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US20090087889A1 - Methods and compositions for growth hydrocarbons in botryococcus sp. - Google Patents

Methods and compositions for growth hydrocarbons in botryococcus sp. Download PDF

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US20090087889A1
US20090087889A1 US11/919,082 US91908206A US2009087889A1 US 20090087889 A1 US20090087889 A1 US 20090087889A1 US 91908206 A US91908206 A US 91908206A US 2009087889 A1 US2009087889 A1 US 2009087889A1
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hydrocarbons
salts
botryococcus
ninsei
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Arthur M. Nonomura
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H13/00—Algae
    • 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
    • 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
    • C12N1/125—Unicellular algae isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae

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  • the present invention is a novel and distinct process for commercial growth of hydrocarbons in photosynthetic organisms while maintaining a biologically exclusive monocultural environment, as for example, in the case of the present invention, from Chlorophyta, Trebouxiophyceae , and particularly Botryococcus species.
  • Botryococcus species have been suggested as potential sources of liquid transport fuels (Wolf, et al. 1985). Academically, Botryococcus species have proven quite attractive for their natural chemistries, but the cost for production of a gallon of renewable transport fuel exceeded the sales price of fossil fuels. A number of different culture conditions have been investigated, but a defined system for competitive growth of transport fuels has not been disclosed previously. Humanity would benefit from development of a reliable system for production of petroleum-type hydrocarbons from a renewable energy source.
  • Botryococcus is a primitive colonial photosynthetic organism, dating from 300 million years ago, and may be regarded as a living fossil; as, indeed, B. balkachicus; B. coorongianus; B. luteus ; and B. palanaensis , are true fossil deposits. Oil shale is populated with botryococcite fossils from which petroleum deposits arose. Shale originates from mud, and in consideration of the fossil record, the methods, compositions and organisms of the present invention, allow for expression of the mud origins of live Botryococcus species, including the following: B. australis, B. braunii, B. braunii var. horridus, B. braunii var.
  • B. braunii var. perarmatus B. braunii var. Showa, B. braunii var. validus, B. calcareous, B. canadensis, B. comperei, B. fernandoi, B. giganteus, B. micromorus, B. neglectus , and B. pila .
  • B. braunii var. perarmatus B. braunii var.
  • B. braunii var. Showa, B. braunii var. validus, B. calcareous, B. canadensis, B. comperei, B. fernandoi, B. giganteus, B. micromorus, B. neglectus , and B. pila .
  • Botryococcus sp. thrived in hundreds times the concentrations of the salts in conventional nutrients.
  • the present invention exploits the aforementioned discovery of the exclusive niche of Botryococcus sp.
  • the present invention relates to methods, compositions and systems for the in vitro growth of hydrocarbons in photosynthetic organisms while maintaining a biologically exclusive monocultural environment, as for example, from Botryococcus species.
  • the environmental tolerances that made Botryococcus sp. the fittest for hundreds of millions of years are defined and utilized in novel systems for growing gasoline on a commercial scale.
  • the present inventor discovered that Botryococcus sp. thrived in many times the concentrations of the salts in conventional nutrients; it out-competes all other life forms by living in a chemically extreme environment of high concentrations of all of its nutrient chemicals.
  • the present invention exploits the aforementioned discovery of the exclusive niche of Botryococcus sp.
  • niche-nutrients include about 200 ppm to about 3% nitrogen, and about 100 ppm to about 15% P 2 0 5 , and about 100 ppm to about 3.5% K 2 0.
  • the nutrient medium is a balanced nutrient salt formulary comprising phosphate salts and including ammonium salts, calcium salts, potassium salts, magnesium salts, sodium salts, phosphoric acid, pyrophosphates, polyphosphates, glycerophosphates, and the like; with soluble potassium phosphates, most highly preferred.
  • the present invention relates to the growth of the Chlorophyta such as Botryococcus sp. in a nutrient medium that includes about 800 ppm to 15% phosphates and about 2 ppm to about 70 ppm soluble zinc.
  • the present invention relates to the growth of photosynthetic organisms such as Botryococcus sp. in a nutrient medium that includes soluble iron, manganese and magnesium at concentrations far greater than conventional phycological nutrients in order to further enhance synthesis of hydrocarbons.
