WO2022198278A1

WO2022198278A1 - Buoyant beads with carbonic anhydrase for algae production

Info

Publication number: WO2022198278A1
Application number: PCT/AU2022/050271
Authority: WO
Inventors: Xiaoyin Xu; Gregory John Oliver MARTIN; Sandra Elizabeth KENTISH
Original assignee: The University Of Melbourne
Priority date: 2021-03-24
Filing date: 2022-03-24
Publication date: 2022-09-29

Abstract

The present invention provides a hydrogel buoyant bead comprising cross-linked carbonic anhydrase, wherein the (hydrated) bead has a density of less than 1.03 g/cm³ (at 20°C). In one embodiment, the bead is a glutaraldehyde crosslinked alginate hydrogel comprising carbonic anhydrase and paraffin oil. The beads may be used in a wide variety of applications but are particularly useful in increasing the uptake of CO₂ into an aqueous medium. This application finds particular use in biomass production where a limiting factor in the ability of the biomass to grow is the limited carbonate concentration in the aqueous medium.

Description

Buoyant Beads with Carbonic Anhydrase for Algae Production

Technical Field

[0001] The present invention relates to a novel hydrogel bead containing cross- linked carbonic anhydrase wherein the hydrated bead has a density of less than 1 .03 g/cm³ (at 20°C), preferably less than 1.00 g/cm³. The invention further relates to a process for the manufacture of beads of this type. The beads may be used in a wide variety of applications but are particularly useful in increasing the uptake of CO2 into an aqueous medium. This application finds particular use in biomass production where a limiting factor in the ability of the biomass to grow is the limited carbonate concentration in the aqueous medium.

Background of Invention

[0002] Reducing atmospheric concentrations of CO2 is a critical challenge of our time. The development of cost-effective technologies to sequester CO2 from the atmosphere is needed. One approach is to use organisms to fix carbon during their growth, resulting in the production of biomass and the depletion of atmospheric CO2.

[0003] Biomass is used for the production of a wide range of organic components of interest to society. This is due to the wide range of organisms that can be cultivated as a biomass including algae, plants (or parts thereof), fungi, bacteria, protists and combinations thereof. Due to the wide variety of organisms that can be cultivated/cultured to produce biomass, there is a correspondingly wide variety of organic components that can be recovered. Accordingly, biomass can be used in the generation of feedstock for bioenergy in the form of oils or carbohydrates as well as in the production of human food and/or animal/aquaculture feed and as chemical precursors for further elaboration.

[0004] An example of a suitable biomass crop for up taking atmospheric CO2 is algae, which are photosynthetic aquatic organisms. Algae have untapped potential for global impact as hyper-productive crops. They can be grown at yields many times greater than current crops requiring only non-arable land, non-potable water, sunlight and CO2. However, the current economics of algae production are not favourable, except for specialised, high-value products that cannot be produced at a scale that will have a global environmental benefit. For algae to be grown commercially for bulk products such as protein feeds and biofuels, major technological production break throughs are required.

[0005] One of the most difficult challenges at present is to provide CO2 to the algae in a cost-effective manner over very large areas of cultivation. Relying on diffusion of CO2 from the atmosphere severely limits productivity as the rate of diffusion is relatively low. Achieving productive algae cultures thus currently requires a concentrated source of CO2 such as flue gas to be pumped into the cultivation medium. At the present time CO2 takes up around 70% of the raw material costs in microalgae cultivation and it therefore presents an attractive area for research in improving the efficiency of algae production. Studies have been investigating strategies to improve CO2 delivery efficiency and decrease CO2 cost, such as bubbling air mixed with a suitable portion of CO2, decreasing the size of gas bubbles to improve mass transfer, and regulating bubbling rate by a pH monitor. Unfortunately, however, these regimes rely on a gaseous CO2 source being chemically captured and transported from large point sources, which limits the location of a microalgae plant within the neighborhood of a CO2 capture plant. In addition, the high energy penalties during CO2 capture, CO2 compression, transport and sparging are yet to be solved, and much of the CO2 is lost back into the atmosphere before being consumed by the algae.

[0006] An abundant, sustainable and close CO2 source is the atmosphere, which is ultimately where CO2 concentrations must be reduced. Unfortunately, however, the natural rate of dissolution of atmospheric CO2 is insufficient for dense-grown algae, making it impractical for industrial application.

[0007] One option to overcome this slow uptake of CO2 would be the use of carbonic anhydrase (CA), a group of enzymes able to rapidly catalyse the conversion of CO2/HCO3 (Eq. 1 ). The conversion shown in equation 1 is in reversible equilibrium and could therefore be used to facilitate the dissolution of atmospheric CO2 into an aqueous medium.

[0008] In analysing the performance of carbonic anhydrase there are two relevant considerations. The first is that the enzyme is relatively expensive meaning that it is desirable to utilise as little of the material as possible. Secondly the ability of carbonic anhydrase to dissolve carbon dioxide from the atmosphere into an aqueous medium reduces dramatically the further the enzyme is from the medium/air interface. As a result of these two factors it is not currently possible to cost effectively disperse carbonic anhydrase in an aqueous medium to facilitate absorption of carbon dioxide. In addition, carbonic anhydrase freely dispersed in an algae culture is susceptible to biodegradation by bacteria and proteolytic enzymes that are present.

[0009] Accordingly, it would be desirable to provide a means by which the uptake of atmospheric carbon dioxide by an aqueous medium could be facilitated.

Summary of Invention

[0010] As a result of the desire to provide an improved way to facilitate the uptake of CO2 by an aqueous medium the present applicants have developed a hydrogel bead that contains cross-linked carbonic anhydrase. The bead is typically buoyant and can therefore float on the surface of most aqueous mediums such that the cross-linked carbonic anhydrase is located at the interface between the aqueous medium and the air (CO2 source) where the enzyme is most effective at solubilising CO2 from the atmosphere above. The use of such a bead ensures that the enzyme is located in the area where it can be most effective in facilitating CO2 uptake into the liquid and also means that the amount of enzyme used can be minimised. This is especially true in an algal processing system that is conducted in a continuous manner as these systems can be configured such that, unlike freely dispersed enzymes, the enzyme-containing beads are retained in the production zone rather than being removed when the biomass is harvested from the system.

