WO2015048423A1 - Souches d'algues tolérantes aux biocides - Google Patents

Souches d'algues tolérantes aux biocides Download PDF

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WO2015048423A1
WO2015048423A1 PCT/US2014/057688 US2014057688W WO2015048423A1 WO 2015048423 A1 WO2015048423 A1 WO 2015048423A1 US 2014057688 W US2014057688 W US 2014057688W WO 2015048423 A1 WO2015048423 A1 WO 2015048423A1
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ppm
acres
liters
fungicide
years
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PCT/US2014/057688
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Matthew Saunders
Adam HANLEY
Melisa Low
Philip Lee
Christopher Yohn
Salvador Lopez
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Sapphire Energy, Inc.
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Publication of WO2015048423A1 publication Critical patent/WO2015048423A1/fr

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    • 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
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/03—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
    • C12N1/125—Unicellular algae isolates
    • 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
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • 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

Definitions

  • This disclosure includes mutagenized strains of niicroalgae that ha ve improved tolerance to a biocide and in particular a fungicide. These mutagenized strains can be grown in liquid systems, such as pools, ponds, and the like. The mutagenized strains' improved tolerance to a biocide results in, for example, an increase in the biomass yield and/or biomass productivity and/or growth of the strains. This disclosure also includes methods for suppressing, inhibiting, or reducing the growth of a fungus in such systems. Such systems are useful for the production of biomass such as
  • niicroalgae for example, a Desmodesmus species.
  • a Desmodesmus species can be, for example, Desrnodesmus armatus.
  • Biomass produced using the mutagenized strains and methods described herein can be used to produce a variety of useful products.
  • the biomass produced is used for the production of oil which can be refined into a variety of products, including, but not limited to, transportation fuels.
  • Microalgae are unicellular non-vascular photosynthetic organisms, producing oxygen by photosynthesis.
  • One group, the microalgae are useful for biotechnology applications for many reasons, including their high growth rate and tolerance to varying environmental conditions.
  • Use of microalgae in a variety of industrial processes for commercially important products has been reported.
  • microalgae have u ses in the production of nutritional supplements, pharmaceuticals, natural dyes, a food source for fish and crustaceans, biological control of agricultural pests, production of oxygen and removal of nitrogen, phosphorus and toxic substances in sewage treatment, and pollution controls, such as biodegradation of plastics or uptake of carbon dioxide.
  • Fuel products such as oil, petrochemicals, and other substances useful for the production of
  • Microalgae can produce 10 to 100 times as much mass as terrestrial plants in a year.
  • Microalgae also produce oils (lipids) and starches that may he converted into hiofuels. These microalgae can grow almost anywhere, although are most commonly found at latitudes between 40 N and 40 S. With more than 100,000 known species of diatoms (a type of microalgae), 40,000 known species of green microalgae, and smaller numbers of other microalgae species, microalgae will grow rapidly in nearly any environment, with almost any kind of water, including marginal areas with limited or poor quality water.
  • Microalgae can store energy in the form of either oil or starch. Stored oil can be as much as 60% of the weight of the microalgae. Certain species which are enhanced in oil or starch production have been identified, and growing conditions have been tested. Processes for extracting and converting these materials to fuels have also been developed.
  • Microalgae such as a Desmodesmus species (for example, Desmodesmus armatus), grown in outdoor ponds are exposed to a diversity of natural predators including, for exampl e, ehytrids, rotifers, and amoebas. Infestations with certain predators are common and often have adverse effects on the pond's biomass productivity, while extreme infestations may even decimate ("crash") an outdoor pond culture.
  • One method to protect the microalgae from natural predators is to use commercially available fungicides, such as OMEGA 3 ⁇ 4 500F (Syngenta; USA) (hereinafter
  • OEGA 1*1 Although the therapeutic doses of biocides are effective to control predators, the biocide itself can have toxic effects on the microalgae and may reduce biomass productivity and/or biomass quality. Thus, microalga strains with improved (increased) tolerance to biocides may help improve field productivity overall, since biocide-tolerant microalga! strains should produce more biomass (improved biomass productivity) than a non-tolerant strains (e.g. a wild type microalga! strain) upon routine pesticide treatments. Thus, there exists a need for the creation, isolation, and characterization of microalga! strains with increased biocide-tolerance that can result in an increased ield of commercially important products, such as fuel products.
  • the invention is directed to an isolated microorganism (OM27) of the species deposited under ATCC Accession No. PTA-120600.
  • the isolated microorganism (OM27) also known herein as a Desmodesmus armatus ATCC No. PTA-120600.
  • the isolated OM27 also known herein as a Desmodesmus armatus ATCC No. PTA-120600.
  • the invention is directed to an isolated microorganism(s) having the characteristics of the
  • microorganism deposited under ATCC Accession No. PTA 120600 (OM27).
  • the characteristics of the species deposited under ATCC Accession No. PTA- 120600 include its growth rate, biomass productivity, in situ productivity, doubling rate, photosynthetic efficiency, photosynthetic health, lipid profiles, FAME content, and its nucleotide sequence or sequences.
  • die invention is directed to an isolated microorganism (OM65) of the species deposited under ATCC Accession No. PTA- 120599.
  • the isolated microorganism (OM65) is also known herein as a Desmodesmus armatus ATCC No. PTA-120599.
  • the isolated microorganism (OM65) is also known herein as a Desmodesmus armatus ATCC No. PTA-120599.
  • microorganism (OM65) associated with ATCC Accession No. PTA-120599 was deposited under the Budapest Treaty on September 24, 2013, at the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA, 201 10-2209.
  • the invention is directed to an isolated microorganism (s) having the characteristics of the
  • microorganism deposited under ATCC Accession No. PTA-120599 (OM65).
  • the characteristics of the species deposited under ATCC Accession No. PTA-120599 include its growth rate, biomass productivity, in situ productivity, doubling rate, photosynthetic efficiency, photosynthetic health, lipid profiles, FAME content, and its nucleotide sequence or sequences.
  • an ultraviolet (UV) mutagenized Desmodesmus strain selected from the group consisting of OM65 and OM27, with an increased tolerance to a fungicide composition as compared to an unmutagenized Desmodesmus strain's tolerance to the fungicide composition, wherein the active ingredient of the fungicide composition is a Fungicide Resistance Action Committee 29 (FRAC 29) fungicide.
  • the FRAC 29 fungicide is any one or more of Fluazinam, Bmapacryl, Meptyldinocap, or Dinocap.
  • the FRAC 29 fungicide is an uncoupler of oxidative phosphorylation.
  • the FRAC 29 fungicide comprises from 10%-20% of the fungicide composition, from 20%-30% of the fungicide composition, from 30%-40% of the fungicide composition, from 40% ⁇ 50% of the fungicide composition, from 50%-60% of the fungicide composition, from 60%-70% of the fungicide composition, from 70%-80% of the fungicide composition, from 80%-90%, or from 90% to 100% of the fungicide composition.
  • UV mutagenized Desmodesmus strain selected from the group consisting of OM65 and OM27 with an increased tolerance to a fungicide composition wherein the active ingredient is Fluazinam, as compared to an unmutagenized Desmodesmus strain's tolerance to the fungicide composition.
  • Fluazinam comprises from 10-20% of the fungicide composition, from 20%-30% of the fungicide composition, from 30%-40% of the fungicide composition, from 40%-50% of the fungicide composition, from 50%-60% of the fungicide composition, from 60%-70% of the fungicide composition, from 70%-80% of the fungicide composition, from 80%-90%, or from 90% to 100%) of the fungicide composition, In another embodiment, both the mutagenized Desmodesmus strain and the unmutagenized
  • Desmodesmus strain are treated with one or more doses of the fungicide composition.
  • any one or more dose of the fungicide composition is from 0.25 ppm to 15 ppm, or 0,01 ppm to 20 ppm.
  • any one or more dose of the fungicide composition is from 0.1 ppm to 20 ppm, from 0.25 ppm to 2.5 ppm, from 0.25 ppm to 5.0 ppm, from 0.25 ppm to 10 ppm, from 0.25 ppm to 12,5 ppm, from 0.25 ppm to 15 ppm, or from 0.25 ppm to 20 ppm.
  • any one or more dose of the fungicide composition is from 0.5 ppm to 5.0 ppm, from 5 ppm to 10 ppm, from 10 ppm to 20 ppm, from 20 ppm to 50 ppm, or from 50 ppm to about 100 ppm.
  • any one or more dose of the fungicide composition is about 0.5 ppm, about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 11 ppm, about 12 ppm, about 13 ppm, about 14 ppm, about 1 ppm, about 16 ppm, about 17 ppm, about 18 ppm, about 19 ppm, or about 20 ppm.
  • both the mutagenized Desmodesmus strain and the unmutagenized Desmodesmus strain are treated with two to six doses of the fungicide composition.
  • the increase in tolerance is defined as any one or more of an increase in: growth rate, biomass productivity, in situ productivity, doubling rate, photosynthetic efficiency, photosynthetic health, or FAME content.
  • the increase in tolerance is defined as an improved color of pigmentation,
  • the increase in tolerance is defined as an increase in growth rate.
  • the increase in tolerance is defined as an increase in biomass productivity.
  • the increase in biomass productivity is 2% to 5%, 5% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 75% to 80%, 85% to 90%, 95% to 100%, 100% to 150%, 150% to 200%, 200% to 250%, 250% to 300%, 300% to 350%, 350% to 400%, or more than 400% ⁇ .
  • biomass productivity is measured in dry weight (DW) grams per liter (g/L).
  • the increase in tolerance is defined as an increase in situ productivity.
  • the increase in in situ productivity is 2% to 5%, 5% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 75% to 80%, 85% to 90%, 95% to 100%, 100% to 150%, 150% to 200%, 200% to 250%, 250% to 300%», 300% to 350%, 350% to 400%, or more than 400%.
  • the increase in in situ productivity is 2% to 5%, 5% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 75% to 80%, 85% to 90%, 95% to 100%, 100% to 150%, 150% to 200%, 200% to 250%, 250% to 300%», 300% to 350%, 350%
  • productivity is about a 20% mcrease, about a 28%), about a 30% increase, or about a 118%» increase,
  • in situ productivity is measured in g/m 2 /day
  • the mcrease in tolerance is defined as an increase in doubling rate.
  • the increase in tolerance is defined as an increase in photosvntlietic efficiency.
  • the photosvntlietic efficiency is determined by Pulse-Amplitude-Modulation (TP AM) fluorometry (Fv/Fm).
  • TP AM Pulse-Amplitude-Modulation
  • Fv/Fm Pulse-Amplitude-Modulation
  • the increase in tolerance is defined as an increase in
  • the photosynthetic health is determined by Pulse- Amplitude-Modulation (PAM) fluorometry (Fv/Fm).
  • PAM Pulse- Amplitude-Modulation
  • Fv/Fm Pulse- Amplitude-Modulation
  • the increase in tolerance is defined as an increase in FAME content.
  • the increase in FAME content is 1.25-fold to two-fold higher, two-fold to three-fold higher, three-fold to four-fold higher, or fourfold to five-fold higher.
  • the mutagenized Desmodesmus species is genetically engineered prior to being mutagenized. In another embodiment, the mutagenized Desmodesmus species is genetically engineered after being mutagenized.
  • a fungus on which the fungicide composition acts is a Chytridiomycoia or a Crypiomycota.
  • the Chytridiomycota is a Chytridiales, a Rhizophylctidales, a Spizellomycetales, a Rhizophydiales , a Lobulomycetales, a Cladochytriales, a Polychytrium, or a Monoblepharidomycetes.
  • an ultraviolet (LTV 7 ) mutagenized Desmodesmus strain selected from the group consisting of OM65 and OM27 with an increased tolerance to OMEGA*, as compared to an unmutagenized Desmodesmus strain's tolerance to OM EGA* ' .
  • both the mutagenized Desmodesmus strain and the unmutagenized Desmodesmus strain are trea ted with one or more doses of OMEGA ⁇ .
  • any one or more dose of OMEGA* is from 0.25 ppm to 15 ppm, or from 0.01 ppm to 20 ppm.
  • any one or more dose of OMEGA* is from 0.1 ppm to 20 ppm, from 0.25 ppm to 2.5 ppm, from 0.25 ppm to 5.0 ppm, from 0,25 ppm to 10 ppm, from 0.25 ppm to 12.5 ppm, from 0.25 ppm to 15 ppm, or from 0.25 ppm to 20 ppm. In other embodiments, any one or more dose of OMEGA* is from 0.5 ppm to 5.0 ppm, from 5 ppm to 10 ppm, from 10 ppm to 20 ppm, from 20 ppm to 50 ppm, or from 50 ppm to about 100 ppm.
  • any one or more dose of OMEGA* is about 0.5 ppm, about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 11 ppm, about 12 ppm, about 13 ppm, about 14 ppm, about 15 ppm, about 16 ppm, about 17 ppm, about 18 ppm, about 19 ppm, or about 20 ppm, In one embodiment, both the mutagenized Desmodesmus strain and the unmutagenized Desmodesmus strain are treated with two to six doses of OMEG A*.
  • the increase in tolerance is defined as any one or more of an increase in: growth rate; biomass productivity; in situ productivity; doubling rate; photosynthetic efficiency; photosynthetic health; or FAME content.
  • the increase in tolerance is defined as an improved color of pigmentation.
  • the increase in tolerance is defined as an increase in growth rate.
  • the increase in tolerance is defined as an increase in biomass productivity.
  • the increase in biomass productivity is 2% to 5%, 5% to 10%, 10% to 15%), 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 75% to 80%, 85% to 90%, 95% to 100%, 100% to 150%, 150% to 200%, 200% to 250%, 250% to 300%, 300% to 350%, 350% to 400%, or more than 400%).
  • biomass productivity is measured in dry w r eight (DW) grams per liter (g/L).
  • the increase in tolerance is deimed as an increase in situ productivity.
  • the increase in in situ productivity is 2% to 5%, 5% to 10%, 10% to 15%, 1 % to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 75% to 80%, 85% to 90%, 95% to 100%, 100% to 1 0%, 150% to 200%, 200% to 250%, 250% to 300%, 300%, to 350%, 350% to 400%, or more than 400%.
  • the increase in in situ productivity is about a 20% increase, about a 28%, about a 30% increase, or about a 1 18% increase.
  • in situ productivity is measured in g/m 2 /day.
  • the increase in tolerance is defined as an increase in doubling rate.
  • the increase in tolerance is defined as an increase in photosynthetic efficiency.
  • the photosynthetic efficiency is determined by Pulse- Amplitude-Modulation (PAM) fluorometry (Fv/Fm).
  • PAM Pulse- Amplitude-Modulation
  • the increase in tolerance is defined as an increase in photosynthetic health.
  • the increase in tolerance is defined as an increase in photosynthetic health.
  • the photosynthetic health is determined by Pulse- Amplitude-Modulation (PAM) fluorometry (Fv Fm).
  • PAM Pulse- Amplitude-Modulation
  • Fv Fm Pulse- Amplitude-Modulation
  • the increase in tolerance is defined as an increase in FAME content.
  • the increase in FAME content is 1 ,25-fold to two-fold higher, two-fold to three-fold higher, three-fold to four-fold higher, or four-fold to five-fold higher.
  • the mutagenized Desmodesmus species is genetically engineered prior to being mutagenized, In another embodiment, the mutagenized Desmodesmus species is genetically engineered after being mutagenized.
  • a fungus on which the fungicide composition acts is a
  • Chytridiomycota or a Cryptomycota.
  • the Chytridiomycota is a
  • Chytridiales a Rhizophylctidales, a Spizellomycetales, a Rhizop ydi les, a Lobulomycetales, a Cladochytriales, a Polychytrium, or a Monohlepharidomycetes .
