GB2626588A

GB2626588A - Carotenoid production methods

Info

Publication number: GB2626588A
Application number: GB2301206.5A
Authority: GB
Inventors: Jovine Raffael; Skardon James
Original assignee: Brilliant Planet Ltd
Current assignee: Brilliant Planet Ltd
Priority date: 2023-01-27
Filing date: 2023-01-27
Publication date: 2024-07-31
Also published as: WO2024157034A1; GB202301206D0

Abstract

A method for producing carotenoids or pigments by culturing algae in land-based mariculture, comprises a first algal culture phase where marine carotenoid-producing microalgae are maintained in an exponential growth phase by successive dilution with seawater and nutrient addition in a series of connected raceway ponds 210. A second phase comprises inducing the algae from the first phase to produce carotenoid pigment by culturing the algae with limited nutrients, high light exposure and/or reducing the dilution rate in the raceway pond. An algal slurry is produced in a harvest stage, and the method also includes an extraction phase where the algal slurry is suspended in an alkyl-ester solvent, such as ethyl acetate, which is separated from the water phase and then evaporated, saponified and filtered. The carotenoids may be xanthophylls. A method of producing copper chlorophyllin salts is also disclosed, in which an organic non-ester is used as the extraction solvent, such as ethanol.

Description

CAROTENOID PRODUCTION METHODS All documents cited herein are incorporated by reference in their entirety. 5 TECHNICAL FIELD The present invention relates to methods for producing carotenoids by culturing algae in land-based mariculture.

BACKGROUND

Natural 0-carotene is commercially isolated from many different sources including hypersaline microalgae, fungus, and fruit. Commercial 13-carotene production includes isolation from DunaIlene sauna (hypersaline chlorophyte microalga), Blakeslee trispora (fungus), Daucus carota (root vegetable) and a large selection of fruit such as Elaeis guineensis (oil palm), Citrus sinensis (oranges) or Man gifera id/ca (mango) among many other fruit and vegetables.

13-carotene is also produced by chemical synthesis since the 1950s through the combination of two 15-carbon phosphonium salts being added on each side of a 10-carbon dialdehyde in the Wittig reaction, with subsequent isomerization to create a symmetric compound (Ernst, Henrich and Keller, 2003). Alternatively, it is produced via the Grignard reaction to combine a long-chain diketone with two methanol molecules. However, large quantities (at very large doses with an LD50 >5,000 mg/kg) of pure chemically synthesized 13-carotene (often used as a pure colorant) have been implicated in exacerbation of UV-carcinogenesis for example (Black et al., 2020). For this reason, biological 13-carotene extracts or those in combination with natural a-tocopherols are preferred for human dietary supplementation, as these show significantly less carcinogenic exacerbation.

Natural 0-carotene extracts often contain a large number of isomers created during biosynthesis, metabolic modification or as the result of the extraction conditions. These include amongst others all-trans, 9-cis, 13-cis, 15-cis, 5,13-cis, 8,9-cis (C-carotene) as well as similar carotenoids such as a-carotene, lycopene, and lutein. Depending on the method of extraction these natural extracts may also contain traces of chlorophyll and its derivatives. Due to the higher degree of intestinal absorption of all-trans 13-carotene relative to other isomers such as 9-cis 0-carotene for example, generally all-trans sources of 13-carotene are preferred for human nutritional supplementation (Johnson et al., 1997).

In humans, 3-carotene is the primary precursor of vitamin A and therefore often referred to as provitamin-A and is implicated in retinal photoreception but also proper functioning of brain health, the immune system, treatment of erythropoietic protoporphyria (sun sensitivity), and in the prevention of metabolic syndrome related diseases. Because of its biomedical value (3-carotene is a high value product for human consumption and there is a well-documented commercial market for natural 13-carotene extracts (Grune et al, 2010).

In Europe 0-carotene has been evaluated as a food additive by the Joint FAO/WHO Expert Committee on Food Additives (JEFCA) in 2001 and by the Scientific Committee on Food (SCF) in 1997 and 2000, assigned an E-number (E160a (ii) -for 0-carotene extracts) and re-evaluated the assignment of Acceptable Daily Intake (ADI) in the Commission Directive 2008/128/EC (for regular consumption <10 mg / day). In the United States the FDA has recognized it as a 'generally recognized as safe' (GRAS) substance for food additives.

Because of the lipophilic nature of 0-carotene, there are multiple commercial extraction methods from biomass including super-critical and sub-critical carbon-dioxide extraction, solvent extraction and oil extraction (Mendes et al, 1995).

Milking' of live microalgae by passing these through food grade oils, where the 0-carotene transfers from the algae to the oil without disrupting the cells, has been evaluated. These extraction methods are often accompanied by pre-treatment methods to mechanically break or disrupt complex plant cell walls such as ultra-sonication, high-pressure homogenization (HPH), shear or grinding. Electro-technology-assisted extraction methods, such as pulsed electric field (PEF), moderate electric field (MEF), high-voltage electric discharges (HVED) or pressurized liquid extraction (PLE), and microwave-assisted extraction (MAE) have also been used. To increase the extraction efficiency, wet or dry biomass is sometimes emulsified in a solvent or solvent mixes often with the addition of process aides such as proteases or emulsification agents (Correa et al, 2020; Gupta et al, 2021).

Many organic solvents such acetone, hexane, chloroform or dichloromethane have been used to extract carotenoids from biomass. Food grade organic solvents such as acetic-esters of C1-C4 alcohols, hot ethanol as well as 'green' solvents like limonene have been used. Similarly, food oils such as olive, canola or other vegetable oils are used for extraction (Ghazi, 1999).

After extraction into an organic phase, many additional purification steps are used to remove carbohydrates, lipids, proteins, water, other carotenoids and pigments. These purification steps often include the preparation of oleoresins, hydrolysis of biological molecules and alkaline saponification. Multiple physical concentration methods through semi-permeable membranes, precipitation and evaporation are common (Correa at al, 2020).

There is a need for more efficient methods for producing high quality natural carotenoid extracts. SUMMARY OF THE INVENTION The invention relates to a production system based on culturing algae in raceway ponds to create a very high-throughput, highly scalable and highly sustainable production and extraction system with the minimum of processing steps to produce very high-quality natural carotenoid extracts.

The inventors' methods combine: (1) high-throughput algal culture, (2) high-throughput harvesting to result in a live slurry of concentrated algal cells, (3) carotenoid extraction from fresh wet biomass and (4) purification and separation of carotenoids with minimal processing steps.

The high-throughput algal culture system comprises two principal phases. In the first phase, algae are maintained in the exponential growth phase by successive dilution with seawater and nutrient addition. The algae typically contain low levels of carotenoids during this phase. In the second phase, the cultivation conditions are adjusted to deliberately induce the formation of carotenoids, typically by stressing the cells through nutrient limitation and/or increasing light exposure. The 'in-sequence' switch from maximizing growth to maximizing carotenoid content at the end of a connected series of raceway ponds results in highly efficient carotenoid production.

The inventors have provided guidance on methods for culturing algae in United Kingdom Patent Application Nos. 2207837.2, 2212805.2 and 2212809.4 (each of which is incorporated herein by reference) The invention provides a method for producing a carotenoid or pigment wherein the method comprises: (a) a first algal culture phase, wherein the first algal culture phase comprises culturing marine carotenoid-producing microalgae in a series of connected raceway ponds, arranged in stages, and wherein the algae are maintained in the exponential growth phase by successive dilution with seawater and nutrient addition, (b) a second algal culture phase, wherein the second algal culture phase comprises culturing algae in a raceway pond that is connected to the series of connected raceway ponds in which algae are cultured in step (a), and wherein the algae are induced to produce the carotenoid or pigment by culturing the algae with limited nutrients, high light exposure and/or reducing the dilution rate in the raceway pond, (c) a harvest phase, wherein the harvest phase comprises harvesting algae from the raceway pond in which algae are cultured in step (b) to produce an algal slurry, and (d) an extraction phase, wherein the extraction phase comprises extracting the carotenoid or pigment from the algal slurry produced in step (c), wherein said extracting comprises: (i) suspending the algal slurry in an alkyl-ester solvent, (ii) separating the alkyl-ester solvent phase from the water phase, (iii) evaporating the alkyl-ester solvent to generate an oleoresin, wherein water is added before or during evaporation, (iv) saponifying the oleoresin to generate a slurry that comprises the carotenoid or pigment, and (v) filtering the slurry to generate a filter cake that comprises the carotenoid or pigment.

In some embodiments, the method is for producing a carotenoid, optionally wherein the carotenoid is beta-carotene.

In some embodiments, the marine carotenoid-producing microalgae are Chlorophyta.

Chlorarachniophyta, Haptophyta, Dinophyta, Euglenophyta, Heterokontophyta (e.g. Chrysophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, Xanthophyceae or Eustigmatophyceae), Cryptophyta, unicellular Rhodophyta, Glaucophyta or Cyanophyta, optionally wherein the marine carotenoid-producing microalgae are: (i) Rhopalodiaceae sp., Hem/au/us sp., Climacodium sp., Skeletonema sp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp. or Nitzschia sp., or (ii) Dunaliella sp.

In some embodiments, the series of connected raceway ponds in step (a) comprises a first stage comprising one or more covered raceway ponds and a second stage comprising one or more stages of open raceway ponds.

In some embodiments, one or more nutrient mineral acids are added during step (a), optionally wherein the one or more nutrient mineral acids are selected from nitric acid, phosphoric acid and silicic acid.

In some embodiments, the algal slurry produced in step (c) is acidified before step (d)(i) is performed, optionally wherein the algal slurry is acidified to pH 4 or lower.

In some embodiments, the alkyl-ester solvent in step (d)(i) is ethyl acetate.

In some embodiments, step (d)(i) further comprises agitating the suspended slurry.

In some embodiments, the algal slurry is suspended in the solvent in step (d)(i) to give a 10-100 g/L dry weight suspension.

In some embodiments, step (d)(ii) is performed by centrifugation, optionally using a three-phase (liquid-solid-liquid) centrifuge, optionally further wherein the separated solid phase is incorporated into protein powder.

In some embodiments, step (d)(ii) further comprises depth filtration of the alkyl-ester solvent phase. In some embodiments, the evaporation in step (d)(iii) is performed by vacuum evaporation.

In some embodiments, the water is added to the alkyl-ester solvent in a volume ratio of 0.2-5 to 1, such as 1:1.

In some embodiments, step d(iii) further comprises recovering the evaporated alkyl-ester solvent. In some such embodiments, step d(iii) further comprises (A) passing the evaporated solvent through one or more drying columns, and/or (B) passing the condensed solvent through one or more activated carbon columns In some embodiments, step d(v) further comprises washing and/or drying the filter cake, optionally wherein said drying is under vacuum.

In some embodiments, step (d)(iv) is performed by adding concentrated alkali solution (e.g. a concentrated alkali metal hydroxide solution), such as NaOH (50 wt%), optionally to a final concentration of 0.5-5 M NaOH (e.g. 1-2.5 M NaOH).

The invention further provides a method for producing a xanthophyll, wherein the method comprises: (a) a first algal culture phase, wherein the first algal culture phase comprises culturing marine carotenoid-producing microalgae in a series of connected raceway ponds, arranged in stages, and wherein the algae are maintained in the exponential growth phase by successive dilution with seawater and nutrient addition, (b) a second algal culture phase, wherein the second algal culture phase comprises culturing algae in a raceway pond that is connected to the series of connected raceway ponds in which algae are cultured in step (a), and wherein the algae are induced to produce the carotenoid or pigment by culturing the algae with limited nutrients, high light exposure and/or reducing the dilution rate in the raceway pond, (c) a harvest phase, wherein the harvest phase comprises harvesting algae from the raceway pond in which algae are cultured in step (b) to produce an algal slurry, and (d) an extraction phase, wherein the extraction phase comprises extracting the carotenoid or pigment from the algal slurry produced in step (c), wherein said extracting comprises: (i) suspending the algal slurry in an alkyl-ester solvent, (i) separating the alkyl-ester solvent phase from the water phase, (iii) evaporating the alkyl-ester solvent to generate an oleoresin, wherein water is added before or during evaporation, (iv) saponifying the oleoresin to generate a slurry that comprises the carotenoid or pigment, (v) filtering the slurry to generate a filter cake that comprises the carotenoid or pigment, (vi) adding an organic non-ester solvent to the filtrate obtained in step (v), (vii) separating the organic non-ester solvent phase from the water phase, (viii) evaporating the organic non-ester solvent to generate an oleoresin that comprises the xanthophyll, and (ix) drying the oleoresin.

In preferred embodiments, step (d)(iv) comprises adding concentrated alkali solution (e.g. a concentrated alkali metal hydroxide solution). In some such embodiments, step (d)(iv) is performed by adding concentrated alkali solution, such as NaOH (50 wt%), optionally to a final concentration of 0.55 M NaOH (e.g. 1-2.5 M NaOH).

In some embodiments, the marine carotenoid-producing microalgae are Chlorophyta, Chlorarachniophyta, Haptophyta, Dinophyta, Euglenophyta, Heterokontophyta (e.g. Chrysophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, Xanthophyceae or Eustigmatophyceae), Cryptophyta, unicellular Rhodophyta, Glaucophyta or Cyanophyta, optionally wherein the marine carotenoid-producing microalgae are: (i) Rhopalodiaceae sp., Hem/au/us sp., Climacodium sp., Skeletonema sp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp. or Nitzschia sp., or (ii) DunaBella sp.

