CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/724,710, filed November 9, 2012, entitled Methods of Culturing Microorganisms in Mixotrophic Conditions; and U.S. Provisional Application No. 61/798,969, filed March 15, 2013, entitled Mixotrophy Systems and Methods, the entire contents of which are hereby incorporated by reference herein.

BACKGROUND

[0002] Microorganisms, such as but not limited to microalgae and cyanobacteria, have gained attention as a viable source for food, fuel, fertilizers, cosmetics, chemicals, and pharmaceuticals due to the ability to grow rapidly and in a variety of conditions, such as a wastewater medium. Each microalgal and cyanobacteria species have a different protein, mineral, and fatty acid profile which makes some species better sources for certain products than other species. Different microalgal and cyanobacteria species may use different sources of energy and carbon. Phototrophic microorganisms use light energy, as well as inorganic carbon (e.g., carbon dioxide), to carry out metabolic activity. Heterotrophic microorganisms do not use light as an energy source and instead use an organic carbon source for energy and carbon to carry out metabolic activity. Mixotrophic microorganisms can use a mix of energy and carbon sources, including light, inorganic carbon (e.g., carbon dioxide) and organic carbon. Each set of culture conditions may be susceptible to growth inhibition and possibly death of the microorganism culture due to contaminating organisms such as bacteria and fungi.

[0003] Heterotrophic and mixotrophic cultures using an organic carbon source may have an increased risk of susceptibility to bacteria, fungi, or other unwanted contaminating organisms that use the organic carbon source as a feed source. Phototrophic microorganism cultures do not use an organic carbon source, but may be grown outside in open, non-axenic conditions for exposure to natural light where contaminating organisms may easily invade the culture. Also, growth of microorganisms in phototrophic conditions may be slower than in cultures that use an organic carbon source, thus making the microorganisms (e.g., microalgae) in phototrophic conditions susceptible to being over taken by contaminating organisms (e.g., bacteria) that have the ability to grow faster or consume all available resources. [0004] Additionally, contaminating bacteria may affect microorganism product formation (e.g., lipids, pigments, proteins) and growth. The proliferation of contaminating bacteria and other contamination organisms in microorganism cultures has proven to be detrimental to the production of microalgae and cyanobacteria if the contaminating bacteria population is not controlled and is allowed to consume resources needed by the microalgae and cyanobacteria. Therefore, there is a need in the art for a method of efficiently culturing microalgae and cyanobacteria under non-axenic conditions which control the contamination and maintain the culture nutrients at levels to maximize growth of the microalgae and cyanobacteria.

SUMMARY

[0005] Embodiments described herein relate generally to methods for reducing the concentration of contaminating organisms in a culture of microorganisms. The methods comprise the use of electro-coagulation and separation to concentrate the microorganisms in a substantially solids-rich fraction and separate the microorganisms from a substantially liquid fraction comprising contaminating organisms.

[0006] In some embodiments of the invention a method of reducing the concentration of contaminating bacteria in a microorganism culture comprises: supplying a culture of microorganisms at a first concentration of the microorganisms in a culture medium comprising non-axenic conditions with at least some contaminating bacteria to an electro-coagulation system, the electro-coagulation system comprising at least one cathode and anode pair, and a power supply for supplying electrical power to the at least one cathode and anode pair;

applying an electrical field to the culture of microorganisms to alter the surface charge of the microorganisms and cause coagulation among the microorganisms, generating a substantially solids-rich fraction comprising coagulated microorganisms, and a substantially liquid fraction comprising contaminating bacteria and culture medium; and separating the substantially solids- rich fraction comprising coagulated microorganisms from the substantially liquid fraction comprising contaminating bacteria and culture medium to produce a mass of microorganisms with a second concentration of the microorganisms higher than the first concentration of the microorganisms and a reduced concentration of bacteria.

[0007] In some embodiments, the separation step comprises decantation, centrifugation, sedimentation, skimming, filtration, dissolved gas flotation, electro-flotation, acoustic energy separation, or foam fractionation. In some embodiments, the method further comprises transferring the mass of microorganisms comprising a reduced concentration of contaminating bacteria to a culturing vessel for inoculation in a culture medium, wherein the culture of microorganisms may be grown in phototrophic, mixotrophic, or heterotrophic conditions. In some embodiments, the method further comprises packaging the mass of microorganisms comprising a reduced concentration of bacteria.

[0008] In some embodiments, the second concentration of coagulated microorganisms is at least 100 times greater than the first concentration of the microorganisms. In some embodiments, the mass of microorganisms with a reduced concentration of contaminating bacteria has a bacteria cell count of a least one log less than the original bacteria count of the culture of microorganisms. In some embodiments, the method further comprises treating the substantially liquid fraction comprising contaminating bacteria and culture medium with at least one method selected from the group consisting of: filtration, UV sterilization, ozone, hydrogen peroxide, and antibiotics. In some embodiments, the method further comprises re- inoculating the mass of microorganisms comprising a reduced concentration of contaminating bacteria in the treated substantially liquid fraction in a culturing vessel.