  • the present invention relates to the growth of the Chlorophyta such as Botryococcus sp. in a nutrient medium that includes phosphate salts, including, ammonium salts, calcium salts, potassium salts, magnesium salts, sodium salts, phosphoric acid, pyrophosphates, polyphosphates, glycerophosphates, and the like, phosphate buffers comprised of monobasic, dibasic, and tribasic salts; citrates; Krebs Cycle carboxylates; and derivatives thereof and the like.
  • phosphate salts including, ammonium salts, calcium salts, potassium salts, magnesium salts, sodium salts, phosphoric acid, pyrophosphates, polyphosphates, glycerophosphates, and the like
  • phosphate buffers comprised of monobasic, dibasic, and tribasic salts; citrates; Krebs Cycle carboxylates; and derivatives thereof and the like.
  • Suitable ranges of nutrients include 0.800 ppm to 30% phosphates, with preferred phosphate concentration of from about 0.800 ppm to about 3% phosphates, about 25 ppm to about 250 ppm soluble magnesium, about 0.3 ppm to about 3 ppm soluble manganese, about 0.3 ppm to about 10 ppm soluble iron, preferably about 5 ppm to about 9 ppm soluble iron, most preferably about 6 ppm to 8 ppm soluble iron, and about 0.2 ppm to about 70 ppm soluble zinc (Zn +2 ).
  • the present invention also relates to a substantially pure culture of Botryococcus braunii var. Showa, strain Ninsei, having the ATCC Accession No. PTA-7441, its parts, and hydrocarbons produced therefrom.
  • FIG. 1 shows the meniscus at the top of a clear glass culture cylinder that is highly populated with colonies of Botryococcus sp. afloat at the surface and in the thin film of water being drawn above the meniscus by capillary action of the mass aggregate of colonies.
  • the culture was lightly tapped just prior to photography to release some colonies into the water below the meniscus to display separated colonies as best as possible, and, as a result, the colonies followed a sine wave pattern into submergence just below the surface. All colonies that were submerged by the wave, floated back again to the meniscus.
  • the unit of scale to the right is in millimeters.
  • the colonies can be cultured on any water-moistened solid surface that can hold and supply nutrients in an aqueous medium, including, paper, plastic, colloids, phycocolloids, agar, agarose, carrageenan, agar substitutes, textiles, gel, mud, soil, earth, shale, clays, foam, ceramics, concrete, brick, metal foam, wovens, nonwovens, and solid and liquid substrates of all types;
  • FIG. 2 illustrates a 5 ⁇ m breadth by 8 ⁇ m height cell of a colony showing an ovoid cell containing nine round hydrocarbon vesicles, a dictyosome, a nucleolus, and a parietal chloroplast;
  • FIG. 3 is a microphotograph of a green hydrocarbon-rich colony showing ovoid protuberant cells, each containing five and more round hydrocarbon vesicles. At the perimeter are seven hydrocarbon droplets extruded from the colony by pressure from the glass cover slip; and
  • FIG. 4 is an exemplary schematic diagram of a hydrocarbon production process of the present invention.
  • FIG. 1 shows floating colonies of Botryococcus sp. with a corresponding 30% hydrocarbon content. Additionally, the hydrocarbon-vesicle counts within cells, as drawn in FIG. 2 , were found to be in direct correlation to hydrocarbon yields, providing microscopy for confirming selection of cells and colonies with high hydrocarbon contents.
  • Colonies in hydrocarbon-enhancement were placed in the dark without supplemental carbon dioxide gas to eliminate artifacts of flotation from bubbles.
  • Environmental conditions were 150 ⁇ E/m 2 /sec PAR to 1700 ⁇ E/m 2 /sec PAR at 25-37° Centigrade.
  • Nutrient chemicals were reagent grade in all laboratory-scale experiments. In field-scale tests, fertilizer grade chemical nutrients were provided. In order to minimize or eliminate ammonia, nitrates were optionally kiln dried prior to admixture.
