Cultivation of phototrophic organisms

Abstract

The present disclosure relates to a method for culturing a phototrophic organism in an aqueous medium under culture conditions including light and a gaseous atmosphere containing carbon dioxide. The culture can be continued. The method is useful for carbon dioxide capture and / or production of organic feedstock useful for downstream processes, such as for producing biofuels and / or biofertilizers. The present disclosure further relates to a CO2-adapted Chlorella vulgaris strain.

Description

[Technical Field]

[0001] Reference to earlier application This application claims the benefit of Australian Provisional Patent Application No. 2023900393, filed February 16, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to a method for culturing a phototrophic organism in an aqueous medium under culture conditions including light and a gaseous atmosphere containing carbon dioxide. The culture can be continued. The method is useful for carbon dioxide capture and / or production of organic feedstock useful for downstream processes, such as for producing biofuels and / or biofertilizers. The present disclosure further relates to a CO2-adapted Chlorella vulgaris strain. [Background technology]

[0003] A major contributor identified to the phenomenon of global warming and its associated environmental problems is carbon dioxide (CO2) emissions. CO2 is primarily a by-product of combustion. CO2 production is largely anthropogenic and occurs in various industrial applications, such as power generation from coal in power plants, and in the use of hydrocarbons, which are typically the major component of fuels burned in combustion devices, such as engines. Exhaust gases emitted from such combustion devices contain CO2 gas, which is currently simply released into the atmosphere. Anthropogenic global carbon dioxide (CO2) emissions have risen by approximately 50% in recent decades. While political, societal, and industrial focus has been on mitigating emissions, this alone is widely viewed as insufficient to curb the continuing adverse impacts on the global environment. Therefore, a greater focus on carbon dioxide removal (CDR) technologies (also known as carbon capture) that can result in net-negative emissions is needed.

[0004] Existing CDR techniques include afforestation and reforestation, soil carbon sequestration, enhanced weathering, ocean fertilization, carbon capture and storage (CCS) and bioenergy, and direct air carbon capture. These techniques have limitations. For example, terrestrial plants have the innate ability to capture CO2 through the photosynthetic process and are typically adapted to atmospheric CO2 concentrations of approximately 0.033%, whereas industrial exhaust gases contain much higher concentrations, usually in the range of 10–25% CO2. Therefore, afforestation and reforestation require large tracts of land to be nearly net negative, which is offset by the release of captured carbon back into the atmosphere as the biomass decays or is burned. There are also biodiversity concerns associated with afforestation. Therefore, afforestation processes have limited practicality and commercial feasibility. Enhanced weathering also has limited commercial feasibility and carries the risk of adverse impacts on biodiversity, water quality, and food security. Ocean fertilization is also limited in its applicability due to its minimal practical impact and enormous material demands. Carbon capture and storage and bioenergy remain the most plausible and scalable pathways to achieving a net-negative carbon economy, but are not without their challenges.

[0005] Several carbon capture methods have been proposed for sequestering CO2 produced by industrial processes, such as coal-fired and other power plants. One method involves reacting CO2 with large quantities of metal oxides, particularly calcium and magnesium oxides, and then burying the resulting carbonates. This method has the disadvantage of requiring a large and continuous supply of minerals. Another method involves removing CO2 from combustion gases, compressing it, transporting it by pipeline to peridotite or serpentinite mines for conversion to carbonates, and then burying it at the mine site. However, CO2 is heavier than air, and transporting compressed CO2 presents material handling challenges. For example, if CO2 is accidentally released during transport, it can remain near the ground and be life-threatening. Other issues can be the availability of mine sites for such burials, as well as the cost and energy consumption of transportation. Other technologies have also been attempted, which can be generally classified as geological, terrestrial, or marine systems, relying on the transport of CO2 to injection sites in geological formations, soils, or ocean repositories. However, as with peridotite or serpentine mining, each of these processes is expensive in terms of capital costs, transportation, and energy consumption. These carbon capture methods also focus on storing carbon rather than converting it into useful products.

[0006] One method that has recently gained attention involves the use of microalgae. Microalgae have the innate ability to capture CO2 at a higher rate than terrestrial plants, and algal biomass is a useful product in a number of downstream industrial processes. However, culturing algae for carbon capture is challenging. Algal growth profiles follow a general path consisting of a long lag phase, during which biomass remains constant or increases slowly, followed by an exponential phase, during which the growth rate rapidly increases, eventually reaching a pre-exponential phase. While the CO2 capture capacity of the biomass increases throughout the exponential phase, the exponential phase is generally short-lived, with the algae transitioning to a stationary phase, during which net growth ceases, followed by a death phase, during which viable biomass rapidly declines. Thus, the exponential phase, during which adequate levels of carbon capture are achieved, is short-lived. Algal cultures are also sensitive to environmental changes and may not grow, or may not grow at an adequate rate, in environments containing carbon dioxide at levels typically found in industrial emissions.

[0007] It is possible to produce algae that are tolerant to high CO2 environments through a process of adaptive laboratory evolution (ALE), which involves exposing algae to an initial low CO2 concentration, followed by a slight increase in the concentration once the algae have stabilized until the desired tolerance is reached. ALE is typically a gradual process, taking at least several months and even years. Furthermore, once a suitable culture is finally produced, it must be grown to a useful commercial scale for CO2 capture, shortly thereafter losing viability at the end of a single exponential growth phase and the process must be repeated. There are also limits to the level of tolerance that can be achieved, frequently lower than the carbon dioxide levels commonly found in industrial emissions.

[0008] It would be advantageous to provide CO2 sequestration technologies that can convert carbon into useful downstream products and / or to provide improved cultivation methods for algae that can be used, particularly in CO2 sequestration processes of industrial emissions.

[0009] With respect to any prior art publications or references referred to herein, it should be understood that such reference does not amount to an admission that those publications form part of the general knowledge in the art in any country. Summary of the Invention

[0010] In a first aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: a) growing a phototrophic organism in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide; and b) when the phototrophic organisms reach a point between the exponential and stationary growth phases, reducing the biomass concentration of the phototrophic organisms in the aqueous medium to a reduced biomass concentration that is equal to or greater than the concentration present during the early exponential growth phase; The present invention provides a method for culturing a phototrophic organism, comprising:

[0011] In some embodiments, reducing the biomass concentration includes removing phototrophic biomass from the aqueous medium. The removed biomass may be referred to as organic feedstock. This may be done in conjunction with the addition of a dilution volume of aqueous medium. The dilution volume of aqueous medium may be freshly prepared or recycled.

[0012] In some embodiments, the method further comprises cycling the reduced biomass concentration, wherein cycling comprises repeating steps a) and b) using the reduced biomass concentration, preferably repeating steps a) and b) multiple times using the reduced biomass concentration for each step b). When the method comprises a cycling step, it may be referred to as a continuous culture method.

[0013] In some embodiments, sources of carbon dioxide include waste gas streams, such as waste gas streams produced by combustion of hydrocarbons (e.g., flue gas), by steam reforming, and / or by biological decomposition.

[0014] In another aspect, the present disclosure provides a method of continuously culturing a phototrophic organism, the method comprising performing the method of the first aspect described herein and cycling the reduced biomass concentration, wherein the cycling comprises repeating steps a) and b) using the reduced biomass concentration, preferably repeating steps a) and b) multiple times using the reduced biomass concentration for each step b).

[0015] In another aspect, the present disclosure provides a method of culturing a phototrophic organism, comprising growing the phototrophic organism in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide, wherein the light is at an intensity of between about 3000 lux and about 7000 lux, and the culture conditions further comprise a photoperiod of between about 12 h and about 20 h.

[0016] In another aspect, the present disclosure provides a culture of a phototrophic organism produced by the methods described herein.

[0017] In another aspect, the present disclosure provides an organic feedstock produced by the methods described herein, wherein reducing the biomass concentration comprises removing a quantity of phototrophic biomass from the aqueous medium, and the organic feedstock comprises the removed phototrophic biomass.

[0018] In another aspect, the present disclosure provides the methods described herein when used in the production of organic feedstocks.

[0019] In another aspect, the present disclosure provides a method for recovering carbon dioxide from a waste gas stream, comprising culturing a phototrophic organism according to the methods described herein, wherein the source of carbon dioxide comprises the waste gas stream.

[0020] In another aspect, the present disclosure provides the methods described herein when used in the recovery of carbon dioxide from waste gas streams.

[0021] In another aspect, the present disclosure provides a CO2-adapted phototrophic organism, wherein the phototrophic organism is selected from one or both of C. vulgaris species strains CS-41 or CCAP211 / 11S, preferably CS-41, and wherein the phototrophic organism is CO2-adapted to a gaseous atmosphere containing a carbon dioxide concentration of 5% or greater. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram of the photobioreactor setup, where the triangle represents the confluence of the CO2 and air inlets and the rotameter is the electronic pressure regulator. [Figure 2-1] Comparison of (a) biomass concentration, (b) pH, and (c) biomass growth rate from the fourth generation (4G) and fifth generation (5G) in 10% CO2 (BBM culture medium, 0.05 g / L inoculum, 12:12 light:dark cycle). [Figure 2-2] (Continued) Comparison of (a) biomass concentration, (b) pH, and (c) biomass growth rate from the fourth generation (4G) and fifth generation (5G) in 10% CO2 (BBM culture medium, 0.05 g / L inoculum, 12:12 light:dark cycle). [Figure 3] (a) Elemental analysis data at 7 days, (b) CO2 biofixation rate, and (c) CO2 concentration in the inlet and outlet gas streams measured by a CO2 sensor for biomass from generations 4 and 5 in 10% CO2 (BBM culture medium, 10% CO2, 0.05 g / L inoculum, 12:12 light:dark cycle). [Figure 4] 1 shows the effect of light intensity on the growth rate of C. vulgaris CS-41 grown in different culture media. [Figure 5] 1 shows the effect of light intensity on biomass production by C. vulgaris CS-41 grown in different culture media. [Figure 6] 1 shows the effect of light intensity on CO2 biofixation by C. vulgaris CS-41 grown in either BBM or BG-11 culture medium. [Figure 7] 1 shows the effect of light intensity on total CO 2 recovery by C. vulgaris CS-41 grown in either BBM or BG-11 culture medium. [Figure 8] Biomass of C. vulgaris CS-41 grown in either BBM or BG-11 culture medium under five photoperiods (12:12, 16:8, 18:6, 20:4, and 24:0 h light / dark) is shown. [Figure 9] Figure 1 shows the dry weight biomass (g / L) of C. vulgaris at different pH (6, 7, 8, and 9) under a constant photoperiod (16:8 h) and light intensity (5000 lux) during 7 days of cultivation in BG-11 and BBM culture media. [Figure 10] Average CO2 fixation rates by C. vulgaris CS-41 grown at various pHs in BBM or BG-11 over a 7-day culture period are shown. [Figure 11] Figure 1 shows total CO2 recovery by C. vulgaris CS-41 grown at various pHs in BBM or BG-11 at 7 days. [Figure 12] Comparison of pre- and post-optimization C. vulgaris growth rates grown in BG-11 culture medium. [Figure 13] The effect of fresh and recycled culture medium on biomass concentration is shown (BBM culture medium, 10% CO, 0.05 g / L inoculum, 12:12 light:dark cycle; note: cycle 1 - test with fresh BBM, cycle 2 - test with used BBM at the end of cycle 1). [Figure 14] 7-day growth curve of C. vulgaris CS-41 under optimized conditions. [Figure 15]Flow diagrams for the continuous circulation methods (CCA) and (CCB). A starter batch culture is grown for 7 days (cycle 0). The biomass from cycle 0 is split into two batches (fresh, CCA; or recycled, CCB) and incubated in low light for an additional 2 days (cycle 1). Half of the biomass is removed for downstream use (e.g., biodegradation), and the remaining batch is replenished with either fresh or recycled medium, as before. This process is repeated until the subsequent batch loses health, as measured by predetermined growth criteria. [Figure 16] Biomass production of C. vulgaris CS-41 when grown under continuous circulation methods (A) CCA (biomass separation and medium refreshment) and (B) CCB (biomass concentration and medium recycling). [Figure 17] A comparison between continuous culture CCA and a batch culture system (BCS) is shown in terms of (A) the amount of CO2 recovery in g / L and (B) the amount of CO2 recovery in total CO2 recovery. [Figure 18] (a) Overview of the overall material balance and optimal operating conditions in the reactor, (b) a simplified flow diagram of the microalgae cultivation process with gas recycle. [Figure 19] 1 is a graph showing biomass accumulation (g / L) at time intervals during the acclimation phase. [Figure 20] 1 is a graph showing the effect of biomass harvest on total biomass (g / L) of a continuous culture process across multiple biomass harvest events. [Figure 21] There are two graphs: Graph A shows the biomass density (g / L) for each harvest interval treatment over time since harvesting began; Graph B shows the total biomass harvested from each of the different treatments (30 min, 1 h, 2 h (duplicate), 3 h, and 4 h harvest intervals). DETAILED DESCRIPTION OF THE INVENTION

