Culturing phototrophic organisms

Abstract

This disclosure relates to a method for culturing a phototrophic organism in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide. The culturing may be continued. The method is useful for capturing carbon dioxide, and / or producing an organic feedstock useful in downstream processes, for example to generate biofuel and / or biofertiliser. The disclosure also relates to a CO2-adapted Chlorella vulgaris species strain.

Description

[0001] Culturing Phototrophic Organisms

[0002] Reference to Earlier Application

[0003] The present application claims the benefit of Australian Provisional Patent Application No. 2023900393, filed on 16 February 2023, the entire contents of which is incorporated herein by reference.

[0004] Technical Field

[0005] This disclosure relates to a method for culturing a phototrophic organism in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide. The culturing may be continued. The method is useful for capturing carbon dioxide, and / or producing an organic feedstock useful in downstream processes, for example to generate biofuel and / or biofertiliser. The disclosure also relates to a CO2-adapted Chlorella vulgaris species strain.

[0006] Background

[0007] A major contributor identified to the phenomenon of global warming and the associated environmental problems is carbon dioxide (CO2) emissions. CO2is mainly a by-product of combustion. The production of CO2is in large part anthropogenic; it occurs in a variety of industrial applications such as the generation of electricity from coal at power plants and in the use of hydrocarbons that are typically the main components of fuels that are combusted in combustion devices, such as engines. Exhaust gas discharged from such combustion devices contains CO2gas, which at present is simply released to the atmosphere. Anthropogenic global carbon dioxide (CO2) emissions have increased by about 50% in the last couple of decades. While political, social and industrial focus has been on reducing emissions, this alone is widely believed to be insufficient to curtail the continuing negative impact on the global environment. Accordingly, a larger focus is required on carbon dioxide removal (CDR) technologies (also referred to as carbon capture methods) that can lead to net negative emissions.

[0008] Existing CDR technologies include afforestation and reforestation, soil carbon sequestration, enhanced weathering, ocean fertilisation, bioenergy with carbon capture and storage, and direct air carbon capture. These technologies have their limitations. For instance, while terrestrial plants have a natural capacity to capture CO2 through the photosynthesis process, they are adapted to atmospheric CO2 concentrations of typically around 0.033%, while industrial discharge gases normally contain much higher concentrations in the area of 10- 25% CO2. Afforestation and reforestation accordingly have a huge land requirement to be even close to net negative, and this is offset by the release of captured carbon back into the atmosphere when the biomass decays or is burned. There are also biodiversity concerns associated with forestation. Forestation process therefore have limited practicality and commercial feasibility. Enhanced weathering is also limited commercial feasibility and is associated with a risk of negatively impacting biodiversity, water quality and food security. Ocean fertilisation is also limited in its applicability, as its practical impact is minimal, and it has a huge material demand. Bioenergy with carbon capture and storage remains the most plausible and scalable pathway to achieve a net negative carbon economy, though it is not without its problems.

[0009] Some carbon capture methods have been proposed for sequestering the CO2produced by industrial processes such as coal-fired and other power plants. Reacting the CO2 with large quantities of metal oxides, particularly with the oxide of calcium and oxide of magnesium, then burying the resulting carbonates is one method. This method has a disadvantage in that it requires a large and continuous supply of minerals. Another method involves removing the CO2from the combustion gas, compressing it, then shipping it by pipeline to a peridotite or serpentinite mine for conversion to a carbonate, before burying it at the mine site. However, for CO2, which is heavier than air, shipment of compressed CO2 presents material handling challenges. For instance, CO2 will stay close to the ground if it is accidentally released during transit and can thus be life threatening. Another problem may be the availability of mine sites for such burying, and the cost and energy consumption of transport. Other techniques, which can be generally classified as geologic, terrestrial, or ocean systems, have been attempted which also rely on transport of CO2to sites for injection into geologic, soil, or ocean repositories. However, like for peridotite or serpentinite mine sites, each of these processes are expensive in terms of capital costs, transport, and energy consumption. These carbon capture methods also focus on storage rather than converting carbon into a useful product. One method which has gained attention recently involves the use of microalgae. Microalgae have a natural ability to capture CO2 at higher rates than terrestrial plants and algal biomass is a useful product in a number of downstream industrial processes. However, algae cultivation for carbon capture is difficult. The growth profile of algae follows a general pathway consisting of a long lag phase where biomass remains constant or increases slowly, before finally reaching a pre-exponential then exponential phase where the growth rate increases rapidly. While the CO2 capture capabilities of the biomass increases through the exponential phase, the exponential phase is generally short lived before the algae transitions to a stationary phase where net growth halts, followed by a death phase where the viable biomass decreases rapidly. The window for suitable levels of carbon capture in the exponential phase is therefore short lived. Algae cultures are also sensitive to environmental changes and may be unable to grow, or grow at a suitable rate, in environments containing carbon dioxide at levels commonly found in industrial discharge.

[0010] It is possible to produce an algae that is tolerant to high-CC environments by a process of adaptive laboratory evolution (ALE) which involves subjecting an algae to an initial low CO2concentration and then, when the algae has stabilised, increasing that concentration slightly, and so-on, until a desired tolerance is reached. ALE is typically a slow process that can take at least several months and even several years. Then, when a suitable culture is finally produced, it must be grown to a useful commercial scale for capturing CO2, and shortly thereafter at the conclusion of one exponential growth phase it loses viability and the process must be repeated. There are also limitations to the level of tolerance achievable, often being lower than the carbon dioxide levels commonly found in industrial discharge.

[0011] It would be advantageous to provide a CO2 sequestration technique that could convert carbon into a useful downstream product, and / or to provide an improved culturing method for algae that may be used in a CO2 sequestration process, especially of industrial discharge.

[0012] It is to be understood that, for any prior art publication or reference that is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art in any country. Summary

[0013] In a first aspect, the present disclosure provides a method of culturing a phototrophic organism, comprising the steps of: 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 organism reaches a point between an exponential growth phase and a stationary growth phase, reducing a biomass concentration of the phototrophic organism in the aqueous medium to a reduced biomass concentration not less than that which existed in an early-exponential growth phase.

[0014] In some embodiments, reducing the biomass concentration comprises removing phototrophic organism biomass from the aqueous medium. The removed biomass may be referred to as an organic feedstock. This may be performed together with the addition of a dilution volume of aqueous medium. The dilution volume of aqueous medium may be prepared fresh or be recycled.

[0015] In some embodiments, the method further comprises cycling the reduced biomass concentration, wherein the cycling comprises repeating steps a) and b) using the reduced biomass concentration, and preferably repeating steps a) and b) a plurality of times using the reduced biomass concentration of each step b). The method when including a cycling step may be referred to as a continued culturing method.

[0016] In some embodiments, a source of the carbon dioxide comprises a waste gas stream, for example a waste gas stream generated by combustion of hydrocarbons (e.g., flue gas), by steam reforming and / or by bio-decomposition.

[0017] In another aspect, the present disclosure provides a method of continued culturing of a phototrophic organism, comprising performing the method of the first aspect as herein described, and cycling the reduced biomass concentration, wherein the cycling comprises repeating steps a) and b) using the reduced biomass concentration, and preferably repeating steps a) and b) a plurality of times using the reduced biomass concentration of each step b). In another aspect, the present disclosure provides a method of culturing a phototrophic organism, comprising growing a phototrophic organism in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide, wherein the light is of an intensity of between about 3000 lux and about 7000 lux, and wherein the culture conditions further comprise a photoperiod of between about 12 h and about 20 h.

[0018] In another aspect, the present disclosure provides a culture of a phototrophic organism produced by a method as herein described.

[0019] In another aspect, the present disclosure provides an organic feedstock produced by a method as herein described, wherein the reducing of the biomass concentration comprises a step of removing a volume of the phototrophic organism biomass from the aqueous medium, and the organic feedstock comprises removed phototrophic organism biomass.

[0020] In another aspect, the present disclosure provides a method as herein described, when used for producing an organic feedstock.

[0021] In another aspect, the present disclosure provides a method of capturing carbon dioxide from a waste gas stream, comprising culturing a phototrophic organism by a method as herein described, wherein a source of the carbon dioxide comprises a waste gas stream.

[0022] In another aspect, the present disclosure provides a method as herein described, when used for capturing carbon dioxide from a waste gas stream.

[0023] 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 strain CS-41 or CCAP211 / 11S, and preferably CS-41, and wherein the phototrophic organism is CO2-adapted to a gaseous atmosphere comprising carbon dioxide concentrations of 5% or greater.

[0024] Brief Description of the Drawings

[0025] Figure 1 is a schematic diagram of a photobioreactor setup, where the triangle joins the CO2and air inlets and the rotameter represents an electronic pressure regulator. Figure 2 shows a comparison of (a) biomass concentration, (b) pH, and (c) biomass growth rate from 10% CO2 4thgeneration (4G) and 5thgeneration (5G) (BBM culture medium, 0.05 g / L inoculum size, 12:12 photo: dark period).

[0026] Figure 3 shows biomass (a) elemental analysis data at day 7, (b) bio-fixation rate of CO2, (c) CO2 concentration in inlet and outlet gas stream measured by CO2 sensors from 10% CO24thgeneration and 5thgeneration (BBM culture medium, 10% CO2, 0.05 g / L inoculum size, 12:12 photo:dark period).

[0027] Figure 4 shows the effect of light intensity on growth rate of C. vulgaris CS-41 grown in different culture medium.

[0028] Figure 5 shows the effect of light intensity on biomass production by C. vulgaris CS-41 grown in different medium.

[0029] Figure 6 shows the effect of light intensity on bio-fixation of CO2 by C. vulgaris CS-41 grown in either BBM or BG-11 culture medium.

[0030] Figure 7 shows the effect of light intensity on total CO2 capture by C. vulgaris CS-41 grown in either BBM or BG-11 culture medium.

[0031] Figure 8 shows the biomass of C. vulgaris CS-41 at five photoperiods (12:12, 16:8, 18:6, 20:4 and 24:0 h light / dark) grown in either BBM or BG-11 culture media.

[0032] Figure 9 shows dry weight biomass (g / L) of C. vulgaris at different pH (6, 7, 8, and 9), under the fixed photoperiod (16:8 h) and light intensity (5000 lux) during the cultivation of 7 days in BG-11 and BBM culture media.

[0033] Figure 10 shows the average CO2 fixation rate over a day 7 cultivation by C. vulgaris CS-41 grown at a range of pH in BBM or BG-11.

[0034] Figure 11 shows total CO2captured at day 7 by C. vulgaris CS-41 grown at a range of pH in BBM or BG-11.

[0035] Figure 12 is a comparison of C. vulgaris growth rate pre and post optimisation grown in BG- 11 culture medium. Figure 13 shows the effect of fresh and reused culture medium on biomass concentration. (BBM culture medium, 10% CO2, 0.05 g / L inoculum size, 12:12 photo:dark period; note: cycle- 1 - trial with a fresh BBM, cycle-2 - trial with the spent BBM at the end of cycle-1).

[0036] Figure 14 is a seven day growth curve of C. vulgaris CS-41 under optimised conditions.

[0037] Figure 15 is a flow diagram of the continued cycling methods (CCA) and (CCB). Starter batch culture is grown for 7 d (cycle 0). Biomass resulting from cycle 0 is split into two batches (fresh, CCA; or recycled, CCB), and incubated at lower light for a further 2 d (cycle 1). Half of the biomass is removed for downstream applications (e.g., bio-decomposition), remaining batch is replenished with either fresh or recycled medium as before. This process is repeated until the continued batch loses integrity as determined by pre-establish growth criteria.

[0038] Figure 16 shows the biomass production of C. vulgaris CS-41 when grown under continued cycling methods (A) CCA (splitting biomass and refreshing medium method) and (B) CCB (concentrating biomass and recycling medium method).

