WO2018053071A1 - Methods of treating wastewater with microalgae cultures supplemented with organic carbon - Google Patents

Abstract

Method of treating wastewater using mixotrophic or heterotrophic cultures of microalgae supplemented with at least one of acetic acid and acetate for increased efficiency in removing substances such as nitrogen, phosphorus, and heavy metals from the wastewater are disclosed herein.

Description

BACKGROUND

[0001] This patent application claims priority to US Provisional Patent Application 62/395,722, filed September 16, 2016, the entirety of which is hereby incorporated by reference.

BACKGROUND

[0002] Microalgae may be used in a variety of applications based on the function of the microalgae. The function of microalgae provides opportunities in wastewater treatment and bioremediation through the ability to utilize components of the waste (e.g., carbon dioxide, nitrogen, phosphorus) as nutrients for growth and metabolite production, and to sequester unwanted elements or high concentrations of elements (e.g., nitrogen, phosphorus, heavy- metals) from liquid mediums for removal. To supply demand for clean water and bioremediation markets, large volumes of microalgae must be grown quickly and at a reasonable cost, or operate at an efficiency level that reduces the residence time for wastewater treatment. Additionally, microalgae resulting from wastewater treatment processes may provide a potentially usable product for markets such as fuels, chemicals, and agricultural products, in addition to the function of remediating waste.

[0003] Phototrophic methods of culturing microalgae traditionally use low cost equipment and provide microalgae with a product profile desired for multiple applications, but do not produce large volumes of biomass at fast rates or remediate water at a high efficiency (e.g., long residence times from low metabolic activity, and large surface area requirements due to light path restrictions on culture depth) and hence lead to large land area requirements. Phototrophic methods are also limited in function and application because the phototrophic operations may be hindered in wastewater with high biochemical oxygen demand (BOD). Also, in phototrophic conditions the net balance of carbon dioxide is negative and requires the carbon dioxide to be externally supplied for any metabolic activity. Additionally, phototrophic methods of culturing microalgae depend on numerous variable drivers in order to assimilate inorganic carbon (e.g. , carbon dioxide), including availability of photosynthetically active radiation (PAR) (i.e., light path) which limits the culture depth and therefore the volume in a given surface area. PAR must also be provided artificially at night to avoid cell respiration, winch adds cost to outdoor cultivation, or else inorganic carbon cannot be assimilated and metabolic activity may be inhibited.

[0004] Mixotrophic and heterotrophic methods of culturing rnicroalgae provide numerous benefits such as higher growth and reproduction rates than phototrophic methods, while providing the most flexibility in the bioremediation and wastewater treatment functions due to the ability to utilize organic carbon as an energy source.

[0005] Culturing rnicroalgae with an organic carbon supply also creates unique opportunities in the wastewater treatment and bioremediation industries, as mixotrophic and heterotrophic rnicroalgae cultures are able to grow quickly and consume larger quantities of nutrients from waste mediums in varying conditions than phototrophic cultures. The balance of fast growth with the aid of an organic carbon source creates the opportunity to uptake increased quantities of elements from the waste medium (e.g., metals, nitrogen, phosphorus, carbon dioxide), and produce a biomass or lipid product for fuel, chemical, and agricultural markets.

SUMMARY

[0006] Embodiments of methods of treating wastewater using mixotrophic or heterotrophic cultures of rnicroalgae supplemented with organic carbon for increased efficiency in removing substances such as nitrogen, phosphorus, and heavy metals from the wastewater. In some embodiments, the organic carbon may comprise at least one of acetate and acetic acid. The organic carbon supplemented cultures of rnicroalgae utilized for treating wastewater may result in benefits over phototrophic cultures of rnicroalgae treating wastewater such as, but not limited to: reducing the residence time for wastewater treatment, increasing the surface to volume ratio of the culture treating the wastewater, reducing the flocculation of the rnicroalgae during the wastewater treatment, reducing the evaporation of ammonium during the wastewater treatment, and reducing the ratio of bacteria to rnicroalgae in the culture.

DESCRIPTION OF THE INVENTION

Definitions

[0007] The term "rnicroalgae" refers to microscopic single cell organisms such as rnicroalgae, cyanobacteria, algae, diatoms, dinoflageiattes, freshwater organisms, marine organisms, or other similar single ceil organisms capable of growth in phototrophic, mixotrophic, or heterotrophic culture conditions. fOOOS] The term "mixotrophic" or "mixotrophy" refers to culture conditions in which light, organic carbon, and inorganic carbon (e.g., carbon dioxide) may be applied to a culture of microalgae. Microalgae capable of growing in mixotrophic conditions have the metabolic profile of both phototrophic and heterotrophic microalgae, 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 microalgae 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. Microalgae in mixotrophic culture conditions may be a net oxygen or carbon dioxide producer depending on the energy source and carbon source utilized by the microalgae. Microalgae capable of mixotrophic growth comprise microalgae with the natural metabolism and ability to grow in mixotrophic conditions, as well as microalgae which obtain the metabolism and ability through modification of cells by way of methods such as mutagenesis or genetic engineering.

|0009] The term "phototrophic" or "phototrophy" refers to culture conditions in which light and inorganic carbon (e.g., carbon dioxide) may be applied to a culture of microalgae. Microalgae capable of growing in phototrophic conditions may use light as an energy source and inorganic carbon (e.g., carbon dioxide) as a carbon source. Microalgae in phototrophic conditions may produce oxygen. The term photoautotrophic is also used synonymously with phototrophic.

[0010] The term "heterotrophic" or "heterotrophy" refers to culture conditions in which organic carbon may be applied to a culture of microalgae in the absence of light. Microalgae capable of growing in heterotrophic conditions may use organic carbon as both an energy source and as a carbon source. Microalgae in heterotrophic conditions may produce carbon dioxide.