[0023] The following is a detailed description of the present disclosure provided to aid those skilled in the art in practicing the disclosure. Those skilled in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure.

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described.

[0025] As used herein, the term "and / or," e.g., "X and / or Y," is understood to mean either "X and Y" or "X or Y," providing clear grounds for either or both meanings.

[0026] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0027] As used herein, the term "about," when in reference to a numerical value or range, allows for some variation in the value or range, for example, within 10% of the stated limits of the range.

[0028] Ranges provided herein are understood to be shorthand notations for all of the values ​​within that range. For example, a range of 1 to 10 is understood to include ranges specified as "between" 1 and 10, and to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Similarly, a range of 1.0 to 2.0 is understood to include any number, combination of numbers, or subrange from the group consisting of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0.

[0029] As used herein, the phrase "at least one of," when used in conjunction with a list of items, means that one or more different combinations of the listed items can be selected, and that only one of the items in the list can be selected. In other words, "at least one of" means that any number or combination of items from the list can be selected. For example, "at least one of item A, item B, and item C" can mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C.

[0030] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", and variations thereof, such as "comprises" and "comprising", will be understood to imply the inclusion of a specified integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0031] Reference will now be made in detail to preferred exemplary embodiments of the present disclosure. While the present disclosure will be described in connection with the exemplary embodiments, it will be understood that it is not intended to limit the disclosure to those embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the present disclosure, as defined by the appended claims.

[0032] phototrophs The present disclosure provides a method of culturing a phototrophic organism comprising growing the phototrophic organism in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide.

[0033] "Phototroph" means an organism that can use light as the primary energy source for metabolism in the presence of water and carbon dioxide to produce energy in the form of carbohydrates by photosynthesis. Examples include species of algae, phytoplankton, bacteria, and protists.

[0034] In a preferred embodiment, the phototrophic organism is an alga. The algae can be identified as a photosynthetic, eukaryotic, cryptogamic aquatic plant. Examples include red algae, green algae, and cyanobacteria, including plant species generally classified as Halvaria and Hacrobia (red algae), which include Heterokontophytes, Dinoflagellates, Haptophytes, and Cryptophytes; Excavata and Rhizaria (green algae), which include Chlorarachniophytes, Euglenids, and Chlorophytes; and Primary Plants and Archaeplastida (cyanobacteria), which include Chlorophytes and Red Algae. Preferably, the phototrophic organism is a microalga. The microalgae can be identified as a unicellular algae, or may otherwise be called a microphyte. Preferably, the microalgae are green algae. Preferably, the microalgae is from the Chlorella species, such as Chlorella autotrophica, Chlorella colonials, Chlorella lewinii, Chlorella minutissima, Chlorella pituita, Chlorella pulchelloides, Chlorella pyrenoidosa, Chlorella rotunda, Chlorella singularis, Chlorella sorokiniana, Chlorella variabilis, Chlorella volutis and Chlorella vulgaris. vulgaris).

[0035] In a preferred embodiment, the microalgae is of the C. vulgaris species, preferably selected from one or both of the C. vulgaris species strains CS-41 and CCAP211 / 11S, most preferably CS-41. A culture of C. vulgaris strain CS-41 is maintained at the Australian National Algae Culture Collection (CSIRO, Hobart, Tasmania, Australia) under CS number "CS-41." A culture of C. vulgaris strain CCAP211 / 11S is maintained at the Culture Collection of Algae and Protozoa (Oban, Scotland, UK) under accession number MG022720 and is considered to be the same strain as CS-41.

[0036] The phototrophic organisms are preferably CO2-adapted, meaning that they can grow under culture conditions that include a gaseous atmosphere containing carbon dioxide at a concentration higher than that of atmospheric CO2 (atmospheric CO2 concentration is typically approximately 0.033%). Methods for producing CO2-adapted phototrophic organisms are described below.

[0037] In preferred embodiments, the phototrophic organism is adapted to a gaseous atmosphere containing a carbon dioxide concentration of at least about 5%, preferably at least about 8%, 9%, or 10%, and may be adapted to a concentration of about 15%, 20%, or 25%. Alternatively or additionally, the phototrophic organism may be adapted to a gaseous atmosphere containing a carbon dioxide concentration of between about 5% and 25%, which may be from about 5%, 6%, 7%, etc. up to 23%, 24%, or 25%. Preferably, the phototrophic organism is adapted to a gaseous atmosphere containing a carbon dioxide concentration of between about 8% and 25%, or between 9% or 10% and 25%, or between 8% and 20%, which may be any one of about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. Phototrophic organisms adapted to carbon dioxide concentrations exceeding atmospheric concentrations are believed to be able to consume greater amounts of carbon dioxide, which is converted by photosynthesis into high-energy production in the form of carbohydrates. Furthermore, carbon dioxide levels between about 8% and 25% are commonly found in waste streams of various industrial processes, and therefore, the methods described herein can be used to sequester carbon dioxide from gaseous atmospheres, as described below.

[0038] At the very least, preferred phototrophic organisms that are capable of consuming greater amounts of carbon dioxide when CO2 adapted may be CO2 adapted much more rapidly than conventional ALE processes and / or may also be CO2 adapted to higher carbon dioxide concentrations than previously thought. For this reason, it is preferred that the phototrophic organism is a microalgae, preferably a microalgae of the Chlorella species, preferably C. vulgaris species, most preferably strain CS-41 or CCAP211 / 11S.

[0039] Accordingly, the present disclosure further provides a CO2-adapted phototrophic organism, wherein the phototrophic organism is selected from one or both of C. vulgaris species strains CS-41 or CCAP211 / 11S, preferably CS-41, and wherein the phototrophic organism is CO2-adapted to a gaseous atmosphere containing a carbon dioxide concentration of 5% or greater.

[0040] Culture method The present disclosure provides a method for culturing phototrophic organisms by growing them in an aqueous medium. As used in this disclosure, the term "aqueous medium" means an aqueous liquid. The aqueous medium may contain nutrients that support the growth of the organism. Preferred characteristics of the aqueous medium are described below.

[0041] The cultivation method may be carried out in a bioreactor, which may generally be a vessel containing phototrophic organisms in an aqueous medium. Numerous bioreactors are known to those skilled in the art. A preferred bioreactor is that disclosed in Australian Provisional Patent Application No. 2023902970, filed September 14, 2023, the entire contents of which are incorporated herein by reference.

[0042] Reducing biomass concentration The present disclosure provides a method of culturing phototrophic organisms, wherein the biomass concentration of the phototrophic organisms in the aqueous medium is reduced to a post-reduced biomass concentration that is equal to or greater than the concentration that existed during the early-exponential growth phase when the phototrophic organisms reach a point between the exponential and stationary growth phases. Other ways of expressing "when the phototrophic organisms reach a point between the exponential and stationary growth phases" are when the phototrophic organisms reach a concentration that corresponds to a point between the exponential and stationary growth phases, or when the phototrophic organisms reach a concentration that exists between the exponential and stationary growth phases.

[0043] Generally, phototrophs, such as microalgae, and in particular Chlorella species such as C. vulgaris, are asexually reproducing, non-motile germplasm (autospores), where cells grow by autospore formation, in which four daughter cells with individual cell walls are formed within the cell wall of a mother cell. After maturation of the newly formed cells, the mother cell wall ruptures, releasing the daughter cells, and the mother cell debris is consumed by the daughter cells as feed. Thus, the culture grows.

[0044] The growth profile of phototrophic organisms, including algae, particularly microalgae, generally follows a sigmoidal pathway consisting of a long lag phase (e.g., autosporulation occurs at a low rate) during which biomass remains constant or increases slowly, followed by a pre-exponential phase during which growth rates transition to higher growth rates in the subsequent exponential phase (e.g., autosporulation occurs at a high rate). The exponential phase is then followed by a second transitional stage during which growth slows and biomass plateaus in the subsequent stationary phase. In the subsequent death phase, biomass declines as organisms die.

[0045] As used herein, the term "exponential phase" refers to the phase of growth of a phototrophic organism occurring between the initial lag phase and the later stationary phase in the organism's sigmoidal growth profile, characterized by a period of rapid growth relative to both the initial lag phase and the later stationary phase. "Early exponential phase" refers to the initial increase in the growth rate of an organism as it transitions from the initial lag phase and includes the transitional period. "Mid-exponential phase" occurs after the early-exponential phase, approximately in the middle of the exponential phase, and includes the "mid-exponential point," which is the midpoint of the mid-exponential phase. "Late-exponential phase" follows the mid-exponential phase and precedes the stationary phase, and is often the period during which the maximum growth rate is observed. "Stationary phase" refers to the stage of growth in which the growth rate of an organism begins to slow and growth plateaus. "Saturation point" occurs during the late-exponential phase, just before the onset of the stationary phase. In the context of culturing phototrophic organisms, "growth phase" can be abbreviated to "phase." In many embodiments, the early-exponential, mid-exponential, and late-exponential phases that make up the exponential phase can be considered as three parts in terms of biomass concentration and / or duration.

[0046] Advantageously, when a phototrophic organism, particularly a microalga, is cultivated in an aqueous medium between the exponential and stationary growth phases and the biomass concentration of the phototrophic organism in the aqueous medium is reduced to a concentration equal to or greater than that present during the early exponential growth phase, the phototrophic organism avoids or substantially avoids the stationary phase and continues to grow without a prolonged initial lag phase. The phototrophic organism quickly adapts to the concentration change in the aqueous medium and returns to the exponential growth phase after the pre-exponential phase, or even continues to grow at a rate consistent with the exponential growth phase. In other words, in this manner, the exponential phase is essentially extended as a result of the reduction in the phototrophic organism concentration, allowing the phototrophic organism to return to an earlier point in the exponential growth phase, for example, from the late exponential phase to the early exponential phase. Thus, the method provides increased biomass production in a shorter time frame.