[0011] Accordingly, in a first aspect the present invention provides a hydrogel bead containing cross-linked carbonic anhydrase, wherein the (hydrated) bead has a density of less than 1 .03 g/cm³ (at 20°C). As the bead has a density of less than 1.03 g/cm³ (at 20°C) the bead will float on an aqueous medium typically used for algal production (seawater) which ensures that the cross-linked carbonic anhydrase is located at the interface between the aqueous medium and atmosphere containing carbon dioxide. In addition, as the bead is a hydrogel bead it absorbs water ensuring that the cross-linked carbonic anhydrase is brought into contact with the aqueous medium. In one embodiment the bead has a density of less than 1.00 g/cm³ (at 20°C) and the bead will float on almost all aqueous medium providing greater flexibility in the media in which algae are produced.

[0012] In a second aspect the present invention provides a method for producing a hydrogel bead comprising cross-linked carbonic anhydrase, wherein the (hydrated) bead has a density of less than 1.03 g/cm³ (at 20°C), the method comprising: (a) forming an aqueous emulsion comprising (i) one or more hydrophilic gel materials (ii) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C and either (iii) cross-linked carbonic anhydrase or (iiia) carbonic anhydrase and (iiib) a crosslinking agent for the carbonic anhydrase; and (iv) water and (b) treating the emulsion to form the hydrogel bead.

[0013] In one embodiment the aqueous emulsion comprises (i) one or more hydrophilic gel materials (ii) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C, (iii) cross-linked carbonic anhydrase and (iv) water.

[0014] In another embodiment the aqueous emulsion comprises (i) one or more hydrophilic gel materials (ii) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C (iiia) carbonic anhydrase and (iiib) a crosslinking agent for the carbonic anhydrase and (iv) water.

[0015] In one embodiment step (b) comprises the steps of: (b1) subjecting the emulsion to gelling conditions to form a gel; and (b2) extruding the gel to form a hydrogel bead. [0016] In another embodiment step (b) comprises injecting the emulsion into a gelling solution to form a hydrogel bead. The gelling solution has a physical or chemical property such that once the emulsion is injected into the gelling solution the hydrogel bead is formed. Once produced the hydrogel bead can be isolated and stored for later use.

[0017] In yet an even further aspect the present invention provides a method of increasing the uptake of CO2 into an aqueous medium the method comprising applying a hydrogel bead of the invention as described above to the surface of the aqueous medium. As the bead contains cross-linked carbonic anhydrase it facilitates the uptake of CO2 by the aqueous medium.

[0018] In yet an even further aspect the present invention provides a method of promoting algae growth in an aqueous medium the method comprising applying a hydrogel bead according to the invention to the surface of the aqueous medium.

Description of the Drawings

[0019] Figure 1 shows a workflow of the preparation of buoyant beads of the invention.

[0020] Figure 2 shows microscopy images of the CA-GA beads: a-b) Optical microscopy images showing a whole bead and the interior of a bead; c-d) CLSM images, c) a dry bead, CA stained with fluorescein isothiocyanate (FITC); d) a wet bead, paraffin stained with Nile red.

[0021] Figure 3 shows SEM images of a bead cross-section using a) secondary electron (SE) detector, b) back-scattered electron (BSE) detector and c-f) energy- dispersive X-ray (EDX) detector, showing element mapping of d) Oxygen e) Calcium f) Carbon. All scale bars represent 200pm.

[0022] Figure 4 shows the residual activity of different Ca beads through repeated assays. [0023] Figure 5 correlation of CO2 hydration activity in Wilbur Anderson units (WAU ) and as the p-NPA hydrolysis rate versus the amount of CA. The x-axis for Figure 5 (a) gives the CA concentration in the sample after dilution with Tris buffer.

[0024] Figure 6 (a) Growth curves and (b) pH of Nannochloropsis sp. growing with different concentrations of free CA. Error bars represent the ranges of duplicate batches. The control is an identical culture without any CA addition.

[0025] Figure 7 shows a) photos and b) absorption spectrum of a CBBG solution after incubating with CA-GA beads or blank beads for 10 d (blank beads is the top line).

[0026] Figure 8 shows Growth rate of Nannochloropsis sp. with CA in different forms.

[0027] Figure 9 shows a) biomass and b) nitrate concentration in Nannochloropsis sp. cultures grown under different conditions as shown in the legend. Error bars represent the ranges of duplicate batches grown under identical conditions.

[0028] Figure 10 shows TIC concentration in the air sparged culture, CA-GA beads (2g) and the equivalent free CA group. Samples were taken on Days 1 ,3 and 7 of the cultivation period. Error bars represent the ranges of duplicate batches.

[0029] Figure 11 shows a) Growth enhancement and b) water evaporation of 200 mL Nannochloropsis sp. culture grown with 2g CA-GA beads in three cycles. Error bars represent the ranges of duplicate batches.

[0030] Figure 12 shows a) Picture and b) growth parameters of Nannochloropsis sp. growing in a mini raceway pond with and without CA-GA beads.

Detailed Description

[0031] In this specification, a number of terms are used that are well known to a skilled addressee. Nevertheless, for the purposes of clarity, a number of terms will be defined.

[0032] Throughout the description and the claims of this specification unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0033] It is to be noted that where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all numerical values or sub-ranges in between these limits as if each numerical value and sub-range is explicitly recited. The statement "about X% to Y%" has the same meaning as "about X% to about Y%," unless indicated otherwise.

