WO2011017565A2

WO2011017565A2 - Methods and compositions for controlling contamination growth in cell cultures

Info

Publication number: WO2011017565A2
Application number: PCT/US2010/044613
Authority: WO
Inventors: Christian P. Ridley; Scott A. Michonski; Nikos B. Reppas; Johan Van Walsem; Brian D. Green; Frank A. Skraly; Dan E. Robertson; James R. Mcintire
Original assignee: Joule Unlimited, Inc.
Priority date: 2009-08-07
Filing date: 2010-08-05
Publication date: 2011-02-10
Also published as: WO2011017565A3

Abstract

The present invention provides compositions and methods for controlling the growth of contaminating microbes in cell cultures, particularly cultures of photosynthetic organisms used to produce bio fuels and other carbon-based products of interest.

Description

METHODS AND COMPOSITIONS FOR CONTROLLING CONTAMINATION

GROWTH IN CELL CULTURES

BACKGROUND

[0001] Recent progress in the development of bio fuel-generating systems has demonstrated a need for methods and compositions that prevent or inhibit the growth of contaminating microorganisms. Such contaminants could diminish the efficiency of systems that rely primarily on recombinant microbial cultures for the generation of fuels.

SUMMARY

[0002] The present invention provides compositions and methods for controlling the growth of contaminating microbes in cell cultures, particularly cultures of photosynthetic organisms used to produce bio fuels and other carbon-based products of interest. Such photosynthetic organisms may be found in nature, or may be genetically engineered to possess desired metabolic properties, such as an increased production of bio fuels.

[0003] In certain embodiments, a method is provided for inhibiting contaminant growth by reducing phosphate levels in the culture medium. For example, the invention provides a method for culturing a photosynthetic microorganism, comprising: inoculating a culture medium with at least one cell of a photosynthetic microorganism, wherein the culture medium comprises 0-35 mg/L phosphate; and incubating said inoculated culture at a temperature of at least 30 degrees Celsius until a desired optical density is reached. Varying amounts of phosphate within the range can used, including 1-15 mg/L phosphate, or approximately 7 mg/L phosphate, or less. In certain embodiments, the photosynthetic organism is a cyanobacterium. In other embodiments, the photosynthetic organism is selected from the group consisting of Synechococcus, Synechocystis,

Cyanobacteriumstanieri, Cyanobium, Gleocapsa, Dunaliellabardawil, Chlamydomonas Spirulina, Chlorell, Botryococcus and Hamatococcuspluvialis .

[0004] In certain related embodiments, the culture is incubated at a temperature between 30 degrees and 40 degrees Celsius, e.g., a temperature of about 37 degrees Celsius. In other embodiments, the photosynthetic organism of interest is a thermophile and the culture is incubated at a temperature between 45 and 80 degrees Celsius. In certain embodiments, the culture is incubated at temperatures between 50 and 80 degrees Celsius, between 55 and 80 degrees Celsius, between 60 and 80 degrees Celsius, between 65 and 80 degrees Celsius or between 70 and 80 degrees Celsius. [0005] Another embodiment provides a method for culturing a photosynthetic microorganism, comprising: growing a culture of cells of said photosynthetic microorganism to a desired optical density in a first culture medium; isolating said cells from said first culture medium; washing said cells with a wash solution, wherein said wash solution comprises less than 35 mg/ml phosphate; isolating the washed cells from said wash solution; resuspending said washed cells in a second culture medium, wherein said second culture medium comprises no more than 35 mg/ml phosphate; and incubating the resuspended cells at a temperature of at least 30 degrees Celsius until a desired optical density is reached. In certain embodiments, the second culture medium comprises 1-15 mg/L phosphate, e.g., approximately 7 mg/L phosphate, or less, i.e., 6 mg/L, 5 mg/L, 4 mg/L, 3 mg/L, 2mg/L or 1 mg/L. In a related embodiment, the photosynthetic organism is a Synechococcus species. In certain related embodiments, the culture is incubated at a temperature between 30 degrees and 40 degrees Celsius, e.g., a temperature of about 37 degrees Celsius, or a temperature of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 degrees Celsius.

[0006] Both of the above-mentioned embodiments result in the inhibition of growth of contaminating microorganisms relative to an otherwise identical method wherein the culture medium comprises amounts of phosphate equal to or greater than 50 mgs/L (e.g., 70, 100, 140 mgs/L or more).

[0007] In other embodiments, the invention provides compositions for inhibiting the growth of contaminants. For example, one embodiment provides a liquid medium for culturing a photosynthetic microorganism, wherein said medium comprises: a buffer, a vitamin, and one or more salts, wherein said salts include one or metal ions selected from the group consisting of calcium, magnesium, iron, copper, manganese, molybdenum, and cobalt; and wherein said medium comprises no more than 7 mg/ml phosphate.