  • Supplementation with 0.1% to 100% carbon dioxide gas or carbonate e.g., 10 mg/ml sodium bicarbonate or potassium bicarbonate
  • Clones of Botryococcus sp. originated from the collections from Nature. Axenic clones, from the Ninsei strain disclosed in provisional application Ser. No. 60/678,711, filed May 6, 2005 and incorporated herein by reference, micropropagated rapidly in light on novel carbon- and nutrient-supplemented solid media. Solids included selections from 0.5% to 1.5% agar, colloids, gelatin, plastic gels, cellulose, plant fibers, synthetic fibers, polymers, woven fabrics, nonwovens, paper, broadcloth, iron, stainless steel, netting, moist glass, brick, concrete, plastic, foams, nylon, and ceramic surfaces.
  • Sunken colonies with very low hydrocarbon content were archived as controls in Showa nutrients, US Patent Plant 6169, and are incorporated by reference herein, to provide controls in experiments. Up to the point of experimentation, the varieties were variously maintained in Showa media; Chu 13 (Chu 1942); other conventional phycological nutrient media; and were supplemented with trace minerals, spring water, or soil-water extracts in aqueous solution. Botryococcus sp. was maintained in defined iron-, zinc- and phosphate-enriched media of the present invention.
  • Colonies of Ninsei are variably-shaped groups of cells held together in the cups of tough sporopollenin-like matrices. Depth of color depends on the light regime, density or culture and physiological state of the colonies. All color designations are made with reference to the Munsell Book of Color. Normal healthy colonies range from 5 GY 7/8 to 2.5 GY 8/12 on the Munsell color chart and these Ninsei colonies, fully pigmented with chlorophylls, may float at the surface of growing cultures with high hydrocarbon content that may reflect golden overtones.
  • cells of the colonies When released from the colonial matrix, cells of the colonies are 5 ⁇ m to 10 ⁇ m spheres often pressed by neighboring cells into irregular shapes. Within the colony, the cells are wedged into an almond-shape between neighboring cells. Deposits of hydrocarbon, 0.1 ⁇ m to 1 ⁇ m in diameter, are present in the cytoplasm, wall, and matrix. An occasional cell of Ninsei exhibits a depression at the outer tip of the cell that most frequently appears in cells with few hydrocarbon vesicles and may be a result of secretion of oils. The name of the strain is, in fact, derived from the urn shape of cells, reminiscent of shapes of large ceramic wares by the artist of Kyoto, ca. 1600 AD, Ninsei.
  • the colonial unit is spherical and aggregates of units contribute to the formation of irregular grape-like clusters observed in large colonies. During rapid growth of the novel strain, colonies are generally smaller than Showa's 50 ⁇ m colonies. In Ninsei, smaller colonies may range from 10 ⁇ m to 45 ⁇ m in diameter. Colonies of 100 or more cells are predominantly composed of irregularly shaped units that fragment into roughly rounded colonies. Ninsei is visually distinguishable from other strains of the variety by its deep green hue, small size attributable to rapid growth, cell structure, and niche. Botryococcus braunii var. Showa, strain Ninsei was deposited at the American Type Culture Collection (Post Office Box 1549, Manassas, Va. 20108) in March 2006 and assigned ATCC No. PTA-7441.
  • hydroponic culture media effectively suppress protistons by being far too highly concentrated for unicellular organisms.
  • Botryococcus species were found to grow in full-strength Hydroponic Solutions and, to the complete surprise of the inventor, in nutrient salt concentrations previously thought to be toxic to plants and to other life.
  • Phosphate salts provided the additional benefit of buffering within a range of pH 6 to pH 7. Furthermore, concentrations of soluble zinc ions (Zn +2 ) were increased one hundred times to two thousand times that of final concentrations found for soluble zinc in vegetable crop production; and soluble iron was increased up to quadruple the concentration used in vegetable solution culture.
  • KwiK The preferred nutrient enrichment solution of the present invention is hereinafter denoted as KwiK (see Table below). Botryococcus sp. was rapidly propagated in KwiK and it should be noted that the preferred mild formulation of KwiK contains over fifty times the total phosphates and many times higher Zn +2 concentrations than Chu 13 (Table Comparing Media, below) and hydroponic media.
  • the present invention comprises trials with Trebouxiophyceaen Botryococcus species; hereinafter Botryococcus sp.
  • Botryococcus sp. The preferred environment for maintenance of colonies required buffering by appropriate concentrations of nutrient phosphate salts and carbonate adjusted to pH 6.3 to pH 6.8, preferably to pH 6.7.