In some embodiments, the alkyl-ester solvent in step (d)(i) is ethyl acetate.

In some embodiments, step (d)(ii) further comprises depth filtration of the alkyl-ester solvent phase.

In some embodiments, the evaporation in step (d)(iii) is performed by vacuum evaporation.

In some embodiments, the organic non-ester solvent in step d(vi) is: (i) a (food grade) aliphatic hydrocarbon, (ii) a (food grade) monoterpene, (iii) (food grade) hexane, (iv) (food grade) limonene, or (v) (food grade) p-cymene.

In some embodiments, the xanthophyll is lutein. In some such embodiments, the marine carotenoid-producing microalgae are Chlorophyta (e.g. Dunaliella sp.) or Chlorarachniophyta.

In some embodiments, the xanthophyll is: (A) diadinoxanthin, optionally wherein the marine carotenoid-producing microalgae are Heterokontophyta (e.g. Chrysophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, or Xanthophyceae), Haptophyta, Dinophyta or Euglenophyta, (B) zeaxanthin, optionally wherein the marine carotenoid-producing microalgae are Chlorophyta, Chlorarachniophyta, Haptophyta, Dinophyta, Euglenophyta, Heterokontophyta (e.g., Chrysophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, or Xanthophyceae), Cryptophyta, unicellular Rhodophyta, Glaucophyta or Cyanophyta, optionally further wherein the marine carotenoid-producing microalgae are: (i) Rhopalodiaceae sp., Hemiaulus sp., Climacodium sp., Skeletonema spp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp., or Nitzschia sp., or (ii) Dunaliella sp., or (iii) Trichodesmium sp., Richelia sp., Calothrix sp., Crocosphaera sp or Candidatus Atelocyanobacterium Thalassa, (C) violaxanthin, optionally wherein the marine carotenoid-producing microalgae are Chlorophyta (e.g. Duna sp.), Chlorarachniophyta, or Heterokontophyta (e.g. Raphidophyceae, Phaeophyceae, or Eustigmatophyceae), (D) neoxanthin, optionally wherein the marine carotenoid-producing microalgae are Euglenophyta, Chlorophyta (e.g. Dunariella sp.) or Chlorarachniophyta, (E) fu coxa nth in, optionally wherein the marine carotenoid-producing microalgae are Heterokontophyta (e.g. Chrysophyceae, Raphidophyceae, Bacillariophyceae, or Phaeophyceae), Haptophyta, or Dinophyta, optionally further wherein the marine carotenoid-producing microalgae are Rhopalodiaceae sp. Hemiaulus sp., Climacodium sp., Skeletonema spp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp. or Nitzschia sp., (F) vaucheriaxanthin, optionally wherein the marine carotenoid-producing microalgae are Heterokontophyta (e.g. Chrysophyceae or Eustigmatophyceae), (G) loroxanthin, optionally wherein the marine carotenoid-producing microalgae are Euglenophyta, Chlorophyta (e.g. Prasinophyceae, Chlorophyceae or Ulvophyceae) or Chlorarachniophyta, (H) siphonaxanthin, optionally wherein the marine carotenoid-producing microalgae are Euglenophyta or Chlorophyta (e.g., Prasinophyceae, Chlorophyceae or Ulvophyceae), (I) one or more of nostoxanthin, echinenone, myxol glycosides and oscillol glycosides, optionally wherein the marine carotenoid-producing microalgae are Cyanophyta, optionally further wherein the marine carotenoid-producing microalgae are Trichodesmium sp., Richelia sp., Calothrix sp., Crocosphaera sp. or Candidatus Atelocyanobacterium Thalassa, (J) one or more of alloxanthin, crocoxanthin and monadoxanthin, optionally wherein the marine carotenoid-producing microalgae are Cryptophyta, (K) violaxanthin fatty acid ester, optionally wherein the marine carotenoid-producing microalgae are Heterokontophyta (e.g., Xanthophyceae), (L) fucoxanthin fatty acid ester, optionally wherein the marine carotenoid-producing microalgae are Haptophyta,

S

(M) peridinin, optionally wherein the marine carotenoid-producing microalgae are Dinophyta, (N) prasinoxanthin, optionally wherein the marine carotenoid-producing microalgae are Chlorophyta (e.g., Prasinophyceae), (0) loroxanthin fatty acid ester, optionally wherein the marine carotenoid-producing microalgae are Chlorophyta (e.g,. Prasinophyceae) or Chlorarachniophyta, or (P) siphonaxanthin fatty acid ester, optionally wherein the marine carotenoid-producing microalgae are Chlorophyta (e.g. Prasinophyceae, Chlorophyceae or Ulvophyceae).

The invention further provides a method for producing copper chlorophyllin salts, wherein the method comprises: (a) a first algal culture phase, wherein the first algal culture phase comprises culturing marine carotenoid-producing microalgae in a series of connected raceway ponds, arranged in stages, and wherein the algae are maintained in the exponential growth phase by successive dilution with seawater and nutrient addition, (b) a second algal culture phase, wherein the second algal culture phase comprises culturing algae in a raceway pond that is connected to the series of connected raceway ponds in which algae are cultured in step (a), and wherein the algae are induced to produce carotene by culturing the algae with limited nutrients, high light exposure and/or reducing the dilution rate in the raceway pond, (c) a harvest phase, wherein the harvest phase comprises harvesting algae from the raceway pond in which algae are cultured in step (b) to produce an algal slurry, and (d) an extraction phase, wherein the extraction phase comprises extracting the carotene from the algal slurry produced in step (c), wherein said extracting comprises: (i) suspending the algal slurry in an alkyl-ester solvent, (ii) separating the alkyl-ester solvent phase from the water phase, (iii) evaporating the alkyl-ester solvent to generate an oleoresin, wherein water is added before or during evaporation, (iv) saponifying the oleoresin to generate a slurry that comprises the carotene, (v) filtering the slurry to generate a filter cake that comprises the carotene, (vi) adding an organic non-ester solvent to the filtrate obtained in step (v), (vii) separating the water phase from the organic non-ester solvent phase, (viii) mixing the water phase with one or more copper salts to precipitate copper chlorophyllin salts, and (ix) collecting the precipitated copper chlorophyllin salts.

In some embodiments, the marine carotenoid-producing microalgae are Chlorophyta. Chlorarachniophyta, Haptophyta, Dinophyta, Euglenophyta, Heterokontophyta (e.g. Chrysophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, Xanthophyceae or Eustigmatophyceae), Cryptophyta, unicellular Rhodophyta, Glaucophyta or Cyanophyta, optionally wherein the marine carotenoid-producing microalgae are: (i) Rhopalodiaceae sp., Hemiaulus sp., Climacodium sp., Skeletonema sp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp. or Nitzschia sp., or (ii) DunaBella sp.

In some embodiments, the alkyl-ester solvent in step (d)(i) is ethyl acetate.

In some embodiments, step d(iii) further comprises recovering the evaporated alkyl-ester solvent. In some such embodiments, step d(iii) further comprises (A) passing the evaporated solvent through one or more drying columns, and/or (B) passing the condensed solvent through one or more activated carbon columns In some embodiments, step (d)(iv) comprises adding concentrated alkali solution (e.g. a concentrated alkali metal hydroxide solution). In some such embodiments, step (d)(iv) is performed by adding concentrated alkali solution, such as NaOH (50 wt%), optionally to a final concentration of 0.5-5 M NaOH (e.g. 1-2.5 M NaOH).

In some embodiments, step d(v) further comprises washing and/or drying the filter cake, optionally wherein said drying is under vacuum.

In some embodiments, step (viii) further comprises acidifying the water phase to pH 2-3 (e.g. by the addition of concentrated hydrochloric acid).

In some embodiments, the one or more copper salts in step (d)(viii) comprise copper sulphate.

In some embodiments, step (d) further comprises: (x) dissolving the precipitated copper chlorophyllin salts in (95%) ethanol, (xi) adjusting the pH to pH 10-12 (e.g. pH 11) by the addition of sodium hydroxide to obtain sodium copper chlorophyllin, and (xii) filtering, washing and drying the sodium copper chlorophyllin.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates an example raceway pond according to the invention.

Figure 2A illustrates an algal cultivation system and depicts a series of connected raceway ponds arranged in stages according to the invention. Figure 2B illustrates a subsection of the algal cultivation system of Figure 2A.

Figure 3 illustrates exemplary methods according to the invention.

Figure 4A is a chromatogram for crude carotenoid extract. Figure 4B is a chromatogram for carotenoid powder 84% all-trans beta carotene. Figure 4C is a chromatogram for BASF Betatene® 30% OLV, 72% all-trans beta carotene.

Figure 5A is a chromatogram for lutein extract (45 min run time). Figure 5B is a chromatogram for lutein extract (60 min run time).

Figure 6 is a chromatogram for crude extract obtained from Skeletonema pseudocostatum.

Figure 7 provides structural formulae for some exemplary carotenoids. This figure is reproduced from Takaichi 2011.

Figure 8 provides a summary of carotenoid distribution in algae. This figure is reproduced from Takaichi 2011. (Key: H, Major carotenoid in most species of the class; L, Low content in most species or major carotenoid in some species. a, a-carotene; 3-carotene; Al, alloxanthin; Cr, crocoxanthin; Da, diatoxanthin; Dd, diadinoxanthin; Ec, echinenone; FA, fatty acid ester; Fx, fucoxanthin; Lo, loroxanthin; Lu, lutein; Mo, monadoxanthin; My, myxol glycosides and oscillol glycosides; Ne, neoxanthin; No, nostoxanthin; Pe, peridinin* Pr, prasinoxanthin; Sx, siphonaxanthin; Va, vaucheriaxanthin; Vi, violaxanthin; Ze, zeaxanthin). Lu, Lo, Lo-FA, Cr, Mo, Pr, Sx and Sx-FA are a-carotene derivatives.

DETAILED DESCRIPTION OF THE INVENTION

S

Carotenes Carotenes are one of the two major divisions of carotenoids. The other division is formed by the xanthophylls. While carotenes are purely hydrocarbons, xanthophylls contain oxygen atoms, typically as a hydroxyl group.

Carotenes are terpenoids assembled from eight isoprene units resulting in 40 branched and double bonded carbon atoms, capped with beta-ionone rings at each end. In nature beta-carotene is biosynthesized from geranylgeranyl pyrophosphate. Like most of the naturally occurring carotenoids, it is strongly colored and highly lipophilic (since it lacks functional groups).

The invention provides a method for producing a carotene, wherein the method comprises: (a) a first algal culture phase, wherein the first algal culture phase comprises culturing marine carotenoid-producing microalgae in a series of connected raceway ponds, arranged in stages, and wherein the algae are maintained in the exponential growth phase by successive dilution with seawater and nutrient addition, (b) a second algal culture phase, wherein the second algal culture phase comprises culturing algae in a raceway pond that is connected to the series of connected raceway ponds in which algae are cultured in step (a), and wherein the algae are induced to produce the carotene by culturing the algae with limited nutrients, high light exposure and/or reducing the dilution rate in the raceway pond, (c) a harvest phase, wherein the harvest phase comprises harvesting algae from the raceway pond in which algae are cultured in step (b) to produce an algal slurry, and (d) an extraction phase, wherein the extraction phase comprises extracting the carotene from the algal slurry produced in step (c), wherein said extracting comprises: (i) suspending the algal slurry in an alkyl-ester solvent, 00 separating the alkyl-ester solvent phase from the water phase, (iii) evaporating the alkyl-ester solvent to generate an oleoresin, wherein water is added before or during evaporation, (iv) saponifying the oleoresin to generate a slurry that comprises the carotene, and (v) filtering the slurry to generate a filter cake that comprises the carotene.

In some embodiments, the alkyl-ester solvent in step d(i) is ethyl acetate. In some embodiments, the alkyl-ester solvent in step d(i) is food-grade ethyl acetate. In some embodiments, the alkyl-ester solvent in step d(i) is 100% food-grade ethyl acetate.

In some embodiments, step (d)(i) further comprises agitating the suspended slurry. This is advantageous when the microalgae are difficult to break open, such as when Dunaliella sauna has formed storage cysts. In contrast, many diatoms have silicate frustules (cell walls) that are similar to a fine mesh, and so do not require agitation to extract carotenoids.

In some embodiments, the algal slurry comprises at least 10% dry weight, wet biomass. In some such embodiments, suspension of the algal slurry in the alkyl-ester solvent (e.g., (100% food-grade) ethyl acetate) provides a 10-100 g/L 40-60 g/L) dry weight suspension.

Advantageously, this facilitates the removal of particulate biomass.

In some embodiments, the water is added to the alkyl-ester solvent in a volume ratio of 0.2-5 to 1, such as 1:1. Advantageously, this reduces the quantity of oleoresin that adheres to vessel walls to facilitate further processing and to avoid a second resuspension process.

In some embodiments, step (d)(iii) is performed by vacuum evaporation. Advantageously, this facilitates performance of the method at lower temperatures, thereby preventing isomerisafion of the beta-carotene Onto cis-17 and cis-19).

