# Patent

## Title
Method of culturing microorganisms using phototrophic and mixotrophic culture conditions

## Abstract

Methods of culturing microorganisms in combinations of phototrophic, mixotrophic, and heterotrophic culture conditions are disclosed. A culture of microorganisms may be transitioned between culture conditions over the life of a culture in various combinations, utilizing various conditions in a sequential manner to optimize the culture for growth or product accumulation.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation International Application No. PCT/US2013/06049, filed Nov. 8, 2013 entitled Mixotrophic, Phototrophic, and Heterotrophic Combination Methods and Systems which claims the benefit of U.S. Provisional Application No. 61/724,710, filed Nov. 9, 2012, entitled Methods of Culturing Microorganisms in Mixotrophic Conditions; U.S. Provisional Application No. 61/798,969, filed Mar. 15, 2013, entitled Mixotrophy Systems and Methods; and U.S. Provisional Application No. 61/883,805, filed Sep. 27, 2013, entitled Mixotrophic, Phototrophic, and Heterotrophic Combination Methods and Systems, the entire contents of which are hereby incorporated by reference herein.


BACKGROUND
Microorganisms, such as microalgae and cyanobacteria, may be cultured in a variety of conditions that affect the microorganisms in multiple ways. With each set of culture conditions having unique advantages and disadvantages, a culturing method using a single set of culture conditions may not provide advantages in all the circumstances a culture of microorganisms may experience over the life of the culture. In the prior art, methods of sequentially culturing microalgae in a first phototrophic (or mixotrophic) stage and then a second heterotrophic stage are used to increase accumulation of medium length carbon chain lipids (e.g., C16:0) that are used for biofuel production. The sequential stages of culture conditions attempt to leverage the low cost of phototrophic methods with the high productivity of heterotrophic methods to increase the efficiency of the biofuel production process. While this sequence may be beneficial for the production of lipids used in biofuels, the method is not optimized for the variety of diverse product markets that may be served by microalgae and cyanobacteria through whole biomass, proteins, pigments, polysaccharides, nutritional fatty acids, and others microorganism products.
Beyond lipid production for biofuels, the culture conditions may be more broadly applicable to enhancing the rate of growth, controlling the level of contaminating organisms (e.g., contaminating bacteria, competing organisms, fungi), product formation and accumulation (e.g., proteins, lipids, pigments), and other aspects relevant to the commercial production of microorganisms. Mixotrophic microorganisms provide the capability to culture microorganisms in a more flexible system utilizing a variety of culture conditions for different purposes or for different functions of the microorganisms. Therefore, there is a need in the art for flexible methods of culturing mixotrophic microorganisms in a plurality of culture conditions which optimize the culture for growth and a diverse product profile.
SUMMARY
Embodiments described herein relate generally to methods for culturing microorganisms in combinations of phototrophic, mixotrophic, and heterotrophic culture conditions. A culture of microorganisms may be transitioned between culture conditions over the life of a culture, utilizing various conditions in a sequential manner to optimize the culture for growth or product accumulation.
In some embodiments of the invention, a method of culturing microorganisms comprises: providing a culture of microorganisms capable of growth in mixotrophic and phototrophic culture conditions; growing the culture of microorganisms in mixotrophic culture conditions; growing the culture of microorganisms in phototrophic culture conditions absent a supply of the organic carbon source; and transitioning the culture of microorganisms between mixotrophic and phototrophic conditions at least once in the life of the culture of microorganisms. The mixotrophic culture conditions comprise: supplying the culture of microorganisms with at least some light; supplying the culture of microorganisms with at least one organic carbon source; and supplying the culture of microorganisms with carbon dioxide. The phototrophic culture conditions comprise: supplying the culture of microorganisms with light and carbon dioxide.
In some embodiments, the microorganisms are selected from the group consisting of microalgae and cyanobacteria. In some embodiments, the at least one organic carbon source is selected 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 stillage, urea, industrial waste solutions, and yeast extracts.
In some embodiments, transitioning the culture of microorganisms between mixotrophic and phototrophic conditions at least once in the life of the culture of microorganisms comprises harvesting a portion of the culture of microorganisms in mixotrophic conditions to inoculate the harvested culture of microorganisms in phototrophic conditions. In some embodiments, transitioning the culture of microorganisms between mixotrophic and phototrophic conditions at least once in the life of the culture of microorganisms comprises harvesting a portion of the culture of microorganisms in phototrophic conditions to inoculate the harvested culture of microorganisms in mixotrophic conditions.
In some embodiments, transitioning the culture of microorganisms between mixotrophic and phototrophic conditions at least once in the life of the culture of microorganisms comprises at least one of decreasing the chlorophyll content, decreasing the sterols content, decreasing the total amino acid content, increasing the carotene content, and increasing the carbohydrate content in the culture of microorganisms by transitioning the culture of microorganisms from phototrophic to mixotrophic conditions before harvest. In some embodiments, transitioning the culture of microorganisms between mixotrophic and phototrophic conditions at least once in the life of the culture of microorganisms comprises at least one of increasing the chlorophyll content, increasing the sterols content, increasing the total amino acid content, decreasing the carotene content, and decreasing the carbohydrate content in the culture of microorganisms by transitioning the culture of microorganism from mixotrophic to phototrophic conditions before harvest.
In some embodiments of the invention, a method of culturing microorganisms comprises: providing a culture of microorganisms in an aqueous medium capable of growth on light as an energy source, carbon dioxide as a carbon source, and at least one organic carbon source as an energy and carbon source; growing the culture of microorganisms in phototrophic conditions, wherein the phototrophic conditions comprise a supply of light as an energy source and carbon dioxide as a carbon source in a phototrophic culturing vessel comprising a volume less than 10 L and a lack of a supply of at least one organic carbon source; harvesting at least a portion of the culture of microorganisms from the phototrophic culturing vessel; inoculating the culture of microorganisms harvested from the phototrophic culturing vessel into a small volume mixotrophic culturing vessel, wherein the small volume mixotrophic culturing vessel comprises a volume of 10 to 100 L and operates in a mixotrophic conditions for growing the culture of microorganisms, wherein the mixotrophic conditions comprise a supply of light as an energy source, carbon dioxide as an inorganic carbon source, and at least one organic carbon source as an energy and carbon source; harvesting at least a portion of the culture of microorganisms form the small volume mixotrophic culturing vessel; inoculating the culture of microorganisms harvested from the small volume mixotrophic culturing vessel into a large volume mixotrophic culturing vessel, wherein the large volume mixotrophic culturing vessel comprises a volume of at least 100 L and operates in mixotrophic conditions for growing the culture of microorganisms; and harvesting the culture of microorganisms from the large volume mixotrophic culturing vessel.
In some embodiments, the phototrophic culturing vessel is closed or open. In some embodiments, the small volume mixotrophic culturing vessel is closed or open. In some embodiments, the large volume mixotrophic culturing vessel is closed or open. In some embodiments, the large volume mixotrophic culturing vessel comprises a volume of 100 to 750,000 L.
In some embodiments of the invention, a method of culturing microorganisms comprises: providing a culture of microorganisms in an aqueous culture medium capable of growth heterotrophic, mixotrophic, and phototrophic culture conditions; disposing the culture of microorganisms in non-axenic conditions in a culturing vessel wherein the culture of microorganisms is exposed to at least some sunlight during daylight hours and no sunlight during night hours, the daylight hours comprising peak hours of sunlight and non-peak hours of sunlight; supplying at least one organic carbon source to the culture of microorganisms during a time period comprising the non-peak hours of sunlight and night hours, the peak hours of sunlight comprising an equal amount of time on either side of a time when the sun is at its zenith and non-peak hours of sunlight comprising all other hours of sunlight during daylight hours; eliminating the supply of at least one organic carbon source during the peak hours of sunlight; and growing the culture of microorganisms for at least two consecutive days.
In some embodiments, the peak hours of sunlight are 6-10 hours. In some embodiments, growing the culture of microorganisms is continued for 2 to 90 consecutive days. In some embodiments, the method further comprises supplying carbon dioxide to the culture of microorganisms during peak hours of sunlight and non-peak hours of sunlight. In some embodiments, the method further comprises detecting the level of sunlight with a photodetector and controlling the supply of organic carbon with a programmable logic controller which receives data from the photodetector.
In some embodiments, a method of culturing microorganisms comprises: providing a culture of microorganisms in an aqueous medium capable of growth on light as an energy source, carbon dioxide as a carbon source, and at least one organic carbon source as an energy and carbon source; transitioning the culture of microorganisms between at least two selected from the group consisting of phototrophic, mixotrophic and heterotrophic culture conditions, at least once in the life of the culture of microalgae; and wherein the transition between at least two selected from the group consisting of phototrophic, mixotrophic, and heterotrophic culture conditions occurs when at least one threshold condition of the culture it met.
The phototrophic culture conditions comprise supplying the culture of microorganisms with light and carbon dioxide in the absence of a supply of an organic carbon source. The mixotrophic culture conditions comprise supplying the culture of microorganisms with light, carbon dioxide, and the at least one organic carbon source. The heterotrophic culture conditions comprise supplying the culture of microorganisms with the at least one organic carbon source in the absence of a supply of light and carbon dioxide. The threshold conditions of the culture comprise: a detected level of dissolved oxygen, a detected level of dissolved carbon dioxide, a detected level of contaminating organisms, a detected level of chlorophyll, a detected temperature level, a detected residual organic carbon level, and complete consumption of a batch of organic carbon.
In some embodiments, the contaminating organisms comprise bacteria or fungi. In some embodiments, the culture of microorganisms transitions from phototrophic to mixotrophic culture conditions when the detected dissolved oxygen level is above a threshold level. In some embodiments, the culture of microorganisms transitions from phototrophic to heterotrophic culture conditions when the detected dissolved oxygen level is above a threshold level. In some embodiments, the culture of microorganisms transitions from mixotrophic to phototrophic culture conditions when the detected dissolved oxygen level is below a threshold level. In some embodiments, the culture of microorganisms transitions from heterotrophic to phototrophic culture conditions when the detected dissolved oxygen level is below a threshold level.
In some embodiments, the culture of microorganisms transitions from mixotrophic to phototrophic culture conditions when the detected dissolved carbon dioxide level is above a threshold level. In some embodiments, the culture of microorganisms transitions from heterotrophic to phototrophic culture conditions when the detected dissolved carbon dioxide level is above a threshold level. In some embodiments, the culture of microorganisms transitions from phototrophic to mixotrophic culture conditions when the detected dissolved carbon dioxide level is below a threshold level. In some embodiments, the culture of microorganisms transitions from phototrophic to heterotrophic culture conditions when the detected dissolved carbon dioxide level is below a threshold level.
In some embodiments, the culture of microorganisms transitions from heterotrophic to mixotrophic culture conditions when the detected chlorophyll level is below a threshold level. In some embodiments, the culture of microorganisms transitions from heterotrophic to phototrophic culture conditions when the detected chlorophyll level is below a threshold level. In some embodiments, the culture of microorganisms transitions from mixotrophic to phototrophic culture conditions when the detected chlorophyll level is below a threshold level. In some embodiments, the culture of microorganisms transitions from phototrophic to mixotrophic culture conditions when the detected chlorophyll level is above a threshold level. In some embodiments, the culture of microorganisms transitions from phototrophic to heterotrophic culture conditions when the detected chlorophyll level is above a threshold level.
In some embodiments, the method further comprises measuring density of the culture and the culture of microorganisms transitions from phototrophic to mixotrophic culture conditions when the detected cell culture density level is above a first threshold level. In some embodiments, the culture of microorganisms transitions from mixotrophic to heterotrophic culture conditions when the detected cell culture density level is above a second threshold level.
In some embodiments, the culture of microorganisms transitions from phototrophic to mixotrophic culture conditions when the detected level of contaminating organisms is below a threshold level. In some embodiments, the culture of microorganisms transitions from phototrophic to heterotrophic culture conditions when the detected level of contaminating organisms is below a threshold level. In some embodiments, the culture of microorganisms transitions from mixotrophic to phototrophic culture conditions when the detected level of contaminating organisms is above a threshold level. In some embodiments, the culture of microorganisms transitions from heterotrophic to phototrophic culture conditions when the detected level of contaminating organisms is above a threshold level.
In some embodiments, the culture of microorganisms transitions from phototrophic to mixotrophic culture conditions when the detected level of residual organic carbon is below a threshold level. In some embodiments, the culture of microorganisms transitions from phototrophic to heterotrophic culture conditions when the detected level of residual organic carbon is below a threshold level.
In some embodiments, the culture of microorganisms transitions from heterotrophic to phototrophic culture conditions when the detected culture temperature is below a threshold level. In some embodiments, the culture of microorganisms transitions from mixotrophic to phototrophic culture conditions when the detected culture temperature is below a threshold level. In some embodiments, the culture of microorganisms transitions from phototrophic to mixotrophic culture conditions when the detected culture temperature is above a threshold level. In some embodiments, the culture of microorganisms transitions from phototrophic to heterotrophic culture conditions when the detected culture temperature is above a threshold level.
In some embodiments, the culture of microorganisms transitions from heterotrophic to phototrophic culture conditions upon the complete consumption of a batch of organic carbon. In some embodiments, the culture of microorganisms transitions from mixotrophic to phototrophic culture conditions upon the complete consumption of a batch of organic carbon.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a graph comparing the product profile of microalgae grown in phototrophic and mixotrophic conditions.
FIG. 2 is a graph comparing the product profile of microalgae grown in phototrophic and mixotrophic conditions.
FIG. 3 is a graph comparing the fatty acid profile of microalgae grown in phototrophic and mixotrophic conditions.
FIG. 4 shows an embodiment of a fermentation vessel.
FIG. 5 is a graph showing the growth rate of Haematococcus in heterotrophic growth conditions.
FIG. 6 is a graph showing the ratio of red and green cysts in a culture of Haematococcus. 



