WO2018039569A1

WO2018039569A1 - Method of recycling culture media from organic carbon supplied microalgae cultures

Info

Publication number: WO2018039569A1
Application number: PCT/US2017/048631
Authority: WO
Inventors: Ravindhar VANNELA; Michael Lamont; Anthony SHAVER; Jennifer LLOYD-RANDOLFI
Original assignee: Heliae Development Llc
Priority date: 2016-08-25
Filing date: 2017-08-25
Publication date: 2018-03-01

Abstract

Methods of recycling culture media in microalgae cultures are described herein. Aspects of the method include centrifugation, filtration, and ozonation to reduce the concentration of bacteria in the culture medium. The method can surprisingly be performed with inclusion of one or more substances that are reduced by the ozone treatment, such as microalgae cultureinhibiting compounds and/or substances that are surprisingly not impacted by ozonation.

Description

METHOD OF RECYCLING CULTURE MEDIA FROM: ORGANIC CARBON SUPPLIED

MICROALGAE CULTURES

BACKGROUND

[0001] This application claims the benefit of U.S. Provisional Application No. 62/379,555, filed August 25, 2016, entitled Method of Recycling Culture Media from Organic Carbon Supplied Microalgae Cultures. The entire contents of all of the foregoing are hereby incorporated by reference herein.

[0002] Culturing microalgae at commercial volumes can produce a large volume of waste culture media when the culture is harvested and the biomass is separated from the culture media. The ability to recycle the culture media would provide economic, environmental, and efficiency benefits by reducing the water usage and nutrient addition for subsequent cultures, but prior attempts to implement recycling of such media in the practice of microalgae culturing processes has resulted in a variety' of challenges. For example, a first challenge that may arise is that the microalgae culture may accumulate contaminating organisms over the life of the culture that are harmful to microalgae and may remain in the culture medium after typical harvesting and biomass separation processes (i .e., dewatering). A second challenge that can arise is that the substances that inhibit the growth of the microalgae may also accumulate over the life of the culture that are harmful to microalgae and may remain in the culture medium after typical dewatering methods. A third challenge is that the turbidity of the culture media separated from the culture may be high and thus limit the availability of light to microalgae in subsequent cultures.

SUMMARY

[0003] Novel and inventive methods of recycling culture media in microalgae cultures receiving a supply of acetate or acetic acid as an organic carbon source are described herein. These inventive methods of recycling may comprise centrifugation, filtration, and ozonation of the culture media to reduce the concentration of bacteria in the culture while either (a) reducing the malic acid content of the microalgae media and/or (b) maintaining the levels of one or more hexose saccharides (such as glucose) of the culture at a level that is substantially the same (no more than 15% different, such as about 12% or less different, about 10% or less different, or about 7.5% or less different) before using the treated culture media in a subsequent microalgae culture, alone or together with acetic acid, if present. [0004] The methods of the invention may be used in a singular or serial application, and the treated culture media may be used in as a portion of or as the entire culture media in a subsequent microalgae culture.

BRIEF DESCRIPTION OF THE FIGURE

[0005] The graph in Figure 1 shows the effect of an exemplar}' ozone treatment on the dry- weight, acetic acid, and malic acid concentrations of a liquid centrate of a microalgae (Chlorella) culture, reflecting an exemplary embodiment of the in vention.

DETAILED DES CRIPTION

[0006] While many wastewater treatment methods involving microalgae have been practiced or have been proposed in the art, methods of recycling microalgal growth media that are sensitive to (a) the retention of specific microalgae nutrients (such as acetic acid, a hexose, or both), (b) microalgae growth inhibitors (such as malic acid), and (c) contamination for microalgae cultures is needed to realize the benefits for recycling culture media in microalgae production processes. The inventive methods provided herein provide, inter alia, effective methods of recycling microalgal growth media.

[0007] To better illuminate the invention, certain terms are defined herein. Such terms are to be construed according to such definitions, unless explicitly contradicted herein or clearly contradicted by context.

[0008] The term "centrate" refers to a liquid phase comprising liquid culture media resulting from processing a culture of suspended microalgae cells in a liquid culture medium through a centrifuge to separate the microalgae cells from the liquid culture medium.

[0009] The terms "mixotrophic" and "mixotrophy" refer to microalgae culture conditions in which light, organic carbon, and inorganic carbon (e.g., carbon dioxide, carbonate, bicarbonate) may be applied to a culture of microorganisms (i.e., are present at some time in the culturing of the microorganisms). Microorganisms capable of growing in mixotrophic condi tions 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.

[0010] Multiple methods of generally treating wastewater exist, including physical separation of solids (e.g., centrifugation, filtration, sedimentation), the application of bleach, the application of peroxides, the application of peracetic acid, the application of ozone (O₃), anaerobic or aerobic digestion, biochemical or chemical oxidation, carbon filtering, and combinations thereof, with the goal of reducing suspended solids, kill pathogenic bacteria, and remove target molecules to enable use of the water for another purpose. Oxidation of dissolved organic materials with ozone has been shown to result in polar and charged molecules. Ozonation has also been shown to result in a decrease in turbidity, improved settleability, a reduction in the number of particles, and a reduction in the population of microorganisms (e.g. bacteria). Ozone may be applied in a variety of manners, including not limited to, infusing ozone into water.

[0011] Filtration comprises a physical separation of particles from a fluid using a variety of filter media with different pore sizes and numbers of pores. Non-limiting examples of filtration for the separation of particles from a fluid comprise cross flow filtration, dead end filtration, membrane filtration, vacuum filtration, surface filtration, solid sieve, screen filters, belt filters, beds of granular material (e.g., sand), hollow fiber filters, fiat sheet/plate filters, spiral wound filters, and paper filters. Selection of the proper filtration system for an application may comprise determining parameters such as, but not limited to, the proper flux (e.g., the volumetric flow rate per unit of filter media), overall porosity of the filter media, resistance of the filter media, rate of filter media fouling, resistance of the particle cake, liquid viscosity, osmotic pressure, transmedia pressure, pressure differential, dynamic viscosity, and filter media surface area. Separation aids may also be used to aid filtration, such as but not limited to, diatomaceous earth. Separation with membrane filtration may occur through diffusion or convectively. [0012] Cross-flow or tangential filtration may be used to reduce the blockages that occur in convective separation of membrane filtration. In cross-flow filtration, the feed flow is tangential to the surface of membrane, which allows the retentate to be removed from the same side of the filter further downstream, whereas the permeate flow is tracked on the other side of the filter. Dead-end filtration differs from cross-flow in that the direct of the fluid flow is normal to the filter surface. Membrane separation process may include, but are not limited to, microfiltratioii, ultrafiltration, nanofiltration, reverse osmosis, electrolysis, dialysis, electfodiaivsis, gas separation, vapor permeation, pervaporation, membrane distillation, and membrane contactors. Pores on filtration media are available in a variety of sizes, such as, 0.1 micron, 0.2, micron, 0.3 micron, 0.5 micron, 0.6 micron, 0.7 micron, 0.8 micron, 0.9 micron, 1 micron, greater than 1 micron, less than 1 nanometer, 1-2 nanometers, 2-50 nanometers, 50- 100 nanometers, and larger.

[0013] Operation of a filtration system may also be influenced by parameters such as, but not limited to, operating pressure, solution concentration, electric potential of the solution, and operating temperature. Depending on the filtration system utilized and the characteristics of the working solution, filtration operations may be conducted in a batch or continuous manner. In the context of recycling culture media (e.g., centrate) for microalgae culturing, the fluid that passes through the filter is used in subsequent cultures, which may also be refeired to as the permeate or filtrate; while the separated particles, which may also be referred to as the retentate, may be used further processed for a particular use or discarded.