In some embodiments, the alkyl-ester solvent is condensed, recaptured and recycled. In some such embodiments, the solvent is passed through a drying column (e.g., a solid phase that selectively absorbs hydrophilic material). This is to ensure that it is dry and clean before reuse. In some such embodiments, the condensed solvent is passed through one or more activated carbon columns. This acts, for example, to remove organic impurities, flavours and off-colour products.

In some embodiments, step (d)(iv) is performed by adding concentrated alkali solution, such as a concentrated alkali metal hydroxide solution (e.g. concentrated NaOH or KOH).

In some embodiments, step (d)(iv) is performed by adding concentrated alkali solution, such as NaOH (50 wt%), optionally to a final concentration of 0.5-5 M NaOH (e.g., 1-2.5 M NaOH).

In some embodiments, step (d)(iv) is performed by adding concentrated alkali solution, such as KOH (50 wt%), optionally to a final concentration of 0.5-5 M KOH (e.g., 1-2.5 M KOH).

In some embodiments, step d(iv) is performed at 40-80°C (e.g., 55-65°C). In some such embodiments, step d(iv) is performed for at least 30 minutes (e.g., 60 minutes). In some such embodiments, step d(iv) is performed for 30-90 minutes (e.g., 45-75 minutes).

In preferred embodiments, the extraction phase is performed under an inert gas (e.g., a nitrogen blanket). This prevents oxidation of the extracts.

In preferred embodiments, the carotene is beta-carotene. In some such embodiments, the marine carotenoid-producing microalgae are Cyanophyta, Glaucophyta, unicellular Rhodophyta, Heterokontophyta, Haptophyta, Dinophyta, Euglenophyta, Chlorarachniophyta or Chlorophyta (e.g., Prasinophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae or Charophyceae). In some such embodiments, the marine carotenoid-producing microalgae are Cyanophyta, Glaucophyta, unicellular Rhodophyta, Heterokontophyta, Haptophyta, Euglenophyta, Chlorarachniophyta or Chlorophyta (e.g., Prasinophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae or Charophyceae).

In some such embodiments, the marine carotenoid-producing microalgae are: (i) Rhopalodiaceae sp., Hem/au/us sp., Climacodium sp., Skeletonema sp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Nayicula sp., Synedra sp. or Nitzschia sp., or (ii) DunaBella sp..

In some such embodiments, the marine carotenoid-producing microalgae are Dunaliege sp., such as Dunaliella saline.

In some embodiments, the carotene is alpha-carotene. In some such embodiments, the marine carotenoid-producing microalgae are Cyanophyta, Cryptophyta, Prasinophyceae, Chlorophyceae or Ulvophyceae. In some such embodiments, the marine carotenoid-producing microalgae are Cryptophyta or Chlorophyceae.

Xanthophylls Xanthophylls are one of the two major divisions of carotenoids. The other division is formed by the carotenes. While carotenes are purely hydrocarbons, xanthophylls contain oxygen atoms, typically as a hydroxyl group.

The invention provides a method for producing a xanthophyll, wherein the method comprises: (a) a first algal culture phase, wherein the first algal culture phase comprises culturing marine carotenoid-producing microalgae in a series of connected raceway ponds, arranged in stages, and wherein the algae are maintained in the exponential growth phase by successive dilution with seawater and nutrient addition, (b) a second algal culture phase, wherein the second algal culture phase comprises culturing algae in a raceway pond that is connected to the series of connected raceway ponds in which algae are cultured in step (a), and wherein the algae are induced to produce carotene by culturing the algae with limited nutrients, high light exposure and/or reducing the dilution rate in the raceway pond, (c) a harvest phase, wherein the harvest phase comprises harvesting algae from the raceway pond in which algae are cultured in step (b) to produce an algal slurry, and (d) an extraction phase, wherein the extraction phase comprises extracting the carotene from the algal slurry produced in step (c), wherein said extracting comprises: (i) suspending the algal slurry in an alkyl-ester solvent, (ii) separating the alkyl-ester solvent phase from the water phase, (iii) evaporating the alkyl-ester solvent to generate an oleoresin, wherein water is added before or during evaporation, (iv) saponifying the oleoresin to generate a slurry that comprises the carotene, (v) filtering the slurry to generate a filter cake that comprises the carotene, (vi) adding an organic non-ester solvent to the filtrate obtained in step (v), (vii) separating the water phase from the organic non-ester solvent phase, (viii) evaporating the organic non-ester solvent to generate an oleoresin that comprises the xanthophyll, and (ix) drying the oleoresin.

In some embodiments, the alkyl-ester solvent in step de) is ethyl acetate. In some embodiments, the alkyl-ester solvent in step d(i) is food-grade ethyl acetate.

The organic non-ester solvent in step d(vi) preferably displays volatility and immiscibility with water. In addition, the solvent is preferably non-hydrolysable under basic conditions.

In some embodiments, the organic non-ester solvent in step d(vi) is an aliphatic hydrocarbon. In some embodiments, the solvent is a food grade aliphatic hydrocarbon.

In some embodiments, the organic non-ester solvent in step d(vi) is a monoterpene. In some embodiments, the solvent is a food grade monoterpene.

In some embodiments, the organic non-ester solvent in step d(vi) is selected from hexane and limonene. In some embodiments, the organic non-ester solvent in step d(vi) is selected from food grade hexane and food grade limonene.

In some embodiments, the organic non-ester solvent in step d(vi) is hexane. In some embodiments, the solvent is food grade hexane.

In some embodiments, the organic non-ester solvent in step d(vi) is limonene. In some embodiments, the solvent is food grade limonene.

In some embodiments, the organic non-ester solvent in step d(vi) is p-cymene. In some embodiments, the solvent is food grade p-cymene.

In some embodiments, the xanthophyll is lutein. In some such embodiments, the marine carotenoid-producing microalgae are Chlorophyta (e.g., Dunaliella sp.) or Chlorarachniophyta. In some such embodiments, the marine carotenoid-producing microalgae are Chlorophyceae, Trebouxiophyceae or Charophyceae.

In some embodiments, the xanthophyll is diadinoxanthin. In some such embodiments, the marine carotenoid-producing microalgae are Heterokontophyta (e.g., Chrysophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, or Xanthophyceae), Haptophyta, Dinophyta or Euglenophyta. In some such embodiments, the marine carotenoid-producing microalgae are Rhopalodiaceae sp. Hemiaulus sp., Climacodium sp., Skeletonema spp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp. or Nitzschia sp.

In some embodiments, the xanthophyll is diadinoxanthin and the marine carotenoid-producing microalgae are Xanthophyceae, Haptophyta, Dinophyta or Euglenophyta.

In some embodiments, the xanthophyll is zeaxanthin. In some such embodiments, the marine carotenoid-producing microalgae are Chlorophyta, Chlorarachniophyta, Haptophyta, Dinophyta, Euglenophyta, Heterokontophyta (e.g., Chrysophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, or Xanthophyceae), Cryptophyta, unicellular Rhodophyta, Glaucophyta or Cyanophyta. In some such embodiments, the marine carotenoid-producing microalgae are (i) Rhopalodiaceae sp., Hemiaulus sp., Climacodium sp., Skeletonema spp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp. or Nitzschia sp., (ii) Dunaliella sp.

In some embodiments, the xanthophyll is zeaxanthin and the marine carotenoid-producing microalgae are Cyanophyta, Glaucophyta, unicellular Rhodophyta, Raphidophyceae or Phaeophyceae.

In some embodiments, the xanthophyll is violaxanthin. In some such embodiments, the marine carotenoid-producing microalgae are Chlorophyta (e.g., Dunaliella sp.), Chlorarachniophyta, or Heterokontophyta (e.g., Raphidophyceae, Phaeophyceae, or Eustigmatophyceae). In some such embodiments, the marine carotenoid-producing microalgae are Chlorophyta (e.g., Prasinophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae or Charophyceae), Phaeophyceae or Eustigmatophyceae.

In some embodiments, the xanthophyll is neoxanthin. In some such embodiments, the marine carotenoid-producing microalgae are Euglenophyta, Chlorophyta (e.g., Dunaliella sp.) or Chlorarachniophyta. In some such embodiments, the marine carotenoid-producing microalgae are Chlorophyta (e.g., Prasinophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae or Charophyceae).

In some embodiments, the xanthophyll is fucoxanthin. In some such embodiments, the marine carotenoid-producing microalgae are Heterokontophyta (e.g., Chrysophyceae, Raphidophyceae, Bacillariophyceae, or Phaeophyceae), Haptophyta, or Dinophyta. In some such embodiments, the marine carotenoid-producing microalgae are Rhopalodiaceae sp. Hemiaulus sp., Climacodium sp., Skeletonema spp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp. or Nitzschia sp.

In some embodiments, the xanthophyll is fucoxanthin and the marine carotenoid-producing microalgae are Chrysophyceae, Bacillariophyceae, Phaeophyceae or Haptophyta.

In some embodiments, the xanthophyll is vaucheriaxanthin. In some such embodiments, the marine carotenoid-producing microalgae are Heterokontophyta (e.g., Chrysophyceae or Eustigmatophyceae).

In some embodiments, the xanthophyll is loroxanthin. In some such embodiments, the marine carotenoid-producing microalgae are Euglenophyta, Chlorophyta (e.g., Prasinophyceae, Chlorophyceae or Ulvophyceae) or Chlorarachniophyta, In some embodiments, the xanthophyll is siphonaxanthin. In some such embodiments, the marine carotenoid-producing microalgae are Euglenophyta or Chlorophyta (e.g., Prasinophyceae, Chlorophyceae or Ulvophyceae). In some such embodiments, the marine carotenoid-producing microalgae are Prasinophyceae.

In some embodiments, the xanthophyll is one or more of nostoxanthin, echinenone, myxol glycosides and oscillol glycosides. In some such embodiments, the marine carotenoid-producing microalgae are Cyanophyta.

In some embodiments, the xanthophyll is one or more of echinenone, myxol glycosides and oscillol glycosides. In some such embodiments, the marine carotenoid-producing microalgae are Cyanophyta.

In some embodiments, the xanthophyll is one or more of alloxanthin, crocoxanthin and monadoxanthin. In some such embodiments, the marine carotenoid-producing microalgae are Cryptophyta.

In some embodiments, the xanthophyll is violaxanthin fatty acid ester. In some such embodiments, the marine carotenoid-producing microalgae are Heterokontophyta (e.g., Xanthophyceae), In some embodiments, the xanthophyll is fucoxanthin fatty acid ester. In some such embodiments, the marine carotenoid-producing microalgae are Haptophyta.

In some embodiments, the xanthophyll is peridinin. In some such embodiments, the marine carotenoid-producing microalgae are Dinophyta.

In some embodiments, the xanthophyll is prasinoxanthin. In some such embodiments, the marine carotenoid-producing microalgae are Chlorophyta (e.g., Prasinophyceae), In some embodiments, the xanthophyll is loroxanthin fatty acid ester. In some such embodiments, the marine carotenoid-producing microalgae are Chlorophyta (e.g., Prasinophyceae) or Chlorarachniophyta.

In some embodiments, the xanthophyll is siphonaxanthin fatty acid ester. In some such embodiments, the marine carotenoid-producing microalgae are Chlorophyta (e.g., Prasinophyceae, Chlorophyceae or Ulvophyceae). In some such embodiments, the marine carotenoid-producing microalgae are Prasinophyceae or Ulvophyceae.

In some embodiments, the marine carotenoid-producing microalgae are Cyanophyta. In some such embodiments, the xanthophyll is one or more of zeaxanthin, nostoxanthin, echinenone, myxol glycosides and oscillol glycosides. In some such embodiments, the xanthophyll is one or more of zeaxanthin, echinenone, myxol glycosides and oscillol glycosides.

In some embodiments, the marine carotenoid-producing microalgae are Glaucophyta. In some such embodiments, the xanthophyll is zeaxanthin.

In some embodiments, the marine carotenoid-producing microalgae are Rhodophyta. In some such embodiments, the xanthophyll is zeaxanthin.

In some embodiments, the marine carotenoid-producing microalgae are Cryptophyta. In some such embodiments, the xanthophyll is one or more of alloxanthin, crocoxanthin and monadoxanthin.

In some embodiments, the marine carotenoid-producing microalgae are Chrysophyceae. In some such embodiments, the xanthophyll is one or more of zeaxanthin, diatoxanthin, diadinoxanthin, fucoxanthin and vaucheriaxanthin. In some such embodiments, the xanthophyll is fucoxanthin.

In some embodiments, the marine carotenoid-producing microalgae are Raphidophyceae. In some such embodiments, the xanthophyll is one or more of zeaxanthin, violaxanthin, diatoxanthin, diadinoxanthin and fucoxanthin. In some such embodiments, the xanthophyll is zeaxanthin.

In some embodiments, the marine carotenoid-producing microalgae are Bacillariophyceae. In some such embodiments, the xanthophyll is one or more of zeaxanthin, diatoxanthin, diadinoxanthin and fucoxanthin. In some such embodiments, the xanthophyll is fucoxanthin.

