill remain bound to the organic molecule during pH change conditions, such as digestion. 6 WO 2013/123032 PCT/US2013/025914 [0014] This chelating process provides more stability to metal ions and reduces the ability of the ions to leach or form soluble precipitates. Following the trace metal absorption, the microalgae cell can uptake the minerals and for peptide complexes., commercially known as "chelated minerals", that may increase the tolerance to high ion concentrations. Alternatively, the mieroalgae cell can reverse the chelation reaction and release the minerals, for example when the pH of the suspension decreases. The reversible properties of the mineral chelation in microalgae provide a clear benefit to its application to the aquafeed industry, for instance the minerals can be released into the fish digestive track in response to a change in the pH. The acid digestion of the fish will release the minerals chelated to the microalgae cell wall, therefore avoiding any unwanted leaching of the essential nutrients in the water before digestion by the fish. Therefore, the minerals will be delivered in the appropriate place and timing to maximize the efficiency of the fish feeding process. [0015] The minerals may be absorbed, reversibly chelated or assimilated by the microalgae through a variety of mechanisms. Examples of such mnechanisms comprise two active absorbing substances in a Chlorella cell wall: the cellulose inicrofibrils and the sporopollenin. Additionally, the imucopolysaccharides covering the cell wall possesses a similar mechanism to the ion exchange resigns that are used to reversibly chelate heavy metals in industrial wastewater treatment. Also, the microalgae can uptake and assimilate the minerals in their organic forns known as "chelated minerals", which further enhances the digestibility of the mmerals by the fish, as opposed to the inorganic form of the minerals most conmionly used in aquafeeds. By binding the supplemental minerals through chelating, assimilating and absorbing, the enriched microalgae are acting as a carrier or vehicle for supplying the minerals to adult fish, WO 2013/123032 PCT/US2013/025914 which is distinigushable from adding trace minerals to a culture of microalgae for the nucroalgae to metabolize. The metabolized minerals provide nutrition to the microalgae cell for growth. whereas the bound minerals provide nutrition directly to the adult fish. [0016] The supplemental minerals may comprise any suitable mineral that may provide nutrition to the microalgae cell and/or an aquatic animal and may come from a variety of sources, including purchased concentrations of the minerals. For example, the supplemental minerals may comprise various sources of boron, bromine, calcium. chloride, clonun, cobalt, copper, fluone, iodine, iron, lithium, niagnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, sodium, silicon, sulphur, vanadium, and/or zinc. The calcium sources may comprise: calcium carbonate (CaCO 3 ); monocalciumn phosphate, monohydrte (Ca(H 2 P04) 2
    .H
    2 0); dicalcium phosphate, anhydrous (CaHP04); dicalciuni phosphate, dihydrate (CaHPO 4 .2H 2 O); tricalcium phosphate (Ca 3(PO4)2); calcium sulphate (CaSO4); bonenmeal; oystershell grit; and ground limestone (CaCO3). The chloride sources may comprise: sodium chloride (NaCl) and potassium chloride (KCI). The chromium sources may comprise: chromium (III) chloride (CrCI-); chromium (I1) chloride, hexahydrate (CrCI' 6H2O); and chromium picolinate (Cr(C 6
    H
    4 NO2)3). The cobalt sources may comprise: cobalt chloride, pentahydrate (CoCI 2 .5H 2 O); cobalt chloride, hexahydrate (CoC126H2O); and cobalt sulphate, monohydrate (CoSO 4 .H2O). The copper sources may comprise: copper sulphate (CuSO4); copper supiate, pentahydrate (CuSO 4 .5H2O); copper chloride (CuCl 2 ); copper (I) oxide (Cu0): and copper (II) hydroxide (Cu(OH) 2 ). The iodine sources may comprise: potassium iodide (KI); potassium iodate (KO1); calcium iodate (Ca(10 3 )2 sodium iodide (Na); and 8 WO 2013/123032 PCT/US2013/025914 ethylenediamine dihydriodide (C2H.N -2HI). The iron sources may comprise: ferrous sulphate, heptahydrate (F'eSO 4 .7H2O); ferrous (H) carbonate (FeCO 3 ); and ferrous oxide (FeO). The magnesium sources may comprise: magnesium chloride (MgCle-.6H 2 O); magnesium oxide (MgO); magnesium carbonate (MgCO); dimagnesium phosphate, trihydrate (MgHPO 4 3H 2 O); magnesium sulphate (MgSO4); and magnesium sulphate, heptahydrate (MgSO 4 7H 2 0). The manganese sources may comprise: manganese oxide (Mn); manganese dioxide (MnO2); manganese carbonate (MnCO3); manganese chloride, tetrahydrate (MnCI2.4H 2 0); manganese sulphate (MnSO 4 ); manganese sulphate, hydrate (MnSO 4 H2; and manganese sulphate. tetrahydrate (MnSO 4 4H 2 0). The molybdemun somuces may comprise: sodium molybdate, dihydrate (Na-2MoO42H 2 0) and sodium molybdate, pentahydrate (NaMO 4 .5H 2 0). The phosphorus sources may comprise: monocalcium phosphate, monohydrate (Ca(H 2 PO4) 2
    .H
    2 0); dicalcium phosphate, anhous (CaHPO4); dicalciumi phosphate, dihydrate (CaHPO 4 .2H20); tricalcium phosphate (Ca 3
    (PO
    4 )2); potassium orthophosphate (K 2
    HPO
    4 ); potassium dihydrogen orthophosphate
    (KH
    2
    PO
    4 ); sodium hydrogen orthophosphate (Na 2 HPO4); sodium dihydrogen orthophosphate, hydrate (NaH 3
    PO
    4
    .H
    2 0); sodi umn dihydrogen orthosphosphate, dihydrate (NaH 3
    PO
    4 .2H 2 O) dimagnesium phosphate, trihydrate (MgHPO 4 .3H,0); and rock phosphate ((Ca(PO 4 )2)3CaF2). The potassium sources may comprise: potassium chloride (KCL); potassium carbonate (K2CO 3 ); potassium bicarbonate (KHCO); potassium acetate (KC 2
    H
    3 02); potassium orthophosphate (K3PO 4 ): and potassium sulphate (K 2 SO4). The selenium sources may comprise: sodium selernite (Na2SeO) and sodium selenate (NaSeO4). The sodium sources may comprise: sodium chloride (NaCI); sodium bicarbonate (NaHCO 3 ); and sodium sulphate (Na 2
    SO
    4 ). The zinc 9 WO 2013/123032 PCT/US2013/025914 sources may comprise: zinc carbonate (ZnCOj): zinc chloride (ZnCLI zinc oxide (ZnO); zinc sulphate (ZnSoz); zinc sulphate. hydrate (ZnSO 4
    .H
    2 O); and zinc sulphate heptahydrate (ZnSO 4 .7H20). In some embodiments, the supplemental minerals are added to de-ionized water and then administered to the microalgae. [0017] Electrodes receiving electrical current in direct, alternating, pulsed or any other form from a power source are known to degrade over time and leach electrode material. Electrodes that are submerged in an aqueous medium will leach the electrode material into the aqueous medium. Applying an electric field to an aqueous culture of microalgae through electrodes submerged in the aqueous culture is also known in the art to cause flocculation among the microalgae by mechanisms such as, but not limited to, changing the surface charge of the microalgae cells to reduce electrostatic repulsion, and the leached electrode material acting as a flocculent or flocculating aid. In some embodiments, electrodes comprised of an electrode material of a desired mineral composition as described above, such as but not limited to copper, zinc, iron, and alloys thereof, are submerged in an aqueous culture comprising mnicroalgae. When current is applied to the electrodes, the electrode material degrades and leaches into the aqueous medium which supplies the supplemental minerals for uptake by the microalgae. The microalgae assinrilate, reversibly chelate, and absorb the leached electrode material to produce a microalgae product enriched with the desired iuineral composition in a non-metabolized forn. In further embodiments, the application of an electric field by the electrodes simultaneously causes flocculation of the nicroalgae which results in a flocculated mass of mineral enriched microalgae. [0018] In an exemplary embodiment of the present invention, a method of making the microalgae product enriched with non-metabolized minerals may comprise growing a 10 WO 2013/123032 PCT/US2013/025914 culture of nicroalgae in a culturing vessel containing an aqueous culture medium and at least one pair electrodes submerged in the aqueous culture medium. The at least one pair of electrodes may comprise in electrode material comprising a mineral specific to a nutritional profile of an animal. The electric current may be applied to the at least one pair of electrodes sufficient to cause the electrode material to leach into the aqueous culture medium. The microalgae may be incubated to facilitate the microalgae assimilating, reversibly chelating., and/or absorbing the electrode material to produce a inicroalgae product enriched with non-metabolized minerals specific to the profile of nutritional requirements of the animal. The microalgae product enriched with non-metabolized minerals may be harvested to separate the microalgae product from the aqueous culture medium. [0019] The enriched microalgae may be adnInistered to the fish in various forms. In some embodiments, the enriched microalgae comprise a suspension of microalgae in water. In some embodiments, the enriched microalgae comprise a paste or cake resulting from dewatermg the microalgae culture to a desired percent of solids. In some embodiments, the enriched microalgae comprises a dried free flowing powder or flakes for use as an ingredient in the dietary mixing and pelletizing. [0020] A method for making a microalgae product enriched with non-metabolized minerals, comprises the steps of: growing a culture of microalgae in an aqueous culture medium; harvesting the microalgae by separating the microalgae from the aqueous culture medium; adding supplemental minerals specific to a profile of nutritional requirements for an animal to the microalgae; incubating the nmicroalgae and the supplemental minerals to facilitate the microalgae assimilating, reversibly chelating, and absorbing the supplemental minerals to produce a microalgae product enriched with non