DETAILED DESCRIPTION
Definitions
The term “microorganism” refers to microscopic organisms such as microalgae and cyanobacteria. Microalgae include microscopic multi-cellular plants (e.g. duckweed), photosynthetic microorganisms, heterotrophic microorganisms, diatoms, dinoflagelattes, and unicellular algae.
The terms “microbiological culture”, “microbial culture”, or “microorganism culture” refer to a method or system for multiplying microorganisms through reproduction in a predetermined culture medium, including under controlled laboratory conditions. Microbiological cultures, microbial cultures, and microorganism cultures are used to multiply the organism, to determine the type of organism, or the abundance of the organism in the sample being tested. In liquid culture medium, the term microbiological, microbial, or microorganism culture generally refers to the entire liquid medium and the microorganisms in the liquid medium regardless of the vessel in which the culture resides. A liquid medium is often referred to as “media”, “culture medium”, or “culture media”. The act of culturing is generally referred to as “culturing microorganisms” when emphasis is on plural microorganisms. The act of culturing is generally referred to as “culturing a microorganism” when importance is placed on a species or genus of microorganism. Microorganism culture is used synonymously with culture of microorganisms.
Microorganisms that may grow in mixotrophic culture conditions include microalgae, diatoms, and cyanobacteria. Non-limiting examples of mixotrophic microorganisms may comprise organisms of the genera: Agmenellum, Amphora, Anabaena, Anacystis, Apistonema, Pleurochyrsis, Arthrospira (Spirulina), Botryococcus, Brachiomonas, Chlamydomonas, Chlorella, Chloroccum, Cruciplacolithus, Cylindrotheca, Coenochloris, Cyanophora, Cyclotella, Dunaliella, Emiliania, Euglena, Extubocellulus, Fragilaria, Galdieria, Goniotrichium, Haematococcus, Halochlorella, Isochyrsis, Leptocylindrus, Micractinium, Melosira, Monodus, Nostoc, Nannochloris, Nannochloropsis, Navicula, Neospongiococcum, Nitzschia., Odontella, Ochromonas, Ochrosphaera, Pavlova, Picochlorum, Phaeodactylum, Pleurochyrsis, Porphyridium, Poteriochromonas, Prymnesium, Rhodomonas, Scenedesmus, Skeletonema, Spumella, Stauroneis, Stichococcus, Auxenochlorella, Cheatoceros, Neochloris, Ocromonas, Porphiridium, Synechococcus, Synechocystis, Tetraselmis, Thraustochytrids, Thalassiosira, and species thereof.
The organic carbon sources suitable for growing a microorganism mixotrophically or heterotrophically may comprise: acetate, acetic acid, ammonium linoleate, arabinose, arginine, aspartic acid, butyric acid, cellulose, citric acid, ethanol, fructose, fatty acids, galactose, glucose, glycerol, glycine, lactic acid, lactose, maleic acid, maltose, mannose, methanol, molasses, peptone, plant based hydrolyzate, proline, propionic acid, ribose, sacchrose, partial or complete hydrolysates of starch, sucrose, tartaric, TCA-cycle organic acids, thin stillage, urea, industrial waste solutions, yeast extract, and combinations thereof. The organic carbon source may comprise any single source, combination of sources, and dilutions of single sources or combinations of sources.
The terms “mixotrophic” and “mixotrophy” refer to culture conditions in which light, organic carbon, and inorganic carbon (e.g., carbon dioxide, carbonate, bi-carbonate) may be applied to a culture of microorganisms. Microorganisms capable of growing in mixotrophic conditions have the metabolic profile of both phototrophic and heterotrophic microorganisms, and may use both light and organic carbon as energy sources, as well as both inorganic carbon and organic carbon as carbon sources. A mixotrophic microorganism may be using light, inorganic carbon, and organic carbon through the phototrophic and heterotrophic metabolisms simultaneously or may switch between the utilization of each metabolism. A microorganism in mixotrophic culture conditions may be a net oxygen or carbon dioxide producer depending on the energy source and carbon source utilized by the microorganism. Microorganisms capable of mixotrophic growth comprise microorganisms with the natural metabolism and ability to grow in mixotrophic conditions, as well as microorganisms which obtain the metabolism and ability through modification of cells by way of methods such as mutagenesis or genetic engineering.
The terms “phototrophic”, “phototrophy”, “photoautotrophy”, “photoautotrophic”, and “autotroph” refer to culture conditions in which light and inorganic carbon (e.g., carbon dioxide, carbonate, bi-carbonate) may be applied to a culture of microorganisms. Microorganisms capable of growing in phototrophic conditions may use light as an energy source and inorganic carbon (e.g., carbon dioxide) as a carbon source. A microorganism in phototrophic conditions may produce oxygen.
The terms “heterotrophic” and “heterotrophy” refer to culture conditions in which organic carbon may be applied to a culture of microorganisms in the absence of light. Microorganisms capable of growing in heterotrophic conditions may use organic carbon as both an energy source and as a carbon source. A microorganism in heterotrophic conditions may produce carbon dioxide.
The term “axenic” describes a culture of an organism that is entirely free of all other “contaminating” organisms (i.e., organisms that are detrimental to the health of the microalgae or cyanobacteria culture). Throughout the specification, axenic refers to a culture that when inoculated in an agar plate with bacterial basal medium, does not form any colonies other than the microorganism of interest. Axenic describes cultures not contaminated by or associated with any other living organisms such as but not limited to bacteria, cyanobacteria, microalgae and/or fungi. Axenic is usually used in reference to pure cultures of microorganisms that are completely free of the presence of other different organisms. An axenic culture of microalgae or cyanobacteria is completely free from other different organisms.
Bacteria that may be present in cultures of microalgae and cyanobacteria comprise, but are not limited to: Achromobacter sp., Acidovorax sp., Acinetobacter sp., Aeromonas sp., Agrobacterium sp., Alteromonas sp., Ancylobacter sp., Aquaspirillum sp., Azospirillum sp., Azotobacter sp., Bacillus sp., Bergeyella sp., Brevundimonas sp., Brochothrix sp., Brumimicrobium sp., Burkholderia sp., Caulobacter sp., Cellulomonas sp., Chryseobacterium sp., Curtobacterium sp., Delftia sp., Empedobacter sp., Enterobacter sp., Escherichia sp., Flavobacterium sp., Gemmatimonas sp., Halomonas sp., Hydrogenophaga sp., Janthinobacterium sp., Lactobacillus sp., Marinobacter sp., Massilia sp., Microbacterium sp., Myroides sp., Pantoea sp., Paracoccus sp., Pedobacter sp., Phaeobacter sp., Phyllobacterium sp., Pseudoalteromonas sp., Pseudomonas sp., Rahnella sp., Ralstonia sp., Rhizobium sp., Rhodococcus sp., Roseomonas sp., Sphingobacterium sp., Sphingomoas sp., Staphylococcus sp., Stenotrophomonas sp., Vibrio sp., and Zobelliae sp.
Bacteria that have a negative or harmful effect on the microalgae and cyanobacteria may be designated as contaminating bacteria. The bacteria that may have a negative or harmful effect on microalgae or cyanobacteria in a culture comprise, but are not limited to: Achromobacter sp., Acidovorax sp., Aeromonas sp., Agrobacterium sp., Alteromonas sp., Aquaspirillum sp., Azospirillum sp., Azotobacter sp., Bergeyella sp., Brochothrix sp., Brumimicrobium sp., Burkholderia sp., Caulobacter sp., Cellulomonas sp., Chryseobacterium sp., Curtobacterium sp., Delftia sp., Empedobacter sp., Enterobacter sp., Escherichia sp., Flavobacterium sp., Marinobacter sp., Microbacterium sp., Myroides sp., Paracoccus sp., Pedobacter sp., Phaeobacter sp., Pseudoalteromonas sp., Pseudomonas sp., Rahnella sp., Ralstonia sp., Rhizobium sp., Rhodococcus sp., Roseomonas sp., Staphylococcus sp., Stenotrophomonas sp., Vibrio sp., Zobelliae sp. and other bacteria which share similar characteristics.
The bacteria that may have a neutral or beneficial effect on microalgae or cyanobacteria in a culture comprise, but are not limited to: Acidovorax sp., Acinetobacter sp., Aeromonas sp., Agrobacterium sp., Alteromonas sp., Ancylobacter sp., Azospirillum sp., Azotobacter sp., Bacillus sp., Brevundimonas sp., Brumimicrobium sp., Burkholderia sp., Caulobacter sp., Cellulomonas sp., Delftia sp., Empedobacter sp., Gemmatimonas sp., Halomonas sp., Hydrogenophaga sp., Janthinobacterium sp., Lactobacillus sp., Marinobacter sp., Pantoea sp., Paracoccus sp., Phaeobacter sp., Phyllobacterium sp., Pseudoalteromonas sp., Pseudomonas sp., Rhizobium sp., Sphingomoas sp., Zobelliae sp. and other bacteria which share similar characteristics. While bacteria in a particular genus generally have the same characteristics, it is recognized that a genus of bacteria with the majority of species generally identified as harmful to microalgae or cyanobacteria may also include a particular species within the genus which is neutral or beneficial to a specific culture of microalgae or cyanobacteria, and vice versa. For example, many species of Pseudomonas have been observed to be harmful to microalgae, however literature has described certain species of Pseudomonas with anti-fungal functionality which may be beneficial to a culture of microalgae or cyanobacteria.
