Cultivating phototrophic objects

Abstract

The present disclosure relates to a method of culturing a phototrophic organism in an aqueous medium under culture conditions comprising light and a gas atmosphere comprising carbon dioxide. The culture can be carried out continuously. The methods may be used to capture carbon dioxide, and / or to produce organic feedstocks that may be used in downstream processes, such as to produce biofuels and / or biofertilizers. The present disclosure also relates to strains of Chlorella species adapted to CO2.

Description

[0001] Citations of previous applications

[0002] This application claims the benefit of Australian Provisional Patent Application No. 2023900393 filed on February 16, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to a method for cultivating phototrophic organisms in an aqueous medium under culture conditions comprising light and a gaseous atmosphere containing carbon dioxide. The culture can be continuous. The method can be used to capture carbon dioxide and / or produce organic feedstock that can be used in downstream processes, such as biofuel and / or biofertilizer production. The present disclosure also relates to strains of the species Chlorella vulgaris adapted to CO₂. Background Art

[0004] The primary cause of what is considered the global warming phenomenon and the associated environmental problems is carbon dioxide (CO2) emissions. CO2 is primarily a by-product of combustion. The production of CO2 is largely anthropogenic; it occurs in various industrial applications, such as the use of coal in power plants to generate electricity, and in the use of hydrocarbons, which are often the main component of fuel burned in combustion devices such as engines. The exhaust gases emitted from such combustion devices contain CO2 gas, which is currently simply released into the atmosphere. Over the past few decades, anthropogenic global carbon dioxide (CO2) emissions have increased by approximately 50%. While the focus of politics, society, and industry has been on reducing emissions, it is generally agreed that this alone is insufficient to curb the ongoing negative impacts on the global environment. Consequently, there needs to be greater attention paid to carbon dioxide removal (CDR) technologies (also known as carbon capture methods) that can result in net negative emissions.

[0005] Existing CDR technologies include afforestation and reforestation, soil carbon sequestration, enhanced weathering, ocean fertilization, bioenergy with carbon capture and storage, and direct air carbon capture. These technologies have their limitations. For example, while terrestrial plants have a natural ability to capture CO₂ through photosynthesis, they are adapted to atmospheric CO₂ concentrations typically around 0.033%, while industrial emissions typically contain much higher concentrations, ranging from 10% to 25%. Afforestation and reforestation require significant land to achieve near-net-negative carbon emissions, which is offset by the release of captured carbon back into the atmosphere when biomass decays or burns. There are also biodiversity concerns associated with afforestation. Consequently, afforestation processes have limited practicality and commercial viability. Enhanced weathering also limits commercial viability and is associated with the risk of negative impacts on biodiversity, water quality, and food security. Ocean fertilization also has limited applicability due to its low actual impact and high material requirements. Bioenergy with carbon capture and storage remains the most plausible and scalable path to a net-negative carbon economy, but it is not without its challenges.

[0006] Several carbon capture methods have been proposed to sequester CO2 produced by industrial processes such as coal-fired power plants. One approach involves reacting CO2 with large quantities of metal oxides, particularly calcium oxide and magnesium oxide, and then burying the resulting carbonates. A disadvantage of this approach is that it requires a large and continuous supply of minerals. Another approach involves removing CO2 from combustion gases, compressing it, and then transporting it via pipeline to peridotite or serpentinite mines to convert it into carbonates, which are then buried at the mine site. However, transporting compressed CO2, which is heavier than air, presents material handling challenges. For example, if the CO2 is accidentally released during transport, it will remain near the surface and could be life-threatening. Another issue may be the availability of mines for such burials, as well as the cost and energy consumption of transportation. Other technologies have been attempted, which can generally be categorized as geological, terrestrial, or marine systems. They also rely on transporting the CO2 to a site for injection into geological, soil, or marine reservoirs. However, like peridotite or serpentinite mines, each of these processes is expensive in terms of capital costs, transportation, and energy consumption. These carbon capture methods also focus on storage rather than converting carbon into useful products.

[0007] One approach that has recently gained attention involves the use of microalgae. Microalgae have a natural ability to capture CO₂ at a higher rate than terrestrial plants, and algal biomass is a useful product in many downstream industrial processes. However, algae cultivation for carbon capture is difficult. The growth curve of algae follows a general path, including a long lag phase in which the biomass remains unchanged or increases slowly, then reaches a pre-exponential phase, followed by an exponential phase in which the growth rate increases rapidly. Although the CO₂ capture capacity of the biomass increases during the exponential phase, the exponential phase is typically short-lived, and the algae then transitions to a stable phase in which net growth ceases, followed by a decay phase in which the living biomass rapidly decreases. Therefore, the window to reach a suitable carbon capture level during the exponential phase is short-lived. Algae cultures are also very sensitive to environmental changes and may not grow or grow at a suitable rate in an environment containing levels of carbon dioxide commonly found in industrial emissions.

[0008] It is possible to produce algae that tolerate high CO2 environments through a process called adaptive laboratory evolution (ALE), which involves subjecting the algae to an initial low CO2 concentration, then slightly increasing that concentration when the algae have stabilized, and so on, until the desired tolerance is achieved. ALE is generally a slow process, taking at least several months or even 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 end of an exponential growth phase, it loses viability and the process must be repeated. There are also limits to the permissible levels that can be achieved, which are generally lower than the carbon dioxide levels commonly found in industrial emissions.

[0009] It would be advantageous to provide CO2 sequestration technologies that can convert carbon into useful downstream products, and / or to provide improved methods of cultivating algae that can be used in CO2 sequestration processes, particularly for sequestering CO2 from industrial emissions.

[0010] It should be understood that, in relation to the mention of any prior art publication or reference herein, such reference does not constitute an admission that the publication forms part of the common general knowledge in the art in any country. Summary of the Invention

[0011] In a first aspect, the present disclosure provides a method for cultivating a phototrophic organism, comprising the steps of:

[0012] a) cultivating the phototrophic organism in an aqueous culture medium under culture conditions comprising light and a gas atmosphere comprising carbon dioxide; and

[0013] b) when the phototrophic organism reaches a point between the exponential growth phase and the stationary growth phase, reducing the biomass concentration of the phototrophic organism in the aqueous culture medium to a reduced biomass concentration that is no less than the biomass concentration that existed during the initial exponential growth phase.

[0014] In some embodiments, reducing the biomass concentration comprises removing the phototrophic organism biomass from the aqueous medium. The removed biomass can be referred to as organic feedstock. This can be done with adding a dilution volume of aqueous medium. The dilution volume of aqueous medium can be freshly prepared or recycled.

[0015] In some embodiments, the method further comprises recycling the reduced biomass concentration, wherein the recycling comprises repeating steps a) and b) using the reduced biomass concentration, and preferably repeating steps a) and b) multiple times using the reduced biomass concentration in each step b). When the method comprises a recycling step, the method can be referred to as a continuous culture method.

[0016] In some embodiments, the source of carbon dioxide comprises a waste gas stream, such as produced by combustion of hydrocarbons (eg, flue gas), by steam reforming, and / or by biological decomposition.

[0017] In another aspect, the present disclosure provides a method for continuously culturing phototrophic organisms, the method comprising performing the method of the first aspect as described herein, and recycling the reduced biomass concentration, wherein the recycling comprises repeating steps a) and b) using the reduced biomass concentration, and preferably repeating steps a) and b) multiple times using the reduced biomass concentration in each step b).

[0018] In another aspect, the present disclosure provides a method of culturing a phototrophic organism, the method comprising culturing the phototrophic organism in an aqueous culture medium under culture conditions comprising light and a gas atmosphere comprising carbon dioxide, wherein the intensity of the light is from about 3000 lux to about 7000 lux, and wherein the culture conditions further comprise a photoperiod of from about 12 hours to about 20 hours.

[0019] In another aspect, the present disclosure provides a culture of a phototrophic organism produced by the methods described herein.

[0020] In another aspect, the present disclosure provides an organic feedstock produced by the methods described herein, wherein reducing the biomass concentration comprises the step of removing a volume of phototrophic organism biomass from the aqueous culture medium, and the organic feedstock comprises the removed phototrophic organism biomass.

[0021] In another aspect, the present disclosure provides a method as described herein for producing an organic feedstock.

[0022] In another aspect, the present disclosure provides a method of capturing carbon dioxide from a waste gas stream, the method comprising culturing a phototrophic organism by a method as described herein, wherein the source of carbon dioxide comprises the waste gas stream.

[0023] In another aspect, the present disclosure provides a method as described herein for capturing carbon dioxide from a waste gas stream.

[0024] In another aspect, the present disclosure provides a CO2-adapted phototrophic organism, wherein the phototrophic organism is selected from one or both of strains CS-41 or CCAP211 / 11S of the Chlorella species, preferably CS-41, and wherein the phototrophic organism is adapted to CO2 in a gas atmosphere comprising a carbon dioxide concentration of 5% or greater. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic diagram of the photobioreactor setup, where the triangle connects the CO2 inlet and the air inlet, and the rotameter represents the electronic pressure regulator.

[0026] Figure 2 Shown are comparisons of (a) biomass concentration, (b) pH, and (c) biomass growth rate from the fourth (4G) and fifth (5G) generations (BBM medium, 0.05 g / L inoculum, 12:12 light:dark cycle) in 10% CO2.

[0027] Figure 3 Shown are (a) elemental analysis data on day 7 of biomass from the fourth and fifth generations under 10% CO2 (BBM medium, 10% CO2, 0.05 g / L inoculum, 12:12 light:dark cycle), (b) biological fixation rate of CO2, and (c) CO2 concentrations in the inlet and outlet gas streams measured by CO2 sensors.

[0028] Figure 4 Shown are the effects of light intensity on the growth rate of Chlorella CS-41 grown in different culture media.

[0029] Figure 5 Shown are the effects of light intensity on biomass production by Chlorella CS-41 grown in different culture media.

[0030] Figure 6 Shown are the effects of light intensity on CO2 biofixation by Chlorella vulgaris CS-41 grown in BBM or BG-11 medium.

[0031] Figure 7 Shown are the effects of light intensity on total CO2 capture by Chlorella vulgaris CS-41 grown in BBM or BG-11 medium.

[0032] Figure 8 Shown are the biomass of Chlorella vulgaris CS-41 grown in BBM or BG-11 medium under five photoperiods (12:12, 16:8, 18:6, 20:4, and 24:0 hours light / dark).

[0033] Figure 9 Shown are the dry weight biomass (g / L) of Chlorella at different pH (6, 7, 8, and 9) during 7-day cultivation in BG-11 and BBM media under a fixed photoperiod (16:8 h) and light intensity (5000 lux).

[0034] Figure 10 Shown are the average CO2 fixation rates of Chlorella CS-41 grown in BBM or BG-11 over a range of pH values ​​after 7 days of cultivation.

[0035] Figure 11 Shown are the total CO2 captured on day 7 by Chlorella CS-41 grown in BBM or BG-11 over a range of pH.

[0036] Figure 12 Comparison of the growth rate of Chlorella vulgaris grown in BG-11 medium before and after optimization.