[0039] Figure 17 is a comparison between continued culture methods CCA and batch culture system (BCS) of the amount of CO2captured (A) as shown in g / L and (B) shown as total CO2captured.

[0040] Figure 18 shows (a) a summary of overall mass balance and optimum operating conditions in the reactor, (b) simple flowsheet of the microalgae cultivation process with recycling of gas.

[0041] Figure 19 is a graph that shows the accumulation of biomass (g / L) during the acclimatisation phase at hourly intervals.

[0042] Figure 20 is a graph that shows the effect of biomass harvesting on the total biomass (g / L) of the continued culture method over multiple biomass harvest events.

[0043] Figure 21 is two graphs: graph A shows the biomass density (g / L) of each harvest interval treatment over time once harvesting commenced; graph B shows the total biomass harvested from each different treatment (harvest intervals of 30 min, 1 h, 2 h (performed in duplicate), 3 h and 4 h. Detailed description

[0044] The following is a detailed description of the disclosure provided to aid those skilled in the art in practicing the present disclosure. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those 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.

[0046] As used herein, the term "and / or", e.g., "X and / or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

[0047] 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.

[0048] The term "about" as used herein, when referring to a numerical value or range, allows for a degree of variability in the value or range, for example within 10% of a stated limit of a range.

[0049] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 10, including a range that is specified to be "between" 1 and 10, is understood to include any number, combination of numbers, or sub-range from the group consisting 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 sub-range from the group consisting 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0.

[0050] As used herein, the phrase "at least one of", when used with a list of items, means that different combinations of one or more of the listed items may be selected, and that only one of the items in the list may be selected. In other words, "at least one of" means any number or combination of items may be selected from the list. For example, "at least one of item A, item B, and item C" may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C.

[0051] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated 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.

[0052] Reference will now be made in detail to preferred and exemplary embodiments of the disclosure. While the disclosure will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the disclosure to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the disclosure as defined by the appended claims.

[0053] Phototrophic Organisms

[0054] The present disclosure provides a method of culturing a phototrophic organism including growing it in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide.

[0055] By a "phototrophic organism" is meant an organism that is capable of using light as a 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.

[0056] In preferred embodiments, the phototrophic organism is an algae. An algae is identifiable as a photosynthetic eukaryotic non-flowering aquatic plant. Examples include red algae, green algae and cyanobacteria, which generally includes plant species classified as Halvaria and Hacrobia (red algae) containing the Heterokonts, Dinoflagellates, Haptophyta and Cryptomonads, as Excavata and Rhizaria (green algae) containing the Chlorarachniophytes, Euglenids and Chlorophyta, and as Primoplantae and Archaeplastida (cyanobacteria) containing the Chlorophyta and Rhodophyta. Preferably, the phototrophic organism is a microalgae. A microalgae is identifiable as a unicellular algae, and may otherwise be referred to as a microphyte. Preferably, the microalgae is a green algae. Preferably, the microalgae is selected from a Chlorella spp., such as from the group consisting of 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.

[0057] In preferred embodiments, the microalgae is C. vulgaris species microalgae, preferably selected from one or both of C. vulgaris species strain CS-41 or CCAP 211 / 11 S, 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 CCAP 211 / 11 S is maintained at the Culture Collection of Algae and Protozoa, Oban, Scotland, UK, under accession no. MG022720, and is postulated to be the same strain as CS-41.

[0058] It is preferred that the phototrophic organism is COz-adapted, meaning that it is able to grow under culture conditions comprising a gaseous atmosphere comprising carbon dioxide, wherein the carbon dioxide concentration is greater than atmospheric CO2 concentrations (atmospheric CO2 concentrations are typically around 0.033%). Methods for producing CO2- adapted phototrophic organisms are described below.

[0059] In preferred embodiments, the phototrophic organism is adapted to a gaseous atmosphere containing a carbon dioxide concentration of at least about 5%, preferably of at least about 8%, 9% or 10%, and may be adapted to concentrations of about 15%, 20% or 25%. Alternatively, or in addition, the phototrophic organism may be adapted to a gaseous atmosphere containing a carbon dioxide concentration of between about 5% and 25%, which may be a concentration of about 5%, 6%, 7% etc. 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 9% or 10% and 25%, or 8% and 20%, which may be any one of about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. It is believed that a phototrophic organism that is adapted to above-atmospheric concentrations of carbon dioxide is able to consume a greater amount of carbon dioxide which, arising from photosynthesis, translates to a higher energy production in the form of carbohydrates. In addition, carbon dioxide levels of between about 8% and 25% are commonly found in waste streams of various industrial processes and accordingly the methods described herein may be used to sequester carbon dioxide from the gaseous atmosphere, as described below.

[0060] At least the preferred phototrophic organisms that are capable of consuming a greater amount of carbon dioxide when CC -adapted, are capable of being CC -adapted in much faster time than traditional ALE processes, and / or are also capable of being CC -adapted to carbon-dioxide concentrations that are higher than those previously thought. It is for this reason that it is preferred that the phototrophic organism is a microalgae, preferably a Chlorella spp., preferably a C. vulgaris species microalgae, and most preferably strain CS-41 or CCAP 211 / 11 S.

[0061] Accordingly, the present disclosure also provides a CC -adapted phototrophic organism, wherein the phototrophic organism is selected from one or both of C. vulgaris species strain CS-41 or CCAP211 / 11S, and preferably CS-41, and wherein the phototrophic organism is COz-adapted to a gaseous atmosphere comprising carbon dioxide concentrations of 5% or greater.

[0062] Culturing Methods

[0063] The present disclosure provides a method of culturing a phototrophic organism by growing it in an aqueous medium. The term "aqueous medium" as used in the present disclosure means a water-based liquid. The aqueous medium may contain nutrients for supporting organism growth. Preferred characteristics of the aqueous medium are described below.

[0064] Culturing methods may be performed in a bioreactor, which may generally be a vessel containing the phototrophic organism in the aqueous medium. Numerous bioreactors are known to those of skill in the art. The preferred bioreactor is that disclosed in Australian Provisional Patent Application No. 2023902970, filed on 14 September 2023, the entire contents of which is incorporated herein by reference. Reducing Biomass Concentration

[0065] The present disclosure provides a method of culturing a phototrophic organism wherein when the phototrophic organism reaches a point between an exponential growth phase and a stationary growth phase, a biomass concentration of the phototrophic organism in the aqueous medium is reduced to a reduced biomass concentration not less than that which existed in an early-exponential growth phase. Another way of expressing that when the phototrophic organism reaches a point between an exponential growth phase and a stationary growth phase, is to say that when the phototrophic organism reaches a concentration corresponding to a point between an exponential growth phase and a stationary growth phase, or that when the phototrophic organism reaches a concentration which exists between an exponential growth phase and a stationary growth phase.

[0066] Generally speaking, phototrophic organisms such as microalgae and especially Chlorella species such as C. vulgaris are non-motile reproductive cells (autospores) which reproduce asexually, whereby cells multiply by autosporulation in which four daughter cells having individual cell walls are formed inside the cell wall of a mother cell. After maturation of the newly formed cells, the mother cell wall ruptures liberating the daughter cells and the debris of the mother cell will be consumed as feed by the daughter cells. A culture thus grows.

[0067] The growth profile of phototrophic organisms, including algae and especially microalgae, follows a generally sigmoidal pathway consisting of a long lag phase wherein biomass remains constant or increases slowly (e.g., where autosporulation occurs at a low rate), before a pre-exponential phase where the growth rate transitions to a high growth rate in a subsequent exponential phase (e.g., where autosporulation occurs at a high rate). The exponential phase is then followed by a second transition phase where the growth rate slows and the biomass plateaus in a subsequent stationary phase. In a later death phase, the biomass decreases as the organism dies.

[0068] As used herein, the term "exponential phase", refers to a stage of growth of the phototrophic organism which occurs in the organism's sigmoidal growth profile between an initial lag phase and a later stationary phase, and is characterised by a period of rapid growth as compared with both the initial lag phase and later stationary phase. An "early exponential phase" refers to the initial increase in growth rate of the organism as it transitions from the initial lag phase, and encompasses the period of transition. The "mid-exponential phase" occurs after the early exponential phase in about the mid-region of the exponential phase and encompasses a "mid-exponential phase point" being the mid-point of the midexponential phase. A "late exponential phase" follows the mid-exponential phase and precedes the stationary phase, and is often during which the maximal growth rate is observed. The "stationary phase" refers to the stage of growth where the organism growth rate begins to slow and where growth plateaus. A "saturation point" occurs during the late exponential phase just prior to commencement of the stationary phase. In the context of culturing a phototrophic organism, a "growth phase" may be referred to as a "phase" for short. In many embodiments, the early, mid and late exponential phases which make up the exponential phase may be thought of as separated into thirds in terms of biomass concentration and / or duration.

[0069] Advantageously, when a culture of a phototrophic organism in an aqueous medium, and especially microalgae, is at a point between an exponential growth phase and a stationary growth phase, and a biomass concentration of the phototrophic organism in the aqueous medium is reduced to a concentration that is not less than that which existed in an early- exponential growth phase, the phototrophic organism avoids or substantially avoids a stationary phase and continually grows without a prolonged initial lag phase. The phototrophic organism quickly adjusts to the altered aqueous medium concentration and returns to an exponential growth phase after a pre-exponential phase, or even continues growing at a rate concordant with the exponential growth phase. Put in other words, in this way, the phototrophic organism may be returned to an earlier point in the exponential growth phase, for example from a late exponential phase to an early exponential phase, as a way of essentially extending the exponential phase as a result of reducing the phototrophic organism concentration. Accordingly, the method provides for an increased biomass production in a shorter time frame.

[0070] The advantage may be efficiently exploited when the point between an exponential growth phase and a stationary growth phase, immediately prior to the biomass concentration of the phototrophic organism in the aqueous medium being reduced, is a point between a mid- exponential phase point and a saturation point, or preferably between about the midexponential growth phase and a saturation point, or in other words is a point in the late exponential phase, and is preferably a point at about a saturation point. As the growth rate and / or biomass is generally highest in the late exponential phase and / or at the saturation point, this allows for a high biomass production before the concentration is reduced. The advantage may further be efficiently exploited when the reduced biomass concentration is not less than that which existed at the beginning of the exponential growth phase, or in other words, the reduced concentration is a concentration which existed in the early exponential phase, and is preferably that which existed in the mid-exponential growth phase, which may be above the mid-exponential phase point, and may even be within the late-exponential phase. A reduced concentration of phototrophic organism, and especially microalgae, as existed in the early exponential phase, and especially in the mid- or late-exponential growth phase, provides for the quickest adjustment to the altered aqueous medium concentration and the quickest return to exponential growth, allowing for even higher biomass production.

[0071] Accordingly, in preferred embodiments, the point between an exponential growth phase and a stationary growth phase immediately prior to the biomass concentration of the phototrophic organism being reduced, is a point between the mid-exponential growth phase and a saturation point, or in other words is a point in the late exponential phase, and is preferably a point at about a saturation point. Also in preferred embodiments, the reduced biomass concentration is not less than that which existed at the beginning of the exponential growth phase, or is a concentration which existed in the early exponential phase, and is preferably that which existed in the mid- or late-exponential growth phase.

[0072] As this provides for a rapid return to exponential growth, in preferred embodiments the method further comprising repeating step a) using the reduced biomass concentration; that is, using the reduced biomass concentration, growing a phototrophic organism in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide. Step a) may be referred to as the "growth step".

[0073] The reduced biomass concentration may be grown in the same bioreactor, or it may be transferred to another. If the concentration is reduced by removing a volume of the phototrophic organism biomass, then the removed biomass may be transferred to a bioreactor for continued growth, or it may be transferred to a storage vessel for use in downstream processes.