In some embodiments, the marine carotenoid-producing microalgae are Phaeophyceae. In some such embodiments, the xanthophyll is one or more of zeaxanthin, violaxanthin, diatoxanthin, diadinoxanthin and fucoxanthin. In some such embodiments, the xanthophyll is one or more of zeaxanthin, violaxanthin and fucoxanthin.

In some embodiments, the marine carotenoid-producing microalgae are Xanthophyceae. In some such embodiments, the xanthophyll is one or more of zeaxanthin, diatoxanthin, diadinoxanthin and violaxanthin fatty acid ester. In some such embodiments, the xanthophyll is one or more of diatoxanthin and diadinoxanthin.

In some embodiments, the marine carotenoid-producing microalgae are Eustigmatophyceae. In some such embodiments, the xanthophyll is one or more of violaxanthin and vaucheriaxanthin. In some such embodiments, the xanthophyll is violaxanthin.

In some embodiments, the marine carotenoid-producing microalgae are Haptophyta. In some such embodiments, the xanthophyll is one or more of zeaxanthin, diatoxanthin, diadinoxanthin, fucoxanthin and fucoxanthin fatty acid ester. In some such embodiments, the xanthophyll is one or more of diadinoxanthin and fucoxanthin.

In some embodiments, the marine carotenoid-producing microalgae are Dinophyta. In some such embodiments, the xanthophyll is one or more of zeaxanthin, diatoxanthin, diadinoxanthin, fucoxanthin and peridinin. In some such embodiments, the xanthophyll is one or more of diadinoxanthin and peridinin.

In some embodiments, the marine carotenoid-producing microalgae are Euglenophyta. In some such embodiments, the xanthophyll is one or more of zeaxanthin, neoxanthin, diatoxanthin, diadinoxanthin, loroxanthin and siphonaxanthin. In some such embodiments, the xanthophyll is diadinoxanthin.

In some embodiments, the marine carotenoid-producing microalgae are Chlorarachniophyta. In some such embodiments, the xanthophyll is one or more of zeaxanthin, violaxanthin, neoxanthin, lutein, loroxanthin and loroxanthin fatty acid ester.

In some embodiments, the marine carotenoid-producing microalgae are Prasinophyceae. In some such embodiments, the xanthophyll is one or more of zeaxanthin, violaxanthin, neoxanthin, lutein, loroxanthin, siphonaxanthin, prasinoxanthin, loroxanthin fatty acid ester and siphonaxanthin fatty acid ester. In some such embodiments, the xanthophyll is one or more of violaxanthin, neoxanthin, siphonaxanthin and siphonaxanthin fatty acid ester.

In some embodiments, the marine carotenoid-producing microalgae are Chlorophyceae. In some such embodiments, the xanthophyll is one or more of zeaxanthin, violaxanthin, neoxanthin, lutein, loroxanthin, siphonaxanthin and and siphonaxanthin fatty acid ester. In some such embodiments, the xanthophyll is one or more of violaxanthin, neoxanthin and lutein.

In some embodiments, the marine carotenoid-producing microalgae are Ulvophyceae. In some such embodiments, the xanthophyll is one or more of zeaxanthin, violaxanthin, neoxanthin, lutein, loroxanthin, siphonaxanthin and siphonaxanthin fatty acid ester. In some such embodiments, the xanthophyll is one or more of violaxanthin, neoxanthin and siphonaxanthin fatty acid ester.

In some embodiments, the marine carotenoid-producing microalgae are Trebouxiophyceae. In some such embodiments, the xanthophyll is one or more of zeaxanthin, violaxanthin, neoxanthin and lutein. In some such embodiments, the xanthophyll is one or more of violaxanthin, neoxanthin and lutein.

In some embodiments, the marine carotenoid-producing microalgae are Charophyceae. In some such embodiments, the xanthophyll is one or more of zeaxanthin, violaxanthin, neoxanthin and lutein. In some such embodiments, the xanthophyll is one or more of violaxanthin, neoxanthin and lutein.

Copper chlorophyllin salts The invention provides a method for producing copper chlorophyllin salts, wherein the method comprises: (a) a first algal culture phase, wherein the first algal culture phase comprises culturing marine carotenoid-producing microalgae in a series of connected raceway ponds, arranged in stages, and wherein the algae are maintained in the exponential growth phase by successive dilution with seawater and nutrient addition, (b) a second algal culture phase, wherein the second algal culture phase comprises culturing algae in a raceway pond that is connected to the series of connected raceway ponds in which algae are cultured in step (a), and wherein the algae are induced to produce carotene by culturing the algae with limited nutrients, high light exposure and/or reducing the dilution rate in the raceway pond, (c) a harvest phase, wherein the harvest phase comprises harvesting algae from the raceway pond in which algae are cultured in step (b) to produce an algal slurry, and (d) an extraction phase, wherein the extraction phase comprises extracting the carotene from the algal slurry produced in step (c), wherein said extracting comprises: (i) suspending the algal slurry in an alkyl-ester solvent, (ii) separating the alkyl-ester solvent phase from the water phase, (iii) evaporating the alkyl-ester solvent to generate an oleoresin, wherein water is added before or during evaporation, (iv) saponifying the oleoresin to generate a slurry that comprises the carotene, (v) filtering the slurry to generate a filter cake that comprises the carotene, (vi) adding an organic non-ester solvent to the filtrate obtained in step (v), (vii) separating the water phase from the organic non-ester solvent phase, (viii) mixing the water phase with one or more copper salts to precipitate copper chlorophyllin salts, and (ix) collecting the precipitated copper chlorophyllin salts.

In some embodiments, the one or more copper salts in step (d)(viii) comprise copper sulphate. In some embodiments, 10% copper sulphate is mixed with the water phase in step (viii), e.g., for at least minutes at 40-80°C to precipitate chlorophyllin salts. In some such embodiments, 10% copper sulphate is mixed with the water phase in step (viii) for 60 minutes at 60°C to precipitate chlorophyllin salts.

In some embodiments, step (d) further comprises: (x) dissolving the precipitated copper chlorophyllin salts in (95%) ethanol, (xi) adjusting the pH to pH 10-12 (e.g., pH 11) by the addition of sodium hydroxide to obtain sodium copper chlorophyllin, and (xii) filtering, washing and drying the sodium copper chlorophyllin. 40 In some embodiments, 0.5-2 M (e.g.,1 M) sodium hydroxide is added in step (d)(xi).

Raceway ponds Land-based mariculture according to the invention comprises culturing algae in at least one raceway pond 100. Preferably the cultivation water for the at least one raceway pond 100 comprises seawater, but other types of water may be used, as discussed herein.

A typical raceway pond is illustrated in Figure 1. Raceway pond 100 is stadium shaped (La, a rectangle with semicircles at a pair of opposite ends), with a partial divide in the centre of the pond 100 (i.e., along the longitudinal axis A) to create a circuit (Le., channel 120) having two longitudinal channel sections 120a, 120b which are joined at opposite ends of the raceway pond 100 by U-bend channel sections 120c, 120d. The stadium shape is defined by side wall 102. The partial divide is defined by divider 104. The side wall 102 and divider 104 may be formed of plastic covered and reinforced walls, fencing posts or earthen berms. Water and algae is retained in the raceway pond 100 by the side wall 102 and the base of the pond (not shown).

At one side of divider 104 (e.g., along a side wall 102) there may be a support structure that serves both as an anchor for a paddlewheel 110 and to anchor the divider 104 between the longitudinal channel sections 120a, 120b. Paddlewheel 110 maintains the flow of water and algae around the circuit. The paddlewheel 110 is typically a variable speed paddlewheel.

At the opposite ends of the raceway pond 100 (Le., at the semicircles of the stadium shape), the channel has U-bend channel sections 120c, 120d. Within the U-bend channel sections 120c, 120d, there are flow diverters 106 to ensure efficient flow throughout the raceway. In particular, flow diverters 106 act to maintain laminar flow of algae and water around the channel 120, especially at the U-bend channel sections 120c, 120d.

The raceway pond 100 further comprises an inlet pipe 108 and a drainpipe 112. The inlet pipe 108 may be connected to a gate-controlled sluice (not shown) for pond intake from either a seawater canal and/or a previous pond. The drainpipe 112 facilitates pond discharge through a second gate-controlled sluice (not shown). The dilution rate of the algae in the raceway pond 100, which is the rate at which water is added to the algae (i.e., to dilute the algae), is determined by the position of the gate-controlled sluice of the inlet pipe 108 and/or the drainpipe 112. For instance, opening the gate-controlled sluice of the inlet pipe 108 and closing the gate-controlled sluice of the drainpipe 112 increases the dilution rate. The dilution volume is the volume of water added to the raceway pond.

In one embodiment, raceway pond 100 may be a covered raceway pond, which is a raceway pond covered by a greenhouse (not shown). 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; both to increase the algal growth rate, and 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 wavelength of light. In one specific embodiment, every raceway pond 100 is covered by a greenhouse.

Alternatively, raceway pond 100 may be an open raceway pond. 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.

Each raceway pond 100 is able to hold a certain volume of water and algae, depending on the depth, width and length of the raceway pond 100. In the context of the invention, 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. An increase in volume of raceway pond 100 is achieved by increased width and/or length of the raceway pond. In a specific embodiment, raceway pond 100 increases in volume through increased width and length. Depth of the raceway pond 100 cannot be increased as easily since changing the depth affects algal solar irradiation.

In a specific embodiment, raceway pond 100 has a width (perpendicular to axis A) to length (along axis A) size ratio of between 1:4 and 1:12, preferably 1:8. At this ratio, there is relatively low head loss at each paddlewheel 110 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 120 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.

In some embodiments, a plurality of raceway ponds 100 may be used in parallel to increase the collective volume of water and algae. The plurality of raceway ponds 100 may form a group. The group may comprise, for example, 2, 4, 6, 8, 10, 12, 14, 16, or 18 ponds. The inlet pipe 108 of each raceway pond 100 of the group may connected to a common seawater canal. Similarly, the drainpipe 112 of each raceway pond 100 of the group may connected to a common outlet.

In one embodiment, a covered raceway pond (or group of covered raceway ponds) has volume of between 50 land 15,000,000 I, for example between 50 land 50,000 I. In a particular embodiment, there is a series of covered raceway ponds, and the volume of covered raceway ponds in the series increases such that there is at least one pond in the series with a volume of (a) 50-1,000 I; 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 (or group of open raceway ponds) has volume of between 1,500,000 land 3,000,0001, in a further embodiment between 6,000,000 land 15,000,0001. 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. As mentioned, paddlewheel 110 may be used to maintain the flow of the algae and water around the raceway pond 100. 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, paddlewheel 100 may be used to achieve a different flow rate of algae and water within a raceway pond based on the physical attributes of the paddlewheel (e.g., number of paddles, size of paddles) and/or the paddlewheel speed (i.e., its rotational speed). The flow rate may be 0.1-0.5 m/minute, e.g., 0.1-0.4 m/minute or 0.1- 0.3 m/minute. In one embodiment, each raceway pond 100 has one or more paddlewheels 110 depending on the degree of agitation that is required. Preferably, each raceway pond 100 has one paddlewheel 110. The paddlewheel 110 may be positioned at any section of the raceway pond, but is preferably positioned close to the inlet pipe 108 of a raceway pond (e.g., where the rectangle section of the stadium shape transitions to a semicircle).

In one embodiment, one or more paddlewheels 110 maintain the flow of the algae and water within raceway pond 100 at a rate of about 0.1-0.5 m/minute, for example 0.1-0.4 m/minute, or 0.1-0.3 m/minute, or 0.15 m/minute, 0.2 m/minute, 0.25 m/minute.

The depth of raceway pond 100 affects algal solar irradiation. Both light intensity and wavelengths 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. However, algae in a shallower raceway pond 100 experiences greater light intensity and a greater proportion of red light than algae in a deeper raceway pond 100. In one embodiment, raceway pond 100 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, lm, 1.5m, 2 m, 3 m, 5 m, or 10 m deep. In one embodiment, a covered raceway pond is 0.1m -lm 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, if the depth of raceway pond 100 is varied along the length of channel 120 (e.g., between longitudinal channel sections 120a, 120b), 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 raceway pond 100 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 longitudinal channel sections 120a, 120b is 0.05-1.0 m, for example 0.05 m, 010 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 longitudinal channel section 120a is 0.2-0.5 m deep, while the second longitudinal channel section 120b is 0.8-1.0 m deep.

As described herein, raceway pond 100 may be lined (not shown). In one embodiment, raceway pond 100 is 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 100 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 a black or white geomembrane. A white coating, for instance from titanium oxide, may be provided over the black geomembrane.

In one embodiment, the colour of the pond liners, or a coating of the pond liners, may be chosen to alter the wavelengths of light received by the algae as light reflects off the base of the ponds. The colour of the liner or coating may selectively decrease exposure of the algae to underwater red (630- 680 nm), far red (700-750 nm) and/or blue (400-450 nm) light. In one embodiment, the liner or coating selectively decreases 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%, 01 40-60%. For example, the liner or coating 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. This is achieved by selecting the colour of the pond liner or coating based on the wavelengths that are to be absorbed.

In a preferred embodiment, the lining or coating is white in order to reflect light off the base of the pond 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 or coating may be used to induce different physiological effects in the algae.

For example, the lining or coating 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 or coated 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).

As an alternative to coloured lining or coating, the side wall 102, base and other structural features within the pond may be coloured. For instance, the side wall 102, base and other structural features may be white.