  • mutagenized Desmodesmus species is OM65. In another embodiment, the mutagenized
  • Desmodesmus species is OM65 and the species was deposited under ATCC Accession No. PTA- 120599 on September 24, 2013.
  • the mutagenized Desmodesmus species is OM27.
  • the mutagenized Desmodesmus species is OM27 and the species was deposited under ATCC Accession No. PTA-120600 on September 24, 2013.
  • Desmodesmus species OM65 that is tolerant to OMEGA* at a concentration of from 0,01 ppm to 20 ppm which is deposited under ATCC Accession No. PT A 120599.
  • the Desmodesmus species OM65 is tolerant to OMEGA* at a concentration of from 0.25 ppm to 2,5 ppm, from 0.25 ppm to 5.0 ppm, from 0.25 ppm to 10 ppm, from 0.25 ppm to 12.5 ppm, from 0.25 ppm to 15 ppm, or from 0.25 ppm to 20 ppm.
  • Desmodesmus species OM27 that is tolerant to OMEGA* at a concentration of from 0.01 ppm to 20 ppm which is deposited under ATCC Accession No. PTA- 120600.
  • the Desmodesmus species OM27 is tolerant to OMEGA ® at a concentration of from 0.25 ppm to 2,5 ppm, from 0.25 ppm to 5.0 ppm, from 0.25 ppm to 10 ppm, from 0.25 ppm to 12.5 ppm, from 0.25 ppm to 15 ppm, or from 0.25 ppm to 20 ppm.
  • OM65 isolated microorganism
  • ATCC American Type Culture Collection
  • OM27 isolated microorganism
  • OM27 deposited under ATCC Accession No. PTA-600120 on September 24, 2013 with the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, CA, 20110, USA,
  • the method further comprises treating the liquid culture system either before or after step b) or both before and after step b) with an effective concentration of a fungicide composition to reduce the growth of the fungus, wherein die active ingredient of the fungicide composition is a Fungicide Resistance Action Committee 29 (FRAC 29) fungicide.
  • the Desmodesmus species is mutagenized using ultraviolet (UV) radiation.
  • the FRAC ' 29 fungicide is any one or more of Fluazinam, Binapacryl, Meptyldinocap, or Dinocap.
  • the FRAC 29 fungicide is Fluazinam.
  • the fungicide composition is OMEGA*.
  • the FRAC 29 fungicide is an uncoupler of oxidative phosphorylation.
  • the FRAC 29 fungicide comprises from 10%-20% of the fungicide composition, from 20%-30% of the fungicide composition, from 30%-4Q% of the fungicide composition, from 40%-50% of the fungicide composition, from 50%- 60% of the fungicide composition, from 60%-70% of the fungicide composition, from 70%-80% of the fungicide composition, from 80% ⁇ 90% or from 90% to 100% of the fungicide composition ,
  • the treating step is a prophylactic treating step.
  • the liquid culture system may be treated with the fungicide composition on multiple occasions. In another embodiment, the liquid culture system is treated one or more times with one or more effective concentrations of the fungicide composition.
  • any one or more of the effecti ve concentrations is from 0.25 ppm to 15 ppm, or 0.01 ppm to 20 ppm. In some embodiments, any one or more of the effective concentrations is from 0.1 ppm to 20 ppm, from 0,25 ppm to 2.5 ppm, from 0.25 ppm to 5.0 ppm, from 0.25 ppm to 10 ppm, from 0.25 ppm to 12.5 ppm, from 0.25 ppm to 15 ppm, or from 0.25 ppm to 20 ppm.
  • any one or more of the effective concentrations is from 0.5 ppm to 5,0 ppm, from 5 ppm to 10 ppm, from 10 ppm to 20 ppm, from 20 ppm to 50 ppm, or from 50 ppm to about 100 ppm.
  • any one or more of the effective concentrations is about 0.5 ppm, about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 1 1 ppm, about 12 ppm, about 13 ppm, about 14 ppm, about 15 ppm, about 16 ppm, about 17 ppm, about 18 ppm, about 19 ppm, or about 20 ppm.
  • the liquid cuiture system is treated two to six times with one or more effective concentrations of the fungicide composition.
  • the fungus in which the fungicide composition acts on is a Chytridiom cota or a Cryptomycot .
  • the Chytridiomycota is a Chytridiales, a Rhizophylctidales, a Spizellomycetales, a Rhizophydiales, a Lobulomycetales, a Cladockytriales, a Polychytrium, or a Monoblepharidomycetes.
  • the reduction in growth is measured by any one or more of growth rate, biomass productivity, in situ productivity, or doubling rate
  • the matagemzed Desmodesmus species is one or both of OM65 and OM27
  • the mutagenized Desmodesmus species is one or both of OM65 and OM27 deposited under ATCC Accession No. PTA-120599 and A.TCC Accession No. PTA-120600, respectively, on September 24, 2013 with the American Type Culture Collection (ATCC).
  • the muiagenized Desmodesmus species is genetically engineered prior to being mutagenized.
  • the mutagenized Desmodesmus species is genetically engineered after being mutagenized.
  • the mutagenized Desmodesmus species is grown in the liquid culture system.
  • the gro wing is for a number of days selected from the group consisting of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 30 or more, 45 or more, 60 or more, 90 or more, 120 or more, 1 80 or more, 250 or more, 500 or more, 1000 or more, 1500 or more, and 2000 or more days after inoculation.
  • the growing is from 4 to 6 days, from 4 to 14 days, or is 96 hours or more, after the inoculation.
  • the growing is from 1 to 30 days, 1 to 40, from 30 to 60 days, from 60-90 days, or from 90-120 days
  • the algae is grown from 1 to 7 days, from 1 week to 2 weeks, from 2 weeks to 3 weeks, from 3 weeks to 1 month, from 1 month to 3 months, from 3 months to 6 months, from 6 months to 9 months, from 9 months to 12 months, from 12 months to 18 months, from 18 months to 2 years, from 2 years to 3 years, from 3 years to 4 years, from 4 years to 5 years, from 5 years to 6 years, from 6 years to 7 years, from 7 years to 8 years, from 8 years to 9 years or from 9 years and 10 years after inoculation.
  • the method further comprises providing one or more additional amounts of the fungicide composition to maintain an effective concentration of the fungicide composition during the growing.
  • the liquid culture system is an open outdoor liquid culture system.
  • the open outdoor culture system has a volume of: at least 20,000 liters, at least 40,000 liters, at least 80,000 liters, at least 100,000 liters, at least 150,000 liters, at least 200,000 liters, at least 250,000 liters, at least 500,000 liters, at least 600,000 liters, or at least 1,000,000 liters; or 10,000 to 20,000 liters, 10,000 to 40,000 liters, 10,000 to 80,000 liters, 10,000 to 100,000 liters, 10,000 to 150,000 liters, 10,000 to 200,000 liters, 10,000 to 250,000 liters, 10,000 to 500,000 liters, 10,000 to 600,000 liters, 10,000 to 1,000,000 liters, 20,000 to 40,000 liters, 20,000 to 80,000 liters, 20,000 to 100,000 liters,
  • the open outdoor liquid culture system has an area selected from the group consisting of at least 0.25 acre, at least 0.5 acre, at least 1.0 acre, at least 1.5 acres, at least 2.0 acres, at least 2.5 acres, at least 5.0 acres, and 7.5 or more acres.
  • the open outdoor liquid culture system has an area selected from the group consisting of 0.25 to 0.5 acres, 0.25 to 1.0 acres, 0.25 to 1.5 acres, 0.25 to 2.0 acres, 0.25 to 2.5 acres, 0.25 to 5.0 acres, 0.25 to 7.5 acres, 0.5 to 1.0 acres, 0.5 to 1.5 acres, 0.5 to 2.0 acres, 0.5 to 2.5 acres, 0.5 to 5.0 acres, 0.5 to 7.5 acres, 1.0 to 1.5 acres, 1.0 to 2.0 acres, 1 .0 to 2.5 acres, 1.0 to 5.0 acres, 1.0 to 7.5 acres, 2.0 to 2.5 acres, 2,0 to 5.0 acres, 2.0 to 7,5 acres, 2.5 acres to 5.0 acres, 2.5 to 7.5 acres, 7,5 to 10 acres, 10 to 15 acres, 1 to 20 acres, 20 to 25 acres, 25 to 30 acres, 30 to 35 acres, 35 to 40 acres, 40 to 45 acres, 45 to 50 acres, 50 to 75 acres, 75 to 100 acres, 100 to 125 acres, 125 to 150 acres, 150 to 175 acres, 175 to 200 acres, 200 to 250 acres,
  • the liquid culture system is a mono-culture.
  • the growing provides a yield of mutagenized Desmodesmus species greater than 0.4 gram per liter (g/L) ash free dry weight (AFDW).
  • the yield is selected from the group consisting of greater than 0.5 g/L, greater than 0.6 g/L, greater than 0.7 g/L, greater than 0.8 g/L, greater than 0.9 g/L, and greater than 1.0 g/L AFDW.
  • the growing provides a yield of the mutagenized Desmodesmus species that is at least 5% greater or at least 10% greater than a yield of an
  • the growing provides a yield of the mutagenized Desmodesmus species selected from the group consisting of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, and 100% greater than the yield of an unmutagenized
  • Desmodesmus species harvested from a second liquid culture system having a fungal infection that has not been provided the effective concentration of the fungicide composition In some
  • the growing provides a yield of the mutagenized Desmodesmus species selected from the group consisting of at least 1 ,25 fold, 1.5 fold. 2.0 fold, 2,5 fold, 5.0 fold, 7,5 fold, 10 fold and 1 fold greater than the yield of an unmutagenized Desmodesmus species harvested from a second liquid culture system having a fungal infection that has not been provided the effective
  • the mutagenized Desmodesmus species in the liquid culture system is harvested, in another embodiment, a fuel product is obtained from the harvested mutagenized Desmodesmus species, In other embodiments, the fuel product is an oil, a petrochemical, or a substance useful in the production of a petrochemical.
  • Pro vided herein is a method of reducing the growth of a fungus in a liquid culture system, comprising: a) providing a liquid culture system; b) inoculating the liquid culture system with at least one mutagenized Desmodesmus species selected from the group consisting of OM27 and OM65; and c) treating the liquid culture system either before or after step b) or both before and after s tep b) with an effecti ve concentration of a fungicide composition to reduce the growth of the fungus, wherein the active ingredient of the fungicide composition is a Fungicide Resistance Action Committee 29 (FRAC 29) fungicide.
  • the Desmodesmus species is mutagenized using ultraviolet (UV) radiation.
  • the FRAC ' 29 fungicide is any one or more of Fluazinam, Binapacryl, Meptyldinocap, or Dinocap.
  • the FRAC 29 fungicide is Fluazinam.
  • the fungicide composition is OMEGA " ".
  • the FRAC 29 fungicide is an uncoupler of oxidative phosphorylation.
  • the FRAC 29 fungicide comprises from 10%-20% of the fungicide composition, from 20%-3()% of the fungicide composition, from 30%-40% of the fungicide composition, from 40%-50%) of the fungicide composition, from 50%-60% of the fungicide composition, from 60%- 70% of the fungicide composition, from 70%-80% of the fungicide composition, from 80%-90%, or from 90% to 100% of the fungicide composition.
  • the treating step is a prophylactic treating step.
  • the liquid culture system is treated one or more times with one or more effective concentrations of the fungicide composition. In other
  • any one or more of the effective concentrations is from 0.25 ppm to 15 ppm, or 0.01 ppm to 20 ppm. in some embodiments, any one or more of the effective concentrations is from 0.1 pni to 20 ppni, from 0.25 ppm to 2.5 ppm, from 0.25 ppm to 5.0 pm, from 0.25 ppm to 10 ppm, from 0.25 ppm to 12.5 ppm, from 0.25 ppm to 15 ppm, or from 0.25 ppm to 20 ppm.
  • any one or more of the effective concentrations is from 0.5 ppm to 5.0 ppm, from 5 ppm to 10 ppm, from 10 ppm to 20 ppm, from 20 ppm to 50 ppm, or from 50 ppm to about 100 ppm.
  • any one or more of the effective concentrations is about 0.5 ppm, about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 1 1 ppm, about 12 ppm, about 13 ppm, about 14 ppm, about 15 ppm, about 16 ppm, about 17 ppm, about 18 ppm, about 19 ppm, or about 20 ppm.
  • the liquid culture system is treated two to six times with one or more effective concentrations of the fungicide composition.
  • the fungus in which the fungicide composition acts on is a Chytridiomycota or a Cryptomycota.
  • the Chytridiomycota is a Chytridiales, a Rhizophylctidales, a Spizellomycetales, a Rhizophydiales, a Lobulomycetales, a Cladochytriales, a Polychytrium, or a Monoblepharidomycetes.
  • the reduction in gro wth is measured by any one or more of growth rate, biomass productivity, in situ productivity, or doubling rate.
  • the mutagenized is measured by any one or more of growth rate, biomass productivity, in situ productivity, or doubling rate.
  • Desmodesmiis species is OM65 and OM27.
  • the mutagenized Desmodesmus species OM65 or OM27 was deposited under ATCC Accession No. PTA-120599, or
  • the mutagenized Desmodesmus species is genetically engineered prior to being mutagenized.
  • the mutagenized Desmodesmus species is genetically engineered after being mutagenized.
  • the mutagenized Desmodesmus species is genetically engineered after being mutagenized.
  • the mutagenized Desmodesmus species is grown in the liquid culture system.
  • the gro wing is for a number of days selected from the group consisting of 1 or more,
  • the growing is from 4 to 6 days, from 4 to 14 days, or is 96 hours or more, after the inoculation.
  • the growing is from 1 to 30 days, 1 to 40, from 30 to 60 days, from 60-90 days, or from 90-120 days.
  • the growing is from 1 to 7 days, from 1 week to 2 weeks, from 2 weeks to
  • the method further comprises providing one or more additional amounts of the fungicide composition to maintain an effective concentration of the fungicide composition during the growing.
  • the liquid culture system is an open outdoor liquid culture system.
  • the open outdoor culture system has a volume of: at least 20,000 liters, at least 40,000 liters, at least 80,000 liters, at least 100,000 liters, at least 150,000 liters, at least 200,000 liters, at least 250,000 liters, at least 500,000 liters, at least 600,000 liters, or at least 1,000,000 liters; or 10,000 to 20,000 liters, 10,000 to 40,000 liters, 10,000 to 80,000 liters, 10,000 to 100,000 liters, 10,000 to 150,000 liters, 10,000 to 200,000 liters, 10,000 to 250,000 liters, 10,000 to 500,000 liters, 10,000 to 600,000 liters, 10,000 to 1,000,000 liters, 20,000 to 40,000 liters, 20,000 to 80,000 liters, 20,000 to 100,000 liters, 20,000 to 150,000 liters, 20,000 to 200,000 liters, 20,000 to 250,000 liters, 20,000 to 500,000 liters, 20,000 to 600,000 liters, 20,000 to 1 1,000,000 liters
  • the open outdoor liquid culture system has an area selected from the group consisting of at least 0,25 acre, at least 0,5 acre, at least 1 ,0 acre, at least 1.5 acres, at least 2.0 acres, at least 2.5 acres, at least 5.0 acres, and 7.5 or more acres.
