Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/12—Unicellular algae; Culture media therefor
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
  • A01G33/00—Cultivation of seaweed or algae
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
  • C12M21/02—Photobioreactors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/18—Open ponds; Greenhouse type or underground installations
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/58—Reaction vessels connected in series or in parallel
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH

Definitions

• the present invention in some embodiments thereof, relates to methods and systems for cultivation of microalgae.
• the global carbon cycle is heavily influenced by the activities of man. For example, the combustion of fuels by man is believed to have resulted in a large increase in the amount of carbon dioxide present in the atmosphere. In the last hundred years, global fossil carbon emissions have increased by more than a factor of ten. As nations around the globe continue to become more industrialized, demands for energy are expected to increase dramatically. As such, in the absence of new technological solutions, it is believed that the trend toward increased fossil carbon emissions will continue.
• Carbon dioxide is considered to be a "greenhouse” gas and is believed to have contributed to global warming trends. Carbon dioxide, along with water vapor, methane, nitrous oxide, and ozone, causes more heat to be retained by the Earth than would otherwise be captured. It is believed that this is due, at least in part, to the observed increase in greenhouse gas concentrations. Further increases in global temperatures may lead to various catastrophic effects including a rising sea level, increased extreme weather events, reduced agricultural yields, glacier retreat, and species extinction, amongst others.
• plants efficiently capture atmospheric carbon through the process of photosynthesis. Using sunlight as energy, plants convert carbon dioxide and water into the precursors of carbohydrates and other plant constituents. Many different types of plants and microorganisms capture considerable amounts of carbon dioxide.
• Algae are photosynthetic organisms that occur in most habitats. They vary from small, single- celled forms to complex multicellular forms. Algae are estimated to generate as much as 80 percent of the Earth's oxygen. It is also estimated that algae fix 90 gigatons of carbon per year.
• open culture systems are open to the atmosphere and are equivalent to common high plants agriculture. They have the advantage of being relatively inexpensive to construct and maintain.
• open culture systems are subject to contamination issues.
• closed culture systems or the photobioreactors are closed to the atmosphere and therefore provide the advantages of a controlled environment, lower evaporative water loss, and fewer contamination issues.
• many closed culture systems require relatively complex structures and therefore have substantially higher construction and operating costs.
• many closed culture systems have issues associated with insufficient light penetration, algae growth on walls, in- situ contamination that is difficult to clean, and poor temperature control.
• the microalgae culture is a monoculture.
• the acidic pH conditions are between 4-5.
• the basic pH conditions are between 9-11.
• a time ratio between step (a) and step (b) is predetermined according to an algal productivity of interest.
• the algal productivity of interest is selected from the group consisting of biomass, carbohydrates, lipids and protein.
• the cultivating is effected in open-body ponds.
• the cultivating comprises enriching the microalgae culture with inorganic carbon.
• the inorganic carbon is comprised in flue gas.
• step (a) or (b) comprises transferring the microalgae culture into a source of flue gas so as to allow flow of flue-gas into the microalgae culture, and returning the microalgae culture into the open-body pond.
• the source of flue-gas comprises a power plant.
• the inorganic carbon comprises calcium carbonate.
• the cultivating is effected at low culture flow.
• the method further comprising:
• step (c) harvesting microalgae from the microalgae culture following step (b).
• the harvesting is effected by a method selected from the group consisting of flocculation, sedimentation, filtration and floatation.
• the method further comprising:
• step (d) recycling a culture medium of the microalgae culture following step (c).
• the microalgae comprises marine microalgae. According to some embodiments of the invention, the microalgae comprises a fresh water microalgae.
• the microalgae culture comprises sea water.
• the microalgae culture comprises fresh water, brackish water and/or wastewater.
• the microalgae is selected from the group consisting of starch-producing algae; chrysolaminarin— producing algae
• the microalgae comprise coccoids.
• the microalgae comprise flagellates.
• a culture medium of the microalgae culture of the step (a) or (b) is augmented to include the following constituent values:
• the microalgae culture of step (a) is augmented to include the following constituent values:
• the microalgae culture of step (a) is augmented to include the following constituent values:
• microalgae culture of step (b) is augmented to include the following constituent values:
• the microalgae culture of step (b) is augmented to include nitrate concentration of 0.5-1 mM.