Series of raceway ponds In one embodiment, the land-based mariculture of the invention comprises culturing algae in a series of connected raceway ponds, arranged in stages. Each of the raceway ponds in the series of connected raceway ponds may be based on raceway pond 100 of Figure 1.

An example of an algal cultivation system 200 comprising a series of connected raceway ponds 210 arranged in stages 210A-230E is illustrated in Figure 2A. Figure 2B shows subsystem 200' of system 200 in further detail. The stages 210A-230E of raceway ponds are connected in such a way so as to allow water and algae to pass directly between raceway ponds 100 in successive stages of the series. However, the connection between the stages 210A-230E of raceway ponds 100 can be closed and each stage 210A-230E of raceway ponds 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 particular, in Figures 2A and 2B, there are three stages of covered raceway ponds, 210A, 210B and 2100 and two stages of open raceway ponds 210D, 210E. However, other numbers of stages of covered raceway ponds and open raceway ponds may be used. In one specific embodiment of the invention the series of connected raceway ponds 210 comprises firstly, one or more stages of covered raceway ponds 210A-2100 and secondly, one or more stages of open raceway ponds 210D-210E. The designations of "firstly" one or more stages of covered raceway ponds 210A-2100 and "secondly" one or more stages of open raceway ponds 210D-210E indicate that within the series of connected ponds 210, the stages comprising covered raceway ponds 210A-2100 will always come before the stages comprising open raceway ponds 210D-210E. Put another way, an open raceway 210A-2100 pond will never be succeeded by a covered raceway pond 210D-210E.

Each stage 210A-210E of the series of connected raceway ponds 210 may comprise one or more raceway ponds 100. Stages having a plurality (or group) of raceway ponds 100 use these ponds in parallel to increase the collective volume of water and algae that is throughput. The system 200 in Figures 2A and 2B, there are 16 ponds in each stage 210A-210E. However, the number of ponds does not have to be the same for each stage, as discussed further herein.

In addition to the series of connected raceway ponds 210, system 200 comprises an intake pipeline 202 to transport water to the system, typically seawater from the ocean. Intake pipeline 202 feeds intake canal 208 which provides each of the raceway ponds in the series of connected raceway ponds 210 with water. Each raceway pond 100 has a connection to supply canal 208 at its respective inlet pipe 108. The supply canal 208 may be elevated so that gravity can be used to transport water to each of the raceway ponds 100 via the inlet pipe 108. The elevated supply canal filled from intake pipeline 202 with high-rate, low-head pumps.

System 200 also comprises a harvest canal 212. Each raceway pond 100 in at least the final stage of the series of connected raceway ponds 210 has a connection to harvest canal 212 at its respective drain pipe 112. In stages other than the final stage, each raceway pond 100 is connected to the next stage of raceway ponds via its respective drainpipe 112. The harvest canal 212 leads to a harvesting building 214, where the algae is collected. The spent water is discharged through discharge pipeline 216. The discharge pipeline is at low elevation so that gravity moves water out of the harvesting building 214. In embodiments where the water is seawater, the intake pipeline 202 is upstream and as far needed from the discharge pipeline 216 to avoid reuptake of already spent seawater.

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 pond(s) 100 in each stage 210A-210E of the series. This increase in collective volume can be achieved by increasing the volume of the individual raceway ponds in each successive stage 210A-210E of the series and/or by increasing the number of raceway ponds in each successive stage 210A-210E of the series. Thus, at each successive stage 210A-210E in the series of raceway ponds 210 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 of water in discrete steps to a maximum volume within a raceway pond 100, before the water and algae are transferred to the subsequent larger pond(s). For example, each pond may be initially filled to a first volume having a first depth (e.g., 0.25 m) where the cells complete a growth cycle. When it is time to increase (e.g., double) the volume of the water, to enable the cells to grow at a low standing stock with natural nutrients, the volume of water of the same pond is increased to a second volume having a second depth (e.g., 0.5 m). After the second growth cycle is complete within that pond, the total volume of the two division stages and the water 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 1,000-2,000 mg Chl a m-3), 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-3). 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.

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 (e.g., stage 210A) is used to seed the raceway pond or raceway ponds of the next stage (e.g., stage 210B), 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 210A-210E 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 some embodiments, the number of open raceway ponds in each stage is the same for each successive stage in the series. For example, the system 200 in Figure 2A and 2B has 16 ponds in the first stage of covered raceway ponds 210A, 16 ponds in the second stage of covered raceway ponds 210B which are larger than the ponds in the first stage of covered raceway ponds 210A, 16 ponds in the third stage of covered raceway ponds 210C which are larger than the ponds in the second stage of covered raceway ponds 210B. There is also 16 ponds in the first stage of open raceway ponds 210D (fourth stage overall) which are larger than the ponds in the third stage of covered raceway ponds 210C, and 16 ponds in the second stage of open raceway ponds 210E (fifth stage overall), which are larger than the ponds in the first stage of open raceway ponds 210E. 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.

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 2A shows a particular layout for a series of connected raceway ponds 210. The advantage of this layout is that it efficiently hugs the coast along the edge of an ocean and enables each raceway pond to have at least one contact with the intake canal 208 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.

In some embodiments, the series comprises at least ten raceway ponds. In some embodiments, the series comprises at least fifteen raceway ponds.

Seed ponds and photobioreactors In one embodiment, the first stage 210A of covered raceway ponds is seeded with algae cultivated in a photobioreactor (PBR) 204 or a seed pond (not shown). The amount of algae that is used for seeding the initial pond at first stage 210A is referred to as the inoculum density.

A PBR 204 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, PBR 204 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, PBR 204 operates in a sterile environment.

A seed pond (not shown) 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 210A of covered raceway ponds.

In one embodiment, the first stage 210A of covered raceway ponds is seeded with algae from a PBR 204 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 210A 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 5 AM to 8 AM. Algae

In some embodiments, a single species of algae is cultured. In some embodiments, more than one species of algae is cultured.

In some embodiments, the marine carotenoid-producing microalgae are Cyanophyta, Glaucophyta, unicellular Rhodophyta, Heterokontophyta, Haptophyta, Dinophyta, Euglenophyta, Chlorarachniophyta or Chlorophyta (e.g., Prasinophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae or Charophyceae). In some such embodiments, the marine carotenoid-producing microalgae are Cyanophyta, Glaucophyta, unicellular Rhodophyta, Heterokontophyta, Haptophyta, Euglenophyta, Chlorarachniophyta or Chlorophyta (e.g., Prasinophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae or Charophyceae).

In some embodiments, the marine carotenoid-producing microalgae are: (i) Rhopalodiaceae sp., Hem/au/us sp., Climacodium sp., Skeletonema sp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp. or Nitzschia sp., or (ii) Dunalielia sp..

In some embodiments, the marine carotenoid-producing microalgae are Dunaliella sp. In some such embodiments, the marine carotenoid-producing microalgae are Dunaliella saline.

In some embodiments, the marine carotenoid-producing microalgae are Skeletonema sp. In some such embodiments, the marine carotenoid-producing microalgae are Skeletonema pseudocostatum.

In some embodiments, the algae comprise diatom-diazotroph assemblages (DDAs).

In some embodiments, the algae comprise diatoms.

In some embodiments, the algae comprise bloom-forming algae.

In some embodiments, the algae comprise r-strategist algae. DDAs

In some embodiments, the algae that are cultured in the method of the invention comprise "diatom-diazotroph assemblages (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 Richelia, Calothrix and other unicellular species similar in morphology to free-living diazotroph Crocosphaera, thrive by forming symbiotic relationships with diatoms like Hemiaulus, Rhizosolenia, Chaetoceros and Climacodium (Mutalipassi et al., 2021; Hilton 2014).

Some of these associations, like Calothrix or the novel unicellular cyanobacteria Candidatus Atelocyanobacterium thalassa (UCYN-A) (Tuo etal., 2017) associate with unicellular algae epiphytically (or on the outside of cells). Other diazotrophic symbionts are intracellular such as 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 Richelia 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 Richelia sp., optionally R. intracellularis.

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. solitaria, 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: Hemiaulus sp., Skeletonema sp., Rhizosolenia sp., Climacodium sp. and Chaetoceros sp.

In some embodiments, the DDAs comprise Hemiaulus sp., optionally H. hauckii, H. indicus, H. membranaceus, or H. sinensis.

In some embodiments, the DDAs comprise Skeletonema sp., optionally S. barbadense, S. costatum, S. cylindraceum, S. meditenaneum, S. punctatum, S. tropicum, or S. pseudocostatum.

In some embodiments, the DDAs comprise Rhizosolenia sp., optionally R. elate, R. acuminata, R. antarctica, R. antennata, R. bergonii, R. clevei, R. curvata, R. cylindrus, R. delicatula, R. minima, R. pugens, R. rob usta, R. rothii, R. stricta, or R. styliformis.

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

In some embodiments, the DDAs comprise Chaetoceros sp., optionally C. social's, 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 Richeha sp., Calothrix sp., Crocosphaera sp. and Candidatus Atelocynaobacterium Thalassa, and the diatoms are selected from Hemiaulus sp., Skeletonema sp., Rhizosolenia sp., Crimacodium sp. and Chaetoceros sp.

In some embodiments, the DDAs comprise an assemblage of Richeria sp. and Hemiaulas sp.

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.

In some instances, the oligotrophic conditions comprise less than 20 pM (e.g., 10 pM) phosphorus and less than 40 pM (e.g., 20 pM) nitrogen. Accordingly, in some embodiments of the invention, the algae comprise DDAs, and nutrient mineral acids are added to a total concentration of less than 60 pM (e.g., less than 30 pM). In some such embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the growth phase to a final concentration of less than 20 pM (e.g., less than 10 pM). In some such embodiments, nitrogen-containing acid (e.g., HNO3) is added in the growth phase to a final concentration of less than 40 pM (e.g., less than 20 pM).

Diatoms In some embodiments, the algae that are cultured in the method of the invention comprise diatoms. In some embodiments, the diatoms are fast-growing diatoms.

In some embodiments, the diatoms are bloom-forming diatoms. For example, diatoms that have (i) the ability to grow exponentially or (ii) a cell division rate that exceeds one division per day.

In some embodiments, the diatoms are selected from Skeletonema sp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp. and Nitzschia sp.

In some embodiments, the diatoms are Skeletonema sp., optionally S. barbadense, S. costatum, S. cylindraceum, S. mediterraneum, S. punctatum, S. tropicum, or S. pseudocostatum.

In some embodiments, the diatoms are Chaetoceros sp., optionally C. sociaris, C. debilis, C. curvisetus, C. muelleri, C. calcitrans, or C. didymus.

In some embodiments, the diatoms are Thalassiosira sp., optionally T. pseudonana, or T. symmetrica.

In some embodiments, the diatoms are Coscinodiscus sp optionally C. wailesii.

In some embodiments, the diatoms are Navicula sp., optionally N. pelliculosa, N. incenta, N. oblonga, N. salinicola, N. ramosissima, N. minima, N. ctyptocephala, or N. trivial/s.

In some embodiments, the diatoms are Synedra sp., optionally S. capitata, S. famelica, S. radians, S. rumpens, or S. ulna.

In some embodiments, the diatoms are Nitzschia sp., optionally N. frigida, N. acicularis, N. amphibia, or N. angustata.

Bloom-forming algae In some embodiments, the algae that are cultured in the method of the invention comprise bloom-forming algae.

In some embodiments, the bloom-forming algae are Dunaliella sp., optionally D. sauna.

In some embodiments, the bloom-forming algae are Rhodomonas sp., optionally R. minuta.

In some embodiments, the bloom-forming algae are Chaetoceros sp., optionally C. socialis, C. debills, C. curvisetus, C. muelleri, C. calcitrans, C. didymus, or C. convolutus.

R-strategist algae In ecology, r-strategist organisms are characterised by their high growth rates in less-crowded ecological niches. They are considered opportunistic organisms and thrive in more unstable and unpredictable environments due to their ability to reproduce rapidly.

Conversely, K-strategist organisms are characterised by a much lower growth rate in an ecological niche which is close to or at the carrying capacity for that organism. They exist in an equilibrium and thrive in more stable or predictable environments, competing for limited resources.

The method of the invention described herein represents a controlled, stable and predictable environment in which algae are cultured. Algae often fit the description of r-strategist organisms, displaying rapid growth rates in often unstable and unpredictable environments.

Therefore, it is surprising to find that algae grow particularly well in the tightly controlled, highly predictable conditions of the method of the invention described herein.

In some embodiments, the algae that are cultured in the method of the invention comprise r-strategist algae.

In some embodiments, the r-strategist algae are cultured in a K-strategist environment. In some embodiments, the r-strategist algae are cultured under K-strategist conditions.

In some embodiments, K-strategist conditions comprise one or more of a stable temperature, a stable salinity level, a stable humidity and a stable duration of sunlight.

First algal culture phase The first algal culture phase comprises culturing marine carotenoid-producing microalgae in a series of connected raceway ponds, arranged in stages, wherein the algae are maintained in the exponential growth phase by successive dilution with seawater and nutrient addition.

As used herein, "nutrient mineral acid" refers to an inorganic acid in which the conjugate base provides nourishment for algae (e.g., the conjugate base is used by algae to survive and grow).