    II
    WO 2013/123032 PCT/US2013/025914 metabolized minerals specific to the profile of nutritional requirements of the animaL In one embodiment, the method may further comprise dewatering the microalgae product enriched with minerals to further reduce the water content of the microalgae product. In another embodiment, the method may further comprise stabilizig the microalgae product enriched with minerals. Additionally, in various embodiments of the present invention, the supplemental minerals may be added to the microalgae before or after the step of harvesting the microalgae. [0021] In some embodiments of the present invention, the mineral supplement composition for an aquatic animal may comprise the microalgae product enriched with at least one mineral from the group consisting of arsenic, chroimniu, cobalt, copper, fluorine, iron, iodine, lead, lithium, manganese, molybdenum, nickel, selenium, silicon, vanadium, zinc in a non-metabolized form. [0022] In another embodiment of the present invention, the nmieral supplement composition for a non-aquatic animal may comprise a microalgae product enriched with at least one mineral from the group consisting of boron, bromine, calcium, chloride, chromium, cobalt, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, sodium. sulphur, vanadium and zinc in a non metabolized form. [0023] In various embodiments of the present invention, the microalgae product may comprise any suitable species of algae and/or microalgae for providing nutrition to an animal. For example, the microalgae product may comprise microalgae that are members of one of the following divisions: Chlorophyta, Cyanophyta (Cyanobacteria), and Heterokontophyta. In some embodiments, the nmicroalgae product may comprise microalgae of the following classes: Bacillariophyceae, Eustigmatophyceae, and 12 WO 2013/123032 PCT/US2013/025914 Chrysophyce-ae. In certain embodiments, the nmicroalgae product may comprise microalgae that are members of one of the following genera: Nannochloropsis; Chlorela. Dunaliella, Scenedesm us, Selenastrum, Oscillatoria, Phormidimn Spirulina, Amphora, and Ochromonas. [0024] In various embodiments of the present invention, the microalgae product may comprise saltwater algal cells such as, but not limited to, marine and brackish algal species. Non-limiting examples of saIltwater algal species include Nannochloropsis species and Duna1/ella species. Saltwater algal cells may be found ini nature in bodies of water such as. but not limited to, seas. oceans, and estuaries. Further, in some embodiments, the nicroalgae product may comprise freshwater microalgal cells such as, but not limited to Scenedesmus species and Haematococcus species. Freshwater microalgal cells may be fond in nature in bodies of water such as, but not limited to, lakes and ponds. [0025] In various embodiments of the present invention, the microalgae product may comprise one or more microalgae species such as, but not limited to: Achnanthes orientalis, Agmenellum .spp., Amphiprora hvaine, Amphora coffetforins., A-mphora coffeiformis var. linea, Amphora coffetformis var. punctata, Amphora cofkeioris var. talori, Amphora cofoi/brns var. tennis Amphora delicatissima, Amphora delica tissima var. capitata, Amphora sp. Anabaena. Ankistrodesmus Ankistrodesmus flcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus branni, Botyococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros graciis, Chaetoceros muelleri, Chaetoceros nmelleri var. subsalsum. Chaetoceros sp., Chlaivdmonas perianuata, Cl/orella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate., Chlorella 13 WO 2013/123032 PCT/US2013/025914 desiccate (h/orella el/ipsoidea, Chlorella emersonii., C/orela fiusca, Cilorella fisca var. vacuolate, Chlorela glucotropha, Chlorella infuslionum, Chlorela infusionun var. actophila, Chlorela infusionum var. auxenophi/a, dChlorelia kessleri, Chlorella lobaphora, Chblorella luteoviridis, Chlorella luteo viridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella mnniata., Chlorella inutissima, Chlorella mutabilis, Chlorela nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorela pringsheimii, Chlorela protothecoides, Chlorella protothecoides var acidicola, Cblorella regularis, Chlorella regulars var. minima, Chlorella regulars var. ubricata, Chlorella reis/gi, Chlorella saccharophila, Chlorela saccharophila var. e/lipsoidea, Chlorella salina, Chlorella simplex Chlorela sorokanian.,' Chlorella sp, Chlorella sphaerica, Chlorella stigmatophora, Cborella vannielli, ('h/arella vu/garis, 'Chlorella vulgaris fo. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. virid/is, Chlorella vwlgaris var, vu/garis, Chlorella vulgaris var. vulgarisjb. tertia, Ch/orella vulgaris var. vulgaris fo. v/vdis, ('h/ore//a xandhe//a, G/ore//a ::opngiensis, ('horella trebouxioides, Chlorella vulgaris, Chlorococcum intfisionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chryssphaera sp, Cricosphaera sp, Crypthecodinium sp., Crypthecodinium cohni Cryptomanas sp., Cycatella cvptica, Cvlatella menegbiniana, Cyclatella sp., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunalella maritime, Dunaliella in/auta, Dunaliella parva, Danaliella peircei, Dunaliella prim olecta, Dunaliel/a salina, Dunaliela terricola, Dunaliella tertiolecta Dunaliella viridis, Dunaiela tertiolecto., Eremiosphaera viridis, Eremosphaera sp. El/ipsoidon sp., Eag/ena spp, Franceia sp., Fragilaria cratonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus p/uvialis, Hvrmenomonas sp.' 14 WO 2013/123032 PCT/US2013/025914 /sochirrsis aoff galbana, tsochtysis galbana, Lepocinclis, Micraciinium, Micractium, Afonorapjhidin minutumfn, Afonoraphidium sp., ANmochloris sp., Nannochloropsis salina, Ntmoch/oropsis ~spavicula ac eptata, Navicla bikanterae, Navicuin peudoteneloides Avicula peticulosfa, Navicula sap rop/hila, Navicula sp., Nephrochloris sp, Nephroselnis sp., Nischia conunmis, Nitzschia alexandrine, NMitt:schia closterhun, Aitschia comuninis, A
    T
    itzschia dissipate, Nitzschia ifustulim, Nitzschia hantzschiain, NItschia inconspicua, Nitzschia intermedia, Nitzschi/a nicrocephala, Nitzschia pusila, Nitzschia pusil/a el/ipt/ca, Nitzschia pusilla Itnonoensis. NI/tzschia quadrangular, Nitzschia sp., Ochronionas sp., Ocyestis parva, Oocysis pusilla, Oocvstis sp,, Oscillatoria innetica, Oscillatoria sp. Oscillatoria subbraevis, Parach/ore/la kesleri, Pascheria acidophila, Pavova sp., Phaeodacty/um triconmtu, Phagus, Phornidumn, PIatvmonas sp., Pleurochrysis camterae, Pleurochysis dentate, Pleurochr-isis sp., Prototheca wickerhamii, Prototheca siagnora, Prototheca portoricensis. Prototheca noriforin/s, Prototheca zopfli, Pseudochlorella aquatica, Pyraminnonas sp., Pyrobrsotrs Rhodococcus opacus, Sarcinoid chtysophyte, Scenedesums arminatus, Schizochvtriun Spiro gyra Spirulina platensis, Stichococcus coccuss sp, Synechocstisf Tagetes erecta, Tageits patua, Tetraedron, Tetrasebmis sp., Tetirasebnis suecica, Tha lassiosirn weisspfogii, and Viridiela frideric/ano. [0026] In various embodiments, the microalgae may be grown in any type of culturing vessel such as, but not limited to, a pond, a raceway pond, a trough, a V-trough, a tank, or a photobioreactor able to contain an aqueous medium. hi some embodiments, the iicroalgae may be grown phototrophically. In some embodinents, the microalgae 15 WO 2013/123032 PCT/US2013/025914 may be grown mixotrophically. In some embodiments, the mnicroalgae may be grown heterotrophically. [0027] During the harvesting step, the microalgae may be separated from the aqueous culture medium. The microalgae may be harvested using ainy method known in the art such as, but not limited to, separation by an adsorptive bubble separation device, centrifuige. dissolved air flotation (DAB), and settling. During the dew'atering step, the harvested microalgae have additional water removed to decrease the water content and increase the solids content of the microalgae product. In some enibodiments, dewatering may comprise the removal of at least some water from the microalgae. The microalgae may be dewatered using any method known in the art such as, but not limited to, electrodewatering, filtration, centrifugation, adsorptive bubble separation, and pressing. In some methods microalgae, harvested or dewatered