[0037] Figure 13 The effect of fresh and reused medium on biomass concentration is shown. (BBM medium, 10% CO2, 0.05 g / L inoculum, 12:12 light:dark cycle; Note: Cycle 1 - experiment with fresh BBM, Cycle 2 - experiment with spent BBM at the end of Cycle 1.

[0038] Figure 14 This is the seven-day growth curve of Chlorella CS-41 under optimized conditions.

[0039] Figure 15 Flowchart of continuous cycle method (CCA) and (CCB). Initial batch culture is grown for 7 days (cycle 0). The biomass produced in cycle 0 is divided into two batches (fresh, CCA or recycled CCB) and cultured for another 2 days under lower light (cycle 1). Half of the biomass is removed for downstream applications (e.g., biodegradation), and the remaining batch is supplemented with fresh culture medium or recycled culture medium as described above. This process is repeated until the successive batches lose integrity as determined by pre-established growth criteria.

[0040] Figure 16 Shown are the biomass yields of Chlorella vulgaris CS-41 when grown under the continuous circulation methods (A) CCA (divide biomass and renew media method) and (B) CCB (concentrate biomass and recycle media method).

[0041] Figure 17 Comparison of the amount of CO2 captured by the continuous culture method CCA and the batch culture system (BCS), (A) shown in g / L, (B) shown in total CO2 captured.

[0042] Figure 18 Shown are (a) a summary of the overall mass balance and optimal operating conditions in the reactor, and (b) a simple flow diagram of the microalgae cultivation process with gas recycling.

[0043] Figure 19 is a graph showing biomass accumulation (g / L) at one-hour intervals during the acclimation phase.

[0044] Figure 20 is a graph showing the effect of biomass harvest on total biomass (g / L) for a continuous culture process over multiple biomass harvest events.

[0045] Figure 21 Two graphs are shown, Panel A shows the change in biomass density (g / L) over time for each harvest interval treatment once harvesting began; Panel B shows the total biomass harvested from each different treatment (harvest intervals were 30 minutes, 1 hour, 2 hours (performed in duplicate), 3 hours, and 4 hours). DETAILED DESCRIPTION

[0046] The following is a detailed description of the present disclosure provided to help those skilled in the art practice the present disclosure. Those skilled in the art may make modifications and variations to the embodiments described herein without departing from the spirit or scope of the present disclosure.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0048] As used herein, the term "and / or," such as "X and / or Y," should be understood to mean "X and Y" or "X or Y," and should be used to provide explicit support for both meanings or either meaning.

[0049] The articles "a" and "an" are used herein to refer to one or to more than one (ie, 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.

[0050] As used herein, the term "about" when referring to a numerical value or range allows for a certain degree of variability in the value or range, for example, within 10% of the stated limit of the range.

[0051] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 10, including a range designated as "between" 1 and 10, is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Similarly, a range of 1.0 to 2.0 is understood to include any number, combination of numbers, or subrange from the group consisting of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0.

[0052] As used herein, when used in conjunction with a list of items, the phrase "at least one of" means that different combinations of one or more of the listed items can be selected, as well as one item from the list. In other words, "at least one of" refers to any number of items or combinations of items that can be selected from the list. For example, "at least one of item A, item B, and item C" can refer to item A; item A and item B; item B; item A, item B, and item C; or item B and item C.

[0053] Throughout the specification and claims that 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 groups of integers or steps but not the exclusion of any other integer or step or groups of integers or steps.

[0054] Reference will now be made in detail to the preferred and exemplary embodiments of the present disclosure. Although the present disclosure will be described in conjunction with the exemplary embodiments, it should be understood that the present disclosure is not intended to limit the present disclosure to those embodiments. On the contrary, the present disclosure is intended to cover alternatives, modifications and equivalents that may be included within the spirit and scope of the present disclosure as defined by the appended claims.

[0055] Phototrophic organisms

[0056] The present disclosure provides a method of culturing a phototrophic organism, the method comprising culturing the phototrophic organism in an aqueous culture medium under culture conditions comprising light and a gas atmosphere comprising carbon dioxide.

[0057] "Phototrophic organisms" refer to organisms that can use light as the primary energy source for metabolism in the presence of water and carbon dioxide to produce energy in the form of carbohydrates through photosynthesis. Examples include algae, phytoplankton, bacteria, and protists.

[0058] In a preferred embodiment, the phototrophic organism is an algae. Algae can be identified as photosynthetic, eukaryotic, non-flowering aquatic plants. Examples include red algae, green algae, and cyanobacteria, which generally include plant species classified as Halvaria and Hacrobia (red algae), containing heterokonts, dinoflagellates, haptophyta, and cryptoalgae; plant species classified as Excavata and Rhizaria (green algae), containing chlororachnophytes, euglena, and chlorophyta; and plant species classified as Primoplantae and Archaeplastida (cyanobacteria), containing the phyla Chlorophyta and Rhodophyta. Preferably, the phototrophic organism is a microalgae. Microalgae can be identified as unicellular algae, and may also be referred to as microphytes. Preferably, the microalgae is a green algae. Preferably, the microalgae is selected from the genus Chlorella, such as selected 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.

[0059] In a preferred embodiment, the microalgae is a microalgae of the species Chlorella, preferably selected from one or both of the strains CS-41 or CCAP 211 / 11S of the species Chlorella, most preferably CS-41. A culture of the Chlorella strain CS-41 is deposited at the Australian National Algae Culture Collection of CSIRO in Hobart, Tasmania, Australia, under the CS reference number "CS-41". A culture of the Chlorella strain CCAP 211 / 11S is deposited at the Culture Collection of Algae and Protozoa in Oban, Scotland, UK, under the accession number MG022720 and is assumed to be the same strain as CS-41.

[0060] Preferably, the phototrophic organism is CO adapted, which means that it is able to grow under culture conditions comprising a gas atmosphere containing carbon dioxide, wherein the carbon dioxide concentration is greater than the atmospheric CO concentration (the atmospheric CO concentration is typically about 0.033%). Methods for producing CO adapted phototrophic organisms are described below.

[0061] In preferred embodiments, the phototrophic organisms are adapted to a gaseous atmosphere containing a carbon dioxide concentration of at least about 5%, preferably at least about 8%, 9%, or 10%, and may be adapted to concentrations of about 15%, 20%, or 25%. Alternatively, or in addition, the phototrophic organisms may be adapted to a gaseous atmosphere containing a carbon dioxide concentration of about 5% to 25%, which may range from about 5%, 6%, 7%, etc., to 23%, 24%, or 25%. Preferably, the phototrophic organisms are adapted to a gaseous atmosphere containing a carbon dioxide concentration of about 8% to 25%, or 9% or 10% to 25%, or 8% to 20%, which may range from about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. It is believed that phototrophic organisms adapted to carbon dioxide concentrations higher than atmospheric carbon dioxide are able to consume greater amounts of carbon dioxide produced by photosynthesis, converting them into a higher energy yield in the form of carbohydrates. Furthermore, carbon dioxide levels of approximately 8% to 25% are commonly found in waste streams from various industrial processes, and thus the methods described herein can be used to sequester carbon dioxide from gaseous atmospheres, as described below.

[0062] At least preferred phototrophic organisms that are capable of consuming greater amounts of CO2 when adapted to CO2 are capable of adapting to CO2 in a much faster time than conventional ALE processes and / or are also capable of adapting to CO2 at higher CO2 concentrations than previously believed. It is for this reason that preferred phototrophic organisms are microalgae, preferably microalgae of the genus Chlorella, preferably of the species Chlorella, most preferably strains CS-41 or CCAP 211 / 11S.

[0063] Therefore, the present disclosure also provides a CO2-adapted phototrophic organism, wherein the phototrophic organism is selected from one or both of strains CS-41 or CCAP211 / 11S of the Chlorella species, preferably CS-41, and wherein the phototrophic organism is adapted to CO2 in a gas atmosphere comprising a carbon dioxide concentration of 5% or higher.

[0064] Cultivation methods

[0065] The present disclosure provides a method for cultivating phototrophic organisms by culturing the phototrophic organisms in an aqueous medium. As used in this disclosure, the term "aqueous medium" refers to a water-based liquid. The aqueous medium may contain nutrients that support the growth of the organisms. Preferred features of the aqueous medium are described below.

[0066] The culture method can be carried out in a bioreactor, which can generally be a container containing an aqueous culture medium containing phototrophic organisms. Many bioreactors are known to those skilled in the art. A preferred bioreactor is the bioreactor disclosed in Australian Provisional Patent Application No. 2023902970, filed on September 14, 2023, the entire contents of which are incorporated herein by reference.

[0067] Reduce biomass concentration

[0068] The present disclosure provides a method for culturing a phototrophic organism, wherein when the phototrophic organism reaches a point between an exponential growth phase and a stationary growth phase, the biomass concentration of the phototrophic organism in an aqueous culture medium is reduced to a reduced biomass concentration that is no less than the biomass concentration that existed during the initial exponential growth phase. Another way of expressing "when the phototrophic organism reaches a point between the exponential growth phase and the stationary growth phase" is when the phototrophic organism reaches a concentration corresponding to a point between the exponential growth phase and the stationary growth phase, or when the phototrophic organism reaches a concentration intermediate between the exponential growth phase and the stationary growth phase.

[0069] Generally speaking, phototrophic organisms, such as microalgae, and particularly species of Chlorella, such as Chlorella, reproduce asexually through non-motile reproductive cells (autospores), whereby the cells reproduce by autospore formation, in which four daughter cells with independent cell walls are formed within the cell wall of a mother cell. After the newly formed cells mature, the cell wall of the mother cell ruptures, releasing the daughter cells, and the fragments of the mother cell are consumed by the daughter cells as feed. The culture thus grows.

[0070] The growth curve of phototrophic organisms, including algae, particularly microalgae, follows a generally sigmoidal path, comprising a long lag phase in which biomass remains constant or increases slowly (e.g., during which the rate of autospore formation is low), followed by a pre-exponential phase in which the growth rate transitions to a higher growth rate in a subsequent exponential phase, e.g., during which the rate of autospore formation is high. The exponential phase is followed by a second transition phase in which the growth rate slows, and the biomass levels off in a subsequent stationary phase. In a subsequent decay phase, the biomass decreases as the organism dies.

[0071] As used herein, the term "exponential phase" refers to the growth phase of a phototrophic organism that occurs between the initial lag phase and the later stationary phase in the organism's S-shaped growth curve and is characterized by a period of rapid growth compared to the initial lag phase and the later stationary phase. The "early exponential phase" refers to the initial increase in growth rate when an organism transitions from the initial lag phase and includes the transition period. The "mid-exponential phase" occurs after the early exponential phase, around the middle of the exponential phase, and includes the "mid-exponential point" which is the midpoint of the mid-exponential phase. The "late exponential phase" follows the mid-exponential phase and precedes the stationary phase, and is generally the period during which the maximum growth rate is observed. The "stationary phase" refers to the growth phase in which the organism's growth rate begins to slow and in which growth tends to stabilize. The "saturation point" occurs in the late exponential phase of growth just before the start of the stationary phase. In the context of cultivating phototrophic organisms, the "growth phase" can be simply referred to as a "phase." In many embodiments, the early exponential phase, the mid-exponential phase, and the late exponential phase, which constitute the exponential phase, can be considered to be divided into three parts based on biomass concentration and / or duration.