In some embodiments, one or more nutrient mineral acids are added in the first algal culture phase. In some embodiments, the one or more nutrient mineral acids are selected from a phosphorus-containing acid, silicon-containing acid, and nitrogen-containing acid.

In some embodiments, a phosphorus-containing acid is added in the first algal culture phase. In general, all algae assimilate phosphorus for survival and growth. Therefore, phosphorus-containing acids are nutritious for any algal species. In some embodiments, the nutrient mineral acid is a phosphorus oxoacid. In some embodiments, the nutrient mineral acid has a phosphate anion as a conjugate base. In some embodiments, the nutrient mineral acid is a phosphoric acid. In some embodiments, the phosphoric acid has the chemical formula H3PO4. In preferred embodiments, the phosphorus-containing acid is phosphoric acid (H3PO4).

In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final concentration of at least 0.15 pM, at least 0.5 pM, at least 1.0 pM, at least 1.5 pM, at least 2.0 pM, at least 5.0 pM, at least 10 pM, or at least 15 pM. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final concentration of 0.15-15 pM (e.g., 0.5-4.5 pM, such as 1-2 pM). In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final concentration of between 0.1 pM and 2.0 pM, between 0.5 pM and 2.0 pM, or between 1.0 pM and 2.0 pM. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final concentration of 1.5 pM.

The concentration of phosphorus-containing acid that is added can be influenced by the algal cell density. Accordingly, in some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) number of thousand algal cells per ml multiplied by 5 to (ii) number of thousand algal cells per ml multiplied by 25. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 7 to (ii) the number of thousand algal cells per ml multiplied by 15. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 10 to (ii) the number of thousand algal cells per ml multiplied by 13.

In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 5. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 10. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 12. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 15. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 20. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 25.

In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 5. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 10. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 12. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 15. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 20. In some embodiments, phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 25.

In some embodiments, the phosphorous-containing conjugate base to the phosphorous-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final concentration of at least 0.15 pM, at least 0.5 pM, at least 1.0 pM, at least 1.5 pM, at least 2.0 pM, at least 5.0 pM, at least 10 pM, or at least 15 pM. In some embodiments, the phosphorous-containing conjugate base to the phosphorous-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final concentration of 0.15-15 pM (e.g., 0.5-4.5 pM, such as 1-2 pM). In some embodiments, the phosphorous-containing conjugate base to the phosphorous-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final concentration of between 0.1 pM and 2.0 pM, between 0.5 pM and 2.0 pM, or between 1.0 pM and 2.0 pM. In some embodiments, the phosphorous-containing conjugate base to the phosphorous-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final concentration of 1.5 pM.

In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) number of thousand algal cells per ml multiplied by 5 to (ii) number of thousand algal cells per ml multiplied by 25. In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 7 to (ii) the number of thousand algal cells per ml multiplied by 15. In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 10 to (ii) the number of thousand algal cells per ml multiplied by 13.

In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 5. In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 10. In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 12. In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 15. In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 20.

In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 25.

In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 5. In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 10. In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 12. In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 15. In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 20. In some embodiments, the phosphorous-containing conjugate base to the phosphorus-containing acid (e.g., H3PO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 25.

In some embodiments, phosphorous is added in the first algal culture phase to a final concentration of at least 0.15 pM, at least 0.5 pM, at least 1.0 pM, at least 1.5 pM, at least 2.0 pM, at least 5.0 pM, at least 10 pM, or at least 15 pM. In some embodiments, phosphorous is is added in the first algal culture phase to a final concentration of 0.15-15 pM (e.g., 0.5-4.5 pM, such as 1-2 pM). In some embodiments, phosphorous is added in the first algal culture phase to a final concentration of between 0.1 pM and 2.0 pM, between 0.5 pM and 2.0 pM, or between 1.0 pM and 2.0 pM. In some embodiments, phosphorous is added in the first algal culture phase to a final concentration of 1.5 pM.

In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) number of thousand algal cells per ml multiplied by 5 to (ii) number of thousand algal cells per ml multiplied by 25. In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 7 to (ii) the number of thousand algal cells per ml multiplied by 15. In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 10 to (ii) the number of thousand algal cells per ml multiplied by 13.

In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 5. In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 10. In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 12.

In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 15. In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 20.

In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 25.

In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 5. In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 10. In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 12. In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 15. In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 20. In some embodiments, phosphorus is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 25.

In some embodiments, a silicon-containing acid is added in the first algal culture phase. Some algal species (e.g., diatoms) assimilate silicon as a component of their cell walls. Therefore, silicon-containing acids are nutritious for algal species with silica cell wells (e.g., diatoms). In some embodiments, the nutrient mineral acid has a silicate anion as a conjugate base. In some embodiments, the nutrient mineral acid is a silicic acid. In some embodiments, the nutrient mineral acid is orthosilicic acid, metasilicic acid, pyrosilicic acid or disilicic acid. In preferred embodiments, the silicon-containing acid is orthosilicic acid (H4SiO4).

In some embodiments, silicon-containing acid (e.g.. H4SiO4) is added in the first algal culture phase to a final concentration of at least 0.3 pM, at least 0.5 pM, at least 1.0 pM, at least 1.5 pM, at least 2.0 pM, at least 5.0 pM, at least 10 pM, at least 15 pM, or at least 30 pM. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final concentration of 0.3-30 pM (e.g., 1-9 pM, such as 2-4 pM). In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final concentration of between 0.3 pM and 5.0 pM, between 0.5 pM and 5 pM, between 1.0 pM and 5.0 pM, between 1.5 pM and 5.0 pM, between 2.0 pM and 5.0 pM, between 2.5 pM and 5.0 pM, between 2.5 pM and 4.5 pM, between 2.5 pM and 4.0 pM or between 2.5 pM and 3.5 pM. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final concentration of 3 pM.

The concentration of silicon-containing acid (e.g., H4SiO4) that is added can be influenced by the algal cell density. Accordingly, in some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) number of thousand algal cells per ml multiplied by 5 to (ii) number of thousand algal cells per ml multiplied by 50. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 15 to (ii) the number of thousand algal cells per ml multiplied by 35. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 20 to (ii) the number of thousand algal cells per ml multiplied by 30. In some embodiments, silicon-containing acid (e.g., H4SO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 22 to (ii) the number of thousand algal cells per ml multiplied by 26.

In some embodiments, silicon-containing acid (e.g.; 1-14SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 5. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 10. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 15. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 20. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 25. In some embodiments, silicon-containing acid (e.g., H4SO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 30. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 40. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 50.

In some embodiments, silicon-containing acid (e.g.. H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 5. In some embodiments, silicon-containing acid (e.g., H48iO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 10. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 15. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 20. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 25. In some embodiments, silicon-containing acid (e.g., H43iO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 30. In some embodiments, silicon-containing acid (e.g., 1-148iO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 40. In some embodiments, silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 50.

In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final concentration of at least 0.15 pM, at least 0.5 pM, at least 1.0 pM, at least 1.5 pM, at least 2.0 pM, at least 5.0 pM, at least 10 pM, or at least 15 pM.

In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final concentration of 0.15-15 pM (e.g., 0.5-4.5 pM, such as 1-2 pM). In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final concentration of between 0.1 pM and 2.0 pM, between 0.5 pM and 2.0 pM, or between 1.0 pM and 2.0 pM. In some embodiments, the final concentration of the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final concentration of 1.5 pM.

In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) number of thousand algal cells per ml multiplied by 5 to (ii) number of thousand algal cells per ml multiplied by 50. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 15 to (ii) the number of thousand algal cells per ml multiplied by 35. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., 1-14SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 20 to (ii) the number of thousand algal cells per ml multiplied by 30. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 22 to (ii) the number of thousand algal cells per ml multiplied by 26.

In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H48iO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 5. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 10. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 15. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 20. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 25. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 30.

In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H48iO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 40. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H43iO4) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 50.

In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 5. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 10. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 15. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 20. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H48iO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 25. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 30. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H4SiO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 40. In some embodiments, the silicon-containing conjugate base to the silicon-containing acid (e.g., H43iO4) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 50 In some embodiments, silicon is added in the first algal culture phase to a final concentration of at least 0.15 pM, at least 0.5 pM, at least 1.0 pM, at least 1.5 pM, at least 2.0 pM, at least 5.0 pM, at least 10 pM, or at least 15 pM. In some embodiments, silicon is added in the first algal culture phase to a final concentration of 0.15-15 pM (e.g., 0.5-4.5 pM, such as 1-2 pM). In some embodiments, silicon is added in the first algal culture phase to a final concentration of between 0.1 pM and 2.0 pM, between 0.5 pM and 2.0 pM, or between 1.0 pM and 2.0 pM. In some embodiments, silicon is added in the first algal culture phase to a final concentration of 1.5 pM.

In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) number of thousand algal cells per ml multiplied by 5 to (ii) number of thousand algal cells per ml multiplied by 50. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 15 to (ii) the number of thousand algal cells per ml multiplied by 35. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 20 to (ii) the number of thousand algal cells per ml multiplied by 30. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 22 to (ii) the number of thousand algal cells per ml multiplied by 26.

In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 5. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 10. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 15. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 20. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 25. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 30. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 40. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 50.

In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 5. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 10. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 15. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 20. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 25. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 30. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 40. In some embodiments, silicon is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 50.

In some embodiments, a nitrogen-containing acid is added in the first algal culture phase. Nitrogen-fixing algae (e.g., diatom-diazotroph assemblages) have a reduced requirement for assimilating nitrogen from the culture medium. Other algal species typically have a much greater need for nitrogen assimilation than for phosphorus assimilation from the culture medium. The average nitrogen to phosphorus ratio in algal biomass is 16:1. In some embodiments, the nutrient mineral acid is a nitrogen acid. In some embodiments, the nutrient mineral acid has a nitrate anion as a conjugate base. In some embodiments, the nutrient mineral acid is nitric acid, nitrous acid or hyponitrous acid. In preferred embodiments, the nitrogen-containing acid is nitric acid (HNO3).

In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final concentration of at least 8 pM, at least 15 pM, at least 30 pM, at least 50 pM, at least 80 pM, at least 100 pM, at least 200 pM, at least 500 pM, or at least 800 pM. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final concentration of 8-800 pM (e.g., 20-320 pM, such as 60-100 pM). In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final concentration of between 50 pM and 100 pM, between 60 pM and 100 pM, between 70 pM and 100 pM, between 80 pM and 100 pM or between 80 pM and 90 pM. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final concentration of 80 pM.

The concentration of nitrogen-containing acid (e.g., HNO3) that is added can be influenced by the algal cell density. Accordingly, in some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) number of thousand algal cells per ml multiplied by 50 to (ii) number of thousand algal cells per ml multiplied by 5000. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 200 to (H) the number of thousand algal cells per ml multiplied by 1500. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 400 to (i) the number of thousand algal cells per ml multiplied by 800. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 600 to (ii) the number of thousand algal cells per ml multiplied by 700.

In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 50. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 100. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 200. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 400. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 500. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 600. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 700. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 1000. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 2000.

In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 50. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 100. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 200. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 500. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 600. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 650. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 700. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 1000. In some embodiments, nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 2000.

In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final concentration of at least 8 pM, at least 15 pM, at least 30 pM, at least 50 pM, at least 80 pM, at least 100 pM, at least 200 pM, at least 500 pM, or at least 800 pM. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final concentration of 8-800 pM (e.g., 20-320 pM, such as 60-100 pM). In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final concentration of between 50 pM and 100 pM, between 60 pM and 100 pM, between 70 pM and 100 pM, between 80 pM and 100 pM or between 80 pM and 90 pM. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final concentration of 80 pM.

In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) number of thousand algal cells per ml multiplied by 50 to (ii) number of thousand algal cells per ml multiplied by 5000. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of (i) the number of thousand algal cells per ml multiplied by 200 to (ii) the number of thousand algal cells per ml multiplied by 1500. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 400 to (ii) the number of thousand algal cells per ml multiplied by 800. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 600 to (ii) the number of thousand algal cells per ml multiplied by 700.

In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 50. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 100. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 200. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 400. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 500. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 600. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 700. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 1000. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 2000.

In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 50. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 100. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 200. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 500. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 600. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 650. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 700. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 1000. In some embodiments, the nitrogen-containing conjugate base to the nitrogen-containing acid (e.g., HNO3) is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 2000.

In some embodiments, nitrogen is added in the first algal culture phase to a final concentration of at least 8 pM, at least 15 pM, at least 30 pM, at least 50 pM, at least 80 pM, at least 100 pM, at least 200 pM, at least 500 pM, or at least 800 pM. In some embodiments, nitrogen is added in the first algal culture phase to a final concentration of 8-800 pM (e.g., 20-320 pM, such as 60-100 pM). In some embodiments, nitrogen is added in the first algal culture phase to a final concentration of between 50 pM and 100 pM, between 60 pM and 100 pM, between 70 pM and 100 pM, between 80 pM and 100 pM or between 80 pM and 90 pM. In some embodiments, nitrogen is added in the first algal culture phase to a final concentration of 80 pM.