[0009] In some embodiments, the at least one cathode and anode pair comprises one electrode disposed within a tube electrode. In some embodiments, the at least one cathode and anode pair comprises electrodes at least partially submerged in a tank. In some embodiments, the electrical field is a pulsed electrical field. In some embodiments, the culture of microorganisms is supplied to the electro-coagulation system at a flow rate of 0.1 to 100 ml/s. In some embodiments, the electrical field is created by the at least one cathode and anode pair by applying a current density between 20 and 60 mA/cm ² . In some embodiments, the electrical power comprises 1 to 50 Amps. In some embodiments, the electrical power comprises 1 to 100 Volts.

[0010] In some embodiments, the at least one cathode and anode pair comprises at least one conductive material selected from the group consisting of: silver, copper, gold, aluminum, zinc, nickel, brass, bronze, iron, lead, platinum, steel, stainless steel, titanium, graphite, grapheme, synthetic graphite, carbon fiber, nano-carbon structures, and carbon deposited on silicon substrates. In some embodiments, the microorganisms comprise at least one selected from the group consisting of microalgae and cyanobacteria.

BRIEF DESCRIPTION OF FIGURES

[0011] FIG. 1 is a graph showing the bacteria count results for a culture of microalgae after an electro-coagulation treatment.

[0012] FIG. 2 is a schematic diagram showing an in line electro-coagulation treatment and separation embodiment.

[0013] FIG. 3 is a graph showing the bacteria count results for a culture of microalgae after an electro-coagulation treatment. [0014] FIG. 4 is a graph showing the bacteria count results for a culture of microalgae after an electro-coagulation treatment.

[0015] FIG. 5 is a graph showing the bacteria count results for a culture of microalgae after an electro-coagulation treatment.

[0016] FIG. 6 is a graph showing the concentration results for a culture of microalgae after an electro-coagulation treatment.

[0017] FIG. 7 is a graph showing the concentration results for a culture of microalgae after an electro-coagulation treatment.

[0018] FIG. 8 is a graph showing the concentration results for a culture of microalgae after an electro-coagulation treatment.

DETAILED DESCRIPTION

Definitions

[0019] The term "microorganism" refers to microscopic organisms such as microalgae and cyanobacteria. Microalgae include microscopic multi-cellular plants (e.g. duckweed), photosynthetic microorganisms, heterotrophic microorganisms, diatoms, dinoflagelattes, and unicellular algae.

[0020] The terms "microbiological culture", "microbial culture", or "microorganism culture" refer to a method or system for multiplying microorganisms through reproduction in a predetermined culture medium, including under controlled laboratory conditions.

Microbiological cultures, microbial cultures, and microorganism cultures are used to multiply the organism, to determine the type of organism, or the abundance of the organism in the sample being tested. In liquid culture medium, the term microbiological, microbial, or microorganism culture generally refers to the entire liquid medium and the microorganisms in the liquid medium regardless of the vessel in which the culture resides. A liquid medium is often referred to as "media", "culture medium", or "culture media". The act of culturing is generally referred to as "culturing microorganisms" when emphasis is on plural

microorganisms. The act of culturing is generally referred to as "culturing a microorganism" when importance is placed on a species or genus of microorganism. Microorganism culture is used synonymously with culture of microorganisms.

[0021] Microorganisms that may grow in mixotrophic culture conditions include microalgae, diatoms, and cyanobacteria. Non-limiting examples of mixotrophic

microorganisms may comprise organisms of the genera: Agmenellum, Amphora, Anabaena, Anacystis, Apistonema, Pleurochyrsis, Arthrospira (Spirulina), Botryococcus, Brachiomonas, Chlamydomonas, Chlorella, Chloroccum, Cruciplacolithus, Cylindrotheca, Coenochloris, Cyanophora, Cyclotella, Dunaliella, Emiliania, Euglena, Extubocellulus, Fragilaria, Galdieria, Goniotrichium, Haematococcus, Halochlorella, Isochyrsis, Leptocylindrus, Micractinium, Melosira, Monodus, Nostoc, Nannochloris, Nannochloropsis, Navicula, Neospongiococcum, Nitzschia., Odontella, Ochromonas, Ochrosphaera, Pavlova,

Picochlorum, Phaeodactylum, Pleurochyrsis, Porphyridium, Poteriochromonas, Prymnesium, Rhodomonas, Scenedesmus, Skeletonema, Spumella, Stauroneis, Stichococcus,

Auxenochlorella, Cheatoceros, Neochloris, Ocromonas, Porphiridium, Synechococcus, Synechocystis, Tetraselmis, Thraustochytrids, Thalassiosira, and species thereof.