microalgae product may be dried by methods known in the art. [0028] In some embodiments, the supplemental minerals added comprise at least one from the group comprising: calcian, chloride, chromium, cobalt, copper, iodine, iron, magnesim, molybdenum, phosphorus, potassium, selenium, sodium, and zinc. hi some embodiments, the supplemental minerals are added to tie microalgae within the culturing vessel, before the microalgae are harvested. In further embodiments, the supplemental minerals are added to the cultuing vessel at a specific time when the incroalgae are in a specified condition or state such as, but not limited to, growth phase, a period of environmental stress, and oil phase. In some enibodiments, the supplemental minerals are added to the microalgae culture after the microalgae have been harvested. In further embodiments, the supplemental minerals are added to the harvested nmicroalgae culture at a specific time duration after the microalgae have been 16 WO 2013/123032 PCT/US2013/025914 harvested. In some embodiments, the supplemental minerals are added to the nuemralgae and incubated for a detennined period of time, at a determined temperature, and at a determined pH. [0029] The timing at which the supplemental minerals are added awd the duration of the incubation period con-esponds to the amount of minerals that are metabolized by the microalgae. When the supplemental minerals are added to the microalgae after harvest from the growing vessel and are no longer in growth phase, the microalgae have less time to metabolize the minerals which results in more binding of the minerals to the nilcroalgae cell walls. The amount of minerals that are bound and reversibly chelated, instead of metabolized may be also related to the proportion or concentration at which the supplemental minerals are added to the microalgae, and other factors such as temperature and pH. [0030] In some embodiments, the supplemental minerals may be added at a specific concentration. In some embodiments, the supplemental minerals may be added in a specific proportion to the amount of microalgal biomass. In some embodiments, the supplemental minerals may be added to the microalgae culture when the nicroalgae culture is at a specific pH. In some embodiments, the supplemental minerals may be added to a specific mass of microalgae. In some embodiments, the supplemental minerals may be given a specific time duration in which to bind to the microalgae by assimilation, reversible chelation, and/or absorption. Reversible chelation may refer to the ionic binding of minerals to a cell wall and/or exo-polysaccharides of the microalgae. Reversible chelation may not require metabolization of the mineral and may be based on an ion exchange process. The process may be reversible and 17 WO 2013/123032 PCT/US2013/025914 therefore may allow the mineral ion to release in the conditions of a pH change, such as in a digestive track. [0031] In some embodiments, the supplemental minerals may be added after the inicroalgae culture is concentrated to a certain concentration of solids by dewatering or other known methods of concentrating a inicroalgae culture. In sonic embodimeits, the supplemental minerals may be added before the mricroalgae culture is concentrated to a certain concentration of solids by dewatering or other known methods of concentrating a microalgae culture. Iii some embodiments, the enriched nicroalgae may be dewatered to a specific concentration of solids. In further embodiments, the dewatered microalgae may comprise a wet solution. In further embodiments, the dewatered microalgae may comprise a microalgal paste. In various embodiments of the present invention, "algal paste" and/or "nicroalgal paste" may refer to a partially dewatered algal or microalgal culture having fluid properties that allow it to flow. Generally an algal or microalgal paste may have a water content of about 90%. [0032] In various enibodinients, the dewatered microalgae may comprise a microalgal cake. An "algal cake" and/or "microalgal cake" may refer to a partially dewatered algal or microalgal culture that lacks the fluid properties of an algal or microalgal paste and/or tends to chimp. Generally an algal or microalgal cake niay have a water content of about 60% or less. [0033] In one embodiment, the dewatered microalgae may comprise a free flowing powder. In some embodiments, the dexvatered microalgae may be stabilized by methods such as, but not limited to. driving, cooling, freeze dying and freezing. [0034] In one non-limiting example of the above method, the microalgae may be harvested from a growing vessel by centrifugation concentrating the microalgae at levels up to
    IS
    WO 2013/123032 PCT/US2013/025914 50-200 g Dry Weight (DW)/iter to produce a microalgal paste. The resulting harvested microalgal paste has supplemental minerals comprising one or more mineral salt (containing minerals such as Se, Fe, Mn, Zn, and Cu) added to the microalgal paste at a concentration less than 15-0.1 g/liter, and is then incubated in an enrichment medium for 15-120 minutes, The incubation is caied out at a temperature of 5-40 degrees C, a pH of 6-12, and orbital shaking at 25-150 rpm. For finther enrichment of the microalgae, the supplemental minerals comprising one or more mineral salts could be added to the culture medium 1-2 days before the microalgae are harvested. The incubated microalgal paste enriched with minerals is batch centrifuged, with the resulting supernatant being recycled back to the enrichment medium. The resulting enriched microalgal solids are stabilized by known methods such as, but not limited to, freezing, refrigerated storage, freeze drying, spray drying, or drum dying to produce an enriched microalgae product. [0035] In some embodiments, the method further comprises the step of feeding the enriched microalgae product directly to any suitable aquatic animal such as an adult fish. The incroalgae product may also be directly fed to other aquatic animals such as, for example, oysters, mollusks, scallops, and/or shrimp. In some embodiments, the method further comprises the step of mixing the enriched microalgae product in an aquafeed comprising additional ingredients. In some embodiments, the method father comprises mixing the microalgae in an aquafeed to comprise a specific percent of the aquafeed. In some embodients, the additional aquafeed ingredients comprise fishimeal or fish oil. [0036] With the above method, which adds supplemental minerals directly to natural incroalgae and feeds the enriched microalgae product directly to adult fish/aquatic 19 WO 2013/123032 PCT/US2013/025914 animals or usin2 the enriched microalgae product directly in an aquafeed mixture, the prior art steps of encapsulating a preparation, feeding the inicroalgae to another live feed (e.g. rotifers) before consuption by fish, and genetic modification of the ucroalgae are not required. Eliminating these steps increases the efficiency of the process of maldig an aquafeed, and reduces the costs associated with the time and resources required to: encapsulate a preparation grow, maintain, and handle an additional live feed; and genetically modify a uicroalgal strain. [0037] The method described above produces an enriched microalgae product for use as an aquafeed for aquatic animals (e.g. adult fish, oysters. mollusks. scallops, and sluhip). The various parameters of the method may be adjusted to produce an aquafeed of a desired composition and Mineral profile matching the nutritional requirements of a specific fish or aquatic animal. [0038] In some embodiments, the resulting enriched microalgae product may be combined in an aquafeed composition comprising a percentage of the enriched mlicroalgae. The level of inclusion in the aquafeed depends on the fish nutritional requirements for the mineral(s) of interest and a mineral's bioaccumulation capacity with the species of nucroalgae used in the above described method. In some embodiments, the enriched microalgae product comprises less than 1% of an aquafeed for adult fish. In some embodiments the enriched microalgae product comprises about 1% of an aquafeed for adult fish. For example, in one embodiment, the microalgae product may comprise less than 1% of microalgae enriched with a profile of assimilated, reversibly chelated, and absorbed minerals specific to the nutritional requirements of an adult fish; and a remainder comprising at least one or more other ingredients from the group consisting of fishmeal and fish oil. In another embodiment, the microalgae product may be an 20 WO 2013/123032 PCT/US2013/025914 annal feed product comprising at least 0.1% to about 1-5% of microalgae enriched with a profile of assimilated, reversibly chelated, and/or absorbed minerals specific to the nutritional requirements of the animal. [0039] While the percent inclusion of enriched microalgae for the application of providing supplemental minerals in an aquafeed is small, such as at least 0.1%, and in some embodiments about 1% or less, using the enriched microalgae in a different application in an aquafeed will change the percent of inclusion. In some embodiments, the uicroalgae are used in dietary applications such as, but not limited to, probiotics, protein supplementation, amino acid supplementation, fatty acid supplementation, vitamin supplementation, and carbohydrate supplementation; and comprise about 5% or less of an aquafeed. In further embodiments, the enriched imcroalgae comprise about 5-10% h of an aquafeed. In some embodiments, the enriched microalgae are used to entirely replace fishneal and comprise about 50-80% ' of an aquafeed. In further embodiments, the enriched algae comprise about 70% of an aquafeed. [0040] Several experiments were perfonned to illustrate various exemplary embodiments of methods for enriching microalgae with essential minerals and/or to illustrate the concept of mineral delivery using microalgae as a vehicle. [0041] Example I [0042]