  • the open outdoor liquid culture system has an area selected from the group consisting of 0.25 to 0.5 acres, 0.25 to 1.0 acres, 0.25 to 1.5 acres, 0.25 to 2.0 acres, 0.25 to 2.5 acres, 0.25 to 5.0 acres, 0.25 to 7.5 acres, 0,5 to 1.0 acres, 0,5 to 1.5 acres, 0.5 to 2,0 acres, 0.5 to 2.5 acres, 0,5 to 5.0 acres, 0,5 to 7.5 acres, 1 ,0 to 1 ,5 acres, 1.0 to 2,0 acres, 1.0 to 2,5 acres, 1.0 to 5,0 acres, 1.0 to 7,5 acres, 2.0 to 2.5 acres, 2.0 to 5.0 acres, 2.0 to 7.5 acres, 2.5 acres to 5,0 acres, 2,5 to 7.5 acres, from 7,5 to 10 acres, from 10 to 15 acres, from 15 to 20 acres, from 20 to 25 acres, from 25 to 30 acres, from 30 to 35 acres, from 35 to 40 acres, from 40 to 45 acres, from 45 to 50 acres, from 50 to 75 acres, from 75 to 100 acres, from 100 to 125 acres, from 125 to
  • the yield is selected from the group consisting of greater than 0.5 g/L, greater than 0.6 g/L, greater than 0.7 g/L, greater than 0,8 g/L, greater than 0.9 g/L, and greater than 1 ,0 g/L AFDW.
  • the growing provides a yield of the mutagenized
  • Desmodesmus species that is at least 5% greater or at least 10% greater than a yield of an
  • the growing provides a yield of the mutagenized Desmodesmus species selected from the group consisting of at least 5%, at least 10%, at least 1 %, at least 20%, at least 25%, at least 50%), at least 75%, and 100%) greater than the yield of an unmutagenized
  • Desmodesmus species harvested from a second liquid culture system having a fungal infection that has not been provided the effective concentration of the fungicide composition are harvested from a second liquid culture system having a fungal infection that has not been provided the effective concentration of the fungicide composition.
  • the growing provides a yield of the mutagenized Desmodesmus species selected from the group consisting of at least 1.25 fold, 1.5 fold, 2.0 fold, 2.5 fold, 5.0 fold, 7.5 fold, 10 fold and 15 fold greater than the yield of an unmutagenized Desmodesmus species harvested from a second liquid culture system having a fungal infection that has not been provided the effective
  • the mutagenized Desmodesmus species in the liquid culture system is harvested.
  • a fuel product is obtained from the harvested mutagenized Desmodesmus species.
  • the fuel product is an oil, a petrochemical, or a substance useful in the production of a petrochemical.
  • Also provided herein is a method of determining the biocide tolerance of a modified (for example, mutated, evolved, or genetically engineered) strain as compared to an unmodified strain, comprising: measuring the Fv/Fm deficit of the modified and unmodified strains, and determining that the strain with a smaller deficit has a higher biocide tolerance than the other,
  • a strain can be genetically engineered by, for example, transforming it with a gene, and other techniques known to one skilled in the art.
  • Figure ⁇ shows results of a 96-well high throughput microliter growth rate assay (MGRA) for nominated clones and aWT Desmodesmus species, as described in Example 2.
  • Figure 2A to Figiire 2D show die results from Validation III (aka “Sawtooth Experiment”) for WT, as described in Example 2.
  • Figure 3A to Figure 3E shows the results from Validation III (aka “Sawtooth Experiment") for OMl 5 ( Figure 3 A), OM25 ( Figure 3B), OM27 (Figure 3C), OM65 ( Figure 3D), and OM82
  • Figure 4 shows the doubling rate of OMl 5, OM25, QM27, OM65, OM82, and WT at a dose of 2.5 ppm of OMEGA* ' , as is described in Example 2.
  • Figure 5 is a photographic image of Validation ⁇ at 3 ppm (Round 6), as described in Example 2.
  • Figure 6A to Figure 6F show Pulse- Amplitude-Modulation (PAM) fluorometry (Fv/Fm) results for outdoor minipotids OMl 5, OM25, OM27, OM65, OM82, and WT, as described in Example 3.
  • PAM Pulse- Amplitude-Modulation
  • FIG. 7 shows total suspended solids (TSS) productivity of ponds comprising WT, OM 15, OM25, OM27, OM65, and OM82, as described in Example 3.
  • Figure 8 shows in situ biomass productivity for WT, OMl 5, OM25, OM27, OM65, and OM82, as is described in Example 3.
  • Figure 9 shows in situ biomass productivity for WT, OM15, OM25, OM27, OM65, and OM82, normalized to WT, as is described in Example 3.
  • Figure 10 shows the data from Figure 8 analyzed by Oneway ANOVA, as is described in Example 3.
  • Figure 11 shows the PAM yield (photosynthetic health) of OM l 5, OM25, OM27, OM65, OM82, and WT during the in situ biomass productivity experiment, as is described in Example 3.
  • Figure 12 shows the "Fv/Fm deficit" for WT, OM15, OM25, OM27, OM65, and OM82, following OMEG A* treatment, as is described in Example 3.
  • Figure 13 is a photographic image of OM65 biomass collected from miniponds, as is described in Example 3.
  • Figure 14A to Figure 14C show fluorescence measurements for OM15, OM25, GM27, O 65, QM82, and WT, as is described in Example 3.
  • Figure 15 shows fatty acid methyl ester (FAME) analysis of biomass from cultures of OM27, O 65, and WT, as is described in Example 3,
  • Figure 16 shows PAM measurements from a "control" WT Desmodesmus species culture that was not subject to a continuous dosing scheme of OMEGA*, as is described in Example 4.
  • Figure 17 shows PAM measurements from a control WT Desmodesmus species culture and cultures of OM27 and OM65, as described in Example 5,
  • Figure 18 shows biomass productivity data analyzed by Oneway ANOVA, as is described in Example 5, WT is on the left, OM27 in the middle, and OM65 on the right,
  • Figure 19 shows a markedly visual difference between an OM27 culture (left) and a control WT Desmodesmus species culture (right), as is described in Example 5.
  • Oxygenic hoto synthetic microalgae and cvanobacteria represent an extremely diverse, yet highly specialized group of micro-organisms that live in diverse ecological habitats such as freshwater, brackish, marine, and hyper-saline, with a range of temperatures and H, and unique nutrient availabilities (for example, as described in Falkowski, P.G., and Raven, J.A., Aquatic Photosynthesis, Maiden, MA: Blackwell Science).
  • One genus of microalgae is Desmodesmus .
  • UV mutagenesis and laboratory selection methods were employed to create and identify non-genetically modified (GM) variants of a Desmodesmus species that have increased or improved tolerance (hyper-tolerance or increased resistance) to the fungicide
  • OMEGA* as compared to the unmutagenized Desmodesmus species wild type (WT) strain when challenged with the same dose(s), dosing regimen, or concentrations of OMEGA*.
  • Improved or increased tolerance to the fungicide OMEGA ® can be shown by any one or more of the parameters described herein. Other parameters known to one skilled in the art can also be used in place of or in combination with any of the following parameters.
  • the variants can, for example, exhibit an increase in productive pond lifetime, an increase in biomass productivity, an increase in in situ productivity, and/or an increase in biomass yield relative to the WT strain when challenged with the same dose(s), dosing regimen, or concentrations of OMEG A*.
  • the variants can, for example, have increased growth rate, increased photosynthetic efficiency (Fv/Fm), increased photosynthetic health, increased F AME content, and/or increased doubling rates as compared to the WT strain when challenged with the same dose(s), dosing regimen, or concentrations of OMEG A*.
  • measuring biomass productivity can be done in using any one or more assays/methods known to one skilled in the art and can be measured in a wide range of units, for example, dry weight (DW) grams per liter (g L). Choosing the appropriate method and units of measurement are well within the ability of one skilled in the art.
  • Growth rate, biomass productivity, and in situ productivity can all be measured using the following exemplary units: grams per liter per day, grams per meter squared per day, OD 75 o nm per day, or colony forming units (cfu) per liter per day.
  • a mutagenized strain is said to have an increased tolerance to a fungicide composition as compared to an unmutagenized strain or other strain if it is more tolerant to the same concentration or dose of the fungicide composition.
  • the term "variant” is used interchangeably throughout the disclosure with “mutant”, “mutagenized strain”, “variant strain”, “evolved strain”, and “evolved mutant strain”.
  • the WT Desmodesmus species is sensitive to therapeutic doses (0.5-2.0 ppm) of OMEGA* (a common fungicide) resulting in a decrease of in situ productivity.
  • OMEGA* is an effective treatment for common chytrid infestations. It would therefore be desirable to have a variant Desmodesmus species strain(s) with improved biomass productivity that is able to sustain a minimal or reduced toxic effect when dosed with OMEGA 1 *,
  • one strain OM65 had biomass productivity that was greater than twice (118%) the productivity of WT when treated with 2ppm OMEGA*.
  • the quality of biomass for the evolved strains was significantly better than WT, when chronically treated with OMEGA*.
  • FAME content of biomass from O EGA*-treated WT miniponds was -50% of normal, while FAME content of select evolved strains was similar to normal FAME content. Therefore, as a result of their innate biocide tolerance, certain evolved strains exhibited improved biomass productivity vs. WT during biocide treatment periods that were required to control pest infestations.
  • An increase in FAME content can be, for example, 1.25-fold to two-fold higher, two-fold to three-fold higher, three-fold to four- fold higher, or four- fold to five-fold higher.
  • An increase in FAME content can be, for example, 0.25-fold to five-fold higher, five-fold to ten-fold higher, tenfold to twenty-fold higher, or twenty-fold to fifty-fold higher, or higher than 50-fold.
  • ATCC deposits are made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of M icroorganisms for the Purpose of Patent Procedure and the
  • the UV -mutated Desmodesmiis species may be a transformed microalgae having one or more exogenous DNA sequences.
  • Desmodesmiis species may have sequences that are endogenous DN A sequences in a recombinant construct.
  • the sequences may be exogenous DNA sequences in a recombinant construct.
  • Fv/Fm is the photosynthetic efficiency.
  • ANOVA is a statistical test used to determine if more than two population means are equal. The test uses the F-distribution (probability distribution) function and information about the variances of each population ( within) and grouping of populations (between) to help decide if variability between and within each population are significantly different.
  • Dunnett's test (method) is a statistical tool known to one skilled in the art and is described, for example, in Dunnett, C. W. (1955), "A multiple comparison procedure for comparing several treatments with a control", Journal of the American Statistical Association, 50: 1096-1 121, and Dunnett, C. W. (1964), “New tables for multiple comparisons with a control", Biometrics, 20:482- 491 . Dunnett's test compares group means, it is specifically designed for situations where all groups
  • the increase in productive pond lifetime, biomass productivity, in situ biomass productivity, or biomass yield can be 2-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40- 45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 75-80%, 85-90%», 95-100%», 100-150%, 150- 200%, 200-250%, 250-300%, 300-350%, 350-400%, or more than 400%.
  • the increase can be about a 20% increase, about a 28%», about a 30%» increase, or about a 1 18% increase.
  • the term "yiel d" refers to the number of microalgae per unit volume at harvest, and may be expressed, for example, as the number of cells per volume of culture, a mass per volume of culture, etc. Yield, used herein may also be expressed as a mass per area of culture.
  • a yield of a harvested mutagenized Desmodesmus species can be greater than 0.4 gram per liter (g/L) AFDW, greater than 0.5 g/L AFDW, greater than 0.6 g/L AFDW, greater than 0.7 g/L AFDW, greater than 0.8 g/L, AFDW, greater than 0.9 g/L AFDW, or greater than 1.0 g/L AFDW.
  • the yield can be from 0.1 g/L AFDW to 2.0 g/L AFDW, from 2.0 g/L AFDW to 5.0 g/L AFDW, or from 5,0 g/L AFDW to 10.0 g L AFDW,
  • the growing of the mutagenized Desmodesmus species can provide a yield that is at least 5% greater or at least 10% greater than a yield of an
  • the growing of the mutagenized Desmodesmus species can provide a yield of the mutagenized Desmodesmus species selected from the group consisting of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%», and 100% greater than the yield of an unmutagenized Desmodesmus species harvested from a second liquid culture system having a fungal infection that has not been provided the effective concentration of the fungicide composition.
  • the growing of the mutagenized Desmodesmus species can provide a yield of the mutagenized Desmodesmus species selected from the group consisting of at least 1.25 fold, 1.5 fold, 2.0 fold, 2.5 fold, 5.0 fold, 7.5 fold, 10 fold and 15 fold greater than the yield of an unmutagenized
  • Desmodesmus species harvested from a second liquid culture system having a fungal infection that has not been provided the effective concentration of the fungicide composition can be used to measure the tolerance of a mutant Desmodesmus species.
  • the doubling time is the period of time required for a quantity to double in size.
  • OMEGA* is a fungicide composition which acts to inhibit cellular energy production.
  • the active ingredient in OMEGA* is Fluazinam: 3-chloro-A'-[3-chloro-2,6-dinitro-4-trifli!oromethyl) phenyl]-5-trifluoromediyl-2-pyridinamine (CA). This active ingredient constitutes 40% of the composition. Other ingredients make up the other 60%.
  • Fluazinam is a highly active fungicide with a broad spectrum including soil borne diseases. Fluazinam is a multi-site fungicide that is a member of the pyridinamine family.
  • OMEG A ® has a mode of action that disrupts the energy production of a fungus. It is listed in FRAC code 29 as an uncoupler of oxidative phosphorylation.
  • OMEGA* contains 4.17 pounds fluzainam per gallon (500 grams per liter).
  • Possible doses of OMEGA* range from 0.0.00 to 0,25 ppm, 0.1 ppm to 20 ppm, from 0.25 ppm to 2.5 ppm, from 0.25 ppm to 5.0 ppm, from 0.25 ppm to 10 ppm, from 0,25 ppm to 12.5 ppms, from 0.25 ppm to 15 ppm, from 0.25 ppm to 20 ppm.
  • Other possible doses range from 0.5 ppm to 5.5 ppm, from 5 ppm to 10 ppm, from 10 ppm to 20 ppm, from 20 ppm to 50 ppm, or from 50 ppm to about 100 ppm.
  • Possible other doses of OMEGA* range from 0.25 to 15 ppm, or from 0.01 to 20 ppm.
  • exemplary doses of OMEG A* can be about 0.5 ppm, about 1 ppm, about 2 ppm, about 3 ppm, about 4ppm, about 5ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 11 ppm, about 12 ppm, about 13 ppm, about 14 ppm, about 1 ppm, about 16 ppm, about 17 ppm, about 18 ppm, about 19 ppm, or about 20 ppm.
  • the variant strains were dosed several times (one to six times) with varying ppm of OMEGA ranging from 0.25 to 3.0 ppm. The final ppm of any culture was not determined. Although the maximum ppm of OMEGA* left at the end of any experiment would total the amount of OMEG A ® put in. However this does not account for any loss of activity of OMEGA ® or removal of OMEGA* during harvesting.
  • a variant strain can be dosed, for example, 1 -3 times, 3-5 times, 6-8 times, 8-10 times, or more than 10 times. Each dose can be the same or different.
  • Mutagenized strains of the disclosure can be treated with one or more doses of a fungicide composition, like OMEGA ® .
  • Mutagenized strains can be treated with one or more additional amounts of a fungicide composition, after the initial treatment, to maintain an effective
  • Any one or more dose of a fungicide composition can be: from 0.25 ppm to 15 ppm, or from 0.01 ppm to 20 ppm; from 0.1 ppm to 20 ppm, from 0.25 ppm to 2.5 ppm, from 0.25 ppm to 5.0 ppm, from 0.25 ppm to 10 ppm, from 0.25 ppm to 12.5 ppm, from 0.25 ppm to 15 ppm, or from 0.25 ppm to 20 ppm; from 0.5 ppm to 5.0 ppm, from 5 ppm to 10 ppm, from 10 ppm to 20 ppm, from 20 ppm to 50 ppm, or from 50 ppm to about 100 ppm; or about 0.5 ppm, about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm,
  • Mutagenized strains of the disclosure can be treated with other fungicide compositions, like OMEGA* ' , wherein an active ingredient is fluazinam.
  • the mutagenized strains of the disclosure can be treated with a fungicide composition in which the active ingredient is a fungicide that is listed in the group Fungicide Resistance Action Committee 29 (FRAC 29) as uncouplers of oxidative phosphorylation.