[0022] Non-limiting examples of microorganism species capable of growth in phototrophic or heterotrophic culture conditions may comprise: Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphora delicatissima, Amphora delicatissima var. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp.,

Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var. subsalsum, Chaetoceros sp., Chlamydomas perigranulata, Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella zofmgiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis,

Eremosphaera sp., Ellipsoidon sp., Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis aff. galbana, Isochrysis galbana, Lepocinclis, Micractinium, Micractinium, Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschia closterium, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis,

Parachlorella kessleri, Pascheria acidophila, Pavlova sp., Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana.

[0023] The organic carbon sources suitable for growing a microorganism mixotrophically or heterotrophically may comprise: acetate, acetic acid, ammonium linoleate, arabinose, arginine, aspartic acid, butyric acid, cellulose, citric acid, ethanol, fructose, fatty acids, galactose, glucose, glycerol, glycine, lactic acid, lactose, maleic acid, maltose, mannose, methanol, molasses, peptone, plant based hydrolyzate, proline, propionic acid, ribose, sacchrose, partial or complete hydrolysates of starch, sucrose, tartaric, TCA-cycle organic acids, thin stillage, urea, industrial waste solutions, yeast extract, and combinations thereof. The organic carbon source may comprise any single source, combination of sources, and dilutions of single sources or combinations of sources.

[0024] The terms "mixotrophic" and "mixotrophy" refer to culture conditions in which light, organic carbon, and inorganic carbon (e.g., carbon dioxide, carbonate, bi-carbonate) may be applied to a culture of microorganisms. Microorganisms capable of growing in mixotrophic conditions have the metabolic profile of both phototrophic and heterotrophic microorganisms, and may use both light and organic carbon as energy sources, as well as both inorganic carbon and organic carbon as carbon sources. A mixotrophic microorganism may be using light, inorganic carbon, and organic carbon through the phototrophic and heterotrophic metabolisms simultaneously or may switch between the utilization of each metabolism. A microorganism in mixotrophic culture conditions may be a net oxygen or carbon dioxide producer depending on the energy source and carbon source utilized by the microorganism. Microorganisms capable of mixotrophic growth comprise microorganisms with the natural metabolism and ability to grow in mixotrophic conditions, as well as microorganisms which obtain the metabolism and ability through modification of cells by way of methods such as mutagenesis or genetic engineering.

[0025] The terms "phototrophic", "phototrophy", "photoautotrophy", "photoautotrophic", and "autotroph" refer to culture conditions in which light and inorganic carbon (e.g., carbon dioxide, carbonate, bi-carbonate) may be applied to a culture of microorganisms.

Microorganisms capable of growing in phototrophic conditions may use light as an energy source and inorganic carbon (e.g., carbon dioxide) as a carbon source. A microorganism in phototrophic conditions may produce oxygen.

[0026] The terms "heterotrophic" and "heterotrophy" refer to culture conditions in which organic carbon may be applied to a culture of microorganisms in the absence of light.

Microorganisms capable of growing in heterotrophic conditions may use organic carbon as both an energy source and as a carbon source. A microorganism in heterotrophic conditions may produce carbon dioxide.

[0027] The term "axenic" describes a culture of an organism that is entirely free of all other "contaminating" organisms (i.e., organisms that are detrimental to the health of the microalgae or cyanobacteria culture). Throughout the specification, axenic refers to a culture that when inoculated in an agar plate with bacterial basal medium, does not form any colonies other than the microorganism of interest. Axenic describes cultures not contaminated by or associated with any other living organisms such as but not limited to bacteria, cyanobacteria, microalgae and/or fungi. Axenic is usually used in reference to pure cultures of microorganisms that are completely free of the presence of other different organisms. An axenic culture of microalgae or cyanobacteria is completely free from other different organisms.

[0028] Bacteria that may be present in cultures of microalgae and cyanobacteria comprise, but are not limited to: Achromobacter sp., Acidovorax sp., Acinetobacter sp., Aeromonas sp., Agrobacterium sp., Alteromonas sp., Ancylobacter sp., Aquaspirillum sp., Azospirillum sp., Azotobacter sp., Bacillus sp., Bergeyella sp., Brevundimonas sp., Brochothrix sp.,

Brumimicrobium sp., Burkholderia sp., Caulobacter sp., Cellulomonas sp., Chryseobacterium sp., Curtobacterium sp., Delftia sp., Empedobacter sp., Enterobacter sp., Escherichia sp., Flavobacterium sp., Gemmatimonas sp., Halomonas sp., Hydrogenophaga sp.,

Janthinobacterium sp., Lactobacillus sp., Marinobacter sp., Massilia sp., Microbacterium sp., Myroides sp., Pantoea sp., Paracoccus sp., Pedobacter sp., Phaeobacter sp., Phyllobacterium sp., Pseudoalteromonas sp., Pseudomonas sp., Rahnella sp., Ralstonia sp., Rhizobium sp., Rhodococcus sp., Roseomonas sp., Sphingobacterium sp., Sphingomoas sp., Staphylococcus sp., Stenotrophomonas sp., Vibrio sp., and Zobelliae sp.