    T
    annochloropsis is cultured following standard procedures known in the art and harvested directly from an aqueous culture medium by centriffigation, without the use of any flocculants. The dry weight of the harvested microalgae is adjusted to 50 g/liter through the addition of a salt water medium. Using the harvested microalgae, an experiment is mn in trip icate using a 250 nil shake flask with 100 ml running volume. 21 WO 2013/123032 PCT/US2013/025914 Salt comprising trace minerals (Zn, Se, Mn, Cu or Fe) is added to the harvested nucroalgae at around Igliter, depending on the type of inorganic mineral. The flasks are incubated at 50 g CDW (cell dry weight) Aannochioropsis microalgae per liter, a temperature of 30 degrees C, a pH of 8, and 120 rpm for time periods of 0, 15, 30, 60, 120 and 180 minutes. The initial pH of the medium is set with NaOH (IM) and H2SO4 (IM). At each sampling point (0, 15, 30, 60, 120, and 180 minutes) a 20 ml sample is taken and centrifuged (3000 g, 30 degrees C, 5 minutes), with the time 0 minutes sample being taken right before the addition of the salt comprising trace minerals. The resulting pellet is freeze dried and the supernatant is frozen. The mineral analysis of the resulting pellet and supernatant is made by atomic adsorption spectrophotometry (AOAC 0968.008 and AOAC 0996.16). The results show the mineral content of the Nannochloropsis samples achieved with the different incubation time periods, which enables a determination of the optional incubation time period for enriching Nannochloropsis with the identified minerals. [0043] Example 2 [0044] Nannochloropsis is enriched according to the method developed in Example 1 and stabilized by freeze drying. Using the enriched nicroalgae, an experiment is run where the freeze dried microalgae biomass is re-suspended in fresh water to achieve 50 g CDWliter. The suspension is mixed with a kitchen blender for 2 minutes to ensure the release of the single microalgae cells to the medium. 100 ml of the suspension is poured into a 250 ml Erlenmneyer flask and placed in an orbital incubator at a temperature of 30 degrees C and 180 rpm. The initial pH of the suspension is 8, and the pH is decreased in a stepwise manier to pH levels of 7, 6, 5, 4, 3 and 2. The decrease in pH is achieved through the addition of 1M solution of NaOH. After 5 22 WO 2013/123032 PCT/US2013/025914 minutes of incubation at each pH levet a 20ml sample of the suspension from each pH set point (8, 7, 6, 5, 4 3, and 2) is centrifuged (3000 g, 30 degrees C, 5 minutes). The resulting pellet and supernatant is freeze cied before performing mineral analysis with atomic adsorption spectrophotometry (AOAC 0968,08 and AOAC 0996.16). The results show the amount of minerals released at each pH level by the enriched microalgae, enabling a determination of the anoiunt of minerals that will be released by the enriched microalgae at pH levels achieved within the digestive systems of animals fed the enriched algae. [0045] Example 3 [0046] Vannochloropsis is enriched with a blend of minerals according to the method developed in Example 1. In this experiment, the blend of minerals added to the microalgae matches the ratios of the nutritional requirements of adult Atlantic salmon. After the incubation period, the microalgae biomass is centrifiged. The resulting pellet and supernatant are analyzed to determine the mineral profile of the microalgae following the chelation process. The mineral profile of the microalgae is then compared to the nutritional requirements of the Atlantic salmon to determile if the ratios of enriclnnent are preserved during the chelation process. Based on the results of the mineral analysis, the interaction between the minerals during the chelation process is determined. The experiment is then repeated with a blend of minerals adjusted to account for the interactions between the minerals during the chelation process to achieve the nutritional requirements of adult salmon. [0047] Example 4 [0048] The mineral enriched microalgal bionass produced according to Example 1 and Example 3 is used to manufacture commercial Atlantic Sahnon pellets according to 23 WO 2013/123032 PCT/US2013/025914 commercial extrusion process. The diets contain 0.5 % microalgae biomass in dry weight to which the minerals are attached. The microalgae ingredient utilizing dietary enrichlent with inorganic mineral salts is used to produce the diet of reference. This diet includes 0.2 % of mineral salts on their composition, at least ten tines more minerals than the microalgae based diets. [0049] The experimental diets were fed to Jivenile Atlantic salons for three consecutive months in triplicate tanks. The fish grew in 1000 liter tanks with a stocking density of 4 kg /m 3 and 12 h light photoperiod. The juveniles were fed "add lihilm" twice a day recirculation of 10 % volume /day . At the beginning, middle, and end of the experiment, each tank was sampled for standard length and body weight gain of the salmon. Blood and muscle samples were taken at the end of the experiment and mineral content on the muscle and blood samples were analyzed by atonlic adsorption spectrophotometry (AOAC 0968.08 and AOAC 0996.16). Sahnon feces, the pellets deposited in the pond, the fresh water, and spent water of the tank were collected to analvze the leaching of minerals into the water medium. [0050] Results showed a similar growth pattern and body mineral content between the mineral enrichment protocols used in the diet, demonstrating the capacity of microalgae to deliver minerals in a water body more efficiently. The experiment demonstrated that the extra minerals used to formulate the diet, containing inorganic mineral, were lost through the leaching into the water body and through the defecation. The process of utilizing microalgae as a mineral enrichment method proved to be more efficient with regards to the overall amount of minerals used and also in terms of maintaining the water quality. 24 WO 2013/123032 PCT/US2013/025914 [0051] While the various embodiments discussed above for the enriched microalgae may be a mnneral supplement in an aquafeed for adult fish, the enriched microalgae may also be a vehicle to provide a tailored mineral profile having a variety of applications. As described above, the inicroalgae may be enriched to produce a variety of nutritional profiles based on the types of minerals added, the concentration of minerals added, the species of microalgae uptaking the minerals, the timing of adding the minerals for uptake by the microalgae, and other factors which may affect the mineral, protein, anmino acid, fatty acid. vitamin, or carbohydrate profiles of the microalgae. Using the above method, the nutritional profile of the microalgae may be tailored for the nutritional requirements of any aquatic and or non-aquatic animals, and used in a nutritional feed for such animals. [0052] In some embodiments, the nutritional profile of the enriched nicroalgae may be customized for the nutritional requirements of non-aquatic animals such as livestock (e.g. cattle and other bovine, swine, chickens, turkeys, goats, bison, sheep, and water buffalo), equine (e.g. horse, donkeys, miles, and zebras), ungulates (e.g. horse, zebra., donkey, cattle/bison, rhinoceros, camel, hippopotamus, tapir, goat, pig, sheep, giraffe, okapi, moose, elk, deer, antelope, and gazelle), pets (e.g. dogs, cats, rabbits, and guinea pigs), poultry (e.g. chickens, turkeys, ducks, geese, and ostriches), game animals (e.g. pheasants and quails), exotic/zoo animals (e.g. non-hInuan primates) and other domesticated animals. The enriched nmicroalgae may be used i various forms (e.g. aqueous solution, paste, cake, powder, flakes, and pellets) within a feed for such animals. The animal feed may comprise a mixed product comprising a certain percent of enriched microalgae with the remainder comprising other ingredients. 