[0011 ] Non-limiting examples of microalgae capable of growing in mixotrophic and heterotrophic culture conditions may comprise: Agmenellum quadruplica um. Amphora coffeae.form.is, Anabaena variabilis, Anacystis niduians, Apistonema sp., Pleurochyrsis sp., Arthrospira (Spirulina), Aurantiochytrium sp., Botryococcus sp., Brachiomonas sp., Chlamyd.om.onas sp., Chlorella sp., Chloroccum sp., Cruciplacolithus neohelis, Cylindrotheca fusiformis, Coenochloris fottii, Cyanophora paradoxa, Cyclotella, Dunaliella, Emiliania hux!eyi, Euglena gracilis, Extubocellu!us spinifer, Fragilaria pinnata, Galdieria sulphuraria, Goniotrichium elegans, Haematococcus pluvialis, Halochlorella rubescens, Isochyrsis galbana, leptocylindrus danicus, Micractinium sp., Melosira octagona c , Monodus subterraneus, Nostoc sp., Nannochloris sp., Nannochloropsis sp., Navicula sp., Neosongiococcum, Nitzschia sp., Odontella aurita, Ochromonas sp., Ochrosphaera neapolitana, Pavlova hither i. Picochlorum sp., Phaeodactylum tricornutum, Pleurochyrsis carterae, Porphyridium omentum, Poteriochromonas malhamensis, Prymnesium parvum, Rhodomonas sp., Schizochytrium sp., Scenedesmus sp., Skeleionema menzelii, Spumetta sp., Stauroneis, Stichococcus sp., Auxenochlorella protothecoid.es, Cheatoceros sp., Neochloris oleabundans , Nespongiococcum sp.. Ocromon s neapolitana, Porphyridium sp.. Synechococcus sp., Synechocysti sp., Tetraselmis sp., Thraustochytrids sp., and Thalassiosira wessflogii.

[0012] Organic carbon sources 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, wastewater from sewage treatment plants, liquid waste from terrestrial animal production systems, liquid waste from bio-digesters, yeast extract, and combinations thereof. Acetate and acetic acid are known to have toxic effects on organisms, including bacteria, at certain concentrations in a culture media, and as such may provide an additional benefit to a culture of microalgae above functioning as a supply of organic carbon.

[0013] Non-limiting examples of suitable microalgae for mixotrophic or heterotrophic growth using acetate or acetic acid as an organic carbon source may comprise organisms of the genera, but are not limited to: Ch!orel!a, Anacystis, Synechococcus, Synechocystis, Neospongiococcum, Chlorococcum, Phaeodactylum, Spirulina, Micractinium, Haematococcus, Nannochloropsis, Brachiomonas, Schizochytrium, Aurantiochytrium, Scenedesmus, and species thereof. Gesieral culturing, harvesting ara extraction

[0014] Microalgae cultured in mixotrophic or heterotrophic conditions may be produced at a high rate while maintaining a product profile suitable for a wide variety of commercial applications. In a mixotrophic culture of microalgae, the microalgae are cultured in an aqueous culture medium contained in a bioreactor with a supply of both an organic carbon source and inorganic carbon source, and at least some light. In a heterotrophic culture of microalgae, the microorganisms are cultured in an aqueous culture medium contained in a bioreactor with a supply of an organic carbon source and inorganic carbon source, but either without light or without the capability for the microalgae to utilize light as an energy source. The bioreactor may comprise any vessel known in the art suitable for microorganism culturing such as, but not limited to, a pond, a raceway pond, a flat panel bioreactor, a tubular bioreactor, a tank, a trough, a fermenter, column bioreactor, and a bag. The microalgae cultures may be mixed by any method known in the art such as, but not limited to, aerators, pumps, paddlewheels, submerged thrusters, and the like.

[0015] The microalgae may utilize organic carbon as an energy source and carbon source for growth and the production of carbon dioxide using the heterotrophic metabolism. A mixotrophic microalgae may also use light as an energy source and inorganic carbon (e.g., residual carbon dioxide, respirated carbon dioxide) as a carbon source for growth and the production of oxygen. The organic carbon may be supplied in batch, fed-batch, semi- continuous, and continuous methods. In some embodiments, acetate or acetic acid may be supplied on demand using a pH auxostat system to provide a supply of organic carbon and control the pH of the culture. The light for mixotrophic cultures may be supplied by natural sources (e.g. solar radiation, sunlight), by artificial sources (e.g. light emitting diodes, fluorescent bulbs), continuously, discontinuous!}⁷, flashing, pulsing, constant intensity, varying intensity, and combinations thereof. Unwanted wavelengths of light may be excluded through filtering and blocking, and preferred wavelengths may be enhanced or provided by filtering, reflecting, or selectively tuning a source of light. In some embodiments, the supply of light may be increased or decreased based on the current supplied to artificial lights, or the activation and deactivation of artificial lights in part or in whole. In some embodiments, the supply of light may be increased or decreased based on the natural or artificial light that is blocked, such as by louvers, covers, awnings, shades, coatings, and sheathes. [0016] The microalgae may consume nutrient during culturing, such as organic carbon, inorganic carbon (e.g., carbon dioxide), oxygen, nitrogen containing compounds, nitrates, sulfates, phosphates, salts, and trace metals. Trace metals may comprise: Iron, Copper, Boron, Molybdenum, Zinc, Cobalt, Magnesium, Manganese, and combinations thereof. In some embodiments, the nutrients may be in a concentrated form. In some embodiments, the nutrients may be in a diluted form or supplemented in the culture medium. In some embodiments the nutrients may be modified for a particular microalgae species. In some embodiments the nutrients may be modified depending on the microalgae culture lifespan, water source, environmental conditions, and reactor design. In some embodiments, probiotic mixtures of beneficial live microorganisms may be added to maintain the balance of microalgae in the culture. In some embodiments, vitamins such as vitamin Bn (cobalamin), vitamin Bi (thiamine), and vitamin B? (biotin) may or not be included in the nutrient mixes.

[0017] In some embodiments, culture parameters such as, but not limited to, pH, temperature, dissolved oxygen, dissolved carbon dioxide, and flow rate, may be monitored and controlled by any method known in the art such as, but not limited to, sensors and programmable logic control systems. In some embodiments the pH, dissolved oxygen, temperature may be controlled within a tight range (e.g., less than 5% of a set point), and in some embodiments the pH, dissolved oxygen, temperature may be controlled within a large range (e.g., greater than or equal to 5% of a set point). In alternate embodiments, a hybrid operation may be used, where large fluctuations (e.g., greater than or equal to 5% of a set point) may be intermittent between times of tight control (e.g., less than 5% of a set point). The culture parameters, nutrients, and organic carbon source in culture conditions may vary between the microalgae species cultured.