[0029] Bacteria that have a negative or harmful effect on the microalgae and cyanobacteria may be designated as contaminating bacteria. The bacteria that may have a negative or harmful effect on microalgae or cyanobacteria in a culture comprise, but are not limited to:

Achromobacter sp., Acidovorax sp., Aeromonas sp., Agrobacterium sp., Alteromonas sp., Aquaspirillum sp., Azospirillum sp., Azotobacter sp., Bergeyella sp., Brochothrix sp.,

Brumimicrobium sp., Burkholderia sp., Caulobacter sp., Cellulomonas sp., Chryseobacterium sp., Curtobacterium sp., Delftia sp., Empedobacter sp., Enterobacter sp., Escherichia sp., Flavobacterium sp., Marinobacter sp., Microbacterium sp., Myroides sp., Paracoccus sp., Pedobacter sp., Phaeobacter sp., Pseudoalteromonas sp., Pseudomonas sp., Rahnella sp., Ralstonia sp., Rhizobium sp., Rhodococcus sp., Roseomonas sp., Staphylococcus sp.,

Stenotrophomonas sp., i¾no sp., Zobelliae sp. and other bacteria which share similar characteristics.

[0030] The bacteria that may have a neutral or beneficial effect on microalgae or cyanobacteria in a culture comprise, but are not limited to: Acidovorax sp., Acinetobacter sp., Aeromonas sp., Agrobacterium sp., Alteromonas sp., Ancylobacter sp., Azospirillum sp.,

Azotobacter sp., Bacillus sp., Brevundimonas sp., Brumimicrobium sp., Burkholderia sp., Caulobacter sp., Cellulomonas sp., Delftia sp., Empedobacter sp., Gemmatimonas sp.,

Halomonas sp., Hydrogenophaga sp., Janthinobacterium sp., Lactobacillus sp., Marinobacter sp., Pantoea sp., Paracoccus sp., Phaeobacter sp., Phyllobacterium sp., Pseudoalteromonas sp., Pseudomonas sp., Rhizobium sp., Sphingomoas sp., Zobelliae sp. and other bacteria which share similar characteristics. While bacteria in a particular genus generally have the same characteristics, it is recognized that a genus of bacteria with the majority of species generally identified as harmful to microalgae or cyanobacteria may also include a particular species within the genus which is neutral or beneficial to a specific culture of microalgae or cyanobacteria, and vice versa. For example, many species of Pseudomonas have been observed to be harmful to microalgae, however literature has described certain species of Pseudomonas with anti-fungal functionality which may be beneficial to a culture of microalgae or cyanobacteria.

[0031] The term "pH auxostat" refers to the microbial cultivation technique that couples the addition of fresh medium (e.g., medium containing organic carbon or acetic acid) to pH control. As the pH drifts from a given set point, fresh medium is added to bring the pH back to the set point. The rate of pH change is often an excellent indication of growth and meets the requirements as a growth-dependent parameter. The feed will keep the residual nutrient concentration in balance with the buffering capacity of the medium. The pH set point may be changed depending on the microorganisms present in the culture at the time. The

microorganisms present may be driven by the location and season where the bioreactor is operated and how close the cultures are positioned to other contamination sources (e.g., other farms, agriculture, ocean, lake, river, waste water). The rate of medium addition is determined by the buffering capacity and the feed concentration of the limiting nutrient and not directly by the set point (pH) as in a traditional auxostat. The pH auxostat is robust but controls nutrient concentration indirectly. The pH level represents the summation of the production of different ionic species and ion release during carbon and nutrient uptake. Therefore the pH level can move either up or down as a function of growth of the microorganisms. The most common situation is pH depression caused by organic acid production and ammonium uptake.

However, for microorganisms growing on protein or amino acid-rich media, the pH level will rise with growth because of the release of excess ammonia.

[0032] The term "harvesting" refers to removing the culture of microorganisms from the culturing vessel and/or separating the microorganisms from the culture medium. Harvesting of microorganisms may be conducted by any method known in the art such as, but not limited to, skimming, draining, dissolved gas flotation, foam fractionation, centrifugation, filtration, sedimentation, chemical flocculation, and electro-dewatering.