25 WO 2013/123032 PCT/US2013/025914 [0053] The nutritional requirements for aquatic and non-aquatic animals, comprising protein, amino acids, fatty acids, vitamins, carbohydrates, macro minerals, and trace mineral requirements may be obtained from a variety of published sources. Such publications and sources of publications on animal nutritional requirements include, but are not limited to, the Merck Veterinary Manual; reports published by the National Research Council (NRC) of the National Academies and papers, conference presentations and webpages published by educational institutions, cooperatives, and extension systems (e.g. North Dakota State University, Alabama Cooperative Extension System, University of Tennessee, and Mississippi State University Extension Service). For example, according to the NRC report on the Nutritional Requirements of Dogs and Cats, the daily reconnended allowance of minerals for an adult dog weighing 33 pounds and consuming 1,000 calories per day comprises: 0.75 g Calcium, 0.75 g Phosphorus, 150 mug Magnesium, 100 ing Sodium, 1 g Potassium, 150 mg Chlorine. 7.5 mg Iron, 1.5 rmg Copper, 15 mg Zinc, 1.2 mg Manganese, 90 jig Selenium, and 220 pg Iodine. An example of the nutritional requirements for a gestating beef cow (in mg mineral per kg dry diet) provided by the NRC report on the Nutritional Requirements of Beef Cattle comprises: 0.10 mg/kg Cobalt, 10 mug/kg Copper, 0.50 mg/kg Iodine, 50 mg/kg Iron, 40 mgikg Manganese, 0.10 mg/kg Selenium, and 30 mg/kg Zinc. These published nutritional requirements can be used with the above described system to produce enriched microalgae products for feeding different animals. [0054] The above method may also be used to produce a fertilizer composition and/or phyto-nutrient product comprising the nicroalgae product enriched with minerals for the nutritional profiles of plants. In one embodiment, the fertilizer composition may 26 WO 2013/123032 PCT/US2013/025914 be configured to be a liquid, a dry flake., and/or a powder. The essential nutrients for plants may include primary nutrients, secondary nutrients, and micronutrients capable of being assimilated, reversibly chelated, and absorbed by microalgae. The primary nutrients that may be enriched into the nicroalgae product include Nitrogen (N). Phosphoms (P), and Potassium (K), The secondary nutrients that may be enriched into the microalgae product include Sulfur (S), Calcim (Ca), and Magnesimn (Mg). The micronutrients that may be enriched into the microalgae product include Zinc (Zn), Iron (Fe), Copper (Cu), Manganese (Mn), Boron (B), Molybdenum (Mo), and Chlorine (Cl) In various embodiments of the present invention, the mnicroalgae product may be enriched with the prUiary nutrients, secondary nutrients, and/or the micronutrients in a non-metabolized forn. In addition to the mineral delivery capability of microalgae, other nutrients such as lipids, amino acids and vitamins can be provided to plants, crops and/or soil by microalgae. In some embodiments, the enriched microalgae ray be used as a fertilizer or an ingredient of a fertilizer for plants, crops and/or soil. In further embodiments, the fertilizer is distributed to plants, crops and/or soil with water through irrigation systems such as, but not limited to, drip lines or spraying. Spraying applications may comprise spraying a solution directly on the plant leaves, plant stems, plant stalks, plant vines, the airspace inumediately proximate to the plant, and/or the ground immediately proximate to the plant. In further embodiments, the fertilizer may be distributed to plants, crops and/or soil in a dry flake or powder forn. Dry flake or powder applications may comprise shaking or sprinkling directly on the leaves, stalk or vine; shaking or sprinkling directly on the ground immediately proximate to the plant; and/or mixing the flakes or powder with the soil in which the plant is growing or will be planted. 2 7 WO 2013/123032 PCT/US2013/025914 [0055] In some enbodiments, the enriched microalgae transfer nutrients from the imicroalgae cell to the plant cells in the leaf system through cytoplasinc streaming. In some embodiments, the enriched microalgae transfer nutrients from the microalgae cell to the plant cells in the root system through cytoplasmic streaming. In father embodinients, the nutrients not transferred from the microalgae cell to the plant cells in the root system through cytoplasmic streaming are released into the soil. [0056] The amount of fertilizer or phyto-nutrient product to use and methods of applying fertilizer and phyto-nutrient products vary based on the condition of the soil, time of year, plant yield, and the type of plant growing in the soil. Recommendations are provided by government entities such as, but not limited to, state university extension systems (e.g. Washington State Extension Programs), local agriculture divisions (e. g. Governunent of Alberta Agriculture and Rural Development), and the Food and Agriculture Organization (FAO) of the United Nations. For example the Alberta Agriculture and Rural Development's recommendation for sufficient nutritional requirements of sprmg wheat in growth stage include: 2.0-3.0% N, 0.26-0.5% P, 1.5 3.0% K, 0.1-0.15% S, 0.1-0.2 % Ca, 0.1-0.15% Mg, 10-15 ppm Zn, 3.0-4.5 ppm Cu, 1 5-20 ppm Fe, 10-15 ppmn Mn, 3-5 ppm B and 0.0 1-0.02 ppm Mo in the whole plant prior to filling. The nmicroalgae mineral profile may also be customized for the nutritional requirements of house plants (e.g. ferns), flowers, agricultural crops (e.g., wheat, corn, grain sorghum, soybeans, canola, milo, barley, sugarcane, pumpkins, rice, cassava. tobacco, hay, potatoes, cotton, beets, strawberries), fruit trees and bushes (e.g., apple, orange, grapefruit, lemon, lime, raspberries, blackberries), nut trees and bushes (e.g. pecan, butterut, walnut, almond, chestnut), fruit vines (e.g. grapes, melons, kiwi), grasses, and residential landscaping plants. 28 WO 2013/123032 PCT/US2013/025914 [0057] One demonstration of the capability of enriched microalgae to deliver nutrients to plants may be provided by the use of enriched Chlorella vuLgaris. When additional Phosphorus is added to the culture medium, Chlorelia algaris is known to be able to assimilate and store between 1.7 and 3.5 times more Phosphors than the Chlore/la vugaris requires, The enriched Chlorella can be administered to plants as a fertilizer or as an ingredient of a fertilizer through a drip line or spray application and supply significant amounts of Phosphorus in a water soluble form, as well as numerous other proteins, amino acids, and micronutrients contained in the nicroalgae. [0058] In another embodiment, the mineral enriched microalgae may be combined in a solution with herbicides and pesticides that are applied to plants, crops, andlor the soil. The combination with herbicides and pesticides allows the nutrients to be supplied to the plants, crops, and/or soil i a single application with pest and weed control benefits. [0059] Several experiments are run to optimize the method of fertilizing plants with mineral enriched nmicroalgae, and to optiize the method of enriching mnicroalgae with the nutritional profile specific to a plant. [0060] Example 5 [0061] The goal of this experiment is to determine the vohune of mineral enriched microalgae fertilizer at which the plant stops uptaking nutrients and the minerals are lost to the soil. Chlorella is enriched with a blend of minerals, including Phosphorus, according to the methods disclosed above. In this experiment, the blend of minerals added to the microalgae matches the ratios of the nutritional requirements of a plant. After the incubation period, a fertilizer solution comprising enriched Chlore/la and water, with a determined concentration of solids (enriched microalgae), is applied to soil in a series WO 2013/123032 PCT/US2013/025914 of paired containers Each pair of containers comprises one container with the contents comprising soil only, and one container with the contents comprising soil and the plant. All other container inputs such as light. air etc., are identical for each container and held constant. Different volumes of the fertilizer solution are administered to each container pair through a drip lrigation system with each volume of fertilizer solution having the same solids concentration. Soil samples from each container are taken before the application of the fertilizer solution, and at time intervals of 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, and 24 hours after the application of the fertilizer solution. The soil samples are analyzed for their mineral composition. The mineral compositions of the soil samples are compared to determine the vohune of fertilizer solution at which the nutrients of the fertilizer solution are no longer transferred to the plant or uptaken by the root system, and remain in the soil. From this experlnent which varies the volume of enriched mineral fertilizer solution used, it is desired to leam the most efficient volume of fertilizer solution in which the delivery of minerals to the plant is maximized, and the loss of minerals and nicroalgae to the soil is ninimized, therefore maximizing the cost effectiveness of the enriched microalgae fertilizer. [0062] The experiment is then repeated using a, fertilizer solution comprising water and mnorganic minerals in place of the fertilizer solution enriched microalgae and water. The results of tie soil sample analysis for both the enriched mnicroalgae fertilizer solution experimental run and the inorganic mineral fertilizer solution experimental run are compared to determine the efficiency increase in delivery of minerals to the plant through the use of imicroalgae as a mineral vehicle as opposed to the use of inorganic minerals. 