• the microalgae culture of step (b) is augmented to include nitrate concentration of 1-5 mM.
• the method further comprises monitoring in the open body ponds a parameter selected from the group consisting of medium pH, medium TDC, medium conductivity, medium salinity, depth, medium temperature, cell number, chlorophyll, carotenoids, biomass and environmental conditions.
• a cultivation system comprising a first body pond which comprises a microalgae culture having an acidic pH between 4-5 and a second open body pond which comprises a microalgae culture having a basic pH between 9-11, wherein the first body pond and the second body pond are connected via a conduit for allowing fluid communication which allows transfer of microalgae culture having the acidic pH from the first body pond to the second body pond.
• the cultivation system further comprises a conduit for introducing inorganic carbon into the first open body pond and optionally the second open body pond.
• the cultivation system further comprises a device for pumping microalgae culture having an acidic pH between 4-5 from the first open body pond to the second open body pond via the conduit.
• FIG. 1 is a graph showing dissolved inorganic carbon species as a function of increasing pH
• FIG. 2 shows a pond system according to some embodiments of the present invention.
• FIG. 3 shows a pond system according to some embodiments of the present invention.
• the present invention in some embodiments thereof, relates to methods and systems for cultivating microalgae.
• Microalgae are one of the most potential sources of bioenergy. In the reports by U.S. Department of Energy, it is mentioned that bio-diesel transformed from the oil produced using the microalgae would fully meet the demand in the American diesel market.
• microalgae also known as open systems and closed systems, respectively.
• open ponds have been associated with contamination, excessive space requirements and limited location possibilities due to climate.
• closed bioreactors are mainly been considered too expensive.
• TDC variable total dissolve carbon
• cultivating refers to growth and expansion of a microalgae culture.
• microalgae refers to aquatic photosynthetic
• the present teachings refer to the use of any species of such microalgae.
• the algae used according to some embodiment of the present teachings are both fresh water and marine species including e.g., coccoides and flagellates.
• the microalgae can be naive or genetically modified to express a protein of interest. Methods of genetically modifying microalgae are well known in the art (some are taught in U.S. 20090010947 and 6,027,900 each of which is hereby incorporated by reference in its entirety).
• the microalgae culture is a monoculture.
• the term "monoculture” refers to a culture in which microalgae (single species or a combination of two or more species) is the dominant species.
• the culture comprises at least 90 % microalgae (e.g., a single species of microalgae) out of total microorganisms in the pond.
• the monoculture is predator-free i.e., it does not include predators of the cultivated microalgae.
• microalgae Any species of microalgae may be cultivated, alone or in various combinations, such as for example Chlorella, Chlamdomonas, Chaetoceros, Spirolina, Dunaliella and Porphyridum.
• the specific algal species is selected according its ability to accumulate a product of interest, such as carbohydrates and lipids, under the indicated defined conditions, as below.
• Table 1 Microalgae Species
• Cultivation of the microalgae can be done is fresh water, brackish water, waste water or sea water. According to a specific embodiment, cultivation is effected in brackish water.
• the water must be in a temperature range that will support the specific algal species being grown.
• Nutrients such as nitrogen (N), phosphorus (P), and potassium (K) can serve as fertilizer for algae.
• Silica and iron, as well as several trace elements, may also be considered important marine nutrients as the lack of one can limit the growth of, or productivity in, a given area.
• water (any water source) can be augmented to provide a basic nutritional medium which comprises NaCl, 10-500 mM, 100-300 mM or specifically, lOOmM; Mg ++ 1-500 mM, 1-100 mM or 1-10 mM or specifically 5mM; K + , 1-20 mM or 1-10 mM and specifically 5mM; Ca +2 , 0.1-500 mM. 0.1-100 mM, 1-100 mM or specifically 50mM; Fe 3+ as available and specifically 2 ⁇ ;
• embodiments of the invention rely on cultivating a microalgae culture under acidic pH conditions (as a first or single stage) that predominantly drive carbonic acid and ammonia assimilation by the microalgae. Uptake of carbonic acid and ammonia by the algae, releases protons to the medium with no need for pH gradient and no need for proton pump.
• the acidic conditions comprise a pH below 5, or according to further specific embodiments, 4-5, 4.5-5 or 4- 4.5. According to a further specific embodiment, the pH range at the acidic conditions is 4-5.