[0072] Advantageously, when the culture of phototrophic organism, especially microalgae in aqueous medium is in a certain point between exponential growth phase and the stable growth phase, and when the biomass concentration of phototrophic organism is reduced to be not less than the concentration that exists in the exponential growth initial stage in the aqueous medium, the phototrophic organism avoids or avoids the stable phase basically, and continues to grow and does not have the initial lag phase of prolongation.The phototrophic organism adapts to the aqueous medium concentration of change rapidly, and returns to the exponential growth phase after the exponential phase early stage, or even continues to grow with the speed consistent with the exponential growth phase.In other words, by reducing the concentration of the phototrophic organism, the phototrophic organism can get back to the early stage of the exponential growth phase, for example gets back to the exponential growth initial stage from the exponential growth later stage, thereby significantly prolongs the exponential growth phase.Therefore, this method can improve the output of biomass in a shorter time.

[0073] Before the biomass concentration of the phototrophic organism in the aqueous medium was about to reduce, when the point between the exponential growth phase and the stable growth phase was the point between the exponential mid-term point and the saturation point or preferably the point between the exponential growth phase mid-term and the saturation point, or in other words the point in the exponential late period, when preferably being in the point near the saturation point, can effectively utilize this advantage.Because growth rate and / or biomass are usually the highest in the exponential growth late period and / or saturation point, this allows to have high biomass output before the concentration reduces.When the biomass concentration that reduces is not less than the concentration that exists when the exponential growth phase begins, or in other words, the concentration that reduces is the concentration that exists in the exponential growth early stage, the concentration that preferably exists in the exponential growth phase mid-term, it can be higher than the exponential mid-term point, and even when in the scope of the exponential late period, can further effectively utilize this advantage.The phototrophic organism (particularly micro-algae) concentration that reduces is as in the exponential early stage, especially in the exponential growth phase mid-term or the concentration in the later period, provides the fastest adaptation to the aqueous medium concentration that changes and returns to exponential growth the fastest, allows even higher biomass output.

[0074] Thus, in a preferred embodiment, the point between the exponential growth phase and the stationary growth phase immediately before the biomass concentration of the phototrophic organism decreases is a point between the middle of the exponential growth phase and the saturation point, or in other words, a point in the late exponential growth phase, and preferably a point near the saturation point. Also in a preferred embodiment, the biomass concentration that decreases is no less than the concentration that existed at the beginning of the exponential growth phase, or the concentration that existed in the early exponential phase, and preferably is the concentration that existed in the middle or late exponential growth phase.

[0075] Because this allows for a rapid return to exponential growth, in a preferred embodiment, the method further comprises repeating step a) using a reduced biomass concentration; that is, culturing the phototrophic organism in an aqueous culture medium under culture conditions comprising light and a carbon dioxide-containing gas atmosphere using the reduced biomass concentration. Step a) may be referred to as a "growth step."

[0076] The reduced biomass concentration can be grown in the same bioreactor or can be transferred to another bioreactor. If the concentration is reduced by removing a volume of phototrophic organism biomass, the removed biomass can be transferred to a bioreactor for continued growth or can be transferred to a storage container for use in downstream processes.

[0077] In some embodiments, the biomass of the present invention can be used to grow at a specific light nutrient concentration.In some embodiments, the biomass of the present invention can be used to grow at a specific light nutrient concentration.In some embodiments, the biomass of the present invention can be used to grow at a specific light nutrient concentration.In some embodiments, the biomass of the present invention can be used to grow at a specific light nutrient concentration.In some embodiments, the biomass of the present invention can be used to grow at a specific light nutrient concentration.In some embodiments, the biomass of the present invention can be used to grow at a specific light nutrient concentration.In some embodiments, the biomass of the present invention can be used to grow at a specific light nutrient concentration.In some embodiments, the biomass of the present invention can be used to grow at a specific light nutrient concentration.In some embodiments, the biomass of the present invention can be used to grow at a specific light nutrient concentration.In some embodiments, the biomass of the present invention can be used to grow at a specific light nutrient concentration. In the case of microalgae, these concentrations can correspond to the mid-exponential range or around the mid-exponential point, or in the late exponential range. Therefore, in a preferred embodiment, and especially for microalgae, the biomass concentration in the aqueous culture medium is reduced by about 0.5 g / L to about 6.0 g / L, preferably from about 1.5 g / L to about 5.5 g / L, preferably from about 2.0 g / L to about 5.0 g / L, preferably from about 2.5 g / L to about 4.5 g / L. The amount by which the biomass concentration in the aqueous culture medium is reduced, i.e., the difference between the phototrophic organism concentration in the aqueous culture medium immediately before removal and the reduced biomass concentration, can be referred to as a "concentration change."

[0078] The duration of the growth step varies for different phototrophic organisms. For microalgae, the duration of at least the first growth step from the initial exponential growth phase to the stationary growth phase can be from about 1.5 to about 4 days, preferably from about 1.5 to about 3 days, and preferably from about 1.8 to about 2.2 days.

[0079] The step of reducing biomass concentration can be carried out by for example adding the aqueous medium of certain volume and / or removing the phototrophic organism biomass of certain volume.The volume of the aqueous medium added can be called " dilution volume ".Removing the phototrophic organism biomass of certain volume also can be called " results ".When only reducing biomass concentration by adding the aqueous medium of certain volume, the combined volume of phototrophic organism biomass and aqueous medium will increase so, and may need extra disposal, for example, transfer to the container of larger size.Therefore, in a preferred embodiment, in order for example to avoid increasing container size, reducing biomass concentration comprises the step of removing the phototrophic organism biomass of certain volume.

[0080] Removing the phototrophic organism biomass also can remove a certain volume of aqueous culture medium.When this happens, and when the volume of the removed phototrophic organism biomass is equal to or less than the volume of the removed aqueous culture medium, reducing the biomass concentration usually will need to add the aqueous culture medium of dilution volume so, and this dilution volume is greater than the volume difference between the removed phototrophic organism biomass and the removed aqueous culture medium.On the other hand, when the volume of the removed phototrophic organism biomass is greater than the volume of the removed aqueous culture medium, reducing the biomass concentration may not need to add the aqueous culture medium of dilution volume so, but can carry out adding the aqueous culture medium of dilution volume, for example, to realize the biomass concentration of the reduction of expectation.

[0081] In a preferred embodiment, reducing the biomass concentration comprises removing a volume of the phototrophic organisms and a volume of the aqueous culture medium and adding a dilute volume of the aqueous culture medium to the remaining biomass.

[0082] A certain volume of aqueous culture medium can be removed together with a certain volume of phototrophic organism biomass or separately. For example, a certain volume of aqueous culture medium can be removed before removing the biomass. This can be achieved by flocculating the biomass and removing the aqueous culture medium as the supernatant. Flocculation methods are well known to those skilled in the art. For example, and relevant to the purpose of this disclosure, flocculation can be achieved by increasing the carbon dioxide concentration in the gas atmosphere. Chemical flocculants can also be used, such as chitosan (also known as deacetylated chitosan).

[0083] The aqueous culture medium of the added dilution volume can comprise the aqueous culture medium that is removed. In other words, the aqueous culture medium can be recycled. Removing the aqueous culture medium as supernatant is a method that can allow the recycling of culture medium. The recycling of aqueous culture medium has advantages aspect cost-effective and ecological sustainable biomass production. The dilution volume can comprise fresh aqueous culture medium at least in part. This can allow at least a portion of aqueous culture medium to be renewed, which can contribute to the growth of residual biomass. The ratio of the biomass that is removed will depend on the relative biomass concentration that exists at the different points in the growth curve stage, and may be different because of specific phototrophic organism. Based on the disclosure, those skilled in the art can use conventional methods to determine the ratio that is removed, while keeping the concentration of biomass to be not less than the concentration that exists in the initial stage of exponential growth.

[0084] As further described below, the phototrophic biomass can be removed in a batch manner. In batch removal, the amount removed is about 30% to about 80%, preferably about 40% to about 65%, and preferably about 45% to about 60%. In the case of phototrophic organisms, and particularly microalgae, these ratios can correspond to the difference between the saturation point concentration and the mid-exponential point concentration when equal volumes of diluted aqueous culture medium are added to the remaining biomass.

[0085] In other words, the concentration change can be from about 30% to about 80%, preferably from about 40% to about 65%, and more preferably from about 45% to about 60%. For example, if the concentration of phototrophic organisms in the aqueous medium immediately before removal is 7.5 g / L and the reduced biomass concentration is 5.0 g / L, this constitutes a 33% concentration change. Similarly, if the concentration of phototrophic organisms in the aqueous medium immediately before removal is 9.0 g / L and the reduced biomass concentration is 2.0 g / L, this constitutes a 78% concentration change, and so on.

[0086] Advantageously, reducing the concentration by removing biomass allows the removed biomass to be used in downstream processes, and by culturing the reduced biomass, which is rapidly regenerated through the exponential growth phase, the biomass can be removed again and again in a relatively rapid, continuous process as feedstock for downstream processes, i.e., steps a) and b) can be repeated one or more times. Reducing the concentration by removing biomass within the parameters described and repeating the growth steps provides the basis for a continuous culture method.

[0087] Continuous culture

[0088] In a preferred embodiment, the method further comprises recycling the reduced biomass concentration, wherein recycling the reduced biomass concentration comprises using the reduced biomass concentration to repeat at least step a), and preferably steps a) and b), and preferably recycling the reduced biomass concentration using the reduced biomass concentration in each previous cycle (i.e., each time step b) (i.e., repeating steps a) and b) multiple times). A method comprising repeating at least step a), and preferably steps a) and b), at least once, preferably multiple times (i.e., more than once) can be referred to as a "continuous culture."

[0089] Continuous culture can be batch or continuous.That is to say, "continuous culture" includes both batch culture and continuous culture."Batch" (also can be referred to as "semi-continuous") refers to that the concentration of the phototrophic organism in the aqueous medium is reduced with discrete steps.For example, when reducing concentration by removing the biomass of the phototrophic organism, batch culture relates to removing and optionally adding the aqueous medium of dilution volume with discrete steps. "Continuous" refers to that the concentration of the phototrophic organism in the aqueous medium is continuously reduced.For example, when reducing concentration by removing the biomass of the phototrophic organism, continuous culture relates to removing and optionally adding the aqueous medium of dilution volume.The phototrophic organism can be recycled repeatedly by this method, without obvious culture efficiency loss, be included under higher than atmospheric carbon dioxide concentration, and especially use microalgae.

[0090] In some embodiments, the biomass of reduction can be recycled at least 2 times, 3 times, 4 times, and preferably at least 5 times, such as 10 times, 20 times, 50 times, 75 times or 100 times or more times, by step a) and b).The biomass of reduction can be recycled many times, even thousands of times.For example, the biomass of reduction can be recycled as many as 125 times, 315 times, 630 times or 1260 times.Express with scope, the biomass of reduction can be recycled 2 times to approximately 1260 times, preferably approximately 20 times to approximately 630 times, and preferably approximately 125 times to 315 times.