[0014] Growing microorganisms, such as microalgae (including cyanobacteria), in mixotrophic or other conditions (e.g. , phototrophic or heterotrophic conditions) can produce a culture media with unique characteristics that may determine which treatment method or methods are the most effective for recycling the culture media for use in a subsequent culture. Microalgae are typically grown in a nutrient media comprising mostly water, but also comprising trace metals, nitrogen, phosphorus, salts, and vitamins. Examples of different liquid culture media for culturing microalgae are available from a variety of public sources and include, but are not limited, formulations designated as BG-11 and f/2 media. Microalgae that may grow in mixotrophic culture conditions, as well as phototrophic and heterotrophic conditions, comprise microalgae and cyanobacteria. Non-limiting examples of mixotrophic microalgae may comprise organisms of the genera: AgmeneUurn, Amphora, Anabaena, Anacystis, Apistonema, Arthrospira (Spirulina), Botryococcus, Brachiomonas. CMamydomonas, Chlorella, Chloroccum, Cruciplacolithus, Cylindrotheca, Coenochloris, Cyanophora, Cyclotella, Dunaliella, Emiliania, Euglena, Extubocellulus, Fragilaria, Galdieria, Goniotrichium, Haematococcus. Halochlorella, Isochyrsis, Leptocylindrus, Micractinium, Melosira, Monodus, Nostoc, Nannochloris, Nannoehloropsis, Navicula, Neospongiococcum, Nitzschia, Odontella, Ochromonas, Ochrosphaera, Pavlova, Picochlorum, Phaeodactylum, Pleurochyrsis, Porphyridium, Poteriochromonas, Prymnesium. Rhodomonas, Scenedesmus, Skeletonema, Spumella, Stauroneis, Stichococcus, AuxenochloreUa, Cheatoceros, Neochloris, Ocromonas, Porphiridium, Synechococcus, Synechocyslis, Tetraselmis, Thraustochytrids, Thalassiosira, and species thereof.

[0015] 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, malic acid, maltose, mannose, methanol, molasses, peptone, plant based hydrolyzate, proline, propionic acid, ribose, sacchrose, partial or complete hydrolysates of starch, sucrose, tartaric, TCA-cyele 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.

[0016] Non-limiting examples of suitable microalgae that may be the primaiy or sole organism in the medium upon which the methods of the invention can comprise organisms of the genera; Chlorella, Anacystis, Synechococcus, Synechocystis , Neospongiococcum,, Chlorococcum, Phaeodactylum., Spirulina, Micractinium, Haematococcus, Nannoehloropsis, Brachiomonas, and species thereof. While non-limiting examples of the inventive method are described throughout comprising cultures of Chlorella, Schizochytrium, and Scenedesmus, the inventive methods may be used for recycling culture media for any microorganism culture in which the microorganism receives one of the critical nutrients (e.g., glucose, acetic acid, or both) and/or accumulates one of the critical inhibitory compounds (e.g., malic or malic acid).

[0017] Growing microalgae in a variety of culture conditions, including in phototrophic, mixotrophic, and/or heterotrophic conditions, may accumulate a number of components in the culture that are detrimental to the health, growth, and proliferation of microalgae. This accumulation may occur in a variety of available bioreactors for culturing microalgae, but may be somewhat more pronounced for mixotrophic cultures due to the availability of both light and organic carbon as energy sources.

[0018] Deleterious components of a microalgae culture may comprise predator organisms, fungi, bacteria, high levels of a toxic substance, or organic acids (e.g., malic acid). Bacteria that may have a negative or harmful effect on microalgae in a culture comprise, but are not limited to: Achromobacter sp., Acidovorax sp., Aeromonas sp., Agrobacterium sp., Alteromonas sp., Aquas pir ilium sp., Azospirillum sp., Azoiohacier sp., Bergeyelia sp. , Brochothrix sp., Brumimicrobium sp., Burkholderia sp., Caulobacter sp., Cellulomonas sp. , Chryseobacterium sp., Curtobacterium sp., Delfiia sp., Empedobacier 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., Vibrio sp., Zobelliae sp. and other bacteria which share similar characteristics.

[0019] There are two sterioisomeric forms of malic acid (C₄H₆O₅), the L- and D-enantiomers). The L-isomer exists naturally, although a mixture of L- and D-malic acid may be produced synthetically. Malate is naturally produced as an intermediate in the Kreb's cycle. The L-malic acid form in microalgae cultures has been confirmed by the inventors using High Performance Liquid Chromatography (HPLC) and mass spectral analysis using soft ionization technique (Maldi-Tof). In one non-limiting example, it was discovered that in acetate/acetic acid fed mixotrophic cultures of ChioreUa, malic acid was present in the culture media as an excreted substance in a variety of conditions include axenic, non-axenic, open, and closed cultures. Additionally, the excretion of malic acid by was also found to occur in ChioreUa cultures at various levels in phototrophic and heterotrophic culture conditions.

[0020] From one perspective, the presence of the malic acid in a culture of malic-acid producing microalgae, such as ChioreUa microalgae, provides a product opportunity, as malic acid is known to have value in the marketplace as a food additive (e.g., flavoring, preservative, chelation). [0021] From another perspecti ve, the excretion of malic acid can be used as a health indicator pointing towards a bottleneck in a microalgae cells metabolism. This metabolic effect is evidenced by the observation of malic acid excretion as having a detrimental effect on the health of the Chlorella microalgae, resulting in decreased growth and longevity for the Chlorella culture. Also, while concentrations of malic acid may have a direct effect on Chlorella, such as toxicity at concentrations of about 3.8 g/L and higher, malic acid may also have indirect effects on the Chlorella. Bacteria are known to excrete and consume malic acid, and in non-axemc cultures of Chlorella the presence of malic acid provides a substrate for contaminating bacteria to proliferate and compete with Chlorella. The proliferation of bacteria may lead to the decline in health of the Chlorella even when the concentration of malic acid does not reach toxic levels (e.g., below a concentration of 3.8 g/L) that directly affect the Chlorella. Therefore, when the growth of a microalgae that is inhibited by malic acid, such as Chlorella, is prioritized, the elimination of malic acid from the culture media contributes to optimizing the malic acid-inhibited microalgae (such as Chlorella) productivity and culture longevity.

[0022] When developing a solution to reduce the water usage and nutrient addition in microalgae cultivation, including mixotrophic and heterotrophic microalgae cultivation, the inventors investigated combinations of methods to determine what was the most effective regarding the key culture media recycling parameters for organic carbon supplied microalgae cultures: 1) bacteria concentration, 2) acetate/acetic acid concentration, 3) hexose (e.g., glucose) concentration, and 4) malic acid concentration. Not all of these factors are present in every type of microalgae culture and, accordingly, not all will be present or implicated in every aspect of the invention. The reduction of the bacteria and malic acid concentration parameters in the culture media can be important in reducing the risk of harm to the subsequent culture of microalgae in the case of malic acid-inhibited microalgae. The maintenance of the acetate/acetic acid concentration in the culture media of certain cultures allows the amount of subsequent nutrient addition (e.g., organic carbon) to be reduced for subsequent acetate/acetic acid fed mixotrophic microalgae cultures. Therefore, in one aspect, the inventors developed a method for recycling culture media wherein the treatment reduces the concentration of bacteria and malic acid while also maintaining the concentration of acetate/acetic acid. Methods that were evaluated, but unsuccessful regarding the effect on the key parameters comprised: the application of bleach only at concentrations of 100 mg/L and 400 mg/L, 0.1 micron filtration only, a combination of filtration and application of bleach, application of peroxide, and application of peracetic acid. Successful methods typically comprise a two-stage treatment method comprising (a) filtration and (b) the application of ozone.

|0023] When evaluating ozonation methods, the parameters found to be relevant that the inventors monitored comprised of: oxidation reduction potential (ORP), conductivity, pH, dissolved oxygen, bacteria petrifiim counts (i.e., bacteria quantification), dry weight, acetate/acetic acid concentration, malic acid concentration, glucose (or hexose) concentration, and concentration of various metals. During ozonation of microalgae culture media, ORP was found to rapidly increase (about 150mV) and then slowly decrease (about 50 mV) over the course of 6 hours, the pH level gradually increased about 1 over the course of 6 hours, and the bacteria count rapidly fell in the first hour of treatment.