• the pH of the culture is predominantly acidified by adding to the culture ammonia 1-5 mM (the uptake of ammonia by the microalgae releases proton (H+) to the culture); additionally, the medium is acidified by flowing carbon dioxide (thereby also enriched with carbon) by using inorganic carbon, such as using flue gas of various sources e.g., power plants.
• the present inventor has realized that cultivation under acidic pH with essentially no nitrate at this stage (below 0.1 nM) has a number of advantages:
• Exemplary embodiments for cultivating under acidic conditions are provided infra.
• Microalgae are grown at low pH below 5 using carbon dioxide (CO 2 ) or flue gases for carbon source and ammonium ions (N3 ⁇ 4 or NH4 + by any source). These conditions maintain high biomass under species selected conditions. Uptake of ammonia by the algae releases proton (H + ) to the medium so at 5mM NH 4 + the medium is acidified. The following parameters are monitored: weather, and temperature. The following parameters are controlled: pH between 4 to 5, total dissolve carbon (TDC) 0.5-5 mM, conductivity between 1 to 20 mS/cm, salinity between 0.1 to 2 M and depth between 5 cm to 40 cm, ammonia l-5mM, and sulfate l-500mM. The following biological parameters are analyzed: cell number 1-300 million/ml, chlorophyll 0.1-30 mg/L, carotenoids 0.02-6 mg/L and biomass 0.005-1.5 g/L (Table 4, Low pH).
• an acidic culture will perform better than an alkaline culture in the production of total biomass (as determined e.g., by total dry weight) carbohydrates and proteins. Conversely, lipid generation is better achieved under alkaline conditions.
• the microalgae culture is cultivated under basic pH conditions that predominantly drive carbonate and nitrate assimilation by the microalgae.
• the extracellular induced carbonic anhydrase allows active uptake of carbon from the medium into the cells while keeping pH gradient between the medium and the intracellular space. Under neutral cellular pH nitrate reductase and protein biosynthesis proceed and function optimally.
• basic pH conditions or “alkaline pH conditions” (also referred to herein as “2 nd stage”) comprise a pH of at least 8, or according to specific embodiments at least 9, 9-11, 8-11. According to a specific embodiment, the pH range at the basic conditions is 9-11. Culturing under basic pH conditions is also advantageous in terms of medium recycling, as further described hereinbelow.
• Culturing under basic conditions comprises outdoor conditions of high pH 9-11 using flue gas, bicarbonate or carbonate (any source, sodium carbonate, calcium carbonate, magnesium carbonate and others), to reach high TDC of at least about 5 mM and nitrate 0.5-2 mM (see Table 5, high pH).
• the alkaline conditions induce the enzyme carbonic anhydrase, an algal outer membrane for most efficient carbon capture at these high pH conditions.
• the induced enzyme CA allows algal cultivation at high TDC (at least 5mM) and high pH for cellular carbohydrates and oil controlled production.
• Nitrate Reductase (NR) Induction in Stage 2 (basic).
• the enzyme nitrate reductase is induced on transferring the algae to medium containing nitrate (all sources) at alkaline pH.
• the ability to control cellular constituents allows the controlled production of cellular products of commercial value.
• step (a) or (b) also referred to herein as time ration between step (a) and step (b)] is governed according to an algal productivity of interest.
• an algal productivity of interest refers to a cellular constituent or total biomass which is of commercial value to an end user.
• algal products include, but are not limited to , carotenoids, antioxidants, fatty acids, enzymes, polymers polysaccharides), peptides, toxins and sterols.
• lipid enrichment is achieved under step (b) (basic stage).
• step (b) basic stage
• nitrate In order to obtain high content of lipids it is necessary to supply limiting concentration of nitrogen which is provided on Stage 2 by nitrate.
• the nitrate content should be below ImM (e.g., 0.1-1 mM) for cellular lipid enrichment.
• ImM e.g., 0.1-1 mM
• cultivation at each step is effected until a concentration of about 1 gram of ash free fry weight algae per liter is obtained.
• the life time cycle of each stage is related to the environmental conditions and varies between 24 to 72 hours.