[0091] For each cycle, steps a) and b) may be as described above. For example, particularly in the case of microalgae:

[0092] The biomass concentration in the aqueous culture medium (i.e., before it is reduced) may be at a concentration of about 2.0 g / L to about 9.0 g / L, preferably about 3.0 g / L to about 8.5 g / L, preferably about 4.5 g / L to about 8.0 g / L, preferably about 5.0 g / L to 7.5 g / L, and preferably about 6.0 g / L to 7.5 g / L;

[0093] The reduced biomass concentration in the aqueous culture medium may be at a concentration of about 2.0 g / L to about 6.5 g / L, preferably about 2.5 g / L to about 6.0 g / L, preferably about 3.0 g / L to 5.5 g / L, and preferably about 3.5 g / L to 5.0 g / L;

[0094] Reducing the biomass concentration preferably comprises removing a volume of the phototrophic organisms and a volume of the aqueous culture medium and adding a dilute volume of the aqueous culture medium to the remaining biomass; and

[0095] • An amount of phototrophic organism biomass of about 30% to about 80%, preferably about 40% to about 65%, and preferably about 45% to about 60% may be removed.

[0096] For at least the second and subsequent circulations carried out by steps a) and b), the duration of the growth step (step a)) will be different because of different phototrophic organisms, and will depend on the biomass concentration of the reduction of the previous circulation, and the point between the exponential growth phase and the stable growth phase that the phototrophic organism reaches before its biomass concentration is reduced again, or in other words, the concentration that the biomass reaches. That is, in general, at least for microalgae, in some embodiments, the duration of the growth step from the initial exponential growth phase to the stable growth phase can be from about 1.5 days to about 4 days, preferably from about 1.5 days to about 3 days, and preferably from about 1.8 days to about 2.2 days. Similarly, if the biomass concentration of the reduction of the previous circulation is a concentration around the mid-exponential point, the duration of the growth step from the mid-exponential point to the stable growth phase or saturation point can be from about 0.7 to about 2 days, preferably from about 0.7 day to about 1.5 days, and preferably from about 0.9 to about 1.1 days.

[0097] That is to say, it has been found that shorter growth phase duration, or in other words, when concentration variation (between the concentration being reduced and the concentration before being about to reduce) is less, some benefits can be produced." short " growth phase duration can be defined as about 24 hours or less than 24 hours." small " concentration variation can be defined as about or less than 1g / L. The benefit of finding is that the concentration of some growth phase points of definition can be made to migrate to higher concentration. This can be, for example, when concentration is reduced to more than the mid-exponential point from the saturation point, or when being reduced to the scope of the exponential late stage from the saturation point. The mid-exponential point and the saturation point can be made to migrate towards higher concentration. For example, in a non-restrictive manner and only for the purpose of illustrating this point, it has been found that, for example, if the mid-exponential growth phase point in a circulation is 4.0g / L, and the stationary phase of this circulation begins at 7.5g / L, and concentration variation is little, such as the biomass concentration reduced is 6.5g / L, so this can provide a higher mid-exponential point (such as 4.1g / L) in subsequent circulation, and begins at the stationary phase (such as 7.6g / L) of higher concentration. The use of short growth step durations (small concentration changes) in subsequent cycles can achieve further increases in concentration at these growth points in yet another subsequent cycle. When the concentration is reduced at least in part by biomass removal, this allows for the removal of larger amounts of biomass from the same total volume of biomass and aqueous medium within the same time period. This allows for the production of more biomass and feedstock in successive cycles. At least for microalgae, it is believed that smaller changes in biomass concentration promote the adaptation of the microalgae to conditions of increased concentration.

[0098] In the method described herein, provide the biomass concentration that reduces at least in the second and subsequent circulation and can be greater than the biomass concentration of the reduction of the previous circulation.This also can provide the biomass concentration at the exponential mid-term point and be greater than the biomass concentration at the exponential mid-term point of the previous circulation.This also can provide the biomass concentration at the saturation point and be greater than the biomass concentration at the saturation point of the previous circulation.In a preferred embodiment, the biomass concentration that reduces corresponds to the concentration of about or above the exponential mid-term point of the previous circulation, preferably about the point that the exponential mid-term ends and the exponential late period begins, and preferably in the scope of the exponential late period.Preferably, the duration of the growth step makes biomass concentration reach about or above the saturation point of the previous circulation.The benefit of doing this is, in continuous circulation, biomass concentration can be made to fluctuate in the exponential late period (i.e. the stage with the maximum growth rate), and further make this stage migrate towards higher concentration, make the production efficiency and the CO2 sequestration capacity of the phototrophic organism maximized.

[0099] Thus, in preferred embodiments, in particular with microalgae, at least in the second and subsequent cycles, the duration of the growing step may be from about 15 minutes to 15 hours, preferably from about 30 minutes to 10 hours or from 30 minutes to 5 hours, and preferably from about 30 minutes to 3 hours or from 30 minutes to 2 hours and from 1 hour to 2 hours.

[0100] Similarly, in preferred embodiments, especially with microalgae, at least in the second and subsequent cycles, the concentration may vary from about 0.5 g / L to 5.0 g / L, preferably from about 0.5 g / L to 4.5 g / L, preferably from about 1.0 g / L to 4.0 g / L, preferably from about 1.5 g / L to 3.5 g / L, and preferably from about 2.0 g / L to 3.0 g / L. The concentration variation may be even smaller, for example, less than 1.0 g / L, for example, from about 0.001 g / L to 1.0 g / L, preferably from about 0.005 g / L to 0.8 g / L, preferably from about 0.01 g / L to 0.6 g / L, preferably from about 0.015 g / L to 0.4 g / L, and preferably from about 0.1 g / L to 0.3 g / L, for a growth step duration of about 30 minutes to 2 hours.

[0101] Similarly, in preferred embodiments, in particular with the use of microalgae, and when the concentration of phototrophic organisms in the aqueous culture medium is reduced by removing biomass, at least in the second and subsequent cycles, an amount of about 0.1% to about 25%, preferably about 0.5% to about 20%, preferably about 15% to about 1% can be removed.

[0102] Similarly, in other words, the concentration change can be from about 0.1% to about 25%, preferably from about 0.5% to about 20%, and more preferably from about 15% to about 1%. For example, if the concentration of phototrophic organisms in the aqueous medium immediately before removal is 7.5 g / L and the reduced biomass concentration is 7.0 g / L, this constitutes a concentration change of 7%. Similarly, if the concentration of phototrophic organisms in the aqueous medium immediately before removal is 7.5 g / L and the reduced biomass concentration is 7.3 g / L, this constitutes a concentration change of 3%, and so on.

[0103] When reducing the biomass concentration involves removing biomass and aqueous culture medium, a reduced concentration above the previous cycle can be achieved by adding a smaller dilution volume of aqueous culture medium than was added in the previous cycle.

[0104] For continuous culture, the number of times of circulation is essentially unlimited, and circulation can be expressed according to the total duration of cultivation or circulation. The duration of circulation can be at least about 12 hours, 1 day, 2 days, 5 days, 7 days or 10 days. The duration of circulation can be the time of reaching several weeks. For example, the duration of circulation can be at most about 40 days, 35 days or 30 days. Expressed in terms of range, the duration of circulation can be about 12 hours to 40 days, preferably about 5 days to 35 days, and preferably about 10 days to 30 days.

[0105] Continuous removal can be by removing biomass alone or removing together with aqueous culture medium.When removing biomass alone, this is used to reduce the concentration of remaining biomass, to promote the sustained growth of phototrophic organisms.When biomass is removed together with aqueous culture medium, the culture medium of dilution volume can be added to the remaining biomass to reduce its concentration.For example, when biomass and aqueous culture medium are removed with the same or higher concentration before removal, then the aqueous culture of dilution volume is added to the remaining biomass to reduce its concentration.Removing the biomass in the aqueous culture medium and adding the aqueous culture medium of dilution volume can be continuous.This can be realized using a flow velocity suitable for removal and addition.Conveniently, the flow velocity for removal and addition can be calibrated according to the biomass growth rate in the exponential growth phase or the exponential growth late stage.For example, the flow velocity for biomass removal can be set to the growth amount of the biological mass removed and the biomass; That is, according to growth rate.For example, for the biomass growth rate of 1.0g / L / day, the flow velocity for removing biomass can be set to remove the biomass of 1.0g / L / day. In some embodiments, the flow velocity that is used to add the aqueous culture medium dilution volume can be set to the removal rate that mates biomass and aqueous culture medium are combined.For the same reason, the flow velocity that is used to add the aqueous culture medium dilution volume can be set to the removal rate that mates biomass and aqueous culture medium are combined.For example, for the removal rate of 2.0g / L / days of biomass and aqueous culture medium combination, the flow velocity that is used to add the aqueous culture medium dilution volume can be set to add 2.0g / L / days.Can monitor and regulate flow velocity as required.Be used to provide the device of continuous flow and setting flow velocity to be known in the art.Example comprises the use and the siphon of container overflow device, pump.

[0106] Continuous culture has the advantage of providing a continuous supply of feedstock for downstream processes, which, combined with the advantage of rapidly regenerating biomass, provides increased capacity for these downstream processes.

[0107] The aqueous medium can be recycled continuously, but preferably only to the point where the recycled aqueous medium continues to promote growth. The phototrophic organism in the culture can produce byproducts, which accumulate in the aqueous medium and can prevent growth at the same rate as in fresh aqueous medium. For example, microalgae byproducts may include dissolved organic matter, such as extracellular polymeric substances. When the aqueous medium was not recyclable, it could be replaced with fresh aqueous medium.

[0108] Gas atmosphere

[0109] The gas atmosphere may contain gases other than carbon dioxide and may contain other substances such as particulates. The other gases may be one or more gases commonly found in air, such as oxygen, nitrogen, water vapor, hydrogen, methane, and inert gases. The other gases may also be one or more gases commonly found in exhaust streams, such as one or more gases commonly found in air, and gases produced by the combustion of hydrocarbons, such as carbon monoxide, nitrogen oxides (NOx), and sulfur oxides (SOx).

[0110] The gaseous atmosphere can be provided by a single source or multiple sources (i.e., two or more). For example, the gaseous atmosphere can be provided by a single source, such as the 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 gaseous atmosphere source can be determined by the concentration of carbon dioxide to which the phototrophic organism adapted to CO2 is adapted. For example, when using a phototrophic organism adapted to CO2 (such as 8% CO2), the gaseous atmosphere provided using a purified CO2 source (which can contain about 95% or more CO2) can be diluted with a dilution gas source (such as air) containing relatively less or no CO2 to provide a gaseous atmosphere comprising about 8% CO2. Similarly, when using a phototrophic organism adapted to CO2, such as 8% CO2, the gaseous atmosphere provided using a waste gas stream containing about 8% CO2 can be provided as a single source of waste gas stream. In some embodiments, the gaseous atmosphere provided using the waste gas stream needs to dilute the CO2 concentration. In this case, it is preferred to provide a dilution gas source. Preferably, the dilution gas source is air. The diluent gas source may also be recycled from the gas atmosphere in which the phototrophic organisms are cultured in the aqueous medium.