The term “pH auxostat” refers to the microbial cultivation technique that couples the addition of fresh medium (e.g., medium containing organic carbon or acetic acid) to pH control. As the pH drifts from a given set point, fresh medium is added to bring the pH back to the set point. The rate of pH change is often an excellent indication of growth and meets the requirements as a growth-dependent parameter. The feed will keep the residual nutrient concentration in balance with the buffering capacity of the medium. The pH set point may be changed depending on the microorganisms present in the culture at the time. The microorganisms present may be driven by the location and season where the bioreactor is operated and how close the cultures are positioned to other contamination sources (e.g., other farms, agriculture, ocean, lake, river, waste water). The rate of medium addition is determined by the buffering capacity and the feed concentration of the limiting nutrient and not directly by the set point (pH) as in a traditional auxostat. The pH auxostat is robust but controls nutrient concentration indirectly. The pH level represents the summation of the production of different ionic species and ion release during carbon and nutrient uptake. Therefore the pH level can move either up or down as a function of growth of the microorganisms. The most common situation is pH depression caused by organic acid production and ammonium uptake. However, for microorganisms growing on protein or amino acid-rich media, the pH level will rise with growth because of the release of excess ammonia.
The term “harvesting” refers to removing the culture of microorganisms from the culturing vessel and/or separating the microorganisms from the culture medium. Harvesting of microorganisms may be conducted by any method known in the art such as, but not limited to, skimming, draining, dissolved gas flotation, foam fractionation, centrifugation, filtration, sedimentation, chemical flocculation, and electro-dewatering.
The term “inoculate” refers to implanting or introducing microorganisms into a culture medium. Inoculate or inoculating a culture of microorganisms in the described culture conditions throughout the specification refers to starting a culture of microorganisms in the culture conditions, as is commonly used in the art of microorganism culturing. The microorganisms that are introduced into a culture medium may be referred to as seed or inoculum.
Overview
Cultures of mixotrophic microorganisms, such as microalgae and cyanobacteria, may be grown solely in mixotrophic conditions receiving an organic carbon supply, an optional inorganic carbon supply (e.g., carbon dioxide, carbonate, bi-carbonate), and at least some light (either intermittent or continuous) from the beginning to end of the life of the culture of the mixotrophic microorganisms. While microorganism cultures may experience growth benefits in a mixotrophic only system, the selection of culture conditions may be reversible and flexible over the life of the culture allowing for the use of a variety of different culture conditions. Such a transition between culture conditions, such as phototrophic, mixotrophic, and heterotrophic, may provide benefits such as, but not limited to, an increase in overall microorganism growth, an increase in initial microorganism growth, manipulation of product formation in the microorganisms, an extension to the life of the culture of microorganisms, and the suppression of contaminating organisms (e.g., bacteria, fungi, competing organisms, invading species of microalgae) in a culture. Phototrophic, mixotrophic, and heterotrophic culturing conditions may be conducted in any suitable culturing vessel or bioreactor such as, but not limited to, a pond, a raceway pond, a trough, a tank, a tubular bioreactor, a flat panel bioreactor, a bag bioreactor, and a column bioreactor.
For example, methods for cultures of microorganisms with transitions between trophic conditions may regulate the feed source for contaminating bacteria (i.e., bacteria which have a negative or harmful effect on a culture of microalgae or cyanobacteria), while allowing the desired microorganisms to continue to grow in the varied conditions. Microorganisms, such as microalgae and cyanobacteria, growth may slow down in phototrophic conditions compared to mixotrophic or heterotrophic conditions, but the contaminating bacteria growth may slow down as well and possibly stop if the bacteria does not have a photosynthetic metabolism. Additionally, microorganism culturing methods with transitions between trophic conditions may regulate the dissolved oxygen level in the microorganism culture, which may negatively affect contaminating bacteria while allowing the microorganisms to continue to grow in the varied conditions. When dissolved oxygen concentrations reach the targeted concentrations and the contaminating bacteria populations have been reduced, the system may be transitioned from phototrophic conditions back to mixotrophic conditions thus creating a cyclic pattern that reduces contaminating bacteria and increases microalgae or cyanobacteria culture longevity.
In product formation, microorganisms may provide a variety of proteins, polysaccharides, lipids, and pigments that are influenced by the culture conditions. The formation of products may be influenced by the energy inputs, carbon inputs, temperature, pH, amount of growth, or stage of growth provided by and dictated by different culture conditions.
Transitions Between Phototrophy and Mixotrophy
Cultures of microorganisms may be transitioned between mixotrophy and phototrophy by controlling the supply of the organic carbon, light, and inorganic carbon (e.g., carbon dioxide, carbonate, bi-carbonate). A mixotrophic culture of microorganisms, such as microalgae and cyanobacteria, may provide inoculum to a phototrophic culture by transferring a portion of the microorganisms harvested from a mixotrophic culture to a culture medium and system for phototrophic growth. Growing a microorganism mixotrophically first allows the culture to take advantage of the boost in initial growth experienced in mixotrophic conditions to provide a sufficient volume of seed or inoculum for a plurality of phototrophic cultures. The transition between culturing methods may also go from phototrophic to mixotrophic conditions, allowing lower performing cultures in phototrophic conditions to receive a boost in productivity through conversion to higher performing mixotrophic conditions that provide both organic carbon and light as energy sources.
Additionally, transitioning a culture between phototrophic and mixotrophic culturing conditions, and vice versa, may also affect the product formation in the microorganism. Pigments (e.g., chlorophyll, carotenoids, phycocyanin), polysaccharides, proteins, amino acids, hormones, lipids, fatty acids, carbohydrates and other products may accumulate differently in the microorganism depending on the culture conditions. Also, a culture growing mixotrophically may be converted to phototrophic growth to allow for photopigment saturation in the microorganisms, leading to increased product formation and/or microorganism health. Therefore control over the microorganism products may be exercised by the choice of culturing conditions and/or the timing of the transition between culturing conditions.
In another embodiment, the level of contaminating organisms (e.g., bacteria, fungi, completing organisms, invading species of microalgae) in a culture of microorganisms may be managed by transitioning between mixotrophy and phototrophy. In some embodiments, the culture of microorganisms may be transitioned from mixotrophy to phototrophy for a period of time (e.g., 24-48 hours), and a harvest may occur when cultures are transitioned to reduce the total organic carbon content in the culture. By reducing the total organic carbon in the culture, the available feed sources for contaminating organisms may be reduced and aid in controlling contaminating populations. The transition from mixotrophy to phototrophy itself may also reduce the organic carbon source in the culture medium without a harvest, and reduce the feed available to contaminating organisms.
EXAMPLE 1
Samples of a Chlorella species were obtained from phototrophic and mixotrophic open pond cultures (pond volume of 570 L) and compared. These samples were harvested by centrifuging, dried using a freeze drier and analyzed for the composition of the algae at POS Bio-Sciences (Saskatoon, Canada). The phototrophically cultured Chlorella was grown under non-axenic conditions using standard procedures known in the art comprising an f/2 culture medium with trace metals, a culture temperature of approximately 28° C., a raceway pond photobioreactor, a supply of sunlight, sparging of air within the culture medium, and sparging of carbon dioxide within the culture medium.
The mixotrophically cultured Chlorella was grown under non-axenic conditions using an acetic acid/pH auxostat feeding system to supply organic carbon in non-axenic conditions for 10 days. The trial was performed with two raceway pond photobioreactors made of PVC, with a cultivable area of 5.6 m2 and a 10 cm light path (i.e., distance of light penetration). Both photobioreactors contained mixotrophic cultures aerated with two 50 cm porous diffusers at 10 liters per minute (LPM) and were located outdoors. The first photobioreactor (Reactor 1) was mixed hydraulically (pump). The second photobioreactor (Reactor 2) was mixed with a paddle wheel. The Chlorella was inoculated at a density of 0.3 g/L in the Reactors 1 and 2. The Chlorella was adapted to the outdoor conditions under CO2/pH control until it attained a density of 0.3-0.4 g/L for the experimental trials. The acetic acid supply occurred using the pH auxotstat drip system which dosed acetic acid when the detected pH level of the mixotrophic cultures was above the set point of 7.5.