[0024] In one non-limiting embodiment, a method may comprise: a) centrifuging a microalgae culture comprising culture media, bacteria, and a population of microalgae cells, to separate the microalgae cells from a centrate comprising a first concentration of bacteria and culture media, wherein the culture media comprises a first concentration of malic acid and a first concentration of acetic acid or acetate; b) filtering the centrate; c) treating the filtered centrate with ozone for an effective amount of time to produce a treated medium, optionally with a second concentration of malic acid that is lower than the first concentration of malic acid, and also a second concentration of bacteria lower than the first concentration of bacteria, and either (1) a concentration of acetate or acetic acid that is substantially the same as the first concentration of acetate or acetic acid or (2) a concentration of hexose that is substantially the same as the first concentration of the hexose in the medium (e.g., glucose). In some embodiments, the microalgae may be Chlorella, but other suitable species can include Schizochytrium and Scenedesmus .

[0025] The term "substantially the same" refers to a change less than or equal to 15%, such as a change of about 12.5% or less, about 10% or less, about 9% or less, or about 7.5% or less.

[0026] In some embodiments, the effective amount of time the filtered centrate may be treated with ozone is less than one hour. In some embodiments, the effective time the filtered centrate may be treated with ozone is less than 30 minutes. In some embodiments, the effective time the filtered centrate may be treated with ozone is less than 15 minutes. In some embodiments, the effective time the filtered centrate may be treated with ozone is at least one hour. In some embodiments, the effective time the filtered centrate may be treated with ozone is in the range of 1-10 hours. In some embodiments, the effective time the filtered centrate may be treated with ozone is in the range of 1-8 hours. In some embodiments, the effective time the filtered centrate may be treated with ozone is in the range of 1 -6 hours. In some embodiments, the effective time the filtered centrate may be treated with ozone is in the range of 1-5 hours. In some embodiments, the effective time the filtered centrate may be treated with ozone is in the range of 1 -4 hours. In some embodiments, the effective time the filtered centrate may be treated with ozone is in the range of 1-3 hours. In some embodiments, the effective time the filtered centrate may be treated with ozone is in the range of 1-2 hours.

|0027] In some embodiments, the method is characterized by being performed with a malic acid-inhibited microalgae. In some aspects, the microalgae is also responsible for production of the inhibitory malic acid. In such aspects, the method can often be characterized by the fact that the malic acid concentration of the ozone-treated culture being substantially less than the malic acid concentration in the culture before treatment. "Substantially less" in this context means at least 10% less. In some embodiments, the second concentration of malic acid may be at least 15% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 20%) less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 25% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 30% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 33% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 40% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 50% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 60% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 67% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 70% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 75% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 80% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 90% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 95% less than the first concentration of malic acid. In some embodiments, the second concentration of malic acid may be at least 99% less than the first concentration of malic acid.

[0028] In some embodiments, acetic acid is a component of the microorganism culture. Acetic acid can sen'e as a growth media (nutrition source) for microalgae and/or as a means for inhibiting contamination of a microalgae culture, or be present for other reasons. In such embodiments, the second concentration of acetic acid (post ozone treatment) may be substantially the same as the first concentration of acetic acid (pre-treatment) in the culture. In some embodiments, the second concentration of acetic acid may be within 10% of the first concentration of acetic acid, in some embodiments, the second concentration of acetic acid may be within 9 % of the first concentration of acetic acid. In some embodiments, the second concentration of acetic acid may be within 8% of the first concentration of acetic acid. In some embodiments, the second concentration of acetic acid may be within 7% of the first concentration of acetic acid. In some embodiments, the second concentration of acetic acid may be within 6% of the first concentration of acetic acid. In some embodiments, the second concentration of acetic acid may be within 5% of the first concentration of acetic acid. In some embodiments, the second concentration of acetic acid may be within 4% of the first concentration of acetic acid. In some embodiments, the second concentration of acetic acid may be within 3% of the first concentration of acetic acid. In some embodiments, the second concentration of acetic acid may be within 2% of the first concentration of acetic acid. In some embodiments, the second concentration of acetic acid may be within 1% of the first concentration of acetic acid. In some embodiments, the second concentration of acetic acid may not be detectably different from the first concentration of acetic acid.

[0029] The ozone treatment methods of the invention typically will result in a reduction in bacterial concentration such that the amount of bacteria in the culture is reduced by a measurable amount, such as at least about 10%, at least about 25%, at least about 50%, at least about 75%, or more, and/or the viability of the microalgae culture is increased (e.g., by measurable increases in the longevity of the culture and/or amount of microalgae cells and/or concentration of microalgae cells in the culture). In some embodiments, the second concentration of bacteria (the concentration of bacteria after treatment) may be at least one order of magnitude lower than the first concentration. In some embodiments, the second concentration of bacteria may be at least two orders of magnitude lower than the first concentration. In some embodiments, the second concentration of bacteria may be at least three orders of magnitude lower than the first concentration. In some embodiments, the second concentration of bacteria may be at least four orders of magnitude lower than the first concentration. In some embodiments, the second concentration of bacteria may be at least five orders of magnitude lower than the first concentration.

[0030] In some embodiments, microalgae be inoculated in at least one subsequent culture in which the culture media of the subsequent culture comprises at least a portion of treated medium (from the first culture), typically where the remainder of the culture media of the subsequent culture comprising fresh culture media that has not been treated or recycled. In some embodiments, such methods ma}'^' further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 5% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 10% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 15% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 20% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at leas t 25% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 30% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 33% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 40% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 50% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 60% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 67% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 70% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 75% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 80% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 90% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising at least 95% of the treated medium. In some embodiments, the method may further comprise inoculating microalgae in at least one subsequent culture with a first recy cled culture medium comprising at least 99% of the treated medium. In some embodiments, the method may further compnse inoculating microalgae in at least one subsequent culture with a first recycled culture medium comprising 100% treated medium.

[0031 ] The method may also be used to treat and recycle culture media multiple times in a series of microalgae cultures. In some embodiments, the method may further comprise repeating the steps of centrifuging, filtering, and treating with ozone the filtered centrate from a first subsequent culture, and inoculating microalgae in a second subsequent culture with a recycled culture medium comprising at least 5% of the treated medium. In some embodiments, the method may further comprise repeating the steps of centrifuging, filtering, and treating with ozone the filtered centrate from a second subsequent culture, and inoculating microalgae in a third subsequent culture with a recycled culture medium comprising at least 5% of the treated medium. In further embodiments, the recycled medium may comprise at least 10% of the treated medium. In further embodiments, the recycled medium may comprise at least 15% of the treated medium. In further embodiments, the recycled medium may comprise at least 20% of the treated medium. In further embodiments, the recycled medium may comprise at least 25% of the treated medium. In further embodiments, the recycled medium may comprise at least 30%» of the treated medium. In further embodiments, the recycled medium may comprise at least 33% of the treated medium. In further embodiments, the recycled medium may comprise at least 40% of the treated medium. In further embodiments, the recycled medium may comprise at least 50% of the treated medium. In further embodiments, the recycled medium may comprise at least 60% of the treated medium. In further embodiments, the recycled medium may comprise at least 67% of the treated medium. In further embodiments, the recycled medium may comprise at least 70% of the treated medium. In further embodiments, the recycled medium may comprise at least 75% of the treated medium. In further embodiments, the recycled medium may comprise at least 80% of the treated medium. In further embodiments, the recycled medium may comprise at least 90% of the treated medium. In further embodiments, the recycled medium may comprise at least 95% of the treated medium. In further embodiments, the recycled medium may comprise at least 99% of the treated medium. In further embodiments, the recycled medium may comprise 100% treated medium.

