GB2615376A - Diazotroph carbon sequestration - Google Patents

Abstract

The invention relates to a method for sequestering carbon dioxide from the atmosphere, wherein the method comprises culturing diazotrophic phytoplankton and/or diatom-diazotroph assemblages (DDAs) in land-based mariculture under oligotrophic conditions. The diazotrophic phytoplankton may be cyanobacteria, Trichodesmium sp, Crocosphaera sp or Calothrix sp, preferably Trichodesmium erthyraerum. The DDAs may be Richelia sp, Calothrix sp, Crocosphaerea sp and Candidatus Atelocyanobacterium Thalassa, Hermialus sp, Skeletonema sp,Rhizosolenia sp, Chaetoceros sp and Climacodium sp. The oligotrophic conditions may comprise low concentrations of nitrogen and phosphorus (eg less than 10µM nitrogen and less than 20µM phosphorus and may further comprise low concentrations of silicates (less than 15µM silicate). The land-based mariculture may comprise one or more raceway ponds. The diazotrophic phytoplankton and/or diatom-diazotroph assemblages (DDAs) may be harvested and buried for carbon sequestration.

Description

DIAZOTROPH CARBON SEQUESTRATION All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to methods for efficient sequestration of carbon by culturing diazotrophic phytoplankton and/or diazotroph-diatom assemblages in land-based mariculture.

BACKGROUND

The current Intergovernmental Panel on Climate Change (IPCC) Synthesis Reports (AR6, UN) have demonstrated that it is difficult to attain the 1.5t or the 2.0t Paris Agreement targets through mitigation measures, or measures that reduce carbon emissions, alone. Demand for energy is increasing as developing and developed nations alike are increasing consumption of primary energy due to population increases and a continuous global increase in living standards. While the installation and utilisation of renewable energies is rapidly increasing, it is not at a rate that is sufficient to provide for both the increased demand for, and the replacement of, thermal energy production.

There is a need for anthropogenic CO2 removal technologies to remove CO2 from the atmosphere, and durably store it, are needed to compensate for residual emissions to reach net zero greenhouse gas emissions.

Previously, a marine phytoplankton cultivation method was designed to work in nutrient rich upwelling seawater locations with very rapidly growing 'bloom' forming organisms. However, there remains a pressing need to develop more cost-efficient methods for sequestering atmospheric CO2.

SUMMARY OF THE INVENTION

The inventors have devised a new method for carbon sequestration that involves culturing specialised organisms that have natural nutrient accumulation capabilities under oligotrophic conditions. These organisms fix many of their own nutrients, instead of consuming them from surface seawater. The inventors' method advantageously increases the mass culture of marine organisms for the purpose of low-cost carbon sequestration, without the negative externalities of land, energy, or freshwater use, at even lower cost than the previous application of these algal raceway ponds. It is an 'additive' technology that dramatically increases Net Primary Productivity (NPP) in large desert areas using abundantly available and underutilised natural inputs. "Additive" here means that this form of algal cultivation enables effective and efficient biomass growth at large scale, where it would not have otherwise grown, without displacing pre-existing primary productivity (or biological growth).

Unlike previous methods that rely on rapid serial dilution of bloom'-forming or 'r-strategist' organisms that thrive during periods of relative nutrient abundance, this invention relies on organisms that concentrate, accumulate, fix and transform nutrients in otherwise unavailable forms to grow in low-nutrient (marine desert) environments. Thus, the present invention focuses on a 'functional niche' approach, promoting resilient, self-sufficient organisms with specific energetic demands to reduce overall input costs.

The invention provides a method for sequestering carbon dioxide from the atmosphere, wherein the method comprises culturing diazotrophic phytoplankton and/or diatom-diazotroph assemblages (DDAs) in land-based mariculture under oligotrophic conditions.

In some embodiments, the diazotrophic phytoplankton are cyanobacteria, such as Trichodesmium sp. (e.g. Trichodesmium etythraeum).

In some embodiments, the DDAs comprise: (i) one or more of the following diazotrophs: Riche//a sp., Calothfix sp., Crocosphaera sp. and Candidatus Atelocynaobacterium thalassa, and/or 00 one or more of the following diatoms: Hemiaulus sp., Skeletonema sp., Rhizosolenia sp., Chaetoceros sp., and Climacodium sp. For example, in some embodiments, the DDAs comprise an assemblage of Richelia sp. and Hemiaulas sp.

In some embodiments, the oligotrophic conditions comprise low concentrations of bioavailable nitrogen and phosphorus. In some embodiments, the oligotrophic conditions further comprise low concentrations of silicate.

In some embodiments, the method further comprises harvesting the diazotrophic phytoplankton and/or DDAs.

The invention further provides diazotrophic phytoplankton and/or diatom-diazotroph assemblages (DDAs) obtained by a method of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: A raceway pond with two channels. Features include an inlet pipe, a paddle wheel, flow diverters, a drain pipe, side walls and dividers. The side walls and dividers may be formed of plastic covered and reinforced walls or berms. The paddlewheel is typically a variable speed paddlewheel. The flow diverters act to maintain laminar flow. The inlet pipe may be connected to a gate controlled sluice for pond intake from either a seawater canal and/or the previous ponds. The drain pipe facilitates pond discharge through a gate controlled sluice.

Figure 2: a series of connected raceway ponds arranged in stages. The schematic outlines: * Intake pipeline * Elevated supply canal filled from intake pipeline with high-rate, low-head pumps * Greenhouse-covered sequential seed ponds filled with filtered seawater to grow inoculum for growth ponds * Uncovered, sequential growth ponds connected with and filled from previous pond plus fresh seawater from supply canal * Harvesting ponds for grow-out before harvesting * Harvesting canal and harvesting building * Low elevation discharge pipeline

DETAILED DESCRIPTION OF THE INVENTION

Diazotrophic phytoplankton As used herein, "diazotrophic phytoplankton" refers to marine and freshwater nitrogen-fixing photosynthetic algae. Preferably, the diazotrophic phytoplankton used in the invention are marine diazotrophic phytoplankton.

In some embodiments, the diazotrophic phytoplankton belong to the Cyanobacteria phylum.

Accordingly, in some embodiments the diazotrophic phytoplankton comprise cyanobacteria.

In preferred embodiments, the diazotrophic phytoplankton is Trichodesmium sp. In some embodiments, the diazotrophic phytoplankton is one or more of T. erythraeum, T. contortum, 7: hildebrandtii, T. radians, T. tenue, and T. thiebautii. In some embodiments, the diazotrophic phytoplankton is T. erythraeum.

In some embodiments, the diazotrophic phytoplankton is Crocosphaera sp., optionally C. watsonii.

In some embodiments, the diazotrophic phytoplankton is Calothrix sp., optionally C. adscendens, C. atricha, C. braunii, C. breviarticulata, C. caespitora, C. confervicola, C. crustacea, C. donnelli, C. elenkinii, C. epiphytic°, C. fusca, C. juliana, C. parasitic°, C. parietina, C. pilosa, C. pulvinata, C. scopulorum, C. scytonemicola, C. simulans, C. so//tar/a, C. stagnalis, C. stellaris, and C. thermal's. DDAs

As used herein, "diatom-diazotroph assemblages (DDAs)" refers to symbioses between diatoms and diazotrophic prokaryotes. In these associations, the diazotrophic prokaryote captures or 'fixes' atmospheric N2 and makes it bioavailable to the diatom symbiont.