• the culture may be enriched with inorganic carbon such that the TDC at the first (acidic stage) is 0.5-5mM, while at the basic stage (second stage) high TDC of at least 5 mM is achieved (e.g., 3-6 mM).
• inorganic carbon Any source of inorganic carbon can be used. These include, carbon dioxide, carbonic acid, bicarbonate and carbonate (see Figure 1).
• flue gas is used as the source of inorganic carbon. According to an embodiment of the invention, flue gas is streamed into the algae culture.
• the microalgae culture is transferred into a source of flue gas (e.g., power plant) so as to allow flow or streaming of flue-gas into the microalgae culture, and returning the microalgae culture into the open-body pond.
• a source of flue gas e.g., power plant
• This embodiment negates the need to transfer the flue-gas to the cultivation area.
• flue gas refers to the exhaust gas from any sort of combustion process (including coal, oil, natural gas, etc.). Flue gas typically includes acid gases such as C0 2 , S0 2 , HC1, S0 3 , and NO x .
• the present invention contemplates removing at least 5 % of the C0 2 present in the flue gas by carbon dioxide fixation, more preferably at least 10 % of the C0 2 present in the flue gas, more preferably at least 20 % of the C0 2 present in the flue gas, more preferably at least 30 % of the C0 2 present in the flue gas, more preferably at least 40 % of the C0 2 present in the flue gas and even more preferably at least 50 % of the C0 2 present in the flue gas.
• the flue gas is streamed (directly) into the algae culture medium.
• the gas may be bubbled at the bottom of the algae culture thereby creating turbulence, mixing the culture so that the algae at the bottom of the culture moves to the top. In this way an airy culture is obtained which comes in contact with the nutrients and oxygen. Further movement in the culture ensures that the algae has sufficient exposure to a light source.
• the flue gas may be streamed above the algae culture (i.e. in a gas headspace).
• C0 2 -rich gas flows in the headspace, C0 2 dissolves into the liquid medium.
• Misters which spray the liquid medium into the gas headspace may be used in some embodiments.
• the culture medium of the microalgae culture of step (a) or (b) is augmented to include the following constituent values:
• microalgae culture of step (a) is augmented to include the following constituent values:
• microalgae culture of step (a) is augmented to include the following constituent values:
• microalgae culture of step (b) is augmented to include the following constituent values:
• said microalgae culture of step (b) is augmented to include nitrate concentration of 0.5-1 mM.
• said microalgae culture of step (b) is augmented to include nitrate concentration of 1-5 mM.
• the open-body pond system comprises a plurality of ponds (e.g., 5. 10, 50 or 100 ponds) of basic and acidic ponds (having the above-defined pH conditions) that are interconnected as further described hereinbelow.
• the open ponds may be each divided outside and inside by ground dikes where each unit of operation is 1-2 hectare in step wise down ground level.
• the pond is about 5-40 cm deep to ensure maximal photosynthesis (deeper water require agitation as described hereinabove).
• the pond may be of 1, 2 5, 10 or 20 hectares.
• water flow between the pools is achieved by leveling down with no agitation or mixing that are typical for an intensive cultivation.
• the ponds may be lined with clay or calcium carbonate, under acidic conditions the latter is used as a carbon dioxide source.
• step (b) Throughout cultivation i.e., at step (a), step (b) or both steps, monitoring a various abiotic/biotic parameters is effected.
• abiotic/biotic parameters include, but are not limited to, culture medium pH, culture medium TDC, culture medium conductivity, culture medium salinity, depth, culture medium temperature, cell number, chlorophyll, carotenoids and biomass.
• the microalgae culture is harvested.
• Cultures can be harvested by continuous centrifugation in a batch-centrifuge or in a "de-sludger” centrifuge at about 3000 g. Generally it is advantageous to harvest each day about half the cells, the remaining culture being diluted with fresh medium. One liter contains about 1 gram ash free dry weight (AFDW).
• AFDW gram ash free dry weight
• Algae can be harvested by gravitational sedimentation.
• the culture is transferred to a conical tank where they settle out.
• About 30 % of the daily growth settles out by gravitation and there a concentration factor of about 25 is attained. Centrifugation of this sediment yields fresh algal paste.
• the algae may be flocculated from the culture medium by the addition of certain salts, such as ferric chloride, aluminum chloride or aluminum sulfate.