[0034] The term “about” as used in the specification means approximately or nearly and in the context of a numerical value or range set forth herein is meant to encompass variations of +/- 10% or less, +/- 5% or less, +/- 1% or less, or +/- 0.1% or less of and from the numerical value or range recited or claimed.

[0035] It is also to be noted that, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context already dictates otherwise.

[0036] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[0037] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[0038] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential. [0039] An “emulsion” is a mixture of two or more liquids that are normally immiscible (unmixable or unblendable), with one of the liquids forming the dispersed phase and the other liquid forming the dispersion medium. Two liquids can form a number of different types of emulsions. By way of example, oil and water can form an oil-in-water emulsion where the oil is the dispersed phase and the water is the dispersion medium. Alternatively, they can form a water-in-oil emulsion where water is the dispersed phase and oil is the dispersion medium. Multiple emulsions are also possible such as a water- in-oil-in-water emulsion or an oil-in-water-in-oil emulsion. In circumstances where there are multiple similar phases in the same emulsion (like the two oil phases in an oil-in- water-in-oil emulsion) each phase may contain a different solute.

[0040] As used herein the term “miscibility” and derivations thereof such as “miscible” refers to the property or ability of two substances to mix in all proportions, or put another way, their ability to fully dissolve in each other at any concentration.

[0041] Accordingly, as used herein the term “immiscible” means that there are certain proportions of the substance where it does not dissolve in a second substance (the substance it is immiscible with). For example, butanone (methyl ethyl ketone) is significantly soluble in water but is still classed as water-immiscible as these two solvents are not soluble in each other in all proportions.

[0042] As used herein the term “oil" refers to any nonpolar chemical substance that is both hydrophobic and lipophilic and may include triesters of glycerol and fatty acids. Oils are typically liquids at room temperature.

Hydrogel Beads

[0043] As discussed above in one embodiment the present invention provides a hydrogel bead comprising containing cross-linked carbonic anhydrase, wherein the (hydrated) bead has a density of less than 1.03 g/cm³ (at 20°C). In one embodiment the (hydrated) bead has a density of less than 1.00 g/cm³ (at 20°C). For freshwater applications it is important that the hydrated bead has a density of less than 1 .00 g/cm³ (at 20°C). For marine applications it is important that the hydrated bead has a density of less than 1.03 g/cm³ (at 20°C). In general, as pure water has a density of approximately 1.00 g/cm³ (at 20°C), almost all aqueous mediums which contain dissolved solids to some degree will have densities in excess of this amount, so less dense beads will work in more dense water columns. The hydrogel beads of the invention typically comprise (a) one or more hydrophilic gel materials (b) cross-linked carbonic anhydrase and (c) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C.

[0044] The hydrogel beads of the present invention typically contain one or more hydrophilic hydrogel materials. The hydrogel bead may contain a single hydrogel material, or it may contain a number of different hydrogel materials that combine to form the final hydrogel. As would be appreciated by a person of skill in the art that there are a number of materials that can be used in the formation of the hydrogel beads of the present invention.

[0045] The hydrogel material may take any of a number of forms although it is typically found to be a polymeric material. In some embodiments the polymeric material is a synthetic polymeric material. In other embodiments the polymeric material is a natural polymeric material or a biopolymer. A number of suitable materials of this type are well known.

[0046] In order to be able to form a hydrogel it is preferred that the hydrogel material is hydrophilic. In addition, it is necessary that the material be able to be cross-linked in order to form a suitable hydrogel. In the manufacture of the hydrogel bead the hydrogel material is typically exposed to a gelling agent that leads to cross-linking of the hydrogel material leading to formation of the hydrogel bead.

[0047] In general, therefore the suitable hydrogel material contains suitable functionality either within the backbone of the material itself or as a pendant reactive group on the backbone of the material that allows the material to be cross-linked in hydrogel production. The reactive group can either be a group that reacts chemically with a gelling or cross-linking agent to form a new chemical bond or it may be a chelating group that can be cross linked with a cation such as a metal cation. [0048] In certain embodiments rather than being chemically cross-linked the hydrogel material is such that it can be physically cross-linked by subjection to certain physical stimulation.

[0049] Examples of suitable materials include: polyethylene glycol (PEG) -thiol, PEG-acrylate, acrylamide, N, N'-bis (acryloyl) citamine (BACy), PEG, polypropylene oxide (PPO) , polyacrylic acid, poly (hydroxyethyl methacrylate) (PHEMA), poly (methyl methacrylate) (PMMA), poly (N-isopropylacrylamide) (PNIPAAm), poly (lactic acid) (PLA), poly (Lactic acid-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly (vinylsulfonic acid) (PVSA), poly (L-aspartic acid), poly (L-glutamic add), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol, acrylates, polymethylene glycol diacrylates, polyethylene glycol diacrylates, trimethylropropane trimethacrylate, ethoxylated trimethylol triacrylates or ethoxylated pentaerythritol tetraacrylates or combinations thereof. In principle any material may be used as long as it can be used in the formation of a hydrogel.

[0050] In one embodiment the gel material is alginate. In one embodiment the gel material is pectin. In one embodiment the gel material is chitosan. In one embodiment the gel material is an alginate derivative.

[0051] The gel material is typically cross-linked in the final hydrogel bead using a gelling agent. As would be appreciated by a skilled worker in the field the identity of the suitable gelling (or cross-linking) agent will be readily determined based on the nature of the chosen hydrogel material. In some embodiments the gelling agent is a cationic species. Suitable cationic species are polyvalent cations. In one embodiment the cation is a +2 species. In one embodiment the cation is a +3 species. In one embodiment the cation is a +4 species.