  • Methods, compositions and systems of the present invention provide means for in vitro growth of transport fuel hydrocarbons.
  • Stock cultures were taken from bottom-dwelling colonies that had been maintained in conventional liquid culture media, typified by approximately 1 ppm to 10 ppm phosphate, 0.01 ppm to 0.3 ppm iron, and 0.01 ppm to 0.5 ppm zinc, as for example, in Showa medium and other phycological media.
  • Aqueous nutrient solutions were placed in sterile culture tubes and flasks and steam sterilized twice for 60 minutes. Following inoculation and growth, colonies were concentrated with overnight settling and approximately one million submerged green colonies were collected from the bottom of culture vessels. Equal volumes were resuspended into replicate culture vessels with equal volumes of enhancement media in the highest light intensities available in preparation for experiments. Control cultures were transferred into equal volumes of Showa medium and placed side by side under identical conditions as controls.
  • the preferred buffered enrichment solution with 80 mM to 120 mM potassium phosphate was developed for the present invention.
  • the solution was adjusted to fall within a range of about pH 6.5 to pH 7 by regulation of approximately equimolar mono- and di-potassium phosphates (MKP and DKP) or other phosphate salts.
  • MKP and DKP equimolar mono- and di-potassium phosphates
  • Phosphates were selected because of the high-energy requirement of adenosine triphosphate, ATP, for metabolism of cellular resources into hydrocarbons.
  • Potassium was selected as the counter-ion of choice because it is a major nutrient that does not precipitate at high concentrations.
  • supplementation with 1 ppm to 90 ppm soluble Zn +2 was critical to acceleration of hydrocarbon chain elongation and 0.1 ppm to 10 ppm soluble iron was essential to photosynthesis. That is, in illuminated cultures in KwiK supplemented preferably with 5 ppm soluble zinc and iron, colonies grew hydrocarbons at an accelerated rate by provision of a controlled upwelling of Kwik power.
  • buoyant colonies of the present invention were characterized by high growth content, upwards of 5% to 50% dry weight of mixed lipids.
  • the preferred environment for maintenance of the floating colonies requires buffering by appropriate concentrations of available 20 mM to 125 mM phosphates, 3 ppm to 10 ppm Fe, and 0.1 ppm to 70 ppm Zn +2 , where 80 mM to 90 mM phosphates with 0.2 ppm to 45 ppm chelated Zn +2 is preferred.
  • Colonies tolerate a broad physiological range from pH 5.5 up to pH 8.3; however, under high light intensity and with carbonate availability, continuous adjustment to maintain pH 6.8 is essential to maintain the solubility of minerals in high concentrations of phosphate.
  • Organic substrates for enhancement of hydrocarbons include 1 mm to 100 mM Krebs Cycle carboxylates, preferably with citrate as an acid component of citrate-phosphate buffer; mevalonates; methionines, preferably adenosyl-methionine; alcohols; and fatty acids.
  • citrate as an acid component of citrate-phosphate buffer
  • mevalonates preferably adenosyl-methionine
  • alcohols preferably adenosyl-methionine
  • fatty acids fatty acids.
  • sequestering agents such as disodium-, diammonium-, and dipotassium-ethylenediaminetetraacetates; citrate; carboxylates; and the like.
  • maintenance of acidic environments assists with solubility of media with high concentrations of phosphate.
  • Agriculturally accepted sources of Zn +2 include, without exclusion of any other zinc salts, zinc sulfate, zinc oxide, zinc carbonate, zinc chloride, zinc citrate, zinc oxysulfate, zinc ammonium sulfate, and zinc nitrate, supplemented by chelation with, for example, salts of EDTA, HEEDTA, NTA, DTPA, EDDHA, and the like.
  • Commercially available 6% to 14% Zn +2 as diammonium EDTA may be alkaline which may be compensated by addition of the monobasic phosphate salt to adjust the final solution to pH 7.
  • Sources of iron include, without exclusion of any other iron supplements, iron sulfate, iron oxide, iron filings, ferric chloride, ferric ammonium citrates, ferrous salts, soil extracts, and supplemented by chelation with, for example, salts of EDTA, HEEDTA, NTA, DTPA, EDDHA, and the like.