[0047] The advantage can be effectively utilized when the point between the exponential and stationary growth phases immediately prior to reducing the biomass concentration of the phototrophic organism in the aqueous medium is between the mid-exponential phase and the saturation point, preferably approximately between the mid-exponential phase and the saturation point, in other words, a point in the late-exponential phase, preferably approximately the saturation point. Since the growth rate and / or biomass are generally greatest in the late-exponential phase and / or the saturation point, high biomass production is possible before reducing the concentration. The advantage can also be effectively utilized when the post-reduction biomass concentration is equal to or greater than the concentration present at the beginning of the exponential growth phase, in other words, the post-reduction concentration is the concentration present in the early-exponential phase, preferably the concentration present in the mid-exponential phase, which may exceed the mid-exponential phase point and may even be within the range of the late-exponential phase. A reduced concentration of phototrophic organisms, particularly microalgae, as present in the early exponential phase, particularly the mid-exponential or late-exponential phase, provides the fastest adaptation to the change in concentration of the aqueous medium and the fastest return to exponential growth, allowing for even greater biomass production.

[0048] Thus, in a preferred embodiment, the point between the exponential and stationary growth phases immediately prior to reducing the biomass concentration of the phototrophic organism is a point between mid-exponential growth and the saturation point, in other words, a point in the late exponential phase, preferably at about the saturation point. In an equally preferred embodiment, the post-reduction biomass concentration is equal to or greater than the concentration present at the start of the exponential growth phase or is the concentration present in the early exponential phase, preferably the concentration present in the mid-exponential or late-exponential growth phase.

[0049] Since this provides a rapid return to exponential growth, in a preferred embodiment, the method further comprises repeating step a) using the reduced biomass concentration; i.e., repeating the step of growing the phototrophic organism in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide using the reduced biomass concentration. Step a) may be referred to as the "growth step."

[0050] The reduced biomass concentration can be grown in the same bioreactor or transferred to another bioreactor. If the concentration is reduced by removing a portion of the phototrophic biomass, the removed biomass can be transferred to a bioreactor for continued growth or to a storage vessel for downstream processing.

[0051] The physical amount of biomass present at various points in the growth profile may vary between specific phototrophic organisms and can be determined by those skilled in the art using routine methods. In a preferred embodiment, and particularly when using microalgae, the biomass concentration in the aqueous medium (i.e., just before reduction) is between about 2.0 g / L and about 9.0 g / L, preferably between about 3.0 g / L and about 8.5 g / L, preferably between about 4.5 g / L and about 8.0 g / L, preferably between about 5.0 g / L and 7.5 g / L, preferably between about 6.0 g / L and 7.5 g / L. In the case of microalgae, these concentrations may correspond approximately to the saturation point. In an equally preferred embodiment, the reduced biomass concentration in the aqueous medium is between about 2.0 g / L and about 6.5 g / L, preferably between about 2.5 g / L and about 6.0 g / L, preferably between about 3.0 g / L and 5.5 g / L, preferably between about 3.5 g / L and 5.0 g / L. In the case of microalgae, these concentrations may correspond to concentrations in the mid-exponential phase, or at about the mid-exponential phase, or in the late-exponential phase. Thus, in a preferred embodiment, and particularly when using microalgae, the biomass concentration in the aqueous medium is reduced between about 0.5 g / L and about 6.0 g / L, preferably between about 1.5 g / L and about 5.5 g / L, preferably between about 2.0 g / L and about 5.0 g / L, preferably between about 2.5 g / L and about 4.5 g / L. The amount by which the biomass concentration in the aqueous medium is reduced, i.e., the difference between the concentration of phototrophic organisms in the aqueous medium immediately before removal and the biomass concentration after reduction, can be referred to as the "concentration change."

[0052] The duration of the growth steps varies among different phototrophic organisms. For microalgae, the duration of the at least one first growth step, from early exponential growth to stationary growth, can be between about 1.5 and about 4 days, preferably between about 1.5 and about 3 days, and preferably between about 1.8 and about 2.2 days.

[0053] The step of reducing the biomass concentration can be performed, for example, by adding an amount of aqueous medium and / or removing an amount of phototrophic biomass. The added amount of aqueous medium can be referred to as a "dilution amount." The removal of an amount of phototrophic biomass can also be referred to as "recovery." If the reduction of the biomass concentration is performed simply by adding an amount of aqueous medium, the combined amount of phototrophic biomass and aqueous medium will increase, which may require further manipulation, for example, transferring to a larger vessel size. Thus, in a preferred embodiment, the reduction of the biomass concentration includes removing an amount of phototrophic biomass, for example, to avoid increasing the vessel size.

[0054] Removal of phototrophic biomass may also remove a quantity of aqueous medium. If this occurs, and if the quantity of phototrophic biomass removed is the same as or less than the quantity of aqueous medium removed, reducing the biomass concentration generally requires the addition of a dilution quantity of aqueous medium greater than the difference in volume between the phototrophic biomass removed and the aqueous medium removed. On the other hand, if the quantity of phototrophic biomass removed is greater than the quantity of aqueous medium removed, reducing the biomass concentration may not require the addition of a dilution quantity of aqueous medium, although addition of a dilution quantity of aqueous medium may be performed, for example, to achieve a desired post-reduction biomass concentration.

[0055] In a preferred embodiment, reducing the biomass concentration involves removing a quantity of aqueous medium as well as a quantity of phototrophic organisms, and adding a diluting quantity of aqueous medium to the remaining biomass.

[0056] The amount of aqueous medium can be removed together with the amount of phototrophic biomass or separately. For example, the amount of aqueous medium can be removed prior to biomass removal. This can be achieved by flocculation of the biomass and removal of the aqueous medium as a supernatant. Methods of flocculation are known to those skilled in the art. For example, for the present purpose, flocculation can be achieved by increasing the carbon dioxide concentration in the gaseous atmosphere. Chemical flocculants, such as chitosan (also called deacetylated chitin), can also be used.

[0057] The diluted portion of aqueous medium added may include the removed aqueous medium. In other words, the aqueous medium may be recycled. Removal of the aqueous medium as a supernatant is one method that may allow for medium recycling. Recycling of aqueous culture medium provides advantages in economically and ecologically sustainable production of biomass. The diluted portion may at least partially comprise fresh aqueous medium. This may allow for at least a partial refreshment of the aqueous medium and may support the growth of the remaining biomass. The percentage of biomass removed depends on the relative biomass concentrations present at various points in the growth profile and may vary between specific phototrophic organisms. The percentage removed while maintaining biomass at a concentration equal to or greater than that present during the early exponential growth phase can be determined by one skilled in the art using routine methods based on the present disclosure.

[0058] Phototrophic biomass can be removed batchwise, as described further below. Batchwise removal removes between about 30% and about 80%, preferably between about 40% and about 65%, and preferably between about 45% and about 60%. For phototrophs, particularly microalgae, these percentages can correspond to the difference between the saturation point concentration and the mid-exponential point concentration when an equal dilution volume of aqueous medium is added to the remaining biomass.

[0059] In other words, the concentration change can be between about 30% and about 80%, preferably between about 40% and about 65%, preferably between about 45% and about 60%. For example, if the concentration of phototrophic organisms in the aqueous medium immediately before removal was 7.5 g / L and the post-reduction biomass concentration was 5.0 g / L, that corresponds to a 33% concentration change. Similarly, if the concentration of phototrophic organisms in the aqueous medium immediately before removal was 9.0 g / L and the post-reduction biomass concentration was 2.0 g / L, that corresponds to a 78% concentration change, etc.

[0060] Advantageously, reduction in concentration by biomass removal allows for the use of the removed biomass in downstream processes and rapid regeneration through the exponential growth phase by culturing the reduced biomass, so that biomass can be repeatedly removed in rapid succession as feedstock for said downstream processes, i.e., steps a) and b) can be repeated one or many times. Repetition of the reduction in concentration by biomass removal and growth steps within the described parameters provides the basis for a continuous culture method.

[0061] Continuous culture In a preferred embodiment, the method further comprises cycling the reduced biomass concentration (wherein cycling the reduced biomass concentration comprises repeating at least step a), and preferably steps a) and b), using the reduced biomass concentration), preferably cycling the reduced biomass concentration multiple times (i.e., repeating steps a) and b) multiple times) using the reduced biomass concentration of each previous cycle (i.e., each step b)). A method comprising repeating at least step a), preferably steps a) and b), at least once, preferably multiple times (i.e., two or more times) may be referred to as "continuous cultivation."

[0062] Continuous culture can be batch or continuous. That is, "continuous culture" includes both batch and continuous culture. "Batch" (also called "semi-continuous") means that the concentration of the phototrophic organism in the aqueous medium is reduced in a discrete step. For example, if the concentration is reduced by removing the phototrophic biomass, batch culture includes its removal and optional addition of diluting amounts of aqueous medium in a discrete step. "Continuous" means that the concentration of the phototrophic organism in the aqueous medium is reduced continuously. For example, if the concentration is reduced by removing the phototrophic biomass, continuous culture includes its continuous removal and optional addition of diluting amounts of aqueous medium. The phototrophic organism can be cycled multiple times through the present method, including at carbon dioxide concentrations above atmospheric concentrations, without noticeable loss of culture efficiency, particularly using microalgae.

[0063] When the culture is batch-based, these cycles are counted as individual steps. In batch-based embodiments, the reduced biomass can be cycled through steps a) and b) at least 2, 3, 4, preferably at least 5 times, e.g., 10, 20, 50, 75, or 100 or more times. The reduced biomass can be cycled many times, even thousands of times. For example, the reduced biomass can be cycled up to 125, 315, 630, or 1260 times. Expressed as a range, the reduced biomass can be cycled between 2 and about 1260 times, preferably between about 20 and about 630 times, preferably between about 125 and 315 times.

[0064] For each cycle, steps a) and b) can be as described above, for example, particularly for microalgae: the biomass concentration in the aqueous medium (i.e., before reduction) can be between about 2.0 g / L and about 9.0 g / L, preferably between about 3.0 g / L and about 8.5 g / L, preferably between about 4.5 g / L and about 8.0 g / L, preferably between about 5.0 g / L and 7.5 g / L, preferably between about 6.0 g / L and 7.5 g / L; the reduced biomass concentration in the aqueous medium can be between about 2.0 g / L and about 6.5 g / L, preferably between about 2.5 g / L and about 6.0 g / L, preferably between about 3.0 g / L and 5.5 g / L, preferably between about 3.5 g / L and 5.0 g / L; The reduction of the biomass concentration preferably involves removing a portion of the aqueous medium as well as a portion of the phototrophic organisms and adding a diluting portion of the aqueous medium to the remaining biomass; and Between about 30% and about 80%, preferably between about 40% and about 65%, preferably between about 45% and about 60% of the phototrophic biomass may be removed.

[0065] For at least the second and subsequent cycles through steps a) and b), the duration of the growth step (step a)) varies between different phototrophic organisms and depends on the post-reduction biomass concentration of the previous cycle and the point between exponential and stationary growth phases that the phototrophic organism reaches before reducing its biomass concentration again, i.e., the concentration to which it grows its biomass. Thus, generally, at least for microalgae, in some embodiments, the duration of the growth step from early-exponential growth to stationary growth can be between about 1.5 and about 4 days, preferably between about 1.5 and about 3 days, and preferably between about 1.8 and about 2.2 days. Similarly, if the post-reduction biomass concentration of the previous cycle is approximately the concentration present at the mid-exponential growth point, the duration of the growth step from mid-exponential growth to stationary growth or saturation can be between about 0.7 and about 2 days, preferably between about 0.7 and about 1.5 days, and preferably between about 0.9 and about 1.1 days.