30 WO 2013/123032 PCT/US2013/025914 [0063] Example 6 [0064] The goal of this experiment is to determine the concentration of mineral enriched microalgae fertilizer at which the plant stops uptaking nutrients and the minerals are lost to the soil. Chlorella is enriched with a blend of minerals inchiding Phosphorus according to the methods disclosed above. In this experiment, the blend of minerals added to the microalgae matches the ratios of the nutritional requirements of a plant. After the incubation period, a series of fertilizer solutions comprising enriched Chlorella and water at different concentrations of solids (enriched inicroalgae) are applied to soil in a series of paired containers. Each pair of containers comprises one container with the contents comprising soil only, and one container with the contents comprising soil and the plant. All other container inputs such as light, air, etc., are identical for each container and held constant. The same volume of the fertilizer solutions are added to each container pair through a drip irrigation system, which each volume of fertilizer solution having different solids concentrations. Soil samples from each container are taken before the application of the fertilizer solution, and at time intervals of 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, and 24 hours after the application of the fertilizer solution. The soil samples are analyzed for their mineral composition. The mineral compositions of the soil samples are compared to determine the concentration of enriched algae at which the nutrients of the fertilizer solution are no longer transferred to the plant or uptaken by the root system, and remain in the soil. From this experiment which varies the concentration of enriched algae in the fertilizer solution. it is desired to learn the most efficient concentration of fertilizer solution in which the delivery of minerals to the plant is maximized, and the loss of nminerals and 31 WO 2013/123032 PCT/US2013/025914 nucroalgae to the soil is minimized, therefore maxinlmizing the cost effectiveness of the enriched microalaae fertilizer. [0065] The experiment is then repeated using a fertilizer solution comprising water and inorganic minerals in place of the fertilizer solution enriched microalgae and water. The results of the soil sample analysis for both the emiched iicroalgae fertilizer solution experimental run and the inorganic mineral fertilizer solution experimental run are compared to determine the efficiency increase in delivery of minerals to the plant through the use of miicroalgae as a mineral vehicle as opposed to the use of inorganic minerals. [0066] Example 7 [0067] The goal of this experiment is to supply a plant with the required nutritional profile using a mineral enriched strain of microalgae. Using the above disclosed method, Chlorella is enriched with a profile of minerals specific to the nutritional requirements of spring wheat in growth stage through the addition of a blend of nitrogen, phosphorus, potassium, sulphur, calcium, magnesiun, zinc, copper, iron, manganese. boron, and molybdenum, to a culture of Chlorella. The nutritional profile specific to the wheat comprises 20-3.0% N. 0.26-0.5% P, 1.5-3.0% K, 0.1-0.15% S, 0.1-0.2 % Ca. 0.1-0.15% Mg, 10-15 ppm Zn, 3.0-4.5 ppm Cu, 15-20 ppm Fe, 10-15 ppm Mn, 3 5 ppm B and 0.01-0.02 ppm Mo in the whole plant prior to filling. After the incubation period, the microalgae biomass is centrifuged. The resulting solids and supenatant are analyzed to determine the mineral profile of the microalgae following the chelation process. The mineral profile of the microalgae is then compared to the nutritional requirements of spring wheat in growth stage to determine if the ratios of enriclunent are preserved during the chelation process. Based on the results of the 32 WO 2013/123032 PCT/US2013/025914 mineral analysis, the interaction between the minerals during the chelation process is determined. The experiment is then repeated with a blend of minerals adjusted to account for the interactions between the minerals during the chelation process to achieve the nutritional requirements of spring wheat in growth stage. The enriched Chlorella cells are administered to the wheat through a spray or drip irrigation system in a fertilizer solution comprising water. [0068] In the foregoing description, the invention has been described with reference to specific exemplary embodiments. Various modifications and changes may be made. however, without departing from the scope of the present invention as set forth. The description and figures are to be regarded in an illustrative manner, rather than. a restrictive one and all such modifications are intended to be included within the scope of the present invention. Accordingly, the scope of the invention should be determined by the generic embodiments described and their legal equivalents rather than by merely the specific examples described above. For example, the steps recited in any method or process embodiment may be executed in any appropriate order and are not limited to the explicit order presented in the specific examples. Additionally, the components and/or elements recited in any system embodiment may be combined in a variety of permutations to produce substantially the same result as the present invention and are accordingly not limited to the specific configuration recited in the specific examples. [0069] Benefits, other advantages and solutions to problems have been described above with regard to particular embodiments. Any benefit, advantage, solution to problems or any element that may cause any particular benefit, advantage or solution to occur or to 3 3 WO 2013/123032 PCT/US2013/025914 become more pronounced, however, is not to be construed as a critical, required or essential feature or component. [0070] The terms "comprises", "comprising", or any variation thereof, are intended to reference a non-exclusive inclusioM, such that a process, method, article, composition, system, or apparatus that comprises a list of elements does not include only those elements recited, but may also include other elements not expressly listed or inherent to such process, method, article, composition, system, or apparatus. Other combinations and/or modifications of the above-described structures, arrangements, applications, proportions, elements, materials or components used in the practice of the present invention, in addition to those not specifically recited, may be varied or otherwise particularly adapted to specific environments, manufacturing specifications, design paramneters or other operating requirements without departing from the general principles of the same. [0071] The present invention has been described above with reference to an exemplary embodiment. However, changes and modifications may be made to the exemplary embodiment without departing from the scope of the present invention. These and other changes or modifications are intended to be included within the scope of the present invention. 34
    权利要求:
    Claims (37)
    [1] 1. A method of making a nicroalgae product enriched with non-metabolized minerals, comprising: a. Growing a culture of microalgae in an aqueous culture medium; b. Harvesting the microalgae by separating the mricroalgae from the aqueous culture medium; c. Adding supplemental minerals specific to a profile of nutritional requirements for an animal to the nicroalgae; d. Incubating the microalgae and the supplemental minerals to facilitate the nicroalgae assimilating, reversibly chelatig, and absorbing the supplemental minerals to produce a nicroalgae product enriched with non-metabolized minferals specific to the profile of nutritional requirements of the animal.
    [2] 2. The method of claim 1 further comprises the step of a. Dewatering the incroalgae product enriched with minerals to further reduce the water content of the microalgae product.
    [3] 3. The method of claim 2 further comprising the step of a. Stabilizing the microalgae product enriched with minerals.
    [4] 4. The method of claim 1, wherein the supplemental minerals are added to the microalgae before harvesting the microalgae. . The method of clim 1, wherein the supplemental minerals are added to the microalgae after harvesting the microalgae.
    [5] 6. The method of claim 1, wherein the animal is an aquatic animal selected from the group consisting of: adult fish, oysters, mollusks, scallops, and shrimp. 35 WO 2013/123032 PCT/US2013/025914
    [6] 7. The method of claim 6, furdier comprising the step of mixing the microalgae with at least one from the group consisting of fishmeal and fish oil, to produce an aquafeed. S. The method of claim 6, wherein the microalgae are fed directly to the aquatic animal.
    [7] 9. The method of claim 7, wherein the aquafeed is fed directly to the aquatic animal.