  • the most highly preferred medium eliminates all sources of ammoniacal nitrogen in order to fully enhance hydrocarbon production of mass cultures. It is the hypothesis of the present invention that hydrocarbon synthesis may be fully optimized by providing nutrients beneficial to photosynthesis, including 50 ppm to 200 ppm magnesium as part of the chlorophyll molecule and about 5 ppm to 10 ppm soluble iron that is essential to electron transport.
  • inclusion of 7 ppm to 9 ppm soluble ferric or ferrous ions in the media accomplishes the same when balanced equally by 0.2 ppm soluble Mn, and with provision of high light intensity illumination, carbon dioxide gas, and 0.2 ppm to 50 ppm Zn +2 , the synthesis of hydrocarbons may be optimized.
  • phosphates For a population of Botryococcus colonies, 2 mM and greater concentrations of phosphates, 500 ppm to 1200 ppm nitrate salt, 500 ppm potassium salt, 3 ppm to 10 ppm Fe, 0.1 ppm to 3 ppm Mn, and 0.1 ppm to 5 ppm Zn +2 are required for long-term growth of hydrocarbons.
  • the recommended and preferred upper limits are 120 mM total phosphates at pH 7 and 50 ppm soluble zinc.
  • For hydrocarbon synthesis supplementations with 25 ppm to 250 ppm soluble magnesium, 0.2 ppm soluble manganese, 5-9 ppm soluble iron, and 0.1 ppm to 50 ppm Zn +2 , are preferred.
  • Trace metals are preferably chelated.
  • the medium is supplemented as specified in KwiK.
  • the preferred method for making ZiP is to mix and sterilize a solution of 160 mM to 400 mM total phosphates and add equal volumes of the phosphate solution to pre-sterilized KwiK resulting in 80 mM to 200 mM total phosphates ZiP solutions with chelated nutrients.
  • the preferred ZiP solution at 88 mM to 150 mM balanced phosphates with 2 ppm to 50 ppm Zn +2 and with 10 ppm to 20 ppm Fe in KwiK supports growth of hydrocarbons.
  • the biosynthesis of hydrocarbons is an energy-intensive pathway that may be accelerated by the availability of very high concentrations of ferrous, ferric, Zn +2 and phosphates of the present invention.
  • the enzymes in this system require phosphate-energy-complexes, such as the Zn +2 -requiring farnesyl pyrophosphate synthase, as demonstrated in the current invention.
  • phosphate-energy-complexes such as the Zn +2 -requiring farnesyl pyrophosphate synthase
  • KwiK Medium adjusted to pH 7 with phosphate buffer Concentration KwiK Component Range Preferred KH 2 PO 4 80-800 ppm 136 ppm K 2 HPO 4 80-1000 ppm 174 ppm KNO 3 500-2500 ppm 570 ppm
  • ZiP medium adjusted to pH 7 with phosphate buffers Concentration ZiP Component Range Preferred Phosphates 0.1% to 3% 0.5% to 0.8% Zn +2 0.2 ppm to 70 ppm 36 ppm to 50 ppm
  • Colonies of Botryococcus sp. were found to live and grow at the surface of the water or on moist substrates, whereas, other general methods of culture involved immersion in water.
  • Colonies grow particularly well in KwiK-supplemented water-moistened solid media under continuous or periodic (e.g., 16:8 h LD) PAR light exposure, with high light intensities up to direct sunlight as high as 500 to 1700 ⁇ E/m 2 /sec in high density cultures and temperatures of 20° to 37°.
  • continuous or periodic e.g. 16:8 h LD
  • growth media for the strains of the present invention include primary, secondary and trace metal plant nutrients.
  • the most highly preferred formula provides balanced N—P—K at many times the concentrations of conventional nutrients.
  • Balanced formulations include nitrate, phosphate and potash sources of fertilizers at rates exceeding hydroponics of flowering plants; as well as the secondary nutrients, Ca +2 , S, and Mg; and soluble micronutrients such as ions of Fe, Mn, Zn +2 , Cu, B, Mo, Co, and Ni.
  • a cocktail of metal ions is preferred for maintenance of growth of hydrocarbons and preferably include 5 mM to 25 mM magnesium, 0.1 ppm to 3 ppm manganese, 3 ppm to 10 ppm iron, and 0.01 mM+2 to 0.1 mM Zn + .