  • FRAC 29 Fungicide Resistance Action Committee 29
  • Members of this group include binapacryl, meptyldinocap, or dinocap, and they are grouped together by FRAC because they have been shown to have the same mode of action.
  • fungicide composition in which the active ingredient is binapacryl, meptyldinocap, or dinocap, could easily test a range of concentrations or doses of a fungicide composition in which the active ingredient is binapacryl, meptyldinocap, or dinocap, to determine the effective concentration or dose of each fungicide composition on any of the variants of the disclosure to control the growth of fungus.
  • a fungicide composition useful in treating the mutagenized strain of the disclosure can comprise as an active ingredient any one or more of fluazinam, binapacryl, meptyldinocap, or dinocap.
  • a fungicide composition comprises at least one active ingredient.
  • a fungicide composition can comprise, for example, 40% active ingredients and 60% other ingredients. The other ingredients can comprise one or more inactive ingredients.
  • Any one active ingredient can constitute about 40% of the fungicide composition.
  • any one active ingredient can constitute about 10-30°/», about 30-60%, about 60-90% or about 90% to 100% of the fungicide composition.
  • the FRAC 29 fungicide can comprise from 10-20% of the fungicide composition, from 20-30% of the fungicide composition, from 30-40% of the fungicide composition, from 40-50% of the fungicide composition, from 50- 60% of the fungicide composition, from 60-70% of the fungicide composition, from 70-80% of the fungicide composition, from 80-90%, or from 90-100% of the fungicide composition.
  • FRAC is a Specialist Technical Group of CropLife International (Formerly Global Crop Protection Federation, GCPF).
  • the purpose of FRAC is to provide fungicide resistance management guidelines to prolong the effectiveness of "at risk” fungicides and to limit crop losses should resistance, occur.
  • the main aims of FRAC are to:l. Identify existing and potential resistance problems. 2. Collate information and distribute it to those involved with fungicide research, distribution, registration and use. 3. Provide guidel ines and advice on the use of fungicides to reduce the risk of resistance developing, and to manage it should it occur. 4.
  • the 2013 FRAC Code List* sorts fungicides by mode of action and assigns them a numerical code (e.g. FRAC Code 29). Below is a subset of the FRAC Code list, specifically the members of FRAC code 29 that include the fungicide fluazinam used in the disclosure.
  • MOA Different letters (e.g. "C") are used to distinguish fungicide groups according to their biochemical mode of action (MOA) in the biosynthetic pathways of plant pathogens.
  • Target Site and Code The biochemical mode of action is given.
  • a grouping can be made due to cross resistant profiles within a group or in relation to other groups.
  • Chemical Group Grouping is based on chemical considerations, Nomenclature is according to the International Union of Pure and Applied Chemistry (IUPAC) and Chemical Abstract Name. Comments on Resistance: Details are given for the (molecular) mechanism of resistance and resistance risk.
  • Culture time “growing time”, “length of growth,” or “time to harvest,” are used interchangeably herein as a measurement from the date of inoculation of a liquid culture system with an algae species.
  • Exemplary culture times can be about 30 days, from 30-60 days, or from 60- 90 days.
  • exemplary growing times are: 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 30 or more, 45 or more, 60 or more, 90 or more, 120 or more, 180 or more, 250 or more, 500 or more, 1000 or more, 1500 or more, or 2000 or more days after the date of inoculation.
  • the growing time can be from 4 to 6 days, from 4 to 14 days, or about 96 hours or more after inoculation of the liquid culture.
  • the growing time can be, for example, from 4 to 6 days, from 4 to 14 days, or is 96 hours or more, after the inoculation.
  • the growing is from 1 to 30 days, 1 to 40, from 30 to 60 days, from 60-90 days, or from 90- 120 days.
  • the growing time can be from 1 to 7 days, from 1 week to 2 weeks, from 2 weeks to 3 weeks, from 3 weeks to 1 month, from 1 month to 3 months, from 3 months to 6 months, from 6 months to 9 months, from 9 months to 12 months, from 12 months to 18 months, from 18 months to 2 years, from 2 years to 3 years, from 3 years to 4 years, from 4 years to 5 years, from 5 years to 6 years, from 6 years to 7 years, from 7 years to 8 years, from 8 years to 9 years or from 9 years and 10 years after inoculation.
  • liquid system refers to a system for culturing a microalgae, such as a Desmodesmus species.
  • a liquid system may include both a closed and an open culture system.
  • An open liquid system may include, for example an open or semi-closed photobioreactor, semi-closed ponds, open ponds, or lakes.
  • a liquid system can be an outdoor liquid system, an indoor liquid system, or a combination of both.
  • “Growing microalgae,” “growing”, “growing the microalgae,” “microalgae growth,” and “culturing the microalgae” as used herein, refer to one or more steps including microalgae in culture to when microalgae are in suspension just prior to the beginning of a harvesting step.
  • reduced growth As used herein, “reduced growth,” “reducing the growth”, “inhibited growth,” “growth reduction”, “a reduction in growth”, and “growth inhibition” relate to the decreased reproduction or division of a pest relative to the amount of reproduction or division of a pest under similar or identical conditions in the absence of any treatment. “Reduced growth,” “inhibited growth,” “growth reduction” and “growth inhibition” may also refer to the killing or death of the pest by the treatment (e.g. fungicide composition).
  • harvesting relates to the removal or isolation of all , or part of microalgae in a culture system, including a liquid culture system. Harvesting may occur continuously from a growing culture, batch-wise or as a total collection of the microalgae at the end of a culture period. A liquid, as a supernatant, siphonate, flow-through or other separated form, may be returned to the liquid culture system. In some instances, relative amount harvested refers to the amount of microalgae remaining compared to the amount contained in the liquid culture system before harvesting.
  • treatment refers to methods or compositions that inhibit the growth of a pest. A treatment may include methods or compositions that kill a pest,
  • pests may be detected using Polymerase Chain Reaction (PGR) to detect ribosomal sequences.
  • PGR Polymerase Chain Reaction
  • ribosomal sequences may include DNA sequence selected from the group consisting of NC_003053 Rhizophydiiim sp. 136 mitochondrion, NC_003048
  • NC 003052 Spizellomyces pimctatus mitochondrion chromosome 1 NC_003061 Spizellomyces pimctatus mitochondrion chromosome 2
  • NC__003060 Spizellomyces pimctatus mitochondrion chromosome 3
  • NC 004760 Harpochytrium sp. JEL94 mitochondrion NC__004624 Monoblepharella sp. JEL I5 mitochondrion, and NC_004623
  • Methods of the present disclosure include methods of detection that may detect a pest present at a level of at least 10 5 cells/ml. In another aspect, the methods of the present disclosure provide for the detection of a pest at a concentration I Q 4 cells ml. In a further aspect, the concentration of pest may be detected at 10" cells/ml . In another aspect, a pest present at a concentration of 10 ⁇ cells/ml or even 10 1 cells/ml may be detected.
  • PCR polymerase chain reaction
  • Methods for performing PCR are known in the art. Nucleic acid analysis by PCR requires sample preparation, amplification, and product analysis. Although these steps are usually performed sequentially, amplification and analysis can occur simultaneously. Quantitative analysis occurs concurrently with amplification in the same tube within the same instrument.
  • the concept of combining amplification with product analysis has become known as "real time' PCR or quantitative PCR (qPCR). See, for example, U.S. Pat. No. 6, 174,670, herein incorporated by reference in its entirety.
  • real-time methods of PCR may be used to detect the presence of a pest in a liquid system (e.g., quantitative PCR).
  • a fluorescent signal accumulates during each amplification cycle.
  • a positive reaction is provided when the fluorescent signal exceeds a threshold level, typically the background fluorescence.
  • the cycle threshold G the number of cycles required to cross the threshold and the Q levels are inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Q level the greater the amount of target nucleic acid in the sample).
  • Real time PCR assays typically undergo 40 cycles of amplification.
  • the Q value may be compared to a standard curve prepared from a serially diluted pest to determine a number of pests/ml of sample.
  • a pest is detected when die C t value is less than 35 cycles for at least one monitoring step.
  • a pest is detected when the Q value is less that 35 cycles for at least two consecutive monitoring steps.
  • a C t value of less than 35 cycles for three consecutive monitoring steps indicates the presence of a pest.
  • the present disclosure further provides for the detection of pest when there is a consistent decrease in the Ct over two or more monitoring steps.
  • a consistent decrease from a C t of 35 or higher to a C t value of 30 or less indicates a need for crop protective action.
  • treatment may be provided to the liquid system within hours of the detection of a pest contamination. In an aspect, treatment may be provided within 2 hours of detection of a pest contamination. In another aspect, treatment may be provided within 4 hours of detection of a pest contamination. In yet another aspect, treatment may be provided within 8 hours of the detec tion of a need for crop protective action. In a further aspect, treatment may be provided within one day of detection of a need for crop protective action. In another aspect, treatment may be provided within 2 days of a need for crop protective action, in an aspect, monitoring and detection of pests may be continuous.
  • the present disclosure provides for methods of reducing the growth of a pest in a liquid culture of microalgae where a liquid system is inoculated with a microalgae, die system is monitored for the presence of a pest, and an effective concentration of a fungicide is pro vided to inhibit the growth of the pest relative to the growth of the pest without the fungicide, and growing the microalgae.
  • the present disclosure further provides for the reduction of viable pests in a liquid system.
  • the present disclosure provides for a method of reducing the growth of a pest where a reduction of the growth of a pest in the presence of an inhibitor is measured relative to the growth of a pest under similar conditions in the absence of an inhibitor.
  • a reduction of the growth of a pest is achie ved by the death of the pest.
  • a reduction of the growth of a pest is achieved by the inhibition of division of the pest.
  • grow h of the pest is reduced by 99%, or more.
  • the growth of the pest is reduced by 95%, or more.
  • the growth of a pest is reduced by 90%, or more.
  • the growth of a pest is reduced by at least 80%.
  • the growth of a pest is reduced by at least 70%. In another aspect, the growth of a pest is reduced by at least 60%, In another aspect, the growth of a pest is reduced by at least 50%. In another aspect, the growth of a pest is reduced by at least 90 to 99%, at least 95 to 99%, at least 80 to 95%, at least 80 to 99%, or 75 to 99%. In yet another aspect, the growth of a pest is reduced no less than 90%, 95% or 99%,
  • the pest may be a member of the fungi kingdom.
  • the pest may be a member of the division Chytridiomycota or Cryptomycota,
  • the pest may be a member of the class Chytridiomycetes .
  • the pest may be a species of Chy iridium spp.
  • Examples of pests of microalgae cultures are members of the fungi kingdom and include the division Blastocladiomycota, Chytridiomycota, Cryptomycota, Glomeromycota, Micro sporidia, Neocallimastigomycota, Ascomycota, or Basidiomycota.
  • a fungus as used herein, includes members of the classes Chytridiomycetes and Monobiepharidomycetes as well as species of Chytridium spp.
  • pests that are members of the fungi kingdom may be identified by molecular phylogeny, for example, using the methods of James et al.
  • a pest may be a member of the Rozella genus of Chytridiomycota.
  • a pest may be a member of the Chytridiales/Rhizophydium clade of Chytridiomycota.
  • a pest may be a member of the Amoeboaphelidium genus.
  • a pest may be phylogenetically related to a clade of the Chytriodmycota division including the
  • the pest may be phylogenetically related to a Rozella spp..
  • pest relates to any undesired biological organism in a sample culture, such as a Desmodesmus culture.
  • Non-limiting examples of pests are bacteria and fungi.
  • a pest may be undesired because it decreases the growth rate of a Desmodesmus culture.
  • a pest may be undesired because it decreases the overall extent o f Desmodesmus growth or the total yield of Desmodesmus per volume of culture.
  • a pest may be undesired because it leads to the death of a Desmodesmus culture.
  • a pest may be undesired because it changes the gene expression of the cultured Desmodesmus .
  • a pest may be population of a single organism or a mixed population,
  • a "fungus,” as used herein, is a member of the fungi kingdom and the division
  • a fungus includes members of the classes Chytridiomycetes and Monoblepharidomycetes as well as species oi " Chyiridium spp,, or any combination of fungi.
  • a fungus as used herein includes members oi ' Chyiridium species included in the Chytridiomycota division of the fungi kingdom including the orders Chytridiales,
  • Rhizophylctidal.es Spizellomycetales, Rhizophydiales, Lobulomycetales, Cladochytriales,
  • concentration of a biocide or fungicide that is sufficient to control the growth, or kill, a pest while providing for the continued growth, or survival, of the growing culture, for example, a
  • Desmodesmus culture in the liquid system Exemplary ranges of doses of a fungicide concentration are provided herein.
  • Chytrids are primitive fungi and are mostly saprophytic (degrading chitin and keratin). Some species are unicellular. As with other fungi, the cell wall in a chytrid is composed of chitin. Many chytrid species are aquatic (mostly found in fresh water). There are approximately 1,000 chytrid species, in 127 genera, distributed among 5 orders. Some chytrid species are parasitic and may infect plants, including microaigae.
  • chytrids included in the present disclosure include Achlyogeton, Allochytridium, Allochytridium expandens, Allochytridium luteum, Allomyces, Allomyces (subgenus), Allomyces attomyces, Allomyces catenoides, Allomyces reticulatus,
  • Amoeboaphelidium protococcarum Alphamycetaceae, Alphamyces, Alphamyces chaetiferum, Amphicypellus, Amphicypellus elegans, Anaeromyces, Anaeromyces elegans, Anaeromyces mucronatus, Angulomycetaceae, Angulomyces, Angulomyces argentinensis, Aquamycetaceae, Aquamyces, Aquamyces chlorogonii, Arnaudovia, Arnaudovia.
  • hyponeustonica Asterophlyciis irregularis, Asterophlyciis sarcoptoides, Batracrochytrium, Batrachochytrium dend.ro batidis, Blastocladia arboraia, Blastocladia cadiica, Blastocladia coronata, Blastocladia cristata,
  • Blastocladia heierosporangia Blastocladia mammilata, Blastocladia picaria, Blastocladia pileota, Blastocladia pusilla, Blastocladia sessilis, Blastocladia spiciformis, Blastocladiella, Blastocladiella anahaenae, Blastocladiella.