    [8] 10. The method of claim 1, wherein the supplemental minerals comprise at least one from the group consisting of: boron, bromine, calciun, chloride, chromimin, cobalt, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, sodium, sulphur, vanadium and zinc.
    [9] 11. The method of claim 1, wherein the microalgae is selected from at least one from the group consisting of: Nannochloropsis, Chlorella, Spirulina, Schizochyrrimn, Crxlpthecodinium. and Scenedesm us.
    [10] 12. The method of claim 1, wherein the supplemental minerals are added at a mineral concentration of less than about 15 to 0.1 g/liter to the harvested microalgae of a concentration of about 50-200 g microalgae DW/liter.
    [11] 13. The method of claim 1. wherein the supplemental minerals and microalgae are incubated for 15-120 minutes.
    [12] 14. The method of claim 1, wherein the supplemental minerals and microalgae are incubated at a temperature of about 5-40 degrees C.
    [13] 15. The method of claim 1, wherein the supplemental minerals and microalgae are incubated at a pH of about 6-12
    [14] 16. The method of claim 1, wherein the supplemental minerals and mnicroalgae are micubated with orbital shaking at about 25-150 rpm.
    [15] 17. The method of claim 1. wherein the supplemental minerals are added to the culture of microalgae 1-2 days before the harvesting of the microalgae. 36 WO 2013/123032 PCT/US2013/025914
    [16] 18. The method of claim 3, wherein the method of stabilizing is selected from the group consisting of: freezing .refigeration, freeze drying, spray drying, or drun drying.
    [17] 19. A mineral supplement composition for an aquatic animal the composition comprising a microalgae product enriched with at least one mineral from the group consisting of arsenic, chromium, cobalt, copper, fluorine, iron, iodine, lead, lithium, manganese, molybdenum, nickel, selenium, silicon, vanadium, zinc in a non-metabolized form.
    [18] 20. The mineral supplement composition of claim 19, wherein the aquatic animal is selected from the group consisting of: adult fish, oysters, mollusks, scallops, and shrimp.
    [19] 21. An aquafeed product for adult fish, comprising less than 1% of microalgae enriched with a profile of assimilated, reversibly chelated, and absorbed minerals specific to the nutritional requirements of an adult fish; and a remainder comprising at least one or more other ingredients from the group consisting of fisluneal and fish oil.
    [20] 22. A mineral supplement composition for a non-aquatic animal, the composition comprising a microalgae product enriched with at least one mineral from the group consisting of boron, bromine, calcium, chloride, chromium, cobalt, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium sodium, sulphur, vanadium and zinc in a non-metabolized form.
    [21] 23. The mineral supplement composition of claim 22, wherein the non-aquatic animal is selected from the group consisting of: poultry, horses, ungulates, game animals, bovine, pets, pigs.
    [22] 24. A fatty acid supplement composition for animals, comprising a microalgae product comprising at least one fatty acid of chain length between C10 and C24 and enriched with at least one mineral from the group consisting of: arsenic, boron, bromine, 37 WO 2013/123032 PCT/US2013/025914 calciun chloride chromium, cobalt, copper, fluorine, iodine, iron, lithium, magnesium, manganese, molybdemun nickel, phosphoms, potassium, selenium, silicon, sodium, sulphur, vanadium and zinc iI a non-metabolized form.
    [23] 25. An animal feed product, comprising at least 0.1% of microalgae enriched with a profile of assimilated, reversible chelated, and absorbed minerals specific to the nutritional requirements of an anial.
    [24] 26. The animal feed product of claim 25, wherein the product comprises about 1-5% of incroalgae enriched with a profile of assimilated, reversible chelated, and absorbed minerals specific to the nutritional requirements of an animal.
    [25] 27. The animal feed product of claim 25, wherein the product comprises about 5-10% ' of microalgae enriched with a profile of assimilated, reversibly chelated, and absorbed minerals specific to the nutritional requirements of an animal.
    [26] 28. The animal feed product of claim 25 , wherein the product comprises about 50-80% of microalgae enriched with a profile of assimilated, reversibly chelated, and absorbed minerals specific to the nutritional requirements of an animal.
    [27] 29. The animal feed product of claim 25, wherein the product comprises about 1% or less of mnicroalgae enriched with a profile of assimilated, reversibly chelated, and absorbed minerals specific to the nutritional requirements of an animal.
    [28] 30. An aqnafeed product for adult fish, comprising: a. Microalgal biomass, wherein the microalgal bionass is a mineral enriched microalgae product providing a mineral profile comprising at least one from the group consisting of i. About 30-170 mg Iron per kg of dry aquafeed; ii. About 1-5 ing Copper per kg of dry aquafeed; 38 WO 2013/123032 PCT/US2013/025914 iii. About 2-20 mg Manganese kg of dry aquafeed; iv. About 15-40 mg Zinc per kg of dry aquafeed; v. About 0.05-1.0 mg Cobalt per kg of dry aquafeed; vi. About 0.15-0.5 mg Selenium per kg of dry aquafeed; vii. About 1-4 mug Iodine per kg of dry aquafeed; and b. At least one or more additional Mredients.
    [29] 31. A dog food product for adult dogs, comprising: a. Microalgal biomass, wherein the inicroalgal biomass is a mineral enriched microalgae product providing a mineral profile comprising at least one floru the group consisting of i. About 0.75 g Calcium per 1000 calories of dog food; ii. About 0.75 g Phosphorus per 1000 calories of dog food; iii. About 150 mg Magnesium per 1000 calories of dog food; iv. About 100 mg Sodium per 1000 calories of dog food v. About 1 g mg Potassium per 1000 calories of dog food; vi. About 150 mg Chlorine per 1000 calories of dog food vii. About 7.5 mg Iron per 1000 calories of dog food; viii. About 1.5 mg Copper per 1000 calories of dog food ix. About 15 mg Zinc per 1000 calories of dog food x. About 1.2 mug Manganese per 1000 calories of dog food; xi. About 90 pg Selenium per 1000 calories of dog food; xii. About 220 pg Iodine per 1000 calories of dog food; and b. At least one or more additional ingredients. 32 A cattle feed product for gestating beef cows, comprising: 39 WO 2013/123032 PCT/US2013/025914 a. Microalgal biomass, wherein the microalgal biomass is a mineral enriched microalgae product providing a mineral profile comprising at least one from the group consisting of i About 0.10 ig Cobalt per kg of dry cattle feed; ii. About 10 mg Copper per kg of dry cattle feed iii. About 0.50 mg Iodine per kg of dry cattle feed; iv. About 50 mg Iron per kg of dry cattle feed v. About 40 mg Manganese per kg of dry cattle feed; vi. About 0.10 mg Selenium per kg of dry cattle feed; vii. About 30 mg Zinc per kg of dry cattle feed; and b. At least one or more additional mredients.
    [30] 33. A fertilizer composition for plants, the composition comprising a. microalgae product enriched with at least one mineral from the group consisting of: nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, zinc. iron, copper, manganese, boron, molybdenum, and chlorine in a non-metabolized form.
    [31] 34. The composition of claim 33, wherein the composition is a liquid.
    [32] 35. The composition of claim 34, wherein the liquid composition is sprayed on at least one selected from the group consisting of: plant leaves, plant stalks, plant vines, the airspace inmiediately proximate to the plant, and the ground immediately proximate to the plant.
    [33] 36. The composition of claim 33, wherein the composition is in the form of di flakes or powder. 40 WO 2013/123032 PCT/US2013/025914
    [34] 37. The composition of claim 36, wherein the dry flakes or powder are applied to at least one selected from the group consisting of. the ground innediately proximate to the plant, and soil in which'a plant is growing or will be planted.
    [35] 38. A method of making a inicroalgae product enriched with non-metabolized minerals, comprising: a. Growing a culture of microalgae in a culturing vessel comprising an aqueous culture medium and at least one pair electrodes submerged in the aqueous culture medium, i. The electrode comprised of an electrode material comprising at least one of a mineral specific to a nutritional profile of an animal; b. Applying an electric current to the at least one pair of electrodes sufficient to cause the electrode material to leach into the aqueous culture medium c. Incubating the microalgae to facilitate the microalgae assimilating, reversibly chelating, and absorbing of the electrode material to produce a microalgae product enriched with non-ietabolized minerals specific to the profile of nutritional requirements of the animal d. Harvesting the microalgae product enriched with non-metabolized minerals to separate the microalgae product from the aqueous culture medium.