[0111] Biomass uses

[0112] The biomass obtained from the growth of phototrophic organisms is used as an organic raw material in many downstream processes. In the presence of water and carbon dioxide, phototrophic organisms use light as the main energy source for metabolism and produce energy in the form of carbohydrates through photosynthesis. Therefore, organic raw materials include simple and complex carbohydrates, such as simple and complex sugars, and biopolymers, such as exopolysaccharides. Therefore, organic raw materials can be used as a nutrient source for downstream applications, for example, in a biodegradation step.

[0113] Exemplary biodecomposition steps that may utilize phototrophic biomass are described in related applications entitled “Methods and Systems for Generating Hydrogen” (PCT Application No. PCT / AU2020 / 050285) and “Biomass Production” (PCT Application No. PCT / AU2022 / 051348), the entire disclosures of each of which are incorporated herein by reference in their entirety.

[0114] Storing CO2

[0115] In addition to producing biomass, the methods described herein can be used to sequester carbon dioxide from a gaseous atmosphere. This offers the advantage of not only producing useful downstream products, but in doing so, the carbon dioxide is captured and converted into useful downstream products.

[0116] In some embodiments, carbon dioxide can be captured from the air.

[0117] Providing a gas atmosphere containing a higher concentration of carbon dioxide than atmospheric CO2 can be used to sequester CO2, resulting in less CO2 in the atmosphere than would otherwise be present. However, in the cultivation of phototrophic organisms, particularly microalgae, high concentrations of CO2 can cause stress to organisms adapted to atmospheric CO2 concentrations, leading to reduced growth. It is for this reason that it is preferred that the phototrophic organisms be CO2-adapted, as described herein, in order to effectively utilize the CO2 sequestration capacity of the phototrophic organisms in the methods described.

[0118] As described above, in preferred embodiments, the phototrophic organisms are adapted to a gas atmosphere containing a carbon dioxide concentration of at least about 5%, preferably at least about 8%, 9%, or 10%, which concentration can range from about 5% to 25%, preferably from about 8% to 25%. Thus, the phototrophic organisms can be adapted to a gas atmosphere containing carbon dioxide at concentrations similar to those found in gas sources from industrial processes, thereby allowing the gas sources used in the method to be derived from industrial processes that generate gas streams containing these carbon dioxide levels. Thus, the method can help reduce the amount of carbon dioxide that these industrial processes may emit into the atmosphere.

[0119] In a preferred embodiment, a gas atmosphere is provided in which the carbon dioxide concentration is similar to the concentration to which the phototrophic organisms are adapted. In other words, in a preferred embodiment, the phototrophic organisms are adapted to the carbon dioxide concentration in the provided gas atmosphere. For example, for CO₂-adapted phototrophs adapted to 8% carbon dioxide or 9%, 10%, 15%, or 20% carbon dioxide, a gas atmosphere is preferably provided in which the carbon dioxide concentration is approximately 8%, 9%, 10%, 15%, or 20%, respectively. In other words, for a provided gas atmosphere with a carbon dioxide concentration of approximately 8%, 9%, 10%, 15%, or 20%, respectively, the CO₂-adapted phototrophs are adapted to 8% carbon dioxide or 9%, 10%, 15%, or 20%, respectively. Carbon dioxide contents of approximately 8% to 25% are commonly found in waste streams from various industrial processes. Therefore, in a preferred embodiment, the source of carbon dioxide comprises a waste gas stream, preferably a waste gas stream generated by hydrocarbon combustion, steam reforming, and / or biodegradation.

[0120] The exhaust gas stream may be filtered to remove particulate matter and / or treated before being applied to the gas atmosphere.

[0121] Optimal culture condition parameters

[0122] Optimal parameters for the growth of phototrophic organisms in a cultivation method have been developed. The described parameters, whether used alone or in combination, help promote faster growth of phototrophic organisms, particularly microalgae.

[0123] Because the phototrophic organism can consume carbon dioxide, the carbon dioxide in the gaseous atmosphere can be replenished to maintain a certain concentration in the gaseous atmosphere. This can be easily achieved using a gas stream. In some embodiments, the flow velocity of the gas stream used is about 0.05 volume / volume per minute (VVM) to about 5VVM, preferably about 0.1VVM to about 2VVM, preferably about 0.1VVM to about 1.0VVM, preferably about 0.1VVM to about 0.5VVM, preferably about 0.1VVM to 0.3VVM, preferably about 0.1VVM to 0.2VVM, and more preferably about 0.15VVM.

[0124] Generally, a culture of a phototrophic organism that has grown through the lag phase and entered the exponential phase is initially present as an inoculum. In a preferred embodiment, a culture containing about 10 6 cells / mL to about 10 10 cells / mL, more preferably about 10 7 cells / mL to about 10 9 cells / mL, and most preferably about 10 8 An inoculum of phototrophic organisms of 1,000 cells / mL is preferably prepared as a cell suspension in an aqueous culture medium.

[0125] In a preferred embodiment, the cultivation method is carried out in a bioreactor having a capacity of at least 1.11 times (i.e., 111%), preferably 1.17 times (i.e., 117%), the volume of the phototrophic organism and the aqueous medium. For example, for a working volume of 900 mL, the capacity of the bioreactor will preferably be at least 1000 mL, preferably at least 1053 mL, respectively. Preferably, the cultivation method is carried out in a bioreactor having a capacity of no more than 2.85 times (i.e., 285%), preferably 2.50 times (i.e., 250%), the volume of the phototrophic organism and the aqueous medium. For example, for a working volume of 900 mL, the capacity of the bioreactor will preferably be no more than about 2565 mL, preferably no more than about 2250 mL, respectively. Alternatively or additionally, preferably, the cultivation method is carried out in a bioreactor having a capacity of about 1.11 to 2.85 times, preferably about 1.17 to 2.50 times, preferably about 1.42 times (i.e. 140%) to 2.00 times (i.e. 200%), preferably about 1.67 times (i.e. 167%) the volume of the phototrophic organism and the aqueous culture medium.

[0126] In other words, in a preferred embodiment, the cultivation method is carried out in a bioreactor, wherein the volume of the phototrophic organisms and the aqueous medium is at least about 35%, and preferably at least about 40%, of the bioreactor capacity. For example, for a bioreactor capacity of 1000 mL, the volume of the phototrophic organisms and the aqueous medium will preferably be at least about 350 mL, and preferably at least about 400 mL. Preferably, the cultivation method is carried out in a bioreactor, wherein the volume of the phototrophic organisms and the aqueous medium is at most about 90%, and preferably at most about 85%, of the bioreactor capacity. For example, for a bioreactor capacity of 1000 mL, the volume of the phototrophic organisms and the aqueous medium will preferably be at most about 900 mL, and preferably at most about 850 mL. Alternatively or additionally, preferably, the cultivation method is carried out in a bioreactor, wherein the volume of the phototrophic organisms and the aqueous medium is between about 35% and 90%, preferably between about 40% and 85%, preferably between about 50% and 70%, and preferably about 60% of the bioreactor capacity. These levels provide a balance between the headspace of the gaseous environment and the volume of the phototrophic organism and aqueous medium for efficient gas exchange between the gaseous atmosphere and the aqueous medium to promote carbon dioxide consumption and growth of the organism.

[0127] The bioreactor can, and preferably is, equipped with a mixing device, such as an impeller, to agitate the aqueous culture medium, thereby aerating the culture, which can further aid in gas exchange. This also helps ensure uniform distribution of light exposure across the phototrophic organism culture. This is particularly applicable when using a low flow rate gas atmosphere, for example, less than about 1.0 VVM.

[0128] Phototrophic organisms, including microalgae, typically thrive under neutral or alkaline conditions. Therefore, in preferred embodiments, the pH of the aqueous medium is maintained at about 5 to about 9, preferably about 6 to about 8, and more preferably about 6.5 to about 7.5, with a pH of about 7 being most preferred. Carbon dioxide that may be dissolved or become dissolved in the aqueous medium can help lower the pH, while consumption of carbon dioxide by the organism can help increase the pH. Additives can be used from time to time as needed to maintain pH; for example, alkaline substances to increase pH and acidic substances to lower pH. Suitable substances are known to those skilled in the art.

[0129] In a preferred embodiment, the temperature is maintained at about 25°C to about 31°C, preferably about 27°C to about 29°C, and more preferably about 28°C.

[0130] Preferably, the aqueous culture medium comprises nutrients. Preferably, the nutrients are selected from one or more, preferably two or three or more, and preferably all of the following group consisting of: boric acid, calcium chloride, cobalt nitrate, copper sulfate, EDTA disodium salt, magnesium sulfate, manganese chloride, potassium phosphate, sodium molybdate, sodium nitrate and zinc sulfate. Aqueous culture media containing these nutrients are known to those skilled in the art or can be prepared by those skilled in the art. In a preferred embodiment, the aqueous culture medium is selected from one or both of Bold Basal medium (BBM) or Blue-Green 11 medium (BG-11), preferably BG-11.

[0131] In a preferred embodiment, the intensity of the light is from about 3000 lux to about 7000 lux, preferably from about 4000 lux to about 6000 lux, and preferably from about 4500 lux to about 5500 lux, or preferably about 5000 lux. Light sources including LED lamps capable of providing these light intensities are known to those skilled in the art.

[0132] Because phototrophic organisms use light to produce energy, the photoperiod (i.e., the duration of light exposure of a phototrophic organism within a 24-hour period) is preferably extended compared to what phototropic organisms typically experience in their natural environment. However, excessively long photoperiods may not promote growth as well as balanced light and dark cycles. Thus, in preferred embodiments, the photoperiod is from about 12 hours to about 20 hours, preferably from about 14 hours to about 18 hours, more preferably from about 15 hours to about 17 hours, and most preferably about 16 hours.

[0133] Among these preferred culture conditions, the preferred combination of light intensity and photoperiod is particularly effective in promoting the growth of larger phototrophic organisms, especially microalgae.

[0134] Thus, the present disclosure provides a method for culturing a phototrophic organism, the method comprising culturing the phototrophic organism in an aqueous culture medium under culture conditions comprising light and a gas atmosphere comprising carbon dioxide, wherein the light has a light intensity of about 3000 lux to about 7000 lux, and wherein the culture conditions further comprise a photoperiod of about 12 hours to about 20 hours. The preferred integers as defined herein also apply to the method, including the use of microalgae, the culture conditions, and the method as a continuous culture method.

[0135] Cultivation equipment and systems

[0136] Apparatus and systems useful for performing the cultivation methods of the present disclosure are described in related applications entitled “Methods and Systems for Generating Hydrogen” (PCT Application No. PCT / AU2020 / 050285) and “Biomass Production” (PCT Application No. PCT / AU2022 / 051348), the entire disclosure of each of which is incorporated herein by reference in its entirety.