[0052] A particularly suitable gelling agent is a metallic cation. Examples of suitable metallic cations include, Ca²⁺, Ba²⁺, Be²⁺, Cd²⁺, Co²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pt2+, Sn²⁺ and Zn²⁺. In one embodiment the gelling agent is Ca²⁺. [0053] The amount of gel material present in the bead may vary depending upon the gel material used. Nevertheless, the amount of gel material in the final hydrogel bead (once hydrated) is typically from 1wt% to 7wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is from 2wt% to 6wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is from 2.5wt% to 5.5wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is from 3.0wt% to 5.0wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is from 3.2wt% to 4.8wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is from 3.4wt% to 4.6wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is from 3.5wt% to 4.5wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is about 3.6 wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is about 3.7 wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is about 3.8 wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is about 3.9 wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is about 4.0 wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is about 4.1 wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is about 4.2 wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is about 4.3 wt%. %. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is about 4.4 wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is about 4.5 wt%. In one embodiment the amount of gel material in the final hydrogel bead (once hydrated) is about 4.6 wt%.

[0054] As stated above the hydrogel beads of the present invention also contain cross-linked carbonic anhydrase. Carbonic anhydrase enzymes are present in many living organisms. Widely accessible carbonic anhydrase sources include bacteria, archaea, algae and red blood cells. Most commercially available carbonic anhydrase are human and bovine, often expressed in bacterial systems. In one embodiment the carbonic anhydrase is human carbonic anhydrase. In one embodiment the carbonic anhydrase is bovine carbonic anhydrase. In one embodiment the carbonic anhydrase is marine algae carbonic anhydrase. In another embodiment the carbonic anhydrase is sourced from any other organism, either directly, or produced via microbial fermentation.

[0055] The amount of cross-linked carbonic anhydrase included in the hydrogel bead (when hydrated) may vary widely as in effect there are no limitations on the amount that may be used. Nevertheless, the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is typically from 0.1 mg/ g to 10 mg/g based on the total weight of the hydrogel bead (once hydrated). In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.1 mg/g to 10.0 mg/g. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.2 mg/g to 9.0 mg/g. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.3 mg/g to 8.0 mg/g. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.4 mg/g to 7.0 mg/g. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.5 mg/g to 6.0 mg/g. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.6 mg/g to 5.0 mg/g. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.7 mg/g to 4.0 mg/g. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.8 mg/g to 3.0 mg/g. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.9 mg/g to 2.5 mg/g. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 1.0 mg/g to 2.0 mg/g. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 1.2 mg/g to 1.8 mg/g.

[0056] The above amounts are expressed in milligrams per gram. For the assistance of the reader these can be expressed in weight percent as follows. The amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is typically from 0.01 wt% to 1 wt% based on the total weight of the hydrogel bead (once hydrated). In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.01 wt% to 1.0 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.02 wt% to 0.9 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.03 wt% to 0.8 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.04 wt% to 0.7 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.05 wt% to 0.6 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.06 wt% to 0.5 wt. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.07 wt% to 0.4 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.08 wt% to 0.3 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.09 wt% to 0.25 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.1 wt% to 0.2 wt% In one embodiment the amount of cross-linked carbonic anhydrase present in the hydrogel bead (once hydrated) is from 0.12 wt% to 0.18 wt%.

[0057] In order to ensure that the carbonic anhydrase is not readily leached from the hydrogel (as it is water soluble) it is cross linked or otherwise bound in some form to increase its bulk and help retain it in the hydrogel bead once formed. There are a number of cross-linking agents known in the art that may be used to cross-link carbonic anhydrase. In one embodiment the cross-linking agent is glutaraldehyde. In one embodiment the crosslinker is tannic acid. Whilst in principle a number of cross-linking agents could be used (such as a combination of the cross linkers described above) in practice it is typical to use a single cross-linking agent for simplicity. The amount of cross linking agent can vary although it is typically used in excess to ensure adequate cross-linking of the carbonic anhydrase. Accordingly it is typical that the weight ratio of cross linking agent to carbonic anhydrase is about 1 :1 . In one embodiment the weight ratio of cross-linking agent: carbonic anhydrase is from 0.2:1 to 10:1. In one embodiment the weight ratio of cross-linking agent: carbonic anhydrase is from 0.4:1 to 8:1. In one embodiment the weight ratio of cross-linking agent: carbonic anhydrase is from 0.6:1 to 6:1. In one embodiment the weight ratio of cross-linking agent: carbonic anhydrase is from 0.7:1 to 5:1 . In one embodiment the weight ratio of cross linking agent: carbonic anhydrase is from 0.8:1 to 4:1. In one embodiment the weight ratio of cross-linking agent: carbonic anhydrase is from 0.8:1 to 3:1. In one embodiment the weight ratio of cross-linking agent: carbonic anhydrase is from 0.8:1 to 2:1. In one embodiment the weight ratio of cross-linking agent: carbonic anhydrase is from 0.8:1 to 1.5:1. In one embodiment the weight ratio of cross-linking agent: carbonic anhydrase is from 0.8:1 to 1 .2:1 .

[0058] As would be appreciated by a skilled worker in the field the density of the hydrated hydrogel is controlled by selection of the materials used to make the hydrogel bead. The end aim is to produce a hydrogel bead wherein the hydrated bead has a density of less than 1 ,03g/cm³ at 20°C, preferably less than 1.OOg/cm³ at 20°C so that the hydrogel bead is buoyant. In one embodiment this is achieved by inclusion of a density modifying material in the hydrogel bead.

[0059] Accordingly, in one embodiment the bead comprises (a) one or more hydrophilic gel materials (b) cross-linked carbonic anhydrase and (c) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C.

[0060] The density modifying agent is one that is incorporated into the hydrogel bead such that the hydrated hydrogel bead has the desired final density. A number of materials may be used in order to produce a final hydrated hydrogel bead with the final desired density. A skilled worker in the field can readily choose a suitable density modifying material based on the identity of the other materials included in the hydrogel.