[0032] In some embodiments, the serial treatment of culture medium for recycling the medium in a series of subsequent microalgae cultures may occur up to two times. In some embodiments, the serial treatment of culture medium for recycling the medium in a series of subsequent microalgae cultures may occur up to three times. In some embodiments, the serial treatment of culture medium for recycling the medium in a series of subsequent microalgae cultures may occur up to four times. In some embodiments, the serial treatment of culture medium for recycling the medium in a series of subsequent microalgae cultures may occur up to five times. In some embodiments, the serial treatment of culture medium for recycling the medium in a series of subsequent microalgae cultures may occur up to six times. In some embodiments, the serial treatment of culture medium for recycling the medium in a series of subsequent microalgae cultures may occur up to seven times. In some embodiments, the serial treatment of culture medium for recycling the medium in a series of subsequent microalgae cultures may- occur up to eight times. In some embodiments, the serial treatment of culture medium for recycling the medium in a series of subsequent microalgae cultures may occur up to nine times. In some embodiments, the serial treatment of culture medium for recycling the medium in a series of subsequent microalgae cultures may occur up to ten times.

[0033] In some embodiments, microalgae cells may be inoculated in a subsequent culture without reducing the microalgae growth rate in dry weight grams per liter per day more than 5% as compared to a control culture of microalgae with 100% newly prepared culture media that has not he treated or recycled. In some embodiments, microalgae cells may be inoculated in a subsequent culture without reducing the microalgae growth rate in dry weight grams per liter per day more than 10% as compared to a control culture of microalgae with 100% newly prepared culture media that has not be treated or recycled. In some embodiments, microalgae cells may be inoculated in a subsequent culture without reducing the microalgae growth rate in dry weight grams per liter per day more than 15% as compared to a control culture of microalgae with 100% newly prepared culture media that has not be treated or recycled. In some embodiments, microalgae cells may be inoculated in a subsequent culture without reducing the microalgae growth rate in dry weight grams per liter per day more than 20% as compared to a control culture of microalgae with 100% newly prepared culture media that has not be treated or recycled. In some embodiments, microalgae cells may be inoculated in a subsequent culture without reducing the microalgae growth rate in dry weight grams per liter per day more than 25% as compared to a control culture of microalgae with 100% newly prepared culture media that has not be treated or recycled. In some embodiments, microalgae cells may be inoculated in a subsequent culture without reducing the microalgae growth rate in dry weight grams per liter per day more than 30%) as compared to a control culture of microalgae with 100% newly prepared culture media that has not be treated or recycled.

[0034] In some embodiments, the centrate may be filtered with a membrane filter. In some embodiments, the pore size of a membrane filter may be in the range of 0.1-1 microns. In some embodiments, the pore size of a membrane filter may be in the range of 0.1-0.5 microns. In some embodiments, the pore size of a membrane filter may be in the range of 0.1 -0.25 microns. In some embodiments, the pore size of a membrane filter may be in the range of 0.1-0.2 microns.

[0035] In some embodiments, the oxidation reduction potential (ORP) of the centrate may be maintained in the range of 500-1000 mV during treatment with ozone. In some embodiments, the oxidation reduction potential (ORP) of the centrate may be maintained in the range of 500- 600 mV during treatment with ozone. In some embodiments, the oxidation reduction potential (ORP) of the centrate may be maintained in the range of 600-700 mV during treatment with ozone. In some embodiments, the oxidation reduction potential (ORP) of the centrate may be maintained in the range of 700-800 mV during treatment with ozone. In some embodiments, the oxidation reduction potential (ORP) of the centrate may be maintained in the range of 800- 900 mV during treatment with ozone. In some embodiments, the oxidation reduction potential (ORP) of the centrate may be maintained in the range of 900-1000 mV during treatment with ozone. In some embodiments, the oxidation reduction potential (ORP) of the centrate may be maintained in the range of 500-750 mV during treatment with ozone. In some embodiments, the oxidation reduction potential (ORP) of the centrate may be maintained in the range of 750- 1000 mV during treatment with ozone. [0036] In some embodiments, the filtered centrate may be treated with an effective amount of ozone to achieve the results described throughout the specification. In some embodiments, the filtered centrate is treated with an amount of ozone in the range of 25-150 mg of ozone per liter of centrate. In some embodiments, the filtered centrate is treated with an amount of ozone in the range of 50-125 mg of ozone per liter of centrate. In some embodiments, the filtered centrate is treated with an amount of ozone in the range of 55-111 mg of ozone per liter of centrate. In some embodiments, the filtered centrate is treated with an amount of ozone in the range of 25-50 mg of ozone per liter of centrate. In some embodiments, the filtered centrate is treated with an amount of ozone in the range of 50-75 mg of ozone per liter of centrate. In some embodiments, the filtered centrate is treated with an amount of ozone in the range of 75- 100 mg of ozone per liter of centrate. In some embodiments, the filtered centrate is treated with an amount of ozone in the range of 100-125 mg of ozone per liter of centrate. In some embodiments, the filtered centrate is treated with an amount of ozone in the range of 125-150 mg of ozone per liter of centrate.

[0037] In another non-limiting embodiment, a composition may comprise: a) a population of microalgae ceils; and b) a culture media comprising at least one of acetic acid and acetate, and at least 5% recycled culture media from a previous microalgae culture, wherein the recycled culture media has been filtered and treated with ozone. In some embodiments, the microalgae may be Chlorella. In some embodiments, the composition may comprise at least 10% recycle culture media. In some embodiments, the composition may comprise at least 15% recycle culture media. In some embodiments, the composition may comprise at least 20% recycle culture media. In some embodiments, the composition may comprise at least 25% recycle culture media. In some embodiments, the composition may comprise at least 30% recycle culture media. In some embodiments, the composition may comprise at least 33% recycle culture media. In some embodiments, the composition may comprise at least 40% recycle culture media. In some embodiments, the composition may comprise at least 50% recycle culture media. In some embodiments, the composition may comprise at least 60% recycle culture media. In some embodiments, the composition may comprise at least 67% recycle culture media. In some embodiments, the composition may comprise at least 70% recycle culture media. In some embodiments, the composition may comprise at least 75% recycle culture media. In some embodiments, the composition may comprise at least 80% recycle culture media. In some embodiments, the composition may comprise at least 90% recycle culture media. In some embodiments, the composition may comprise at least 95% recycle culture media. In some embodiments, the composition may comprise at least 99% recycle culture media. In some embodiments, the composition may comprise 100% recycle culture media. In other embodiments the same amount of recycle culture media is used but the microalgae composition of the culture primarily or entirely is composed of Schizochytrium, Scenedesmus, or another microalgae genus than Chlorella.

[0038] In another non-limiting embodiment, a method of reducing the addition of fresh media and nutrients to a non-axenic mixotrophic culture of microalgae may comprise: a) providing a culture comprising a population of microalgae cells and at least some bacteria in a culture medium comprising water, nutrients, malic acid, and at least one of acetic acid and acetate; b) centrifuging the culture to separate the microalgae cells from a centrate comprising a first concentration of bacteria and centrifuged culture media, wherein the centrifuged culture media comprises a first concentration of malic acid and a first concentration of at least one of acetic acid and acetate; c) filtering the centrate to form a filtered centrate; d) treating the filtered centrate with ozone for an effective amount of time to produce a treated medium with a second concentration of malic acid lower than the first concentration of malic acid, a second concentration of bacteria lower than the first concentration of bacteria, and a concentration of at least one of acetate and acetic acid substantially similar to a first concentration of at least one of acetate and acetic acid; e) inoculating the microalgae cells in a first subsequent culture with a first recycled culture medium comprising at least a portion of the treated medium and the remainder comprising of freshly prepared culture media; and f) supplying less of at least one of acetate and acetic acid to the first subsequent culture than a control culture would receive which comprises 100% freshly prepared culture media. In some embodiments, the cultures may be mixotrophic or heterotrophic cultures supplied with at least one of acetate or acetic acid as the organic carbon source. In some embodiments, the microalgae may be Chlorella.