[0008] The invention also provides a method for reducing or inhibiting the growth of contaminating microorganisms in a culture of photosynthetic cells, comprising adding one or more inhibitory microorganisms to said culture, wherein said inhibitory microorganisms do not substantially interfere with the growth or activity of said photosynthetic cells.

DETAILED DESCRIPTION

[0009] The invention provides methods and compositions for preventing or inhibiting the growth of contaminating microorganisms in a culturing apparatus or container, e.g., a bioreactor, containing an organism of interest from which optimized growth and/or production of a carbon-based product of interest is desired. In preferred embodiments, the organism is a photosynthetic organism that produces carbon-based products of interest, e.g., bio fuels or other organic compounds, using light and carbon dioxide as starting materials. Without being limited to any particular mechanism or theory of action, a method of preventing or inhibiting the growth of contaminating microorganisms is provided which requires the use of limiting amounts of phosphate ion (PO₄ ^3").

[0010] One skilled in the art will be aware of many "standard" culture media recipes for growing photo synthetic microorganisms. Typically these recipes include a buffer, salt, and various trace elements, e.g., metals such as iron and magnesium, vitamin cofactors, and phosphate. Any phosphate in these recipes, and in the methods and compositions of the present invention, may be provided in various forms, e.g., sodium phosphate, potassium phosphate, etc. For purposes of this disclosure, the term "phosphate" refers to PO₄ ^3", having a molecular weight (MW) of 94.97. Thus, a salt such as monopotassium phosphate, with a molecular weight of 136.1, is 69.8% phosphate by weight.

[0011] A typical concentration of monopotassium phosphate found in standard media (e.g., JB 2.1, as set forth in the Examples, below) is about 200 mg/ml or 1.47 mM (= 140 mg/ml PO₄ ^3"). A typical concentration of monopotassium phosphate found in a contaminant- inhibitory culture medium contains 25%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or less than that found in standard media (5% of 200mg/L KH₂PO⁴ is 10mg/L or 73.5nM KH₂PO⁴), with lower concentrations preferred. An ideal concentration is one which prevents contaminants from growing but does not substantially affect the growth or desired metabolic activity (e.g., biofuel production) of the cultured photosynthetic organism of interest.

[0012] Organisms may be cultured in the contaminant-inhibitory, phosphate-depleted medium for as long as desired. The temperature of incubation may vary, depending on the desired rate of growth of the photosynthetic organism (or organisms) of interest. For example, many mesophiles prefer temperatures between 15 and 40 degrees Celsius. Diverse organisms such as E. coli and Synechococcus sp. PCC 7002 each grow maximally at temperatures of approximately 37 degrees Celsius. However, thermophiles such as

Thermosynechococcus elongatus grow well at much higher temperatures, e.g., around 55 degrees Celsius. The contaminant-inhibiting methods of the present invention may be applied to organisms which thrive under a variety of conditions.

[0013] Examples of microbes which are known to contaminate cultures of photosynthetic organisms growing in media such as, e.g., JB 2.1, include Pseudomonas and Microbacterium species. The unwanted growth of these contaminating microbes and others may be inhibited by the use of the methods and compositions described herein. [0014] Photosynthetic organisms of interest which may be cultured under the contaminant-inhibiting conditions described herein include, without limitation, eukaryotic plants and algae, as well as prokaryotic cyanobacteria, green sulfur bacteria, green non sulfur bacteria, purple sulfur bacteria, and purple non sulfur bacteria.

[0015] Plants include but are not limited to the following genera: Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia and Zea.

[0016] Algae and cyanobacteria include but are not limited to the following genera: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira,

Ascochloris, Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritractus, Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema,

Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus, Chrysolepidomonas, Chrysolykos,

Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella,

Chrysostephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus,

Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospertnum, Cylindrotheca, Cymatopleura, Cymbella,

Cymbellonitzschia, Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermocarpa, Dertnocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus,

Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum,

Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis, Diplostauron,

Distrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis,

Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium,

Gloeocapsa, Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria,

Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum,

Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia,

Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitonia, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon,

Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia,

Juranyiella, Karayevia, Kathablepharis, Katodinium, Kephyrion, Keratococcus,

Kirchneriella, Klebsortnidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion, Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium, Micr actinium, Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis,

Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema, Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria, Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora, Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium,

Pocillomonas, Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma, Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate,

Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion,

Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum,

Pseudostaurosira, Pseudotetrastrum, Pteromonas, Punctastruata, Pyramichlamys,

Pyramimonas, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiococcus,

Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria,

Rhabdodertna, Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium,

Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum,

Stauerodesmus, Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea,

Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia,

Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira, Thamniochaete,

Thorakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella,

Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox,

Volvulina, Westella, Woloszynskia, Xanthidium, Xanthophyta, Xenococcus, Zygnema,

Zygnemopsis, and Zygonium. A preferred Synechococcus strain is Synechococcus

sp. PCC 7002.