The mixotrophic cultures were harvested as needed when the culture density reached 1.5 g/L. The initial mixotrophic culture medium was a BG-11 medium with sodium acetate (1 g/L) supplemented in the initial medium. Natural sunlight was supplied to the mixotrophic cultures, with the average photoperiod in May 2012 for Gilbert, Ariz. being about 14.5 h. The temperature was controlled by cooling coils at 28° C. The pH controller of the pH auxostat system, as previously described, was set at 7.5. Temperature, pH and dissolved oxygen were measured continuously. Acetic acid consumption was monitored by the acetic acid feed tank level daily. Dry weights were taken three times (n=3) daily and nitrate levels were taken daily. Spin down to measure residual acetate (200 ml) and biomass was performed every two days.
Contamination observation (400×, 1000× oil immersion-phase contrast micrograph and cell cytometry with bacterial dying) was performed every two days, including a measurement of bacterial contamination by flow cytometry. For bacterial contamination measurement, to each 1 ml sample, 1 μl BacLightTM Green bacterial stain (Invitrogen, Eugene, Oreg., USA) was added, and samples were incubated at room temperature in the dark for 30 to 60 minutes. After incubation, samples were analyzed on a BD FACSAriaTM (BD Biosciences, San Jose, Calif., USA) and populations of bacteria and microalgae were gated based on BacLightTM fluorescence and chlorophyll autofluoresence.
FIGS. 1-3 compare the compositions of the Chlorella produced in mixotrophic and phototrophic cultures as determined by POS analytical methods, and illustrate the differences in product formation that result from the two culturing methods. Referring to FIG. 1, the fat percent of the mixotrophically cultured microalgae was about 13% of dry weight, while the phototrophically cultured microalgae was approximately 2.6 times lower at about 5%. The carbohydrates percent showed similar results with the mixotrophic microalgae comprising about 48% and the phototrophic microalgae being about 1.4 times lower at about 35%. The protein accumulation represented by the total amino acids content in dry weight was lower in the mixotrophically cultured microalgae.
FIG. 2 compares additional high value components in the Chlorella samples. FIG. 2 shows that the phototrophically cultured microalgae had about 12 times more chlorophyll content than the mixotrophically cultured microalgae. The xanthophylls and tocopherols were slightly higher in the mixotrophic culture than the phototrophic culture.
As shown in the fatty acid composition comparison of FIG. 3, concentration of monounsaturated fatty acids almost tripled from about 6% of total fatty acid in the phototrophic culture to about 16% in the mixotrophic culture. Particularly, the oleic acid concentration increased significantly from about 2% for the phototrophic culture to about 14% for the mixotrophic culture. However, the saturates also doubled in the phototrophic culture as compared to the mixotrophic culture, specifically palmitic acid increased from about 8% in the phototrophic culture to about 19% in the mixotrophic culture. The concentration of palmitoleic acid was slightly higher in the phototrophically cultured microalgae than in the mixotrophically cultured microalgae. Also, the palmitic acid concentration was much more than the palmitoleic acid concentration, whereas the stearic acid was almost negligible compared to the oleic acid and polyunsaturated forms in the mixotrophically cultured microalgae. Overall, the microalgae cultured in mixotrophic conditions may be more valuable as a food or fuel precursor due to higher concentration of unsaturated fatty acids (for food, nutraceutical applications) and a high concentration of palmitic acid (for fuel applications).
Several key deductions may be made from the compositional analysis: 1) as observed in the example, the mixotrophic conditions provide the advantage of producing high levels of carotenes (similar to a phototrophic culture), and high growth rates (similar to a heterotrophic culture); and 2) the ability to transition from phototrophic to mixotrophic culturing conditions and vice versa, provides flexibility in the product profile of the microalgae. For example, the chlorophyll content may be reduced significantly by transitioning from phototrophic to mixotrophic culture conditions just before harvest, which neutralizes the product coloring. Such neutralization of product coloring may be desirable in food and feed applications. Transitioning the culture between phototrophy and mixotrophy may similarly increase or decrease the product content as demonstrated in FIGS. 1-3.
Multi-Stage Culturing with Phototrophy and Mixotrophy
In some embodiments, a multi-stage commercial method of culturing microorganisms, such as microalgae and cyanobacteria, may comprise a transition between culturing conditions in combination with multiple stages in a single culture condition. In some embodiments, a commercial method may have different culture volumes for different culture conditions at different stages. In some embodiments, a commercial method may have different culture volumes for different stages of the same culture conditions.
In some embodiments, a commercial method of culturing microorganisms may comprise growing a culture of microorganisms in phototrophic conditions in a first stage, and growing the culture of microorganisms in mixotrophic conditions in at least one subsequent stage. In a preferred embodiment, the subsequent stages in mixotrophic conditions may comprise at least two stages. In some embodiments, the phototrophic conditions of the first stage comprise a phototrophic culturing vessel with a volume less than 10 L. In some embodiments, the culture of microorganisms may be harvested from the phototrophic culturing vessel and used to inoculate a small volume mixotrophic culturing vessel comprising a volume of 10 to 100 L in a culturing stage using mixotrophic conditions. In some embodiments, the culture of microorganisms may be harvested from the small volume mixotrophic culturing vessel and used to inoculate a large volume mixotrophic culturing vessel comprising a volume greater than 100 L in a culturing stage using mixotrophic conditions. In some embodiments, the large volume mixotrophic culturing vessel comprises a volume between 100 and 750,000 L. The culture of microorganisms may be harvested from the large volume mixotrophic culturing vessel for downstream processing such as, but not limited to, dewatering, extraction, esterification, transesterification, hydrotreatment. In some embodiments, the culturing vessels may be open culturing vessels such as, but not limited to, troughs, ponds and raceway ponds. In some embodiments, the culturing vessels may be closed culturing vessels such as, but not limited to, tanks, column bioreactors, bag bioreactors, column bioreactors, and tubular bioreactors.
In some embodiments, a culture of microorganisms may be cultured mixotrophically for 3-14 days before harvesting the entire volume, and the harvested volume may be split among up to 10 bioreactors of equal volume (i.e., essentially diluting each culture by up to 90%). The plurality of bioreactors may culture the microorganisms in phototrophic conditions for 1-4 days to develop photopigments (e.g., chlorophyll, carotenoids), lipids, proteins, and other compounds using the phototrophic metabolism. The time period of culturing phototrophic and mixotrophic stages may be species specific and dependent on growth rate or contamination risks.
Cultures may then be harvested in their entirety or harvested partially (i.e., 10%-90% harvest) and then transitioned to be cultured in mixotrophic conditions to increase growth. The process may be repeated among multiple series of bioreactors. The process allows for final product curing while producing seed (i.e., inoculum) for the next bioreactor. This process also allows for: reduced cost through less seed production; conditioning of microorganisms for outdoor production as it completes each stage of bioreactors; bioreactors which may reseed themselves after a harvest; and modifying the water quality of the culture by eliminating organic carbon during phototrophic growth. In some embodiments, bacteria that have been identified as providing a positive or neutral effect on the microorganism culture may be added in any one of or combination of stages for multiple purposes such as enhancement of the culture growth, health, and product formation.
Transitions Between Heterotrophy and Mixotrophy
A culture of heterotrophic microorganisms may be axenically produced in fermenters (i.e., heterotrophic bioreactors) at a large scale using convention fermentation technology. The heterotrophically produced microorganisms may be used to inoculate a mixotrophic or phototrophic culture. The utilization of bacterial free or low-bacteria (<104 cells/mL) inoculum may help prevent the colonization of contaminating bacteria in non-axenic mixotrophic cultures in larger production-scale reactors (>500 L volume). Additionally, it has been observed in some species that when phototrophically cultured microalgae is utilized to inoculate a mixotrophic culture, the mixotrophic culture may experience a longer lag period (e.g., 1-100 hours) in the initial growth than microalgae which was previously cultured with at least some organic carbon due to the stress experienced by the cells in phototrophic culturing conditions. Therefore, using heterotrophically cultured microalgae as inoculum for a mixotrophic culture may mitigate the growth lag experienced in mixotrophic cultures inoculated with phototrophically cultured inoculum. In some embodiments, a culture of microorganisms may be grown axenically in a fermenter vessel to a high density in a first step and then seeded in large outdoor bioreactors that are open and capable of operation in phototrophic and mixotrophic culture conditions.
EXAMPLE 2