[0039] In another aspect, the invention provides methods that comprise (a) concentrating a microalgae culture (e.g., centrifuging the microalgae culture) comprising an initial culture media, bacteria, and a population of microalgae cells, to separate the microalgae cells from a centrate, wherein the centrate comprises (1) a first concentration of bacteri a (BC) and the used or "spent" culture media, the spent culture media comprising a first malic acid concentration (MAC) and/or a first hexose concentration (HC). The centrate may optionally be filtered as described elsewhere herein. Either way, the method comprises treating the centrate (or filtered centrate) with ozone for an effective amount of time to produce a treated medium characterized in having a second concentration of bacteria that is lower than the first concentration of bacteria (typically by at least one order of magnitude, and sometimes at least two orders of magnitude). The treated centrate will also have (a) a second MAC that is lower than the first MAC, (b) a second HC that is substantially the same as the first HC, or (c) a second MAC that is lower than the first MAC and a second HC that is substantially the same as the first HC. The meaning of the phrases "substantially the same" and "substantially lower" are provided elsewhere in this Description.

[0040] The inventors have surprisingly discovered that such methods are possible by uncovering the impacts of ozone on these compounds in the context of spent microalgae culture / centrate. For example, malic acid is surprisingly reduced in concentration by ozone treatment. This is advantageous in the culturing of a malic acid-inhibited microalgae (such as Chlorella), as in such microalgae the presence of higher amounts of malic acid (e.g., about 8 g/L or higher) will inhibit continued growth of the culture. The malic acid content of a spent culture can be reduced at least 50%, at least 60%, or even at least 65% (such as at least 67%) by the application of ozonation according to the inventive methods described herein. However, although possessing a somewhat similar chemical structure in terms of having a number of hydroxy 1 groups, other compounds, such as hexoses, for example glucose, are not reduced by the application of ozone in the context of microalgae culturing, particular with respect to ozone treatment of spent media / centrate. Based on these discoveries the inventors have invented new methods of effectively re-using large portions of spent microalgal culture at substantial scale, as exemplified by the examples provided herein, which results in a more sustainable and cost effective operation.

[0041] In those aspects of the invention in which spent media is filtered, generally any suitable level and type of filtration may be applied. In some cases, it can be advantageous to have spent media filtered with a membrane filter, wherein the membrane filter has a pore size of 0.1-0.5 microns.

[0042] In some contexts, the methods of the invention are performed in a setting in which the spent culture / centrate further comprises acetic acid (AC), such that the culture comprises a first acetic acid concentration (AAC) and a post-treatment or second acetic acid concentration. The inventors find that acetic acid, although in many ways more similar to malic acid than a hexose, is also not subject to a substantial reduction in amount after ozone treatment. Thus, in a particularly advantageous aspect, the methods described herein can be applied to cultures comprising both acetic acid and a hexose, which each contribute to the growth of the microalgae to be cultured with these nutrients, while not being substantially lower in spent media / centrate after ozonation.

[0043] Based on experimentation and scientific insight it appears that the inventive methods can be performed with very different microalgae. The inventors have demonstrated, or contemplate, that the method can be performed with microalgae from the genera Chlorella, Schizochytrium, and Scenedesmus. The inventors have also, surprisingly, discovered that the method can be performed on media that is markedly different from and has a substantially lower concentration of water than traditional microalgae growth media. For example, a waste stream that can be used as a growth medium, such as cola waste material, may contain less than 85%, less than 90%, less than 92%, less than 95%, or less than 97% water.

[0044] The methods of the invention can be applied to recycle a range of different amounts of spent material. In one aspect, at least 20% or at least 25%) of the treated medium is recycled (e.g., at least 30%, 35%, or 40% of the medium is recycled). In some aspects, at least 50% of the treated medium can be recycled. In some cases, more than 65%, 2/3rds, 75%, 80%, 85%, 90%, or even more than 99% (such as 100%) of the media used for new growth of microalgae in the method is obtained from the treatment and recycling of spent media. Remarkably, the inventors have determined that spent medium can be subjected to 2, 3, 4, 5, or more rounds of treatment and recycling, with up to a substantially amount (e.g., at least about 25%) of such multiply -treated media being used for new microalgae culture growth in subsequent rounds of culturing, while not exhibiting significant losses in terms of microalgae growth rates in such cultures containing such multiple treatment media (e.g., no losses observed of more than 20% in growth rate).

[0045] The application of ozone in practicing the inventive methods described herein can be performed using any parameter or set of conditions that is suitable for the desired outcome of the treatment. In one aspect, the effective amount of time the centrate or filtered centrate is treated with ozone is at least 1 hour; however, the effective time can be significantly longer, such as up to 3, 4, 5, 6, 7, 8, 9, or 10 hours. The result of the ozonation can also vary. In some embodiments it is advantageous that ozonation results in the oxidation reduction potential of the centrate achieving or being maintained in the range of 800-900 mV during and/or after treatment with ozone. In another aspect, the spent media, such as filtered centrate, is treated with an amount of ozone in the range of 25-150 mg ozone/L media, such as about 50-125 mg ozone/L media, for example 55 to 1 11 rag of ozone per liter media (e.g., filtered centrate).

[0046] Those skilled in the art will recognize that in addition to various methods of culturing microoganisms, such as microalgae, the invention also provides a number of new and useful compositions, which are produced by the application of the inventive methods described above. For example, in one aspect the invention further provides a composition comprising (a) a population of microalgae cells (such as a population of Chlorella, Schyzochytrium, Spirulina, or Scenedesmus cells, in a culture media that comprises (a) a hexose (e.g., glucose), (b) a microalgae inhibitory compound that is susceptible to reduction through ozonatoin, such as malic acid, and/or (c) an acetic acid (or similar substance, such as acetate, which in another aspect can be in the place of acetic acid in the various methods described herein), in which at least 25% of the culture media of the culture is derived from spent media (previously microalgae culture) and such recycled media has been subjected to ozone treatment, and optionally filtered. In such compositions the composition can be characterized similar to any of the above-described methods. For example, the composition can be characterized in having a low amount of malic acid as compared to in the spent culture, prior to ozone treatment and/or the composition can be characterized in having an amount of a hexose, such as glucose, that is substantially the same as the level of such substance in the original microalgae growth medium. Advantageously, these methods hold true at large scales, such as more than 1,000 L, more than 2,000 L, more than 2,500 L, more than 3,000 L, more than 5,000 L, more than 7,500 L or even more than 10,000 L of culture and/or recycled culture.

Examples

[0047] Embodiments of the invention are exemplified and additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of any aspect of the invention described herein.

|0048] The strain of Chlorella used in the initial following examples provides an exemplar}' embodiment of the invention but is not intended to limit the invention to a particular strain of microalgae. Analysis of the DNA sequence of the exemplary strain of Chlorella in the NCBI 18s rDNA reference database at the Culture Collection of Algae at the Uni versity of Cologne (CCAC) showed substantial similarity (i.e., greater than 95%) with multiple known strains of Chlorella and Micractinium. Those of skill in the art will recognize that Chlorella and Micractinium appear closely related in many taxonomic classification trees for microalgae, and strains and species may be re-classified from time to time. While the exemplary microalgae strain is referred to in the instant specification as Chlorella, it is recognized that microalgae strains in related taxonomic classifications with similar characteristics to the exemplar}' microalgae strain would reasonably be expected to produce similar results. Accordingly, the inventors contemplate several of the methods described herein and exemplified below can be performed with strains from the genus Micractinium, the genus Chlorella, or both.