[0033] The term "inoculate" refers to implanting or introducing microorganisms into a culture medium. Inoculate or inoculating a culture of microorganisms in the described culture conditions throughout the specification refers to starting a culture of microorganisms in the culture conditions, as is commonly used in the art of microorganism culturing. The microorganisms that are introduced into a culture medium may be referred to as seed or inoculum. Electro-coagulation overview

[0034] The contaminating organism (e.g., bacteria, fungi) population in a phototrophic, mixotrophic, or heterotrophic microorganism culture comprising a primary microalgae or cyanobacteria may be controlled by limiting the available feed sources for the contaminating bacteria. Limiting the feed sources may comprise limiting the amount of free nutrients (e.g., organic carbon, nitrates, phosphates), in the culture medium, as well as eliminating the fouled or precipitated biomass from the culturing vessel. In addition, the primary microorganisms and contaminating organisms may be separated through electro-coagulation methods, and the separated primary microorganisms (e.g., microalgae) may be re-suspended in new or treated culture medium comprising a lower concentration of contamination organisms, such as bacteria and fungi. An electric field may be applied to a culture of microorganisms to induce coagulation of the microorganisms using any suitable power source known in the art in connection with electrodes comprising at least one cathode and anode pair of any known conductive material known in the art. Suitable conductive electrode materials may comprise but are not limited to, conductive metals and metal alloys (e.g., silver, copper, gold, aluminum, zinc, nickel, brass, bronze, iron, lead, platinum, steel, stainless steel, titanium), conductive polymers, conductive carbon allotropes (e.g., graphite, grapheme, synthetic graphite, carbon fiber, nano-carbon structures, carbon deposited on silicon substrates), and conductive coatings. The electrodes may administer the electric field in any electro-coagulation device (sometimes referred to as an electro-dewatering device) configuration such as, but not limited to, submerged electrode devices, partially submerge electrode devices, opposing flat plate electrode devices, devices with an electrode disposed within another tube electrode (e.g., cylindrical tube, rectangular tube), and combinations thereof. The power source may supply direct current, alternating current, or electrical pulses to the electrodes to produce a constant electrical field or a field comprising electric pulses.

[0035] In some embodiments, the characteristics of the electrical field or electrical pulses may be tuned based on parameters of the microorganism culture and electro-coagulation system such as, but not limited to cell density of the culture, temperature of the culture, salinity level of the culture, pH level of the culture, species of microorganism in the culture, species of contaminating organism in the culture, flow rate of the culture within the system, electrode material, culture volume, and spacing between the anode and the cathode. In some embodiments, the frequency of the pulses may comprise least about 500 Hz, 1 kHz, 2 kHz, or kHz. In some embodiments, the frequency may comprise less than 200 kHz, 80 kHz, 50 kHz, 30 kHz, 5 kHz, or 2 kHz; or may comprise any combination of the foregoing maximum and minimum frequencies accordingly. In some embodiments, the electrical pulse duration may range from 1 nanosecond to 100,000 nanoseconds, 1 to 1,000 nanoseconds, 1 to 500 nanoseconds, or 10 to 300 nanoseconds; allowing for frequencies of about 10 kHz to 1,000,000 kHz. In some embodiments, the electrical pulses may be provided by a pulse generator.

[0036] In some embodiments, the operating voltage of the electro-coagulation system may comprise IV to 500 KiloVolts, and preferably between 1 and 100 Volts. In some

embodiments, the operating current may comprise 1 to 100 Amps, and preferable between 1 and 50 Amps. In some embodiments, the operating current density may comprise 20-60 mA/cm ² . In some embodiments, the amplitude of the electrical field to which the

microorganism culture is exposed to may range from 0.05 V/cm to 100,000 kV/cm, and preferably between 0.05 V/cm and 1,000 kV/cm. In some embodiments, the peak power may comprise at least 500 kW, or at least 1 megawatt. In some embodiments, the energy delivered by the electrical field may range from 0.1 to 100 Joules, and preferably from 1 to 10 joules.

[0037] In some embodiments, the spacing between the anode and cathode may comprise 0.5 to 200 mm. In some embodiments, the culture of microorganisms flows through the electro-coagulation system at 0.1 to 100 ml/s. In some embodiments, the culture of microorganisms may be stationary within the electro-coagulation system and does not flow through. The residence time of the culture in the system may depend on the flow rate, whether the system is operating in batch or continuous mode, the strength of the electrical field, the composition of the culture medium, the species of microorganisms, and may range from fractions of a second in a continuous flow through system with a high flow rate to 120 minutes for a batch system with a stationary volume.

[0038] The electrical field applied to the culture of microorganisms may neutralize the surface charge of the microorganisms, causing coagulation. Coagulation of microalgae suing electrical fields is known in the art, but the ability to separate microalgae from contaminating bacteria using the application of electrical field and separation technology was an unexpected result. The differences in the characteristics of microorganism cells (e.g., microalgae or cyanobacteria) and bacteria cells, such as surface area, result in the electrical field affecting microorganisms and bacteria cells differently at different electrical parameters. The electrical parameters, such as current, voltage, and pulse duration may be used to tune the electrical field to cause the microorganisms to coagulate while the contaminating organisms (e.g., bacteria) remain in suspension in the culture media, thus forming a substantially solids-rich fraction comprising coagulated microorganisms and a substantially liquid fraction comprising contaminating organisms suspended in aqueous culture medium. The substantially solids-rich fraction comprising coagulated microorganisms may be separated from the substantially liquid fraction by methods known in the art such as, decantation, dissolved gas floatation, electro- flotation, foam fractionation, centrifugation, skimming, sedimentation, acoustic energy separation, and filtration (i.e., membrane filtration). The separated, coagulated microorganisms may be re-suspended (e.g., re-inoculated) in new culture medium for continued growth in a culture to form a culture comprising a reduced concentration of contaminating organisms.