Haematococcus is known to produce Astaxanthin, with the concentration of Astaxanthin within the cells increasing when the Haematococcus cells are cultured in conditions with at least some light. Most of the Chlorophyceae class of microalgae have developed a phototrophic metabolism, but also evolved developing a heterotrophic metabolism that may be capable of growth in the absence of light with a source of organic carbon.
In one non-limiting embodiment illustrating the potential use of heterotrophy to produce axenic seed for a phototrophic or mixotrophic culture, a fermentation vessel may be utilized to create axenic Haematococcus seed for large outdoor open pond bioreactors reactors receiving at least some light. As shown in FIG. 4, a fermentation vessel may comprise tubing, air filters, media filter, a sampling devise and quick connectors that will be sterilized in an autoclave and carried to a flow hood for preparation. The media will be sterilized through filtration by pumping the media into the fermentation vessel through a 0.1 micron sterile filter. The inlet air will also be sterilized before entering the fermentation vessel. After media supply, the fermentation vessel will be inoculated with an axenic Haematococcus culture grown in flasks. Glucose, sucrose, acetic acid, and/or sodium acetate will be used as the organic carbon source in the fermentation vessel.
The experimental data in FIG. 5 shows that a fermentation vessel utilizing an organic carbon feed of sodium acetate for Haematococcus will reach a concentration of 600,000 cells/ml and will be ready to be transfer the culture of Haematococcus to a larger culturing vessel, such as an open outdoor pond. If the fermentation is not supplied with more nutrients the Haematococcus cells may encyst. The system may need to be replenished with more acetate and nutrients to get higher cell densities. Without an additional batch of organic carbon the cells will start to encyst on about the third day of growing due to a depletion of nutrients and a low dissolved oxygen level. A low rate of mixing with air will be used as Haematococcus may not be able to handle high rates of mixing with air. A fermentation vessel with a higher capacity of mass transfer through the supply of more dissolved oxygen and mixing, along with more nutrient addition, may produce higher cell densities of 2 to 2.5 million cells/ml or 1.5 g/l of biomass. The higher cell densities will allow for the utilization of larger inoculums in open outdoor ponds for mixotrophic or phototrophic culturing. The Astaxanthin which accumulates in the Haematococcus cells in the mixotrophic or phototrophic culture phase will be extracted from the Haematococcus cells using any known method of pigment extraction such as, but not limited to, solvent extraction and supercritical carbon dioxide extraction.
In another embodiment, a culture of mixotrophically or phototrophically produced microorganisms may inoculate a heterotrophic culture. The mixotrophically or phototrophically produced culture inoculating a heterotrophic culture may provide a targeted product in the microorganism or a higher culture density in a shorter time period. As was discussed above with mixotrophic and phototrophic cultures, mixotrophic and heterotrophic cultures may also accumulate pigments (e.g., chlorophyll, carotenoids), proteins, lipids, and other products differently in the microorganism depending on the culturing methods, which allows for the culturing methods to target selected products.
EXAMPLE 3
Haematococcus is known to produce Astaxanthin in culturing conditions with at least some light. The Astaxanthin production is typically induced in nitrogen limited cultures and under high light intensity. The process for Astaxanthin production from Haematococcus in phototrophic conditions is time consuming and requires a large physical footprint to allocate the relatively low cell density of the culture for exposure to the light (e.g., short light path in shallow raceway ponds).
Alternatively, as shown in FIG. 6, the increase in red cysts after 96 hours demonstrates the Astaxanthin accumulation phase may be induced under heterotrophic conditions using the smaller physical footprint of a fermentation vessel (as compared to a short light path of shallow raceway ponds). Once the biomass of Haematococcus is produced in ponds phototrophically over a period of 1-7 days, the cells will be concentrated between 3 and 500 times, and transferred to a fermentation vessel (i.e., heterotrophic bioreactor) that will be supplied with an organic carbon source as an energy and carbon source. The carotenogenesis process which forms the Astaxanthin will occur in the fermentation vessel under strictly heterotrophic conditions. The cells will then increase their formation of pigments and the number of red cysts over time (i.e., the heterotrophic induction phase). The cultures using a fermentation vessel will have a foot print approximately 85 times smaller than the phototrophic ponds used in the “reddening” or cyst cell formation stage ponds.
Transitions Between Phototrophy, Mixotrophy, and Heterotrophy
The transitioning of a culture of microorganisms, such as microalgae and cyanobacteria, between culture conditions may comprise controlling light exposure, carbon dioxide supply, and organic carbon supply for the culture. When transitioning to phototrophy: the exposure of the culture of microorganisms to light may be increased to a high light level, the supply of carbon dioxide to the culture may be increased to a higher level, and the supply of organic carbon will cease. When transitioning to mixotrophy: the exposure of the culture of microorganisms to light may be increased (i.e., transition from heterotrophy) or decreased (i.e., transition from phototrophy) to a low light level, the supply of carbon dioxide may be increased (i.e., transition from heterotrophy) or decreased (i.e., transition from phototrophy), and the organic carbon source will be supplied. When transitioning to heterotrophy: the exposure of the culture of microorganisms to light will cease, the supply of carbon dioxide will cease, and the organic carbon source will be supplied. In some embodiments, different wavelengths, photoperiods, and intensities of light may be applied in phototrophic and mixotrophic conditions, such as but not limited to monochromatic (single light wavelengths) light wavelengths between 200-800 nm (e.g., red wavelengths, blue wavelengths), combinations of various light wavelengths and intensities of light wavelengths between 200-800 nm, constant light, and flashing light. In some embodiments high light levels may comprise light above 400 μmol/m2 s, and low light levels may comprise light below 400 μmol/m2 s, however the sensitivity to and utilization efficiency of light will vary between species, and thus high and low light levels may be species specific.
In some embodiments, a culture may transition from phototrophy to mixotrophy to heterotrophy. In some embodiments, a culture may transition from heterotrophy to mixotrophy to phototrophy. In some embodiments, a culture may transition from phototrophy to heterotrophy to mixotrophy. In some embodiments, a culture may transition from mixotrophy to phototrophy to heterotrophy. In some embodiments, a culture may transition from mixotrophy to heterotrophy to phototrophy. In some embodiments, a culture may transition from heterotrophy to phototrophy to mixotrophy. In some embodiments, the transitions of the culture of microorganisms between culture conditions may be cyclic or follow a pattern.
Transition Based on Sunlight Levels
In some embodiments, the transition between trophic conditions may correspond to the light/dark cycle of the sun. Phototrophic cultures of microorganisms may lose growth achieved during the high light conditions of daylight through respiration as the availability of light is limited during the night and hours of low sunlight during sunrise and sunset. The use of artificial lights to provide 24 hour lighting to maintain phototrophic growth may add costs to the system and reduce the overall energy efficiency of the microorganism culturing system, which is undesirable in a commercial setting. However, transitioning the culture to mixotrophy in the low light portions of sunset and heterotrophy during the night (i.e., absence of light) may preserve the growth achieved through phototrophy during the day, and possibly add additional growth. In some embodiments, the culture may be transitioned from heterotrophy to mixotrophy in the low light conditions of sunrise following night time, and to phototrophy during the high light conditions of daylight. In some embodiments, the culture may transition from phototrophy to mixotrophy during the low light conditions of sun set following the high light conditions of daylight, and to heterotrophy during night time.
In some embodiments, the culture of microorganisms may be fed organic carbon outside of the peak hours of sunlight, and no organic carbon during the peak hours of sunlight. The peak hours of sunlight defined as an equal amount of time on either side of the time when the sun is at its zenith. In some embodiments, the peak hours of sunlight may comprise 6-10 hours. Night may be defined as the time period of no sunlight between when the sun has completely set and when the sun begins to rise. In some embodiments, a culture may transition from phototrophy to mixotrophy to heterotrophy in the time frames corresponding to the peak hours of sunlight (phototrophy), the non-peak hours of sunlight (mixotrophy), and night (heterotrophy). In some embodiments, a culture may transition from heterotrophy to mixotrophy to phototrophy in the time frames corresponding to night (heterotrophy), the non-peak hours of sunlight (mixotrophy), and the peak hours of sunlight (phototrophy). In some embodiments, a culture may transition from phototrophy to mixotrophy to heterotrophy to mixotrophy to phototrophy in a repeating cycle based on the light/dark cycle of the sun and peak hours of sunlight. In some embodiments, the organic carbon may be added at night when the sun sets, and when the sun begins to rise organic carbon is no longer added to the system. In some embodiments, the culture of microorganisms may be cultured with a method using combinations of phototrophic, mixotrophic, and heterotrophic culture conditions for at least two consecutive days, and in preferred embodiments for 2 to 90 consecutive days.