  • Olduvaiensis Caulochytriaceae subramanium, Caulochytrium, Caulochytrium gloeosporii, Caulochytrium protostelioides, Caulochytrium protosteloides var. Vulgaris, Chytridiaceae, Chytridiales, Chytridiomycetes, Chytridiomycota, Chytridium, Chytridium adpressum, Chytridium aggregatum, Chytridium apophysatum, Chytridium brevipes, Chytridium cejpii, Chytridium chlorobotryis, Chytridium citriforme, Chytridium closterii, Chytridium codicola, Chytridium coleochaetes, Chytridium confervae, Chytridium comiculatum, Chytridium cresentum, Chytridium deltanum, Chytridium fusiforme, Chytridium gibbosum, Chy
  • Chytridium telmatoskenae Chytridium turbinatum, Chytriomyces, Chytriomyces angularis, Chytriomyces annulatus, Chytriomyces confervae, Chytriomyces cosmarii, Chytriomyces elegans, Chytriomyces gilgaiensis, Chytriomyces heliozoicola, Chytriomyces hyalinus, Chytriomyces hyalinus var. Granulatus, Chytriomyces laevis, Chytriomyces macro-operculatus, Chytriomyces macro-operculatus var. Hirsutus, Chytriomyces mammilifer, Chytriomyces mortierellae,
  • Chytriomyces reticulatus Chytriomyces reticulosporus, Chytriomyces rhizidiomycetis,
  • Chytriomyces rotoruaensis Chytriomyces suburceolatus, Chytriomyces vallesiacus, Chytriomyces verrucosus, Chytriomyces willoughbyi, Cladochytriales, Cladochytriaceae, Cladochytrium aureum, Cladochytrium granulatum, Cladochytrium indicum, Cladochytrium novoguineense, Cladochytrium replicatum, Cladochytrium salsuginosum, Clydea, Clydea vesicula, Coelomomycetaceae,
  • Dangeardiana Dangeardiana apiculata, Dangeardiana eudoririae, Dangeardiana leptorrhiza, Dangeardiana sporapiculata, Dictyomorpha, Dictyomorpha dioica, Dictyomorpha dioica var, Pythiensis, Diplochytridium, Diplochytridiiim aggregation, Diplochytridium hrevipes, Diplochytridium cejpii, Diplochytridium chlorobotryis, Diplochytridium citriforme, Diplochytridium codicola, Diplochytridium gibbosum, Diplochytridium inflaxum, Diplochytridium isthmiophilum, Diplochytridium kolianum,
  • Diplophlyctis asteroidea, Diplophlyctis buttermerensis, Diplophlyctis chitinophila, Diplophlyctis complicata, Diplophlyctis nephrochytrioides, Diplophlyctis
  • Entophlyctis Entophlyctis apiculata, Entophlyctis bulligera, Entophlyctis bulligera var, Brevis, Entophlyctis caudiformis, Entophlyctis con ervae-glomeratae, Entophlyctis crenata, Entophlyctis fdamentosa, Entophlyctis helioformis, Entophlyctis tohata, Entophlyctis luteolus, Entophlyctis mammilliformis, Entophlyctis molesta, Entophlyctis obsciira, Entophlyctis reticutospora, Entophlyctis rhizina, Entophlyctis sphaerioides, Entophlyctis texana, Entophlyctis variabilis, Entophlyctis variabilis, Entophlyctis variabilis, Entophlyctis
  • Harpochytrium hedenii Harpochytrium hyalothecae
  • Harpochytrium intermedium Harpochytrium monae
  • Harpochytrium natrophilum Harpochytrium ornithocephalum
  • Mitochytridi m regale Monoblepharidomycetes, Monoblepharidales, Monoblepharidaceae, Monoblepharella, Monoblepharis micrandra, Monoblepharis thalassinosus, Monophagus,
  • Neocallimastigomycota NeocaUimastigomycetes, NeocaHimastix, NeocaHimastix frontalis, NeocaHimastix hurleyensis, NeocaHimastix joyonii, NeocaHimastix patriciarum, NeocaHimastix variabilis, Nephrochytrium bipes, Nephrochytrium buttermerense, Nephrochytrium complicatum, Nephrochytrium sexuale, Nowakowskiella crassa, Nowakowskiella delica, Nowakowskiella elegans, Nowakowskiella granulata, Nowakowskiella keratinophila, Nowakowskiella methistemichroma, Nowakowskiella moubasherana, Nowakowskiella multispora, Nowakowskiella multispora, Nowakowskiella multispora, Nowakowskiella multispora, Nowakowskiella multispora, Nowakowskiella multispora, Nowakowskiella multispora, Nowakowskiella multispora, Nowakowskiella multispor
  • Phlyctochytrium aureliae Phlyctochytrium californicum
  • Phlyctochytrium chandleri Phlyctochytrium aureliae, Phlyctochytrium californicum, Phlyctochytrium chandleri,
  • Phlyctochytrium circulidentatum, Phlyctochytrium cystoferum, Phlyctochytrium dichotomum, Phlyctochytrium dissolutum, Phlyctochytrium furcatum, Phlyctochytrium hirsutum, Phlyctochytrium incrustans, Phlyctochytrium indicum, Phlyctochytrium irregulare, Phlyctochytrium kniepii, Phlyctochytrium lackeyi, Phlyctochytrium macrosporum, Phlyctochytrium mangrovii, Phlyctochytrium marilandicum, Phlyctochytrium megastomum, Phlyctochytrium mucosum,
  • Phlyctochytrium paras items Phlyctochytrium peruvianum, Phlyctochytrium ptanicorne,
  • Phlyctochytrium plurigihbosum Phlyctochytrium powhatanense, Phlyctochytrium punctatum, Phlyctochytrium recurvastomum, Phlyctochytrium rhizopenicillium, Phlyctochytrium
  • Rhizophlyctis oceanis Rhizophlyctis oceanis var. Floridaensis, Rhizophlyctis petersenii var.
  • Rhizophydium achnanthis Rhizophydium algavorum, Rhizophydium anatropum, Rhizophydium aridroaioctes, Rhizophydium angulosum, Rhizophydium armulatum, Rhizophydium aphanomycis, Rhizophydium aureum, Rhizophydium biporosum, Rhizophydium blastocladianum, Rhizophydium blyttio ycerum, Rhizophydium brevipes var.
  • Rhizophydium brooksianum Rhizophydium bumilleriae, Rhizophydiu capiltaceum, Rhizophydium clavatum, Rhizophydium coleochaetes, Rhizophydium collapsum, Rhizophydium conchiforme, Rhizophydium condylosum, Rhizophydium contractophilum, Rhizophydium coralloidum, Rhizophydium dentatum, Rhizophydium difficile, Rhizophydium digitatum, Rhizophydium dubium, Rhizophydium echinocystoides, Rhizophydium ellipsoiaium, Rhizophydium fragilariae, Rhizophydium fugax, Rhizophydium gonapodyanum, Rhizophydium hispidulosum, Rhizophydium kariitigii, Rhizophydium lagenaria, Rhizophydium laterale, Rhizophydium lenelangeae, Rhizophydium littoreum, Rhizophydium macroporosum, Rhizophydium man
  • Rhizophydium rion Rhizophydium tetragenum, Rhizophydium tuhulatum, Rhizophydium ubiquetum, Rhizophydium undatum, Rhizophydium undulafum, Rhizophydium urcelolatum, Rhizophydium venezuelensis, Rhizophydium venustum, Rozella, Rozella blastocladiae, Rozella coleochaetis, Rozella diplophlyctidis, Rozella itersoniliae, Rozella longicollis, Rozella
  • M ethods for culturing organisms of the present disclosure are known in the art, for exampl e, in Vonshak, A. Spirulina Platensis (Arthrospira): Physiology, Cell-Biology And Biotechnology. 1997. CRC Press, Andersen, A. Algal Culturing Techniques. 2005. Elsevier Academic Press, Chen et al.
  • a liquid of a liquid culture system of the present disclosure may be a defined or undefined media.
  • the liquid may include untreated water.
  • the untreated water may be water obtainable from a natural source such as a river, lake, aquifer, ocean or a pond, in another aspect, the liquid may be brackish water having an osmolaritv between 0.5 and 30 grams of salt per liter.
  • the liquid may be salt water.
  • the water may be recycled water obtainable from a sewage or waste water treatment lant, or waste water from an industrial process such as power production and the like.
  • the untreated water may be aquifer water.
  • the untreated water may be aquifer water that is not suitable for agriculture.
  • the aquifer water may be aquifer water with an elevated total dissolved solids (TDS).
  • a liquid of a liquid culture system may be supplemented with nutrients that benefit the growth of the microalgae.
  • the liquid may be supplemented with CO 2 to enhance the grow h of the microalgae.
  • the CO? may be introduced into the liquid system by bubbling with air or C0 2 . Bubbling with C0 2 can be, for example, at 1% to 5% CO 2 .
  • CO 2 can be delivered to the liquid system as described herein, for example, by bubbling in C0 2 from under the surface of the liquid containing the microalgae.
  • sparges can be used to inject CO? into the liquid. Spargers are, for example, porous disc or tube assemblies that are also referred to as bubblers, carbonators, aerators, porous stones and diffusers.
  • the CO? may be introduced into the liquid system as a liquid.
  • the liquid may be supplemented with C0 2 to increase the concentration of C0 2 in the liquid to 20 parts-per-million (ppm), or more.
  • the liquid may be supplemented with CO? to increase the concentration of CO?, in the liquid to 25 ppm, or more.
  • the liquid may be supplemented with CO 2 to increase the concentration of C0 2 in the liquid to 30 ppm, or more, in another aspect, the liquid of the liquid system may be supplemented with CO 2 to increase the concentration of CO? in the liquid to 35 ppm, or more.
  • a liquid system may be supplemented with C0 2 to maintain the pH of the liquid system.
  • microalgae photosynthesize they drive the pH of a liquid system up. If at any time the pH surpasses an upper limit of a threshold, CO? is added to the pond until the pH decreases to the specified range.
  • a liquid system inoculated with microalgae is supplemented with C0 2 to maintain a pH of 8.8 to 9,2.
  • the pH of a liquid system is monitored as a proxy for the amount of C0 2 available for photosynthesis.
  • a liquid system being provided with CO? may have a pH defined as an upper limit.
  • the upper pH limit may be 9.2.
  • the upper pH limit may be 9.4.
  • upper limit for pH may be set at 9.4,
  • the upper limit for pH may be set at 9.6.
  • the upper limit for pH may be set at 9.8.
  • the upper limit for pH may be set at 10.2, 10.4 or 10.6.
  • a liquid sy stem being provided with C0 2 may have a pH defined as a lower limit.
  • C0 2 supply is terminated to the liquid system when the pH drops below a pre-defined threshold in order to raise the pH.
  • the threshold may be a pH of 8.8.
  • the threshold may be 9.8.
  • the threshold may be 9.0.
  • the threshold may be 9.2.
  • the threshold may be 9.4, In yet another aspect, the threshold may be 9.6.
  • the present disclosure provides for the addition of C0 2 to maintain a pH within a range with the threshold and limit pH values being set accordingly. It is further understood that different species of microalgae ha ve different preferred pH ranges for optimal growth.
  • the threshold and limit pH values may be determined experimentally to maximize the photosynthesis and growth of microalgae in a liquid culture system.
  • the pH range may be maintained between 8.8 and 9.2.
  • the pH range may be maintained between 8.8 and 9.4.
  • the pH may be maintained between 8.8 and 9.6.
  • the pH may be maintained between 8,8 and 9,8.
  • the pH range may be between 9,8 and 10.2.
  • the pH may be between 9.6 and 10,2.
  • the pH may be between 9.4 and 10.2,
  • Nutrients that can be used in the systems described herein, or known in the art, include, for example, nitrogen, phosphorus, and trace metals.
  • nitrogen may supplemented in the form of ammonia or ammonium.
  • ammonium is provided as ammonium sulfate or ammonium chloride.
  • the nitrogen supplement may be provided as urea.
  • the supplemental nitrogen may be provided as nitrate or nitric acid.
  • the supplemental nitrogen may be provided as a mixture, for example as a mixture of urea and ammonium nitrate, also known as URAN.
  • the nitrogen may be provided as potassium nitrate (KN03).
  • the nitrogen may be provided as sodium nitrate (NaN03).
  • a liquid culture system of the present disclosure may be supplemented with trace metals.
  • Supplements of trace metals may include salts of iron (Fe), magnesium (Mg), potassium ( ), calcium (Ca), cobalt (Co), copper (Cu), manganese (Mn), molybdenum (Mo), zinc (Zn), vanadium (V) or boron (B).
  • the trace metal may be supplied in the form of a nitrate (N03-) or ammonium (NH4+) salt.
  • potassium may be added as potassium chloride or potassium sulfate.
  • potassium may be added to the liquid system as potassium nitrate.
  • the nutrients can come, for example, in a solid form or in a liquid form.
  • the nutrients are in a solid form they can be mixed with, for example, fresh or salt water prior to being delivered to the liquid system containing the organism, or prior to being delivered to a culture system.
  • a nutrient is applied in a manner that minimizes the potential of osmotic stress to the cells.
  • nutrient additions are done over an extended period of time,
  • the nutrients may be diluted prior to being applied to a pond,
  • a liquid culture system of the present disclosure may be maintained at a preferred pH depending on the microalgae.
  • a neutral pH may 3 ⁇ 4 e maintained.
  • the pH may be maintained between a pH of 6.5 and 7.5,
  • an alkaline pH may be maintained, for example, a pH of 10.
  • an alkaline pH in the range of 8.0 to 11.0 may be maintained.
  • the pH of the liquid system may be acidic, for example, a pH of 6,0.
  • an acidic pH of the liquid system may be a pH from about 4,0 to about 6.5,
  • Microalgae can be cultured in defined media known in the art, such as min-70, M-medium, or Tris acetate phosphate (TAP) medium.
  • Microalgae can be grown on a defined minimal medium (for example, high salt medium (HSM), modified artificial sea water medium (MASM), or F/2 medium) with light as the sole energy source.
  • HSM high salt medium
  • MASM modified artificial sea water medium
  • F/2 medium F/2 medium
  • the microalgae can be grown in a medium (for example, TAP medium), and supplemented with an organic carbon source.
  • Organisms can grow naturally in fresh water or marine water.
  • Culture media for freshwater microalgae can be, for example, synthetic media, enriched media, soil water media, and solidified media, such as agar.
  • Various culture media have been developed and used for the isolation and cultivation of fresh water microalgae and are described in Watanabe, M.W. (2005). Freshwater Culture Media. In R.A. Andersen (Ed.), Algal Culturing Techniques (pp. 13-20).
  • Culture media for marine microalgae can be, for example, artificial seawater media or natural seawater media, Guidelines for the preparation of media are described in Harrison, P.J. and Berges, J.A. (2005). Marine Culture Media. In R.A, Andersen (Ed.), Algal Culturing Techniques (pp. 21-33). Elsevier Academic Press.
  • Desmid (e.g., Scenedesmus and Des modes mus ) media may be: 1.929 g/L sodium bicarbonate, 0.1 g/L urea, 2.3730 g/L sodium sulfate, 0,52 g/L sodium chloride, 0.298 g/L potassium chloride, 0.365 g/L magnesium sulfate, 0.084 g/L sodium fluoride, 0.035 niL/L 75% phosphoric acid, 0.018 g/L Librel ⁇ Fe-Lo (BASF), 0.3 mL/L 20X iron stock solution (20X iron stock solution: 1 g/L sodium ethylenediaminetetraacetic acid (EDTA) and 3.88 g/L iron chloride) and 0.06 mL/L 100X trace metal stock solution (100X trace metal stock solution: 1 g/L sodium ethylenediaminetetraacetic acid, 7.2 g/L manganese chloride, 2,09 g/L
  • Organisms may be grown in outdoor open water, such as ponds, the ocean, seas, rivers, waterbeds, marshes, shallow pools, lakes, aqueducts, and reservoirs.
  • the organism When grown in water, the organism can be contained in a halo-like object comprised of lego-like particles.
  • the halo-like object encircles the organism and allows it to retain nutrients from the water beneath while keeping it in open sunlight.
  • the microalgae ca be grown in open and/or closed systems that can vary in volume over a wide range. Closed systems can include reservoir structures, such as ponds, troughs, or tubes, which are protected from the external environment and have controlled temperatures, atmospheres, and other conditions. Closed systems may obtain the light required for photosynthesis artificially or naturally. For some embodiments, the microalgae may be grown in the absence of light and/or in the presence of an organic carbon source.
  • microalgae growth reservoirs can include a carbon dioxide source and a circulating mechanism configured to circulate microalgae within the microalgae growth reservoirs.
  • closed growth environments or reservoirs include closed bioreactors.
  • an open microalgae culture system at least one aspect of the liquid system is open to the environment.
  • An open liquid system may be provided with light for photosynthesis artificially or naturally.
  • the microalgae may be grown in the absence of light and/or in the presence of an organic carbon source.
  • natural light is often used.
  • An open system allows the free exchange of nutrients and products, for example oxygen and carbon dioxide with the air.
  • One way to achieve large surface growth areas is in large ponds or in a captive marine environment.