[0039] Thus an electro-coagulation system may be used to both concentrate the microorganisms and reduce the concentration of contaminating organisms in the

microorganism culture. The separated culture medium comprising contaminating organisms may be further treated with filtration, UV sterilization, ozone, hydrogen peroxide, or antibiotics to clean or purify the culture medium before recycling the culture medium for use in another microorganism culture. In some embodiments, the coagulated microorganisms may be re- inoculated in the same culture medium after further treatment to clean or purify the culture medium.

[0040] In some embodiments, the electro-coagulation system may operate in a continuous manner with a steady flow of microorganism culture passing through the system. In some embodiments, the electro-coagulation system may operate in a batch manner by processing a define volume of microorganism culture discontinuously. In some embodiments, the separated and coagulated microorganisms may be transferred to another culturing vessel. In some embodiments, the separated and coagulated microorganisms may be transferred to another dewatering device, such as a centrifuge or filtration, for further removal of water from the microorganisms. Using electro-coagulated microorganisms as a feed for other dewatering devices may increase the efficiency of the other dewatering devices due to the increased size of the coagulated microorganisms compared to the size of individual microorganism cells. In some embodiments, the separated and coagulated microorganisms may be packaged for shipment. In the packaging and shipping context, the reduction in contaminating organisms may be advantageous for extending the shelf life of the packaged microorganisms, reducing the odor of the packaged microorganisms, and increasing the available product markets for the packaged microorganisms where a certain level of bacteria content may be prohibit entry into the market. By electro-coagulating the microorganisms and separating the coagulated microorganisms from the culture medium, microorganism biomass may be quickly shipped at reduced volume and weight compared to shipping the biomass in the suspended culture medium state. [0041] The microorganisms may be cultured in a culturing vessel comprising a variety of conditions before and/or after a treatment to reduce the concentration of contaminating organisms. In some embodiments, the culture of microorganisms may be cultured in phototrophic conditions comprising a supply of light and a supply of inorganic carbon (e.g., carbon dioxide) before and/or after electro-coagulation treatment. In some embodiments, the culture of microorganisms may be cultured in mixotrophic conditions comprising a supply of light, a supply of organic carbon, and a supply of inorganic carbon (e.g., carbon dioxide) before and/or after electro-coagulation treatment. In some embodiments, the culture of

microorganisms may be cultured in heterotrophic conditions comprising a supply of organic carbon in the absence of light before and/or after electro-coagulation treatment. The supply of light may be natural (e.g., sunlight), any suitable artificial light known in the art (e.g., light emitting diodes, fluorescent bulbs, incandescent bulbs), or a combination thereof. A culturing vessel for culturing microorganisms in phototrophic, mixotrophic, or heterotrophic culturing conditions may comprise any suitable culturing vessel known in the art such as, but not limited to, a tank, a trough, a pond, a raceway pond, a column bioreactor, a flat panel bioreactor, a bag bioreactor, and a tubular bioreactor

[0042] The culture medium may comprise a combination of trace metals and nutrients specific to the nutritional requirements of the microorganism species. Non-limiting examples of such culture mediums known in the art may comprise f/2 medium and BG- 1 1 medium. In some embodiments, the culture of microorganisms may comprise a freshwater culture. In some embodiments, the culture of microorganisms may comprise a saltwater culture. In some embodiments a chemical flocculent or aggregating agent for improving the electro-conductivity within the aqueous culture of microorganisms may be used with the electro-coagulation system to add the formation of coagulated masses and may comprise, but not limited to, salt, alum, aluminum chlorohydrate, aluminum sulfate, calcium oxide calcium hydroxide, iron (III) chloride, iron (II) sulfate, polyacrylamide, polyDADMAC, sodium aluminate, sodium silicate, chitosan, Moringa oleifera seeds, papain, strychnos seeds, isinglass, and combinations thereof.

[0043] The use of electro-coagulation is one non-limiting example demonstrating how the daily or continuous harvesting of a culture of microorganisms contributes to controlling population of contaminating organisms. A method of harvesting and purging a culture of microorganisms for contamination control may also be performed through known methods of harvesting or separating the microorganisms from a culture such as, but not limited to, decantation, foam fractionation, dissolved gas flotation, electro-flotation, centrifugation, skimming, sedimentation, acoustic energy, filtration and chemical flocculation. When a harvesting and purging method is used the addition of the organic carbon source in a mixotrophic or heterotrophic culture will need to be adjusted accordingly.