[0074] The physical amounts of biomass which exist at various points in the growth profile phases may vary between specific phototrophic organisms and is determinable by one of skill in the art using routine methods. In preferred embodiments, and especially with microalgae, the biomass concentration in aqueous medium (i.e., immediately before it is reduced) is a concentration of 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, and preferably between about 6.0 g / L and 7.5 g / L. In the case of microalgae, these concentrations may correspond to about a saturation point. Also in preferred embodiments, the reduced biomass concentration in aqueous medium is a concentration of 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, and preferably between about 3.5 g / L and 5.0 g / L. In the case of microalgae, these concentrations may correspond to within the mid-exponential phase or at about the mid-exponential phase point, or within the late exponential phase. Accordingly, in preferred embodiments, and especially with microalgae, the biomass concentration in aqueous medium is reduced by 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, and preferably between about 2.5 g / L and about 4.5 g / L. The amount that the biomass concentration in aqueous medium is reduced, i.e., the difference between the concentration of the phototrophic organism in the aqueous medium immediately prior to removal and the reduced biomass concentration, by may be referred to as the "concentration change".

[0075] The duration of the growing step will vary between different phototrophic organisms. For microalgae, the duration of at least a first growing step, from an early-exponential growth phase to a stationary growth phase, may be for a time of between about 1.5 to about 4 days, preferably between about 1.5 days to about 3 days, and preferably between about 1.8 to about 2.2 days. The step of reducing the biomass concentration may be performed by, for example, adding a volume of aqueous medium and / or by removing a volume of the phototrophic organism biomass. An added volume of aqueous medium may be referred to as a "dilution volume". Removing a volume of the phototrophic organism biomass may also be referred to as a "harvest". When reducing the biomass concentration is performed solely by adding a volume of aqueous medium, then the combined volume of phototrophic organism biomass and aqueous medium will increase and may require additional handling, e.g., transfer to a larger vessel size. Accordingly, in preferred embodiments, to e.g., avoid increasing vessel size, reducing the biomass concentration comprises a step of removing a volume of the phototrophic organism biomass.

[0076] Removing phototrophic organism biomass may also remove a volume of aqueous medium. When this occurs, and when the volume of phototrophic organism biomass removed is the same or less than the volume of aqueous medium removed, then reducing the biomass concentration will generally require adding a dilution volume of aqueous medium that is greater than the difference in volume between the removed phototrophic organism biomass and the removed aqueous medium. On the other hand, when the volume of phototrophic organism biomass removed is greater than the volume of aqueous medium removed, then reducing the biomass concentration may not require adding a dilution volume of aqueous medium, though adding a dilution volume of aqueous medium may be performed, e.g., to achieve a desired reduced biomass concentration.

[0077] In preferred embodiments, reducing the biomass concentration comprises removing a volume of the phototrophic organism as well as a volume of the aqueous medium and adding a dilution volume of aqueous medium to the remaining biomass.

[0078] A volume of the aqueous medium may be removed along with or separately to a volume of the phototrophic organism biomass. For instance, a volume of the aqueous medium may be removed prior to removing the biomass. This may be achieved by flocculating the biomass and removing the aqueous medium as supernatant. Methods of flocculation are known to those of skill in the art. For instance, and relevantly for present purposes, flocculation may be achieved by increasing the carbon dioxide concentration in the gaseous atmosphere. Chemical flocculants may also be used, for example chitosan (which may also be referred to as deacetylated chitin).

[0079] The dilution volume of aqueous medium added may comprise removed aqueous medium. In other words, the aqueous medium may be recycled. Removing the aqueous medium as supernatant is one way which may allow for the medium to be recycled. Recycling of aqueous culture medium confers an advantage in the cost-effective and ecologically sustainable production of biomass. The dilution volume may at least in part comprise fresh aqueous medium. This may allow for at least a portion of the aqueous medium to be refreshed which may assist with growth of the remaining biomass. The proportion of biomass which is removed will depend on the relative biomass concentrations which exist at various points of the growth profile phases and may vary between specific phototrophic organisms. The proportion removed while retaining biomass at a concentration not less than that which existed in an early-exponential growth phase is determinable based on the present disclosure by one of skill in the art using routine methods.

[0080] The phototrophic organism biomass may be removed in a batch-wise fashion as described further below. In batch-wise removal, an amount of between about 30% and about 80%, preferably between about 40% and about 65%, and preferably between about 45% and about 60%, is removed. In the case of phototrophic organisms and especially microalgae, these proportions may correspond to the difference between a saturation point concentration and mid-exponential point concentration, when an equal dilution volume of aqueous medium is added to the remaining biomass.

[0081] Put another way, the concentration change may be between about 30% and about 80%, preferably between about 40% and about 65%, and preferably between about 45% and about 60%. For example, when the concentration of the phototrophic organism in the aqueous medium immediately prior to removal is 7.5 g / L, and the reduced biomass concentration is 5.0 g / L, this constitutes a concentration change of 33%. Similarly, when the concentration of the phototrophic organism in the aqueous medium immediately prior to removal is 9.0 g / L, and the reduced biomass concentration is 2.0 g / L, this constitutes a concentration change of 78%, etc. To advantage, reducing concentration by removing biomass allows for the removed biomass to be utilised in downstream processes, and that by culturing the reduced biomass it regenerates quickly through an exponential growth phase, and biomass can be removed again and again in relatively quick succession as a feedstock for said downstream processes, i.e., steps a) and b) may be repeated once or a number of times. By reducing concentration by removing biomass within the described parameters and repeating the growing step provides the basis for a continued culturing method.

[0082] Continued Culturing

[0083] In preferred embodiments, the method further comprising cycling the reduced biomass concentration, wherein the cycling of the reduced biomass concentration comprises repeating at least step a), and preferably steps a) and b), using the reduced biomass concentration, and preferably cycling the reduced biomass concentration a plurality of times (i.e. repeating steps a) and b) a plurality of times) using the reduced biomass concentration of each previous cycle (i.e. of each step b)). A method comprising repeating at least step a), and preferably steps a) and b), at least once, and preferably a plurality of times (i.e., more than once) may be referred to as "continued culturing".

[0084] The continued culturing may be batch-wise or continuous. That is, "continued culturing" encompasses both batch-wise and continuous culturing. By "batch-wise" (which may also be referred to as "semi-continuous") is meant that the concentration of the phototrophic organism in the aqueous medium is reduced in a discreet step. For instance, when the concentration is reduced by removing phototrophic organism biomass, batch-wise culturing involves the removal, and optional addition of a dilution volume of aqueous medium, in a discreet step. By "continuous" is meant that the concentration of the phototrophic organism in the aqueous medium is reduced continuously. For instance, when the concentration is reduced by removing phototrophic organism biomass, continuous culturing involves the removal, and optional addition of a dilution volume of aqueous medium, continuously. Phototrophic organisms may be cycled through the method multiple times without noticeable loss of culture efficiency, including at above-atmospheric carbon dioxide concentrations, and especially using microalgae. When the culturing is batch-wise, these cycles will be countable as discreet steps. In batch- wise embodiments, the reduced biomass may be cycled through steps a) and b) at least 2, 3, 4 and preferably at least 5 times, such as 10, 20, 50, 75 or 100 times or more. The reduced biomass may be cycled a large number of times, even into the thousands. For example, the reduced biomass may be cycled up to 125 times, 315 times, 630 times or 1260 times. Expressed as a range, the reduced biomass may be cycled between 2 and about 1260 times, preferably between about 20 and about 630 times, and preferably between about 125 and 315 times.

[0085] For each cycle, the steps a) and b) may be as hereinbefore described. For instance, and especially in the case of microalgae, the:

[0086] • biomass concentration in aqueous medium (i.e., before it is reduced) may be a concentration of 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, and preferably between about 6.0 g / L and 7.5 g / L;

[0087] • the reduced biomass concentration in aqueous medium may be a concentration of 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, and preferably between about 3.5 g / L and 5.0 g / L;

[0088] • reducing the biomass concentration preferably comprises removing a volume of the phototrophic organism as well as a volume of the aqueous medium and adding a dilution volume of aqueous medium to the remaining biomass; and

[0089] • an amount of between about 30% and about 80%, preferably between about 40% and about 65%, and preferably between about 45% and about 60%, of the phototrophic organism biomass may be removed.

[0090] For at least second and subsequent cycles through steps a) and b), the duration of the growing step (step a)) will vary between different phototrophic organisms and will depend on the reduced biomass concentration of the previous cycle, and the point between an exponential growth phase and a stationary growth phase which the phototrophic organism reaches before its biomass concentration is again reduced - or put another way, the concentration to which the biomass is grown. That said, generally speaking, for at least microalgae, and in some embodiments, the duration of the growing step, from an early- exponential growth phase to a stationary growth phase, may be for a time of between about 1.5 to about 4 days, preferably between about 1.5 days to about 3 days, and preferably between about 1.8 to about 2.2 days. Similarly, if the reduced biomass concentration of the previous cycle is that which existed at about the mid-exponential phase point, then the duration of the growing step, from a mid-exponential growth phase point to a stationary growth phase or saturation point, may be for a time of between about 0.7 to about 2 days, preferably between about 0.7 days to about 1.5 days, and preferably between about 0.9 to about 1.1 days.

[0091] That said, it has been found that a short growth phase duration - or put another way, when the concentration change (between the reduced concentration and the concentration immediately prior to reduction) is small - certain benefits arise. A "short" growth phase duration may be defined as about or less than 24 hours. A "small" concentration change may be defined as about or less than 1 g / L. The found benefits are that the concentrations defining certain growth phase points may be made to migrate towards higher concentration. This may be, for example, when the concentration is reduced from a saturation point to above a mid-exponential phase point, or from a saturation point to within the late-exponential phase. The mid-exponential phase point and saturation point may be made to migrate towards a higher concentration. For instance, without limitation and solely to illustrate the point, it has been found that, for example, if the mid-exponential growth phase point of a cycle was 4.0 g / L, and the stationary phase of that cycle began at 7.5 g / L, and the concentration change is small, say the reduced biomass concentration is 6.5 g / L, then this may provide a mid-exponential growth phase point in the subsequent cycle that is higher, of say 4.1 g / L, and a stationary phase beginning at a higher concentration, say 7.6 g / L. Using a short growing step duration (small concentration change) in the said subsequent cycle may achieve another lift in the concentrations defining these growth phase points in yet another subsequent cycle. When reducing the concentration is performed at least in part by biomass removal, this allows for a greater amount of biomass to be removed in the same timeframe and from the same overall volume of biomass and aqueous medium. This allows for greater production of biomass and feedstock across sequential cycles. At least for microalgae, it is believed that smaller changes in biomass concentration encourage the microalgae to adapt to increasing concentration conditions.

[0092] In the methods described herein, this provides, at least in second and subsequent cycles, that the reduced biomass concentration may be greater than the reduced biomass concentration of the previous cycle. This may also provide that the concentration of the biomass at the mid-exponential phase point is greater than the concentration of the biomass at the midexponential phase point of the previous cycle. This may also provide that the concentration of the biomass at the saturation point is greater than the concentration of the biomass at the saturation point of the previous cycle. In preferred embodiments, the reduced biomass concentration corresponds to about or above the mid-exponential phase point concentration of the previous cycle, preferably at about the point where the mid-exponential phase ends and the late-exponential phase begins, and preferably within the late- exponential phase. Preferably, the duration of the growth step is such that the biomass concentration reaches about or above the saturation point of the previous cycle. The benefit of this is that the biomass concentration may, in sequential cycles, be caused to hover around the late-exponential phase being the phase of greatest growth rate, and further causing this phase to migrate towards higher concentrations, maximising the production efficiency and CO2sequestration capability of the phototrophic organism.