In some embodiments, the DDAs comprise diazotrophic cyanobacteria.

Diazotrophic cyanobacteria like Riche/la, Calothrix and other unicellular species similar in morphology to free-living diazotroph Crocosphaera, thrive by forming symbiotic relationships with diatoms like Hem/au/us, Rhizosolenia, Chaetoceros and Climacodium (Mutalipassi et a/., 2021; Hilton 2014). Some of these associations, like Calothrix or the novel unicellular cyanobacteria Candidatus Atelocyanobacterium thalassa (UCYN-A) (Tuo et a/., 2017) associate with unicellular algae epiphytically (or on the outside of cells). Other diazotrophic symbionts are intracellular such as Richelia.

In some embodiments, the DDAs comprise marine diazotrophic cyanobacteria. In some embodiments, the marine diazotrophic cyanobacteria are selected from Richelia sp., Calothrix sp., Crocosphaera sp. and Candidatus Atelocynaobacterium Thalassa. In some embodiments, the marine diazotrophic cyanobacteria are Riche//a sp. In some embodiments, the marine diazotrophic cyanobacteria are Calothrix sp. In some embodiments, the marine diazotrophic cyanobacteria are Crocosphaera sp. In some embodiments, the marine diazotrophic cyanobacteria are Candidatus Atelocynaobacterium Thalassa.

In some embodiments, the DDAs comprise RicheHa sp., optionally R. intracellular/s.

In some embodiments, the DDAs comprise Calothrix sp., optionally one or more of C. adscendens, C. atricha, C. braunii, C. breviarticulata, C. caespitora, C. confervicola, C. crustacea, C. donnelli, C. elenkinii, C. epiphytica, C. fusca, C. juliana, C. parasitica, C. parietina, C. pilosa, C. pulvinata, C. scopulorum, C. scytonemicola, C. simulans, C. so//tar/a, C. stagnalis, C. stellaris, and C. thermal's.

In some embodiments, the DDAs comprise Crocosphaera sp., optionally C. watsonii.

In some embodiments, the DDAs comprise one or more of the following diatoms: Hem/au/us sp., Skeletonema sp., Rhizosolenia sp., Climacodium sp. and Chaetoceros sp In some embodiments, the DDAs comprise Hem/au/us sp., optionally H. hauckii, H. indicus, H. membranaceus, or H. sinensis.

In some embodiments, the DDAs comprise Skeletonema sp., optionally S. barba dense, S. costatum, S. cylindraceum, S. mediterraneum, S. punctatum, S. trop/cum, or S. pseudocostatum.

In some embodiments, the DDAs comprise Rhizosolenia sp., optionally R. elate, R. acuminate, R. antarctic°, R. antennata, R. bergonii, R. clevei, R. curvata, R. cylindrus, R. delicatula, R. minima, R. pugens, R. robusta, R. rothii, R. stricta, or R. sty//form/s.

In some embodiments, the DDAs comprise Climacodium sp., optionally C. biconcavum or C. frauenfeldianum.

In some embodiments, the DDAs comprise Chaetoceros sp., optionally C. socialis, C. debilis, C. curvisetus, C. muelleri, C. calcitrans, or C. didymus, In some embodiments, the DDAs comprise an assemblage of marine diazotrophic cyanobacteria and diatoms. In some embodiments, the marine diazotrophic cyanobacteria are selected from Riche//a sp., Calothrix sp., Crocosphaera sp. and Candidatus Atelocynaobacterium Thalassa, and the diatoms are selected from Hem/au/us sp., Skeletonema sp., Rhizosolenia sp., Climacodium sp. and Chaetoceros sp.

In some embodiments, the DDAs comprise an assemblage of Riche//a sp. and Hem/au/as sp.

Oligotrophic conditions The inventors have found that culturing diazotrophic phytoplankton and/or DDAs in land-based mariculture (e.g. in a raceway pond) under oligotrophic conditions results in especially efficient carbon sequestration. Oligotrophic conditions are nutrient poor conditions. For example, oligotrophic conditions may comprise low concentrations of bioavailable nitrogen and phosphorus. These oligotrophic conditions are preferably present within a raceway pond or a series of connected raceway ponds.

Advantageously, sites that are adjacent to nutrient-poor water (e.g. coastal sites adjacent to oligotrophic seawater) may be used for land-based mariculture according to the invention. These sites would not have been deemed appropriate for previous algae-based carbon sequestration methods without substantial nutrient supplementation. Another advantage of culturing diazotrophic phytoplankton and/or DDAs in oligotrophic conditions is that these organisms are particularly well-adapted to growing under such conditions. Thus, these organisms will grow efficiently under oligotrophic conditions and will out-compete organisms that have higher nutrient requirements (e.g. DunaIlene saline). Accordingly, culturing diazotrophic phytoplankton and/or DDAs under oligotrophic conditions may advantageously create a near-monoculture that dominates all other life forms in the land-based mariculture. Overall, mariculture of diazotrophic phytoplankton and/or DDAs at low-quality locations is more cost-effective than culturing bloom'-forming algae under nutrient-rich conditions at such locations.

In some embodiments, the oligotrophic conditions comprise a low concentration of nitrogen. In some embodiments, the oligotrophic conditions comprise a low concentration of nitrogen and phosphorus.

In some embodiments, the oligotrophic conditions comprise a low concentration of bioavailable nitrogen. In some embodiments, the oligotrophic conditions comprise a low concentration of bioavailable nitrogen and phosphorus.

Accordingly, in some embodiments the oligotrophic conditions comprise less than 20 pM, less than 10 pM or less than 5 pM phosphorus.

Accordingly, in some embodiments the oligotrophic conditions comprise less than 40 pM, less than 20 pM, less than 15 pM, less than 10 pM or less than 5 pM nitrogen.

In some embodiments, the oligotrophic conditions comprise less than 20 pM (e.g. 10 pM) phosphorus and less than 40 pM (e.g. 20 pM) nitrogen.

In some embodiments, the oligotrophic conditions comprise less than 10 pM (e.g. 5 pM) phosphorus and less than 5 pM (e.g. 1 pM) nitrogen.

In some embodiments, DDAs (e.g. assemblages comprising Skeletonema sp.) are cultured and the oligotrophic conditions comprise less than 20 pM phosphorus and less than 30 pM silicate.

In some embodiments, DDAs (e.g. assemblages comprising Skeletonema sp.) are cultured and the oligotrophic conditions comprise less than 10 pM phosphorus and less than 15 pM silicate.

In some embodiments, DDAs comprising Hemiaulas sp. (e.g. assemblages of Riche/la sp. and Hem/au/as sp.) are cultured and the oligotrophic conditions comprise less than 4 pM phosphorus, less than 2 pM nitrogen and less than 20 pM silicate.

In some embodiments, DDAs comprising Hemiaulas sp. (e.g. assemblages of Riche/la sp. and Hem/au/as sp.) are cultured and the oligotrophic conditions comprise less than 2 pM phosphorus, less than 1 pM nitrogen and less than 10 pM silicate.

In some embodiments, diazotrophic phytoplankton (e.g. Trichodesmium sp.) are cultured and the oligotrophic conditions comprise less than 20 pM phosphorus and less than 40 pM nitrogen.

In some embodiments, diazotrophic phytoplankton (e.g. Trichodesmium sp.) are cultured and the oligotrophic conditions comprise less than 10 pM phosphorus and less than 20 pM nitrogen.