[0061] In certain embodiments the density modifying material is a hydrophobic material. In certain embodiments the density modifying material is a hydrophobic liquid. In certain embodiments the density modifying material is a hydrophobic organic liquid. In certain embodiments the density modifying material is an oil. A number of suitable oils may be used including mineral oils and edible oils. In one embodiment the density modifying material is a mineral oil. In one embodiment the density modifying material is an edible oil. [0062] In principle any oil could be used as long as it has low density and is hydrophobic. In certain embodiments it is preferred that the oil is non-volatile (to ensure that it remains in the hydrogel) and non-toxic (to ensure that it does not negatively impact biological systems such as algae).

[0063] In this regard paraffin oil seems particularly suitable due its low density (0.8 g/mL). Silicone oil is less suitable with a density of 0.97 g/mL. Edible oils such as canola oil are also less suitable, being more biodegradable with a density of 0.92 g/mL. In one embodiment therefore the density modifying agent is paraffin oil.

[0064] The amount of density modifying material present in the hydrated beads of the present invention may vary as the amount of density modifying agent is selected to ensure that the final hydrated hydrogel bead has the desired density. As such the amount used will depend on the nature of the hydrogel material and the nature of the density modifying material.

[0065] In general, however, the amount of density modifying material will be present in the hydrated hydrogel bead in an amount of 5wt% to 50wt% based on the total weight of the bead. In one embodiment the amount of density modifying material in the hydrated hydrogel bead is from 10wt% to 30wt% based on the total weight of the bead. In one embodiment the amount of density modifying material in the hydrated hydrogel bead is from 12wt% to 27wt% based on the total weight of the bead. In one embodiment the amount of density modifying material in the hydrated hydrogel bead is from 14wt% to 24wt% based on the total weight of the bead. In one embodiment the amount of density modifying material in the hydrated hydrogel bead is from 16wt% to 21wt% based on the total weight of the bead. In one embodiment the amount of density modifying material in the hydrated hydrogel bead is from 17wt% to 19wt% based on the total weight of the bead.

[0066] The hydrated hydrogel beads may vary in size with the size of the bead typically being determine by the method used to fabricate the bead. The hydrated beads are typically from 0.1mm to 50.0mm in diameter. In one embodiment the hydrated beads are from 0.1 mm to 40.0 mm in diameter. In one embodiment the hydrated beads are from 0.1 mm to 30.0 mm in diameter. In one embodiment the hydrated beads are from 0.1 mm to 20.0 mm in diameter. In one embodiment the hydrated beads are from 0.1 mm to 10.0 mm in diameter. In one embodiment the hydrated beads are from 0.3 mm to 9.0 mm in diameter. In one embodiment the hydrated beads are from 0.5 mm to 8.0 mm in diameter. In one embodiment the hydrated beads are from 0.7 mm to 7.0 mm in diameter. In one embodiment the hydrated beads are from 0.8 mm to 6.0 mm in diameter. In one embodiment the hydrated beads are from 1.0 mm to 5.0 mm in diameter.

Fabrication of the hydrogel beads

[0067] The present invention also provides a method for producing hydrogel beads as discussed above. Accordingly the present invention provides a method for producing a hydrogel bead comprising cross-linked carbonic anhydrase, wherein the (hydrated) bead has a density of less than 1.03 g/cm³ (at 20°C), the method comprising (a) forming an aqueous emulsion comprising (i) one or more hydrophilic gel materials (ii) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C and either (iii) cross-linked carbonic anhydrase or (iiia) carbonic anhydrase and (iiib) a crosslinking agent for the carbonic anhydrase; and (iv) water and (b) treating the emulsion to form the hydrogel bead.

[0068] In essence the carbonic anhydrase can be either-cross linked prior to its use in the formation of the emulsion; or free carbonic anhydrase and a crosslinking agent for the carbonic anhydrase can be added in the formation of the emulsion. The reaction between carbonic anhydrase and the crosslinking agent is typically so fast that even when this is done the carbonic anhydrase will be cross-linked during emulsion formation or during the step of treating the emulsion to form the hydrogel bead.

[0069] In one embodiment the emulsion comprises (i) one or more hydrophilic gel materials (ii) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C and (iii) cross-linked carbonic anhydrase and (iv) water.

[0070] In yet another embodiment the emulsion comprises (i) one or more hydrophilic gel materials (ii) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C (iiia) carbonic anhydrase and (iiib) a crosslinking agent for the carbonic anhydrase and (iv) water.

[0071] As would be appreciated treating the emulsion to form a hydrogel bead may involve either subjecting the emulsion to a physical or chemical reaction to form a gel which is either simultaneously or sequentially then turned into a hydrogel bead. In some embodiments the gelling or the hydrophilic gel material involves temperature elevation. In some embodiments the gelling or the hydrophilic gel material involves a pH change. In some embodiments the gelling or the hydrophilic gel material involves reaction with a gelling agent.

[0072] Treating the emulsion to form the hydrogel bead may be carried out as either a one or two step process. Accordingly, the emulsion may firstly be converted into a gel followed by conversion of the gel into a bead or, alternatively, gelling of the emulsion may occur simultaneously with bead formation.

[0073] In one embodiment step (b) comprises the steps of:(b1) subjecting the emulsion to gelling conditions to form a gel; and (b2) extruding the gel to form a hydrogel bead.

[0074] In one form of this embodiment the emulsion is either subjected to heat or reaction with a pH modifier or a gelling agent to form a gel. Once formed the gel is then extruded to form hydrogel beads of the required size. In order to facilitate bead formation, the gel is typically extruded into an aqueous solution so as to facilitate formation of substantially spherical beads. As will be appreciated once a gel is formed there are a number of potential ways in which the gel can be converted into the required hydrogel beads.

[0075] In one embodiment step (b) comprises injecting the emulsion into a gelling solution to form a hydrogel bead.