[0066] Therefore, it has been found that a short growth phase duration—in other words, a small change in concentration (between the concentration after reduction and the concentration immediately before reduction)—results in a particular benefit. A "short" growth phase duration can be defined as about 24 hours or less. A "small" change in concentration can be defined as about 1 g / L or less than 1 g / L. The benefit found is that the concentration defining a particular growth phase point can be shifted to a higher concentration. This can be, for example, when the concentration is reduced from the saturation point to above the mid-exponential point or from the saturation point to within the late-exponential phase. The mid-exponential point and saturation point can be shifted toward higher concentrations. For example, and not by way of limitation, it has been found that if the mid-exponential growth point of a cycle is 4.0 g / L, the stationary phase of that cycle begins at 7.5 g / L, and a small concentration change, e.g., a post-reduction biomass concentration of 6.5 g / L, can result in a higher mid-exponential growth point in the subsequent cycle, e.g., 4.1 g / L, and a stationary phase beginning at a higher concentration, e.g., 7.6 g / L. The use of a shorter growth step duration (small concentration change) in the subsequent cycle can achieve further increases in the concentrations that define those growth points in yet other subsequent cycles. When the concentration reduction is achieved at least in part by biomass removal, it allows for the removal of a larger amount of biomass in the same time frame and from the same total volume of biomass and aqueous medium, which allows for the production of more biomass and feedstock over successive cycles. At least with respect to microalgae, small changes in biomass concentration are believed to encourage the microalgae to adapt to increasing concentration conditions.

[0067] In the method described herein, it is provided that in at least the second and subsequent cycles, the post-reduction biomass concentration can be higher than the post-reduction biomass concentration of the previous cycle. It can further provide that the biomass concentration at the mid-exponential phase point is higher than the biomass concentration at the mid-exponential phase point of the previous cycle. It can also provide that the biomass concentration at the saturation point is higher than the biomass concentration at the saturation point of the previous cycle. In a preferred embodiment, the post-reduction biomass concentration corresponds to a concentration above the mid-exponential phase or mid-logarithmic beginning of the previous cycle, preferably the concentration at the point where the mid-exponential phase ends and the late-exponential phase begins, preferably the concentration within the late-exponential phase. Preferably, the duration of the growth step is such that the biomass concentration reaches a concentration at or above the saturation point of the previous cycle. The benefit of this is that in successive cycles, the biomass concentration can stabilize around the late-exponential phase, which is the phase of maximum growth rate, and then shift this phase to a higher concentration, maximizing the production efficiency and CO2 sequestration capacity of phototrophic organisms.

[0068] Thus, in preferred embodiments, particularly using microalgae, at least in the second and subsequent cycles, the growth step duration may be between about 15 minutes and 15 hours, preferably between about 30 minutes and 10 hours, or between 30 minutes and 5 hours, preferably between about 30 minutes and 3 hours, or between 30 minutes and 2 hours, and between 1 hour and 2 hours.

[0069] Similarly, in preferred embodiments, particularly using microalgae, the concentration change in at least the second and subsequent cycles can be between about 0.5 g / L and 5.0 g / L, preferably between about 0.5 g / L and 4.5 g / L, preferably between about 1.0 g / L and 4.0 g / L, preferably between about 1.5 g / L and 3.5 g / L, preferably between about 2.0 g / L and 3.0 g / L. The concentration change can even be smaller, e.g., less than 1.0 g / L, e.g., between about 0.001 g / L and 1.0 g / L, preferably between about 0.005 g / L and 0.8 g / L, preferably between about 0.01 g / L and 0.6 g / L, preferably between about 0.015 g / L and 0.4 g / L, preferably between about 0.1 g / L and 0.3 g / L, for a growth step duration of about 30 minutes to 2 hours.

[0070] Similarly, in preferred embodiments, particularly when using microalgae and the concentration of phototrophs in the aqueous medium is reduced by biomass removal, amounts of between about 0.1% and about 25%, preferably between about 0.5% and about 20%, preferably between about 15% and about 1%, can be removed in at least the second and subsequent cycles.

[0071] Similarly, in other words, the concentration change can be between about 0.1% and about 25%, preferably between about 0.5% and about 20%, preferably between about 15% and about 1%. For example, if the concentration of phototrophic organisms in the aqueous medium immediately before removal was 7.5 g / L and the post-reduction biomass concentration was 7.0 g / L, that corresponds to a 7% concentration change. Similarly, if the concentration of phototrophic organisms in the aqueous medium immediately before removal was 7.5 g / L and the post-reduction biomass concentration was 7.3 g / L, that corresponds to a 3% concentration change, etc.

[0072] When the reduction of biomass concentration involves the removal of biomass and aqueous medium, a post-reduction concentration higher than the post-reduction concentration of the previous cycle can be achieved by adding a smaller dilution volume of aqueous medium than was added in the previous cycle.

[0073] For continuous culture, the number of cycles is essentially infinite, and cycling may instead be expressed in terms of the total duration of culture or cycling. The cycling duration may be for a period of at least about 12 hours, 1 day, 2 days, 5 days, 7 days, or 10 days. The cycling duration may be for a period of several weeks. For example, the cycling duration may be up to about 40 days, 35 days, or 30 days. Expressed as a range, the cycling duration may be for a period of between about 12 hours and 40 days, preferably between about 5 and 35 days, and preferably between about 10 and 30 days.

[0074] The continuous removal can be the removal of biomass alone or together with the aqueous culture medium. When biomass alone is removed, it serves to reduce the concentration of the remaining biomass, which is suitable for promoting the continued growth of phototrophic organisms. When biomass is removed together with the aqueous culture medium, a dilution amount of culture medium can be added to the remaining biomass to reduce its concentration. For example, when the biomass and aqueous culture medium are removed at a concentration equal to or greater than that present before removal, a dilution amount of aqueous culture medium can be added to the remaining biomass to reduce its concentration. Both the removal of biomass in the aqueous culture medium and the addition of the dilution amount of aqueous culture medium can be continuous. This can be achieved using appropriate flow rates for removal and addition. Advantageously, the flow rates for removal and addition can be calibrated to the biomass growth rate in the exponential or late exponential growth phase. For example, the flow rate of biomass removal can be set so that the amount of biomass removed matches the amount of biomass growth, i.e., according to the growth rate. For example, for a biomass growth rate of 1.0 g / L / day, the flow rate of biomass removal can be set to remove 1.0 g / L / day of biomass. Similarly, the flow rate of aqueous culture medium dilution volume addition can be set to match the rate of removal of the combined biomass and aqueous culture medium. For example, for a combined biomass and aqueous culture medium removal rate of 2.0 g / L / day, the flow rate of aqueous culture medium dilution volume addition can be set to add 2.0 g / L / day. Flow rates can be monitored and adjusted as needed. Means for providing continuous flow and set flow rates are known in the art. Examples include vessel overflow means, the use of pumps, and siphons.

[0075] Continuous culture has the advantage of providing a continuous feedstock for downstream processes, with the combined advantage of rapid biomass renewal, providing the ability to upscale said downstream processes.

[0076] The aqueous culture medium can be continuously recycled, preferably only to the point where the recycled aqueous medium continues to promote growth. Phototrophic organisms in culture can produce by-products that accumulate in the aqueous culture medium and can prevent growth at the same rate as would occur in fresh aqueous culture medium. For example, by-products of microalgae can include dissolved organic matter, such as extracellular polymeric substances. If the aqueous culture medium is not recyclable, it can be replaced with fresh aqueous culture medium.

[0077] Gaseous atmosphere The gaseous atmosphere may contain gases other than carbon dioxide and may contain other substances, such as particulates. The other gases may be one or more of the gases commonly found in air, such as oxygen, nitrogen, water vapor, hydrogen, methane, and the noble gases. The other gases may also be one or more of the gases commonly found in waste gas streams, such as one or more of the gases commonly found in air, as well as gases produced by the combustion of hydrocarbons, such as carbon monoxide, nitrogen oxides (NOx), and sulfur oxides (SOx).

[0078] The gaseous atmosphere can be provided by a single source or multiple sources (i.e., two or more). For example, the gaseous atmosphere can be provided by a single source, such as a waste gas stream from an industrial process, or a combination of a waste gas stream and another source, such as an air source, or a combination of a purified carbon dioxide source and another source, such as an air source. The source of the gaseous atmosphere can be determined by the concentration of carbon dioxide to which the phototrophic organism is CO2-adapted. For example, when using phototrophic organisms that are CO2-adapted to about 8% CO2, the gaseous atmosphere provided using a purified CO2 source (which may contain about 95% or more CO2) can be diluted with a dilution gas source containing relatively little or no CO2, such as air, to provide a gaseous atmosphere containing about 8% CO2. Similarly, when using phototrophic organisms that are CO2-adapted to about 8% CO2, the gaseous atmosphere provided using a waste gas stream containing about 8% CO2 can be provided as a single source, the waste gas stream being the gaseous atmosphere. In some embodiments, the gaseous atmosphere provided using the waste gas stream requires dilution of the CO concentration, in which case a dilution gas source is preferably provided. Preferably, the dilution gas source is air. The dilution gas source may also be recycled from the gaseous atmosphere under which the phototrophic organisms are grown in aqueous media.

[0079] Use of biomass Biomass obtained from the growth of phototrophic organisms is useful as an organic feedstock in many downstream processes. Phototrophic organisms use light as the primary energy source for metabolism in the presence of water and carbon dioxide to produce energy in the form of carbohydrates by photosynthesis. Thus, organic feedstocks include simple and complex carbohydrates, e.g., mono- and complex sugars, and biopolymers, e.g., exopolysaccharides. Thus, organic feedstocks are useful as nutrients for downstream applications, e.g., biodegradation steps.

[0080] One exemplary biological degradation process for which phototrophic biomass is useful is described in related applications entitled "Process and system for generating hydrogen" (PCT Application No. PCT / AU2020 / 050285) and "Production of Biomass" (PCT Application No. PCT / AU2022 / 051348), the entire disclosures of each of which are incorporated herein by reference in their entirety.

[0081] CO2 sequestration In addition to producing biomass, the methods described herein can be used to sequester carbon dioxide from the gaseous atmosphere, which provides the advantage that not only do they produce useful downstream products, but in doing so, the carbon dioxide is captured and converted into useful downstream products.

[0082] In some embodiments, carbon dioxide may be recovered from air.

[0083] A gaseous atmospheric supply containing carbon dioxide concentrations above atmospheric concentrations can be used to sequester carbon dioxide, resulting in less atmospheric carbon dioxide than would otherwise be present. However, in phototrophic cultures, particularly microalgae, high concentrations of CO can cause stress to organisms adapted to atmospheric carbon dioxide concentrations, resulting in reduced growth. For this reason, to efficiently utilize the carbon dioxide sequestration capabilities of phototrophic organisms in the present methods, it is preferred that the phototrophic organisms be CO-adapted as described herein.