  • Total phosphates may be in the range from 2 mM to 150 mM phosphates.
  • Sources of typical solution culture nutrients are, for example, selected from myriad and various available compounds as generally accepted and known by those in the art.
  • Botryococcus sp. may have adapted to the chemical extremes of exclusive concentrations of phosphate, up to 3% in vitro, and as high as crystalline in Nature. Zinc and manganese have long histories of being formulated into human medications for their germ-fighting benefits and, thus, very high concentrations of Mn and Zn +2 provide clear competitive advantages for Botryococcus sp. to survive where other microorganisms die. Flotation enables it to be transported to live at the surface or edge of the exclusive moist solid medium. Botryococcus sp. occupies the defined niche of the present invention. The preponderance of mixed hydrocarbons, when accumulated in high cellular concentrations, function as naturally effective ultraviolet sunlight blocking agents, necessary for survival on land; and as such, the whole organism as well as its extract may be utilized in topical sun block formulae.
  • the preferred formulation of ZiP contains over 300 times the phosphate and 100 times the zinc concentrations of Chu 13 (Table Comparing Media, above).
  • the process system of the present invention is schematically depicted in FIG. 4 , wherein the mud niche is mimicked by provision of a continuously moistened solid medium such as a fabric beltway that is sufficiently tight in its weave to prevent the colonies from slipping through.
  • a continuously moistened solid medium such as a fabric beltway that is sufficiently tight in its weave to prevent the colonies from slipping through.
  • Nitex® Broadcloth 25 to 50 micron Nitex® Broadcloth is an appropriate selection.
  • Nitex® Broadcloth 10 microns to 600 microns is the material of choice for plankton nets.
  • the fabric belt is inoculated with Botryococcus spp. and growth is maintained by continuous misting with carbon dioxide gas-supplemented KwiK and natural solar illumination. Initially, gas-carbonation assists by sustaining acidity that prevents loss of metallic nutrients to precipitation.
  • Different oleomic strains, varieties and species may be interspersed in the culture.
  • the nutrient mist is replaced with a 10 mM bicarbonate-supplemented ZiP mist.
  • bicarbonate raises the alkalinity of the nutrient solution, thus, provision of an oleomic environment is dependent upon metering appropriate buffering agents into the culture environment to maintain solubility of nutrients.
  • the colonies are allowed sufficient time to ripen by visually monitoring hydrocarbon-vesicle counts within live cells taken through random samplings. At the determined time of maximum vesicle count exceeding 8 hydrocarbon-vesicles per cell in a given plane, as per FIG.
  • hydrocarbons are harvested from live cells by applying aqueous solutions or phytobland organic solvents as 30 PSI to 100 PSI pressurized misting sprays for 1 to 45 minutes. Applying pressure to exude hydrocarbons from cells was photographically recorded in FIG. 3 , where exogenous droplets of oil droplets were visually observed in vivo.
  • pressurized water is applied to the mat following a design that supplies sufficient hydrological shear to press hydrocarbons out of the colonies while keeping the cells alive; therefore, the least pressure that forces exudation of oils is preferred and is dependent on the thickness of the mat. The cycle is repeated until the inoculum is exhausted.
  • the formulations and methods of the present invention may be applied to virtually any variety of living organism that metabolizes hydrocarbons, most preferably photosynthetic organisms. These photosynthetic organisms include protistans, bacteria, and plants. Plants include innumerable agricultural and horticultural species and varieties, known arts to those in the field.
  • the methods of the present invention are amenable to batch processing of captive hydrocarbon vesicles.
  • Sheared botryococcenes allow the possibility of the continuous harvest of products.
  • industrial mimicry of Nature's competitive advantage represents an improvement on systems suited to the production and harvest of renewable hydrocarbons.
  • Botryococcenes are the natural product of choice as a starting material for a number of hydrocarbon based products, such as petrochemicals, pharmaceuticals, and fuels.