In some embodiments, diazotrophic phytoplankton (e.g. Trichodesmium sp.) are cultured and the oligotrophic conditions comprise less than 5 pM phosphorus and less than 10 pM nitrogen.

In some embodiments, diazotrophic phytoplankton (e.g. Trichodesmium sp.) are cultured and the oligotrophic conditions comprise less than 1 pM phosphorus and less than 10 pM nitrogen.

In some embodiments, the oligotrophic conditions are present on seeding diazotrophic phytoplankton and/or DDAs into the land-based mariculture and the oligotrophic conditions are maintained until harvesting of the diazotrophic phytoplankton and/or DDAs.

Raceway ponds Preferably, the land-based mariculture of the invention comprises culturing the diazotrophic phytoplankton and/or diatom-diazotroph assemblages (DDAs) in one or more raceway ponds.

Preferably the one or more raceway ponds comprise seawater.

A typical raceway pond is illustrated in Figure 1. Raceway pond is oblong shaped, with a partial divide in the middle of the pond (along the longitudinal axis) to create a circuit with at least two channels. At one end of this divide there is a support structure that serves both as an anchor for the paddlewheel, the platform for the CO2 equipment and to anchor the divider between the raceway channels. At the other end of the divide is a second flow diverter to ensure efficient flow throughout the raceway. Paddle wheel maintains the flow of water and algae around the circuit.

In one embodiment, a covered raceway pond is a raceway pond covered by a greenhouse. The primary purpose of the greenhouse is to protect the seed algae from being contaminated by windborne or bird-borne contaminants. Secondly the greenhouse is used to raise the temperature of the algal growth environment; firstly to increase the algal growth rate, and secondly to inactivate competing or deleterious organisms that might otherwise contaminate the algae or foul the equipment. The greenhouse can also be used to selectively shade or change the illumination colour of the algae to induce a desirable physiological state by altering the light wavelength ratios. In one specific embodiment, every covered raceway pond is covered by a greenhouse.

An open raceway pond has no external cover, and as such is fully exposed to the ambient atmosphere, while a covered raceway pond (which may be fully or partially covered) allows partial or complete control of the temperature and light environment.

In one embodiment, each covered and/or open raceway pond has two channels. As raceway ponds increase in volume they have increased width and/or length and/or number of channels. In a specific embodiment, as open raceway ponds increase in volume they have increased width and length. . In a specific embodiment, both covered and open raceway ponds have a width to length aspect ratio of between 1:4 and 1:12, preferably 1:8. At this ratio, there is relatively low head loss at each paddlewheel pumping station as the water circulates around the bends, favourable economy of the construction materials (straight walls are easier to build than the bends), the reduction of wind influence (wind fetch) as it blows across the pond (to prevent potentially unmixed zones). The width of approximately 30 metres per channel also reduces meandering flow to maintain turbulent flow.

Information regarding the application of computational fluid dynamics to raceway pond design can be found in Kusmayadi, 2020.

Paddlewheels may be used to maintain the flow of the algae and water around the covered and open raceway ponds. This is energy efficient whilst ensuring thorough mixing and the exposure of the algae to relatively low shear. This enables effective exchange of gases within the ambient air with the algae growth medium. According to the species being cultivated, different rate paddle wheels may be used. The paddlewheel rate may be 5-40 cm/sec, e.g. 10-30 cm/sec, or 15-25 cm/sec. In one embodiment, each covered or open raceway pond preferably has 1 or 2, paddlewheels depending on the degree of agitation that is required. Preferably, each raceway pond has one paddlewheel. The paddlewheel may be positioned at any section of the raceway pond, but is preferably positioned closer to the narrower end of a raceway pond.

In one embodiment, one or more paddlewheel maintains the flow of the algae and water within a covered and/or open raceway pond at a rate of about 5-40 cm/sec, for example 10-30 cm/sec, or 15-25 cm/sec or 20 cm/sec, 25 cm/sec or 30 cm/sec.

In one embodiment, a covered raceway pond has volume of between 50 I and 15,000,000 I, for example between 50 I and 50,000 I. In a particular embodiment, the volume of the covered raceway ponds in the series increases such that there is at least one pond in the series with a volume of (a) 50-1,0001; at least one covered raceway pond in the series with a volume of (b) 100 1-30,000 I; and at least one pond in the series with a volume of (c) 5,000 -12,000,000 I. In one embodiment, these ponds are linked in a linear fashion, such that there is one pond at each stage in the series.

In one embodiment, an open raceway pond has volume of between 1,500,0001 and 3,000,000 I, in a further embodiment between 6,000,000 I and 15,000,000 I. In a still further embodiment, the volume of the open raceway ponds in the series increases such that there is at least one open raceway pond in the series with a volume of (a) 360,000-720,000 I; at least one pond in the series with a volume of (b) 1,500,000-3,000,000 I; and at least one pond in the series with a volume of (c) 6,000,000-15,000,000 I. The depth of a raceway pond affects algal solar irradiation. Both light intensity and the light wavelength ratio are altered by increasing water depth. In general, far red, red and ultraviolet light are absorbed the most rapidly by the water and a blue and green light penetrate the furthest. In one embodiment, a covered or open raceway pond is between 0.05 m and 10 m deep, for example, 0.05 m, 0.10 m, 0.20 m, 0.30 m, 0.40 m, 0.50m, 0.60 m, 0.70 m, 0.80 m, 0.90 m, 1m, 1.5m, 2 m, 3m, 5 m, or 10 m deep. In one embodiment, a covered raceway pond is 0.1m -1m deep, for example 0.1m, 0.2m, 0.3m, 0.4m, or 0.5m deep. In one embodiment, an open raceway pond is 0.25 or 0.3 m -1 m deep, for example 0.25 m, 0.3 -0.4m, 0.5 m, 0.75 or 1 m deep. Preferably an open raceway pond is less than 1 m deep. At this depth there is sufficient outgassing of 02 to help reduce oxidative stress, also at this depth there is sufficient exposure to dissolve atmospheric CO2.

Raceway ponds known in the art are also usually uniform in depth. However, the present inventor has discovered that if the depth of a raceway pond is varied along the length of a channel or between adjacent channels, a change in the flow rate results. Furthermore, the exposure of the algae to solar irradiation is varied, with algae in shallower areas of the raceway pond experiencing greater light intensity and a greater proportion of red light than algae in deeper areas. Photosynthetic output can be affected by the light ratio, especially at dawn and dusk, and as discussed herein the depth of the water alters the light wavelength ratio.

Thus, in one embodiment, the depth of an open raceway pond is non-uniform, resulting in a change in algal exposure to light both in terms of light intensity and light wavelength ratio and/or a change in the rate of gas exchange within the non-uniform section of the raceway pond. In one embodiment, the difference between the depth in adjacent channels is 0.05-1.0 m, for example 0.05 m, 0.10 m, 0.2 m, 0.3 m, 0.4 m, 0.5 m, 0.6 m, 0.7 m, 0.8 m, 0.9 m or 1.0 m. For example, one channel is 0.2- 0.5 m deep, while the adjacent channel is 0.8-1.0 m deep.