[0084] As noted above, in preferred embodiments, the phototrophic organisms are adapted to a gaseous atmosphere containing a carbon dioxide concentration of at least about 5%, preferably at least about 8%, 9%, or 10%, which may be between about 5% and 25%, preferably between about 8% and 25%. Thus, the phototrophic organisms may be adapted to a gaseous atmosphere containing carbon dioxide at concentrations similar to those found in gas sources from industrial processes, thereby enabling the gas source used in the present method to be from an industrial process that produces a gas stream containing those levels of carbon dioxide. Thus, the present method may assist in reducing the amount of carbon dioxide that those industrial processes might otherwise release into the atmosphere.

[0085] In a preferred embodiment, the gaseous atmosphere is supplied with a carbon dioxide concentration similar to that to which the phototrophic organisms are adapted. In other words, in a preferred embodiment, the phototrophic organisms are adapted to the carbon dioxide concentration of the supplied gaseous atmosphere. For example, for phototrophic organisms CO2-adapted to 8% carbon dioxide, or 9%, 10%, 15%, or 20% carbon dioxide, the gaseous atmosphere is preferably supplied with a carbon dioxide concentration of about 8%, 9%, 10%, 15%, or 20%, respectively. In other words, for a supplied gaseous atmosphere having a carbon dioxide concentration of about 8%, 9%, 10%, 15%, or 20%, the phototrophic organisms CO2-adapted to 8% carbon dioxide, or 9%, 10%, 15%, or 20% carbon dioxide, respectively. Carbon dioxide levels between about 8% and 25% are commonly found in waste streams of various industrial processes. Thus, in a preferred embodiment, the source of carbon dioxide comprises a waste gas stream, preferably a waste gas stream produced by the combustion of hydrocarbons, for example by steam reforming and / or by biological decomposition.

[0086] The waste gas stream may be filtered to remove particulate matter and / or otherwise treated before being applied to the gaseous atmosphere.

[0087] Preferred culture parameters Preferred parameters suitable for the growth of phototrophic organisms in this culture method have been developed. The described parameters, both alone and in combination, help promote more favorable growth of phototrophic organisms, particularly microalgae.

[0088] Because phototrophic organisms can consume carbon dioxide, the carbon dioxide in the gaseous atmosphere can be replenished to maintain a constant concentration in the gaseous atmosphere. This is conveniently achieved using a gas flow. In some embodiments, the gas flow is used at a flow rate of between about 0.05 VVM (volumes per minute) and about 5 VVM, preferably between about 0.1 VVM and about 2 VVM, preferably between about 0.1 VVM and about 1.0 VVM, preferably between about 0.1 VVM and 0.5 VVM, preferably between about 0.1 VVM and 0.3 VVM, preferably between about 0.1 VVM and 0.2 VVM, and more preferably about 0.15 VVM.

[0089] Generally, a culture of phototrophic organisms grown through a lag phase to an exponential phase was initially present as an inoculum. In a preferred embodiment, about 10 6 cells / mL to approximately 10 10 between about 10 cells / mL, more preferably about 10 7 cells / mL to approximately 10 9 Between 10 cells / mL, most preferably about 10 8 An inoculum of phototrophic organisms containing cells / mL is used, preferably as a cell suspension in aqueous medium.

[0090] In a preferred embodiment, the cultivation method is carried out in a bioreactor having a volume of at least 1.11 times (i.e., 111%), preferably 1.17 times (i.e., 117%) the volume of the phototrophic organism and the aqueous medium. For example, for a 900 mL working volume, the bioreactor volume is preferably at least 1000 mL, preferably at least 1053 mL, respectively. Preferably, the cultivation method is carried out in a bioreactor having a volume of no more than 2.85 times (i.e., 285%), preferably no more than 2.50 times (i.e., 250%) the volume of the phototrophic organism and the aqueous medium. For example, for a 900 mL working volume, the bioreactor volume is preferably no more than about 2565 mL, preferably no more than about 2250 mL, respectively. Alternatively or additionally, the cultivation method is preferably carried out in a bioreactor having a volume between about 1.11 and 2.85 times, preferably between about 1.17 and 2.50 times, preferably between about 1.42 times (i.e., 140%) and 2.00 times (i.e., 200%), preferably about 1.67 times (i.e., 167%) the volume of the phototrophic organism and aqueous medium.

[0091] In other words, in a preferred embodiment, the cultivation method is carried out in a bioreactor in which the volumes of the phototrophic organisms and aqueous medium are at least about 35%, preferably at least about 40%, of the bioreactor volume. For example, for a 1000 mL bioreactor volume, the volumes of the phototrophic organisms and aqueous medium are preferably at least about 350 mL, preferably at least about 400 mL, respectively. Preferably, the cultivation method is carried out in a bioreactor in which the volumes of the phototrophic organisms and aqueous medium are at most about 90%, preferably at most about 85%, of the bioreactor volume. For example, for a 1000 mL bioreactor volume, the volumes of the phototrophic organisms and aqueous medium are preferably at most about 900 mL, preferably at most about 850 mL, respectively. Alternatively or additionally, the culture method is preferably carried out in a bioreactor in which the volume of phototrophic organisms and aqueous medium is between about 35% and 90%, preferably between about 40% and 85%, preferably between about 50% and 70%, preferably about 60% of the bioreactor volume. These levels provide a balance of headspace for the gaseous environment and the volume of phototrophic organisms and aqueous medium suitable for efficient gas exchange between the gaseous atmosphere and the aqueous medium to promote carbon dioxide consumption and organism growth.

[0092] The bioreactor may be, and preferably is, equipped with a mixing device, e.g., an impeller, to agitate the aqueous medium to aerate the culture, which can further aid gas exchange. It can also help ensure even distribution of light irradiation throughout the phototrophic culture, especially when low flow rates of gaseous atmosphere, e.g., less than about 1.0 VVM, are used.

[0093] Phototrophic organisms, including microalgae, often grow under neutral or alkaline conditions. Thus, in a preferred embodiment, the aqueous medium is maintained at a pH between about 5 and about 9, preferably between about 6 and about 8, more preferably between about 6.5 and about 7.5, and most preferably at a pH of about 7. Carbon dioxide, which may be dissolved or dissolve in the aqueous medium, can contribute to a decrease in pH, while consumption of carbon dioxide by the organisms can contribute to an increase in pH. pH can sometimes be maintained using additives, as needed; for example, alkaline substances to increase pH and acidic substances to decrease pH. Suitable substances are known to those skilled in the art.

[0094] In a preferred embodiment, the temperature is maintained between about 25°C and about 31°C, preferably between about 27°C and about 29°C, more preferably at about 28°C.

[0095] Preferably, the aqueous medium contains nutrients. Preferably, the nutrients are selected from one or more, preferably two or more, more preferably all, of the group consisting of boric acid, calcium chloride, cobalt nitrate, copper sulfate, disodium EDTA, magnesium sulfate, manganese chloride, potassium phosphate, sodium molybdate, sodium nitrate, and zinc sulfate. Aqueous media containing these nutrients are known to those skilled in the art or can be prepared by those skilled in the art. In a preferred embodiment, the aqueous medium is selected from one or both of Bold Basal Medium (BBM) and Blue-Green 11 Medium (BG-11), preferably BG-11.

[0096] In preferred embodiments, the light has an intensity of between about 3000 lux and about 7000 lux, preferably between about 4000 lux and about 6000 lux, preferably between about 4500 lux and about 5500 lux, or preferably about 5000 lux. Light sources, including LED lights, capable of providing these light intensities are known to those skilled in the art.

[0097] Because phototrophs use light to generate energy, the photoperiod, i.e., the duration of light to which the phototrophs are exposed within a 24-hour period, is preferably extended compared to the photoperiod that the phototrophs typically encounter in their natural environment. However, a photoperiod that is too long can not promote growth, as can a photoperiod that is balanced with dark periods. Therefore, in a preferred embodiment, the photoperiod is between about 12 hours and about 20 hours, preferably between about 14 hours and about 18 hours, more preferably between about 15 hours and about 17 hours, and most preferably about 16 hours.

[0098] Of these preferred culture conditions, the combination of preferred light intensity and photoperiod is particularly effective in promoting more favorable growth of phototrophic organisms, particularly microalgae.

[0099] Thus, the present disclosure provides a method for culturing a phototrophic organism, comprising growing the phototrophic organism in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide, wherein the light has a light intensity of between about 3000 lux and about 7000 lux, and the culture conditions further comprise a photoperiod of between about 12 h and about 20 h. The preferred integers specified herein are applicable to the present methods, including the use of microalgae, culture conditions, and methods that are otherwise continuous culture methods.

[0100] Culture equipment & culture system Apparatus and systems that may be used to practice the culture methods of the present disclosure are described in related applications entitled "Process and system for generating hydrogen" (PCT Application No. PCT / AU2020 / 050285) and "Production of Biomass" (PCT Application No. PCT / AU2022 / 051348), the entire disclosures of each of which are incorporated herein by reference in their entirety.

[0101] A basic system may include a bioreactor suitable for culturing phototrophic organisms. The reactor may be a closed system in communication with a gas stream containing carbon dioxide and air. The gas stream may be derived from a waste gas stream, e.g., flue gas, from an industrial process. The bioreactor may be in liquid communication with one or more other reactors and / or storage vessels. The or each bioreactor may include multiple sensors, including pH sensors, temperature sensors, reactor level sensors, sensors monitoring biomass production, and preferably has regulators to control and maintain each monitored parameter. [Example]

[0102] Various embodiments are described herein with reference to the following non-limiting examples.

[0103] Example experiments were conducted using Chlorella vulgaris strain CS-41 obtained from the Australian National Algae Culture Collection (CSIRO, Hobart, Tasmania, Australia). To initiate an experiment, a pre-established volume of algae at a known concentration was added to a closed vessel containing sterile culture medium, and assays were performed in triplicate. Once parameters were optimized (e.g., performing best in terms of biomass production rate), the optimized culture conditions were maintained for future experiments.

[0104] First culture treatment First, a C. vulgaris CS-41 stock was resuscitated from -80°C and recultured in a 1 L Erlenmeyer flask containing a working volume of 400 mL in BG-11 medium at 28°C. The culture was exposed to an LED light source to provide an irradiance of approximately 4000 lux and allowed to reach stationary phase under 10% (v / v) CO2 and 90% air.

[0105] Once the cultures had acclimated to the culture conditions, inocula for the experiments described in this set of examples were prepared under sterile conditions in pre-established volumes at known concentrations. The initial culture conditions used for the experiments in this example (unless otherwise specified) are shown in Table 1.

[0106] [Table 1]

[0107] A number of these conditions were investigated and improved as described below.

[0108] General analysis method The readout parameters evaluated in the experiments were pH, cell concentration, biomass weight, and CO2 uptake. The methods for evaluating each parameter are summarized below. The biomass used in these experiments relates to the C. vulgaris produced. Therefore, the biomass in these experiments illustrates the production of organic feedstock.

[0109] Biomass growth assessment was performed daily as described below. The optical density (OD) of all diluted pellets was measured at a wavelength of 683 nm using a UV-Vis spectrophotometer. The diluted pellets were transferred separately to three 1.5 mL Eppendorf tubes and centrifuged at 4700 rpm for 10 minutes. The supernatant was discarded, and the pellets in the Eppendorf tubes were dried overnight at 50°C. Biomass calibration curves for each batch were obtained by plotting the dry cell weight (DCW) versus the absorbance spectrum of the biomass.