  • a raceway pond can be used as a microalgae growth reservoir in which microalgae are grown in shallow circulating ponds with constant movement around the raceway and constant extraction or skimming off of mature microalgae.
  • microalgae are grown in non-circulating pools.
  • microalgae cultures can become host to other biological organisms that can decrease the production of microalgae by competing for nutrients. Pest organisms are a significant problem for the efficient production of commercial products of interest by microalgae. In other cases, infection of a microalgae culture can completely destroy production either by competition or by parasitism , Non-limiting examples of pests are bacteria and fungi.
  • organisms can be grown in containers wherein each container comprises one or two organisms, or a plurality of organisms.
  • the containers can be configured to float on water.
  • a container can be filled by a combination of air and water to make the container and the organism(s) in it buoyant.
  • An organism that is adapted to grow in fresh water can thus be grown in salt water (i.e., the ocean) and vice versa. This mechanism allows for automatic death of the organism if there is any damage to the container.
  • Culturing techniques for microalgae include those described, for example, in Freshwater Culture Media. In R.A. Andersen (Ed,), Algal Culturing Techniques. Elsevier Academic Press, herein incorporated by reference in its entirety,
  • photosynthetic organisms like microalgae, require sunlight, C0 2 and water for growth, they can be cultivated in, for example, open ponds and lakes, However, these open systems are more vulnerable to contamination with a pest than a closed system.
  • One challenge with using an open system is that the organism of interest may not grow as quickly as a pest. This becomes a problem when a pest invades the liquid environment in which the organism of interest is growing, and the invading pest has a faster grow h rate and takes over the system.
  • Another approach to growing an organism is to use a semi-closed system, such as covering the pond or pool with a structure, for example, a "greenhouse-type' ' ' structure. While this can result in a smaller system, it addresses many of the problems associated with an open system.
  • the advantages of a semi-closed system are that it can allow for a greater number of different organisms to be grown, it can allow for an organism to be dominant over an invading organism by allowing the organism of interest to out compete the invading organism for nutrients required for its growth, and it can extend the growing season for the organism. For example, if the system is heated, the organism can grow year round.
  • a variation of the pond system is an artificial pond, for example, a raceway pond.
  • the organism, water, and nutrients circulate around a "racetrack.”
  • Paddiewheels provide constant motion to the liquid in the racetrack, allowing for the organism to be circulated back to the surface of the liquid at a chosen frequency.
  • Paddiewheels also provide a source of agitation and oxygenate the system.
  • These raceway ponds can be enclosed, for example, in a building or a greenhouse, or can be located outdoors. It will be apparent to one skilled in the art, that other designs of artificial ponds may be used in addition to raceway ponds and that other means of motivating liquid other than paddiewheels, such as pumps, may also be used.
  • Some of the organisms which may be grown in the l iquid systems described herein are halophilic.
  • some microalgae can grow in ocean water and salt lakes (salinity from 30- 300 parts per thousand) and high salinity media (e.g., artificial seawater medium, seawater nutrient agar, brackish water medium, seawater medium, etc.).
  • a halophilic organism may be transformed with any vectors known in the art.
  • a halophilic organism may be transformed with a vector which is capable of insertion into the nuclear genome and which contains nucleic acids which encode a flocculation moiety (e.g., an anti-cell-surface-protein antibody, a carbohydrate binding protein, etc.).
  • Transformed halophilic organisms may then be grown in high-saline environments (e.g., salt lakes, salt ponds, high-saline media, etc.) to produce the products (e.g., isoprenoids, fatty acids, biomass degrading enzymes, etc.), or biomass, of interest.
  • a flocculation moiety may be non-functional under high salinity conditions.
  • flocculation may be induced by lowering the salinity (e.g., by diluting the liquid environment).
  • the floccuiation moiety may be functional under high salinity conditions and floccuiation may be controlled by increasing the salinity of the medium, isolation of any products of interest produced by the organism may involve removing a transformed organism from a high-saline environment prior to extracting the product from the organism. In instances where the product is secreted into the surrounding environment, it may be necessary to desalina te the liquid environment prior to any further processing of the product.
  • Large scale culture can be conducted in a photobioreactor, semi-closed ponds, open ponds, or lakes. Multiple batches of small scale culture can be seeded into one large-scale culture vessel. The ratio of seeding volume to receiving volume can be determined at the time of seeding according to parameters such as optical density and growth rate of the small scale culture(s).
  • autoclaving adding nutrients to recycled media, evaluating the condition of recycled media, and measuring the H, salt, and conductivity of the media can be performed.
  • quality control is performed.
  • Quality control criteria may include sampling and screening for contamination, strain divergence, growth kinetics, oxygen level, nitrogen level, salinity of the liquid, pH of the liquid media, sampling of growing cells for oil content measurement, dry weight/wet weight ratio, and optical density of the culture.
  • the present disclosure also provides for liquid systems having a controlled temperature.
  • the temperature of the liquid system is maintained between 15 °C and 32 °C.
  • the temperature of the system is kept above 15 °C, In yet another aspect, the temperature of the system is not allowed to exceed 32 °C, In an aspect, the temperature of the system is kept below 25 °C.
  • the temperature may be from 0 to 35 °C, from 5 to 35 °C, from 10 to 35 °C, 15 to 35 °C, from 20 to 35 °C, from 25 to 35 °C, and from 30 to 35 °C. In yet another aspect, the temperature may be maintained at greater than 5 C 'C.
  • the temperature may be maintained at greater than 10 °C. In an aspect, the temperature may be maintained at greater than 15 °C. In an aspect, the temperature may be maintained at greater than 20 °C or greater than 30 °C.
  • the present disclosure also provides for liquid systems having a temperature determined by the environment.
  • the microalgae may be grown in liquid culture systems of different volumes.
  • the microalgae can be grown, for example, in small scale laboratory liquid systems.
  • Small scale labora tory systems refer to cultures in volumes of less than about 6 liters.
  • the small scale laboratory culture may be I liter, 2 liters, 3 liters, 4 liters, or 5 liters.
  • the small scale laboratory culture may be less than one liter.
  • the small scale laboratory culture may be 100 milliliters or less.
  • the culture may be 10 milliliter or less.
  • the liquid culture may be 5 milliliters or less.
  • the liquid culture may be 1 milliliter or less.
  • the liquid culture systems may be large scale cultures, where large scale cultures refers to growth of cultures in volumes of greater than about 6 liters, or greater than about 10 liters, or greater than about 20 liters. Large scale growth can also be growth of cultures in volumes of 50 liters or more, 100 liters or more, or 200 liters or more. Large scale gro wth can be growth of cultures in, for example, ponds, containers, vessels, or other areas, where the pond, container, vessel, or area that contains the culture is for example, at least 5 square meters, at least 10 square meters, at least 200 square meters, at least 500 square meters, at least 1,500 square meters, at least 2,500 square meters, in area, or greater,
  • the volume of Hquid culture may be at least 20,000 liters.
  • the volume of liquid can be up to 40,000 liters.
  • the volume of liquid can be up to 80,000 liters.
  • the volume of liquid can be up to 100,000 liters.
  • the volume of liquid can be up to 150,000 liters.
  • the volume of liquid can be up to 200,000 liters.
  • the volume of liquid can be up to 250,000 liters,
  • the volume of liquid can be up to 500,000 liters.
  • the volume of liquid can be up to 600,000 liters.
  • the volume of liquid can be up to 1,000,000 liters,
  • the very large scale liquid system may be from 10,000 to 20,000 liters, In an aspect, the very large scale liquid system may be from 10,000 to 40,000 liters or from 10,000 to 80,000 liters. In another aspect, the very large scale liquid system may be from 10,000 to 100,000 liters or from 10,000 to 150,000 liters. In yet another aspect, the liquid system may be from 10,000 to 200,000 liters or from 10,000 to 250,000 liters. The present disclosure also includes liquid systems from 10,000 to 500,000 liters or from 10,000 to 600,000 liters. The present disclosure further provides for liquid systems from 10,000 to 1,000,000 liters.
  • the liquid culture system may be from 20,000 to 40,000 liters or from 20,000 to 80,000 liters. In another aspect, the liquid system may be from 20,000 to 100,000 liters, In yet another aspect, the liquid system may be from 20,000 to 150,000 liters or from 20,000 to 200,000 liters. In another aspect, may be from 20,000 to 250,000 liters. In another aspect, the liquid system may be from 20,000 to 500,000 liters. In another aspect, the liquid system may be from 20,000 to 600,000 liters, In another aspect, the liquid system may be from 20,000 to 1,000,000 liters,
  • the liquid culture system may be from 40,000 to 80,000 liters. In another aspect, the liquid system may be from 40,000 to 100,000 liters. In another aspect, the liquid system may be from 40,000 to 150,000 liters. In another aspect, the liquid system may be from 40,000 to 200,000 liters. In another aspect, the liquid system may be from 40,000 to 250,000 liters. In another aspect, the liquid system may be from 40,000 to 500,000 liters. In another aspect, the liquid system may be from 40,000 to 600,000 liters. In another aspect, the liquid system may be from 40,000 to 1 ,000,000 liters.
  • the liquid system may be from 80,000 to 100,000 liters. In another aspect, the liquid system may be from 80,000 to 150,000 liters. In another aspect, the liquid system may be from 80,000 to 200,000 liters. In another aspect, the liquid system may be from 80,000 to 250,000 liters. In another aspect, the liquid system may be from 80,000 to 500,000 liters. In another aspect, the liquid system may be from 80,000 to 600,000 liters. In another aspect, the liquid system may be from 80,000 to 1,000,000 liters.
  • the liquid system may be from 100,000 to 150,000 liters. In another aspect, the liquid system may be from 100,000 to 200,000 liters. In another aspect, the liquid system may be from 100,000 to 250,000 liters. In another aspect, the liquid system may be from 100,000 to 500,000 liters. In another aspect, the liquid system may be from 100,000 to 600,000 liters. In another aspect, the liquid system may be from 100,000 to 1,000,000 liters,
  • the liquid system may be from 200,000 to 250,000 liters. In another aspect, the liquid system may be from 200,000 to 500,000 liters. In another aspect, the liquid system may be from 200,000 to 600,000 liters. In another aspect, the liquid system may be from 200,000 to 1,000,000 liters. In another aspect, the liquid system may be from 250,000 to 500,000 liters. In another aspect, the liquid system may be from 250,000 to 600,000 liters. In another aspect, the liquid system may be from 250,000 to 1,000,000 liters. In another aspect, the liquid system may be from 500,000 to 600,000 liters, or 500,000 to 1 ,000,000 liters.
  • the liquid system may be a pond, either natural or artificial,
  • the artificial pond may be a raceway pond.
  • the organism, water, and nutrients circulate around a "racetrack.”
  • Paddlewheels provide constant motion to the liquid in the racetrack, allowing for the organism to be circulated back to the surface of the liquid at a chosen frequency.
  • Paddlewheels also provide a source of agitation and oxygenate the system.
  • CO ? may be added to a liquid system as a feedstock for photosynthesis through a CO ? injection system.
  • These raceway ponds can be enclosed, for example, in a building or a greenhouse, or can be located outdoors. In an aspect, an outdoor raceway liquid system may be enclosed with a cover, or exposed.
  • Raceway ponds are usually kept shallow because the organism needs to be exposed to sunlight, and sunlight can only penetrate the pond water to a limited depth.
  • the depth of a raceway pond can be, for example, about 4 to about 12 inches.
  • the volume of liquid that can be contained in a raceway pond can be, for example, about 200 liters to about 600,000 liters.
  • the raceway ponds can be operated in a continuous manner, with, for example, CO ? and nutrients being constantly fed to the ponds, while water containing the organism is removed at the other end.
  • the ponds may have a surface area of at least 0.25 of an acre. In another aspect, the pond may be at least 0.5 acre or at least 1.0 acre. In yet another aspect, the pond may be at least 1.5 acres or at least 2.0 acres.
  • the liquid system may be a pond of at least 2.5 acres or at least 5.0 acres. In an alternative aspect, the pond may be at least 7.5 acres or at least 10 acres.
  • the pond may have a surface area of at least 12 acres, at least 15 acres, at least 18 acres, at least 20 acres, at least 25 acres, at least 30 acres, at least 35 acres, at least 40 acres, at least 45 acres, at least 50 acres, at least 100 acres, at least 200 acres, at least 300 acres, at least 400 acres or at least 500 acres.
  • the surface area of a pond may be from 0.25 to 0,5 acres or 0.25 to 1.0 acres.
  • the liquid system may be a pond of 0.25 to 1.5 acres or 0.25 to 2.0 acres.
  • the pond may be from 0.25 to 2.5 acres, 0.25 to 5.0 acres or 0.25 to 7.5 acres.
  • the liquid system may be a pond of 0.5 to 1.0 acres, 0.5 to 1 .5 acres, 0.5 to 2.0 acres, 0.5 to 2.5 acres, 0.5 to 5.0 acres or 0.5 to 7.5 acres.
  • the liquid system may cover an area of 1 .0 to 1.5 acres or 1.0 to 2.0 acres.
  • the liquid system may be a pond of 1.0 to 2.5 acres or 1 ,0 to 5.0 acres. In yet another aspect, the liquid system may be a pond of 1.0 to 7.5 acres or 2.0 to 2.5 acres. In another aspect the pond may be from 2.0 to 5,0 acres or 2.0 to 7.5 acres.
  • the pond may range from 2.5 to 5.0 acres, 2.5 to 7.5 acres, 2,5 to 10 acres, 5 to 12 acres, 5 to 15 acres, 5 to 18 acres, 5 to 20 acres, 10 to 25 acres, 10 to 30 acres, 10 to 35 acres, 10 to 40 acres, 10 to 45 acres, 10 to 50 acres, 10 to 15 acres, 15 to 20 acres, 20 to 25 acres, 25 to 30 acres, 30 to 35 acres, 35 to 40 acres, 40 to 45 acres, 45 to 50 acres, 50 to 75 acres, 75 to 100 acres, 100 to 125 acres, 125 to 150 acres, 150 to 175 acres, 175 to 200 acres, 200 to 250 acres, 250 to 300 acres, 300 to 350 acres, 350 to 400 acres, 400 to 450 acres, or 450 to 500 acres in area,
  • a photobioreactor is a bioreactor which incorporates some type of light source to provide photonic energy input into the reactor.
  • the term photobioreactor can refer to a system closed to the environment and having no direct exchange of gases and contaminants with the environment.
  • a photobioreactor can be described as an enclosed, illuminated culture vessel designed for controlled biomass production of prototrophic liquid cel l suspension cultures.
  • Examples of photobioreactors include, for example, glass containers, plastic tubes, tanks, plastic sleeves, and bags
  • Examples of light sources that can be used to provide the energy required to sustain photosynthesis include, for example, fluorescent bulbs, LEDs, and natural sunlight. Because these systems are closed everything that the organism needs to grow (for example, carbon dioxide, nutrients, water, and light) must be introduced into the bioreactor.
  • Photobioreactors despite the costs to set up and maintain them, have several advantages over open systems, they can, for example, prevent or minimize contamination, permit axenic organism cultivation of monocultures (a culture consisting of only one species of organism), offer better control over the culture conditions (for example, pH, light, carbon dioxide, and temperature), prevent water evaporation, lower carbon dioxide losses due to out gassing, and permit higher cell concentrations.
  • Photobioreactors can be set up to be continually harvested (as is with the majority of the larger volume cultivation systems), or harvested one batch at a time (for example, as with polyethlyene bag cultivation).
  • a batch photobioreactor is set up with, for example, nutrients, an organism (for example, microalgae), and water, and the organism is allowed to grow until the batch is harvested.
  • a continuous photobioreactor can be harvested, for example, either continually, daily, or at fixed time intervals.
  • High density photobioreactors may be used and include those that are described in, for example, Lee, et al., Biotech. Bioeiigineering 44: 1161-1167, 1994.