Example 1

[0044] A method of controlling a population of contaminating organisms (e.g., bacteria, fungi) in a microalgae culture comprises the application of an electric field to separate the microalgae from the bacteria. An electrical field was applied to a mixotrophic culture of Chlorella sp. to separate the microalgae from the bacteria by coagulating the microalgae using the following procedure. First, 1 liter of the microalgae culture was taken in a 1 L glass beaker. Second, two aluminum electrode plates 0.46 m wide and 1.83 m tall were immersed in the aqueous culture of microalgae in the 1 L beaker. Third, electricity was passed through the electrodes to get to 6 Amp current and the time period of the exposure to the electric field was adjusted to reach approximately 2 Wh/g. Fourth, the culture was centrifuged at 100 g force for 5 minutes, and a solids-rich fraction comprising microalgae was re-suspended in 1 L of deionized water. The electro-coagulation process was repeated two additional times. The final coagulated microalgae were re-suspended in fresh culture medium to produce a microalgae culture with a reduced bacteria population.

[0045] Microalgae cultures from previous electro-coagulation trials have been successfully regrown after being electro-coagulated using the same method described above. The surface area of the microalgae is less than the bacteria, and it was found that more power was needed to neutralize the charge on the surface of the microalgae than the bacteria. These characteristics lead to the bacteria remaining in suspension while the microalgae coagulated at given conditions of current, voltage, and time. Although the coagulated microalgae and suspended bacteria were separated by centrifugation in the experiment, other methods of separation may be used including separated by decantation and floatation by gas bubbles. A sample of the supernatant from the separation step had a much larger concentration of bacteria than coagulated microalgae. An analysis of the bacterial count of the microalgae culture at various times was performed using Petrifilm™ (3M Company, Minnesota, USA) and was performed consistent with methods of bacterial counting known in the art. As shown in the FIG. 1, the bacterial analysis clearly showed that there was a significant reduction in the bacteria from the initial feed to the first pass through the electro-coagulation treatment, and from the first pass through electro-coagulation treatment to the third pass through electro-coagulation treatment. All three experimental runs displayed in FIG. 1 showed this same benefit of reduced bacteria count even though the starting bacterial population of the feed culture for each experimental ranged rather widely. In-line embodiment

[0046] On a larger scale the separation of microorganisms and contaminating organisms may be accomplished incorporating an inline electro-dewatering device and a membrane filter in a culturing system as shown in FIG. 2. The number of washes may depend upon by the residence time of the microorganism culture in the system and the flow rate of the pump. In FIG. 2, P-l represents the flow path of the microorganism culture comprising at least some contaminating organisms in the bioreactor (i.e., culturing vessel) being pumped to the in-line electro-dewatering unit (i.e., electro-coagulation unit) for the application of electric fields to the microorganism culture to produce an electro-coagulation effect on the microorganisms. The electro-coagulated microorganism culture may be pumped along flow path P-7 to a continuous centrifuge such as, but not limited to, an Alfalaval (Lund, Sweden) or GEA Westfalia

(Nuremberg, Germany) or other disc stack centrifuge, where the coagulated microorganism solids-rich fraction is separated from the aqueous culture medium comprising at least some contaminating organisms and may return to the bioreactor along flow path P-4. The supernatant may then be filtered through a filter P-2 such as, but not limited to, a microfiltration membrane (from various vendors such as Pall, GE, Graver, etc.) to separate the contaminating organisms from the cleaned aqueous culture medium. The cleaned aqueous culture medium may be returned to the bioreactor along flow path P-5. Optionally, the microorganism culture may bypass the electro-coagulation treatment and return to the bioreactor. Also, the centrifuge stage may be optional depending on the selectivity of the membrane to separate the contaminating organisms. A controlled pore Nucleopore membrane Whatman (Kent, UK) may be used to separate the contaminating organisms and the coagulated microorganisms.

Example 2

[0047] An in-line electro-coagulation device was used to process a culture of Chlorella sp. comprising a suspension of Chlorella and contaminating bacteria. The in-line electrocoagulation system was an Algae Appliance Model 4 produced by OrginOil, Inc. (5645 West Adams Blvd, Los Angeles, CA 90016). The device used aluminum and stainless steel based electrodes (i.e., anodes and cathodes) to produce an electrical field with electromagnetic pulses. The microalgae were processed through the system in a two stage process. The first stage comprised a pass through where the electrical field is applied to the flowing microalgae culture as the culture passes through the first stage. The apparatus of the pass through in the first stage comprised a cathode formed by an outer cylinder, and an anode formed by an inner cylinder disposed within the outer cylinder. A power source supplied electricity to the cathode and anode as the culture medium flowed through the annulus formed by the anode and cathode, thereby exposing the culture to a pulsed electrical field.