[0093] Accordingly, in preferred embodiments, especially using microalgae, at least in 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 30 minutes and 5 hours, and preferably between about 30 minutes and 3 hours or 30 minutes and 2 hours and 1 hour and 2 hours.

[0094] Similarly, in preferred embodiments, especially using microalgae, at least in second and subsequent cycles, the concentration change may 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, and preferably between about 2.0 g / L and 3.0 g / L The concentration change may be even smaller, for example for growth step durations of between about 30 minutes and 2 hours, of less than 1.0 g / L, for example of 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, and preferably between about 0.1 g / L and 0.3 g / L.

[0095] Similarly, in preferred embodiments, especially using microalgae, and when the concentration of the phototrophic organism in the aqueous medium is reduced by removal of biomass, at least in second and subsequent cycles, an amount of between about 0.1% and about 25%, preferably between about 0.5% and about 20%, and preferably between about 15% and about 1%, may be removed.

[0096] Similarly, and put another way, the concentration change may be between about 0.1% and about 25%, preferably between about 0.5% and about 20%, and preferably between about 15% and about 1%. For example, when the concentration of the phototrophic organism in the aqueous medium immediately prior to removal is 7.5 g / L, and the reduced biomass concentration is 7.0 g / L, this constitutes a concentration change of 7%. Similarly, when the concentration of the phototrophic organism in the aqueous medium immediately prior to removal is 7.5 g / L, and the reduced biomass concentration is 7.3 g / L, this constitutes a concentration change of 3%, etc.

[0097] When reducing the biomass concentration involves removal of biomass and aqueous medium, a reduced concentration that is greater than that of the previous cycle may be achieved by adding a smaller dilution volume of aqueous medium than was added in the previous cycle.

[0098] For continuous culturing, the number of cycles will be essentially ad infinitum and the cycling may instead be expressed in terms of a total duration of culturing or cycling. The cycling duration may be for a time of at least about 12 hours, 1 day, 2 days, 5 days, 7 days or 10 days. The cycling duration may be for a time as long as several weeks. For example, the cycling duration may be for up to about 40 days, 35 days, or 30 days. Expressed as a range, the cycling duration may be for a time of between about 12 hours and 40 days, preferably between about 5 days and 35 days, and preferably between about 10 days and 30 days. The continuous removal may be by removal of biomass alone or together with aqueous culture medium. When biomass alone is removed, this serves to reduce the concentration of the remaining biomass for promoting continued growth of the phototrophic organism. When biomass is removed together with aqueous culture medium, a dilution volume of culture medium may be added to the remaining biomass to reduce its concentration. For example, when biomass and aqueous culture medium is removed at a same or greater concentration as existed prior to removal, then a dilution volume of aqueous culture medium is added to the remaining biomass to reduce its concentration. The removal of biomass in aqueous culture medium and addition of a dilution volume of aqueous culture medium may both be continuous. This may be achieved using suitable flow rates for the removal and addition. Conveniently, flow rates for removal and addition may be calibrated against the biomass growth rate in the exponential or late-exponential growth phase. For example, the flow rate for biomass removal may be set to match the amount of biomass removed with the amount of biomass growth; i.e., as per the growth rate. For instance, for a biomass growth rate of 1.0 g / L / day, the flow rate for removal of biomass may be set to remove 1.0 g / L / day of biomass. By the same token, the flow rate for addition of aqueous culture medium dilution volume may be set to match the rate of removal of combined biomass and aqueous culture medium. For instance, for a removal rate of 2.0 g / L / day of combined biomass and aqueous culture medium, the flow rate for addition of aqueous culture medium dilution volume may be set to add 2.0 g / L / day. Flow rates may be monitored and adjusted as required. Means for providing continuous flow and set flow rates are known in the art. Examples include vessel overflow means, the use of pumps, and syphoning.

[0099] Continuous culturing has the advantage of providing a continuous feedstock for downstream processes. Coupled with the advantage of regenerating the biomass quickly provides for an upscaling capability of the said downstream processes.

[0100] The aqueous culture medium may be continually recycled, though preferably only to the point that the recycled aqueous medium continues to promote growth. Phototrophic organisms in culture may produce by-products which accumulate in the aqueous culture medium and which may prevent growth at the same rate as may occur in fresh aqueous culture medium. For example, microalgal by-products may include dissolved organic matter such as exo-polymeric substances. When the aqueous culture medium is not recyclable, it may be replaced with fresh aqueous culture medium.

[0101] Gaseous Atmosphere

[0102] The gaseous atmosphere may contain gases other than carbon dioxide, and may contain other matter such as particulates. The other gases may be one or more of those commonly found in air, such as oxygen, nitrogen, water vapour, hydrogen, methane and noble gases. The other gases may also be one or more of those commonly found in waste gas streams, such as one or more of the gases commonly found in air, as well gases generated by combustion of hydrocarbons such as carbon monoxide, nitrogen oxides (NOx) and sulphur oxides (SOx).

[0103] The gaseous atmosphere may be provided by a single source or a plurality of sources (i.e., two or more). For example, the gaseous atmosphere may be provided by a single source such as a waste gas stream of 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 sources of the gaseous atmosphere may be determined by the concentration of carbon dioxide to which the phototrophic organism is CO2-adapted. For example, when using a phototrophic organism that is CO2- adapted to say 8% CO2, a gaseous atmosphere which is provided using a purified CO2source (which may comprise about 95% or more CO2) may be diluted with a dilution gaseous source which contains comparatively little or no CO2, such as air, to provide a gaseous atmosphere which comprises about 8% CO2. Similarly, when using a phototrophic organism that is CO2- adapted to say 8% CO2, a gaseous atmosphere which is provided using a waste gas stream which comprises about 8% CO2may be provided as a single source being the waste gas stream. In some embodiments gaseous atmospheres which are provided using a waste gas stream require dilution of the CO2concentration, in which case it is preferred that a dilution gaseous source is provided. Preferably the dilution gaseous source is air. The dilution gaseous source may also be recycled from the gaseous atmosphere under which the phototrophic organism is grown in aqueous medium. Biomass Uses

[0104] The biomass resulting from the growth of phototrophic organisms is useful as an organic feedstock in many downstream processes. Phototrophic organisms use light as a primary energy source for metabolism in the presence of water and carbon dioxide to produce energy in the form of carbohydrates, by photosynthesis. The organic feedstock accordingly includes simple and complex carbohydrates, such as simple and complex sugars and biopolymers such as exopolysaccharides. The organic feedstock is therefore useful as a nutrient source for downstream applications, for example in a bio-decomposition step.

[0105] An exemplary bio-decomposition step for which the phototrophic organism biomass is useful is described in the 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 disclosure of each of which is hereby incorporated by reference in their entirety.

[0106] Sequestering CO2

[0107] In addition to producing biomass, the methods described herein may be used to sequester carbon dioxide from the gaseous atmosphere. This confers the advantage of not only producing a useful downstream product, but also that in doing so carbon dioxide is captured and converted into a useful downstream product.

[0108] In some embodiments, the carbon dioxide may be captured from the air.

[0109] Providing a gaseous atmosphere containing an above-atmospheric carbon dioxide concentration may be used to sequester carbon dioxide resulting in less carbon dioxide in the atmosphere than may otherwise be present. However, in phototrophic organism cultivation, and especially microalgae, a high concentration of CO2can cause stress to organisms that are adapted to atmospheric carbon dioxide concentrations, resulting in reduced growth. It is for this reason that it is preferred that the phototrophic organism is CO2-adapted as described herein, to make efficient use of the carbon dioxide sequestration capabilities of the phototrophic organisms in the methods. As described above, in preferred embodiments the phototrophic organism is adapted to a gaseous atmosphere containing carbon dioxide concentrations of at least about 5%, preferably at least about 8%, 9% or 10%, which may be a concentration of between about 5% and 25%, preferably between about 8% and 25%. The phototrophic organism may thus be adapted to a gaseous atmosphere comprising carbon dioxide at a similar concentration as may be found in a gas source from an industrial process, and thus allow for a gas source used in the methods to be from an industrial process that produces a gas stream containing these levels of carbon dioxide. The method may thus assist to reduce the amount of carbon dioxide that these industrial processes may otherwise emit into the atmosphere.

[0110] In preferred embodiments, the gaseous atmosphere is provided with a concentration of carbon dioxide that is similar to the concentration to which the phototrophic organism is adapted. Or put in other words, in preferred embodiments, the phototrophic organism is adapted to a carbon dioxide concentration of a provided gaseous atmosphere. For instance, for a phototrophic organism that is COz-adapted to 8% carbon dioxide, or 9%, 10%, 15% or 20% carbon dioxide, the gaseous atmosphere is preferably provided with a concentration of carbon dioxide that is about 8%, 9%, 10%, 15% or 20%, respectively. Or put in other words, for a provided gaseous atmosphere having a concentration of carbon dioxide that is about 8%, 9%, 10%, 15% or 20%, the phototrophic organism that is COz-adapted to 8% carbon dioxide, or 9%, 10%, 15% or 20% carbon dioxide, respectively. Carbon dioxide levels of between about 8% and 25% are commonly found in waste streams of various industrial processes. Accordingly, in preferred embodiments, the source of the carbon dioxide comprises a waste gas stream, preferably a waste gas stream generated by combustion of hydrocarbons, for example by steam reforming and / or by bio-decomposition.

[0111] The waste gas stream may be filtered to remove particulate matter and / or otherwise processed prior to being applied to the gaseous atmosphere.

[0112] Preferred Culture Condition Parameters

[0113] Preferred parameters for growth of the phototrophic organisms in the culturing methods have been developed. The described parameters, alone and more so in combination, assist to promote greater phototrophic organism growth, especially of microalgae. As the phototrophic organism may consume carbon dioxide, the carbon dioxide in the gaseous atmosphere may be replenished to maintain a certain concentration in the gaseous atmosphere. This is conveniently achieved using a gas flow. In some embodiments, a gas flow is used with a flow rate of between about 0.05 volume / volume per minute (WM) and about 5 WM, preferably between about 0.1 WM and about 2 VMM, preferably between about 0.1 WM and about 1.0 VMM, preferably between about 0.1 WM and 0.5 WM, preferably between about 0.1 WM and 0.3 WM, preferably between about 0.1 WM and 0.2 WM, and more preferably of about 0.15 WM.

[0114] Generally speaking, a culture of a phototrophic organism that has grown through a lag phase and into an exponential phase was originally present as an inoculum. In preferred embodiments, an inoculum of phototrophic organisms containing between about 106cells / mL and about 1010cells / mL, more preferably between about 107cells / mL and about 109cells / mL, and most preferably about 108cells / mL, is used, preferably as a cell suspension in an aqueous medium.

[0115] In preferred embodiments the culturing methods are performed in a bioreactor having a capacity of at least 1.11 times (i.e., 111% of) the volume of phototrophic organism and aqueous medium, and preferably 1.17 (i.e. 117%) times. For instance, for a working volume of 900 mL, the capacity of the bioreactor will preferably be at least 1000 mL, and preferably at least 1053 mL, respectably. Preferably, the culturing methods are performed in a bioreactor having a capacity of no more than 2.85 times (i.e., 285% of) the volume of phototrophic organism and aqueous medium, and preferably 2.50 (i.e. 250%) times. For instance, for a working volume of 900 mL, the capacity of the bioreactor will preferably be no more than about 2565 mL, and preferably no more than about 2250 mL, respectably. Alternatively, or in addition, preferably the culturing methods are performed in a bioreactor having a capacity of between about 1.11 and 2.85 times the volume of phototrophic organism and aqueous medium, preferably between about 1.17 times and 2.50 times, preferably between about 1.42 times (i.e., 140%) and 2.00 times (i.e. 200%), and preferably of about 1.67 times (i.e. 167%).