As described herein, the raceway ponds of the present invention may be lined. In one embodiment, covered and open raceway ponds are lined with an impermeable material. If seawater is used to culture the algae, a clay lining may be used to prevent saltwater intrusion onto the land. In one embodiment, a plastic waterproof lining is used instead of, or in addition to, a clay lining. In a particular embodiment, each raceway pond is lined with 10-20 cm of clay and a 2-5 cm, e.g. about 3 cm or specifically 8, 10 or 12 mm, robust synthetic liner such as white HDPE geomembrane.

In one embodiment, the colour of the pond liners may also be chosen to alter the light wavelength ratio experienced by the algae as it reflects off the bottom of the ponds. The colour of the liner may selectively increase or decrease exposure of the algae to underwater red (630-680 nm), far red (700750 nm) and/or blue (400-450 nm) light. In one embodiment, the liner selectively increases the exposure to underwater red (630-680 nm), far red (700-750 nm) and/or blue (400-450 nm) light by 10-90%, 20-80%, 30-70%, or 40-60%. For example, the liner may selectively absorb up to 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the incident underwater red (630-680 nm), far red (700-750 nm) and/or blue (400-450 nm) light before the remaining light is reflected through the algal growth environment again.

In a second embodiment, the lining is white in order to reflect light off the bottom and maximise the light available for photosynthesis and encourage algal growth especially in the low density cultures and/or shallow cultures less than 30cm deep, where light will penetrate the depth of the medium. In some instances, black liners may be used to deliberately increase the temperature of the cultivation medium.

Different colours of lining may be used to induce different physiological effects in the algae. For example, the lining may be entirely red, blue or green. Alternatively, a single stage in the sequence of ponds can be coloured blue, for example at the point where the seed algal growth has been synchronised in the initial covered ponds, to reinforce cellular growth synchronisation and increase growth before cell division, just before the cells are introduced into the open growth ponds so that multiple divisions then occur in the growth pond. Similarly, the growth pond can be lined in blue to stimulate the migration of chloroplasts to the outside of the cells, to promote maximum photosynthesis (Kraml & Hermann, 1991; Furukawa et al., 1998).

Series of raceway ponds In one embodiment, the land-based mariculture of the invention comprises culturing the diazotrophic phytoplankton and/or diatom-diazotroph assemblages (DDAs) in a series of connected raceway ponds, arranged in stages. The raceway ponds are connected in such a way so as to allow water and algae to pass directly between raceway ponds in successive stages of the series. However, the connection between raceway ponds can be closed and each raceway pond can be an isolated growth environment. The flow of water and algae between successive stages in the series is unidirectional, Le. the passage of algae and water through the connected series of raceway ponds is one-way and algae and water are not re-circulated.

In one specific embodiment of the invention the series of connected raceway ponds comprises firstly, one or more stages of covered raceway ponds and secondly, one or more stages of open raceway ponds. The designations of "firstly" one or more stages of covered raceway ponds and "secondly" one or more stages of open raceway ponds indicate that within the series of connected ponds, the stages comprising covered raceway ponds will always come before the stages comprising open raceway ponds. Put another way, an open raceway pond will never be succeeded by a covered raceway pond.

The present inventor has found that successive dilution in a semi-continuous cultivation manner, which maintains a low algal cell density, is beneficial for maintaining algae in the exponential growth phase.

Successive dilution can be achieved in two ways.

In the first way dilution is achieved by increasing the collective volume of the raceway ponds in each stage of the series. This increase in collective volume can be achieved by increasing the volume of the individual raceway ponds in each successive stage of the series and/or by increasing the number of raceway ponds in each successive stage of the series. Thus, at each successive stage in the series of raceway ponds of the present invention, each individual raceway pond has a volume greater than the volume of the individual raceway ponds in the preceding stage of the series; and/or each raceway pond is immediately succeeded by a greater number of raceway ponds, wherein the collective volume of the raceway ponds in any given stage exceeds the collective volume of the raceway ponds of the preceding stage.

In the second way, dilution is achieved by increasing the volume in stages to a maximum volume within a pond, before the water and algae are transferred to the subsequent larger pond. In the embodiment outlined in the model and table below, each pond is initially filled to a depth of 0.25 m where the cells complete a growth cycle. Wien it is time to double the volume of the water, to enable the cells to grow at a low standing stock with natural nutrients, the depth of the same pond is increased to 0.5 m. After the second growth cycle is complete within that pond, the total volume of the two division stages and the seawater is transferred to the next larger, subsequent pond. In other embodiments, the pond is filled in subsequent stages to 0.25 m, 0.5 m, 0.75 m and 1 m depth in four sequential 'within-pond' dilutions before it is transferred to the next larger pond.

This 'stacking' of ponds is possible because of the relatively low standing stock (or low cellular concentration) of the algae in comparison to other cultivation systems. In other commercial algal growth systems (where cells are grown at a density resulting between 1,000-2000, mg Chl a m4), the high cell density results in gas exchange limitations and self-shading. However in this method, neither of these are critical concerns, because the cell densities are significantly lower for the first 12-16 growth cycles resulting in 50, 100, 200, 300, 400, 500 mg Chl a m-s). At these Chl a concentrations, the ponds can be run without cell shading and the natural capacity of the seawater to absorb and buffer gases as well as exchange gases with the atmosphere enables cell growth.

In the context of this invention, the volume of a raceway pond is defined as its capacity (i.e. the volume of fluid a raceway pond is capable of holding) rather than the volume of fluid actually held in the raceway pond at any given time. When algae and water are transferred from a raceway pond (covered or open) in one stage of the series to one or more raceway ponds (covered or open) in the next stage of the series, either the algae and water are transferred to a single raceway pond with a greater volume, or the algae and water are divided between a number of raceway ponds with a larger collective volume. Each transfer of the algae and water from one stage of the series to the next stage in the series thus involves dilution of the algae or is preceded by dilution of the algae in the current stage of ponds.

In one embodiment, when the algae and water from one stage in the series is used to seed the raceway pond or raceway ponds of the next stage, it is transferred to the raceway pond or raceway ponds of larger volume in the next stage of the series. Water is added to, or is already present in, the raceway ponds to be seeded, such that the final volume of fluid within the raceway ponds after seeding is equal to its capacity. This seeding step results in the algae being diluted and a low algal cell density can thus be maintained. Maintaining this constant low cell density of algae prevents the problems associated with traditional high-density algal culture, such as quorum sensing, biofilm formation and other (often unpredictable) algal stress responses.

In an alternative embodiment, the algae are diluted prior to being transferred to the next stage of ponds. In this embodiment, fresh seawater is added to the current stage of ponds to dilute the algae.

The successive dilution of the algae at each seeding step should not be taken to mean that the algal cell density at each stage in the series is successively reduced. Since the algae multiply rapidly, despite the successive dilutions at each seeding step the approximate cell density of algae in each stage of raceway ponds going through the series may increase, decrease or remain the same.

In a specific embodiment, there are at least two stages of covered raceway ponds in the series of connected raceway ponds. For example, there may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 stages of covered raceway ponds. In one embodiment, there are 2-10 stages of covered raceway ponds, 4-8 stages of covered raceway ponds, or 5 stages of covered raceway ponds. In a preferred embodiment, the covered raceway ponds are preferably greenhouse covered and are connected in linear succession, wherein there is one covered raceway pond at each stage in the series of covered raceway ponds. In this embodiment, each covered raceway pond is at least 2 times, for example 2 to 5 times, the volume of the covered raceway pond of the previous stage in the series. In a specific embodiment, each covered raceway pond is 2, 3, 4, or 5 times volume of the covered raceway pond of the previous stage in the series. In a preferred embodiment, each covered raceway pond is 5 times volume of the covered raceway pond of the previous stage in the series.