In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) number of thousand algal cells per ml multiplied by 50 to (ii) number of thousand algal cells per ml multiplied by 5000. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 200 to (ii) the number of thousand algal cells per ml multiplied by 1500. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 400 to (ii) the number of thousand algal cells per ml multiplied by 800. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 600 to (H) the number of thousand algal cells per ml multiplied by 700.

In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 50. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 100.

In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 200. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 400.

In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 500. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 600. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 700.

In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 1000. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as being at least the number of thousand algal cells per ml multiplied by 2000.

In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 50. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 100. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 200. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 500. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 600. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 650. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 700. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 1000. In some embodiments, nitrogen is added in the first algal culture phase to a final nanomolar concentration calculated as the number of thousand algal cells per ml multiplied by 2000.

In some embodiments, one or more, two or more, three or more, four or more, or five or more nutrient mineral acids are added in the first algal culture phase.

In some embodiments, the algae are diatoms, and a phosphorus-containing acid (e.g., H3PO4) a silicon-containing acid (H4SiO4), and a nitrogen-containing acid (e.g., HNO3) are added in the first algal culture phase.

In some embodiments, the algae are diatom-diazotroph assemblages, and a phosphorus-containing acid (e.g., H3PO4) and a silicon-containing acid (H4SiO4) are added in the first algal culture phase.

In some embodiments, the algae are bloom-forming algae, and a phosphorus-containing acid (e.g., H3PO4) and a nitrogen-containing acid (e.g., HNO3) are added in the first algal culture phase.

In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is at least 5 pM, at least 10 pM, at least 25 pM, at least 50 pM, at least 85 pM, at least 150 pM, at least 300 pM, at least 500 pM, or at least 850 pM. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is 5-850 pM (e.g., 25-150 pM, such as 80-90 pM). In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is between 50 and 100 pM, between 60 and 100 pM, between 70 and 100 pM, between 80 and 100 pM or between 80 and 90 pM.

In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being within the range of: (i) number of thousand algal cells per ml multiplied by 50 to (ii) number of thousand algal cells per ml multiplied by 5000. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 200 to (ii) the number of thousand algal cells per ml multiplied by 1500. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 400 to (ii) the number of thousand algal cells per ml multiplied by 800. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being within the range of: (i) the number of thousand algal cells per ml multiplied by 600 to (ii) the number of thousand algal cells per ml multiplied by 700.

In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being at least the number of thousand algal cells per ml multiplied by 50. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being at least the number of thousand algal cells per ml multiplied by 100. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being at least the number of thousand algal cells per ml multiplied by 200. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being at least the number of thousand algal cells per ml multiplied by 400. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being at least the number of thousand algal cells per ml multiplied by 500. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being at least the number of thousand algal cells per ml multiplied by 600. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being at least the number of thousand algal cells per ml multiplied by 700. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being at least the number of thousand algal cells per ml multiplied by 1000. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as being at least the number of thousand algal cells per ml multiplied by 2000.

In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as the number of thousand algal cells per ml multiplied by 50. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as the number of thousand algal cells per ml multiplied by 100. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as the number of thousand algal cells per ml multiplied by 200. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as the number of thousand algal cells per ml multiplied by 500. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as the number of thousand algal cells per ml multiplied by 600. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as the number of thousand algal cells per ml multiplied by 650. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as the number of thousand algal cells per ml multiplied by 700. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as the number of thousand algal cells per ml multiplied by 1000. In some embodiments, the final concentration of total nutrient mineral acid(s) after addition of nutrient mineral acid(s) is calculated as the number of thousand algal cells per ml multiplied by 2000.

In some embodiments, nutrient mineral acids are added in the first algal culture phase to achieve a pH value in the range of pH6.5-7.5.

Second algal culture phase The second algal culture phase comprises culturing algae in a raceway pond that is connected to the series of connected raceway ponds in which algae are cultured in the first algal culture phase, wherein the algae are induced to produce carotene by culturing the algae with limited nutrients, high light exposure and/or reducing the dilution rate in the raceway pond.

In some embodiments, the algae are induced to produce carotene by culturing the algae with limited nutrients. In some such embodiments, seawater is added to the raceway pond without nutrient supplementation.

In some embodiments, the algae are induced to produce carotene by culturing the algae with high light exposure. Beta-carotene may be produced by algae as a photoprotective response.

In some embodiments, the algae are induced to produce carotene by culturing the algae by reducing the dilution rate in the raceway pond. Diluting the algae more slowly reduces the light path, and so increases the relative light exposure of the cells, which can induce beta-carotene production as a photoprotective response.

Algal flow management and operational parameters 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.

In preferred embodiments, the algae are cultured in natural seawater. The use of this water source presents advantages over hypersaline evaporative brine or other high-salt sources of water. For example, seawater is abundantly available worldwide and not constrained to specific geographies where there are evaporative basins or salt flats available.

Once the seawater has circulated through at least one raceway pond 100 (e.g., the series of connected raceway ponds 210), 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, algae are first cultivated in at least one stage of covered raceway ponds (210A-210C). The covered raceway ponds (210A-210C) allow for the control of the algae growth environment. The water used to fill the covered raceway ponds (210A-210C) may be filtered or otherwise treated to remove competing and deleterious organisms before being introduced into the covered raceway ponds.

In one embodiment, greenhouses 206 are used to cover the raceway ponds 100 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 transitioning into the exponential growth phase. Passive and/or active ventilation may be used in greenhouse 206 to regulate the temperature. As passive ventilation, greenhouse 206 may have walls that have a mesh netting on the inside and are movable outside which can be shut to retain more heat inside the greenhouse (e.g., in winter) or moved to allow free air moment (e.g., in summer). For the active ventilation, if a certain temperature threshold is exceeded, vents turn on to remove heat from greenhouse 206.

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 (e.g., from stage 210A to stage 210B, from stage 210B to 210C). 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 (i.e., have a residence time) in each stage of covered raceway ponds 210A, 210B, 210C 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 210A, 210B, 210C for two days, before being transferred to a raceway pond of larger volume. In one embodiment, algae remain in a covered raceway pond 210A, 2108, 20C 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 210D. 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 210D. 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 (i.e., the dilution rate or dilution volume may be increased) by introducing additional water into the current stage of ponds (210A-210E), increasing the volume of water contained within that stage. Additional water may be added by opening the sluice gate of the inlet pipe 108 of a raceway pond 100. 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 210D, 210E are successively diluted by increasing the volume or number of open raceway ponds in each stage of the series (e.g., from stage 210D to 210E). 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 (i.e., have a residence time) in each stage of open raceway ponds 210D, 210E 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 open raceway ponds in the series (e.g., from stage 210D to 210E). In a specific embodiment, algae remain in a stage of open raceway ponds 210D for 48 hours before being transferred to the next stage of raceway ponds 210E in the series. In one embodiment, algae remain in a stage of an open raceway pond 210D, 210E 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 stage of an open raceway pond 210D, 210E 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 204 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 210A-210E in the series. Following the transfer of the algae and water from one stage to seed the next stage in the series (e.g., from stage 210A to 210B, from 210B to 210C, from 210C to 210D, from 210D to 210E), the volume of fluid within each raceway pond 100 is equal to the capacity of the raceway pond 100. In one embodiment, the entire volume of water in a raceway pond 100 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 210 in 24 hours is greater than 20- 250% of the entire volume of the series of covered and open connected raceway ponds 210, 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 210 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 Harvesting the algae is preferably performed by a method that can be applied to large amounts of water with relatively low energy investment and capital expenditure cost.

Accordingly, 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.

Alternatively, 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.

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.

Advantageously, these harvesting methods facilitate the concentration of algal cells without exposure to excess shear or breakage. This facilitates, e.g." the harvest of algal cells that have not formed rigid cysts.

Advantageously, these harvesting methods do not require the addition of coagulants or flocculants or other expensive forms of cell concentration such as settling tanks or dissolved air flotation.

Advantageously, the algal slurry produced by these harvesting methods may contain the original cultivation water and may not require refrigeration. The cells are still vital in the slurry and the dewatering does not typically damage the cells, ensuring high product quality.

In some embodiments, harvesting the algae produces a 500-1000-fold concentrated algal slurry. This slurry is more concentrated than that which can be obtained by conventional dewatering tools such as continuous flow centrifugation or microtubule cross-flow micro-filtration.

In some embodiments, the algal slurry is further dewatered in a centrifuge. The centrifuge is preferably a low-shear centrifuge. Advantageously, this ensures that the cells are not lysed or disrupted while the extracellular seawater is removed. In some embodiments, this results in an algal slurry that contains at least 10% dry weight, wet biomass. In some embodiments, this results in an algal slurry that contains 10-15% dry weight, wet biomass.

In some embodiments, after the algal slurry has been further dewatered in a (low-shear) centrifuge, the algal slurry is refrigerated and then subjected to a continuous flow centrifuge. Advantageously, this ensures a consistent flow of biomass is available for extraction.

In some embodiments, the algal slurry is acidified (e.g., to pH <4, such as pH <3.5) prior to the extraction phase. This is advantageous for microalgae that contain a high level of soluble proteins or soluble starches (like chrysolaminarin) because acidification hydrolyses these large molecules to ensure that they do not bind to the carotenoid extracts. Such microalgae include Skeletonema pseudocostatum, some diazotrophic bacteria, and Chlorella sp..

Maintenance of the raceway ponds When algae is cultured in a series of connected raceway ponds, such 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 100. 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 200. 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. Individual raceway ponds in parallel can also 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 maintenance step 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 maintenance step can be performed once a month, and every several maintenance steps 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.

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 or entities.

EXAMPLES

Example 1

Dunaliella saline was grown in a connected series of 15 raceway ponds. The first fourteen ponds were kept under optimal growth conditions for D. sauna by successive dilution with nutrient-rich seawater in the early mornings and H3PO4 addition in the late afternoons. Overall, the nutrients added were 70-110 micromolar nitrate, 6-8 micromolar phosphate and 1 nanomolar iron (Ill) salt (e.g. ferric chloride). Under these conditions, D. sauna was grown on a three-day doubling cycle, such that cell concentration doubled in three days per unit volume of cultivation water. In these ponds, D. sauna was present as 'green' (low beta-carotene content) cells. In the fifteenth pond, the algae were subjected to nutrient limitation. In particular, seawater was added to this pond without additional supplementation of nitrogen or phosphorus. This resulted in the cells converting to 'orange' (high beta-carotene content) cells.

The algae were then harvested using filter mesh screens that have a pore size of 23 microns. These are hydrophilic, polyester woven mesh screens that can be applied to tangential flow filtration or rotary drum screens. The relatively gently concentration process very rapidly creates a 500-1,000-fold concentration of the cells in the original cultivation water without the need for refrigeration.

The cell concentrate (having 2% -3% dry weight) was further dewatered in a low-shear centrifuge that ensures that the cells are not lysed or disrupted while the extracellular seawater is removed. This centrifuge produced a paste that is 12% -13% dry weight, wet biomass. This paste was refrigerated and the biomass was subjected to a continuous flow centrifuge to ensure a consistent flow of biomass was available for extraction.

All subsequent steps were performed under a nitrogen blanket to prevent oxidation of the extracts.

Extraction of all pigments is performed by suspending approximately 417 g / L of the 12% paste in 100% food grade ethyl acetate, to give a 50 g IL dry weight suspension, and agitated for 10 minutes. At this point carotenoids, pigments, some lipids and solvent soluble components have been dissolved in the ethyl acetate. The water phase contains cellular debris and the remainder of the biomass as well as the intracellular salt from Dunaliella sauna.

The two phases were separated and the water phase is pumped to solar drying and long-term disposal in landfill The ethyl acetate was cleaned of all particulate biomass by means of depth filtration and the solvent was evaporated with vacuum assisted evaporation to create an oleoresin.

At this point, 1 volume of water is added to a volume of ethyl acetate extract. When the ethyl acetate evaporates, the beta-carotene forms a precipitate in water. This reduces the quantity of oleoresin which adheres to vessel walls to facilitate further processing and to avoid a second resuspension process.

The ethyl acetate was condensed, recaptured and recycled. It was passed through a drying column to ensure that it was dry and clean before reuse.

The oleoresin was saponified by the addition of a 50% sodium hydroxide solution, to give a final concentration of 1 mole per liter, for 1 hour at 60 °C with agitation. The beta-carotene is not water soluble and forms a slurry that is filtered and the filter cake is washed with water and subsequently dried to produce the primary product.

The watery alkali filtrate contains lutein which was available for further processing by transferring it into an organic non-ester solvent, that was re-evaporated, condensed and recycled to form a second oleoresin rich in lutein. This was similarly dried and collected as a second product.

The water phase containing sodium magnesium chlorophyllin was acidified to pH 2-3 by the addition of concentrated hydrochloric acid and is mixed with 10% copper sulfate at 60 °C for 1 hour to precipitate chlorophyllin salts. The precipitate was collected and dissolved in 95% ethanol and the pH is adjusted to 11 by the addition of 1M sodium hydroxide. The sodium copper chlorophyllin was filtered, washed and dried as a third product. The watery filtrate was disposed of as the wet biomass remnants above.

Chromatograms for the crude carotenoid extract and the purified carotenoid powder at the end of the process are provided in Figures 4A and 4B, respectively. A chromatogram for a reference carotene product, BASF Betatenee 30% OLV, 72% all-trans beta carotene is provided in Figure 4C.