    [36] 39. The method of claim 38, wherein the electrical current is direct, alternating, or pulsed.
    [37] 40. The method of claim 38, wherein minerals comprise at least one from the group consisting of boron, bromine, calcium, chloride, clhromium, cobalt, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, sodium, sulphur, vanadium and zinc. 41
    类似技术:
    公开号 | 公开日 | 专利标题
    US20160340264A1|2016-11-24|Microalgae as a mineral vehicle in aquafeeds
    Colman et al.1987|Feeding pathways and environmental constraints in waste-fed aquaculture: balance and optimization
    Saeid et al.2013|Biomass of Spirulina maxima enriched by biosorption process as a new feed supplement for swine
    RU2613224C2|2017-03-15|Growth additive for intensive agricultural and livestock production
    CN102690152B|2013-10-09|Liquid fertilizer with magnetic energy
    CN107082687A|2017-08-22|A kind of nano-sized carbon is Se-enriched or mixture fertilizer
    CA2785531A1|2011-06-30|Organically chelated mineral compositions and methods thereof
    CN103689284B|2015-08-19|A kind of bighead organic debris cenobium bait and preparation method thereof
    CN102524547B|2014-05-14|Tourmaline mineral feed additive
    CN103960531B|2016-01-27|Reduce the method for breeding of partly raising nitrogen phosphorus heavy metals emission in traditional black swine excrement scattered
    Ktita et al.2010|The use of seaweeds | for lamb fattening during drought periods
    CN107751590A|2018-03-06|A kind of mutton sheep TRM fermented feed and preparation method thereof
    Tai et al.2001|Future prospects of duck production in Asia
    Gowda et al.2015|Azolla | as a green feed supplement for dairy cattle-an on farm study
    CN101946857A|2011-01-19|Method for preparing poultry feed additive compound mineral substance
    CN105543146A|2016-05-04|Preparation method of photosynthetic bacterium water aqua and special culture medium of photosynthetic bacterium water aqua
    JPH06305871A|1994-11-01|Fermented fertilizer and its production
    RU2501303C2|2013-12-20|Method of production of feed additive for poultry
    CN105532572B|2018-11-06|A kind of production method of richness organic zinc-iron duck&#39;s egg
    CN104719677B|2017-11-10|A kind of sturgeon mixed feed that rubber seedcake utilization rate is improved using yolk powder
    Vaikosen et al.2010|Yield and chemical composition of Chlorella species cultivated in pig, poultry and cow dungs in Southern Nigeria
    El Semary2017|Algae and chain aquaculture: An approach towards sustainable agriculture
    Garg et al.2003|Macro and micro-mineral status of feeds and fodders in Kota district of Rajasthan
    Masters et al.2009|The mineral content of river saltbush | changes when sodium chloride in the irrigation solution is increased
    CN104291995A|2015-01-21|Special chemical fertilizer for beans and preparation method of special chemical fertilizer
    同族专利:
    公开号 | 公开日
    CA2864277A1|2013-08-22|
    US20130205850A1|2013-08-15|
    US20160340264A1|2016-11-24|
    WO2013123032A1|2013-08-22|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    CN108125018A|2018-02-01|2018-06-08|秦烽曦|A kind of Animal nutrition material and its application|JPS57159447A|1981-03-30|1982-10-01|Shikamitsu Honsha:Kk|Production of veterinary nutrition-promoting preparation|
    GB9226182D0|1992-12-16|1993-02-10|Biotechna Ltd|Binding of metals and metalloids by microorganisms|
    FR2827301B1|2001-07-13|2004-10-01|Blue Energy Laboratoire|METHODS OF FIXING / COMPLEMENTATION OF BIOLOGICALLY ACTIVE MOLECULES BY CYANOBACTERIA AND MICROALGAE OR FRACTIONS OF CYANOBACTERIA AND MICROALGAE|
    WO2003079810A1|2002-03-19|2003-10-02|Advanced Bionutrition Corporation|Microalgal feeds containing arachidonic acid and their production and use|
    EP1873233B1|2005-04-12|2013-09-25|Denso Corporation|Microalgae and process for producing hydrocarbon|
    WO2010089864A1|2009-02-04|2010-08-12|クロレラ工業株式会社|Selenium-containing unicellular microalgae for animal plankton feeds and method of culturing selenium-containing animal planktons using the same|US9428425B2|2012-09-20|2016-08-30|Core Intellectual Properties Holdings, Llc|Methods and compositions for treating soil and plants|
    US8747916B1|2012-10-30|2014-06-10|Donald M. Smith|Selecting, producing, and feeding whole algae as a feed supplement for cattle and bison to produce meat high in omega 3's for human health|
    WO2015072539A1|2013-11-18|2015-05-21|株式会社カネカ|Culture feed for aquatic organisms|
    FR3019559B1|2014-04-03|2018-01-05|Fermentalg|PROCESS FOR CULTIVATION OF MICROALGUES OF THE GENUS AURANTIOCHYTRIUM IN A SODIUM-CHLORIDE REDUCED CULTURE MEDIUM FOR THE PRODUCTION OF DHA|
    FR3019466B1|2014-04-08|2018-04-06|Phyco-Biotech|SILICON ENRICHED MICROALGUE IN A WATER-SOLUBLE FORM|
    MX2017005274A|2014-10-21|2018-01-24|M Smith Donald|Feeding algae to cattle at low doses to produce high omega 3 levels in beef.|
    CL2014003348A1|2014-12-09|2016-09-02|Oceaneos Environmental Solutions Inc|Non-radioactive iron isotopes and method of use to track marine species|
    EP3232790B1|2014-12-16|2020-02-05|Heliae Development LLC|Mixotrophic chlorella-based composition, and methods of its preparation and application to plants|
    US11259527B2|2015-08-17|2022-03-01|Heliae Development, Llc|Haematococcus based compositions for plants and methods of application|
    CN105075952B|2015-09-09|2018-07-31|大连市水产技术推广总站|Using the method for Copepods during the net cage seedling nursery of biological prevention and control stichopus japonicus sea area|
    EP3346841A4|2015-09-11|2019-02-13|Heliae Development, LLC|Microalgae based compositions and methods for application to plants|
    CN105638534A|2016-03-07|2016-06-08|五河县金满塘生态种养殖家庭农场|Method for freshwater aquaculture of high-quality penaeus vannamei boone|
    CN106626027A|2017-03-06|2017-05-10|漳州市烨发工贸有限公司|Ceramic chain type full-automatic production line glaze spraying machine|
    WO2018199723A2|2017-04-28|2018-11-01|한남바이오 주식회사|Selenium resistive novel microalgae|
    FR3073860A1|2017-11-23|2019-05-24|Fermentalg|ANTI-ADHESION CULTURE MEDIUM|
    CN108719672B|2018-06-04|2021-12-31|福建省农业科学院农业生态研究所|Selenium-rich feed special for free-range laying hens and use method thereof|
    CN108733985B|2018-06-08|2021-06-25|中国科学院城市环境研究所|Method for determining key environmental parameters for restricting arsenic accumulation capacity of microalgae|
    CN108902026A|2018-06-27|2018-11-30|高邮市普德家庭农场|A kind of cultural method of selenium-rich duck|
    CN109566860A|2018-12-29|2019-04-05|大连海洋大学|One seed oyster special compound feed|
    NL2024465B1|2019-12-16|2021-09-02|Redipro B V|Composition of natural ingredients for nutrition of indoor- and outdoor fields of grass, method of manufacturing such a composition, and use of such a composition|
    法律状态:
    2017-02-09| MK1| Application lapsed section 142(2)(a) - no request for examination in relevant period|
    优先权:
    申请号 | 申请日 | 专利标题
    US201261598235P| true| 2012-02-13|2012-02-13||
    US61/598,235||2012-02-13||
    US201261601970P| true| 2012-02-22|2012-02-22||
    US61/601,970||2012-02-22||
    PCT/US2013/025914|WO2013123032A1|2012-02-13|2013-02-13|Microalgae enriched with trace minerals|
    [返回顶部]