[0116] In other words, in preferred embodiments the culturing methods are performed in a bioreactor wherein the volume of phototrophic organism and aqueous medium is at least about 35% of bioreactor capacity, and preferably at least about 40%. For instance, for a bioreactor capacity of 1000 mL, the volume of phototrophic organism and aqueous medium will preferably be at least about 350 mL, and preferably at least about 400 mL, respectably. Preferably, the culturing methods are performed in a bioreactor wherein the volume of phototrophic organism and aqueous medium is at most about 90% of bioreactor capacity, and preferably at most about 85%. For instance, for a bioreactor capacity of 1000 mL, the volume of phototrophic organism and aqueous medium will preferably be at most about 900 mL, and preferably at most about 850 mL, respectably. Alternatively, or in addition, preferably the culturing methods are performed in a bioreactor wherein the volume of phototrophic organism and aqueous medium is between about 35% and 90% of bioreactor capacity, preferably between about 40% and 85%, preferably between about 50% and 70%, and preferably of about 60%. These levels provide for a balance of head space for the gaseous environment, and volume of phototrophic organism and aqueous medium, for efficient gas exchange between the gaseous atmosphere and the aqueous medium for promoting carbon dioxide consumption and organism growth.

[0117] The bioreactor may be, and is preferably, fitted with a mixing device, such as an impeller, to agitate the aqueous medium to aerate the culture, which may further assist with gas exchange. This may also assist to ensure an even distribution of exposure to light through a phototrophic organism culture. This especially applies when a low flow rate of gaseous atmosphere is used, for example of less than about 1.0 WM.

[0118] Phototrophic organisms, including microalgae, often thrive in neutral or alkaline conditions. Accordingly, in preferred embodiments, the aqueous medium is maintained at a pH of between about 5 and about 9, preferably between about 6 and about 8, and more preferably between about 6.5 and about 7.5, most preferably at a pH of about 7. Carbon dioxide which may be or become dissolved in the aqueous medium may contribute to lowering the pH, while the consumption of carbon dioxide by the organism may contribute to increasing the pH. The pH may be maintained using additives from time to time as required; for example, an alkaline substance to raise pH and an acidic substance to lower pH. Suitable substances are known to those of skill in the art. 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, and more preferably at about 28°C.

[0119] Preferably, the aqueous medium comprises nutrients. Preferably the nutrients are selected from one or more, preferably two or three or more, and more preferably all, of the group consisting of boric acid, calcium chloride, cobalt nitrate, cupric sulphate, EDTA disodium salt, magnesium sulphate, manganese chloride, potassium phosphate, sodium molybdate, sodium nitrate and zinc sulphate. Aqueous media containing these nutrients are known to or may be prepared by those of skill in the art. In a preferred embodiment, the aqueous medium is selected from one or both of Bold Basal medium (BBM) or Blue-Green 11 medium (BG-11), preferably BG-11.

[0120] In preferred embodiments, the light is of an intensity of between about 3000 lux and about 7000 lux, preferably between about 4000 lux and about 6000 lux, and 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 of skill in the art.

[0121] As phototrophic organisms use light to produce energy, the photoperiod i.e., the duration of light the phototrophic organisms are exposed to within a 24 h period, is preferably elongated as compared with what may be typically encountered by the phototropic organism in its natural environment. However, a photoperiod that is too long may not promote growth as well as a photoperiod that is balanced with a dark period. Accordingly, in preferred embodiments the photoperiod, is between about 12 h and about 20 h, preferably between about 14 h and about 18 h, more preferably between about 15 h and about 17 h, and most preferably of about 16 h.

[0122] Of these preferred culture conditions, the combination of the preferred light intensity and photoperiod are particularly effective at promoting greater phototrophic organism growth, especially of microalgae.

[0123] Accordingly, the present disclosure provides a method of culturing a phototrophic organism, comprising growing a phototrophic organism in an aqueous medium under culture conditions comprising light and a gaseous atmosphere comprising carbon dioxide, wherein the light is of a light intensity of between about 3000 lux and about 7000 lux, and wherein the culture conditions further comprise a photoperiod of between about 12 h and about 20 h. The preferred integers as specified herein are otherwise applicable to this method, including the use of microalgae, culture conditions and the method being a continued culturing method.

[0124] Culturing Apparatus & Systems

[0125] Apparatus and systems which may be used for performing the culturing methods of the present disclosure are described in the 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 disclosure of each of which is hereby incorporated by reference in their entirety.

[0126] A basic system may comprise a bioreactor suitable for culturing phototrophic organisms. Said reactor may be in a closed system in communication with a gas stream that comprises carbon dioxide and air. The gas stream may be derived from a waste gas stream of an industrial process, e.g., flue gas. The bioreactor may be in liquid communication with one or more other reactors and / or storage vessels. The, or each, bioreactor may include numerous sensors including pH sensors, temperature sensors, reactor level sensors, and sensors to monitor biomass generation, preferably with regulators and controls to maintain each monitored parameter.

[0127] Examples

[0128] Various embodiments will be described herein with reference to the following non-limiting examples.

[0129] The experiments of the examples were performed using Chlorella vulgaris strain CS-41, which was obtained from the Australian National Algae Culture Collection (CSIRO Hobart, Tasmania, Australia). To start the experiments, pre-established volumes of algae, with known concentration, were added in closed vessels containing sterile culture medium and the assays were performed in triplicate. Once a parameter has been optimised, e.g., best performing in terms of biomass production rate, that optimised culture condition is maintained for future experiments. Initial Culture Handling

[0130] Initially, C. vulgaris CS-41 stocks were resuscitated from -80°C and re-cultured in 1 L conical flask with a working volume of 400 mL in BG-11 medium at 28 °C. The culture was exposed to a LED light source to provide the irradiance of approximately 4000 lux until the stationary phase was attained under an 10% (v / v) concentration of CO2 and 90% air.

[0131] Once the culture was acclimatised to culture conditions, the inoculum for the experiments described in the present set of examples was prepared in pre-established volumes, with known concentration under sterile conditions. The initial culture conditions used in the experiments of the example (unless stated otherwise) are given in Table 1.

[0132] Table 1. Initial culture conditions.

[0133] Inoculum concentration 106cells / mL

[0134] Culture medium BBM or BG-11a

[0135] Aeration Yes

[0136] Flow rate 0.15-1 VVMb

[0137] Flow composition 10% CO2 + 90% air pH 7

[0138] Photoperiod 12 h

[0139] Light intensity 4,000 lux

[0140] Temperature 28°C

[0141] Runtime 7 daysaBlue-green (BG) medium 11bWM, gas volumetric flow rate per unit volume of culture medium.

[0142] A number of these conditions were investigated and improved as described below.

[0143] General Analytical Methods

[0144] The read-out parameters evaluated in the experiments were: pH, cell concentration, biomass weight and CO2 absorption. The methodologies for evaluating each parameter is summarised below. As used for these experiments, biomass refers to the C. vulgaris generated. Accordingly, biomass in these examples illustrates the generation of an organic feedstock.

[0145] Biomass growth assessment was performed daily as described below. The optical density (OD) of all the diluted pellets was measured using a UV-Vis spectrophotometer at a wavelength of 683 nm. The diluted pellets were separately transferred into 3 x 1.5 mL Eppendorf tubes and allowed to centrifuge at 4700 rpm for 10 minutes. The supernatants were discarded, and the pellets in the Eppendorf tubes were dried overnight at 50°C. The absorbance spectra versus dry cell weight (DCW) of the biomass were plotted to obtain a biomass standard curve for every batch.

[0146] Microalgae cultures were enumerated at the end of the 7-day cultivation time. The microalgae cultures were poured into 3 x 50 mL centrifuge tubes and centrifuged at 4700 rpm for 10 minutes to separate the supernatant and pellet. Serial dilution was performed on the pellets resuspended in a known volume of deionized distilled water. The cells present in an appropriate dilution were counted using a Neubauer chamber aid in optical microscope with 400x increase magnification.

[0147] An elemental analyser was used to analyse the content of carbon, hydrogen, and nitrogen in the biomass. A known volume of culture was withdrawn at the beginning and the end of the cultivation cycle, the biomass was harvested by centrifugation and analysed for carbon, hydrogen, and nitrogen content. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) was used to analyse the element present in liquid medium, separated after centrifugation.

[0148] The pH of the cultures was analysed every day using a pH meter. The pH of the cultures was also maintained within the specified range (7.0 ± 0.5) using 0.1 M NaOH. Two CO2 sensors (model no. GMP251) using Vaisala Insight PC Software were installed into the inlet and outlet gas lines as shown in Figure 1 to record CO2 concentration every 5 seconds for the entire cultivation cycle. The change in the CO2 concentration recorded by these sensors was also used to establish mass balance and calculate the bio-fixation of CO2.

[0149] Example 1. Adaptive Laboratory Evolution

[0150] The tolerance of C. vulgaris CS-41 to high concentrations of CO2 was gradually increased through an artificial stepwise adaptive laboratory evolution (ALE) process. The microalgae were cultured in a bioreactor system setup as shown schematically in Figure 1. The reactor was placed in a photo chamber and surrounded by LED light source. Pure CO2 and air were supplied, and flow rates were controlled using flow control valves to achieve a specific CO2concentration in the gas inlet line. CO2 sensors were placed at the gas inlet and outlet lines to continuously measure CO2concentration. Except for varying CO2concentrations, the cultures were grown under the conditions described in Table 1.

[0151] Increased tolerance to CO2 by C. vulgaris CS-41 was initiated by exposing the microalgae culture to ambient air with 0.03 vol% CO2for 7 days. A known concentration of the acclimatised algal culture was used as inoculum for the ALE process. A 400 mL of the working volume of 1000 mL of the bioreactor was used for the ALE of the C. vulgaris strain. After the 7-day cultivation cycle, the algae were then exposed to a 3 vol% CO2 stream. Then, sequential cultivation cycles of subsequent generations were repeated with gradual increase in the CO2 concentration up to 10%.

[0152] Biomass was analysed 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:

[0153] Equation 1.

[0154] Biomass production = (g / L / day), where Cbio represents the biomass concentration in the medium in g / L.

[0155] Equation 2.

[0156] Specific growth rate (Day ).

[0157] Specific growth rate represents the growth rate after achieving a constant biomass concentration.

[0158] The biomass growth data resulting from each cultivation cycle with different CO2 concentrations are summarised in Table 2. At 8% and 10%, the cultivation cycles were conducted at least twice to evaluate the performance of different generations (G).

[0159] Table 2. CS-41 growth at varying CO2 concentration (BBM, 12:12 photo:dark period)

[0160] Cycle CO2 Biomass concentration (g / l) Biomass Specific vol% day-0 day- day- day-3 day-4 day-5 day-6 day-7 productivity Growth Rate

[0161] 1 2 (g / l / day) (Day1)

[0162] 1 0.03% 0.015 0.018 0.025 0.034 0.038 0.041 0.048 0.05 0.005 0.172

[0163] (air)

[0164] 2 3% IG 0.015 0.015 0.06 0.119 0.123 0.181 0.271 0.476 0.0658 0.494

[0165] 3 5% 1 G 0.015 0.062 0.104 0.108 0.134 0.194 0.255 0.334 0.0455 0.443

[0166] 4 8% 1 G 0.025 0.035 0.062 0.082 0.096 0.166 0.212 0.236 0.0316 0.393

[0167] 5 8% 2G 0.025 0.032 0.077 0.161 0.172 0.251 0.301 0.38 0.0521 0.461 6 10% 1 G 0.025 0.036 0.075 0.106 0.141 0.245 0.308 0.34 0.0464 0.445

[0168] 7 10% 2G 0.032 0.047 0.093 0.144 0.201 0.257 0.369 0.428 0.059 0.478

[0169] 8 10% 3G 0.04 0.068 0.115 0.21 0.284 0.419 0.563 0.689 0.0962 0.546

[0170] 9 10% 4G 0.05 0.121 0.249 0.377 0.543 0.635 0.821 1.044 0.147 0.606

[0171] 10 10% 5G 0.05 0.144 0.251 0.375 0.508 0.641 0.837 1.055 0.148 0.607

[0172] At low CO2concentrations (3 and 5%), the cultures were adaptively evolved in a first generation with the biomass concentration increasing until day 5. However, biomass productivity and specific growth rate decreased from cultures grown in the presence of 3% CO2to 5% CO2. This may be due to a slowing of cell division on days 6 and 7 compared to the initial 5 days of the cultivation cycle.