In a further specific embodiment, there are at least two stages of open raceway ponds in the series of connected raceway ponds. For example, there may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20 or 25 stages of open raceway ponds. In one embodiment, there are 2-10 stages of open raceway ponds, 4-6 stages of open raceway ponds, or 5 stages of open raceway ponds. In one specific embodiment, there are more open raceway ponds than closed raceway ponds in the series.

In a particular embodiment, the number of open raceway ponds in each stage increases at each successive stage in the series. In this embodiment, each open raceway pond is connected to two or more open raceway ponds in the next stage in the series, and the collective volume of each of the two or more open raceway pond exceeds the volume of the raceway pond in the preceding stage. Therefore, in this particular embodiment, the algae and water in one open raceway pond is diluted into two or more open raceway ponds when the algae and water are transferred between stages in the series.

In a specific embodiment, the number of open raceway ponds in each stage of the series doubles. Therefore, in this embodiment the number of raceway ponds at each stage in the series increases exponentially. For example, the number of open raceway ponds at each stage increases as follows: 1, 2, 4, 8, 16, 32, 64, 128. In this embodiment, as the number of open raceway ponds at each stage increases, so does the volume of each individual raceway pond. In a preferred embodiment, the volume of the individual open raceway ponds in one stage is at least two times, preferably five times, the volume of the individual open raceway ponds in the previous stage. In an alternative embodiment, the volume of the individual open raceway ponds at each stage in the series remains the same, although the collective volume of the raceway ponds increases with each stage as the number of raceway ponds increases.

The algae and water may be transferred between raceway ponds in successive stages of the series without any external force, for example it may be transferred under the influence of gravity. However, the algae and water will, on occasion when the local topography does not permit the use of gravity transfers, be pumped from one raceway pond to another, using any suitable pumping means that does not shear the cells.

Figure 2 illustrates the layout of a specific algal cultivation system, including the seawater pipe that is used to fill an elevated seawater canal. This seawater canal feeds a series of raceway ponds. Each pond has a connection to at least the elevated seawater canal and a lower lying subsequent pond through a gate controlled sluice pond discharge. The series of ponds commences with small covered seed ponds to grow the inoculums. Each subsequent pond has at least twice the capacity of the previous pond to hold the entirety of the volume of the previous pond and the equivalent volume of unused seawater. The layout in figure 2 has two stages with four channels including the "return channel"). One set of raceway pond with eight channels, including the "return channel, and one set with 10 channels including the "return channels". This final set drains into two large and deep harvesting ponds. The advantage of this oblong layout is that it efficiently hugs the coast along the edge of an ocean and enables each pond to have at least one contact with the elevated seawater canal and a discharge into the next larger and lower pond. This layout enables the water transfer within the entire pond system to rely on gravity feeds and requires a minimum of piping, while enabling easier maintenance of the ponds. This layout also takes advantage of the frequently encountered natural gradient along coastlines where distance from the shore commonly results in a slight increase in elevation. Finally, the intake manifold is upstream and as far as possible from the discharge system to avoid reuptake of already spent seawater.

Seed ponds and photobioreactors In one embodiment, the first stage of covered raceway ponds is seeded with algae cultivated in a photobioreactor (PBR) or a seed pond.

A PBR achieves a highly controlled environment within the reactor to maintain an uncontaminated stock culture. In order to prevent contamination with competing organisms, bacterial and viral infection, or predatory organisms that reduce the yield or availability of the seed algae, a PBR preferably uses sand and membrane filtered, pre-treated and decontaminated seawater. The exchange of gases is carefully controlled, for example by sparging or bubbling CO2 into the reactor, and removing excess 02. The addition of nutrients and the removal of waste products is also carefully controlled. Preferably, a PBR operates in a sterile environment.

A seed pond is an open or closed pond, preferably a closed raceway pond, in which the growth conditions for the algae can be controlled. The seed pond is used to grow a population of algae sufficient to seed the first stage of covered raceway ponds.

In one embodiment, the first stage of covered raceway ponds is seeded with algae from a PER or seed pond in the early morning, e.g. from one hour before to two hours after dawn to enable the algae to exploit their new growth environment, right after they have divided in the predawn hours, for example 1-2 hours before dawn. In a particular embodiment, the first stage of covered raceway ponds is seeded between 1-2 hours before and 1-2 hours after dawn, e.g. between 1 hour before and 2 hours after dawn, or in a specific embodiment when dawn is at 6 AM, the seeding occurs between the hours of SAM to 8 AM.

Algal flow management Algae may be cultivated in seawater, hypersaline water, desalination brine, brackish water, wastewater or freshwater. The choice of water for the culture medium will depend on the algae being grown. Algae will be grown in water that replicates their natural growth environment.

Once the seawater has circulated through the series of connected raceway ponds, it is cleaned of algae and returned to the warmer ocean surface water, down-current at a distance from the intake to avoid intake of water that has already been used for cultivation of the algae.

In one embodiment according to the present invention, algae are first cultivated in at least one stage of covered raceway ponds. The covered raceway ponds allow for the control of the algae growth environment. The water used to fill the covered raceway ponds may be filtered or otherwise treated to remove competing and deleterious organisms before being introduced into the covered raceway ponds.

In one embodiment, greenhouses are used to cover the raceway ponds and as a result the algae environment is maintained at a higher than ambient temperature. In this embodiment, the temperature within the covered raceway ponds is between 18 °C and 32 °C, for example between 28 °C and 34 °C. This will substantially inactivate organisms acclimated to temperatures of 14°C - 18°C when they are pumped from depth off-shore. Advantageously, covered raceway ponds are less susceptible to contamination, either by bacteria or viruses, or by potentially competing organisms. The relatively controlled environment of the covered raceway pond promotes the algae transifioning into the exponential growth phase.

As the algal cellular density increases, the algae are successively diluted, preferably by being transferred between stages in the series of covered raceway ponds to covered raceway ponds of successively larger volume. This successive dilution maintains a relatively low cell density of algae, for example between 100,000 cells/ml and 2,000,000 cells/ml, for example about 350,000 cells/ml.

Each transfer of the algae to seed a covered raceway pond in the next stage of the series (i.e. the successive dilution of the algae) can be timed to match the growth rate of the algae, or cellular resource requirements. In one embodiment, algae reside in each stage of covered raceway ponds for 2 hours to 10 days, for example 2 hours, 3, hours, 5 hours, 12 hours, 24 hours, 36 hours, 2 days, 3, days or 5 days. In one embodiment, the algae reside in each covered raceway pond for two days, before being transferred to a raceway pond of larger volume. In one embodiment, algae remain in a covered raceway pond for a length of time sufficient for the algae cellular population to at least double, for example 2 hours, 4 hours, 12 hours, 24 hours, 36 hours or 48 hours. In a specific embodiment, algae remain in a covered raceway pond for 24 hours before being transferred to the next stage in the series.

Once the algae enter the exponential growth phase, or when sufficient quantities of algae have been cultivated, the algae are transferred to the first stage of open raceway ponds. In one embodiment, the algae and water are transferred in volumes of 1,000 Ito 3,000,000 I, for example 360,000 Ito 720,000 I, into the first stage of open raceway ponds. The transfer of a relatively large bolus of algae is intended to seed the open raceway ponds to populate the growth environment with a large excess of several orders of magnitude of the product algae relative to any surviving organisms that were within the source water used in the open raceway pond thereby establishing a robust population.