Transition Based on Threshold Condition
In some embodiments, the time a culture of microorganisms spends in a culture condition before transitioning to another culture condition may be dictated by the occurrence of at least one threshold condition such as, but not limited to, a detected level of culture cell density, a detected level of dissolved oxygen, a detected level of dissolved carbon dioxide, a detected pH level, a detected level of contaminating organisms, a detected level of chlorophyll, complete consumption of a batch of nutrients, complete consumption of a batch of organic carbon, a detected temperature level, a detected residual nitrate level, and a detected residual organic carbon level. In some embodiments, the transition to another culture condition may occur when the detected level is below a threshold level or set point. In some embodiments, the transition to another culture condition may occur when the detected level is above a threshold level or set point.
In some embodiments, the transition from phototrophy to mixotrophy or heterotrophy may occur when the detected dissolved oxygen level is above a threshold level. A transition from phototrophy to mixotrophy or heterotrophy in a high dissolved oxygen states allows the microorganism to consume some of the oxygen to reduce an inhibiting effect on the microorganisms' growth that may occur in phototrophic culture conditions with high dissolved oxygen. In some embodiments, the transition from mixotrophy or heterotrophy to phototrophy may occur when the detected dissolved oxygen level is below a threshold level.
A transition from mixotrophy or heterotrophy, where the microorganisms consume oxygen, to phototrophy may allow for the oxygen in the culture to be replenished through photosynthetic activity instead of from an external source and enable further heterotrophic growth in a subsequent culturing stage. In some embodiments, the dissolved oxygen threshold level may range from 0.1 mg O2/L to about 30 mg O2/L. In some embodiments, the dissolved oxygen level may be decreased to 6 mg O2/L-0.1 mg O2/L in phototrophic conditions to reduce potential contamination prior to switching to mixotrophic conditions. In some embodiments, the level of dissolved oxygen may be increased to 10 mg O2/L-30 mg O2/L to reduce contamination prior to switching the culture to mixotrophic conditions.
In some embodiments, the transition from mixotrophy or heterotrophy to phototrophy may occur when the detected carbon dioxide level is above a threshold level or set point. Microorganisms in mixotrophic or heterotrophic culture conditions may produce carbon dioxide to a saturation point which slows the mixotrophic or heterotrophic growth, but may boost phototrophic growth where carbon dioxide is consumed. The saturation level of carbon dioxide that inhibits growth and consumption rate of carbon dioxide may be species specific. In some embodiments, the transition from phototrophy to mixotrophy or heterotrophy may occur when the detected carbon dioxide level is below a threshold level. The transition from phototrophy, wherein carbon dioxide is consumed, to mixotrophic or heterotrophic conditions of net carbon dioxide production may allow for the carbon dioxide level of the culture to be replenished through metabolic activity instead of from an external source. Replenishing the carbon dioxide level may enable further photosynthetic growth in a subsequent culturing stage.
In some embodiments, the transition from heterotrophy to mixotrophy or phototrophy, or the transition from mixotrophy to phototrophy, may occur when the detected level of chlorophyll is below a threshold level or set point. Microorganisms in heterotrophic conditions comprising an absence of light may produce less chlorophyll than cultures exposed to at least some light. The chlorophyll level in the microorganisms may be increased by transitioning from heterotrophic to phototrophic or mixotrophic conditions comprising at least some light when the detected chlorophyll level is low, or from mixotrophic to phototrophic for better utilization of high light conditions when the chlorophyll level is low. In some embodiments, the transition from phototrophy or mixotrophy to heterotrophy may occur when the detected level of chlorophyll is above a threshold level.
While some level of chlorophyll may be desirable for certain product markets, transitioning the culture from phototrophy or mixotrophy to heterotrophy may maintain the chlorophyll at a desired level or reduce the chlorophyll content in a final culture stage. In some embodiments, the light exposure may initially comprise lower energy light (e.g., wavelengths between 500 nm-800 nm) and/or lower levels of intensities slowly increased over time. In some embodiments, the light exposure may initially comprise higher energy light (e.g., wavelengths between 200 nm-500 nm) and/or lower levels of intensities slowly increased over time. In some embodiments, the light exposure may initially comprise higher energy light (e.g., wavelengths between 200 nm-500 nm) and/or higher levels of intensities slowly increased over time.
In some embodiments, the transition from phototrophy to mixotrophy may occur when the detected level of culture cell density is above a first threshold level or set point. The first threshold level may comprise a culture density at which self shading begins to inhibit growth, such as but not limited to a density of 1 g/L for some species. In further embodiments, the transition from mixotrophy to heterotrophy may occur when the detected level of the culture density is above a second threshold level. The second threshold level may comprise a culture density at which the mixing regime is no longer capable of exposing all cells of the culture to at least some light, such as but not limited to 2 g/L for some species.
A culture of microorganisms which starts out in phototrophic conditions may utilize the low cell culture density to minimize self-shading and maximize exposure to light for photosynthetic activity. As the cell culture density increases to a first threshold level due to the phototrophic growth and with an accompanying increase in self-shading, the growth of the microorganism culture may be sustained by transitioning to mixotrophic conditions which require less light and can also supply energy to the microorganisms through an organic carbon source. As the cell culture density increases to a second threshold level due to the mixotrophic growth and with an accompanying increase in self-shading, the growth of the microorganism culture may be sustained by transitioning to heterotrophic conditions which requires no light and supplies energy to the microorganisms through an organic carbon source. The culture densities at which the growth stagnates for certain growth conditions may be species specific.
In some embodiments, the transition between culture conditions may occur when the pH level is below a threshold level or set point. In some embodiments, the transition between culture conditions may occur when the pH level is above a threshold level. In some embodiments, the pH level ranges from about 6 to about 9. In other embodiments, the pH level ranges from about 1 to about 5. The pH level of a culture may be influenced by a variety of factors in the culture such as, but no limited to, the inorganic carbon, the organic carbon, and the metabolic activity of the microorganism. Therefore, the pH level may be used as an indicator to initiate a transition to different culture conditions, which use different types or amounts of carbon and alters the metabolic activity of the microorganism to maintain the microorganisms in optimal culture conditions for growth or product accumulation.
In some embodiments, the transition from phototrophy to mixotrophy or heterotrophy may occur when the detected level of contaminating organisms is below a threshold level or set point. The organic carbon provided in mixotrophic and heterotrophic conditions may provide a feed source for contaminating organisms, and thus allowing the transition to mixotrophy or heterotrophy from phototrophy only when the detected level of contaminating organisms is low may keep the contaminating organisms population under control. In some embodiments, the transition from mixotrophy or heterotrophy to phototrophy may occur when the detected level of contaminating organisms is above a threshold level. The transition to phototrophy may reduce the organic carbon available as feed to the contaminating organisms and help keep the contaminating organism population under control. In some embodiments, the desired level of contaminating organisms is less than 25%, 20%, 10%, or 5% of the total cells in a culture. Methods of detecting contaminating organisms may comprise any method known in the art such as, but not limited to, observation of a sample under a microscope, automated nucleic acid sequencing, oil immersion-phase contrast micrograph and cell cytometry with bacterial dying, and quantitative polymerase chain reaction (QPCR).