[0018] The microalgae may be harvested from the aqueous culture by any known method and equipment such as, but not limited to, centrifuge, an electrodewatering device, fiocculation, membrane, filter, settling, skimming, and dissolved air flotation. In some embodiments, the whole microalgae may be used in a product or method. In some embodiments, the harvested microalgae may be further processed to extract products such as, but not limited to, proteins, lipids, pigments, antioxidants, polysaccharides, hormones, recombmantly produced products, and extracted biomass. In some embodiments, at least one product may be extracted from the mi croalgae for use in a product or method. Extraction methods may include any method known in the art for extracting products from microalgae such as, but not limited to, solvent extraction, lysing, grinding, pressing, milking, electrical field application, electroporation, enzymatic, sonic, ultrasonic, osmotic pressure, and supercritical fluid (e.g., carbon dioxide) methods.

|0019] As described throughout the specification, microalgae may experience different phases over the life of a culture. The terms growth phase and oil phase are commonly used in the art of microalgae culturing. In a growth phase, the culture of microalgae experience cell division and an increase in the cell density of a culture. Microalgae in a growth phase may comprise a product profile rich in protein, but also comprise lipids and pigments. In an oil phase, the culture of microalgae may experience stress, such as a reduction in nitrogen, an increase in temperature, or a change in some other environmental parameter, and the rate of growth and cell division may be reduced as the microorganism increases the accumulation of lipids. Microalgae in an oil phase may comprise a product profile rich in lipids, but also comprise protein and pigments.

General methods of mixotrophic microalgae production

[0020] Methods of producing microalgae by mixotrophic production methods that can be applied with the inventive embodiments described herein in the production of microalgae are known in the art. Representative of such methods are those described in International (PCTj Patent Application WO 2014/074769, the entirety of which is hereby incorporated by reference, and which can be performed with the Chlorell microalgae described therein as well as in other mixotrophic microalgae organisms.

General cultivation of microorganisms in wastewater

[0021] Wastewater may come from a variety of sources, such as: municipal sewage, agriculture livestock operations (e.g., swine, cattle, poultry), agriculture crop runoff, food processing operations, mining operations, slaughterhouse operations, landfill leachate, power plants, industrial operations (e.g., carpet manufacturing), and effluent from anaerobic or aerobic digestion. Each source of wastewater may comprise a different composition of constituents such as, but not limited to, salts, alkalis, minerals, dyes, colorants, nitrates, phosphates, ammonia, metals/metalloids (e.g., copper, uranium, cobalt, chromium, cadmium, arsenic), metal ions, minerals, carbohydrates, organic carbon, inorganic carbon, bacteria, microorganisms, and other organic molecules. Also, the composition of wastewater from the same source may fluctuate over time, varying the type and quantity of constituents. [0022] Using wastewater as a culture medium for microaigae has its own set of challenges that must be accounted for when designing a culturing method. One challenge may be that the wastewater may have a high level of biochemical oxygen demand (BOD), which may inhibit the population growth of aerobic microorganisms such as phototrophic microaigae if the oxygen produced by the microaigae during the day cannot satisfy the demands of the wastewater and respiration activity of the microaigae. Another challenge may be that the inconsistent composition of the wastewater may not always provide the correct profile of nutrients for the microorganisms, resulting in stressing the microorganisms in the form of organic shock. Additional competition from other microorganisms and bacteria in the wastewater for resources when culturing a specific strain or consortium of strains of microaigae should also be considered when designing a culturing method that comprises wastewater. The pH of the wastewater medium may also have to be adjusted to be conducive to microaigae of the culture, depending on the source of the wastewater and the species of microaigae.

[0023] In the art, microaigae have been used as a secondary treatment for wastewater. Typically, the wastewater first goes through a primar ' treatment stage comprising UV sterilization, filtration, sedimentation, aerobic digestions, anaerobic digestion, activated sludge treatment, or combinations thereof. The output from the primary treatment stage may be used as a culture medium for microaigae in the secondary treatment stage where constituents of the wastewater are removed by the microaigae through uptake, assimilation, and consumption. The microaigae may then be separated from the aqueous medium and further processed for applications such as methane production, fertilizers, and biofuels through methods such as, but not limited to, extraction, esterifi cation, transesterification, hydrotreatment, and pvrolysis. The treated aqueous medium may go through a tertiary treatment stage, such as UV sterilization, to further condition the aqueous medium for another use or purpose. Microaigae may also be used in treatment methods for untreated wastewater and diluted wastewater.

[0024] Microaigae used in a secondary treatment may comprise microaigae strains prospected from the wastewater source or may comprise a genus known to proliferate in wastewater conditions such as, but not limited to Chlorella, Micraciinium, Chlamydomonas , Chlorococcum, and Scenedesmus. Microaigae cultured in wastewater may be cultured as a monoculture comprising a single strain or species, or as a consortium of at least two strains or species cultured together. Some species of microaigae have also been found to assimilate or uptake more constituents (e.g., phosphorus, metals) tha the microorganism may consume (i.e., luxury consumption) which may be particularly advantageous in wastewater treatment and bioremediation applications. Microalgae cultures in wastewater may also be supplemented with nutrients or organic carbon to facilitate metabolic activity. The wastewater treatment or bioremediation method utilizing microalgae may comprise a batch, semi-continuous, or continuous culturing method, and may be cultured in any bioreactor known in the art such as, but not limited to, flasks, ponds, raceway ponds, tubular bioreactors, flat panel bioreactors, bags, rotating wheel bioreactors, algal turf surfaces, lagoons, column bioreactors, tanks, and troughs.