The algae may be diluted by introducing additional water into the current stage of ponds, increasing the volume of water contained within that stage. Alternatively, the algae may be diluted by transferring the algae to the next stage of ponds and mixing the algae with water already present in those ponds.

Algae in an open raceway pond are successively diluted by increasing the volume or number of open raceway ponds in each stage of the series. This serial dilution maintains the algae at a low enough cell density to sustain exponential growth. In one embodiment, the algae are successively diluted to maintain a cell density of 50,000 cells/ml to 100,000 cells/ml, for example 200,000 cells/ml to 250,000 cells/ml. This approach is completely different to current methods of cultivating algae in which the algae are grown to artificially high densities often reaching cell densities of over 1 million cells/ml.

In one embodiment, algae reside in each stage of raceway ponds for 2 hours to 5 days, for example 2 hours, 3, hours, 5 hours, 12 hours, 24 hours, 36 hours, 2 days, 3, days or 5 days before being transferred to the next stage of raceway ponds in the series. In a specific embodiment, algae remain in a raceway pond for 48 hours before being transferred to the next stage of raceway ponds in the series. In one embodiment, algae remain in a raceway pond for a length of time sufficient for the algae cellular population to at least double, i.e. for one round of cell division to take place. In a specific embodiment, the algae remain in a raceway pond for a length of time sufficient for one, two, three, four or five rounds of cell division to take place. This may be, for example, 2 hours, 4 hours 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours.

This successive dilution of algae maintains a low cell density which advantageously maintains the exponential growth phase, thereby increasing productivity. Furthermore, the problems associated with high-density algal culture methods such using traditional raceway ponds and PBRs are avoided.

With each successive dilution of the algae, water is added to, and/or is already present in, the covered and open raceway ponds in the next stage in the series. Following the transfer of the algae and water from one stage to seed the next stage in the series, the volume of fluid within each covered and open raceway pond is equal to its capacity. In one embodiment, the entire volume of water in an open and/or covered raceway pond is replaced every 2 hours to 8 days, or every 4 hours to 8 days, preferably every 24 to 72 hours. In a preferred embodiment, the volume of water passing through the series of connected covered and open raceway ponds in 24 hours is greater than 20- 250% of the entire volume of the series of covered and open connected raceway ponds, e.g. greater than 30% of the entire volume of the series of covered and open connected raceway ponds, for example greater than 100% for both seed and growth ponds. This exchange rate of water is much higher than in traditional raceway ponds, where the water replacement rate is usually around 0 - 20% in 24 hours, in order to match the growth rate of the cells and rate of evaporation.

This high volume of water exchange has several advantages. In traditional raceway ponds a high cell density of algae is maintained and any contamination can potentially render the entire raceway pond un-harvestable. However, the series of raceway ponds of the present invention is inherently resilient, as small degrees of contamination do not matter since all of the contaminants are inevitably washed out of the series of ponds, and none of the product algae is reintroduced into the series of raceway ponds.

Harvesting the algae In one embodiment, the method of the invention further comprises the step of harvesting the algae.

In some embodiments, the algae are harvested by tangential flow filtration. In some such embodiments, the tangential flow filtration is performed using filters comprising hydrophilic simple weave (irradiated) polyester fabric.

In some embodiments, the algae are harvested by filtering with a rotary mesh screen. In some such embodiments, the screen comprises a (pore-sized) mesh and a filter comprising hydrophilic (irradiated) polyester fabric.

In some embodiments, diazotrophic phytoplankton that are chain-forming cyanobacteria, such as Trichodesmium sp. (e.g. Trichodesmium erythraeum), are cultured. In some such embodiments, the cyanobacteria are harvested by tangential flow filtration. In some such embodiments, the tangential flow filtration is performed using filters comprising hydrophilic (irradiated) polyester fabric.

In some embodiments, diazotrophic phytoplankton that are chain-forming cyanobacteria, such as Trichodesmium sp. (e.g. Trichodesmium erythraeum), are cultured. In some such embodiments, the cyanobacteria are harvested by filtering with a rotary mesh screen. In some such embodiments, the screen comprises a (pore-sized) mesh and a filter comprising hydrophilic (irradiated) polyester fabric.

In some embodiments, DDAs comprising a chain-forming diatom (e.g. Hemiauluas sp.) are cultured and the algae are harvested by tangential flow filtration. In some such embodiments, the tangential flow filtration is performed using filters comprising hydrophilic (irradiated) polyester fabric.

In some embodiments, DDAs comprising a chain-forming diatom (e.g. Hemiauluas sp.) are cultured and the algae are harvested by filtering with a rotary mesh screen. In some such embodiments, the screen comprises a (pore-sized) mesh and a filter comprising hydrophilic (irradiated) polyester fabric.

Suitable rotary mesh screens include (simple weave) polyester screens that have a pore size of 10-200 pM, preferably a pore size of 20-120 pM. In some embodiments, these screens have been irradiated. Advantageously, this makes the screen more hydrophilic, which improves the filtration rate. Suitable rotary mesh screens may comprise a surfactant coating, such as SAATIcare HyphylTM.

A further example of a suitable rotary mesh screens is monofilament polyester fabric, such as SUPREX (EXTRIS).

In the context of rotary mesh screens, the thinner the fibre strands, the larger the percentage of open area for filtration, which the inventors have found to be advantageous for harvest filtration of algae. Accordingly, in some embodiments, the average fibre strand diameter in a rotary mesh screen less than 50 pM.

Accordingly, in some embodiments, the rotary mesh screens have a pore size of 10-200 pM and an average fibre strand diameter of less than 50 pM.

Accordingly, in some embodiments, the rotary mesh screens have a pore size of 20-120 pM and an average fibre strand diameter of less than 50 pM.

In some embodiments, the harvested algal slurry (or algal concentrate) is solar dried and buried to sequester carbon in the ground.

Maintenance of the raceway ponds The methods of the invention that comprise culturing diazotrophic phytoplankton and/or DDAs in a series of connected raceway ponds typically produce sequential batches of algae as opposed to the steady state continuous harvesting of algae that is traditionally utilised in algal culture. This means that batches of algae can be separated according to need. For example, as the algae pass through the series of connected ponds, trace contamination from the air or other sources may occur, particularly in the open raceway ponds. However, the series of connected raceway ponds of the present invention is inherently resilient, because small degrees of contamination do not matter as none of the product algae is reintroduced and all of the contaminants are eventually washed out of the system. Furthermore, in one embodiment the dilution of the algae through the repeat addition of seawater also dilutes any contaminants present.

Raceway ponds in the series are connected to allow algae and water to flow from one raceway pond to another, but each pond can be isolated when required. This is particularly useful to allow the ponds to be cleaned to remove sediment or biofilms. Therefore, between batches of algae in different raceway ponds, where algae are transferred every 2, 3, 4, 5, 6, 8, 10, 12, 24, 36 hours, 2 days, 3 days, 5 days apart, a cleaning shift can be introduced wherein each raceway pond in the series is sequentially pumped dry, cleaned, for example with truck-mounted rotating brushes, and flushed with water. A cleaning cycle can be performed once a month, and every several cleaning cycles an extra day may be introduced to add a day for drying the ponds. Equipment can be cleaned with 0.0001-0.01% peroxyacetic acid or similar disinfectants such as hypochloric acid before it is seeded with algae from the raceway pond in the preceding stage in the series, and topped up with fresh water. In this manner, a running cleaning wave can travel through the entire series of connected covered and open raceway ponds (and the harvesting ponds if desired). This may be scheduled according to the prevalence of oceanic contaminants coming in with the fresh seawater or environmental perturbations such as rainfall or sandstorms.