Example 1

[0049] An experiment was performed to determine the effectiveness of using recycled culture media from mixotrophic cultures of Chlorella in subsequent cultures of Chlorella. The mixotrophic culture conditions for each culture comprised of a supply of light and a supply of acetic acid as the organic carbon source. The first culture media treatment in the experiment comprised using 100% water from the municipal supply (City of Gilbert, Gilbert, Arizona USA) to serve as an untreated control for the initial inoculum media and the media added to increase volume after partial harvests. The second culture media treatment in the experiment comprised using a 50%/50% mixture of water from the municipal supply and the filtered centrate of culture media from a previous culture that was also subjected to ozone treatment after filtration. The 5Q%/'50% mixtures were used for the initial inoculum media and the media added to increase volume after partial harvests. The third culture media treatment in the experiment comprised 100% water from the municipal source as the initial inoculum media, and 100% filtered centrate of culture media from a previous culture that was also subjected to ozone treatment after filtration for the media added to increase volume after partial harvests.

[0050] The first three culture treatments were operated in a fed-batch operation with partial harvests. For the fed batch cultures, the cultures were inoculated at 1 g/L, and partially harvested when the culture density reached about 5 g/L to reduce the culture density to about 3 g/L. The filtration treatment comprised filtering the culture media through a 0.1 micron membrane filter (Graver Technologies, Glasgow, Delaware USA). The previous Chlorella culture had been in an open outdoor reactor for 16 days prior to filter treatment. The ozone treatment comprised applying ozone to the culture media in a bubble column container. The ozone was supplied by a Model CD2000P ozone generator (ClearWater Tech LLC, San Luis Obispo, California USA), set to produce 30 g/hour of ozone and a target cycle time of 50 nig O3 per minute per liter, resulting in an ozone treatment time of about 6 hours. Results of the ozone treatment of the centrate are shown in FIG. 1, with the initial value corresponding to a sample taken at 0.5 hours, the middle value corresponding to a sample taken at 2.5 hours, and the final value corresponding to a value taken at 5 hours.

[0051] As show in FIG. 1 , the acetic acid concentration was maintained substantially constant throughout the treatment, but the malic acid content dropped from over 1600 mg/L to less than 50 mg/L in 2.5 hours. These results indicated that the saturation time for ozone in the centrate treated in the bubble column reactor was about 2 hours.

[0052] Light (natural diurnal), dissolved oxygen (greater than 2.0mg/L), temperature (about 25°C), and pH (about 7.5) conditions were identical for all treatments. All cultures were grown in outdoor, open pond bioreactors. The cell dry weight, bacteria count, residual acetic acid level, residual malic acid level, nitrogen concentration, and dissolved oxygen concentration were monitored for the cultures. Results are provided in Tables 1-7.

Table I

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

[0053] As shown in Tables 1-7, the third treatment comprising 100% municipal ("city") water initially and ozone treated centrate after partial harvests demonstrated the greatest dry weight values and growth rate. This demonstrated that the use of recycled culture media in a mixotrophic Chlorella culture can achieve comparable growth to a culture only using municipal water.

Example 2

[0054] An experiment was performed to determine the effectiveness of using recycled culture media from mixotrophic cultures of Chlorella in subsequent cultures of Chlorella. The mixotrophic culture conditions for each culture comprised of a supply of light and a supply of acetic acid as the organic carbon source. The first culture media treatment in the experiment comprised using 100% water from the municipal supply (City of Gilbert, Gilbert, Arizona USA) to serve as an untreated control for the initial inoculum media and the media added to increase volume after partial harvests. The second culture media treatment in the experiment comprised using a 50%/50% mixture of water from the municipal supply and the filtered centrate of culture media from a previous culture for the initial inoculum media and the media added to increase volume after partial harvests. The third culture media treatment in the experiment comprised using a 50%/50% mixture of water from the municipal supply and the filtered centrate of culture media from a previous culture that was also subjected to ozone treatment after filtration for the initial inoculum media and the media added to increase volume after partial harvests. Two replicates of each treatment were performed in this experiment.

[0055] The three culture treatments were operated in a fed-batch operation with partial harvests. The cultures were inoculated at 1 g/L, and partially harvested when the culture density reached about 5 g/L to reduce the culture density to about 3 g/L. Light (natural diurnal), dissolved oxygen (greater than 2.0mg/L), temperature (about 25°C), and pH (about 7.5) conditions were identical for all treatments. The filtration treatment comprised filtering the culture media through a 0.1 micron membrane filter (Graver Technologies, Glasgow, Delaware USA). The previous Chlorella culture had been in an open outdoor reactor for 19 days prior to filter treating the culture media for this experiment. The ozone treatment comprised applying ozone to the culture media in a bubble column container. The ozone was supplied by a Model CD2000P ozone generator (ClearWater Tech LLC, San Luis Obispo, California USA), set to produce 30 g/hour of ozone and a target cycle time of 50 mg O₃ minutes/liter, resulting in an ozone treatment time of about 4-6 hours. The acetic acid, malic acid, and bacteria content of the culture media from a previous Chlorella culture were quantified before and after ozone treatment, and the results are shown in Table 8. Table 8

[0056] All cultures were grown in outdoor, open pond bioreactors. The cell dry weight, bacteria count, residual acetic acid level, residual malic acid level, nitrogen concentration, and dissolved oxygen concentration were monitored for the cultures. Results are shown in FIGS. 9-1 1.

[0057] As show in Tables 9-11 , below, the residual acetic acid consumption and accumulation rates were similar for the 100% municipal water and the 50% municipal water/50% filtered- ozoned treatments. The malic acid concentration started higher in the 50% municipal water/50% filtered treatment and spiked, while the malic acid in the treatment that also recei ved ozone treatment stayed comparable to the 100% municipal water treatment. Bacterial contamination increased until dominance in the 50% municipal water/50% filtered treatment at about day 8, while the other treatments made it to about day 11. The mean growth rate (g/L/day) for each treatment over the first 10 days was 1.06 g/L/day for the 100% municipal water, 0.794 g/L/day for the 50% municipal water/50% filtered-ozoned, and 0.78 for the 50% municipal water/50% filtered. This demonstrated that the use of recycled culture media that has been subjected to both filtration and ozonation in a mixotrophic CnioreUa culture can achieve comparable growth to a culture only using municipal water, and resulted in better growth than a one stage treatment consisting of filtration only.

Table 9

Table 10

Table 11

Example 3

[0058] An experiment was performed to determine the effectiveness of the serial recycling of culture media from mixotrophic cultures of ^'Chlorella in subsequent cultures of Chlorella. The mixotrophic culture conditions for each culture comprised of a supply of light and a supply of acetic acid as the organic carbon source. Throughout the experiment, a treatment comprising 100% water from the municipal supply (City of Gilbert, Gilbert, Arizona USA) to serve as an untreated control for the initial inoculum media and the media added to increase volume after partial harvests for comparison to the recycled media treatments. The plan for the other treatments was to: 1) start with a culture inoculated into at treatment comprising ! 00% water from the municipal supply, 2) grow the culture from a density of 1 g/L to 6 g/L, 3) conduct a partial harvest to reduce the culture density to 3 g/L, 4) growth the culture to a density of 6 g/L and harvest the entire culture using a Cena z41 centrifuge (Eppendorf, Hamburg, German7), 5) separate the culture media from the microalgae cells using a 0. 1 micron membrane filter (Graver Technologies, Glasgow, Delaware USA), 6) treating the centrate with ozone in a bubble column container for 3 hours, 7) and inoculating filtered and ozonated centrate in a new- culture as a 50% blend with municipal water, 8) and repeat steps 2-7 six times. Two replicates of each culture were performed for this experiment,

[0059] The cultures were inoculated at 1 g/L. Light (natural diurnal), dissolved oxygen (greater than 2.0mg/L), temperature (about 25°C), and pH (about 7.5) conditions were identical for all treatments. The ozone treatment comprised applying ozone to the culture media in a bubble column container. The ozone was supplied by a Model CD2000P ozone generator (ClearWater Tech LLC, San Luis Obispo, California USA), set to produce 30 g/hour of ozone and a target cycle time of 50 mg O₃ minutes/liter. All cultures were grown in outdoor, open pond bioreactors. The cell dry weight, bacteria count, residual acetic acid level, residual malic acid level, nitrogen concentration, and dissolved oxygen concentration were monitored for the cultures.