[0110] At the end of the 7-day incubation period, the microalgae cultures were inventoried. The microalgae cultures were poured into 3 x 50 mL centrifuge tubes and centrifuged at 4700 rpm for 10 minutes to separate the supernatant from the pellet. Serial dilutions were performed on the pellets, which were resuspended in known volumes of deionized distilled water. The cells present in the appropriate dilutions were counted under a light microscope at 400x magnification using a Neubauer chamber.

[0111] An elemental analyzer was used to analyze the carbon, hydrogen, and nitrogen contents of the biomass. At the beginning and end of the culture cycle, a known volume of the culture was taken, and the biomass was recovered by centrifugation and analyzed for carbon, hydrogen, and nitrogen contents. Inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to analyze the elements present in the liquid medium separated after centrifugation.

[0112] The culture pH was analyzed daily using a pH meter. The culture pH was further maintained within the specified range (7.0 ± 0.5) using 0.1 M NaOH. Two CO2 sensors (model number GMP251) using Vaisala Insight software were attached to the inlet and outlet gas lines as shown in Figure 1 to record CO2 concentrations every 5 seconds throughout the culture cycle. The changes in CO2 concentration recorded by these sensors were also used to establish a mass balance and calculate CO2 biofixation.

[0113] [Example 1] Adaptive Laboratory Evolution The tolerance of C. vulgaris CS-41 to high concentrations of CO2 was gradually increased through an artificial stepwise adaptive laboratory evolution (ALE) process. Microalgae were cultivated in a bioreactor system setup, as shown schematically in Figure 1. The reactor was placed in a photochamber and surrounded by an LED light source. Pure CO2 and air were supplied, and the flow rate was controlled using a flow control valve to achieve a specific CO2 concentration in the gas inlet tube. CO2 sensors were placed in the gas inlet and outlet tubes to continuously measure the CO2 concentration. Except for fluctuations in CO2 concentration, the culture was grown under the conditions described in Table 1.

[0114] The increased tolerance to CO2 by C. vulgaris CS-41 was initiated by exposing the microalgae culture to ambient air containing 0.03% CO2 by volume for 7 days. A known concentration of acclimated algae culture was used as the inoculum for the ALE process. A 400 mL working volume of a 1000 mL bioreactor was used for the ALE of the C. vulgaris strain. After the 7-day culture cycle, the algae were exposed to a 3% CO2 flow by volume. Next-generation sequential culture cycles were then repeated with increasing CO2 concentrations up to 10%.

[0115] Biomass was analyzed as described above in the General Analytical Methods section. Biomass production and specific growth rate (Table 2) were calculated as shown in Equations 1 and 2 below.

[0116]

number

[0117]

number

[0118] The biomass growth data resulting from each culture cycle using different CO concentrations are summarized in Table 2. At 8% and 10%, culture cycles were performed at least twice to evaluate the performance of different generations (G).

[0119] [Table 2]

[0120] At low CO2 concentrations (3 and 5%), cultures adaptively evolved in the first generation with increasing biomass concentrations by day 5. However, biomass productivity and specific growth rate decreased from cultures grown in the presence of 3% CO2 to cultures grown in the presence of 5% CO2. This may be due to a slowdown in cell division on days 6 and 7 compared to the first 5 days of the culture cycle.

[0121] Biomass productivity was observed to decrease after each subsequent increase in CO2 concentration from 3% to 8%. This may indicate that the cells were initially stressed but were able to slowly adapt with each successive generation. This is evident from the fact that the 2G run at 8% exceeded the biomass productivity of the 1G run at 5%. A similar pattern of higher biomass productivity from subsequent generations was observed at 10% CO2, where the culture cycle was carried out over five generations.

[0122] The variations in biomass concentration, pH, and growth rate from the 10% fourth-generation run and the 10% fifth-generation run are compared in Figure 2. In this experimental study, each culture cycle was performed in triplicate, and the variation in the error bars represents the variation in the results of separate tests under the same operating conditions. Little, if any, day-to-day variation in biomass concentration and pH values ​​was observed in the 10% fourth-generation and fifth-generation runs. Furthermore, the average biomass productivity values ​​were 0.147 g / L / day and 0.148 g / L / day, respectively, and the specific growth rates were 0.606 and 0.607, respectively. Because the biomass productivity values ​​remained approximately constant for two consecutive runs, the cultures resulting from the fifth generation cultured at 10% CO2 concentration were considered to have adapted to the appropriate CO2 volume percentage.

[0123] Biomass at the end of the 7-day cycle from 10% of the 5G runs was analyzed for its carbon, hydrogen, and nitrogen content using the elemental analyzer described in the General Analytical Methods section. The results of this analysis are shown in Figure 3.

[0124] The carbon content of the total biomass was used to calculate the CO2 biofixation rate over the culture cycle (Equation 3).

[0125]

number

[0126] Figure 3(b) shows that the calculated carbon and CO2 biofixation rates were the same (0.2645 g / L / day of CO2) for generations 4G and 5G grown in the presence of 10% CO2. The CO2 biofixation rate was calculated from the CO2 sensor data, shown as a trace in Figure 3(c). The CO2 concentrations in the inlet and outlet gas streams were plotted over the last 4 days of the 10% CO2 5G culture cycle. The large square peak represents the 12-h photoperiod, while some fluctuations during the photoperiod were reported due to rapid fluctuations in the upstream gas line. Based on the gas stream concentration data, the CO2 biofixation rate was calculated to be 0.269 g / L / day. Both values, calculated from biomass analysis on the one hand and CO2 sensor data on the other, agreed with a difference of approximately 1.7%, confirming the validity of the elemental analysis method. In the following experimental trials, elemental analysis was used to calculate the CO2 biofixation rate in the process.

[0127] [Example 2] Comparison of culture media Two culture media, BG-11 and BBM, were evaluated for optimal growth performance of microalgae using C. vulgaris strain CS-41. The experimental outputs used to evaluate culture medium performance were biomass production and CO2 fixation. The compositions of each BG-11 and BBM are listed in Table 3.

[0128] [Table 3]

[0129] Biomass production was quantified by separating the phototrophic source material from the liquid medium by filtration under vacuum using filter paper (GF / C:1.2) and drying at 30-35 °C in a hot air oven. Biomass was also measured spectrophotometrically using standard optical density techniques and compared to the early growth curve of established growth characteristics for strain CS-41. The culture conditions used for this experiment are listed in Table 1.

[0130] Total CO2 biofixation was measured at 0 and 7 days. To measure CO2 levels, gas samples were collected from the reactor used to perform the photosynthesis step. The samples were collected in a gas collection bag, and the composition of the gas samples was analyzed by GC-MS (Agilent 7890B GC) equipped with a thermal conductivity detector (TCD). Argon / helium was used as the carrier gas, with a 15 mL min -1 The temperatures of the oven, detector, and injector were 190°C, 210°C, and 160°C, respectively.

[0131] The results of the 7-day culture medium comparison experiment are shown in Table 4. C. vulgaris CS-41 was able to grow and fix CO2 in both types of media. BG-11 supported higher levels of biomass production and CO2 fixation than BBM. In this example, CS-41 biofixed 0.593 g / L / day of CO2 compared to 1.029 g / L / day of CO2 when grown in BG-11 or BBM media, respectively.

[0132] [Table 4]

[0133] [Example 3] Light intensity optimization The optimal light intensity for biomass production and CO2 fixation was determined for C. vulgaris strain CS-41. Except for light intensity and photoperiod (16 hours light:8 hours dark), all other culture conditions were as listed in Example 1 and Table 1.

[0134] Growth curve analysis based on spectrophotometric measurements of optical density demonstrates that light intensity induces a growth lag phase. Figure 4 shows that optimizing the light intensity applied during the photosynthesis step can reduce the impact of the lag phase on growth rate and overall biomass production.

[0135] In separate experiments conducted under the same conditions, the light intensity range investigated consisted of 3000 lux to 5500 lux. The photobioreactor system was surrounded by an LED light source with light intensity control, configured as shown in the schematic diagram in Figure 1. Both biomass productivity and biofixation rate increased from 3000 lux to 5000 lux, as shown in Figures 5 and 6, respectively. However, further increase in light intensity to 5500 lux resulted in a decrease in biomass productivity. These results indicate that in all experimental conditions, 5000 lux resulted in the maximum CO2 biofixation rate of 1.37 g / L / day. The results of the light intensity optimization study indicate that a light intensity of 5000 lux is optimal for biomass production (Figure 5), CO2 fixation rate (Figure 6), and total carbon capture (Figure 7) by C. vulgaris strain CS-41 when grown in either BBM or BG-11 culture medium.

[0136] [Example 4] Photoperiod optimization In this study, the microalga C. vulgaris CS-41 was used, and five photoperiods (12:12; 16:8; 18:6; 20:4, and 24:0 h light / dark) were tested to optimize the photoperiod at 4000 lux in each culture medium (BG-11 and BBM). All other culture conditions were the same as those described in Example 1 and Table 1.

[0137] As shown in Figure 8, across all photoperiods tested, microalgae grown in BG-11 medium demonstrated higher biomass productivity than cultures grown in BBM medium. However, BG-11 and BBM accumulated the least biomass at 12:12 h and 24 h light / dark photoperiods, estimated at 1.866 g / L and 0.389 g / L, respectively. This finding revealed that photoperiod is not only species-specific but also influenced by the culture medium used. Furthermore, it was evident that long photoperiods, such as 18:6:20:4 and 24 h light / dark, can adversely affect the biomass productivity of C. vulgaris in BBM compared to BG-11, demonstrating that C. vulgaris can tolerate photooxidative stress more efficiently in BG-11 than in BBM (Figure 8). Nevertheless, both culture media accumulated maximum biomass at a 16:8 h light / dark photoperiod, reported as 3.894 g / L and 2.321 g / L for BG-11 and BBM, respectively.

[0138] [Example 5] Optimization of culture pH In this example, C. vulgaris CS-41 was grown in culture media adjusted to different pH levels. This experiment was conducted to select the optimal pH of the culture medium to achieve the highest biomass productivity during 7 days of culture under a constant photoperiod (16:8 h) and light intensity (5000 lux). All culture conditions, except for photoperiod, light intensity, and pH, were performed as described in Example 1 and Table 1. Biomass productivity, CO2 fixation rate, and total CO2 recovery were measured as described above in the General Analytical Methods section. pH was monitored and adjusted daily.

[0139] The results reported in Figure 9 show that maximum biomass, as determined by dry weight measurements, was achieved at 7 days at pH 7 in either BG-11 or BBM culture media. However, BG-11 exhibited higher overall biomass production compared to cultures grown in BBM, except at pH 9, where BBM-grown cultures accumulated more biomass than cultures produced in BG-11. This may indicate that BBM has a greater buffering effect on alkaline solutions than BG-11. The CO2 fixation rate and total CO2 recovery at each pH tested were measured on day 7, and the results of each measurement are shown in Figures 10 and 11, respectively.

[0140] Based on the data generated in this example, pH 7 was identified as the optimal pH for C. vulgaris culture conditions, providing maximum biomass productivity and CO2 fixation, and considering the adaptability of the culture medium, both media tested (BBM and BG-11) performed optimally at pH 7.