In some embodiments, the transition from phototrophy to mixotrophy or heterotrophy may occur when the detected residual organic carbon level is below a threshold level or set point. The organic carbon provided in mixotrophic and heterotrophic conditions may provide a feed source for contaminating organisms, and thus allowing the transition to mixotrophy or heterotrophy from phototrophy only when the detected residual organic carbon level is low may keep the contaminating organisms population under control by only having enough organic carbon in the culture for the microalgae or cyanobacteria to consume. In some embodiments, the transition from mixotrophy or heterotrophy to phototrophy may occur when the detected residual organic carbon level is above a threshold level. The transition to phototrophic conditions with no organic carbon feed may reduce the organic carbon available as feed to the contaminating organisms and help keep the contaminating organism population under control. In some embodiments, the detection of other residual nutrients (e.g., nitrates) is a trigger for when to transition between culture conditions may be used in a similar as that described above for organic carbon to achieve the goal of minimizing the feed source available to contaminating organisms. In some embodiments, the level of residual organic carbon and other nutrients may comprise less than 1,000 ppm, 500 ppm, 250 ppm, 200 ppm, 100 ppm, or 50 ppm. Consumption rate of organic carbon, nitrates, or other nutrients may be species specific.
Similarly, the transition between culture conditions may also be dictated by consumption of a batch of nutrients, nitrates, and/or organic carbon. In some embodiments, the culture of microorganisms may transition from mixotrophy or heterotrophy to phototrophy when a batch of organic carbon has been completed consumed. The complete consumption of a batch of nutrients, nitrates, and organic carbon may be indicated by a detection of residual nutrients, nitrates or organic carbon a negligible amounts (e.g. 25 ppm or less).
In some embodiments, the transition from heterotrophy to mixotrophy or phototrophy may occur when the temperature of the culture is below a threshold level or set point. In some embodiments, the transition from phototrophy to mixotrophy or heterotrophy may occur when the temperature of the culture is above a threshold level. In some embodiments, the temperature of the culture ranges from about 10° C. to about 30° C. In other embodiments, the temperature of the culture ranges from about 30° C. to about 50° C. The temperature of a culture may be influenced by a variety of factors in the culture such as, but no limited to, the light and the metabolic activity of the microorganism. A transition from heterotrophy (i.e., no light) or mixotrophy (i.e., low light) to phototrophy (i.e., high light) may increase the temperature of the culture due to the heat from the supplied light energy. A transition from phototrophy (i.e., high light) to heterotrophy (i.e., no light) or mixotrophy (i.e., low light) may decrease the temperature of the culture due to the heat from the supplied light energy. Therefore, the temperature of a culture may be used as an indicator to initiate a transition to different culture conditions in which different amounts of light may influence the culture temperature and alter the metabolic activity of the microorganism.
When transitioning a culture between trophic conditions based on sunlight levels or threshold conditions, the transition may be automated by parameter detection equipment (e.g., probes, sensors), a computer, and programmable logic controller (PLC) receiving input from parameter detection equipment and controlling valves and actuators. In some embodiments, a photodiode or photodetector and photovoltaic panel may be used in combination to transition a culture from phototrophic to mixotrophic or heterotrophic conditions in an automated manner. The photodiode or photodetector may detect light that is below a threshold value or set point which indicates low light conditions such as, but not limited to, cloudy days, rainy days, non-peak sunlight hours, and night. When light is detected below the threshold value, the PLC may transition the culture from phototrophic culture conditions to mixotrophic or heterotrophic conditions by controlling the aspects of the system such as, but not limited to, light supply/exposure, inorganic carbon supply, organic carbon supply, dissolved oxygen, and mixing.
In some embodiments, the organic carbon supply rate and the inorganic carbon supply rate may be monitored and controlled by the PLC to maintain the culture conditions in a net carbon dioxide usage state as the PLC transitions the culture between conditions over a time period. Maintaining a net carbon dioxide usage state may be useful in carbon remediation applications of microorganism cultures. In some embodiments, a dissolved oxygen (DO) probe may be used as a proxy for a light or photo sensors. The DO probe may monitor the DO concentration, and enable the inference by the PLC that when the DO level is rising the light may be available for phototrophic conditions and initiate a transition to phototrophy. Also, when the DO level stops rising, light may not be available for phototrophic conditions and the PLC may initiate a transition to mixotrophic or heterotrophic conditions.
In some embodiments, a culture operating under phototrophic conditions utilizing carbon dioxide as inorganic carbon may transition to mixotrophic conditions with the addition of an organic carbon source in the bioreactor. The organic carbon source may be added in a cyclical pattern spaced in minutes to hours via feedback control from a detected culture parameter (e.g., pH, nitrate, total organic carbon, optical density, dissolved oxygen, bacteria contamination). Such culture parameters may be measured and monitored by probes and sensors known in the art and available commercially from companies such as YSI.
In some embodiments, the organic carbon may be added in a batch method where a particular amount of the organic carbon is added to support about 24 hours of growth (dependent on species), then the bioreactor may run for about 24 hours in phototrophic conditions with no organic carbon, and this process may be repeated. In some embodiments, the organic carbon may be added in a method where mixotrophic conditions operate for about 7-10, transitioned to phototrophic conditions for about 2 days, and then transitioned back to mixotrophic conditions. The methods described for adding organic carbon in this specification may be used individually or in combination.
Bioreactor Systems with Multiple Zones
In some embodiments, the transition between culture conditions may be not be dictated solely by culture parameters, but instead may also be dictated by the configuration of a culturing vessel or bioreactor system. In some embodiments, a bioreactor system may have organic carbon supply devices disposed in some portions and no organic carbon supply devices in other portions. In some embodiments, a bioreactor system may provide varying levels of light exposure to the culture of microalgae in some portions and block all exposure to light in other portions. In some embodiments, a bioreactor system may have carbon dioxide supply devices disposed in some portions and no carbon dioxide supply devices in other portions.
By circulating the microorganism culture through portions of a bioreactor system with different combinations of organic carbon supply devices, carbon dioxide supply devices, and exposure to light, the microorganism culture may be transitioned between phototrophic, mixotrophic, and heterotrophic zones of culture conditions. In some embodiments, at least one phototrophic culture zone may comprise at least one carbon dioxide supply device, no organic carbon supply device, and may be configured to expose the culture of microorganisms to at least some light. In some embodiments, the phototrophic zone may comprise an organic carbon supply device which may be deactivated during phototrophic culture conditions. In some embodiments, at least one mixotrophic culture zone may comprise at least one carbon dioxide supply device, at least one organic carbon supply device, and may be configured to expose the culture of microorganisms to at least some light. In some embodiments, at least one heterotrophic culture zone may comprise an organic carbon supply device, no carbon dioxide supply device, and may be configured to block the microorganisms from exposure to light. In some embodiments, the bioreactor system may be configured to allow the microorganisms to consume substantially all organic carbon supplied to the microorganisms in a mixotrophic or heterotrophic zone before the culture of microorganisms enter a phototrophic zone.
In some embodiments, a circulation system such as, but not limited to, at least one pump and fluid conduit may circulate the culture throughout the bioreactor system and plurality of zones. In some embodiments, the circulation of the microorganism culture between the at least one phototrophic, mixotrophic, and heterotrophic zones may be dictated by a detected culture parameter such as, but not limited to, cell culture density, dissolved oxygen, dissolved carbon dioxide, pH, contamination, chlorophyll content, nutrient levels, and temperature. In some embodiments the bioreactor system may comprise adjustable artificial lights and light blocking devices (e.g., louvers, covers, awnings, photovoltaic panels), controllable organic carbon supply devices, and controllable carbon dioxide supply devices, which may transition a single zone in the bioreactor system between functions as a phototrophic, mixotrophic, and heterotrophic zone.
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