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Cited By (12)

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US20110021850A1 (en) * 2007-12-07 2011-01-27 Nigel Stewart Battersby Renewable base oil composition
WO2011058573A1 (fr) * 2009-11-13 2011-05-19 Agharkar Research Institute Of Maharashtra Association For The Cultivation Of Science Préservation de biomatériaux
WO2013161832A1 (fr) 2012-04-24 2013-10-31 富士フイルム株式会社 Procédé de culture d'une micro-algue, biofilm formé sur la surface d'un liquide par ledit procédé de culture, biomasse et huile toutes deux obtenues à partir dudit biofilm, procédé de collecte dudit biofilm, procédé de production de combustible de biomasse, micro-algue apte à former un biofilm sur une surface d'un liquide, biofilm formé sur une surface d'un liquide à l'aide de ladite micro-algue, et biomasse et huile toutes deux obtenues à partir dudit biofilm
JP2013226063A (ja) * 2012-04-24 2013-11-07 Fujifilm Corp 微細藻類の培養方法、該培養方法により液面上に形成されたバイオフィルム、該バイオフィルムから得られるバイオマス及びオイル、該バイオフィルムの回収方法、並びにバイオマス燃料の製造方法
JP2013226062A (ja) * 2012-04-24 2013-11-07 Fujifilm Corp 液面上にバイオフィルムを形成可能な微細藻類、該微細藻類により液面上に形成されたバイオフィルム、並びに該バイオフィルムから得られるバイオマス及びオイル
WO2014088010A1 (fr) * 2012-12-07 2014-06-12 富士フイルム株式会社 Procédé de récolte de phytoplancton à partir de microalgues sur une surface liquide, et de réalisation d'une mise en culture dans un récipient de culture séparé, dans le cadre d'un procédé de mise en culture de microalgues sur une surface liquide
JP2014113083A (ja) * 2012-12-07 2014-06-26 Fujifilm Corp 液面上での微細藻類の培養方法において、液面上の微細藻類を基板に転写回収した後、別の培養容器で培養を行う方法
WO2015041350A1 (fr) * 2013-09-20 2015-03-26 富士フイルム株式会社 Nouveau procédé pour la culture de cellules adhérentes dans une région formée entre du gel de polymère absorbant l'eau et un substrat, procédé pour la fabrication de biomasse et nouvelle microalgue
WO2015041349A1 (fr) * 2013-09-20 2015-03-26 富士フイルム株式会社 Procédé de culture flottante de microalgues à la surface d'un liquide utilisant des microalgues sur la surface de fond en tant qu'algues d'ensemencement, procédé de production de biomasse algale, et microalgue
WO2015041351A1 (fr) * 2013-09-20 2015-03-26 富士フイルム株式会社 Procédé de culture de microalgues améliorant le rapport de la teneur en huile, procédé de production de biomasse algale, et nouvelle microalgue
JP2015057991A (ja) * 2013-09-20 2015-03-30 富士フイルム株式会社 貫通部位を有する構造体を用いる、微細藻類の液面浮遊培養法及び回収方法
JP2015057990A (ja) * 2013-09-20 2015-03-30 富士フイルム株式会社 液面浮遊培養による多段培養法

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KR101090361B1 (ko) 2009-02-02 2011-12-07 디브이에스 코리아 주식회사 미세조류를 이용한 탄화수소의 제조방법
US20120171734A1 (en) * 2009-07-01 2012-07-05 The Regents Of The University Of California Extraction of extracellular terpenoids from microalgae colonies
CA2774166A1 (fr) 2009-09-15 2011-03-24 Wwcc Limited Molecule d'acide nucleique codant pour une synthase triterpenoide
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US20110021850A1 (en) * 2007-12-07 2011-01-27 Nigel Stewart Battersby Renewable base oil composition
WO2011058573A1 (fr) * 2009-11-13 2011-05-19 Agharkar Research Institute Of Maharashtra Association For The Cultivation Of Science Préservation de biomatériaux