[0141] [Example 6] Optimizing inoculation cell density The number of cells present in the inoculum used to initiate the C. vulgaris CS-41 culture for the photosynthesis step affects the growth rate and final biomass achieved. This experimental example was designed to evaluate a range of starting inoculum concentrations (0.006 g / L, 0.015 g / L, 0.025 g / L, 0.050 g / L, and 0.133 g / L). Culture conditions were performed as described in Example 1, Table 1, except for the following culture conditions: BBM culture medium, 10% CO2, and a 16:8 (light:dark) photoperiod. The experimental outputs measured to determine the optimal inoculum were biomass productivity, CO2 fixation rate, and total CO2 recovery. These parameters were analyzed as described in the General Analytical Methods section above. The results of the study are summarized in Table 5.

[0142] [Table 5]

[0143] In general, larger inocula more effectively tolerated environmental stress and acquired adaptation. Environmental stress can prevent some cell populations from effectively undergoing cell division. Therefore, when exposed to environmental stress, the number of cells in the inoculum may be more likely to survive stressful conditions compared with smaller inocula. However, it was observed that larger inocula (0.025–0.133 g / L) of the adapted C. vulgaris strain exhibited a slower growth rate than smaller inocula (0.015 g / L), demonstrating that C. vulgaris strain CS-41, which has acclimated to higher-than-atmospheric CO2 levels, is strongly adapted to growth under high CO2 stress. At the same time, the larger inocula tested resulted in lower biomass productivity, which may be due to the lack of early light penetration compared to smaller inocula. The data presented in Table 5 indicate that the adapted, optimized strain (high CO2-tolerant cells) can divide rapidly and reach maximum biomass at an inoculum of 0.015 g / L. This inoculum was identified as the optimal amount for achieving maximum growth rate (i.e., 0.652 g / L / d), while cultures with a lower inoculum (0.006 g / L) showed a lower growth rate (i.e., 0.514 g / L / d). After the optimum (0.015 g / L), growth rate and biomass productivity decreased with increasing inoculum size. However, at a very high inoculum size (0.133 g / L), growth rate remained low, but biomass productivity was relatively higher than the previous low inoculum size of 0.05 g / L. The data presented in Table 5 clearly show that CO2 fixation is directly related to biomass productivity, while growth rate is inversely related to inoculum size.

[0144] [Example 7] Batch cultivation (single cycle) of C. vulgaris under optimized conditions After optimizing the culture condition parameters as described in Examples 1-6, the optimized culture conditions are summarized in Table 6.

[0145] [Table 6]

[0146] Cultures grown under pre-optimized culture conditions were compared to cultures grown under optimized conditions. The comparisons, summarized in Table 7, compare the output measures: biomass production at 7 days, biomass production rate, carbon fixation over the 7-day period, CO2 fixation rate, and total CO2 recovery on the final day.

[0147] The cultivation method developed as a result of the optimization process resulted in cultures that achieved higher levels of biomass in a shorter time and improved CO2 fixation over a 7-day period. The improved growth rates are shown in Figure 12.

[0148] [Table 7]

[0149] The optimized single batch cultivation system (BCS) conditions described in Examples 1-7 were used as a starting point to develop a continuous cultivation process for the photosynthesis step.

[0150] [Example 8] Growth of microalgae cultures in recycled culture medium In this study, experiments were conducted using fresh and used BBM medium. The results are shown in Figure 13. It was found that fresh BBM medium produced 1.055 g / L of biomass over a 7-day duration, with a biomass productivity of 0.144 g / L / day, while reuse of BBM produced 0.865 g / L, with a biomass productivity rate of 0.116 g / L / day. This implies that biomass productivity decreased by approximately 19% in the second cycle of reused medium. In addition to the potential inhibitory effect of DOC, it is recognized that some nutrient components in the culture medium are depleted after each growth cycle, thereby reducing nutrient availability in subsequent growth cycles. The biomass produced by C. vulgaris CS-41 when grown on recycled medium was deemed acceptable for further development in a continuous cycle cultivation system.

[0151] [Example 9] Continuous culture method To develop a continuous circulation method for cultivating C. vulgaris CS-41 in a photobioreactor, a master seed stock of the microalgae was recovered from storage at -80°C using the optimized culture conditions presented in Table 6. The cell density was 10 4 Once the number of cells / mL was reached, the culture was transferred to a larger volume as the working stock. The working stock was checked for contamination before use and refreshed monthly from the master seed stock. All experimental batches were prepared from the working stock by aliquoting 10 8 Cells / mL of biomass were inoculated and incubated under the culture conditions presented in Table 6.

[0152] An example of a typical growth curve for C. vulgaris CS-41 during the optimization process is shown in Figure 14. A lag phase can be observed between days 5 and 6, while exponential growth can be seen to occur between days 6 and 7. The exponential growth phase is ideal for continuous production, especially if the culture can be reset to day 6 each day to allow the algae cells to double.

[0153] This experiment was designed to evaluate and compare two different continuous circulation (CC) methods for the growth of microalgae at a laboratory scale. The two CC method experiments were performed in parallel. The first culture cycle was grown under the optimized growth conditions presented in Table 6. The two culture methods are described in Table 8. Method A (CCA) provides the basis for developing a continuous siphon process for use with liquid biomass, where the liquid biomass is siphoned every two days and new fresh medium is returned to the reactor (refresh). Method B (CCB) provides the basis for developing a continuous siphon process for use with solid biomass and evaluates the use of recycled medium (recycle).

[0154] In this example, two experiments were conducted in parallel, as described in Table 8. The starting biomass density of the CCA was analyzed and calculated to be 2.803 g / L. The biomass used to start the CCA and CCB was prepared from the same batch circulation system (BCS) (cycle 0). The BCS biomass was agglomerated using high-concentration CO2 gas, which effectively concentrated the biomass. The supernatant was siphoned from the reactor and transferred to a separate container. The remaining biomass concentrate was divided into two equal parts. One portion was removed from the reactor for use as a feedstock in downstream applications, such as biodegradation steps to produce biofuel and / or biofertilizer. The supernatant was returned to the original reactor and recycled as culture medium for the remaining biomass portion. The starting biomass density for method CCB was recorded as 2.201 g / L. A schematic diagram of the CCA method (biomass splitting and medium refreshing) and the CCB method (biomass concentration and splitting and medium recycling) is shown in Figure 15.

[0155] [Table 8]

[0156] The preliminary CC methods CCA and CCB had final daily growth rates of 1.287 g / L / d and 1.4705 g / L / d, respectively, and final densities of 5.377 g / L and 5.142 g / L, respectively (Figure 16). The growth rates for both CCA and CCB were significantly greater than those of BCS, which was 0.750 g / L / d under pre-optimized conditions or 0.88 g / L / d under optimized culture conditions (Table 7). Both CC methods significantly outperformed BCS in terms of growth rate and carbon recovery rate.

[0157] In addition to measuring biomass production from the CCA and CCB processes, the CO2 capture rate (CCR) was also analyzed using the methods presented in the General Analytical Methods section above and in Example 1. The carbon capture rate of each of the CCA and CCB processes was compared to BCS. Using industry standard calculations to estimate CO2 capture rate per gram of biomass for each culture method tested, i.e., BCS, CCA, and CCB, the theoretical levels of biomass production and CO2 capture achievable in scaled-up culture volumes and long-term production are summarized in Table 9.

[0158] Methods for achieving economical and sustainable CO2 sequestration at commercial levels, such as CC utilizing phototrophic sources in the photosynthesis step, are desirable to meet industrial needs and improve the environmental sustainability of industrial implementation.

[0159] [Table 9]

[0160] The significant increase in CCR seen in continuous culture is likely due to the larger proportion of microalgae remaining in the continuous culture system after each biomass splitting event. Unlike the BCS method, where the entire biomass is harvested in one event, the remaining phototrophic source continues to fix CO2 into sugars. Subsequent BCS batches would then require at least four days, starting with the initial inoculum, for the phototrophic source to enter effective exponential growth phase. In contrast, the CC method avoids the need to start a culture cycle with an initial inoculum and instead immediately resets the biomass to early exponential phase.

[0161] For example, the biomass concentration from days 0 to 4 for BCS is approximately less than 0.5 g / L / d, whereas the equivalent time points for CC culture, i.e., days 0 (day 7, cycle 1) and 4 (day 11, cycle 1), have biomass levels of at least 2.8 g / L / d. Therefore, CC operation has (always) a larger amount of biomass to fix CO2, doubling the carbon capture capacity of BCS every six days (Figure 17). Moreover, the performance of CC over BCS is further highlighted during scaling of volume and days of operation. As shown in Table 10, the theoretical increase in CC biomass production and CO2 capture capacity was calculated for increasing reactor scale and extending the culture duration.

[0162] [Table 10]

[0163] [Example 10] Industrial implementation of photosynthetic steps The overall mass balance under optimal operating conditions (BG-11 culture medium, 0.015 g / L inoculum, 1 VVM gas flow rate, 16:8 light:dark ratio, and 5000 lux light intensity) is summarized in Figure 18(a). These optimal conditions resulted in a biofixation rate of 1.37 g CO2 / L / day. Assuming that the CO2 biofixation rate remains constant throughout the cultivation process, the inlet CO2 flow in this process was 40 ml / min (equivalent to 0.0748 g / min, CO2 density = 1.87 kg / m).3 ), and the corresponding outlet CO2 flow can be calculated as 39.7965 ml / min (0.07442 g / min). This further implies that the conversion of CO2 to biomass per pass is approximately 0.50%. This conversion level is significantly lower than industrial processes, where conversion varies from 20 to 100%. Due to the lower conversion, biofixation of CO2 on an industrial scale would require large-scale gas recycling.

[0164] To calculate the mass balance of the gas streams in this process, a simplified flow diagram including gas stream recycling, shown in Figure 18(b), was developed and simulated in Aspen-Hysys. In the flow diagram, the photobioreactor was implemented as a conversion reactor with a CO2-to-biomass conversion rate of 0.50%. This assumes that the CO2 conversion rate achieved in laboratory experiments will be achieved in a large-scale photobioreactor. The adjustment block (ADJ-2) in the flow diagram was used to adjust the fresh air flow rate to reach 10% by volume of the homogenized gas going to the reactor, and the recycle of unreacted gas was achieved using a purge stream.

[0165] Flow diagrams were simulated based on a 100 kg / day fresh CO2 inlet (stream 1) and a 1% purge of recycle gas (stream 6). The converged mass balances from the flow diagram simulations are summarized in Table 11. The converged results showed less than 0.1% discrepancy between the inlets (streams 1 and 2) and outlets (streams 4 and 6). Note that the experimental data in this study is valid for a gas flow rate of 1 VVM, so the volumetric flow rate of stream 3 implies the required liquid holdup in the photobioreactor. Therefore, the required liquid holdup for the simulated infrastructure is 3.545 m 3 It is noteworthy that the amount of CO2 released during the purge (Stream 6) is 95.22 kg / day compared to 4.78 kg / day of biofixation, even at a low purge ratio of 1%.

[0166] [Table 11]

[0167] [Example 11] Evaluation of continuous culture methods The study in this example was conducted to assess the reproducibility of the continuous harvest method and to investigate the effect of multiple harvest cycles on algal biomass production.

[0168] The photosynthesis step was carried out for a total of 40 days, including an initial 8-day acclimation period. Accumulated biomass and pH were measured at regular intervals over the 40-day study using the methods described above. Biomass was harvested every two days, and algal cultures were grown using the CCA method described herein. The aqueous culture medium maintained a pH between 6 and 7 throughout the study.