Exemplary methods In one embodiment, the invention provides a method for sequestering carbon dioxide from the atmosphere, wherein the method comprises culturing Trichodesmium sp. in a connected series of raceway ponds under oligotrophic conditions, and wherein the method further comprises harvesting Trichodesmium sp., wherein the series of raceway ponds comprises a first stage comprising one or more covered raceway ponds and a second stage comprising one or more open raceway ponds, wherein the oligotrophic conditions comprise low concentrations of phosphorus and nitrogen (e.g. less than 20 pM phosphorus and less than 40 pM nitrogen), and wherein the oligotrophic conditions are present on seeding Trichodesmium sp. into the first stage of raceway ponds, and wherein the oligotrophic conditions are maintained until harvesting of the Trichodesmium sp.

In one embodiment, the invention provides a method for sequestering carbon dioxide from the atmosphere, wherein the method comprises culturing Trichodesmium sp. in a connected series of raceway ponds under oligotrophic conditions, and wherein the method further comprises harvesting Trichodesmium sp., wherein the series of raceway ponds comprises a first stage comprising one or more covered raceway ponds and a second stage comprising one or more open raceway ponds, wherein the oligotrophic conditions comprise low concentrations of phosphorus and nitrogen (e.g. less than 1 pM phosphorus and less than 10 pM nitrogen), and wherein the oligotrophic conditions are present on seeding Trichodesmium sp. into the first stage of raceway ponds, and wherein the oligotrophic conditions are maintained until harvesting of the Trichodesmium sp.

In one embodiment, the invention provides a method for sequestering carbon dioxide from the atmosphere, wherein the method comprises culturing assemblages of Richelia sp. and Hemiaulas sp. in a connected series of raceway ponds under oligotrophic conditions, and wherein the method further comprises harvesting assemblages of Richelia sp. and Hem/au/as sp., wherein the series of raceway ponds comprises a first stage comprising one or more covered raceway ponds and a second stage comprising one or more open raceway ponds, wherein the oligotrophic conditions comprise low concentrations of phosphorus and nitrogen (e.g. less than 20 pM phosphorus, less than 40 pM nitrogen and less than 20 pM silicate), and wherein the oligotrophic conditions are present on seeding the assemblages of Richelia sp. and Hem/au/as sp. into the first stage of raceway ponds, and wherein the oligotrophic conditions are maintained until harvesting of the assemblages of Riche//a sp. and Hem/au/as sp.

In one embodiment, the invention provides a method for sequestering carbon dioxide from the atmosphere, wherein the method comprises culturing assemblages of Richelia sp. and Hemiaulas sp. in a connected series of raceway ponds under oligotrophic conditions, and wherein the method further comprises harvesting assemblages of Richelia sp. and Hem/au/as sp., wherein the series of raceway ponds comprises a first stage comprising one or more covered raceway ponds and a second stage comprising one or more open raceway ponds, wherein the oligotrophic conditions comprise low concentrations of phosphorus and nitrogen (e.g less than 2 pM phosphorus, less than 1 pM nitrogen and less than 10 pM silicate), and wherein the oligotrophic conditions are present on seeding the assemblages of Richelia sp. and Hem/au/as sp. into the first stage of raceway ponds, and wherein the oligotrophic conditions are maintained until harvesting of the assemblages of Richelia sp. and Hem/au/as sp.

In one embodiment, the invention provides a method for sequestering carbon dioxide from the atmosphere, wherein the method comprises culturing diatom-diazotroph assemblages (e.g. assemblages comprising Skeletonema sp.) in a connected series of raceway ponds under oligotrophic conditions, and wherein the method further comprises harvesting the diatom-diazotroph assemblages, wherein the series of raceway ponds comprises a first stage comprising one or more covered raceway ponds and a second stage comprising one or more open raceway ponds, wherein the oligotrophic conditions comprise low concentrations of phosphorus and nitrogen (e.g less than 20 pM phosphorus, less than 40 pM nitrogen and less than 20 pM silicate), and wherein the oligotrophic conditions are present on seeding the diatom-diazotroph assemblages into the first stage of raceway ponds, and wherein the oligotrophic conditions are maintained until harvesting of the diatom-diazotroph assemblages.

In one embodiment, the invention provides a method for sequestering carbon dioxide from the atmosphere, wherein the method comprises culturing diatom-diazotroph assemblages (e.g. assemblages comprising Skeletonema sp.) in a connected series of raceway ponds under oligotrophic conditions, and wherein the method further comprises harvesting the diatom-diazotroph assemblages, wherein the series of raceway ponds comprises a first stage comprising one or more covered raceway ponds and a second stage comprising one or more open raceway ponds, wherein the oligotrophic conditions comprise low concentrations of phosphorus and nitrogen (e.g. less than 2 pM phosphorus, less than 1 pM nitrogen and less than 10 pM silicate), and wherein the oligotrophic conditions are present on seeding the diatom-diazotroph assemblages into the first stage of raceway ponds, and wherein the oligotrophic conditions are maintained until harvesting of the diatom-diazotroph assemblages.

General The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y. The term "about" in relation to a numerical value x is optional and means, for example, x+10%.

The various steps of the methods may be carried out at the same time or at different times, in the same geographical location or in different geographical locations, e.g. countries, and by the same or different people of entities

EXAMPLES

Various aspects and embodiments of the invention are described below in more detail by way of example. It will be appreciated that modification of detail may be made without departing from the scope of the invention.

Example

An example of the cells daily carbon, phosphorus and silicate quota (pM d-1) for Trichodesmium and Hem/au/as sp., growing at a rate of 0.35 d-1 and 1.0 d-1 respectively, at biomass levels as expected in the cultivation ponds.

Model Organism Seawater DIC Phosphorus Silicate Diazotrophic phytoplankton Trichodesmium sp. 530 5 -DDA Hemiaulas / 1480 14.0 10.2 Richelia sp.

Based on the nutrient demand of most coastal phytoplankton, in contrast, filtered natural seawater with supplemented nutrients (f/2 medium) used to grow most coastal organisms consists of 882 pM of N, 36.2 pM of P, 106 pM of Si (Guillard & Ryther). This demonstrates how different the nutrient requirements are for the N fixing organisms.

Example 2

This example provides a direct comparison of culturing Dunaliella saline (a chlorophyte that is incapable of fixing nitrogen), Trichodesmium sp. (a diazotrophic phytoplankton) and Hemiaulas-Richelia (a DDA) in the same pond system. The dilution train involves seeding the algae into a greenhouse-covered raceway pond, transitioning through a series of open raceway ponds (in which each pond is five times larger than the subsequent pond), and then harvesting from the final pond in the series. In this system, the harvesting pond is 12,000 m2.