[0076] As would be appreciated by a skilled worker in the field the conditions required to form the hydrogel bead (i.e. the gelling conditions) may vary widely depending upon the hydrophilic gel materials chosen. In general, a skilled worker in the field will be able to determine the appropriate gelling conditions based on the hydrogel material chosen. In general, therefore, the gelling solution will have a physical or chemical property that is selected based on the identity of the hydrophilic gel material such that injection of the emulsion into the gelling solution leads to the formation of a hydrogel bead. The property may be a physical property such as temperature, pressure or pH or it may be a chemical property of the gelling solution such as the presence in the gelling solution of a gelling agent. In some embodiments the gelling or the hydrophilic gel material involves temperature elevation of the gelling solution. In some embodiments the gelling or the hydrophilic gel material involves a pH change and as such the gelling composition is at a pH suitable to lead to gel formation. In some embodiments the gelling or the hydrophilic gel material involves reaction with a gelling agent present in the gelling solution.

[0077] It is preferred that the emulsion is injected into the gelling solution in the form of droplets to facilitate formation of the hydrogel beads.

[0078] A first step in the method is the formation of an emulsion containing the components of the bead as described herein before. Accordingly, the first step in the method involves the formation of an aqueous emulsion comprising (i) one or more hydrophilic gel materials (ii) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C and either (iii) cross-linked carbonic anhydrase or (iiia) carbonic anhydrase and (iiib) a crosslinking agent for the carbonic anhydrase and (iv) water.

[0079] This emulsion may be formed in any way known in the art although it typically involves addition of all ingredients in the appropriate amounts to water followed by agitation to form the desired emulsion.

[0080] As stated previously examples of suitable gel materials include: polyethylene glycol (PEG) -thiol, PEG-acrylate, acrylamide, N, N'-bis (acryloyl) citamine (BACy), PEG, polypropylene oxide (PPO) , polyacrylic acid, poly (hydroxyethyl methacrylate) (PHEMA), poly (methyl methacrylate) (PMMA), poly (N-isopropylacrylamide) (PNIPAAm), poly (lactic acid) (PLA), poly (Lactic acid-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly (vinylsulfonic acid) (PVSA), poly (L-aspartic acid), poly (L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol acrylates, polymethylene glycol diacrylates, polyethylene glycol diacrylates, trimethylropropane trimethacrylate, ethoxylated trimethylol triacrylates or ethoxylated pentaerythritol tetraacrylates or combinations thereof. In principle any material may be used as long as it can be used in the formation of a hydrogel.

[0081] In one embodiment the gel material is alginate. In one embodiment the gel material is pectin. In one embodiment the gel material is chitosan. In one embodiment the gel material is an alginate derivative. The amount of the gel material in the emulsion may vary with the amount typically being from 1 wt% to 3 wt% based on the total weight of the emulsion although this will vary depending upon the hydrogel material used. In one embodiment the amount of gel material in the emulsion is from 1.3wt% to 2.9 wt% of the total weight of the emulsion. In one embodiment the amount of gel material in the emulsion is from 1.6wt% to 2.8 wt% of the total weight of the emulsion. In one embodiment the amount of gel material in the emulsion is from 1.9wt% to 2.7 wt% of the total weight of the emulsion. In one embodiment the amount of gel material in the emulsion is from 2.0% to 2.6 wt% of the total weight of the emulsion. In one embodiment the amount of gel material in the emulsion is from 2.2wt% to 2.5 wt% of the total weight of the emulsion. In one embodiment the amount of gel material in the emulsion is about 2.4 wt% of the total weight of the emulsion.

[0082] The emulsion also contains a density modifying agent that is immiscible with the one or more hydrophobic gel materials. In certain embodiments the density modifying material is a hydrophobic material. In certain embodiments the density modifying material is a hydrophobic liquid. In certain embodiments the density modifying material is a hydrophobic organic liquid. In certain embodiments the density modifying material is an oil. A number of suitable oils may be used including mineral oils and edible oils. In one embodiment the density modifying material is a mineral oil. In one embodiment the density modifying material is an edible oil.

[0083] In principle any oil could be used in the formation of the emulsion as long as it has low density and is hydrophobic. In certain embodiments it is preferred that the oil is non-volatile (to ensure that it remains in the hydrogel once formed) and non-toxic (to ensure that it does not negatively impact biological systems such as algae).

[0084] In this regard paraffin oil seems particularly suitable due its low density (0.8 g/mL). Silicone oil is less suitable with a density of 0.97 g/mL. Edible oils such as canola oil are also less suitable, being more biodegradable with a density of 0.92 g/mL. In one embodiment therefore the density modifying agent is paraffin oil.

[0085] The amount of density modifying material in the emulsion will vary although it is typically present in an amount of 5wt% to 50wt% based on the total weight of the emulsion. In one embodiment the amount of density modifying material in the emulsion is from 6.0wt% to 40wt% based on the total weight of the emulsion. In one embodiment the amount of density modifying material in the emulsion is from 7wt% to 30wt% based on the total weight of the emulsion. In one embodiment the amount of density modifying material in the emulsion is from 8wt% to 20wt% based on the total weight of the emulsion. In one embodiment the amount of density modifying material in the emulsion is from 9wt% to 12wt% based on the total weight of the emulsion.

[0086] The emulsion also contains a carbonic anhydrase or cross-linked carbonic anhydrase as discussed above. If carbonic anhydrase is added prior to cross linking the amount of carbonic anhydrase present in the emulsion is typically from 0.01 wt% to 5 wt% based on the total weight of the emulsion. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.01 wt% to 4.0 wt%. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.01 wt% to 3.0 wt%. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.01 wt% to 2.0 wt%. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.01 wt% to 1.0 wt%. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.02 wt% to 0.9 wt%. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.03 wt% to 0.8 wt%. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.04 wt% to 0.7 wt%. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.05 wt% to 0.6 wt%. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.06 wt% to 0.5 wt%. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.07 wt% to 0.4 wt%. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.08 wt% to 0.3 wt%. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.09 wt% to 0.25 wt%. In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.1 wt% to 0.2 wt% In one embodiment the amount of carbonic anhydrase present in the emulsion is from 0.12 wt% to 0.18 wt%.