  • Other types of bioreactors such as those for sewage and waste water treatments, are described in, Sawayama, et al., Appl. Micro. Biotech., 1:729-731, 1994.
  • Additional examples of photobioreactors are described in, U.S. Appl. Publ. No, 2005/0260553, U.S. Pat. No. 5,958,761, and U.S. Pat. No, 6,083,740.
  • organisms such as microalgae may be mass-cultured for the removal of heavy metals (for example, as described in Wilkinson, Biotech, Letters, 1 1 :861 -864, 1989), hydrogen (for example, as described in U.S. Patent Application Publication No, 2003/0162273), and pharmaceutical compounds from a water, soil, or other source or sample.
  • Organisms can also be cultured in conventional fermentation bioreactors, which include, but are not limited to, batch, fed-batch, cell recycle, and continuous fermenters. Additional methods of culturing organisms and varia tions of the methods described herein are known to one of skil l in the art.
  • the present disclosure further provides for harvesting of the microalgae grown in the liquid system.
  • Harvesting may be accomplished by methods known to one of skill in the art including collection of the microal gae in w tole or in part .
  • harvesting may be accomplished by removing portions of the growing culture and separating the microalgae from the liquid.
  • harvesting may be accomplished by continuous flow methods, for example, using a continuous flow centrifuge,
  • microalgae separation of the microalgae from the liquid may be accomplished by methods known to one of ordinary skill in the art, In one aspect, die microalgae may be allowed to settle by gravity and the overlying liquid remo ved. In another aspect, the microalgae may be harvested by centrifugation of the microalgae containing culture, In an aspect, centrifugation of the liquid culture may be performed in batch mode, using a fixed volume centrifuge. In a different aspect, batch harvesting of the microalgae may be accomplished using a continuous flow r centrifuge. In another aspect, the microalgae may be harvested continuously from the growing culture by continuous flow centrifugation.
  • harvesting of the microalgae grown in the liquid system may be facilitated by flocculation.
  • Methods for inducing floccuiation include those that can be found in U.S. Patent Publication No, US 201 1/0159595, Application No. 13/001027, hereby incorporated in its entirety by reference,
  • the flocculate may be separated from the culture liquid by gravity, centrifugation or other physical method known to those of skill in the art.
  • the flocculate may be separated from the culture liquid by dissolved air flotation (DAT),
  • harvesting includes separating at least 90% of the microalgae from the liquid culture to produce a microaigae depleted liquid.
  • at least 95% of the microaigae are removed from the liquid culture.
  • at least 97% of the microaigae are removed from the liquid culture.
  • at least 99% of the microaigae are removed from the liquid culture.
  • 50% or more of the microaigae are removed.
  • 75%) or more of the microaigae are removed from the liquid culture.
  • 80%) of more of the microaigae are removed from the liquid culture.
  • the liquid culture can have less than 30% of the microaigae remaining after harvesting, in a further aspect, less than 25% of the microaigae remained after harvesting. In a further aspect, less than 5% of the microaigae remained after harvesting. In a further aspect, less than 2.5% of the microaigae remained after harvesting. In an aspect, less than 1% of the microaigae remain after harvesting.
  • harvesting of microaigae from the growmg culture may be performed on a part of the total liquid culture.
  • the part of the liquid culture is removed and the microaigae are harvested.
  • at least 2 percent of a total volume of a liquid culture is removed and the microaigae harvested.
  • at least 2.5 % of the total volume of the liquid culture containing the growing microaigae is removed and the microaigae harvested.
  • at least 5% or at least 7.5% of the total volume of the liquid culture containing the gro wing microaigae is removed for harvesting.
  • at least 10% or at least 12.5% of the total volume of the liquid culture containing the growing microaigae is removed for harvesting.
  • at least 15% or at least 20% of the total volume of the liquid culture containing the growing microaigae is removed for harvesting.
  • the amount of liquid removed for harvesting may range from 2 to 15% or from 2 to 20% of the total volume of the liquid culture. In a further aspect, from 2.5 to 5 %) or from 2.5 to 7.5%» of the total liquid culture volume may be removed for harvesting. In an aspect, the amount of liquid removed for harvesting may be from 2.5 to 10% or from 2.5 to 12.5% of the total growing culture volume.
  • the amount removed may range from 2.5 to 15% or from 2.5 to 20%. In a further aspect, from 5 to 7.5% or from 5 to 10% of the culture volume may be removed for harvesting. In an aspect, from 5 to 12.5%, from 5 to 15%», or even from 5 to 20% of the total volume of liquid culture may be harvested. In another aspect, the amount of harvested culture may be from 7.5 to 1 Q%> or from 7.5 to 12.5% of the total culture volume. In an aspect, the amount of liquid remo ved for harvesting my range from 7.5 to 15% or from 7.5 to 20% of the culture volume, in yet another aspect, 10 to 12,5% or 10 to 15% of the culture volume may be removed from harvesting. In an aspect, 10 to 20% of the total volume of a liquid culture may be removed for harvesting of the growing microalgae.
  • harvesting may he conducted continuously from the growing culture of microalgae.
  • removal of the microalgae maintains the c ulture in a logarithmic phase of microalgae growth.
  • One of skill in the art understands that the when growing in a logarithmic phase, the number of microalgae double within a time period. The time period for microalgae doubling depends on the environment of the growing microalgae.
  • the determination of growth rates and phases of microalgae growth are known in the art. For example, in Sode et al, "On-line monitoring of marine cyanobacterial cultivation based on phycocyanin fluorescence," J. Biotechnology 21 :209-217 ( 1991), Torzillo et al., "On-Line
  • a portion of the liquid culture may be removed for harvesting and the portion replaced so that the total volume of the liquid culture remains within a narrow range.
  • the amount of liquid removed during continuous harvesting is up to 1000 gallons per hour.
  • the amount removed during continuous harvesting may be 1% of the total v olume per hour.
  • up to 5% of the volume per day may be removed during a continuous harvesting.
  • up to 15% of the volume per day may be removed during a continuous harvesting.
  • up to 33% of the volume per day may be removed during a continuous harvesting.
  • an indoor system may be a greenhouse, In an aspect, a greenhouse may receive natural light. In another aspect a greenhouse may be artificially lighted, in yet another aspect, natural light may be supplemented by artificial light.
  • artificial light may be fluorescent light.
  • One source of energy is fluorescent light that can be placed, for example, at a distance of about 1 inch to about two feet from the organism.
  • types of fluorescent lights includes, for example, cool white and daylight. If the lights are turned on and off at regular intervals (for example, 12: 12 or 14: 10 hours of light: dark) the cells of some organisms will become synchronized.
  • the logarithmic growth phase is characterized by log-linear growth of the organism when the density or cell number is plotted on a logarithmic scale versus time. The 'doubling' time is used to characterize this phase of growth. Both extrinsic environmental factors and intrinsic factors control the doubling time of an organisms. Those of skill in the art recognize that the rate of doubling can be limited by the necessity of initiating and completing successive rounds of DNA synthesis and genome replication.
  • Extrinsic factors play important roles in the gro wth of microalgae including the presence of nutrients, the temperature, the H, and the availability of light for photosynthesis.
  • Methods of growing and optimizing the growth of microalgae are known in the art, for example in Vonshak, A. Spirulina Platensis Arthrospira:
  • the rate of doubling decreases in a phase called “late log-phase.” Growth decreases due to limiting nutrients (for example, lack of C0 2 , lack of a carbon source etc.) or is due to factors secreted by the growing organisms (e.g., quorum sensing).
  • the number of microorganisms stops increasing and the culture enters a stationary phase. In some aspects, the microorganisms may initiate
  • microorganisms may have changes in gene expression including both increases and decreases in expression. Removal of microorganisms in the stationary phase and inoculation of a fresh culture often results in a lag phase prior to entry into a logarithmic growth phase.
  • the doubling time (e.g. doubling rate) during growth in the logarithmic phase can depend on a number environmental conditions. Among the factors it is recognized that the nutrients and media conditions significantly affect growth.
  • microaigae can be autotrophic and are therefore less susceptible to the presence of carbon based food sources.
  • One of ordinary skill in the art would understand that the availability of nitrogen affects microaigae growth. Decreased nitrogen leads to longer doubling times, or even entry into stationary phases. Increased nitrogen availability may result in decreased doubling time.
  • a growing liquid culture can be monitored for changes in the environmental conditions to maintain or optimize logarithmic phase growth. Production of microaigae is optimized when growth is logarithmic.
  • the growth of the c ulture proceeds through different growth p hases
  • a liquid culture is inoculated and proceeds from a lag phase to the logarithmic phase to the stationary phase.
  • logarithmically growing microaigae are provided such that there is no lag phase of growth.
  • logarithmic phase is maintained by harvesting microaigae.
  • logarithmic phase is maintained by supplementing the liquid culture system that is limited for one or more nutrients.
  • a logarithmic growth phase is maintained by harvesting microaigae and supplementing the liquid culture system.
  • a liquid after harvest can be monitored and nutrients added prior to returning the liquid culture system.
  • a liquid culture system can be supplied with fresh media, for example water, and logarithmic phase maintained.
  • a fresh media may contain nutrients necessary to maintain the logarithmic phase of microaigae growth.
  • microaigae depleted liquid can be further purified to remove contaminants to maintain logarithmic growth.
  • the liquid culture is treated with fungicide, such as Fluazinam, during the logarithmic phase.
  • the liquid culture is treated during the lag phase.
  • the liquid culture is treated during the stationary phase.
  • the microalgae are harvested from the liquid culture during logarithmic phase.
  • the microalgae are harvested from the liquid culture during late logarithmic phase,
  • the microalgae are harvested from the liquid culture during stationary phase.
  • algae growth is maintained at an optimal density for logarithmic growth.
  • the optimal density may he determined experimentally for a strain of microalgae.
  • testing for the presence of a pest need not be conducted at any particular phase of growth.
  • a pest such as a fungus (e.g. a chytrid)
  • testing for the presence of a pest may be performed before inoculation of the liquid system with a microalga.
  • testing may be performed during the lag phase of microalgae growth.
  • testing may be performed during logarithmic growth or at late logarithmic growth,
  • testing may be performed at a stationary phase of a microalgae growth cycle.
  • testing may be performed throughout each stage of a microalgae growth cycle,
  • treatment may be performed before inoculation of the liquid system with microalgae.
  • treatment may be performed during the lag phase of microalgae growth.
  • treatment may be performed during logarithmic growth or at late logarithmic growth.
  • treatment may be performed at a stationary phase of a microalgae growth cycle.
  • treatment may be performed throughout each stage of a microalgae growth cycle.
  • a liquid culture is grown for 15 or more days. In another aspect, a liquid culture is grown for 30 or more days. In an aspect, a liquid culture is grown for 45 or more days. In another aspect, a liquid culture is grown 60 or more, or 90 or more days. In yet another aspect, growth time may be 120 or more, or 180 or more days. In an aspect, a liquid culture may be maintained 250 or more, or 500 or more days. In yet another aspect, growth of a liquid culture may be continued for 1000 or more, 1500 or more, or 2000 or more days after inoculation of the liquid culture. The culture may be maintained, with fungicide treatments of the present disclosure for an indefinite amount of time.
  • the present disclosure provides for treatments of a liquid system.
  • Treatments may include physical methods to control the growth of, or kill a pest present in a liquid system. Physical methods may include, as non limiting examples, filtration, heating, cooling and irradiation.
  • the present disclosure provides for treatments of a liquid system including the addition of compositions that control the growth of, or kill a pest.
  • the treatment may be provided upon detecting the presence of a pest.
  • the treatment may be provide upon the detection of a fungus in a liquid sy stem.
  • the treatment may be prophylactic and the treatment may be provided during any stage of growth of the microalgae.
  • the treatment can, for example, be applied to the liquid in the system prior to inoculation with the strain, simultaneously applied to the liquid with the strain, or after inoculation of the liquid with the strain.
  • Treatments of the present disclosure include adding one fungicide, wherein an a ctive ingredient is Fluazinam, and at least one additional fungicide, to a liquid culture system.
  • a fungicide comprising Fluazinam can be provided alone or in combination with any one or more of the fungicides listed below in Table 1, to a liquid culture system.
  • a fungicide comprising Binapacryl as an active ingredient can be provided alone or in combination with any one or more of the fungicides listed below in Table I, to a liquid culture system.
  • a fungicide comprising Dinocap as an active ingredient can be provided alone or in combination with any one or more of the fungicides listed below in Table 1, to a liquid culture system.
  • a fungicide comprising Meptyldinocap as an active ingredient can be provided alone or in combination with any one or more of the fungicides listed below in Table 1, to a liquid culture system.
  • Binapacryl, Meptyldinocap, Dinocap, and Fluzinam are all FRAC29 fungicides with the same mechanism of action.
  • a fungicide may be a chemical compound.
  • the fungicide may further contain non-active (inactive) ingredients that may aid in dissolving or dispensing the active ingredient.
  • Fungicides may be known in the art or may be developed to kill or inhibit a pest. Non- limiting examples of fungicides of the present disclosure are presented in Table 1,
  • Treatments of the present disclosure include providing one or more fungicides presented in Table 1.
  • a first effective concentration of fungicide may be provided to a liquid system upon detection of a first pest.
  • an effective concentration of second fungicide may be provided to a liquid system after the effective concentration of the first fungicide and upon detection of a pest.
  • a fungicide is selected to have a different mechanism of action than a first fungicide.
  • a third fungicide may be provided as a treatment of a liquid system after the effective treatment of a first and second fungicide.
  • a first, second and third fungicide may be rotated to ensure effective control of a pest(s) in a liquid culture system and to avoid the development of fungicide resistance in a liquid culture system.
  • a combination of two fungicides may be provided upon detection of a first pest.
  • a third fungicide may be provided where the first and second fungicide combination does not control a pest of the liquid system.
  • a first fungicide may be an inhibitor of respiration that uncouples oxidative phosphorylation (e.g. dinocap, fSuazinani, meptyldinocap, and/or binapacryi) and a second fungicide may be a quinone outside inhibitor of respiration.
  • a first fungicide may be an inhibitor of respiration that uncouples oxidative phosphorylation and a second fungicide may have muiti site contact activity.
  • the treatments may be performed at a specified time of the day.
  • the treatment may be conducted in the morning.
  • the treatment may be conducted at mid-day.
  • the treatment may be performed at or near sunset.
  • treatment may be performed at night.
  • treatment may be performed at two periods each day, for example in the morning and again in the evening.
  • treatment may occur during the day and a second monitoring may occur at night,
  • the present disclosure provides for the treatment of a liquid system to minimize the formation of concentration gradients.
  • an amount of treatment is calculated based on the volume of a liquid system and prepared in a volume of the media (e.g. the culture media of the liquid system) to prepare a concentrated treatment stock.
  • a concentrated treatment stock may be slowly added to a liquid system.
  • concentrated treatment stock is added behind a paddle wheel of a raceway pond system.
  • the concentrated treatment stock is dispersed by spraying of a liquid system.
  • the concentrated treatment stock is added to a water return line of a circulation pump,
  • the present disclosure provides for the treatment of a liquid system with a fungicide of the pyridinamine family.
  • the pyridinamine may be fiuazinam (phenyl-pyridinamine or 3- iloro-N-[3- iloro-2,6 iinitro-4-(tri ⁇
  • fluazinam may be provided as a first fungicide treatment of i liquid system. In another aspect, fluazinam is provided as a second fungicide treatment. In an aspect, fluazinam may be provided as a third fungicide treatment. In yet another aspect, fluazinam may be provided as fourth treatment or a fifth treatment. In another aspect, fluazinam may be provided as sixth treatment or a seventh treatment. In other embodiments, fluazinam may be administered in combination with one or more fungicides either separately by being administered contemporaneously with the one or more fungicides, or as part of a mixture of fungicides.