• certain salts such as ferric chloride, aluminum chloride or aluminum sulfate.
• a concentration of about 0.1 mM to 0.5 mM ferric chloride, or about 0.1 to 0.5 mM aluminum chloride or about 0.1 mM to 0.5 mM aluminum sulfate results in the flocculation of the microalgae and the settled out cells may be harvested after about one hour. The sediment is centrifuged to yield algal paste.
• the algae can be concentrated by cross-flow filtration through a cross-flow filtration system of the type supplied by A. T. Ramot Plastics Ltd., Tel-Aviv, which comprises a plurality of porous plastic tubings assembled in a modular filtration unit containing bundles of many tubes. About 75 liters of algae culture can be concentrated per hour yielding a concentration by a factor of 15. Centrifugation yields algal paste.
• the algae biomass is further pressed and partially dehydrated according to the specifications of the end user for biofuel (biodiesel and bio-ethanol) downstream processing.
• the dry algae contains at optimum conditions about 2-3 folds higher protein content than lipids when produced under acidic conditions (see Table 2) and about the same or 20-30 % higher lipid content than protein content, when grown in stage 2.
• the product of interest can be extracted using any method known in the art.
• Glycerol extraction can be effected using any method known in the art, such as described in details in U.S. 4,115,949 which is hereby incorporated by reference in its entirety.
• Proteinous residue can be extracted using cyclohexane (1: 1 by volume) as further detailed in 4,115,949 (supra).
• the present invention also contemplates a cultivation system.
• cultivation system 10 comprises a first body pond 12 which comprises a microalgae culture 14 having an acidic pH between 4-5 and second open body pond 16 which comprises a microalgae culture 18 having a basic pH between 9-11, wherein first body pond 12 and second body pond 16 are connected via conduit 20 allowing fluid communication which allows transfer of culture 14 from first body pond 12 to second body pond 14.
• Cultivation system 10 may further comprise conduit 22 for introducing inorganic carbon from inorganic carbon supply vessel 46 into first open body pond 12 and optionally second open body pond 16.
• Cultivation system 10 may further comprise pump 24 for pumping microalgae from first open body pond 12 to second open body pond 16 via the conduit 20.
• Cultivation system 10 may further comprise a conduit 11 leading water from source of water 26 via water softener 28, (if seawater is used), into first open body pond 12.
• Nutrients are supplied into first open body pond 12 or second open body pond 16 via conduit 32 from liquid nutrient supply vessel 30.
• Part of the liquid nutrient medium is returned via conduit 34 to first open body pond 12, while part of culture 14 flows via conduit 20 to second open body pond 16.
• Basic culture 18 is flowed from second open body pond 16 via conduit 36 to separator 38 and via conduit 40 to product reservoir 42, which is an algae paste of about 50 % water content.
• Part of the nutrient liquid is returned to second open body pond 16 via conduit 44.
• Cultivation system 100 shows another embodiment to the transfer of algae into the flue gas source and its returned upon TDC enrichment ( Figure 3).
• Cultivation system 10 comprises a first body pond 112 which comprises a microalgae culture 114 having an acidic pH between 4-5 and a second open body pond 116 which comprises a microalgae culture 118 having a basic pH between 9-11, wherein first body pond 112 and second body pond 114 are connected via conduit 120 allowing fluid communication which allows transfer of microalgae culture 114 from first body pond 112 to second body pond 114.
• Cultivation system 110 further comprises at least one conduit 122 for transferring microalgae culture 114 or 118 into a source of flue gas such that flue gas is streamed into the cultivation medium and further cultivation system 110 comprises at least one conduit 124 for returning microalgae culturell4 or 118 being TDC rich into first body pond 112 or second body pond 114.
• compositions, 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.
• the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.
• the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
• 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.
• 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.
• treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
• Table 2 Content of Cellular Ingredients under the Two Biphasic Stages in Starch Algae and in Chrysolaminarin Algae
• Chlorophyll (pg/cell) 0.1-0.2 0.1-0.2 0.05-0.1 0.05-0.1
• Carotenoids (pg/cell) 0.02-0.04 0.02-0.04 0.01-0.02 0.01-0.02
• Chrysolaminarin (mg/g none 400-500 none 250-300 dry weight)
• Lipids 100-200 100-200 200-350 300-350

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