[0025] The pH of microalgae cultures utilizing wastewater are known to be controlled using carbon dioxide or acetic acid. Carbon dioxide supplied to any microalgae culture, including cultures providing wastewater treatment, in phototrophic or mixotrophic culturing conditions may be supplied by sparging or surface contact from sources such as air, sequestered carbon dioxide, flue gases, stack gases, fossil fuel combustion, exhaust, and waste gases from anaerobic digestion, furnaces, boilers, and steam generators. Some combustion, exhaust, waste, flue or stack gases may comprise carbon dioxide, nitrogen, water vapor, oxygen, particulate matter, nitrogen oxides, and sulfur oxides. When combustion, exhaust, waste, flue or stack gases are used in a microalgae culture, the treatment with microorganisms provides a bioremediation function by utilizing some constituents of the gases (e.g., carbon dioxide, oxygen, nitrogen oxides, sulfur oxides) and producing oxygen or carbon dioxide with fewer impurities. The bioremediated gases exiting the microalgae culture may be released into the atmosphere or captured for further processing or use.

[0026] Oxygen may also be supplied to microalgae cultures operating in mixotrophic or heterotrophic conditions. In some embodiments, the oxygen may be supplied through the sparging of an oxygen containing gas (e.g., air) into the aqueous culture medium, infusion from aerators, and combinations thereof. In some embodiments, the amount of oxygen dissolved in the aqueous culture medium may be enough to maintain a desired level of metabolic activity in the microorganisms. In some embodiments, the supply of oxygen and carbon dioxide may be supplied by the mixotrophic microalgae themselves instead of through an external source. When the mixotrophic microalgae use their phototrophic and heterotrophic metabolisms in a balanced manner, the microorganisms may produce both oxygen and carbon dioxide for consumption by the microorganisms within the culture. [0027] In some embodiments, aerators are used to provide both infusion of gases (e.g., oxygen) into the aqueous microaigae culture and turbulent mixing of the microaigae culture. One non- limiting example of an aerator for use in a microaigae culture is the Aire-02® Series 275 Aspirator Aerator (Aeration Industries International, Chaska, MN USA). Such aerators comprise an electric motor drive above the culture media surface mounted on a float, a hollow shaft extending at an angle from above the culture media surface into the culture, and a propeller disposed at end of the shaft which is submerged within the culture media. The motor is coupled to and drives the shaft and propeller. The propeller thrusts the aqueous culture media past a diffuser at the end of the shaft to induce a pressure differential in the hollow shaft, drawing air through intake holes in the shaft above the culture media surface down through the rotating hollow shaft and diffuser into the microaigae culture. While the aerators contribute to the turbulent mixing and infusion of oxygen, the previously described devices and methods of supplying nutrients, supplying organic carbon, and controlling pH may be used in conjunction with such aerators.

[0028] Nutrient and energy requirements by microaigae, such as light, organic carbon, inorganic carbon, oxygen, nitrates, and phosphates, may vary depending on the species of the microaigae and nutrient source. The examples above provide non-limiting examples of the nutrient and energy requirements for culturing in phototrophic, mixotrophic, and heterotrophic culturing conditions. In some embodiments, the desired nitrogen level for a culture of microorganisms in a growth phase may be 200-2000 ppm of total nitrogen, with the desired nitrogen level for oil phase being less. In some embodiments, the desired phosphorus level may be 5 to 20 times less than the desired nitrogen level. In some embodiments, the desired level of organic carbon may comprise 0.1 to 5% of the culture medium, and may be supplied at a rate commensurate with the microorganisms' consumption rate. In some embodiments, the organic carbon supply rate may decrease when the microorganism culture density is greater than about 5-6 grams/liter.

Method of producing treated water

[0029] Microaigae may be used in the treatment of wastewater to produce a treated water composition that may be useable in a wide range of applications. W astewater used as a culture medium for microaigae may comprise an initial concentration of constituents such as nitrates, phosphates, organic compounds, and metals which need to be removed to repurpose the water. Wastewater from different sources, such as industrial, municipal, and agricultural sources, may have different types and levels of constituents.

|003θ] Microaigae in mixotrophic and heterotrophic culturing conditions (i.e., utilizing an organic carbon source as an energy source) have the ability to uptake, assimilate, and consume constituents in the wastewater culture medium, as well as grow at a fast rate and at a higher culture density than microorganisms in phototrophic culturing conditions. The faster growth rate and higher culture density of the microaigae receiving organic carbon may intensify and speed up the wastewater treatment process (i.e. , reduce residence time), thus producing a quantifiable advantage over the use of phototrophic culturing conditions. The microaigae may be separated from the treated wastewater medium after uptake, assimilation, and consumption of constituents to produce a treated water product with a reduced concentration of constituents. The microaigae may be separated from the treated water using any known harvesting device or method such as, but not limited to, dissolved gas flotation, filtration, membrane separation, centrifugation, and eiectrodewatering. Once separated from the microaigae, the treated water product may be used for a variety of purposes such as, but not limited to, agricultural irrigation, cooling system, washing, culture medium for mieroalgai biomass production, and the like.

[0031] Additionally, the use of mixotrophic or heterotrophic culture conditions provides another advantage over phototrophic culture conditions through the ability to grow in low or no light conditions, which reduces the importance of high surface to volume reactors. In phototrophic culturing conditions, a high surface to volume ratio for the photobioreactor may be necessary to ensure that the microaigae are exposed to a sufficient amount of light through mixing to provide energy for photosynthesis. With the ability of mixotrophic microaigae to utilize organic carbon as an energy source, the importance of light in the culturing process may be reduced and allow the surface to volume ratio of the bioreactor to be reduced. With a lower surface to volume ratio, the volume of the microaigae culture may be increased to allow for the treatment of larger volumes of wastewater in the same area! footprint and time frame. Also, with the reduced need for light exposure, the mixing in the bioreactor may not be dictated by exposing the microaigae to light and instead provide a mixing regime that makes gases and organic carbon available to the microaigae.

[0032] In some embodiments, a primary treatment method for treating the wastewater may precede the treatment of wastewater with microaigae. In some embodiments, the primary treatment method may comprise filtration, UV sterilization, aerobic digestion, and anaerobic digestion. In some embodiments, the treated water product resulting from the treatment with microalgae may be further treated in a tertiary treatment method. In some embodiments, the tertiary treatment method may comprise UV sterilization. In some embodiments, the wastewater treatment method may comprise supplying inorganic carbon (e.g. , carbon dioxide) to the culture of microalgae.