[0017] Green non-sulfur bacteria include but are not limited to the following genera:

Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and

Thermomicrobium. Green sulfur bacteria include but are not limited to the following genera:

Chlorobium, Clathrochloris, and Prosthecochloris .

[0018] Purple sulfur bacteria include but are not limited to the following genera:

Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium,

Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis. Purple non-sulfur bacteria include but are not limited to the following genera: Phaeospirillum,

Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas,

Rhodothalassium, Rhodospirillum, Rodovibrio, and Roseospira.

[0019] The invention is equally applicable to natural or genetically engineered organisms. In addition, organisms that are not naturally photosynthetic but have been genetically engineered to be capable of photosynthesis may be cultured according to the phosphate-depletion methods described herein.

[0020] In another embodiment, the invention provides methods of co-culturing a photosynthetic organism of interest {e.g., any of the organisms listed above) with one or more

"inhibitory microorganisms." An "inhibitory microorganism" is defined herein as one which inhibits the growth of undesired contaminant microorganisms but does not substantially adversely affect the growth or other desired property (e.g., production of a bio fuel or other organic compound) of the co-cultured photosynthetic organism of interest.

[0021] The following Examples are presented to further illustrate various embodiments of the invention, and are not intended to be limiting. One skilled in the art will recognize variations that do not depart from the spirit and scope of the invention.

EXAMPLES

Example 1: Inhibition of contaminant growth in cultures of a photosynthetic microbe

[0001] Materials and Methods: For all experiments requiring Synechococcus sp. PCC 7002 culture, the inoculum was grown in JB 2.1. Table 1 lists the recipe for 1 Liter of JB 2.1 culture media. Table 1: Enhanced Media (JB 2.1)

[0002] Each of the above ingredients may be varied by ± 10% without substantially affecting the growth of the cultured photosynthetic organism. After the above ingredients are mixed in a 2 liter flask, they are filter sterilized into an autoclaved 1 liter bottle using a 0.22 μM pore size filter. Sterile technique should be used during the preparation. The pH of the media should be within 7.9-8.0 to avoid precipitation which occurs a higher pHs. One skilled in the art will recognize that minor variations in each of the listed concentrations may be tolerated without adversely affecting growth of a particular culture.

[0003] After a desirable inoculum optical density (OD) was achieved, the culture was spun down, the supernatant discarded and the culture was re-suspended in the media to be tested. After re-suspension the culture was spun down again, the supernatant discarded a second time and the inoculum was then re-suspended in the desired media. This method allowed a high level of confidence that the majority of media from the original culture had been removed. For all experiments, growth of contaminants was measured by plating on Luria broth ("LB") agar and growing overnight at 37° C in the absence of light. The resulting cell colonies were then counted and compared. [0004] Evaporation was corrected in all flasks of Synechococcus sp. PCC 7002 by the correction of weight via filter sterilized MiIIiQ H2O. This prevented a concentration of contaminant cells from occurring due to evaporation.

[0005] Results: This experiment demonstrates that low levels of phosphate (e.g.,

KH₂PO₄) mitigate contamination in cultures of Synechococcus sp. PCC 7002. Ten flasks of

Synechococcus sp. PCC 7002 culture (IL) were made and inoculated with known contaminants (referred to herein as SMl, SM2, SM4, SM5 and SM6). Five flasks contained media with 200mg/L KH₂PO₄ and five contained media without added KH₂PO₄. The flasks were then grown in the light at 37⁰C overnight (21 hours). Contaminant growth was then compared by plating.

[0006] The results for this experiment can be seen in Table 2 below.

Table 2: Effect of KH₂PO₄ Depletion on Growth

[0007] The data in Table 2 shows that the absence of phosphate substantially diminishes the ability of SMl and SM4 to grow in the absence of phosphate. SMl (flask 6) may have had a slight amount of growth without phosphate but the colony increase was insignificant when compared to the positive control for SMl (flask 1). SM4 seemed to have a decrease in contaminant viability when left in a phosphate free media after 21 hours. The positive control for SM4 grew as expected with a doubling time of 2 hours. The remaining flasks had an overgrowth of colonies from the initial time point and the resulting plates were uncountable.