[0137] The basic system can include a bioreactor suitable for cultivating phototrophic organisms. The reactor can be in a closed system that is communicated with a gas stream that comprises carbon dioxide and air. The gas stream can be a waste gas stream from an industrial process, such as flue gas. The bioreactor can be in liquid communication with one or more other reactors and / or storage containers. The bioreactor or each bioreactor can include many sensors, including pH sensors, temperature sensors, reactor content sensors, and sensors that monitor biomass production, preferably with regulators and controllers to maintain the parameters of each monitoring.

[0138] Example

[0139] Various embodiments are described herein with reference to the following non-limiting examples.

[0140] The experiments in the examples were conducted using the Chlorella vulgaris CS-41 strain, obtained from the Australian National Culture Collection of Algae (CSIRO, Hobart, Tasmania, Australia). To initiate the experiment, a predetermined volume of algae at a known concentration was added to a sealed container containing sterile culture medium. Triplicate experiments were performed. Once parameters were optimized, for example, achieving optimal biomass production rate, these optimized culture conditions were maintained for future experiments.

[0141] Initial culture treatment

[0142] Initially, the Chlorella CS-41 stock strain was revived from -80°C and re-cultivated in BG-11 medium in a 1 L Erlenmeyer flask with a working volume of 400 mL at 28°C. The culture was exposed to an LED light source to provide an irradiance of approximately 4000 lux until reaching a steady state under a concentration of 10% (v / v) CO2 and 90% air.

[0143] Once the cultures were adapted to the culture conditions, the inocula used for the experiments described in the examples were prepared aseptically at predetermined volumes at known concentrations. The initial culture conditions used in the experiments of the examples (unless otherwise stated) are given in Table 1.

[0144] Table 1. Initial culture conditions.

[0145]

[0146] a Blue-green (BG) medium 11

[0147] b VVM, volumetric flow rate of gas per unit volume of culture medium.

[0148] A number of these conditions were explored and refined as described below.

[0149] General analytical methods

[0150] The readout parameters evaluated in the experiments were: pH, cell concentration, biomass weight, and CO2 uptake. The methods for evaluating each parameter are summarized below. As used in these experiments, biomass refers to the produced Chlorella vulgaris. Therefore, the biomass in these examples illustrates the production of organic feedstock.

[0151] As described below, biomass growth assessment is carried out every day.Use UV-Vis spectrophotometer to measure the optical density (OD) of all diluted sediments at 683nm wavelength.Diluted sediment is transferred to 3 × 1.5mL Eppendorf tubes respectively, and centrifuged 10 minutes with 4700rpm.Discard supernatant, the sediment in Eppendorf tube is dried overnight at 50 ℃.Draw the curve of the absorption spectrum of biomass and dry cell weight (DCW), to obtain the biomass standard curve of each batch.

[0152] At the end of the 7-day culture period, the microalgae culture was counted. The microalgae culture was poured into 3 x 50 mL centrifuge tubes and centrifuged at 4700 rpm for 10 minutes to separate the supernatant and precipitate. Serial dilutions were performed on the precipitate, which was resuspended in known volumes of deionized distilled water. Cells present in the appropriate dilutions were counted using a Neubauer chamber assist device under a light microscope at 400x magnification.

[0153] Elemental analyzer is used to analyze the content of carbon, hydrogen and nitrogen in biomass.At the beginning and end of cultivation cycle, take out the culture of known volume, harvest biomass by centrifugation and analyze carbon, hydrogen and nitrogen content.Use inductively coupled plasma optical emission spectroscopy (ICP-OES) to analyze the elements present in the liquid culture medium separated after centrifugation.

[0154] The pH of the culture was analyzed daily using a pH meter. The pH of the culture was maintained within the specified range (7.0 ± 0.5) using 0.1 M NaOH. Figure 1 As shown, two CO2 sensors (model GMP251) using Vaisala Insight PC software were installed in the inlet and outlet gas lines to record the CO2 concentration every 5 seconds throughout the cultivation cycle. The changes in CO2 concentration recorded by these sensors were also used to determine the mass balance and calculate the biological fixation of CO2.

[0155] Example 1. Adaptive laboratory evolution

[0156] Through artificial stepwise adaptive laboratory evolution (ALE) process, the tolerance of Chlorella CS-41 to high concentration of CO2 was gradually enhanced. Figure 1 Microalgae are cultivated in a bioreactor system schematically shown. The reactor is placed in a light chamber and surrounded by an LED light source. Pure CO2 and air are supplied, and flow control valves are used to control the flow rates to achieve a specific CO2 concentration in the gas inlet line. CO2 sensors are placed at the gas inlet and outlet lines to continuously measure CO2 concentration. In addition to varying the CO2 concentration, the culture was grown under the conditions described in Table 1.

[0157] Increased CO2 tolerance in Chlorella CS-41 was initiated by exposing the microalgae culture to ambient air containing 0.03% CO2 by volume for 7 days. A known concentration of the adapted algae culture was used as inoculum for the ALE process. A 1000 mL bioreactor with a 400 mL working volume was used for ALE of the Chlorella strain. Following the 7-day culture period, the algae was then exposed to a 3% CO2 flow by volume. Successive culture cycles were then repeated with the CO2 concentration gradually increased to 10% for subsequent generations.

[0158] Biomass was analyzed as described in the General Analytical Methods section above. Biomass yield and specific growth rate (Table 2) were calculated as shown in Equations 1 and 2 below:

[0159] Equation 1.

[0160]

[0161] Among them C bio Express the biomass concentration in the culture medium in g / L.

[0162] Equation 2.

[0163]

[0164] The specific growth rate represents the growth rate after a constant biomass concentration is reached.

[0165] Table 2 summarizes the biomass growth data obtained from each cultivation cycle at different CO2 concentrations. At least two cultivation cycles were performed at 8% and 10% to evaluate the performance of different generations (G).

[0166] Table 2. CS-41 growth under different CO2 concentrations (BBM, 12:12 light:dark cycle)

[0167]

[0168] Under low CO2 concentrations (3% and 5%), the cultures evolved adaptively in the first generation until the biomass concentration increased on day 5. However, biomass productivity and specific growth rate decreased when the cultures were grown in the presence of 3% CO2 to 5% CO2. This was likely due to slower cell division on days 6 and 7 compared to the first 5 days of the culture cycle.

[0169] It was observed that biomass productivity decreased after a continuous increase in CO₂ concentration from 3% to 8%. This may indicate that the cells were initially stressed but were able to slowly adapt over subsequent generations. This was evident because the biomass productivity of 2G at 8% exceeded that of 1G at 5%. A similar pattern of higher biomass productivity over successive generations was observed at 10% CO₂, where the culture cycle continued for five generations.

[0170] Figure 2 Compare the biomass concentration, pH change and growth rate of 10% 4th generation and 10% 5th generation.In this experimental work, each cultivation cycle is repeated three times, and the variation of error bar represents the variation of single test result under identical operating conditions.In 10%4G and 10%5G, almost no daily variation of biomass concentration and pH value is observed.In addition, average biomass productivity value is respectively 0.147g / L / day and 0.148g / L / day, and specific growth rate is respectively 0.606 and 0.607.Because biomass productivity value is roughly constant in two consecutive operations, the culture obtained by cultivating 5 generations under 10%CO2 concentration is considered to be adapted to suitable CO2 volume %.

[0171] At the end of the 7-day period of the 10% 5G run, the biomass was analyzed for carbon, hydrogen, and nitrogen content using an elemental analyzer as described in the General Analytical Methods section. The results of this analysis are given in Figure 3 Shown in.

[0172] The carbon content of the total biomass was used to calculate the biological fixation rate of CO2 during the cultivation cycle using the following equation (Equation 3).

[0173] Equation 3:

[0174] Biological fixation rate of CO2 (g / L / day) =

[0175]

[0176] where X C represents the carbon mass fraction in the biomass sample, and MW represents the molecular weight in g / mol.

[0177] Figure 3 (b) shows that for the 4G and 5G generations grown in the presence of 10% CO2, the calculated carbon and CO2 biofixation rates are the same (0.2645 g / L / day CO2). The CO2 biofixation rate was calculated based on the CO2 sensor data. Figure 3The traces in (c) are shown. The CO2 concentrations in the inlet and outlet gas streams are plotted for the last 4 days of a 10% 5G culture cycle. The large square peak indicates a 12-hour photoperiod, however some fluctuations in the photoperiod are reported due to sudden fluctuations in the upstream gas line. Based on the gas stream concentration data, the biological fixation rate of CO2 was calculated to be 0.269 g / L / day. These two values, one calculated from biomass analysis and the other from CO2 sensor data, are consistent with a difference of ~1.7%, validating the elemental analysis method. In the experiments below, elemental analysis was used to calculate the biological fixation rate of CO2 during the process.

[0178] Example 2. Comparison of culture media

[0179] Two media types, BG-11 and BBM, were evaluated for optimal microalgae growth performance using the Chlorella vulgaris strain CS-41. The experimental outputs used to evaluate media performance were biomass yield and CO₂ fixation. The compositions of BG-11 and BBM are listed in Table 3.

[0180] Table 3. Composition of BBM and BG-11 culture media in 1 L of deionized distilled water

[0181]

[0182]

[0183] Biomass yield was quantified by separating the phototrophic source material from the liquid culture by vacuum filtration using filter paper (GF / C: 1.2), drying in a hot air oven at 30°C-35°C, and determining the weight of the biomass. Biomass was also measured spectrophotometrically using standard optical density techniques and compared to an earlier growth curve of strain CS-41, which has established growth characteristics. The culture conditions used for this experiment are shown in Table 1.

[0184] Total CO2 biofixation was measured on day 0 and day 7. To measure CO2 content, gas samples were collected from the reactor used for the photosynthesis step. The samples were collected in gas collection bags and analyzed for their composition by GC-MS (Agilent 7890B GC) equipped with a thermal conductivity detector (TCD). As carrier gas, argon / helium was used at a flow rate of 15 mL min -1 The temperatures of the oven, detector, and injector were 190°C, 210°C, and 160°C, respectively.

[0185] The results of the 7-day culture medium comparison experiment are shown in Table 4. Chlorella CS-41 was able to grow and fix CO2 in both culture medium types. BG-11 supported higher levels of biomass production and CO2 fixation than BBM. In this example, CS-41 biofixed 1.029 g / L / day of CO2 and 0.593 g / L / day of CO2 when grown in BG-11 or BBM culture medium, respectively.

[0186]

[0187] Example 3. Optimization of light intensity

[0188] The optimal light intensity for biomass production and CO2 fixation of Chlorella strain CS-41 was determined. Except for the light intensity and photoperiod (16 hours light: 8 hours dark), all other culture conditions were as listed in Example 1 and Table 1.

[0189] Growth curve analysis based on spectrophotometric measurement of optical density demonstrated the effect of light intensity on the growth lag phase. Figure 4 It was shown that optimizing the light intensity applied during the photosynthesis step can reduce the impact of the lag phase on growth rate and overall biomass production.