[0033] In some embodiments, the microalgae separated from the treated water product may be used in another wastewater treatment or bioremediation process. In some embodiments, the microalgae separated from the treated water product may be further processed in a downstream process such as extraction, esterification, transesterification, pyrolysis, and hydrotreatment to produce an extracted or treated product such as lipids, chemicals, or biofuels.

Fisrther Embodiments

[0034] In some embodiments, the wastewater to be treated by microalgae may comprise a nitrogen concentration below 500 ppm. In some embodiments, the wastewater to be treated by microalgae may comprise a nitrogen concentration below 400 ppm. In some embodiments, the wastewater to be treated by microalgae may comprise a nitrogen concentration below 300 ppm. In some embodiments, the wastewater to be treated by microalgae may comprise a nitrogen concentration below 200 ppm. In some embodiments, the wastewater to be treated by microalgae may comprise a nitrogen concentration below 100 ppm. In some embodiments, the wastewater to be treated by microalgae may comprise a nitrogen concentration below 50 ppm. In some embodiments, the nitrogen of the wastewater comprises nitrates. In some embodiments, the nitrogen of the wastewater comprises ammonium. In some embodiments, the nitrogen of the wastewater comprises ammonia. In some embodiments, the presence of ammonia may prevent the accumulation in the wastewater of acetate that occurs when microalgae consume NCb as a nitrogen source.

[0035] In some embodiments, the wastewater to be treated by microalgae may comprise a phosphorus concentration below 50 ppm. In some embodiments, the wastewater to be treated by microalgae may comprise a phosphorus concentration below 40 ppm. In some embodiments, the wastewater to be treated by microalgae may comprise a phosphorus concentration below 30 ppm. In some embodiments, the wastewater to be treated by microalgae may comprise a phosphorus concentration below 20 ppm. In some embodiments, the wastewater to he treated by microalgae may comprise a phosphorus concentration below 10 ppm. In some embodiments, the wastewater to be treated by microalgae may comprise a phosphorus concentration below 5 ppm.

[0036] In some embodiments, the wastewater to be treated by microalgae may comprise a concentration of solids below^" 10%. In some embodiments, the wastewater to be treated by microalgae may comprise a concentration of solids below 9%. In some embodiments, the wastewater to be treated by microalgae may comprise a concentration of solids below 8%. In some embodiments, the wastewater to be treated by microalgae may comprise a concentration of solids below 7%. In some embodiments, the wastewater to be treated by microalgae may comprise a concentration of solids below 6%. In some embodiments, the wastewater to be treated by microalgae may comprise a concentration of solids below 5%. In some embodiments, the wastewater to be treated by microalgae may comprise a concentration of solids below 4%». In some embodiments, the wastewater to be treated by microalgae may comprise a concentration of solids below 3%. In some embodiments, the wastewater to be treated by microalgae may comprise a concentration of solids below 2%. In some embodiments, the wastewater to be treated by microalgae may comprise a concentration of solids below 1%. In some embodiments, the wastewater to be treated by microalgae may comprise a concentration of solids below 0.5%.

|0037] In some embodiments, the wastewater to be treated may be supplemented with an organic carbon source to supply energy for microalgae in mixotrophic or heterotrophic conditions. In some embodiments, the wastewater to be treated may be supplemented with at least one of acetate and acetic acid as a supply of organic carbon at a concentration of less than 20 g/L. In some embodiments, the wastewater to be treated may be supplemented with at least one of acetate and acetic acid as a supply of organic carbon at a concentration of less than 15 g/L. In some embodiments, the wastewater to be treated may be supplemented with at least one of acetate and acetic acid as a supply of organic carbon at a concentration of less than 12 g/L. In some embodiments, the wastewater to be treated may be supplemented with at least one of acetate and acetic acid as a supply of organic carbon at a concentration of less than 10 g/L. In some embodiments, the wastewater to be treated may be supplemented with at least one of acetate and acetic acid as a supply of organic carbon at a concentration of less than 8 g/L. In some embodiments, the wastewater to be treated may be supplemented with at least one of acetate and acetic acid as a supply of organic carbon at a concentration of less than 6 g/L. In some embodiments, the wastewater to be treated may be supplemented with at least one of acetate and acetic acid as a supply of organic carbon at a concentration of less tha 4 g/L. In some embodiments, the wastewater to be treated may be supplemented with at least one of acetate and acetic acid as a supply of organic carbon at a concentration of less than 2 g/L,

[0038] In some embodiments, the supplemental at least one of acetate and acetic acid may be supplied as a batch at the beginning of the wastewater treatment process with microalgae. In some embodiments, the supplemental at least one of acetate and acetic acid may be supplied as a batch after the wastewater treatment process with microalgae has begun. In some embodiments, the supplemental at least one of acetate and acetic acid may be supplied continuously during the wastewater treatment process by the microalgae. In some embodiments, the supplemental at least one of acetate and acetic acid may be supplied as a batch multiple times during the wastewater treatment process by microalgae. In some embodiments, the at least one of acetate and acetic acid supplied to a culture of microalgae treating wastewater may be produced by a fermentation or digestion process (e.g., anaerobic or aerobic digestion of waste).

[0039] In some embodiments, the temperature of the microalgae culture treating wastewater may be in the range of 5 to 35 °C. In some embodiments, the temperature of the microalgae culture treating wastewater may be in the range of 5 to 10 °C. In some embodiments, the temperature of the microalgae culture treating wastewater may be in the range of 10 to 15 °C. In some embodiments, the temperature of the microalgae culture treating wastewater may be in the range of 15 to 20 °C. In some embodiments, the temperature of the microalgae culture treating wastewater may be in the range of 20 to 25 °C. In some embodiments, the temperature of the microalgae cuiture treating wastewater may be in the range of 25 to 30 °C. In some embodiments, the temperature of the microalgae culture treating wastewater may be in the range of 30 to 35 °C.