[0169] reproducibility The amount of biomass accumulated throughout the study is shown in Table 12 across three replicate experiments.

[0170] [Table 12]

[0171] The acclimation period was repeated to assess hourly biomass accumulation over the initial 7-day culture period under optimized culture conditions. Hourly biomass measurements are shown in Figure 19. Each time point represents the average of two data points collected from duplicate experiments.

[0172] Multiple Recovery Cycles Further studies were conducted to evaluate the effect of multiple harvest cycles on biomass production during continuous cultivation. Biomass (g / L) was measured as described above. Interestingly, as the number of harvest events increased (Event 1, day 7; Event 2, day 11; Event 3, day 15), the cultures maintained a higher overall biomass (see Figure 20, harvest event on day 15). Figure 20 shows duplicate studies (Rep1 and Rep2) conducted over 18 days under optimized culture conditions. That is, removal of a portion of the microalgal biomass did not cause the total microalgal biomass to drop to the same extent as it did as a result of the previous harvest event, as shown in Figure 20. As a result of harvest events 1 and 2, the biomass dropped to approximately 3.5 g / L. After the third harvest event on day 15, the biomass dropped to approximately 4.7 g / L. After each of the three harvest events, a maximum biomass of approximately 7.5 g / L was achieved between each harvest event and the time before the subsequent harvest event. It was observed that a smaller reduction in biomass concentration allowed the culture to reach maximum biomass in a shorter time without entering stationary phase.

[0173] Collection interval The optimized continuous culture method (Table 6) with a CO2 flow rate of 0.15 VVM was used to investigate the effect of harvest event frequency on biomass production. The purpose of this study was to compare different harvest intervals and monitor the stability of biomass density after harvest initiation. The batches included in this study and the harvest interval times for each treatment are listed in Table 13. At each harvest event for each treatment, a portion of the biomass was harvested by siphoning 5% of the working volume. The biomass density over time (g / L) and the total biomass recovered (g) are shown in Figures 21(A) and 21(B), respectively. A 30-minute harvest interval resulted in the greatest total biomass yield, while biomass density continued to decline throughout the harvest phase. Therefore, a 2-hour harvest interval resulted in the optimal stability of biomass density (an improvement over the 30-minute or 1-hour harvest interval treatments, see Figure 21(A)). Similarly, the 2-hour harvest interval treatment resulted in improved total recovery compared to the 3-hour and 4-hour harvest interval treatments, but biomass density was comparable. A compromise between biomass density stability and yield suggests that a harvest interval of 2 hours is optimal.

[0174] [Table 13]

Claims

1. a) growing a phototrophic organism in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide; and b) reducing the biomass concentration of the phototrophic organisms in the aqueous medium when the phototrophic organisms reach a point between the exponential and stationary growth phases to a post-reduced biomass concentration that is equal to or greater than the concentration present during the early exponential growth phase. A method for culturing a phototrophic organism, comprising:

2. 2. The method of claim 1, wherein the phototrophic organism is an alga, preferably a microalga, preferably a microalga selected from Chlorella species, preferably a microalga of the species Chlorella vulgaris, preferably selected from one or both of the C. vulgaris species strains CS-41 or CCAP211 / 11S, preferably CS-41.

3. The phototrophic organism is CO ２ 3. The method according to claim 1 or 2, which is adaptive and preferably adapted to a gaseous atmosphere containing a carbon dioxide concentration of at least about 5%, preferably at least about 8%, which may be between about 8% and 25%.

4. 4. The method according to any one of claims 1 to 3, wherein the point between the exponential growth phase and the stationary growth phase is a point between the mid-exponential growth phase point and the saturation point, preferably between beyond the mid-exponential growth phase and the saturation point, preferably in the late exponential phase.

5. 5. The method of claim 1, wherein the reduction of the biomass concentration comprises removing a quantity of phototrophic biomass in the aqueous medium and adding a dilution quantity of aqueous medium, optionally including a preliminary step of removing a quantity of the aqueous medium.

6. 6. The method of claim 5, wherein the preliminary step of removing a quantity of the aqueous medium is performed and the diluted quantity of aqueous medium that is added comprises the removed aqueous medium.

7. 7. The method of any one of claims 1 to 6, wherein the biomass concentration is between about 2.0 g / L and about 9.0 g / L, preferably between about 3.0 g / L and about 8.5 g / L, preferably between about 4.5 g / L and about 8.0 g / L, preferably between about 5.0 g / L and 7.5 g / L, preferably between about 6.0 g / L and 7.5 g / L.

8. 8. The method of any one of claims 1 to 7, wherein the reduced biomass concentration is the concentration that existed at about or beyond the mid-exponential growth point.

9. 9. The method of any one of claims 1 to 8, wherein the reduced biomass concentration is between about 2.0 g / L and about 6.5 g / L, preferably between about 2.5 g / L and about 6.0 g / L, preferably between about 3.0 g / L and 5.5 g / L, preferably between about 3.5 g / L and 5.0 g / L.

10. 10. The method of any one of claims 1 to 9, further comprising repeating step a) using the reduced biomass concentration.

11. 10. The method of claim 1, further comprising cycling the reduced biomass concentration, wherein the cycling comprises repeating steps a) and b) using the reduced biomass concentration, preferably repeating steps a) and b) multiple times using the reduced biomass concentration for each step b).

12. 12. The method of claim 11, wherein the plurality of times is between 2 and about 1260 times, preferably between about 20 and about 630 times, preferably between about 125 and 315 times.

13. 7. The method of claim 1, further comprising cycling the reduced biomass concentration, wherein the cycling comprises repeating steps a) and b) multiple times using the reduced biomass concentration of each step b), wherein the duration of step a) is short or the concentration change of step b) is small.

14. 14. The method of claim 13, wherein the duration of step a) during the series of cycles is short or the concentration change of step b) is small.

15. 15. The method of claim 13 or 14, wherein the short duration is between about 30 minutes and 5 hours, preferably between about 30 minutes and 3 hours, preferably between about 30 minutes and 2 hours, preferably between about 1 hour and 2 hours.

16. 15. The method of claim 13 or 14, wherein the small concentration change is between about 0.001 g / L and 1.0 g / L, preferably between about 0.005 g / L and 0.8 g / L, preferably between about 0.01 g / L and 0.6 g / L, preferably between about 0.015 g / L and 0.4 g / L, preferably between about 0.1 g / L and about 0.3 g / L.

17. 13. The method according to any one of claims 1 to 12, wherein the step of removing the amount of phototrophic biomass is batchwise, and preferably an amount between about 30% and about 80%, preferably between about 40% and about 65%, preferably between about 45% and about 60% is removed.

18. 7. The method of claim 5 or 6, wherein the step of removing the amount of phototrophic biomass is a continuous removal of phototrophic biomass from the aqueous medium.

19. 20. The method of claim 18, wherein the continuous removal of phototrophic biomass is done together with aqueous culture medium, and diluting amounts of aqueous culture medium are continuously added to the remaining biomass.

20. 20. The method of claim 19, wherein the continuous removal of phototrophic biomass together with the aqueous culture medium and the continuous addition of dilution volumes of aqueous culture medium are performed using removal and addition flow rates calibrated to match the biomass growth rate.

21. 21. The method of any one of claims 1 to 20, wherein the gaseous atmosphere is provided with a higher concentration of carbon dioxide than air, preferably the concentration of carbon dioxide is at least about 5%, preferably at least about 8%, which may be at a concentration between about 8% and 25%.

22. 22. The method according to any one of claims 1 to 21, wherein the light has an intensity of between about 3000 lux and about 7000 lux, preferably between about 4000 lux and about 6000 lux, preferably between about 4500 lux and about 5500 lux, or preferably about 5000 lux.

23. 23. The method of any one of claims 1 to 22, wherein the culture conditions further comprise a photoperiod of between about 12 h and about 20 h, preferably between about 14 h and about 18 h, preferably between about 15 h and about 17 h, preferably about 16 h per 24 h period.

24. 24. The method of any one of claims 1 to 23, wherein the source of carbon dioxide comprises a waste gas stream, preferably a waste gas stream produced by combustion, by steam reforming and / or by biological decomposition of hydrocarbons.

25. The culture conditions are a) a bioreactor in which the volume of phototrophic organisms and aqueous medium is between about 35% and 90%, preferably between about 40% and 85%, preferably between about 50% and 70%, preferably about 60% of the volume of the bioreactor; b) the gaseous atmosphere further comprises air; c) the gaseous atmosphere is preferably supplied as a continuous stream having a flow rate of between about 0.05 VVM and about 5 VVM, preferably between about 0.1 VVM and about 2 VVM, preferably between about 0.1 VVM and about 1.0 VVM, preferably between about 0.1 VVM and 0.5 VVM, preferably between about 0.1 VVM and 0.3 VVM, preferably between about 0.1 VVM and 0.2 VVM, preferably about 0.15 VVM; d) a pH of the aqueous medium between about 5 and 9, preferably between about 6 and 8, preferably between about 6.5 and 7.5, preferably about 7; e) a temperature between about 25°C and about 31°C, preferably between about 27°C and about 29°C, preferably about 28°C 25. The method of any one of claims 1 to 24, further comprising one or more of the group consisting of:

26. 26. The method of any one of claims 1 to 25, wherein the aqueous medium comprises one or more nutrients, preferably said nutrients selected from one or more, preferably two or more, preferably all, of the group consisting of boric acid, calcium chloride, cobalt nitrate, copper sulphate, EDTA disodium salt, magnesium sulphate, manganese chloride, potassium phosphate, sodium molybdate, sodium nitrate and zinc sulphate.

27. 27. The method of any one of claims 1 to 26, wherein the aqueous medium is selected from one or both of BBM and BG-11, preferably BG-11.

28. 10. A method for continuously culturing a phototrophic organism, comprising the steps of performing the method of claim 1 and cycling the reduced biomass concentration, comprising repeating steps a) and b) using the reduced biomass concentration.

29. 30. The method of claim 28, further comprising repeating steps a) and b) multiple times, each using the reduced biomass concentration of step b).

30. 1. A method for culturing a phototrophic organism, comprising growing the phototrophic organism in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide, wherein the light has a light intensity of between about 3000 lux and about 7000 lux, and the culture conditions further comprise a photoperiod of between about 12 hours and about 20 hours.

31. 31. The method of any one of claims 1 to 30 when used for the recovery of carbon dioxide from a waste gas stream.

32. 31. The method of any one of claims 1 to 30 when used in the production of organic feedstock.

33. 31. A culture of a phototrophic organism produced by the method of any one of claims 1 to 30.

34. 31. An organic feedstock comprising removed phototrophic biomass produced by the method of any one of claims 1 to 30, wherein the reduction of the biomass concentration comprises removing phototrophic biomass from the aqueous medium.

35. 31. A method for recovering carbon dioxide from a waste gas stream comprising culturing a phototrophic organism by the method of any one of claims 1 to 30, wherein the source of carbon dioxide comprises a waste gas stream.

36. C. vulgaris species strain CS-41 or CCAP211 / 11S, preferably CS-41, and is resistant to CO2 in a gaseous atmosphere containing a carbon dioxide concentration of 5% or more. ２ Adapted, CO ２ Adaptive phototrophs.

37. CO in a gaseous atmosphere containing a carbon dioxide concentration of at least about 8%, which may be between about 8% and 25%. ２ 36. The CO of claim 35 ２ Adaptive phototrophs.

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