[0190] In separate experiments conducted under the same culture conditions, the light intensity range explored included 3000 lux to 5500 lux. The photobioreactor system was surrounded by an LED light source with controlled light intensity, which was Figure 1 The configuration is shown in the diagram in the figure. Figure 5 and Figure 6 As shown, from 3000 lux to 5000 lux, the biomass productivity and biofixation rate increased respectively. However, further increase in light intensity to 5500 lux resulted in a decrease in biomass productivity. The results showed that 5000 lux resulted in the highest CO2 biofixation rate (1.37 g / L / day) under all experimental conditions. The results of the light intensity optimization study showed that a light intensity of 5000 lux was effective for the biomass yield ( Figure 5 ), CO2 fixation rate ( Figure 6 ) and total carbon capture ( Figure 7 ) is the best.

[0191] Example 4. Optimization of photoperiod

[0192] In this study, five sets of photoperiods (12:12; 16:8; 18:6; 20:4 and 24:0 hours light / dark) were tried to optimize the light exposure time of microalgae Chlorella vulgaris CS-41 at 4000 lux in each culture medium (BG-11 and BBM). All other culture conditions were the same as those described in Example 1 and Table 1.

[0193] like Figure 8 As shown, in all photoperiod groups tested, microalgae grown in BG-11 medium exhibited higher biomass productivity than cultures grown in BBM medium. However, BG-11 and BBM accumulated the least biomass in 12:12 hour and 24 hour light / dark photoperiods, respectively, accounting for 1.866 g / L and 0.389 g / L. This finding reveals that photoperiod is not only species-specific, but is also affected by the culture medium used. In addition, it is clear that higher photoperiods, such as 18:6; 20:4 and 24 hours of light / darkness, have a greater adverse effect on the biomass productivity of Chlorella in BBM than in BG-11, suggesting that Chlorella can tolerate photooxidative stress more effectively in BG-11 than in BBM ( Figure 8 However, both media accumulated the most biomass under a 16:8 h light / dark photoperiod, with BG-11 and BBM producing 3.894 g / L and 2.321 g / L, respectively.

[0194] Example 5. Optimization of culture pH

[0195] In this example, Chlorella CS-41 was grown in culture media adjusted to different pH levels. This experiment was conducted to select the optimal pH of the culture media to achieve the highest biomass productivity during a 7-day culture period under a fixed photoperiod (16:8 hours) and light intensity (5000 lux). Except for the photoperiod, light intensity, and pH, all culture conditions were 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. pH was monitored and adjusted daily.

[0196] Figure 9 The results reported in show that the most biomass was obtained at pH 7, as determined by dry weight measurements, in both BG-11 and BBM medium. However, when compared to cultures grown in BBM, BG-11 showed higher biomass production overall, except at pH 9, where BBM-grown cultures accumulated more biomass than cultures produced in BG-11. This may indicate that BBM has a greater buffering effect on alkaline solutions than BG-11. The CO2 fixation rate and total CO2 captured were measured at each tested pH on day 7, with each measurement reported in Table 1. Figure 10 and Figure 11 Shown in.

[0197] Based on the data generated in this example, pH 7 was identified as the optimal pH for Chlorella culture conditions, providing maximum biomass productivity and CO2 fixation, and the flexibility of the culture medium allowed each culture medium tested (BBM and BG-11) to perform best at pH 7.

[0198] Example 6. Optimization of inoculation cell density

[0199] The number of cells present in the inoculum used to initiate the photosynthesis step of a Chlorella CS-41 culture will affect the growth rate and the resulting biomass. This experimental example was designed to evaluate a range of starting inocula (0.006 g / L, 0.015 g / L, 0.025 g / L, 0.050 g / L, and 0.133 g / L). In addition to the following culture conditions: BBM medium, 10% CO2, and a photoperiod of 16:8 (light:dark), other culture conditions were as described in Example 1, Table 1. The experimental outputs measured to determine the optimal inoculum size were biomass productivity, CO2 fixation rate, and total CO2 captured. These parameters were analyzed as described in the General Analytical Methods section above. The results of this study are summarized in Table 5.

[0200] Table 5. Effect of inoculum size on biomass growth rate (BBM medium, 10% CO2, 16:8 light: dark cycle)

[0201]

[0202]

[0203] In general, before adaptation is achieved, higher inoculum sizes are more tolerant to environmental stress. Environmental stress can prevent some cell populations from successfully undergoing cell division. Therefore, when exposed to environmental stress, the cell number in the inoculum may be more likely to survive under stress conditions compared to a smaller inoculum size. However, the higher inoculum sizes (0.025 to 0.133 g / L) of the Chlorella strains observed to be adapted showed a lower growth rate than the smaller inoculum size (0.015 g / L), indicating that the Chlorella strain CS-41, which has undergone adaptation to CO2 higher than atmospheric levels, is fully adapted to high CO2 concentration pressure and thrives. Meanwhile, the higher inoculum sizes tested resulted in lower biomass productivity. This may be because, compared to a lower inoculum size, insufficient light penetration occurred earlier. The data presented in Table 5 show that the strains adapted and optimized (high CO2 tolerant cells) can divide quickly and produce the most biomass under 0.015 g / L inoculum size. The inoculation of CO2 was determined to be the best inoculum size of 0.652 g / L / day, and the culture lower than the inoculum size of the best inoculum size (0.006 g / L) showed lower growth rate (0.514 g / L / day). After optimum value (0.015 g / L), growth rate and biomass productivity decreased along with the increase of inoculum size. However, under very high inoculum size (0.133 g / L), although growth rate was still lower, compared to the smaller inoculum size 0.05 g / L next door, its biomass productivity was relatively higher. From the data presented in Table 5, it is clear that CO2 is fixed and has a positive correlation with biomass productivity, while growth rate and inoculum size are negatively correlated.

[0204] Example 7. Batch culture of Chlorella under optimized conditions (single cycle)

[0205] After optimizing the culture condition parameters as described in Examples 1-6, the optimized culture conditions are summarized in Table 6.

[0206] Table 6. Optimized culture conditions of Chlorella CS-41 in the photosynthesis step

[0207]

[0208] Cultures grown under pre-optimization conditions were compared to cultures grown under optimized conditions. The comparison results, summarized in Table 7, compared the following output criteria: biomass yield on day 7, biomass production rate, carbon fixed over 7 days, CO2 fixation rate, and total CO2 captured on the final day.

[0209] The cultivation method developed as a result of the optimization process resulted in the culture reaching higher levels of biomass in a shorter period of time as well as improved CO2 fixation within a 7-day cycle. Figure 12 Shown in.

[0210] Table 7. Comparison of culture performance and culture condition improvement before and after study

[0211]

[0212] The optimized batch culture system (BCS) conditions described in Examples 1-7 were used as a starting point for developing a continuous culture method for the photosynthesis step.

[0213] Example 8. Cultivation of microalgae cultures in recycled culture medium

[0214] In the current study, experiments were performed using fresh and spent BBM medium. Figure 13 presented in . It was found that fresh BBM medium produced 1.055g / L of biomass over a duration of 7 days, with a biomass productivity of 0.144g / L / day, while reused BBM produced 0.865g / L, with a biomass productivity of 0.116g / L / day. This infers that the biomass productivity dropped by about 19% in the second cycle of reusing the medium. In addition to the potential inhibitory effect of DOC, it is recognized that some nutrient components in the medium will be depleted after each growth cycle, thereby reducing the availability of nutrients in subsequent growth cycles. The biomass produced by Chlorella CS-41 when grown on recycled medium is considered acceptable for continued cultivation in a continuous circulation culture system.

[0215] Example 9. Continuous culture method

[0216] To develop a continuous circulation method for cultivating Chlorella CS-41 in a photobioreactor, a master seed stock of the microalgae stored at -80°C was revived using the optimized culture conditions listed in Table 6. Once the cell density reached 10 4 The culture was transferred to a larger volume as the working stock. The working stock was checked for contamination before use and was refreshed monthly from the original germplasm stock. All experimental batches were inoculated with 10 8 cells / mL of biomass from a working stock and incubated under the culture conditions listed in Table 6.

[0217] An example of a typical growth curve for Chlorella CS-41 during the optimization process is given in Figure 14 A lag phase can be observed between days 5 and 6, while exponential growth can be observed between days 6 and 7. The exponential growth phase is ideal for continuous production, especially if the culture can be reset daily until day 6 to allow the algal cells to double.

[0218] This experiment is designed to evaluate and compare two different continuous circulation (CC) methods of microalgae growth under laboratory scale.Two CC method experiments are carried out in parallel.The first culture cycle grows under the optimized growth conditions listed in Table 6.Two culture methods are described in Table 8.Method A (CCA) provides the basis for the continuous siphon process for developing liquid biomass, wherein the liquid biomass is siphoned once every two days, and new fresh culture medium is transported to the reactor (renewal).Method B (CCB) provides the basis for the continuous siphon process for developing solid biomass, and evaluates the use (recirculation) of the culture medium of recirculation.

[0219] In this example, two experiments were performed in parallel as described in Table 8. The starting biomass density of CCA was analyzed and calculated to be 2.803 g / L. The biomass used to start CCA and CCB was prepared from the same batch of circulating system (BCS) (cycle 0). The BCS biomass was flocculated using high concentration CO2 gas that effectively concentrated the biomass. The supernatant was siphoned out of the reactor and transferred to a separate container. The remaining biomass concentrate was divided into two equal parts. A portion was taken out from the reactor and used as a raw material for downstream applications, such as a biodegradation step for the production of biofuels and / or biofertilizers. The supernatant was returned to the original reactor and recycled as a culture medium for the remaining biomass portion. The starting biomass density of method CCB was recorded as 2.201 g / L. Schematic diagrams of the CCA (diverting biomass and renewing culture medium) and CCB (concentrating diverted biomass and recycling culture medium) methods are shown in Figure 15 Shown in.

[0220] Table 8. Description of two continuous circulation culture methods for microalgae growth.

[0221]

[0222]

[0223] The final daily growth rates of the preliminary CC methods CCA and CCB were 1.287 g / L / day and 1.4705 g / L / day, respectively, and the final densities were 5.377 g / L and 5.142 g / L, respectively. Figure 16 Compared to BCS, CCA and CCB showed significantly higher growth rates, reaching 0.750 g / L / day under pre-optimization conditions or 0.88 g / L / day under optimized culture conditions (Table 7). Both CC methods significantly outperformed BCS in terms of growth and carbon capture efficiency.

[0224] In addition to measuring the biomass yield obtained by the CCA and CCB methods, the CO2 capture rate (CCR) was analyzed using the methods listed in the General Analytical Methods section above and Example 1. The carbon capture rates of the CCA and CCB methods were compared with BCS. Using industry standard calculations to estimate the CO2 capture rate per g of biomass, the theoretical levels of biomass yield and CO2 capture that can be achieved in expanded culture volume and long-term production for each culture method tested, namely BCS, CCA, and CCB, are summarized in Table 9.

[0225] Methods to achieve economically sustainable CO2 sequestration at a commercial level, such as CC utilizing phototrophic sources during the photosynthesis step, are desirable to meet industrial needs and improve the environmental sustainability of industrial practices.