[0173] It was observed that biomass productivity decreased after every successive increase in CO2concentration from 3% to 8%. This may indicate the cells were stressed initially, but were able to slowly adapt with subsequent generations. This is evident as 8% 2G run surpassed the biomass productivity of 5% 1G. A similar pattern of higher biomass productivity from successive generations was observed at 10% CO2, where the cultivation cycles were performed for five generations.

[0174] The biomass concentration, variation in pH and growth rate from 10% 4th generation and 10% 5th generation runs are compared in Figure 2. In this experimental work, each cultivation cycle was conducted in triplicate and variation in error bars represents variation in the results of separate trials with the same operating conditions. In 10% 4G and 5G, little if any daily variation in biomass concentration and pH values was observed. Furthermore, the average biomass productivity values were 0.147 g / L / day and 0.148 g / L / day and specific growth rates were 0.606 and 0.607 respectively. As the biomass productivity values were approximately constant for two consecutive runs, the culture resulting from 5 generations of being cultivated at 10% CO2concentration was considered to be adapted to a suitable CO2vol%.

[0175] The biomass at the end of the 7-day cycle from 10% 5G run was analysed for its carbon, hydrogen and nitrogen content using an elemental analyser as described in the general analytical methods section. The result of this analysis is shown in Figure 3.

[0176] The carbon content of the total biomass was used to calculate the bio-fixation rate of CO2 in the cultivation cycle using the following equation (Equation 3). Equation 3:

[0177] Bio-fixation rate of CO2(g / L / day) = where Xc represents carbon mass fraction in the biomass samples and M.W. represents molecular weight in g / mol.

[0178] Figure 3 (b) shows the calculated bio-fixation rate of carbon and CO2was the same (0.2645 g / L / day of CO2) for generation 4G and 5G grown in the presence of 10% CO2. The bio-fixation rate of CO2was calculated from the CO2sensor data, shown in the trace in Figure 3 (c). The CO2concentration in the inlet and outlet gas streams was plotted for the last 4 days of the 10% 5G cultivation cycle. Large square peaks represent 12 h photoperiods, whereas some fluctuations during the photoperiod were reported due to sudden fluctuations in the upstream gas line. Based on the gas stream concentration data, the bio-fixation rate of CO2was calculated as 0.269 g / L / day. Both the values, one calculated from biomass analysis and the other from CO2sensor data, were consistent with a discrepancy of - 1.7%, validating the elemental analysis method. In the following experimental trials, elemental analysis was used to calculate the bio-fixation rate of CO2in the process.

[0179] Example 2. Culture Medium Comparison

[0180] Two culture media types, BG-11 and BBM, were evaluated for optimal microalgal growth performance using C. vulgaris strain CS-41. Experimental outputs used to evaluate culture medium performance were biomass production and CO2fixation. The composition of each BG-11 and BBM are listed in Table 3.

[0181] Table 3. Compositions of BBM and BG-11 culture media in 1 L deionized distilled water

[0182] Chemicals BBM (mg / L) BG11 (mg / L)

[0183] Boric Acid 11.42 2.86

[0184] Calcium Chloride, Anhydrous 18.87 27.18

[0185] Cobalt Nitrate«6H2O 0.49 0.049

[0186] Cupric Sulfate«5H2O 1.57 0.079

[0187] EDTA, Disodium Salt 63.61 1

[0188] Ferrous Sulfate«7H2O 4.98 —

[0189] Magnesium Sulfate, Anhydrous 36.63 75

[0190] Manganese Chloride«4H2O 1.44 1.81 Potassium Hydroxide 31.0 —

[0191] Potassium Phosphate, Dibasic 75.0 40

[0192] Potassium Phosphate, Monobasic 175.0 —

[0193] Sodium Chloride 25.0 —

[0194] Sodium Molybdate 1.19 0.39

[0195] Sodium Nitrate 250.0 1500

[0196] Zinc Sulfate«7H2O 8.82 0.222

[0197] Ferric Ammonium Citrate - 6

[0198] Sodium Carbonate Anhydrous - 20

[0199] Citric Acid Anhydrous - - - 6

[0200] 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 dried in hot air oven at 30-35°C and the weight of the biomass was determined. The biomass was also measured spectrophotometrically using a standard optical density technique and compared to an earlier growth curve of established growth characteristics for strain CS-41. The culture conditions used for this experiment are shown in Table 1.

[0201] Total CO2 bio-fixation was measured at day 0 and day 7. To measure CO2 levels, gas samples were collected from the reactor used to perform the photosynthesis step. Samples were collected in a gas collecting bag and the compositions of these gas samples were analysed by GC-MS (Agilent 7890B GC) equipped with thermal conductivity detector (TCD). As a carrier gas, argon / helium was applied with the flow rate of 15 mL min-1. The temperature of the oven, detector, and injector were 190°C, 210°C, and 160°C, respectively.

[0202] 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 media types. BG-11 supported higher levels of biomass production and CO2 fixation than BBM. In this example, CS-41 bio-fixed 1.029 g / L / day CO2 compared to 0.593 g / L / day CO2 when grown in BG-11 or BBM medium, respectively. Batch Day O Day 7 Photosynthetic fixation Total CO2bio- CO2bio-

[0203] ID Biomass Carbon Carbon Biomass Carbon Carbon of carbon (g C / L) in 7 fixation (g fixation

[0204] (g / L) (%) (g / L) (g / L) (%) (g / L) days CO2 / L) in 7 days (g / L / day)

[0205] A B X = (A x D E Y = D x E Z = Y - X TC = (44.01 / 12) x TC / 7

[0206] B) / 100 Z

[0207] BG11 0.015 50.46 0.007569 3.894 50.64 1.9719216 1.9643526 7.20426316 1.029

[0208] BBM 0.015 48.75 0.0073125 2.321 49.1 1.139611 1.1322985 4.15270475 0.593

[0209] Table 4. Results of comparison of culture media for optimal biomass production and carbon fixation.

[0210] Example 3. Optimisation of Light Intensity

[0211] 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 performed as listed in Example 1 and Table 1.

[0212] Growth curve analysis based on spectrophotometric measurements of optical density demonstrate light intensity effects growth lag phases. Figure 4 shows that optimising the light intensity applied during the photosynthesis step can reduce the impact of a lag phase on growth rate and overall biomass production.

[0213] In a separate experiment performed under the same culture conditions, the light intensity range investigated comprised 3000 lux to 5500 lux. The photobioreactor system was surrounded by an LED light source with light intensity controls, configured as shown in the schematic diagram in Figure 1. Both biomass productivity and bio-fixation rate increased from 3000 to 5000 lux as shown in Figure 5 and Figure 6, respectively. However, a further increase in light intensity to 5500 lux resulted in a decrease in biomass productivity. The results indicate, 5000 lux resulted in the highest bio-fixation rate of CO2 of 1.37 g / L / day in all experimented conditions. The results of the light intensity optimisation studies 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.

[0214] Example 4. Optimisation of Photoperiod

[0215] In this study, five sets of photoperiods (12:12; 16:8; 18:6; 20:4 and 24:0 h light / dark) were attempted to optimize light exposure times in each culture medium (BG-11 and BBM) at 4000 lux with microalgae C. vulgaris CS-41. All other culture conditions were the same as those conditions described in Example 1 and Table 1. As shown in Figure 8, in all sets of photoperiods tested, the microalgae grown in BG-11 medium demonstrated higher biomass productivity than the culture grown in BBM medium. However, BG-11 and BBM accumulated least biomass in 12:12 h and 24 h light / dark photoperiod which were accounted as 1.866 g / L and 0.389 g / L respectively. This finding revealed that photoperiod is not only species specific but is also influenced by the culture medium used. Further, it is evident that higher photoperiod such as 18:6; 20:4 and 24 h light / dark can affect biomass productivity of C. vulgaris in BBM more adversely than BG-11 which demonstrates that C. vulgaris can tolerate photo-oxidative stress in BG-11 more efficiently than BBM (Figure 8). Nevertheless, both culture media accumulated maximum biomass in 16:8 h light / dark photoperiod which were reported as 3.894 g / L and 2.321 g / L in BG-11 and BBM respectively.

[0216] Example 5. Optimisation of Culture pH

[0217] In this example, C. vulgaris CS-41 was grown in culture medium adjusted to different pH levels. This experiment was conducted to select the optimum pH of the culture medium to achieve the highest biomass productivity during the 7 days of cultivation at fixed photoperiod (16:8 h) and light intensity (5000 lux). Except for photoperiod, light intensity and pH, all culture conditions were performed as described in Example 1 and Table 1. Biomass productivity, CO2 fixation rate and total CO2 captured were measured as described in the General Analytical Methods section above. The PH was monitored and adjusted daily.

[0218] The results reported in Figure 9 show that the most biomass, as determined by dry weight measurements, was achieved at pH 7 in either BG-11 or BBM culture medium at day 7. However, BG-11 showed higher biomass production overall, when 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 against alkaline solutions than BG-11. The CO2fixation rate and total CO2captured at each pH tested was measured on day 7, the results for each measurement are shown in Figure 10 and Figure 11, respectively.

[0219] Based on the data generated in this example, pH 7 was identified as the optimal pH for C. vulgaris culture conditions, providing maximal biomass productivity and CO2fixation, and flexibility of culture medium given the each medium tested (BBM and BG-11) both performed optimally at pH 7.

[0220] Example 6. Optimisation of Inoculum Cell Density

[0221] The number of cells present in the inoculum used to initiate a C. vulgaris CS-41 culture for the photosynthesis step will affect the growth rate and final biomass achieved. This experimental example was designed to evaluate a range of starting inoculums (0.006 g / L, 0.015 g / L, 0.025 g / L, 0.050 g / L and 0.133 g / L). With the exception of the following culture conditions: BBM culture medium, 10% CO2, photoperiod 16:8 (light:dark), the culture conditions were performed as described in Example 1, Table 1. The experimental outputs measured to determine the optimal inoculum were biomass productivity, CO2 fixation rate and total CO2 captured. These parameters were analysed as described in the General Analytical Methods section above. The results of this study are summarised in Table 5.