EP2843037A4 (fr) * 2012-04-24 2015-11-18 Fujifilm Corp Procédé de culture d'une micro-algue, biofilm formé sur la surface d'un liquide par ledit procédé de culture, biomasse et huile toutes deux obtenues à partir dudit biofilm, procédé de collecte dudit biofilm, procédé de production de combustible de biomasse, micro-algue apte à former un biofilm sur une surface d'un liquide, biofilm formé sur une surface d'un liquide à l'aide de ladite micro-algue, et biomasse et huile toutes deux obtenues à partir dudit biofilm
WO2013161832A1 (fr) 2012-04-24 2013-10-31 富士フイルム株式会社 Procédé de culture d'une micro-algue, biofilm formé sur la surface d'un liquide par ledit procédé de culture, biomasse et huile toutes deux obtenues à partir dudit biofilm, procédé de collecte dudit biofilm, procédé de production de combustible de biomasse, micro-algue apte à former un biofilm sur une surface d'un liquide, biofilm formé sur une surface d'un liquide à l'aide de ladite micro-algue, et biomasse et huile toutes deux obtenues à partir dudit biofilm
JP2013226063A (ja) * 2012-04-24 2013-11-07 Fujifilm Corp 微細藻類の培養方法、該培養方法により液面上に形成されたバイオフィルム、該バイオフィルムから得られるバイオマス及びオイル、該バイオフィルムの回収方法、並びにバイオマス燃料の製造方法
JP2013226062A (ja) * 2012-04-24 2013-11-07 Fujifilm Corp 液面上にバイオフィルムを形成可能な微細藻類、該微細藻類により液面上に形成されたバイオフィルム、並びに該バイオフィルムから得られるバイオマス及びオイル
AU2013253520B2 (en) * 2012-04-24 2016-12-01 Fujifilm Corporation Method for culturing microalgae, biofilm formed on liquid surface by the culturing method, biomass and oil obtained from the biofilm, method for collecting the biofilm, method for producing biomass fuel, microalgae capable of forming biofilm on liquid surface, biofilm formed on liquid surface using the microalgae, and biomass and oil obtained from the biofilm
WO2014088010A1 (fr) * 2012-12-07 2014-06-12 富士フイルム株式会社 Procédé de récolte de phytoplancton à partir de microalgues sur une surface liquide, et de réalisation d'une mise en culture dans un récipient de culture séparé, dans le cadre d'un procédé de mise en culture de microalgues sur une surface liquide
JP2014113083A (ja) * 2012-12-07 2014-06-26 Fujifilm Corp 液面上での微細藻類の培養方法において、液面上の微細藻類を基板に転写回収した後、別の培養容器で培養を行う方法
JP2014113082A (ja) * 2012-12-07 2014-06-26 Fujifilm Corp 液面上での微細藻類の培養方法において、液面上の微細藻類から種藻を採取し、別の培養容器で培養を行う方法
WO2015041350A1 (fr) * 2013-09-20 2015-03-26 富士フイルム株式会社 Nouveau procédé pour la culture de cellules adhérentes dans une région formée entre du gel de polymère absorbant l'eau et un substrat, procédé pour la fabrication de biomasse et nouvelle microalgue
JP2015057991A (ja) * 2013-09-20 2015-03-30 富士フイルム株式会社 貫通部位を有する構造体を用いる、微細藻類の液面浮遊培養法及び回収方法
JP2015057990A (ja) * 2013-09-20 2015-03-30 富士フイルム株式会社 液面浮遊培養による多段培養法
JP2015192649A (ja) * 2013-09-20 2015-11-05 富士フイルム株式会社 オイル含有率を向上させた微細藻類の培養方法、藻類バイオマスの製造方法、及び新規微細藻類
JP2015192647A (ja) * 2013-09-20 2015-11-05 富士フイルム株式会社 底面の微細藻類を種藻として用いる、微細藻類の液面浮遊培養方法、藻類バイオマスの製造方法、及び微細藻類
JP2015192648A (ja) * 2013-09-20 2015-11-05 富士フイルム株式会社 吸水性高分子ゲルと基板間で形成される領域での新規付着培養方法、バイオマスの製造方法、及び新規微細藻類
WO2015041351A1 (fr) * 2013-09-20 2015-03-26 富士フイルム株式会社 Procédé de culture de microalgues améliorant le rapport de la teneur en huile, procédé de production de biomasse algale, et nouvelle microalgue
WO2015041349A1 (fr) * 2013-09-20 2015-03-26 富士フイルム株式会社 Procédé de culture flottante de microalgues à la surface d'un liquide utilisant des microalgues sur la surface de fond en tant qu'algues d'ensemencement, procédé de production de biomasse algale, et microalgue

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EP1920062A4 (fr) 2011-04-20
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US20060265800P1 (en) 2006-11-23
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