[0008] The effect that phosphate had on SMl and SM4 is likely due to the fact that, without phosphate, DNA cannot be replicated due to an inability to form phosphodiester bonds. Phosphate is also critical for the cell membrane in the form of phospholipids. The need for phosphate also affected the growth of Synechococcus sp. PCC 7002, albeit in a much less dramatic fashion. Cultures containing phosphate grew 2 to 3 times more quickly than those without.

[0009] The foregoing description of the embodiments of the invention has been presented for the purpose of illustration; it is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the above disclosure.

[0010] Finally, the language used in the specification has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. It is therefore intended that the scope of the invention be limited not by this detailed description, but rather by any claims that issue on an application based hereon. Accordingly, the disclosure of the embodiments of the invention is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the following claims.

Claims

What is claimed is:

1. A method for culturing a photosynthetic microorganism, comprising:

inoculating a culture medium with at least one cell of said photosynthetic microorganism, wherein said culture medium comprises 0-35 mg/L phosphate;

and incubating said inoculated culture at a temperature of at least 30 degrees

Celsius until a desired optical density is reached.

2. The method of claim 1, wherein said culture medium comprises 1-15 mg/L phosphate.

3. The method of claim 1, wherein said culture medium comprises approximately 7 mg/L phosphate or less.

4. The method of claim 1 , wherein said photosynthetic organism is selected from the group consisting of Synechococcus, Synechocystis, Cyanobacteriumstanieri, Cyanobium, Gleocapsa, Dunaliellabardawil, Chlamydomonas Spirulina, Chlorell, Botryococcus and Hamatococcuspluvialis .

5. The method of claim 1, wherein said photosynthetic organism is a cyanobacterium.

6. The method of claim 5, wherein said photosynthetic organism is a Synechococcus species.

7. The method of claim 1, wherein said culture is incubated at a temperature between 30 degrees and 40 degrees Celsius.

8. The method of claim 1 , wherein said culture is incubated at a temperature of about 37 degrees Celsius.

9. The method of claim 1, wherein said photosynthetic organism is a thermophile and said culture is incubated at a temperature between 45 and 80 degrees Celsius.

10. A method for culturing a photosynthetic microorganism, comprising:

growing a culture of cells of said photosynthetic microorganism to a desired

optical density in a first culture medium;

isolating said cells from said first culture medium;

washing said cells with a wash solution, wherein said wash solution comprises less than 35 mg/ml phosphate; isolating the washed cells from said wash solution;

resuspending said washed cells in a second culture medium, wherein said second culture medium comprises no more than 35 mg/ml phosphate;

and incubating the resuspended cells at a temperature of at least 30 degrees

Celsius until a desired optical density is reached.

11. The method of claim 10, wherein said second culture medium comprises 1-15 mg/L phosphate.

12. The method of claim 10, wherein said second culture medium comprises

approximately 7 mg/L phosphate.

13. The method of claim 10, wherein said photosynthetic organism is selected from the group consisting of Synechococcus, Synechocystis, Cyanobacteriumstanieri, Cyanobium, Gleocapsa, Dunaliellabardawil, Chlamydomonas Spirulina, Chlorell, Botryococcus and Hamatococcuspluvialis

14. The method of claim 10, wherein said photosynthetic organism is a cyanobacterium.

15. The method of claim 14, wherein said photosynthetic organism is a Synechococcus species.

16. The method of claim 10, wherein said cultures are incubated at a temperature between 30 degrees and 40 degrees Celsius.

17. The method of claim 10, wherein said cultures are incubated at a temperature of about 37 degrees Celsius.

18. The method of claim 10, wherein said photosynthetic organism is a thermophile and said cultures are incubated at a temperature between 45 and 80 degrees Celsius.

19. The method of any of claims 1-18, wherein said method inhibits the growth of any contaminating microorganisms relative to an otherwise identical method wherein the culture medium comprises amounts of phosphate greater than 50 mgs/L.

20. A liquid medium for culturing a photosynthetic microorganism, wherein said medium comprises: a buffer, a vitamin, and one or more salts, wherein said salts include one or metal ions selected from the group consisting of calcium, magnesium, iron, copper, manganese, molybdenum, and cobalt; and wherein said medium comprises no more than 7 mg/ml phosphate.

21. A method for reducing or inhibiting the growth of contaminating microorganisms in a culture of photosynthetic cells, comprising adding one or more inhibitory microorganisms to said culture, wherein said inhibitory microorganisms do not substantially interfere with the growth or activity of said photosynthetic cells.