[0087] In certain embodiments the emulsion also contains a cross-linking agent for the carbonic anhydrase.

[0088] As stated above there are a number of cross-linking agents known in the art that may be used to cross-link carbonic anhydrase. In one embodiment the crosslinking agent is glutaraldehyde. In one embodiment the cross-linking agent is tannic acid. The amount of cross-linking agent can vary although it is typical that the weight ratio of cross-linking agent to carbonic anhydrase is about 1 :1. In one embodiment the weight ratio of cross linking agent: carbonic anhydrase is from 0.2:1 to 10:1. In one embodiment the weight ratio of cross linking agent: carbonic anhydrase is from 0.4:1 to 8:1. In one embodiment the weight ratio of cross linking agent: carbonic anhydrase is from 0.6:1 to 6:1. In one embodiment the weight ratio of cross linking agent: carbonic anhydrase is from 0.7:1 to 5:1 . In one embodiment the weight ratio of cross linking agent: carbonic anhydrase is from 0.8:1 to 4:1. In one embodiment the weight ratio of cross linking agent: carbonic anhydrase is from 0.8:1 to 3:1. In one embodiment the weight ratio of cross linking agent: carbonic anhydrase is from 0.8:1 to 2:1. In one embodiment the weight ratio of cross linking agent: carbonic anhydrase is from 0.8:1 to 1.5:1. In one embodiment the weight ratio of cross linking agent: carbonic anhydrase is from 0.8:1 to 1.2:1.

[0089] If cross-linked carbonic anhydrase is added amount of cross-linked carbonic anhydrase present in the emulsion is typically from 0.01 wt% to 5 wt% based on the total weight of the emulsion. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.01 wt% to 4.0 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.01 wt% to 3.0 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.01 wt% to 2.0 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.01 wt% to 1.0 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.02 wt% to 0.9 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.03 wt% to 0.8 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.04 wt% to 0.7 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.05 wt% to 0.6 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.06 wt% to 0.5 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.07 wt% to 0.4 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.08 wt% to 0.3 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.09 wt% to 0.25 wt%. In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.1 wt% to 0.2 wt% In one embodiment the amount of cross-linked carbonic anhydrase present in the emulsion is from 0.12 wt% to 0.18 wt%.

[0090] Once an emulsion has been formed containing the required components the emulsion is then subjected to gelling conditions to form the hydrogel bead. The exact gelling conditions chosen will depend upon the nature of the hydrophilic gel materials and may involve subjecting the emulsion to temperature changes such as heating, pH changes such as acidification or to chemical reaction such as by adding a solution containing a gelling agent. The gel thus formed may then be extruded to form the required hydrogel beads.

[0091] In certain embodiments the emulsion is converted into a hydrogel bead by injecting the emulsion into a gelling solution. In one embodiment the emulsion is injected into a gelling solution at an elevated temperature to produce a hydrogel bead. In one form of this embodiment the gelling solution is at a temperature of at least 30°C. In one form of this embodiment the gelling solution is at a temperature of at least 40°C. In one form of this embodiment the gelling solution is at a temperature of at least 50°C. In one form of this embodiment the gelling solution is at a temperature of at least 60°C. [0092] In one embodiment emulsion is injected into a gelling solution at an acidic pH to produce a hydrogel bead. In one form of this embodiment the gelling solution has a pH of less than 6.5. In one form of this embodiment the gelling solution has a pH of less than 6.0. In one form of this embodiment the gelling solution has a pH of less than 5.5. In one form of this embodiment the gelling solution has a pH of less than 5.0. In one form of this embodiment the gelling solution has a pH of less than 4.5.

[0093] In one embodiment the emulsion is injected into a gelling solution containing a gelling agent to produce a hydrogel bead.

[0094] The identity of the gelling agent can be readily determined by a skilled worker in the field based on the nature of the chosen hydrogel material. In some embodiments the gelling agent is a cationic species. Suitable cationic species are polyvalent cations. In one embodiment the cation is a +2 species. In one embodiment the cation is a +3 species. In one embodiment the cation is a +4 species.

[0095] A particularly suitable gelling agent is a metallic cation. Examples of suitable metallic cations include, Ca²⁺, Ba²⁺, Be²⁺, Cd²⁺, Co²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pt2+, Sn²⁺ and Zn²⁺. In one embodiment the gelling agent is Ca²⁺ When a cationic gelling agent is used any of a number of suitable anionic counter ions may be used. In one embodiment the anionic counter ion is the chloride ion. Accordingly, if the gelling agent is Ca²⁺ it is preferred that the solution be a solution of CaCl2.

[0096] The solution containing the gelling agent typically contains from 1wt% to 9wt% of the gelling agent. In one embodiment the solution containing the gelling agent contains from 2wt% to 8wt% of the gelling agent. In one embodiment the solution containing the gelling agent contains from 3wt% to 7wt% of the gelling agent. In one embodiment the solution containing the gelling agent contains from 4wt% to 6wt% of the gelling agent. In one embodiment the solution containing the gelling agent contains from 4.5wt% to 5.5wt% of the gelling agent. In one embodiment the solution containing the gelling agent contains about 5wt% of the gelling agent.

[0097] The emulsion is may be injected into the solution using any technique known. A suitable technique is to extrude the emulsion through a needle into the solution containing the gelling agent. This leads to gelation and formation of the buoyant beads. The beads can be collected and stored for later use, further processing or transportation.

Use of Hydrogel Beads

[0098] As stated previously the hydrogel beads can be utilized to increase the uptake of CO2 into an aqueous medium wherever that finds application. Accordingly, in yet an even further aspect the present invention provides a method of increasing the uptake of CO2 into an aqueous medium, the method comprising applying a hydrogel bead of the invention as described above to the surface of the aqueous medium. In essence the beads could be used in any scenario where it is desirable to increase the uptake of CO2 by an aqueous medium.