[0226] Table 9. Carbon capture efficiency of Chlorella CS-41 obtained under different culture systems and different culture methods

[0227]

[0228] The significant increase in CCR seen in continuous cultures is likely due to the fact that a larger proportion of the microalgae remains in the continuous culture system after each biomass diversion event. The remaining phototrophic source continues to fix CO2 into sugars, unlike the BCS method, where the entire biomass is harvested in a single event. Subsequent BCS batches will begin with the initial inoculum and require at least four days for the phototrophic source to enter an effective exponential growth phase. In contrast, the CC method does not require the initiation of a culture cycle from the initial inoculum, but instead immediately resets the biomass to an early exponential phase.

[0229] For example, from day 0 to day 4, the biomass concentration in the BCS was less than approximately 0.5 g / L / day, whereas at the equivalent time points in the CC culture, day 0 (day 7, cycle 1) and day 4 (day 11, cycle 1), the biomass level was at least 2.8 g / L / day. Thus, the CC operation had a higher amount of biomass-fixed CO2 (at any time) and doubled the amount of carbon captured by the BCS every six days ( Figure 17 Furthermore, the advantages of CC over BCS were further demonstrated during the expansion of culture scale and the extension of operation days. The theoretical increase in CC biomass production and CO2 capture capacity was calculated for the expansion of reactor scale and the extension of culture time, as shown in Table 10.

[0230] Table 10. Comparison of theoretical biomass yield and CO2 captured in batch and continuous culture systems during the photosynthesis step.

[0231]

[0232] Example 10. Industrial Implementation of the Photosynthesis Step

[0233] The overall mass balance under the optimal operating conditions (BG-11 medium, 0.015 g / L inoculum, 1 VVM gas flow rate, 16:8 light:dark cycle ratio, and 5000 lux light intensity) is summarized in Figure 18 (a). These optimal conditions resulted in a biofixation rate of 1.37 g CO2 / L / day. The biofixation rate of CO2 was assumed to remain constant throughout the culture, during which the inlet CO2 flow was 40 ml / min (equivalent to 0.0748 g / min, CO2 density = 1.87 kg / m 3 ), the corresponding outlet CO2 flow can be calculated to be 39.7965 ml / min (0.07442 g / min). This further infers a single-pass conversion of CO2 to biomass of approximately 0.50%. This conversion level is significantly lower than that achieved in industrial processes, where conversion rates range from 20% to 100%. Due to this low conversion rate, industrial-scale CO2 biofixation would require significant gas recycle.

[0234] Figure 18 (b) shows a flow chart for a simple gas flow recycling process, developed and simulated in Aspen-Hysys to calculate the mass balance of the gas flows in the process. In this flow chart, the photobioreactor is implemented as a conversion reactor, in which the conversion rate of CO2 to biomass is 0.50%. Here, it is assumed that the CO2 conversion rate achieved in the laboratory experiments will be achieved in a large-scale photobioreactor. The adjustment block (ADJ-2) in the flow chart is used to adjust the flow rate of fresh air to achieve 10% by volume of homogenized gas entering the reactor, and a purge gas flow is used to achieve the recycling of unreacted gases.

[0235] The simulation flowsheet was based on a process flow of 100 kg / day of fresh CO2 inlet (stream 1) and a 1% recycle gas purge (stream 6). The converged mass balances from the flowsheet simulation are summarized in Table 11. In the converged results, the differences between the inlet (streams 1 and 2) and outlet (streams 4 and 6) were less than 0.1%. Note that the volumetric flow rate of stream 3 is used to infer the required liquid holdup in the photobioreactor, as the experimental data for the current study were valid at a gas flow rate of 1 VVM. Therefore, the required liquid holdup for the simulation basis was 3.545 m 3 It is also worth noting that even at a low purge rate of 1%, the amount of CO2 emitted in the purge (stream 6) is 95.22 kg / day, while the biological fixation is 4.78 kg / day.

[0236] Table 11. Mass balance of microalgae cultivation process under gas recirculation

[0237]

[0238] Example 11. Evaluation of continuous culture method

[0239] The studies in this example were conducted to evaluate the reproducibility of a continuous harvesting process and to explore the effects of multiple harvesting cycles on algal biomass yield.

[0240] The photosynthesis step was carried out for a total of 40 days, including an initial 8-day acclimation period. Accumulated biomass and pH were measured regularly throughout the 40-day study, according to the methods described above. Biomass was harvested every 2 days, and algal cultures were grown according to the CCA method as described herein. The pH of the aqueous medium was maintained between 6 and 7 throughout the study.

[0241] Reproducibility

[0242] The amount of biomass accumulated throughout the study of three replicates is shown in Table 12.

[0243] Table 12. Accumulation of microalgal biomass when grown in continuous culture for 40 days

[0244]

[0245] Under the optimized culture conditions, the adaptation period was repeated to assess hourly biomass accumulation during the initial 7-day culture period. Hourly biomass measurements were performed in Figure 19 Each time point represents the mean of two data points collected from duplicate experiments.

[0246] Multiple harvest cycles

[0247] A further study was conducted that was designed to evaluate the effect of multiple harvest cycles on biomass yield during continuous cultivation. 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 , harvest event on day 15). Figure 20 Replicate studies (Rep 1 and Rep 2) performed over 18 days under optimized culture conditions are shown. That is, removing a portion of the microalgal biomass does not cause the total biomass of the microalgae to drop as low as the result of the previous harvest event, as shown in FIG. Figure 20As shown in Figure 2. As a result of gathering the crops event 1 and 2, biomass dropped to about 3.5g / L. After the third gathering the crops event on the 15th day, biomass dropped to about 4.7g / L. After each gathering in the three gathering the crops event, between each gathering the crops event and before the gathering the crops event subsequently, the maximum biomass of about 7.5g / L was reached. It is less to observe that the biomass concentration reduces, allowing the culture to obtain the maximum biomass in a shorter time, and the culture does not enter the stationary phase.

[0248] Harvest interval

[0249] At CO 2 flow rate is under the situation of 0.15VVM, use the continuous culture method (table 6) of optimization, explored the influence of the frequency of gathering the crops event on biomass yield.The purpose of this research is to compare different gathering the crops intervals, and monitor the stability of the biomass density that begins in case of gathering the crops.The batch that comprises in this research and the gathering the crops interval time of each processing are listed in table 13.For each processing, come to the crops part biomass by siphoning 5% working volume in each gathering the crops event. Figure 21 (A) and (B) show the biomass density (g / L) and total biomass harvested (g) over time, respectively. While a 30-minute harvest interval produced the highest total biomass yield, biomass density steadily decreased throughout the harvest period. Thus, a 2-hour harvest interval provided the best biomass density stability (higher than the 30-minute or 1-hour harvest interval treatments, see Figure 21 (A)). Similarly, when compared to the 3 hour and 4 hour harvest interval treatments, the 2 hour harvest interval treatment produced an increased total harvest, but with comparable biomass density. Balancing biomass density and yield stability indicated that a 2 hour harvest interval was optimal.

[0250] Table 13.

[0251]

[0252]

Claims

1. A method for cultivating a phototrophic organism, comprising the following steps: a) cultivating the phototrophic organism in an aqueous culture medium under culture conditions comprising light and a gas atmosphere comprising carbon dioxide; as well as b) when the phototrophic organism reaches a point between the exponential growth phase and the stationary growth phase, reducing the biomass concentration of the phototrophic organism in the aqueous culture medium to a reduced biomass concentration that is no less than the biomass concentration that existed during the initial 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 the genus Chlorella, preferably a microalgae of the species Chlorella, preferably selected from one or both of the strains CS-41 or CCAP211 / 11S of the species Chlorella, preferably CS-41.

3. The method according to claim 1 or 2, wherein the phototrophic organism is adapted to CO2, preferably to a gas atmosphere containing a carbon dioxide concentration of at least about 5% and preferably at least about 8%, said concentration being able to be a concentration of about 8% to 25%.

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

5. The method according to any one of claims 1 to 4, wherein reducing the biomass concentration comprises the following steps: A volume of phototrophic organism biomass is removed from the aqueous culture medium and a dilution volume of aqueous culture medium is added, optionally including a preliminary step of removing a volume of aqueous culture medium.

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

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

8. The method of any one of claims 1 to 7, wherein the reduced biomass concentration is a concentration present around or above the mid-exponential growth point.

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

10. The method of 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 recycling the reduced biomass concentration, wherein the recycling comprises repeating steps a) and b) using the reduced biomass concentration, and preferably repeating steps a) and b) multiple times using the reduced biomass concentration in step b) each time.

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

13. The method according to any one of claims 1 to 6, further comprising recycling the reduced biomass concentration, wherein the recycling comprises repeating steps a) and b) multiple times using the reduced biomass concentration in each step b), wherein the duration of step a) is short or the concentration variation of step b) is small.

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

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

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

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

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

19. The method of claim 18, wherein the continuous removal of the phototrophic organism biomass is accompanied by the removal of the aqueous culture medium and the dilution volume of the aqueous culture medium is continuously added to the remaining biomass.

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

21. The method according to any one of claims 1 to 20, wherein the gas atmosphere is provided with a carbon dioxide concentration higher than the concentration of carbon dioxide in air, preferably wherein the concentration of carbon dioxide is at least about 5%, preferably at least about 8%, said concentration being able to be in the range of about 8% to 25%.

22. The method according to any one of claims 1 to 21, wherein the intensity of the light is about 3000 lux to about 7000 lux, preferably about 4000 lux to about 6000 lux, preferably about 4500 lux to 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 about 12 hours to about 20 hours, preferably about 14 hours to about 18 hours, preferably about 15 hours to about 17 hours, or preferably about 16 hours per 24 hours.

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

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

26. The method according to any one of claims 1 to 25, wherein the aqueous culture medium comprises one or more nutrients, preferably wherein the nutrients are selected from one or more, preferably two or three or more, preferably all, of the following: boric acid, calcium chloride, cobalt nitrate, copper 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 culture medium is selected from one or both of BBM and BG-11, preferably BG-11.

28. A method for the continuous cultivation of phototrophic organisms comprising performing the method of claim 1 and recycling the reduced biomass concentration, wherein the recycling comprises repeating steps a) and b) using the reduced biomass concentration.

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

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

31. The method of any one of claims 1 to 30 for capturing carbon dioxide from a flue gas stream.

32. The method of any one of claims 1 to 30, which is used to produce an organic feedstock.

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

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

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

36. A CO2-adapted phototrophic organism, wherein the phototrophic organism is selected from one or both of strains CS-41 or CCAP211 / 11S of the species Chlorella, preferably CS-41, and wherein the phototrophic organism is adapted to CO2 in a gas atmosphere comprising a carbon dioxide concentration of 5% or greater.

37. The CO2 adapted phototrophic organism of claim 35, wherein the phototrophic organism is adapted to CO2 in a gas atmosphere containing a carbon dioxide concentration of at least about 8%, which concentration can be in a concentration of about 8% to 25%.

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