REFERENCES

Bhumibharnon, 0., Sittiphuprasert, U., Boontaveeynwat. N. and Praiboon, J., 2003. The optimum use of s,alinity, nitrate and pond depth for ft-carotene production of Dunaliella salina. Agriculture and Natural Resources, 37(1), pp.84-89.

Black, H.S., Boehm, F., Edge, R. and Truscott, TO., 2020. The benefits and risks of certain dietary carotenoids that exhibit both anti-and pro-oxidative mechanisms-A comprehensive review. Antioxidants, 9(3), p.264.

Correa, P.S., Marais Junior, WG; Nilartins, A.A., Gaetano, N.S. and Mata, TM., 2020 flicroalgae biornolecules: Extraction, separation and purification methods. Processes, 9(1), p.10.

Ernst, H., Henrich, K Keller, A., 2003., Method for producing carotenoids, U620060106257A1 Furukawa, T., Watanaba, M. and Shihira-lshikawa, I. Green and blue-light-mediated chloroplast migration in the centric diatom Pleurosira Iaevis Protoplasma, 203;214-220 (1998).

Ghazi A 1999. Extraction of carotene from orange peels Food/Nahrung, 43(4), pp.274-277.

Grime, T., Lietz, G., Palou, A., Ross, AC., Stahl, VV., Tang, G., Thurnharn, D., Yin; S.A. and Blesalski, H.K., 2010. [3-Carotene is an important vitamin A source for humans. The Journal of nutrition, 140(12), pp.22685-22855.

Gupta, AK., Seth, K., Maheshwari, K., Earoliya, P.K., Aeena, M., Kumar, A. and Vinayak, V., 2021. Biosynthesis and extraction of high-value carotenoid from algae. Frontiers in Blosoience-Landmark, 26(6), pp.171-190.

Johnson, E.J., Qin, J., Krinsky, NJ. and Russell, R.M., 1997. p-Carotenek n human serum, breast milk and buccal mucosa cells after continuous oral doses of all-trans and cis [3-carotene. The Journal of nutrition, 127(10), pp.1993-1999.

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.

Mendes, RI., Fernandes, HI., Coeihr, Reis, E.C., Cabral, WM., Novais, J M. and Palavra. AF., 1995. Supercritical CO2 extraction of carotenoids and other lipids from Chlorella vulgaris. Food chemistry, 53(1), pp.99-103.

Takaichi, S. Carotenoids in Algae: Distributions, Biosyntheses and Functions, 2011, Marine Drugs 9(6):1101-1118.

Wolf, L., Cummings, T., Muller, K., Reppke, M., Volkmar, M. and Weuster-Botz, D., 2021. Production of p-carotene with Dunaliella salina CCAP19/18 at physically simulated outdoor conditions. Engineering in life sciences, 21(3-4), pp.115-125.

Claims

CLAIMS1. A method for producing a carotenoid or pigment wherein the method comprises: (a) a first algal culture phase, wherein the first algal culture phase comprises culturing marine carotenoid-producing microalgae in a series of connected raceway ponds, arranged in stages, and wherein the algae are maintained in the exponential growth phase by successive dilution with seawater and nutrient addition, (b) a second algal culture phase, wherein the second algal culture phase comprises culturing algae in a raceway pond that is connected to the series of connected raceway ponds in which algae are cultured in step (a), and wherein the algae are induced to produce the carotenoid or pigment by culturing the algae with limited nutrients, high light exposure and/or reducing the dilution rate in the raceway pond, (c) a harvest phase, wherein the harvest phase comprises harvesting algae from the raceway pond in which algae are cultured in step (b) to produce an algal slurry, and (d) an extraction phase, wherein the extraction phase comprises extracting the carotenoid or pigment from the algal slurry produced in step (c), wherein said extracting comprises: (i) suspending the algal slurry in an alkyl-ester solvent, (ii) separating the alkyl-ester solvent phase from the water phase, (iii) evaporating the alkyl-ester solvent to generate an oleoresin, wherein water is added before or during evaporation, (iv) saponifying the oleoresin to generate a slurry that comprises the carotenoid or pigment, and (v) filtering the slurry to generate a filter cake that comprises the carotenoid or pigment.
2. The method of claim 1, wherein the method is for producing a carotenoid, optionally wherein the carotenoid is beta-carotene.
3. The method of claim 1 or claim 2, wherein the marine carotenoid-producing microalgae are Chlorophyta, Chlorarachniophyta, Haptophyta, Dinophyta, Euglenophyta, Heterokontophyta (e.g. Chrysophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, Xanthophyceae or Eusfigmatophyceae), Cryptophyta, unicellular Rhodophyta, Glaucophyta or Cyanophyta, optionally wherein the marine carotenoid-producing microalgae are: (i) Rhopalodiaceae sp., Hem/au/us sp., Climacodium sp., Skeletonema sp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp. or Nitzschia sp., or (ii) Dunaliella sp.
4. The method of any one of claims 1-3, wherein the series of connected raceway ponds in step (a) comprises a first stage comprising one or more covered raceway ponds and a second stage comprising one or more stages of open raceway ponds.
5. The method of any one of claims 1-4, wherein one or more nutrient mineral acids are added during step (a), optionally wherein the one or more nutrient mineral acids are selected from nitric acid, phosphoric acid and silicic acid
6. The method of any one of claims 1-5, wherein the algal slurry produced in step (c) is acidified before step (d)(i) is performed, optionally wherein the algal slurry is acidified to pH 4 or lower.
7. The method of any one of claims 1-6, wherein the alkyl-ester solvent in step (d)(i) is ethyl acetate.
8. The method of any one of claims 1-7, wherein: (A) step (d)(i) further comprises agitating the suspended slurry, and/or (B) algal slurry is suspended in the solvent in step (d)(i) to give a 10-100 g/L dry weight suspension.
9. The method of any one of claims 1-8, wherein step (d)(ii): (A) is performed by centrifugation, optionally using a three-phase (liquid-solid-liquid) centrifuge, optionally further wherein the separated solid phase is incorporated into protein powder, and/or (B) further comprises depth filtration of the alkyl-ester solvent phase.
10. The method of any one of claims 1-9, wherein the evaporation in step (d)(iii) is performed by vacuum evaporation.
11. The method of any one of claims 1-10, wherein the water is added to the alkyl-ester solvent in a volume ratio of 0.2-5 to 1, such as 1:1.
12. The method of any one of claims 1-11, wherein step d(iii) further comprises recovering the evaporated alkyl-ester solvent, and optionally: (A) passing the evaporated solvent through one or more drying columns, and/or (B) passing the condensed solvent through one or more activated carbon columns.
13. The method of any one of claims 1-12, wherein step d(v) further comprises washing and/or drying the filter cake, optionally wherein said drying is under vacuum.
14. The method of any one of claims 1-13, wherein step (d)(iv) is performed by adding concentrated alkali solution (e.g a concentrated alkali metal hydroxide solution), such as NaOH (50 wt%), optionally to a final concentration of 0.5-5 M NaOH (e.g. 1-2.5 M NaOH).
15. A method for producing a xanthophyll, wherein the method comprises performing steps (a)-(d) in any one of claims 1-13, wherein step (d)(iv) comprises adding concentrated alkali solution (e.g. a concentrated alkali metal hydroxide solution), and wherein step (d) further comprises: (vi) adding an organic non-ester solvent to the filtrate obtained in step (v), (vii) separating the organic non-ester solvent phase from the water phase, (viii) evaporating the organic non-ester solvent to generate an oleoresin that comprises the xanthophyll, and (ix) drying the oleoresin.
16. The method of claim 15, wherein the organic non-ester solvent in step d(vi) is: (i) a (food grade) aliphatic hydrocarbon, (ii) a (food grade) monoterpene, (iii) (food grade) hexane, (iv) (food grade) limonene, or (v) (food grade) p-cymene.
17. The method of claim 15 or claim 16, wherein the xanthophyll is lutein.
18. The method of claim 17, wherein the marine carotenoid-producing microalgae are Chlorophyta (e.g. Dunaliella sp.) or Chlorarachniophyta.
19. The method of claim 15 or claim 16, wherein the xanthophyll is: (A) diadinoxanthin, optionally wherein the marine carotenoid-producing microalgae are Heterokontophyta (e.g. Chrysophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, or Xanthophyceae), Haptophyta, Dinophyta or Euglenophyta, (B) zeaxanthin, optionally wherein the marine carotenoid-producing microalgae are Chlorophyta, Chlorarachniophyta, Haptophyta, Dinophyta, Euglenophyta, Heterokontophyta (e.g., Chrysophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, or Xanthophyceae), Cryptophyta, unicellular Rhodophyta, Glaucophyta or Cyanophyta, optionally further wherein the marine carotenoid-producing microalgae are: (i) Rhopalodiaceae sp., Hemiaulus sp., Climacodium sp., Skeletonema spp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp., or Nitzschia sp., or (ii) Dunaliella sp., or (iii) Trichodesmium sp., Richelia sp., Calothrix sp., Crocosphaera sp or Candidatus Atelocyanobacterium Thalassa, (C) violaxanthin, optionally wherein the marine carotenoid-producing microalgae are Chlorophyta (e.g. Dunaliella sp.), Chlorarachniophyta, or Heterokontophyta (e.g. Raphidophyceae, Phaeophyceae, or Eustigmatophyceae), (D) neoxanthin, optionally wherein the marine carotenoid-producing microalgae are Euglenophyta, Chlorophyta (e.g. Dunaliella sp.) or Chlorarachniophyta, (E) fucoxanthin, optionally wherein the marine carotenoid-producing microalgae are Heterokontophyta (e.g. Chrysophyceae, Raphidophyceae, Bacillariophyceae, or Phaeophyceae), Haptophyta, or Dinophyta, optionally further wherein the marine carotenoid-producing microalgae are Rhopalodiaceae sp. Hemiaulus sp., Crimacodium sp., Skeletonema spp., Chaetoceros sp., Thalassiosira sp., Coscinodiscus sp., Navicula sp., Synedra sp. or Nitzschia sp., (F) vaucheriaxanthin, optionally wherein the marine carotenoid-producing microalgae are Heterokontophyta (e.g. Chrysophyceae or Eustigmatophyceae), (G) loroxanthin, optionally wherein the marine carotenoid-producing microalgae are Euglenophyta, Chlorophyta (e.g. Prasinophyceae, Chlorophyceae or Ulvophyceae) or Chlorarachniophyta, (H) siphonaxanthin, optionally wherein the marine carotenoid-producing microalgae are Euglenophyta or Chlorophyta (e.g., Prasinophyceae, Chlorophyceae or Ulvophyceae), (I) one or more of nostoxanthin, echinenone, myxol glycosides and oscillol glycosides, optionally wherein the marine carotenoid-producing microalgae are Cyanophyta, optionally further wherein the marine carotenoid-producing microalgae are Trichodesmium sp., Richelia sp., Calothrix sp., Crocosphaera sp. or Candidatus Atelocyanobacterium Thalassa, (J) one or more of alloxanthin, crocoxanthin and monadoxanthin, optionally wherein the marine carotenoid-producing microalgae are Cryptophyta, (K) violaxanthin fatty acid ester, optionally wherein the marine carotenoid-producing microalgae are Heterokontophyta (e.g., Xanthophyceae), (L) fucoxanthin fatty acid ester, optionally wherein the marine carotenoid-producing microalgae are Haptophyta, (M) peridinin, optionally wherein the marine carotenoid-producing microalgae are Dinophyta, (N) prasinoxanthin, optionally wherein the marine carotenoid-producing microalgae are Chlorophyta (e.g., Prasinophyceae), (0) loroxanthin fatty acid ester, optionally wherein the marine carotenoid-producing microalgae are Chlorophyta (e.g,. Prasinophyceae) or Chlorarachniophyta, or (P) siphonaxanthin fatty acid ester, optionally wherein the marine carotenoid-producing microalgae are Chlorophyta (e.g. Prasinophyceae, Chlorophyceae or Ulvophyceae).
20. The method of any one of claims 15-19, wherein step (d)(iv) is performed by adding concentrated alkali solution, such as NaOH (50 wt%), optionally to a final concentration of 0.5-5 M NaOH (e.g. 1-2.5 M NaOH).
21. A method for producing copper chlorophyllin salts, wherein the method comprises performing steps (a)-(d) in any one of claims 1-15, and wherein step (d) further comprises: (vi) adding an organic non-ester solvent to the filtrate obtained in step (v), (vii) separating the water phase from the organic non-ester solvent phase, (viii) mixing the water phase with one or more copper salts to precipitate copper chlorophyllin salts, and (ix) collecting the precipitated copper chlorophyllin salts.
22. The method of claim 21, wherein step (viii) further comprises acidifying the water phase to pH 2-3 (e.g. by the addition of concentrated hydrochloric acid).
23. The method of claim 21 or claim 22, wherein the one or more copper salts in step (d)(viii) comprise copper sulphate.
24. The method of any one of claims 21-23, wherein step (d) further comprises: (x) dissolving the precipitated copper chlorophyllin salts in (95%) ethanol, (xi) adjusting the pH to pH 10-12 (e.g. pH 11) by the addition of sodium hydroxide to obtain sodium copper chlorophyllin, and (xii) filtering, washing and drying the sodium copper chlorophyllin.