  • Fungicide such as Fluazinam
  • the fungicides may be introduced by methods known in the art.
  • the fungicides may be introduced as a solid.
  • the fungicides may be introduced after solvation in an appropriate solvent.
  • a solvent may be water.
  • the fungicide may be dissolved in an alcohol.
  • the alcohol may be methanol.
  • the alcohol may be ethanol.
  • the fungicide may be prepared in acetonitrile.
  • the fungicide may be prepared in acetone.
  • the fungicide may be dissolved in the culture medium used to grow the microalgae. In an aspect, the effect of the solvent on the organism or organisms is minimized.
  • the present disclosure provides for the introduction of fungicides such as Fluazicam, at an effective concentration. Effective concentrations may be determined according to manufacturer's instructions or may be determined empirically. An effective concentration of a fungicide is not toxic to the microalgae being cultured in the liquid system. Methods to determine toxicity are known in the art and include serial dilutions of a test fungicide in a growing liquid culture of microalgae. Fungicides may begin to show growth effects on microalgae in the exemplar ⁇ ' ranges provided in Table 2.
  • microalgae may have different ranges of toxicity that may be determined by growth of a microalga in the presence of a serial dilution of a fungicide.
  • the ranges provided below can easily be widened by one skilled in th art by conducting appropriate experiments.
  • a fungicide may be toxic to a microalgae if the growth of a microalgae is decreased in a given concentration range.
  • an effective concentration of fungicide may cause a decrease in microalgae growth but causes a greater reduction in the gro wth of a pest.
  • OM27 is the same strain as SE 70179, and OM65 is the same strain as SE 70181.
  • a diverse mutant library was created as follows: the UV intensity that kills 90% of the WT ' population (typically -50 mJ for 2.0*10 A 8 cells) was determined. In addition, the UV intensity that kills 99% of the WT population (up to 200 mi ) was also determined. Batches of WT cultures at UV intensities that range between the 90% and 99% killing intensities were mutagenized. It was assumed that many of the surviving cells will carry >1 mutation per genome. Mutagenized cultures were allowed to recover in culture medium and an equal proportion of live cells from each UV intensity treatment were pooled to create a final library. It was assumed that the higher intensity- treatments could introduce more mutations per genome or different types of mutations compared to the lower intensity treatments, thus creating a diverse library of mutants. Downstream selective screens were then used to identify clones with desirable traits that are different from WT.
  • a rapid and straightforward method to isolate biocide is to screen a genetically diverse population using an inhibitory concentration (IC) of a given biocide.
  • IC inhibitory concentration
  • 100% of WT clones will not survive the IC treatment; while in contrast, some resistant mutants may survive IC treatment thus enabling their isolation in the laboratory.
  • individual mutants with desirable phenotypes from a diverse mutated library may be isolated on solid media containing an IC of a particular biocide.
  • clones can also be isolated by screening in liquid medium containing an IC of a particular biocide. Screening in liquid medium, rather than solid medium, may yield mutants with growth characteristics that would be more translatable to pond conditions.
  • IC of OMEG A* for the WT Desmodesmus species was identified from a dilution series experiment ranging from 1 ppm to 210 ppm in culture medium using 5 mL tubes.
  • the inhibitor ⁇ ' concentration of OMEGA* determined for the WT Desmodesmus species was 100 ppm with a 4-day incubation period.
  • each isolated colony is derived from a single surviving cell. Cultures propagated from an isolated colony are considered axenic. Also, since time of original isolation, the strain was propagated from a single clone after sorting by fluorescence activated cell sorting (FACS). Axenic describes the state of a culture in which only a single species is present, it is a biologically pure culture.
  • BGRA biocide growth rate assay
  • MGRA microliter growth rate assay
  • a logistic grow h model was fit to the data series to estimate the growth rate parameter (r) in permissive medium.
  • Results of the 96-well HTP microliter growth rate assay (MGRA) for nominated clones and the WT Desmodesmus species is shown in Figure 1.
  • the y-axis represents growth rate parameter (r) and the x-axis represents different evolved mutant strains and wild type strains (from left to right: OM15, OM25, OM27, OM65, OM82, WT1 , WT2, WT3, and WT4 (WT1-4 are four separate replicates of the WT Desmodesmus species)).
  • Each bar represents triplicate estimates for the gro wth rate parameter (r) from a logistic growth curve. All of the e vol ved mutant strains exhibited a normal growth rate (r) in permissive media in a microliter format when compared to the wild-type strain(s).
  • the Validation III experiment is similar to the BGRA format described above, but was modified to allow for a semi-continuous harvest scheme in a 96-wel! deep block.
  • This scheme allowed for fresh biocide (OMEGA*) to be replenished at each cutback event, which was critical since the active ingredient of the biocide is expected to decay over the course of several days.
  • the semi-continuous cutback scheme was maintained for 4 cutback events ("rounds"), with two additional dosing events (rounds 5 and 6).
  • each round had six different doses of OMEGA*.
  • the doses were 0.0 ppm, 0.25 ppm, 0.5 ppm, 1.0 ppm, 1.25 ppm, and 1.5 ppm.
  • the doses were 0.0 ppm, 0.5 ppm, 1.0
  • Results from the Validation III experiments are shown for several WT cultures ( Figure 2A to Figure 2D) and for OM15 (Figure 3A), O 25 (Figure 3B), OM27 (Figure 3C), OM65 ( Figure 3D), and OM82 ( Figure 3E).
  • each data point represents three biological replicates in 96-weil deep blocks.
  • the Y-axis is OD750 and the x-axis is time in hours.
  • the WT cultures do not recover compared to, for example, OM65 at 3ppm OMEGA ⁇ (see Figure 3D).
  • FIG. 4 represents the doubling rate for each variant clone and several WT clones at a dose of 2.5 ppm OMEGA*; the y-axis represents doubling rate and the x-axis represents various evolved mutant strains and WT strains. Based on this analysis, OM 15 and OM82 were selected for minipond testing (see Table 3 below)
  • MP cultures were grown until the first MP of the experiment reached a TSS of 0,7g/L, and then all MPs were harvested at that time (even if some MPs have not reached 0.7g/ ' L themselves) to a TSS of 0.4g/L.
  • all MPs were treated with OMEGA* at an increased dose of 1 .0 ppm.
  • all cultures were grown until the first MP reached a TSS of 0.7g/L, and then all MPs were harvested to a TSS of 0.4g/L.
  • all MPs were treated with OMEGA* at an increased dose of 2.0 ppm. All cultures were then grown until the first MP reached a TSS of 0.7g/L.
  • the growth-harvest-treatment cycle continued for a total of -30 plus days including an additional final harvest and dosing at 2.0 ppm.
  • Harvested MPs w r ere replenished after each harvest with fresh culture media, rather than recovered culture media.
  • Data was collected for each mutated and wild type strain throughout the course of the 30 plus day experiment. Standard field measurements were taken on a regular basis, such as dry weigh, OD, Fv/Fni, etc.).
  • the first is a dose of 0.5 ppm of OMEG A*; the second is a dose of 1 ppm of OMEGA*; the third is a dose of 2 ppm of OMEGA* 1 ; and the fourth is a dose of 2 ppm of OMEGA* 1 .
  • Figure 7 shows biomass productivity of ponds comprising the WT strain and the different evolved mutant strains (O 15, OM25, OM27, OM65, and OM82).
  • the y-axis is DW (g/L) and the x-axis is days of experiment. Pigment loss was shown in the WT over time.
  • Downward-facing arrows represent dosing events. From left to right, the first arrow is a dose of 0.5 ppm of OMEGA*, the second arrow is a dose of 1 ppm of OMEGA*, the third arrow is a dose of 2 ppm of OMEGA*, and the forth arrow is a dose of 2 ppm of OMEGA*.
  • each point represents the mean of replicate miniponds for a given strain. Different strains are represented by different shapes; WT is represented by squares.
  • In situ productivity (g m 2 /day) of each MP was calculated using the TSS measurements including a time dimension (days), In situ productivity was calculated for all M Ps during the time span of the 1 ppm treatment ("Round 1": days 4- 14) separately from the time span of the 2 ppm treatment ("Round 2": days 16-25). Given the experimental design, by which all MPs are harvested simultaneously at the point when the first MP of the cohort reaches trigger TSS (0.7 g/L), some MPs will have lower TSS than others at harvest time.
  • Differential TSS values among MPs at the shared harvest times may reflect differential in situ productivities given the shared time- span and OMEGA ®' trea tment across the MPs. Many of the evolved strains showed higher DW (g L) measurements relative to WT at each of the harvesting time points (for example, day 2, 14, and 25).
  • each two-column set represents a 1 ppm OMEGA* dose
  • the right hand column of each two-column set represents a 2 ppm OMEGA* dose
  • the data is viewed in a relati ve fashion by normalizing all productivity estimates to the WT estimates.
  • the left hand column of each two-column set represents a 1 ppm OMEGA* ' dose
  • the right hand column of each two-column set represents a 2 ppm OMEG A* dose.
  • the y-axis is normalized in situ productivity.
  • OM25, OM27, OM65, and QM82 had higher in situ productivity when compared to WT.
  • MPs from two strains were consistently the first to reach trigger TSS in the group (i.e. OM27 at Ippm and OM65 at 2ppm).
  • the calculated in situ productivities reflect that OM65 exhibited higher biomass productivity compared to WT both at 1 ppm and 2 ppm ( Figure 8).
  • the evolved strain OM65 was more than twice as productive as WT under the same conditions. This represents 1 18% higher in situ productivity versus WT (p-va!ue 0.019 by Dunnett's comparison of means test).
  • OM65 trends towards about a 20% improvement versus WT at 1 ppm. Note, that as may be expected, all strains exhibited lower productivities in the presence of OMEGA* than would be typical for cultures that are not treated with biocide. However, for example, the significantly improved relative performance of OM65 when treated with OMEGA* validates the hypothesis that OM65, along with other mutated strains, indeed exhibits a hyper-tolerance of OMEGA* relative to WT. For example, even at I ppm, the in situ, productivity of OM65 is ⁇ 28°/» higher than WT. Also noteworthy is that OM27 displayed in situ productivity similar to OM65 ( Figure 8).
  • Figure 10 shows the data from Figure 8 analyzed by Oneway ANOVA.
  • the product of OM65 is significantly greater than that of WT, with a p-Value of 0.0142.
  • the means comparison with a control (WT) using Dunnett's Method yielded a p-Value of 0.019.
  • Figure 10 represents a dose of 2 ppm of OMEGA*.
  • the y-axis is in situ productivity (g/m 2 /day).
  • Downward- facing arrows represent dosing events. From left to right, the first arrow is a dose of 0.5 ppm of OMEGA*, the second arrow- is a dose of 1 ppm of OMEG A*, the third arrow is a dose of 2 ppm of OMEG A ® , and the forth arrow is a dose of 2 ppm of OMEGA*.
  • each point represents the mean of replicates for a given strain. Different strains are represented by different shapes; WT is represented by a square.
  • the Fv/Fm deficit (y-axis) of OM65 and OM27 was less than half the F v/Fm deficit of WT.
  • the Fv/Fm deficit (y-axis) of OM15, OM25, and OM82 was less than the Fv/Fm deficit of WT,
  • the left hand column of each two-column set represents a 1 ppm OMEGA* dose
  • the right hand column of each two-column set represents a 2 ppm OMEGA* dose.
  • Figure 14A to Figure 14C show that normal pigmentation (including chlorophyll) was severely impacted in the WT strains and to a lesser extent in the variant strains.
  • Figure 16 represents PAM measurements from an independent minipond (RW100) of the WT Desmodesm s species.
  • the y-axis is Fv/Fm (each "notch" is an increment of 0.1; 0, 0.1, 0.2, 0,3, 0,4, 0.5, 0.6, 0.7 and 0.8) and the x-axis is days of experiment (each notch is a two-day increment; 0, 2, 4, 6, 8, 10, 12, 14, and 16).
  • a chytrid infection was detected in the independent minipond (RWIOO) and a Ippm dose of OMEGA 3 was administered.
  • the experiment was initiated by transferring culture from existing R.W ponds as described in Table 4 below. Upon initial inoculation, all RWs were grown using standard cultivation methods to allow for acclimation to the new environment. Typically, a RW would not be treated with biocide unless a chytrid infection is detected. However, for the purpose of experimental design, which was aimed to demonstrate biocide tolerance specifically, cultures were treated, at different times throughout the experiment, with predetermined concentrations of OMEGA* in the absence of an identified chytrid infection.
  • OMEGA*' application was performed independent of harvest operations. RWs were grown with an intended density range of 0.45 - 0,65 g/L, Harvest was completed per standard operating methods returning culture media during biomass removal. Data was collected for each mutated (OM27, OM65) and wild type strain throughout the course of the 21 day experiment. Standard field measurements were taken on a regular basis, such as dr weight, OD, Fv/Fm, etc.).
  • Biomass productivity was determined as a measure of the harvested product removed from the pond during the course of the 21 day field trial. All three ponds, OMEGA*' tolerant mutants plus control, were harvested on day 13, two weeks after the first 1 ppm OMEGA* treatment.
  • OM27 A culture of OM27 is shown in the left hand pond and a culture of WT is shown in the right hand pond.
  • OM 27 is green and healthy and WT is brown and unhealthy.
  • QM65 had the same results and looked similar to OM27 when compared to WT.

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Abstract

Le présent exposé porte sur des souches mutagénisées de microalgues, présentant une tolérance améliorée à un biocide. Ces souches mutagénisées peuvent croître dans des systèmes liquides tels que des piscines, des étangs et analogues. La tolérance améliorée des souches mutagénisées à un biocide conduit à une augmentation du rendement de la biomasse et/ou de la productivité de la biomasse des souches. L'exposé porte aussi sur des procédés pour faire croître des algues dans des systèmes de culture liquides, et sur des procédés pour supprimer, inhiber ou réduire la croissance d'un champignon dans ces systèmes. La biomasse produite par utilisation des souches mutagénisées et des procédés décrits dans l'invention peut être utilisée pour produire toute une gamme de produits utiles, y compris, mais sans s'y limiter, des combustibles de transport.
PCT/US2014/057688 2013-09-27 2014-09-26 Souches d'algues tolérantes aux biocides WO2015048423A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2013056166A1 (fr) * 2011-10-14 2013-04-18 Sapphire Energy, Inc. Utilisation de fongicides dans des systèmes liquides

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013056166A1 (fr) * 2011-10-14 2013-04-18 Sapphire Energy, Inc. Utilisation de fongicides dans des systèmes liquides

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
GALLOWAY, R.E.: "Selective Conditions and Isolation of Mutants in Salt-Tolerant, Lipid- Producing Microalgae", JOURNAL OF PHYCOLOGY, vol. 26, 1990, pages 752 - 760 *
HU, G. ET AL.: "Enhanced Lipid Productivity and Photosynthesis Efficiency in a Desmodesmus sp. Mutant Induced by Heavy Carbon Ions", PLOS ONE, vol. 8, April 2013 (2013-04-01), pages E60700 *
IOKI, M. ET AL.: "Isolation of herbicide-resistant mutants of Botryococcus braunii", BIORESOURCE TECHNOLOGY, vol. 109, 2012, pages 300 - 303 *
MAHAN, K.M. ET AL.: "Controlling fungal contamination in Chlamydomonas reinhardtii cultures", BIOTECHNIQUES, vol. 39, 2005, pages 457 - 458 *
SAUNDERS, M.A.: "Microalgae Strain-Improvement by Evolutionary Engineering for Superior Biofuels Production", 7 October 2013 (2013-10-07), Retrieved from the Internet <URL:http//www.algaebiomass.org/wp-contents/gallery/2012-algae-biomass-summit/2010/06/Saunders_Mathewl.pdf> [retrieved on 20141118] *

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