## Claims

1. A method of culturing microorganisms, comprising:
a. Providing a culture of microorganisms in an aqueous culture medium capable of growth on light as an energy source, carbon dioxide as a carbon source, and at least one organic carbon source as an energy and carbon source;
b. Decreasing at least one of a total fat, oleic acid, and palmitic acid as a percentage of cell dry weight in the culture of microorganisms by transitioning the culture of microorganisms from mixotrophic culture conditions to phototrophic culture conditions, wherein:
i. Phototrophic culture conditions comprise a supply of light, a supply of carbon dioxide, and the absence of a supply of an organic carbon source; and
ii. Mixotrophic culture conditions comprise a supply of light, and a supply of at least one organic carbon source;

c. Wherein the transition from mixotrophic culture conditions to phototrophic culture conditions occurs when at least one threshold condition of the culture is met, the at least one threshold condition comprising:
i. A detected level of dissolved oxygen;
ii. A detected level of contaminating organisms; and
iii. A detected level of cell culture density.

2. The method of claim 1, wherein the culture of microorganisms comprises at least one selected from the group consisting of microalgae and cyanobacteria.

3. The method of claim 2, wherein the microalgae comprises Chlorella.

4. The method of claim 1, wherein the at least one organic carbon source is selected 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 stillage, urea, industrial waste solutions, and yeast extracts.

5. The method of claim 1, wherein the transition between culture conditions is automated by a computer and programmable logic controller receiving input from at least one sensor detecting the at least one threshold condition to control at least one of the supply of light, supply of carbon dioxide, and supply of the at least one organic carbon source.

6. The method of claim 1, wherein the culture of microorganisms transitions from mixotrophic to phototrophic culture conditions when the detected dissolved oxygen level is below a threshold level.

7. The method of claim 1, wherein the culture of microorganisms transitions from mixotrophic to phototrophic culture conditions when the detected level of contaminating organisms is above a threshold level.

8. The method of claim 1, wherein the culture of microorganisms transitions from mixotrophic to phototrophic conditions when the detected level of cell culture density is above a threshold value.

9. The method of claim 1, wherein the decrease of total fat as percentage of dry weight in the culture of microorganisms by transitioning from mixotrophic culture conditions to phototrophic culture conditions is up to 62%.

10. The method of claim 1, wherein the decrease of oleic acid as percentage of dry weight in the culture of microorganisms by transitioning from mixotrophic culture conditions to phototrophic culture conditions is up to 86%.

11. The method of claim 1, wherein the decrease of palmitic acid as percentage of dry weight in the culture of microorganisms by transitioning from mixotrophic culture conditions to phototrophic culture conditions is up to 58%.

12. The method of claim 1, wherein the transition from mixotrophic culture conditions to phototrophic culture conditions further results in a suppression of the contaminating organisms below a level of 25% of total cells in the culture.

13. The method of claim 6, wherein the threshold level of dissolved oxygen is in the range of 0.1 to 30 mg O2/L.

14. The method of claim 13, wherein the threshold level of dissolved oxygen is in the range of 0.1 to 6 mg O2/L.

15. The method of claim 13, wherein the threshold level of dissolved oxygen is in the range of 10 to 30 mg O2/L.