Upwelling Diazotrophic phytoplankton DDA Dunaliella Trichodesmium Hemia ulas-Species salina sp. Richelia Growth cycle (days) 3 5 2 Growth rate (d-1) 0.4 0.25 0.56 Chl a at harvest (pg L-1) 86 150 150 Cells at harvest (cells/mL) 196,452 NA 368,749 POC:Biomass 0.44 0.44 0.44 Biomass at harvest (g m2) 41.5 29.8 30 In this comparison, the inventors determined that the nutrient supplement costs per sequestered tonne of CO2 in the Trichodesmium system are substantially lower than those associated with the D. salina system. The Hemiaulas-Richelia system also exhibited improved nutrient supplement costs per sequestered tonne of 002.

REFERENCES

AR6: httos://www.iQcc.ch/reportlsixth-assessment-report-cycle Benemann, J.R. & Oswald, W.J. Systems and Economic Analysis of Microalgae Ponds for Conversion of CO2 to Biomass. Final Report to the Pittsburgh Energy Technology Center (1996) Furukawa, T., Watanaba, M. and Shihira-Ishikawa, I. Green and blue-light-mediated chloroplast migration in the centric diatom Pleurosira laevis Protoplasma, 203;214-220 (1998) Hilton J.A., Ecology and Evolution of Diatom Associated Cyanobacteria through genetic analysis, June 2014, PhD Dissertation, UC Santa Cruz Kraml, M. and Herrmann, H. Red-blue interaction in Mesotaenium chloroplast movement-blue seems to stabilize the transient memory of the phytochrome signal. Photochem. Photobiol., 53:255259 (1991) Kusmayadi, A., Suyono E. A., Nagarajan, D., Chang, J.-S. and Yen, H.W., Application of computational fluid dynamics (CFD) on the raceway design for the cultivation of microalgae: a review, 2020, Journal of Industrial Microbiology and Biotechnology, 47(4-5):373-382.

Mutalipassi M., Riccio G., Mazzella V., Galasso C., Somma E., Chiarore A., de Pascale D. and Zupo V., Symbioses of Cyanobacteria in Marine Environments: Ecological Insights and Biotechnological Perspectives, 2021, Mar. Drugs 19:227-256 Tuo S-H., Lee Chen Y-L., Chen H-Y., Chen T-Y., Free-living heterocystous cyanobacteria in the tropical marginal seas of the western North Pacific, 2017, J. Plankton Res. 39(3):404-422 Weissman, J.C. and Goebel, R.P. Design and Analysis of Pond Systems for the Purpose of Producing Fuels. Solar Energy Research Institute, Golden Colorado (1987)

Claims (23)

1. CLAIMS1. A method for sequestering carbon dioxide from the atmosphere, wherein the method comprises culturing diazotrophic phytoplankton and/or diatom-diazotroph assemblages (DDAs) 5 in land-based mariculture under oligotrophic conditions.
2. 2. The method of claim 1, wherein the method comprises culturing diazotrophic phytoplankton in land-based mariculture under oligotrophic conditions.
3. 3. The method of claim 1 or claim 2, wherein the diazotrophic phytoplankton are cyanobacteria.
4. 4. The method of claim 2 or claim 3, wherein the diazotrophic phytoplankton is Trichodesmium sp., Crocosphaera sp., or Calothrix sp.
5. 5. The method of claim 4, wherein the diazotrophic phytoplankton is Trichodesmium sp.
6. 6. The method of claim 5, wherein the diazotrophic phytoplankton is one or more of Trichodesmium erythraeum, Trichodesmium contortum, Trichodesmium hildebrandtii, Trichodesmium radians, Trichodesmium tenue, and Trichodesmium thiebautii.
7. 7. The method of claim 6, wherein the diazotrophic phytoplankton is Trichodesmium erythraeum.
8. 8. The method of any one of claims 1-7, wherein the method comprises culturing DDAs in land-based mariculture under oligotrophic conditions.
9. 9. The method of claim 8, wherein the DDAs comprise one or more of the following diazotrophs: Richelia sp., Calothrix sp., Crocosphaera sp. and Candidatus Atelocynaobacterium thalassa.
10. 10. The method of claim 8 or claim 9, wherein the DDAs comprise one or more of the following 30 diatoms: Hemiaulus sp., Skeletonema sp., Rhizosolenia sp., Chaetoceros sp., and Climacodium sp.
11. 11. The method of claim 10, wherein the DDAs comprise Hemiaulus sp.
12. 12. The method of claim 11, wherein the DDAs comprise an assemblage of Richelia sp. and Hemiaulas sp.
13. 13. The method of any one of claims 1-12, wherein the oligotrophic conditions comprise low concentrations of nitrogen and phosphorus (e.g. less than 10 pM nitrogen and less than 20 pM 40 phosphorus).
14. 14. The method of claim 13, wherein the oligotrophic conditions further comprise low concentrations of silicate (e.g. less than 15 pM silicate).
15. 15. The method according to any preceding claim, wherein the land-based mariculture comprises one or more raceway ponds.
16. 16. The method according to any preceding claim, wherein the land-based mariculture comprises culturing the diazotrophic phytoplankton and/or DDAs in a series of connected 50 raceway ponds, arranged in stages.
17. 17. The method according to claim 16, wherein the series of connected raceway ponds comprises a first stage comprising one or more covered raceway ponds and a second stage comprising one or more stages of open raceway ponds.
18. 18. The method according to any preceding claim, wherein the method further comprises harvesting the diazotrophic phytoplankton and/or DDAs.
19. 19. The method of claim 18, wherein the oligotrophic conditions are present on seeding 10 diazotrophic phytoplankton and/or DDAs into the land-based mariculture and the oligotrophic conditions are maintained until harvesting of the diazotrophic phytoplankton and/or DDAs.
20. 20. The method of claim 1, wherein the method comprises culturing Trichodesmium sp. in a connected series of raceway ponds under oligotrophic conditions, and wherein the method 15 further comprises harvesting Trichodesmium sp., wherein the series of raceway ponds comprises a first stage comprising one or more covered raceway ponds and a second stage comprising one or more open raceway ponds, wherein the oligotrophic conditions comprise low concentrations of phosphorus and nitrogen (e.g less than 20 pM phosphorus and less than 40 pM nitrogen), and wherein the oligotrophic conditions are present on seeding Trichodesmium sp. into the first stage of raceway ponds, and wherein the oligotrophic conditions are maintained until harvesting of the Trichodesmium sp.
21. 21. The method of claim 1, wherein the method comprises culturing assemblages of Richelia sp. 25 and Hemiaulas sp. in a connected series of raceway ponds under oligotrophic conditions, and wherein the method further comprises harvesting assemblages of Richelia sp. and Hem/au/as sp., wherein the series of raceway ponds comprises a first stage comprising one or more covered raceway ponds and a second stage comprising one or more open raceway ponds, wherein the oligotrophic conditions comprise low concentrations of phosphorus and nitrogen (e.g less than 20 pM phosphorus, less than 40 pM nitrogen and less than 20 pM silicate), and wherein the oligotrophic conditions are present on seeding the assemblages of Richelia sp. and Hem/au/as sp. into the first stage of raceway ponds, and wherein the oligotrophic conditions are maintained until harvesting of the assemblages of Richelia sp. and Hem/au/as sp.
22. 22. The method of any one of claims 19-21, wherein the method further comprises burying the harvested diazotrophic phytoplankton and/or DDAs for carbon sequestration.
23. 23. The method of claim 22, wherein a slurry or a concentrate of the harvested diazotrophic phytoplankton and/or DDAs is solar dried prior to burial.