[0060] The acetic acid, malic acid, and bacteria content of the centrate from the fifth and six cultures were quantified before and after ozone treatment, and the results are shown in Table 12.

Table 12

[0061 ] As shown in Table 1 1, the ozone treatment for cultures 5 and 6 demonstrated the ability of the method to maintain acetic acid concentration, and reduce malic acid concentration and bacteria counts. The reduction in bacteria was at least two orders of magnitude for each treatment. The acetic acid concentration was within 100 mg/L for all treatments. The reduction in malic acid for treatments 5A and B were at least 90% and at least 67%, The oxidation reduction potential (ORP) for the recycled centrate is shown in Table 13

Table 13

|0062] The growth rate results are shown in Table 14.

Table 14

[0063] The results of the experiment showed that recycled media outperformed the control in cultures 3, 5 and 7. The cultures using recycled media had higher bacteria count and a higher ratio of bacteria to microalgae. The cultures using recycled media had higher nitrate levels than the control in cultures 2-4, but were similar in cultures 5-6. Acetic acid levels were higher for the cultures using recycled media in cultures 5-7. Malic acid levels were consistently lower in the control cultures.

Example 4

[0064] To exemplily application of certain methods of the invention with other microalgae strains the following experiment was performed with microalgae that was characterized as belonging to the genus Schizochytrium.

Schizochytrium Treatments:

3. Control flasks that had only fresh growth media made in sterile water with lx growth

nutrients

2. Flasks that had 50% ozonated growth media or 50% cola waste water (v/v) with lx growth nutrients added (the cola waste water was obtained from a major cola soda manufacturer located in the United States)

3, Flasks that had 75% ozonated growth media or 75% cola wastewater (v/v) with 1 x growth nutrients added

[0065] Results:

* Both cola waste and spent Schizochytrium bioreactor (pond) media water were effectively ozonoated. See Table 15 (oxidation reduction potential/ORP data), below.

Table 15. Oxidation-reduction potential (ORP) as a function of ozonation process time.

• Cola waste syrup was ozonated without any ioss of sugars. Pre-treatment and post-treatment glucose levels were 246 g/L and 249 g/L. respectively, reflecting essentially no loss (1.2% loss, below a 5% loss, 2.5% loss, or even 2% loss) of glucose.

• In addition, acetate loss in the Schizochymum spent water was very low. Specifically, pre- treatment and post-treatment acetate levels were 1090.5 and 1030, respectively (reflecting a loss of 5.5%, which is well below a 15%, 10%, or 7.5% loss of acetate).

[0066] Schizochytrium was regrown in spent water (water obtained from media once used for growth) after it was ozonated at 50% and 75% strength (v/v). The 50% strength ozonated treatment performed well as compared to the control that had no ozonated water. Similarly, 50% strength cola wastewater treated Schizochymum performed at part with the control that had no cola waste. The actual growth results from this portion of the experiment are reflected in Tables 16 and 17, below.

[0067] Table 16. Schizochytrium growth (dry weight) in response to strength of ozonated spent water (50% and 75%, v/v) as a function of run time.

[0068] Table 17. Schizochytrium growth (dry weight) in response to strength of cola waste water (50% and 75%, v/v) as a function of run time.

Example 5

[0069] The following is a predictive experiment that can be performed with microalgae of the genus ozone Scenedesmus spent water (along with nutrients) with a goal of recycling the water and nutrients to reuse in Scenedesmus cultures.

Planned Treatments:

[0070] The experimental procedure that will be carried out is expected to be as follows:

• Fill reactors with water source as directed in treatments,

• Confirm < 1 ppm free chlorine and 0 ppm ozone before adding media components and algae.

• Grow cultures for 5 days (to approximately 6g/L) under mixotrophic conditions with acetate as a carbon source. 50% Partial Harvest on Day 5. Volume up with the same water blend used during inoculation. Grow cultures for 5 days (to approximately 6g/L)

• Centrifuge 400L of MV 1-4 culture on the Cena Z41 Eppendorf centrifuge. Discard algae and save centrate. Store at room temp overnight. Ozone 100L of cen Irate following for three hours. Record start and end times and take pictures. Collect ORP data.

• Sample each batch for petrifilm (2mL), acetic and malic acid (2mL), and 50mL for DW at end of experiment.

• Plate petrifilms and turn in other samples.

• Store at 5 degrees C until next inoculation.

Set Points: Not all parameters are controlled in each unit.

Expected Results:

[0071] Table 18. An example of oxidation-reduction potential (ORP) as a function of ozonation process time.

[0072] Table 19. An example of anticipated Scenedesmus sp. growth (dry weight) in response to strength of ozonated spent water (50% and 75%, v/v) as a function of run time.

[0073] Table 20, An example of residual acetic acid and malic acid amounts in pre and post ozonation spent water obtainable from Scenedesmus ponds.

Aspects of the Invention

[0074] In one non-limiting embodiment, a method may comprise: a) Centrifuging a microaigae culture comprising culture media, bacteria, and a population of microaigae cells, to separate the microaigae cells from a centrate comprising a first concentration of bacteria and culture media, wherein the culture media comprises a first concentration of malic acid and a first concentration of acetic acid; b) filtering the centrate; and c) treating the filtered centrate with ozone for an effective amount of time to produce a treated medium with a second concentration of malic acid lower than the first concentration of malic acid, a second concentration of bacteria lower than the first concentration of bacteria, and a concentration of acetic acid substantially the same as the first concentration of acetic acid. In some embodiments, the microaigae may comprise Chlorella.

[0075] In some embodiments, the effecti v e amount of time the filtered centrate is treated with ozone may be at least one hour. In some embodiments, the effective amount of time the filtered centrate is treated with ozone may be in the range of 1-6 hours. In some embodiments, the second concentration of malic acid may be at least 67% less than the first concentration. In some embodiments, the second concentration of bacteria may be at least two orders of magnitude lower than the first concentration. In some embodiments, the method may further comprise inoculating microaigae cells in a first subsequent culture with a first recycled culture medium comprising at least 50% of the treated medium.

[0076] In some embodiments, the method may further comprise repeating the steps of centrifuging, filtering, and treating with ozone the filtered centrate from the first subsequent culture, and inoculating microaigae cells in a second subsequent culture with a second recycled culture medium comprising at least 25% of the treated medium. In some embodiments, the method may further comprise repeating the steps of centrifuging, filtering, and treating the filtered centrate with ozone from the second subsequent culture, and inoculating microaigae cells in a third recycled culture medium comprising at least 25% of the treated medium. In some embodiments, the method may further comprise repeating the steps of centrifuging, filtering, and treating the filtered centrate from a subsequent culture with ozone, and inoculating microaigae cells in a further subsequent culture with a recycled culture medium comprising at least 25% of the treated medium up to five times without reducing the microaigae growth rate in dry weight grams per liter per day more than 20% as compared to a control culture with 100% newly prepared culture media that has not be treated or recycled.