[0040] In some embodiments, the pH of the microalgae culture treating wastewater may be in the range of 5-1 1. In some embodiments, the pH of the microalgae culture treating wastewater may be in the range of 5-6. In some embodiments, the pH of the microalgae cuiture treating wastewater may be in the range of 6-7. In some embodiments, the pH of the microalgae culture treating wastewater may be in the range of 7-8. In some embodiments, the pH of the microalgae culture treating wastewater may be in the range of 8-9. In some embodiments, the pH of the microalgae culture treating wastewater may be in the range of 9-10. In some embodiments, the pH of the microalgae culture treating wastewater may be in the range of 10-11. In some embodiments, the pH of the microalgae culture treating wastewater may be in the range of 5- 8. In some embodiments, the pH of the microalgae culture treating wastewater may be in the range of 5-7.

[0041] In some embodiments, the salinity of the microalgae culture treating wastewater may be in the range of 0 to 60 ppt. In some embodiments, the salinity of the microalgae culture treating wastewater may be in the range of 0 to 10 ppt. In some embodiments, the salinity of the microalgae culture treating wastewater may be in the range of 10 to 20 ppt. In some embodiments, the salinity of the microalgae culture treating wastewater may be in the range of 20 to 30 ppt. In some embodiments, the salinity of the microalgae culture treating wastewater may be in the range of 30 to 40 ppt. In some embodiments, the salinity of the microalgae culture treating wastewater may be in the range of 40 to 50 ppt. In some embodiments, the salinity of the microalgae culture treating wastewater may be in the range of 50 to 60 ppt.

|0042] In some embodiments, the carbon dioxide may be bubbled or sparged into the microalgae culture treating wastewater. In some embodiments, air (i.e., gas containing carbon dioxide and oxygen) may be introduced into the microalgae culture treating wastewater with aearators. In some embodiments, the dissolved oxygen concentration of the microalgae culture treating wastewater may be in the range of 0 to 100% saturation or 0 to 12 mg O2/L. In some embodiments, the dissolved oxygen concentration of the microalgae culture treating wastewater may be maintained in the range of 0 to 1 mg O2/L. In some embodiments, the dissolved oxygen concentration of the microalgae culture treating wastewater may be maintained in the range of 1 to 2 mg O2/L. In some embodiments, the dissolved oxygen concentration of the microalgae culture treating wastewater may be maintained in the range of 2 to 3 mg O2/L. In some embodiments, the dissolved oxygen concentration of the microalgae culture treating wastewater may be maintained in the range of 3 to 4 mg O2/L. In some embodiments, the dissolved oxygen concentration of the microalgae culture treating wastewater may be maintained in the range of 4 to 5 mg O2/L. In some embodiments, the dissolved oxygen concentration of the microalgae culture treating wastewater may be maintained in the range of 5 to 6 mg O2/L. In some embodiments, the dissolved oxygen concentration of the microalgae culture treating wastewater may be maintained in the range of 6 ίο 7 mg O2/L. In some embodiments, the dissolved oxygen concentration of the microalgae culture treating wastewater may be maintained in the range of 7 to 8 mg O2/L. In some embodiments, the dissolved oxygen concentration of the microalgae culture treating wastewater may be maintained in the range of 8 to 9 mg O2/L. In some embodiments, the dissolved oxygen concentration of the microalgae culture treating wastewater may be maintained in the range of 9 to 10 mg O2/L. In some embodiments, the dissolved oxygen concentration of the microalgae culture treating wastewater may be maintained in the range of 10 to 11 mg O2/L. In some embodiments, the dissolved oxygen concentration of the rnicroalgae culture treating wastewater may be maintained in the range of 1 1 to 12 mg O2/L.

|0043] In some embodiments, the density of the microalgae culture treating wastewater may be in the range of 0.1 -1 g dry weight per liter. In some embodiments, the initial density of the microalgae culture treating wastewater may be in the range of 0.1 -1 g dry weight per liter, but the culture density may increase to densities up to 30 g/L. In some embodiments, the density of the microalgae culture treating wastewater may be in the range of 1-5 g dry weight per liter. In some embodiments, the density of the microalgae culture treating wastewater may be in the range of 5-10 g dry weight per liter. In some embodiments, the density of the microalgae culture treating wastewater may be in the range of 10-15 g dry weight per titer. In some embodiments, the density of the microalgae culture treating wastewater may be in the range of 15-20 g dry weight per liter. In some embodiments, the density of the microalgae culture treating wastewater may be in the range of 20-25 g dry weight per titer. In some embodiments, the density of the microalgae culture treating wastewater may be in the range of 25-30 g dry weight per liter.

[0044] In some embodiments, the residence time of the microalgae culture treating wastewater may be in the range of 0.5-20 days. In some embodiments, the residence time of the microalgae culture treating wastewater may be in the range of 15-20 days. In some embodiments, the residence time of the microalgae culture treating wastewater may be in the range of 10-15 days. In some embodiments, the residence time of the microalgae culture treating wastewater may^¬ be in the range of 5- 10 days. In some embodiments, the residence time of the microalgae culture treating wastewater may be in the range of 1-5 days. In some embodiments, the residence time of the microalgae culture treating wastewater may be in the range of 1 -10 days. In some embodiments, the residence time of the microalgae culture treating wastewater may be in the range of 0.5-1 days. In some embodiments, the residence time of the microalgae culture treating wastewater may be at least 1 day. In some embodiments, the residence time of the microalgae culture treating wastewater may be at least 5 days. In some embodiments, the residence time of the microalgae culture treating wastewater may be at least 10 days. In some embodiments, the residence time of the microalgae culture treating wastewater may be up to 5 days. In some embodiments, the residence time of the microalgae culture treating wastewater may be up to 10 days. In some embodiments, the residence time of the microalgae culture treating wastewater may be up to 15 days. In some embodiments, the residence time of the microalgae culture treating wastewater may be up to 20 days.