[0048] The second stage comprised an electro-flotation chamber wherein the where an electrical field is applied to the stationary microalgae culture in a chamber which was filled with the microalgae culture exiting the first stage pass through. The apparatus of the second stage electro-flotation chamber comprised a tank with a series of submerged anode and cathode pairs. A power source supplied electricity to the anode and cathode pairs which produced bubbles in the tank for floating coagulated microalgae to the liquid surface where the microalgae was separated from the chamber by skimming. The coagulated microalgae removed from the electro-coagulation device after the second stage was analyzed for the quantity of bacteria and the concentration of the coagulated microalgae using the same methods listed in Example 1. During the tests it was observed that as the microalgae volume in the stages increased, the detected electrical resistance of the culture increased and caused the voltage applied by the electrical field to decrease

[0049] In the first test performed, stage one used a flow rate of an aqueous Chlorella culture of 1 gallon per minute (gpm) (equivalent to 63.09 ml/s), a DC voltage of 2.5 V, and a current of 8.1 amps (average that fluctuated based on resistance). Stage two was a 40 minute treatment with a direct current (DC) voltage of 3.8 V when the chamber was empty and 1.2 V when the chamber was full. The power supply used for stage two of the first test did not have a read out for current. The results of the first test are shown in FIGS. 3 and 6.

[0050] In the second test performed, stage one used a flow rate of an aqueous Chlorella culture of 1 gallon per minute (gpm) (equivalent to 63.09 ml/s), a DC voltage of 5 V, and a current of 17 amps (average that fluctuated based on resistance). Stage two was a 60 minute treatment with a DC voltage of 3.8 V when the chamber was empty and 10.9 V when the chamber was full. The power supply used for stage two on the second test did not have a read out for current. The results of the second test are shown in FIG. 4.

[0051] In the third test performed, stage one used a flow rate of an aqueous Chlorella culture of 1 gallon per minute (gpm) (equivalent to 63.09 ml/s), a DC voltage of 50 V, and a current of 17 amps (average that fluctuated based on resistance). Stage two was a 40 minute treatment with a DC voltage of 10 V (average that fluctuated based on resistance) and a current of 22 amps (average that fluctuated based on resistance). The results of the third test are shown in FIGS. 5 and 7. The device settings for the third test were used with two runs of the Chlorella culture, as shown as Tl and T2 in FIG. 7, to confirm the concentration results. [0052] In the fourth test performed, stage one used a flow rate of an aqueous Chlorella culture of 1 gallon per minute (gpm) (equivalent to 63.09 ml/s), a DC voltage of 50 V, and a current of 18 amps (average that fluctuated based on resistance). Stage two was a 30 minute treatment with a DC voltage of 17 V (average that fluctuated based on resistance) and a current of 30.9 amps (constant). In stage two of the fourth test the power supply was set to the maximum setting which let to varying voltages and constant current. The results of the fourth test are shown in FIG. 8.

[0053] As shown in FIGS. 3-5, the comparison of the initial (feed) and treated bacteria levels of the Chlorella culture demonstrate that the separated and coagulated microalgae resulting from the electro-coagulation processing showed a significant reduction in bacteria in the magnitude range of one log. As shown in FIG. 3, the bacterial count for the microalgae culture decreased from 2.36 X 10 ⁵ to 3.43 X 10 ⁴ after the electro-coagulation treatment and separation. As shown in FIG. 4, the bacterial count for the microalgae culture decreased from 1.61 X 10 ⁶ to 8.49 X 10 ⁴ after the electro-coagulation treatment and separation. As shown in

8 7

FIG. 5, the bacterial count for the microalgae culture decreased from 1.47 X 10 to 2.63 X 10 after the electro-coagulation treatment and separation.

[0054] As shown in FIGS. 6-8, the comparison of the initial (feed) and treated cell concentrations of the separated and coagulated microalgae resulting from the electrocoagulation processing showed a significant increase in concentration of the microalgae in the range of 100 times the initial concentration. As shown in FIG. 6, the concentration of microalgae increased from 0.61 g/L to 63.7 g/L after the electro-coagulation treatment and separation. As shown in FIG. 7, the concentration of microalgae increased started at 0.59 g/L for both Tl and T2, and increased to 46.00 g/L for Tl and 48.5 g/L for T2 after the electrocoagulation treatment and separation. As shown in FIG. 8, the concentration of microalgae increased from 0.42 g/L to 48.40 g/L after the electro-coagulation treatment and separation.

[0055] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims. References:

• US 2013/0288329 Al ;

• OriginOil Algae Appliance brochure, http://www.ori moil.com/products/origirioij- algae-appliance/model-4.html . Accessed 1 1/29/201 1.