[0077] In some embodiments, the microaigae cells may be inoculated in a culture comprising at least 50% of the treated medium and the remainder comprising fresh culture media. In some embodiments, the first recycled medium may comprise at least 75% of the treated medium. In some embodiments, the first recycled culture medium may comprise 100% of the treated medium. In some embodiments, the centrate may be filtered with a membrane filter. In some embodiments, the membrane filter may have a pore size of 0.1-0.5 microns. In some embodiments, an oxidation reduction potential of the centrate may be maintained in the range of 800-900 mV during treatment with ozone. In some embodiments, the filtered centrate may be treated with an amount of ozone in the range of 55 to 1 1 1 mg of ozone per liter of centrate.

[0078] In another non-limiting embodiment, a composition may comprise: a population of microalgae cells; and a culture media comprising a least one of acetic acid and acetate, and at least 25% recycled culture media from a previous microalgae culture, wherein the recycled culture media has been filtered and treated with ozone. In some embodiments, the microalgae may comprise Chlorella. In some embodiments, the composition may further comprise at least some bacteria.

[0079] In another non-limiting embodiment, a method of reducing the addition of fresh media and nutrients to a non-axenic culture of microalgae may comprise: a) providing a culture of microalgae comprising a supply of at least one of acetate or acetic acid as an organic carbon source, a population of microalgae cells, and at least some bacteria in a culture medium comprising water, nutrients, malic acid, and acetic acid; b) centnfuging the culture to separate the microalgae cells from a centrate comprising a first concentration of bacteria and centrifuged culture media, wherein the centrifuged culture media comprises a first concentration of malic acid and a first concentration of acetic acid; c) filtering the centrate to form a filtered centrate; d) treating the filtered centrate with ozone for an effective amount of time to produce a treated medium with a second concentration of malic acid lower than the first concentration of malic acid, a second concentration of bacteria lower than the first concentration of bacteria, and a concentration of acetic acid substantially the same as the first concentration of acetic acid; e) inoculating microalgae cells in a first subsequent culture supplied with at least one of acetate or acetic acid with a first recycled culture medium comprising at least 25% of the treated medium and the remainder comprising of freshly prepared culture media; and f) supplying less of at least one of acetate and acetic acid to the first subsequent culture than a control culture supplied with at least one of acetate or acetic acid would receive which comprises 100% freshly prepared culture media. In some embodiments, the microalgae may comprise Chlorella.

[0080] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were indi vidually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein.

[0081] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0082] Unless otherwise stated, ah exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplar}' values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate). All provided ranges of values are intended to include the end points of the ranges, as well as values between the end points.

[0083] The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having," "including," or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of, "consists essentially of, or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0084] All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

[0085] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [0086] The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

[0087] This invention includes ah modifications and equivalents of the subject matter recited in the claims and/or aspects appended hereto as permitted by applicable law.

References:

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* Chen, et al. Employment of organic acids to enhance astaxanthin formation in

heterotrophic Chlorella zofingiensis. Journal of Food Processing and Preservation. 33 (2009) 271-284

* US201 10129906

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* US20130244310

Claims

CLAIMS What is claimed is:

1. A method comprising:

a. Centrifuging a microalgae culture comprising culture media, bacteria, and a population of microalgae cells, to separate the microalgae cells from a centrate comprising a first concentration of bacteria (BC) and culture media, the culture media comprising a first malic acid concentration (MAC) and/or a first hexose concentration (HC);

b. Filtering the centrate; and

c. Treating the filtered centrate with ozone for an effective amount of time to produce a treated medium characterized in having a second concentration of bacteria lower than the first concentration of bacteria and (a) a second MAC that is lower than the first MAC, (b) a second HC that is substantially the same as the first HC, or (c) a second MAC that is lower than the first MAC and a second HC that is substantially the same as the first HC,

2. The method of claim I, wherein the culture comprises a first acetic acid concentration (AAC) in the medium before ozone treatment and the second AAC in the treated medium is substantially the same as the first AAC,

3. The method of claim 1 or claim 2, wherein the hexose is glucose.

4. The method of any one of claims 1-3, further comprising inoculating the microalgae cells in a first subsequent culture with a first recycled culture medium comprising at least 50% of the treated medium.

5. The method of any one of claims 1-4, wherein the effective amount of time the

filtered centrate is treated with ozone is at least 1 hour.

6. The method of claim 5, wherein the effective amount of time the filtered centrate is treated with ozone for up to 6 hours.

7. The method of any one of claims 1-6, wherein the second concentration of bacteria is at least two orders of magnitude lower than the first concentration.

8. The method of any one of claims 1-7, wherein the medium comprises malic acid and the second MAC is at least 67% less than the first MAC.

9. The method of any one of claims 1-8, further comprising repeating the steps of centrifuging, filtering, and treating with ozone the filtered centrate from the first subsequent culture, and inoculating microalgae cells in a second subsequent culture with a second recycled culture medium comprising at least 25% of the treated medium.

10. The method of claim 9, further comprising repeating the steps of centrifuging,

filtering, and treating the filtered centrate with ozone from the second subsequent culture, and inoculating microalgae cells in a third subsequent culture with a third recycled culture medium comprising at least 25% of the treated medium.

1 1. The method of claim 9, further comprising repeating the steps of centrifuging,

filtering, and treating the filtered centrate from a subsequent culture with ozone, and inoculating microalgae cells in a further subsequent culture with a recycled culture medium comprising at least 25% of the treated medium up to five times without reducing the microalgae growth rate in dry weight grams per liter per day more than 20% as compared to a control culture of microalgae with 100% newly prepared culture media that has not be treated or recycled.

12. The method of claim 9, wherein the microalgae cells are inoculated in a culture

comprising at least 50% of the treated medium.

13. The method of claim 12, wherein the first recycled culture medium comprises at least 75% of the treated medium.

14. The method of claim 13, wherein the first recycled culture medium comprises 100% of the treated medium.

15. The method of any one of claims .1 -14, wherein the centrate is filtered with a

membrane filter, w herein the membrane filter has a pore size of 0.1-0.5 microns.

16. The method of any one of claims 1-15, wherem an oxidation reduction potential of the centrate is maintained in the range of 800-900 mV during treatment with ozone.

17. The method of any one of claims 1-15, wherein the filtered centrate is treated with an amount of ozone in the range of 55 to 111 mg of ozone per liter of centrate.

18. The method of any one of claims 1 -17, wherein the microalgae comprises microalgae of the genus selected from Chlorella, Schizochyirium, and Scenedesmus.

19. A composition comprising:

a. A population of microalgae cells: and

b. Culture media comprising:

i. At least one of acetic acid and acetate; and ii. At least 25% recycled culture media from a previous microalgae culture, wherein the recycled culture media has been filtered and treated with ozone.

20. A method of reducing the addition of fresh media and nutrients to a non-axenic

culture of Chiorella:

a. Providing a culture of microalgae comprising a supply of at least one of acetate or acetic acid as an organic carbon source, a population of Chiorella cells, and at least some bacteria in a culture medium comprising water, nutrients, malic acid, and acetic acid;

b. Centrifuging the culture to separate the Chiorella cells from a centrate

comprising a first concentration of bacteria and centnfuged culture media, wherein the centrifuged culture media comprises a first concentration of malic acid and a fi rst concentration of acetic acid;

c. Filtering the centrate to form a filtered centrate;

d. Treating the filtered centrate with ozone for an effective amount of time to produce a treated medium with a second concentration of malic acid lower than the first concentration of malic acid, a second concentration of bacteria lower than the first concentration of bacteria, and a concentration of acetic acid substantially the same as the first concentration of acetic acid; e. Inoculating Chiorella cells in a first subsequent culture supplied with at least one of acetate or acetic acid with a first recycled culture medium comprising at least 25% of the treated medium and the remainder comprising of freshly prepared culture media; and

f. Supplying less of at least one of acetate and acetic acid to the first subsequent culture than a control culture supplied with at least one of acetate or acetic acid would receive which comprises 100% freshly prepared culture media.