[0045] In some embodiments, the culture depth of the microalgae culture treating wastewater may be in the range of 0.5 to 10 m. In some embodiments, the culture depth of the microalgae culture treating wastewater may be in the range of 0.5 to 1 m. In some embodiments, the cul ture depth of the microalgae culture treating wastewater may be in the range of 1 to 2 m. In some embodiments, the culture depth of the microalgae culture treating wastewater may be in the range of 2 to 3 m. In some embodiments, the culture depth of the microalgae culture treating wastewater may be in the range of 3 to 4 m. In some embodiments, the culture depth of the microalgae culture treating wastewater may be in the range of 4 to 5 m. In some embodiments, the culture depth of the microalgae culture treating wastewater may be in the range of 5 to 8 m. In some embodiments, the culture depth of the microalgae culture treating wastewater may be in the range of 8 to 10 m. in some embodiments, the culture depth of the microalgae culture treating wastewater may be at least 0.5 m. In some embodiments, the culture depth of the microalgae culture treating wastewater may be at least 1 m. In some embodiments, the culture depth of the microalgae culture treating wastewater may be at least 2 m. In some embodiments, the culture depth of the microalgae culture treating wastewater may be at l east 5 m.

[0046] In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased volume to surface area ratio of at least 10% compared to a phototrophic culture of microalgae treating wastewater. In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased volume to surface area ratio of at least 20% compared to a phototrophic culture of microalgae treating wastewater. In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased volume to surface area ratio of at least 25% compared to a phototrophic culture of microalgae treating wastewater. In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased volume to surface area ratio of at least 50% compared to a phototrophic culture of microalgae treating wastewater. In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased volume to surface area ratio of at least 100% compared to a phototrophic culture of microalgae treating wastewater.

|0047] In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased volume to surface area ratio of at least 200% compared to a phototrophic culture of microalgae treating wastewater. In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased volume to surface area ratio of at least 300% compared to a phototrophic culture of microalgae treating wastewater. In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased volume to surface area ratio of at least 400% compared to a phototrophic culture of microalgae treating wastewater. In some embodiments, a method of treating wastewater with a culture of microalgae suppiemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased volume to surface area ratio of at least 500% compared to a phototrophic culture of microalgae treating wastewater.

[0048] In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased rate of N, P, or heavy metal uptake of at least 2 times compared to a phototrophic culture of microalgae treating wastewater. In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased rate of N, P, or heavy metal uptake of at least 3 times compared to a phototrophic culture of microalgae treating wastewater. In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased rate of N, P, or heavy metal uptake of at least 4 times compared to a phototrophic culture of microalgae treating wastewater. In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in an increased rate of N, P, or heavy metal uptake of at least 5 times compared to a phototrophic culture of microalgae treating wastewater.

[0049] In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in a decrease of microalgae flocculation (i.e., auto or self-flocculation of the microalgae) in the wastewater compared to a phototrophic culture of microalgae treating wastewater due to the drop in pH induced by the organic carbon, which correlates to an increased ability of the organic carbon supplied microalgae to uptake, assimilate, and consume N, P, or heavy metals compared to a phototrophic culture of microalgae. In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in a decrease in the evaporation of ammonia (i.e., off-gassing from aqueous culture) compared to a phototropic culture of microalgae treating wastewater due to the drop in pH induced by the supply of organic carbon. In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic carbon source such as, but not limited to, acetate or acetic acid, may result in a decrease of the bacteria: microalgae ratio. In some embodiments, a method of treating wastewater with a culture of microalgae supplemented with an organic may comprise supplying the culture of microalgae with a combination of an organic carbon source and an acid such as, but not limited to, hydrochloric acid, to produce the effect of maintaining the pH in a desired range while also supplying energy through the organic carbon source.

Aspects of the Invention

[0050] In one non-limiting embodiment, a method of treating wastewater may comprise: providing a volume of wastewater comprising, a first solids concentration of less than 1%, a first nitrogen (N) concentration of less than 100 ppm N, a first phosphoms (P) concentration of less than 10 ppm, and a culture depth of at least 0.5 m; inoculating a culture of microalgae in the wastewater; supplying the culture with an organic carbon source comprising at least one of acetate and acetic acid; culturing the microalgae for an effective amount of time to reduce the concentration of N and P from the wastewater to a secon concentration of N and P that is less than the first concentratoin of N and P; and separating the microaigae from the wastewater to produce a volume of wastewater with the second concentration of N and P. In some embodiments, the nitrogen may he in the form of at least one of nitrate and ammonium.

[0051] In some embodiments, the method may further comprise supplying the culture with light comprising photosyntheticaily active radiation (PAR). In some embodiments, the microaigae may comprise at least one from the group consisting of: Chlorella, Anacysiis, Synechococcus , Synechocystis, Neospongiococcum, Chlorococcum, Phaeodactylum, Spirulina, Micractinium, Haematococcus, Narmochloropsis, Brachiomonas, Schizochytrium, Aurantiochytrium, Scenedesmus . In some embodiments, the effective amount of time to reduce the first concentrations of N and P from the wastewater to the second concentrations of N and P may be 1 -10 days. In some embodiments, the effective amount of time to reduce the first concentration of N and P from the wastewater to the second concentrations of N and P may result in a rate of N and P reduction at least 2 times faster than a phototrophic microaigae culture.

|0052] In some embodiments, the organic carbon source comprising at least one of acetate and acetic acid may be produced by anaerobic digestion. In some embodiments, a volume to surface area ratio of the microaigae culture receiving the supply of organic carbon to reduce the first concentrations of N and P from the wastewater to the second concentrations of N and P may result in a volume to surface ratio of at least 100%» times larger than a phototrophic microaigae culture performing the same reduction in N and P.

[0053] In another non-limiting embodiment, a method of minimizing the flocculation of micraolgae when treating wastewater may comprise; providing a volume of wastewater comprising at least one from the group consisting of nitrogen, phosphorus, or heavy metals; inoculating a culture of microaigae in the wastewater; and supplying the culture with an organic carbon source comprising at least one of acetate and acetic acid, wherein the culture of microaigae has less flocculation than a phototrophic culture of microaigae inoculated in the wastewater.

[0054] In another non-limiting embodiment, a method of minimizing the evaporation of ammonium when treating wastewater with micraolgae amy comprise: providing a volume of wastewater comprising ammonium; inoculating a culture of microalgae in the wastewater; and supplying the culture with an organic carbon source comprising at least one of acetate and acetic acid, wherein the cutlure of microalgae has less ammonium evaporation than a phototrophic culture of microalgae inoculated in the wastewater.