[0222] Table 5. Effect of inoculum size on biomass growth rate (BBM culture medium, 10% CO2, 16:8 photo: dark period) >

[0223] Inoculum size

[0224] Day 0.006 g / L 0.015 g / L 0.025 g / L 0.050 g / L 0.133 g / L

[0225] 0 0.006 0.015 0.025 0.050 0.133

[0226] 1 0.017 0.21 0.327 0.144 0.177

[0227] 2 0.029 0.37 0.498 0.251 0.327

[0228] 3 0.051 0.67 0.77 0.375 0.515

[0229] 4 0.098 0.79 0.92 0.508 0.615

[0230] 5 0.126 0.96 1.18 0.641 0.716

[0231] 6 0.179 1.114 1.287 0.837 0.979

[0232] 7 0.22 1.439 1.315 1.055 1.266

[0233] Biomass productivity (g / L / day) 0.0305 0.203 0.184 0.1435 0.1618

[0234] Growth rate (g / L / d) 0.514 0.652 0.566 0.435 0.321

[0235] Bio-fixation of CO2 (g / L / day) 0.038 0.290 0.281 0.261 0.275

[0236] In general, higher inoculum size tolerated environmental stress more successfully before acquiring adaptation. Environmental stress can prevent some of the cell population from undergoing cell division successfully. Hence, when exposed to environmental stress, a cell number in inoculum may be more likely to survive the stressful conditions when compared to a smaller inoculum size. However, it was observed that higher inoculums (0.025 to 0.133 g / L) of the adapted C. vulgaris strain showed a lower growth rate than smaller inoculum (0.015 g / L), which demonstrated that C. vulgaris strain CS-41 that had undergone acclimatisation to higher than atmospheric levels of CO2, was firmly adapted to thrive under high CO2 concentration stress. Simultaneously, the higher inoculums tested resulted in lower biomass productivity. This may be due to the earlier scarcity of light penetration when compared to the lower inoculum size. The data presented in Table 5 shows that the adapted and optimised strain (high CO2 tolerant cells) can divide rapidly and achieve the maximum biomass at 0.015 g / L inoculum size. This inoculum was identified as the optimal size to achieve the highest growth rate (i.e., 0.652 g / L / d), while the culture with inoculum size (0.006 g / L) lower than the optimum size reflected lower growth rate (i.e., 0.514 g / L / d). Growth rates and biomass productivity decrease with an increase of inoculum size after the optimum value

[0237] (0.015 g / L), however at very high inoculum size (0.133 g / L) the biomass productivity was relatively higher than the immediately smaller inoculum size 0.05 g / L though the growth rate was still low. It is clear from the data presented in Table 5 that the CO2 fixation is directly related to biomass productivity while the growth rate is inversely related to inoculum size. Example 7. Batch culture (single cycle) of C. vulgaris under optimised conditions

[0238] After optimisation of culture condition parameters as described in examples 1 -6, the optimised culture conditions are summarised in Table 6.

[0239] Table 6. Optimised culture conditions of C. vulgaris CS-41 in the photosynthesis step _

[0240] Parameter Optimised culture condition

[0241] Culture medium BBM or BG-11

[0242] Working culture volume 40% of total bioreactor capacity

[0243] Inoculum volume 1% of working culture total volume

[0244] Inoculum concentration 108cells / mL

[0245] Aeration Yes

[0246] Flow rate 0.15-1.0 WM

[0247] Flow composition 10% CO2and 90% air pH 7

[0248] Photoperiod 16 h (8 h dark)

[0249] Light intensity 5000 lux

[0250] Temperature 28°C

[0251] Runtime >7 d

[0252] Final product Liquid biomass (phototrophic source)

[0253] Concentration 107cells / mL Cultures grown under the pre-optimisation culture conditions were compared with cultures grown under the optimised conditions. The comparison as summarised in Table 7, compares the output criteria of: biomass production at day 7, biomass production rate, carbon fixed over 7 days, CO2 fixation rate and total CO2 captured in final day.

[0254] The culture method developed as a result of the optimisation process resulted in a culture that achieved higher levels of biomass in a shorter time period and improved CO2fixation over a 7 day period. The improved growth rate is shown in Figure 12.

[0255] Table 7. Comparison of culture performance, pre- and post- culture condition improvement investigations

[0256] Comparison criteria Original Optimised Change conditions conditions

[0257] Media type BG11 BG11

[0258] Biomass after 7 days (g / L) 4.044 6.280 fi 53.3%

[0259] Biomass production rate (g / L / d) 0.576 0.884 fi 53.5%

[0260] Carbon fixed over 7 days (g / L) 7.48 11.42 fi 52.7%

[0261] Carbon dioxide fixation rate (g / L / d) 1.07 1.64 fi 53.3%

[0262] The optimised single batch culture system (BCS) conditions as described in examples 1 -7, was used as a starting point for developing a continued culture process for the photosynthesis step.

[0263] Example 8. Growing Microalgal Culture in Recycled Culture Medium

[0264] In the current study, experiments were conducted using fresh and spent BBM medium. The results are presented in Figure 13. It was found that the fresh BBM medium produced 1.055 g / L biomass in a duration of 7 days with 0.144 g / L / day of biomass productivity, while the reuse of the BBM produced 0.865 g / L with the rate of 0.116 g / L / day of biomass productivity. This infers that biomass productivity decreased by - 19% in the second cycle of reuse medium. Besides the potential inhibitory effect of DOCs, it is recognised that some nutrient components in the culture medium will be depleted after each growth cycle, thus reducing the availability of nutrients in subsequent growth cycles. The biomass generated by C. vulgaris CS-41 when grown on recycled media was deemed acceptable to pursue further development in a continued cycle culturing system.

[0265] Example 9. Continued culturing methods

[0266] For the purpose of developing a continued cycling method for the cultivation of C. vulgaris CS-41 in a photobioreactor, a master seed stock of the microalgae was revived from -80°C storage using optimised culture conditions set out in Table 6. Once the cell density reached 104cells / mL, the culture was transferred into a larger volume as an operational stock. Operational stocks were checked before use for contamination and were refreshed monthly from the master seed stock. All experimental batches were seeded with 108cells / mL of biomass from operational stocks and incubated under culture conditions set out in Table 6.

[0267] An example of a typical growth curve of C. Vulgaris CS-41 during the optimisation 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 continued manufacturing, particularly if the culture could be reset to day 6 every day for the algal cells to re-double.

[0268] This experiment was designed to evaluate and compare two different continued cycling (CC) methods for the growth of microalgae at a laboratory scale. The two CC method experiments were run in parallel. The first culture cycle was grown under the optimised growth conditions as set out in Table 6. The two culture methods are described in Table 8. Method A (CCA) provides the basis for the development of a continuous syphoning process for use with liquid biomass, whereby the liquid biomass is syphoned every two days and new, fresh medium is returned to the reactor (refresh). Method B (CCB) provides the basis for the development of a continuous syphoning process for use with solid biomass and evaluates the use of recycled media (recycle)

[0269] In this example, the two experiments were run in parallel as described in Table 8. The starting biomass density for CCA was analysed and calculated to be 2.803 g / L. The biomass used to start CCA and CCB were prepared from the same batch cycling system (BCS) (cycle 0). The BCS biomass was flocculated using a high concentration of CO2 gas effectively concentrating the biomass. The supernatant was syphoned from the reactor and transferred to a separate vessel. The remaining biomass concentrate was split into two equal portions. One portion was removed from the reactor for use as a feedstock in a downstream application, e.g., a biodecomposition step for the production of biofuel and / or biofertiliser. The supernatant was returned to the original reactor and recycled as culture media for the portion of biomass that remained. The starting biomass density for method CCB was recorded at 2.201 g / L. A schematic diagram of the CCA (splitting biomass and refreshing media) and CCB (concentrating and splitting biomass and recycling media) methods is shown in Figure 15.

[0270] Table 8. Description of two continued cycling culture methods for the growth of microalgae.

[0271] Experimental Method A (CCA) Method B (CCB) step

[0272] Step 1. Cycle 0

[0273] - single batch Prepare a BCS culture under optimised culturing conditions for 7 starter culture. days. The biomass was divided into two equal portion that were used to commence cycle 1 for each CCA and CCB (step 2)

[0274] Step 2. Cycle 1 One of two equal portions from One of two equal portions

[0275] - split culture step 1 was transfered to a new from step 1 was transfered to from step 1, reactor. Half the volume was a new reactor and exposed to split biomass removed and the same volume high CO2 concentrations to and add of fresh medium was added cause the algae to flocculate medium back into the original reactor. and settle. The supernatant

[0276] (refresh, CCA; or The removed portion may be was transferred to a new recycle CCB). used to commence a new vessel. culture cycle, or may be used Half the remaining biomass for downstream applications, was removed for downstream e.g., as a feedstock for bio- applications, e.g., as a decomposition. feedstock for biodecomposition.

[0277] The supernatant was returned (recycled) to the same reactor to support growth of the remaining biomass.

[0278] Step 3. Grow Continue culture conditions for a further 2 days under lower cycle 1 culture. light conditions (4000 lux).

[0279] Step 4. Monitor Measure biomass density after 2 d of growth of cycle 1. Biomass growth after 2 d. density levels determine an end point of growth cycle 1.

[0280] Step 5. Cycle 2 If step 4 biomass achieves pre-established growth thresholds

[0281] (generally after 2 d), repeat steps 2 to 4 Step 6. Cycle n Repeat steps 2 to 4 to continue continued culture cycling until + 1 original source batch loses integrity.

[0282] The final growth rate per day of the preliminary CC methods CCA and CCB were 1.287 g / L / d and 1.4705 g / L / d, respectively and the end density were 5.377 g / L and 5.142 g / L, respectively (Figure 16). The growth rate for both CCA and CCB is significantly higher compared to the BCS at 0.750 g / L / d under pre optimised conditions, or 0.88 g / L / d under optimised culture conditions (Table 7). Both CC methods significantly outperformed BCS in terms of growth and carbon capture rate.

[0283] In addition to measuring the biomass production resulting from CCA and CCB methods, the CO2capture rate (CCR) was also analysed using the methodology set out in the General Analytical methods section above and Example 1. The carbon capture rate of each of the CCA and CCB methods were compared to BCS. Using industry standard calculations for estimating CO2capture rate per g of biomass for each culturing method tested, i.e., BCS, CCA and CCB, the theoretical levels of biomass production and CO2capture that could be achieved at scaled up culture volumes and long-term production are summarised in Table 9.

[0284] A method to achieve economical and sustainable CO2sequestration at commercial levels, e.g., CC utilising a phototrophic source in a photosynthesis step is desirable for meeting the needs of industry and improving the environmental sustainability of industrial practices.

[0285] Table 9. Carbon capture rate achieved by C. vulgaris CS-41 when grown in different culture systems and different culture methods _

[0286] Culture system Culture method CCR (g / L / d)

[0287] Batch BCS 1.37

[0288] Continued CCA (split and refresh) 4.23

[0289] Continued CCB (concentrate and recycle) 4.16

[0290] The significant increase in CCR seen in continued culturing is likely due to a larger proportion of microalga remaining in the continued culture system after each biomass division event. The remaining phototrophic source continues to fix CO2into sugars, unlike the BCS method, where the entire biomass is harvested in one event. A subsequent BCS batch would then start from the initial inoculum and require at least 4 days for the phototrophic source to enter begin an effective exponential growth phase. By contrast, the CC method by pass the need to start the culture cycle from the initial inoculum and instead instantly resets the biomass to an early exponential phase.

[0291] For example, day 0 to 4 biomass concentration of BCS is less than approximately 0.5 g / L / d, whereas the equivalent time point in CC cultures, i.e., day 0 (day 7, cycle 1) and day 4 (day

[0292] 11, cycle 1) the biomass level is at least 2.8 g / L / d. As such, the CC operation has a higher amount of biomass fixing CO2 (at all times) and double the amount of carbon captured by BCS every six days (Figure 17). Furthermore, the proficiency of CC over BCS is exaggerated further during scaling of volume and days of operation. The theoretically increased capacity of CC to produce biomass and capture CO2 has been calculated for increased reactor scale and extended culturing duration, as shown in Table 10.

[0293] Table 10. Comparison of theoretical biomass production and CO2captured by batch and continued culture systems in the photosynthesis step.

[0294] Theoretical Culture Reactor size output system 1 L (g) 100 L (g) 100,000 L (g)

[0295] . Batch 0.75 74.80 74,800

[0296] Da'ly biomassCCA 1 2g 128 70 QprOdUCedCCB 1.47 147.05 147,050 / , , . Batch 273.02 27,302 27,302,000

[0297] Yearly biomassCCA

[0298] Pr°UCSCCB 536.73 53,673 53,673,250

[0299] Batch 1.3 130 13,000

[0300] Daily CO2 captured CCA 4.23 423 42,300

[0301] CCB 4.16 416 41,600

[0302] Batch 501.7 50,169 50,169,361 ear yCCA 1,544.0 154,400 154,400,000

[0303] Cap U reCCB 1,518.4 151,840 151,840,000 Example 10. Industrial Implementation of the Photosynthesis Step

[0304] Overall mass balance at optimum operating conditions (BG-11 culture medium, 0.015 g / L inoculum, 1 WM gas flow rate, 16:8 photo: dark period ratio, and 5000 lux light intensity) is summarised in Figure 18(a). These optimum conditions resulted in the bio-fixation rate of 1.37 g CO2 / L / day. Assuming that the bio-fixation rate of CO2remains constant throughout the cultivation process, the inlet CO2stream is 40 ml / min (equivalent to 0.0748 g / min, CO2density = 1.87 kg / m3) and the corresponding outlet CO2stream can be calculated as 39.7965 ml / min (0.07442 g / min) in the process. This further infers that per pass conversion of CO2to biomass is -0.50%. This conversion level is significantly lower than in industrial processes where the conversion varies from 20 to 100%. Due to lower conversion, the bio-fixation of CO2 at an industrial scale would require a large recycling of gases.

[0305] A simple flowsheet with recycle of gas stream is shows in Figure 18(8b) was developed and simulated in Aspen-Hysys to calculate the mass balance of gas streams in the process. In the flowsheet, the photobioreactor was implemented as a conversion reactor with 0.50% conversion of CO2to biomass. Here, the assumption is that the CO2conversion rate achieved in the laboratory experiments would be achieved in a large-scale photobioreactor. The adjusting block (ADJ-2) in the flowsheet was used to adjust the flow rate of fresh air to achieve 10 vol% of homogenised gas to the reactor and recycling of unreacted gas was implemented using a purge stream.

[0306] The flowsheet was simulated with the basis to process 100 kg / day fresh CO2inlet (stream-1) and 1% purge of recycled gas (stream-6). The converged mass balance from the flowsheet simulations is summarised in Table 11 In the converged results, the discrepancy between inlets (streams 1 and 2) and outlets (streams 4 and 6) was less than 0.1 %. Note that the volumetric flow rate of stream-3 infers the liquid holdup required in the photobioreactor as the experimental data of the current study are valid at 1 WM gas flow rate. Thus, the required liquid holdup for the simulated basis is 3.545 m3. It is also noteworthy that the amount of CO2emitted in the purge (stream-6) is 95.22 kg / day against bio-fixation of 4.78 kg / day even at a low purge ratio of 1%.

[0307] Table 11. Mass balance for a microalgae cultivation process with recycling of gases _

[0308] Stream number

[0309] 1 2 3 4 5 6 7 8

[0310] Mass flow (kg / day) 100.00 562.07 6577.64 4.78 6572.85 657.29 5915.57 5915.57

[0311] Vol. flow (m3 / day) 53.74 460.56 5104.80 2.57 5102.40 510.24 4591.20 4591.20

[0312] Vol. frac.

[0313] CO21 0 0.10 1 0.10 0.10 0.10 0.10

[0314] Air 0 1 0.90 0 0.90 0.90 0.90 0.90

[0315] Mass frac.

[0316] CO21 0 0.15 1 0.15 0.15 0.15 0.15 Air 0.85 0 0.85 0.85 0.85 0.85

[0317] Example 77. Evaluation of continued culture method

[0318] The studies in this example were performed to evaluate the reproducibility of the continued harvesting method and to investigate the effect of multiple harvest cycles on algal biomass production.

[0319] The photosynthesis step was performed over a total of 40 days, including an initial 8 day acclimatisation period. The accumulated biomass and pH was measured at regular intervals throughout the 40 day study according to the methods described above. Biomass was harvested every 2 days and the algae culture was grown according to the CCA as described herein. The aqueous culture medium maintained a pH of between 6-7 throughout the study.

[0320] Reproducibility

[0321] The amount of biomass accumulated throughout the study across three replicate experiments is shown in Table 12.

[0322] Table 12. Accumulation of microalgal biomass when grown by continued culture over 40 days

[0323] Under optimised culture conditions, the acclimatisation period was repeated to evaluate hourly biomass accumulation over the initial seven day culture period. The hourly biomass measurements are shown in Figure 19. Each time point represents the average of two data points collected from duplicate experiments.

[0324] Multiple harvest cycles

[0325] A further study designed to evaluate the impact of multiple harvest cycles on biomass production during continued culturing was performed. Biomass (g / L) was measured as described above. Interestingly, it was observed that as the number of harvest events increased (event 1, day 7; event 2, day 11; event 3, day 15), the culture maintained an overall higher biomass (see Figure 20, day 15 harvest event). Figure 20 shows duplicate studies (Rep

[0326] 1 and Rep 2) performed over 18 days under optimised culture conditions. That is, the removal of a portion of microalgal biomass did not cause the total biomass of microalgae to drop as low as it had as a result of previous harvest events, 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, the maximum biomass of approximately 7.5 g / L was reached between each harvest events and prior to the subsequent harvest event. It was observed that reducing the biomass concentration by less, allowed the culture to achieve maximal biomass in less time without the culture entering a stationary phase.

[0327] Harvest interval

[0328] Using the optimised continued culturing method (Table 6) with a CO2flow rate of 0.15 WM, the impact of the frequency of harvesting events on biomass production was investigated. The purpose of this study was to compare different harvest intervals and monitor the stability of biomass density once harvesting begins. The batches included in this study and the harvest interval times for each treatment are listed in Table 13. Portions of biomass were harvested by syphoning 5% of the working volume at each harvesting event for each treatment. The biomass density over time (g / L) and the total biomass harvested (g) are shown Figure 21 (A) and (B), respectively. While 30 minute harvesting intervals yielded the highest total biomass yield, the biomass density steadily decreased throughout the harvesting phase. As such, the

[0329] 2 hour harvest interval provided the optimal stability of biomass density (improved over 30 minutes or 1 hour harvest interval treatments, see Figure 21 (A)). Similarly, the 2 hour harvest interval treatment yielded improved total harvest when compared to the 3 and 4 hour harvest interval treatments, but with comparable biomass densities. Balancing stability of biomass density and yield suggests a 2 hour harvesting interval is optimal. Table 13.

[0330] Batch No. Harvest time intervals (h)

[0331] 1 1

[0332] 2 2

[0333] 3 0.5

[0334] 3 2

[0335] 3 3

[0336] 3 4

Claims

What is claimed is:

1. A method of culturing a phototrophic organism, comprising the steps of: 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 organism reaches a point between an exponential growth phase and a stationary growth phase, reducing a biomass concentration of the phototrophic organism in the aqueous medium to a reduced biomass concentration not less than that which existed in an early- exponential growth phase.

2. The method according to claim 1, wherein the phototrophic organism is an algae, preferably a microalgae, preferably a microalgae selected from a Chlorella spp., preferably a Chlorella vulgaris species microalgae, preferably selected from one or both of C. vulgaris species strain CS-41 or CCAP211 / 11S, and preferably CS-41.

3. The method according to claim 1 or 2, wherein the phototrophic organism is CO2- adapted, preferably adapted to a gaseous atmosphere containing carbon dioxide concentrations of at least about 5% and preferably at least about 8% which may be a concentration of between about 8% and 25%.

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

5. The method according to any one of claims 1 to 4, wherein the reducing of the biomass concentration comprises a step of removing a volume of the phototrophic organism biomass in the aqueous medium and adding a dilution volume of aqueousmedium, optionally with the preliminary step of removing a volume of the aqueous medium.

6. The method according to claim 5, wherein the preliminary step of removing a volume of the aqueous medium is performed, and the dilution volume of aqueous medium added comprises removed aqueous medium.

7. The method according to any one of claims 1 to 6, wherein the biomass concentration is a concentration of 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, and preferably between about 6.0 g / L and 7.5 g / L.

8. The method according to any one of claims 1 to 7, wherein the reduced biomass concentration is a concentration which existed at about or above a mid-exponential growth phase point.

9. The method according to any one of claims 1 to 8, wherein the reduced biomass concentration is a concentration of 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, and preferably between about 3.5 g / L and 5.0 g / L.

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

11. The method according to any one of claims 1 to 9, further comprising cycling the reduced biomass concentration, wherein the cycling comprises repeating steps a) and b) using the reduced biomass concentration, and preferably repeating steps a) and b) a plurality of times using the reduced biomass concentration of each step b).

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

13. The method according to any one of claims 1 to 6, further comprising cycling the reduced biomass concentration, wherein the cycling comprises repeating steps a) andb) a plurality of times using the reduced biomass concentration of each step b), wherein a duration of a step a) is short, or a concentration change of a step b) is small.

14. The method according to claim 13, wherein a duration of a step a) is short, or a concentration change of a step b) is small, in a sequence of cycles15. The method according to claim 13 or claim 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, and preferably between about 1 hour and 2 hours.

16. The method according to claim 13 or claim 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, and preferably between about 0.1 g / L and 0.3 g / L.

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

18. The method according to claim 5 or claim 6, wherein the step of removing a volume of the phototrophic organism biomass is a continuous removal of phototrophic organism biomass from the aqueous medium.

19. The method according to claim 18, wherein the continuous removal of phototrophic organism biomass is together with aqueous culture medium, and a dilution volume of aqueous culture medium is continually added to the remaining biomass.

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

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

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

23. The method according to 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, or preferably of about 16 h, per 24 h period.

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

25. The method according to any one of claims 1 to 24, wherein the culture conditions further comprise one or more of the group consisting of: a) a bioreactor, wherein a volume of phototrophic organism and aqueous medium is between about 35% and 90% of the bioreactor capacity, preferably between about 40% and 85%, preferably between about 50% and 70%, and preferably about 60%; b) the gaseous atmosphere further comprises air; c) the gaseous atmosphere is provided as a continuous flow, preferably with a flow rate of between about 0.05 WM and about 5 WM, preferably between about 0.1 WM and about 2 VMM, preferably between about 0.1 WM and about 1.0 VMM, preferably between about 0.1 WM and 0.5 WM, preferably between about 0.1 WM and 0.3 WM, preferably between about 0.1 WM and 0.2 WM, and preferably of about 0.15 WM; d) a pH of the aqueous medium of between about 5 and 9, preferably between about 6 and 8, and preferably of about 6.5 and 7.5, or preferably about 7; ande) a temperature of between about 25 °C and about 31°C, preferably between about 27°C and about 29°C, and preferably of about 28°C.

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

27. The method according to 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. A method of continued culturing of a phototrophic organism, comprising performing the method of claim 1, and cycling the reduced biomass concentration, wherein the cycling comprises repeating steps a) and b) using the reduced biomass concentration.

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

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

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

32. The method of any one of claims 1 to 30, when used for producing an organic feedstock.

33. A culture of a phototrophic organism produced by the method of any one of claims1 to 30.

34. An organic feedstock produced by the method of any one of claims 1 to 30, wherein the reducing of the biomass concentration comprises a step of removing phototrophic organism biomass from the aqueous medium, and the organic feedstock comprises removed phototrophic organism biomass.

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

36. A COz-adapted phototrophic organism, wherein the phototrophic organism is selected from one or both of C. vulgaris species strain CS-41 or CCAP211 / 11S, and preferably CS-41, and wherein the phototrophic organism is COz-adapted to a gaseous atmosphere comprising carbon dioxide concentrations of 5% or greater.

37. The COz-adapted phototrophic organism of claim 35, wherein the phototrophic organism is COz-adapted to a gaseous atmosphere containing carbon dioxide concentrations of at least about 8% which may be a concentration of between about 8% and 25%.

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