[0055] In another non-limiting embodiment, a method of minimizing the bacteria to microalgae ratio when treating wastewater with microalgae comprising: providing a volume of wastewater comprising at least one form the group consisting of nitrogen, phosphorus, and heavy metals, and bacteria; inoculating a culture of microalgae in the wastewater; and supplying the culture with an organic carbon source comprising at least one of acetate and acetic acid, wherein the culture of microalgae has a lower bacteria: microalgae ratio than a phototrophic culture of microalgae inoculated in the wastewater.

[0056] In another non-limiting embodiment, a method of treating wastewater with a cutlure of microalgae comprises: providing a volume of wastewatwer comprising at least one from the group consisting of nitrogen, phosphorus, and heavy metals; inoculating a culture of microalgae in the wastewater; and supplying the cutlure with an organic carbon source in combination with an effective amount of hydrochloric acid. In some embodiments, the effective amount of hydrochoric acid may maintain the pH of the culture of microalgae in the wastewater in the range of 5-8.

[0057] In some embodiments, the organicc arbon source may comprise at least one from the group consisting of: 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 still age, urea, industrial waste solutions, wastewater from sewage treatment plants, liquid waste from terrestrial animal production systems, liquid waste from bio-digesters, and y east extract. In some embodiments, the method may further comprise supplying the culture with light comprising photosynthetically active radiation (PAR).

[0058] In some embodiments, the microalgae may comprise at least one form the group consisting of: Agmenellum, Amphora, Anabaena, Anacystis, Apistonema, Pleurochyrsis, Arthrospira, Aurantiochytrium, Botryococcus, Bracniomonas, Chlamydomonas, Chloreda, Chloroccum, Cruciplacolithus, Cylindrotheca, Coenochloris, Cyanophora, Cyclotella, Dunaliella. Emiliania, Euglena, Extubocelliilus. Fragilaria, Galdieria, Goniotrichium, Haematococcus, Halochlorella, Isochyrsis, , Micractinium, Melosira, Monodus, Nostoc, Nannochloris, Nannochioropsis, Navicula, Neosongiococcum, Nitzschia, Odontella, Ochromonas, Ochrosphaera, Pavlova, Picochlorum, Phaeodactylum, Pleurochyrsis, Porphyridium, Poteriochromonas, Prymnesiwn, Rhodomonas, Schizochytrium, Scenedesmus, Skeletonema, Spumella, Stauroneis, Stichococcus, Auxenochlorella, Cheatoceros, Neochloris, Nespongiococcum. Ocromonas, Porphyridium, Synechococcus, Synechocysti, Telraselmis , Thraustochytrids, and Thalassiosira.

[0059] In some embodiments, the culture of rnicroaigae in the wastewater may reduce a first concentrations of nitrogen and phosphorus from the wastewater to a second lower concentrations of nitrogen and phosphorus at a rate at least 2 times faster than a phototrophic rnicroaigae culture. In some embodiments, a volume to surface area ratio of the rnicroaigae culture receiving the supply of organic carbon to reduce the first concentrations of nitrogen and phosphorus from the wastewater to the second concentrations of nitrogen and phosphorus may result in a volume to surface ratio of at least 100% times larger than a phototrophic rnicroaigae culture performing the same reduction in nitrogen and phosphorus.

[0060] In some embodiments, the wastewater may further comprise ammonium. In some embodiments, the culture of rnicroaigae may have less ammonium evaporation than a phototrophic culture of rnicroaigae inoculated in the wastewater. In some embodiments, the wastewater may further comprise bacteria. In some embodiments, the culture of rnicroaigae may have a lower bacteria: microaglae ratio than a phototrophic culture of rnicroaigae inoculated in the wastewater.

[0050] 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 individually 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. [0051] 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.

[0052] Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary 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.

[0053] 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).

[0054] All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

[0055] The use of any and all examples, or exemplar}- 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.

[0056] 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.

[0057] This invention includes all 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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Claims

CLAIMS What is claimed is:

1. A method of treating wastewater, comprising:

a. Providing a volume of wastewater comprising:

i. A first solids concentration of less than 1%;

ii. A first nitrogen (N) concentration of less than 100 ppm N;

iii. A first phosphorus (P) concentration of less than 10 ppm; and iv. A culture depth of at least 0.5 m;

b. Inoculating a culture of microalgae in the wastewater;

c. Supplying the culture with an an amount of an organic carbon source comprising at least one of acetate and acetic acid that is capable of detectably promoting growth of the microalgae, inhibiting growth of bacteria in the culture, or a combination thereof;

d. Culturing the microalgae for an effective amount of time to reduce the concentration of N and P from the wastewater to a second concentration of N and P that is less than the first concentration of N and P; and

e. Separating the microalgae from the wastewater to produce a volume of

wastewater with the second concentration of N and P.

2. The method of claim 1, wherein the microalgae comprises at least one from the group consisting of: Chlorella, Anacystis, Synechococcus, Synechocystis ,

Neospongiococcum, Chlorococcum, Phaeodactylum, Spirulina, Mic actinium, Haematococcus, Nannochloropsis, Brachiomonas, Schizochytrium, Aurantiochytrium, and Scenedesmus.

3. The method of claim 1 , wherein the effective amount of time to reduce the first

concentrations of N and P from the wastewater to the second concentrations of N and P is 1-10 days.

4. The method of claim ! , wherein the effective amount of time to reduce the first

concentration of N and P from the wastewater to the second concentration of N and P results in a rate of N and P reduction at least 2 times faster than the reduction that would be obtained from a similar microalgae culture grown under phototrophic conditions.

5. The method of any one of claims 1-4, wherein the organic carbon source comprising at least one of acetate and acetic acid is produced by anaerobic digestion.

6. The method of claim 1 , wherein a volume to surface area ratio of the microalgae culture receiving the supply of organic carbon to reduce the first concentrations of N and P from the wastewater to the second concentrations of N and P results in a volume to surface ratio of at least 100% times larger than a phototrophic microalgae culture performing the same reduction in N and P.

7. The method of any one of claims 1-6, wherein the method comprises treating the culture with an amount of hydrochloric acid that is effective for maintaining the pH of the culture of microalgae in the wastewater in the range of 5-8.