[0099] One particularly suitable application for the beads of the present invention is in the area of algal culture where a rate limiting step on the growth of the algae is the ability of the aqueous medium in which the algae is being cultured to absorb CO2. An additional benefit of applying this technology to algae cultivation is that the algae can maintain a concentration gradient of CO2 to allow sustained uptake of CO2 from the atmosphere by consuming the dissolved CO2 via photosynthesis.

[0100] The applicants note that there are a large number of algal species that have been cultivated/cultured to form biomass and a great diversity of algal that have yet to be cultivated or isolated. Algae include both microalgae (microscopic in size) and macroalgae/filamentous algae that are observable without a microscope. Examples of microalgae that may be used include species in genera such as Nannochloropsis, Chlorella, Haematococcus, Dunaliella, Scenedesmus, Isochrysis, Phaeodactylum, Chlamydomonas, Navicula, Porphyridium, Botryococcus and Thraustochytrium. Examples of macroalgae that may be used include Porphyra, Macrocystis, Spirogyra, Ulva, Sargassum, Augophyllum, and Oedogonium. In addition to eukaryotic algae, blue-green algae/cyanobacteria (photosynthetic bacteria) can be used including for example Spirulina, Microcytis, Anabaena, Prochlorococcus, Nostoc and Synechocytis. In principle the beads of the present invention could be used in the cultivation of any of these species. [0101] As would be appreciated by a skilled worker in the field, in general the cultivation of algal biomass typically involves the culturing of the algae (either freshwater, brine or marine) in a suitable culturing media selected based on the characteristics of the algae. Typically, this will comprise of a source of water of the appropriate salinity (e.g. fresh water, brackish water, seawater, or hypersaline water) supplemented with nutrients (e.g. sources of nitrogen, phosphorous, minerals, trace elements and possibly vitamins). The exact media selected will vary on the algae type as would be well appreciated by a skilled worker in the art.

[0102] The algal species can be cultivated in a wide variety of cultivation systems ranging from large open pond systems such as raceway ponds or disk reactors through to algae turf scrubbers, through to smaller systems. The choice of system will in general depend upon the scale of the cultivation facility, the capital costs, the specific requirements of the species to be produced, and the factors relating to the production location and other process variables such as available space and energy requirements.

[0103] Cultivation of the algal species in these ways may involve the use of natural sunlight or it may involve subjecting the culture to artificial light to allow indoor cultivation or to intensify or lengthen the period of exposure of the culture system to light to increase production.

[0104] The beads of the present invention are added to the culture medium such that the beads, being buoyant cover a portion of the surface of the aqueous medium. As would be appreciated there is somewhat of a trade-off between increasing the uptake of CO2 by increasing the surface area of the aqueous medium covered and still ensuring there is adequate light reaching the algae through the surface. In one embodiment the beads cover from 1% to 50% of the surface of the aqueous medium. In one embodiment the beads cover from 5% to 40% of the surface of the aqueous medium. In one embodiment the beads cover from 10% to 30% of the surface of the aqueous medium. In one embodiment the beads cover from 15% to 25% of the surface of the aqueous medium.

[0105] In general algae are cultured at temperatures in the range of 5°C to 40°C although depending on the climate and the algal species chosen it is not unknown for culture temperatures to go below or to exceed this for limited periods. The temperatures under which the biomass is cultured can vary geographically and temporally, particularly for outdoor cultures as is well known in the art. For indoor cultures the temperature can readily be selected and controlled by the skilled worker based on the identity of the algal species chosen. Accordingly, in one embodiment the method is carried out at a temperature of from 5°C to 40°C. In one embodiment the method is carried out at a temperature of from 15°C to 35°C. Accordingly, in one embodiment the method is carried out at a temperature of from 20°C to 30°C. In one embodiment the method is carried out at a temperature of from 25°C to 28°C.

[0106] The invention will now be illustrated by way of examples; however, the examples are not to be construed as being limitations thereto.

Examples

Materials

[0107] Bovine Erythrocyte CA (BCA), glutaraldehyde (GA, 25%), para-nitrophenyl acetate (p-NPA), para-nitrophenol (p-NP), thiamine hydrochloride, Nile red, fluorescein isothiocyanate (FITC), vitamin B12, CUSO4-5H2O, MnCl2-4H2O and Na2MoO4-2H2O were purchased from Sigma-Aldrich (Castle Hill, Australia). Paraffin liquid, ferric citrate and selenous acid were obtained from Ajax Finechem (Sydney, Australia). Acetonitrile, citric acid, and KH2PO4-2H2O were from Merck (Kenilworth, United States). Tris(hydroxymethyl)aminomethane (Tris), sodium alginate, hydrochloric acid (HCI, 1 N), CaCl2, NaNOa, and COCI2-6H2O were purchased from Chem-Supply (Gillman, Australia). All chemicals were used as purchased unless specifically mentioned. 50 mM Tris buffer was prepared with Tris and purified water (Millipore Elix) and adjusted to pH 8.0 by titrating with 1 N HCI.

[0108] A marine strain of microalgae, Nannochloropsis salina, was obtained from the University of Melbourne Culture Collection. This species has been found to utilize HCOg as a carbon source.²³ MF medium²⁴ was prepared as the algae culture medium by dissolving 33.4 g Red Sea Coral Pro Salt and 1 mL nutrient stock solution in 1 L purified water. The nutrient stock solution contained 200 g/L NaNOa, 15.8 g/L KH2PO4, 9.0 g/L ferric citrate, 9.0 g/L citric acid, 6.4 mg/L CuSO*, 12.9 mg/L ZnSO4, 6.0 mg/L C0CI2, 127 mg/L MnCIz, 7.2 mg/L NaaMoO*. 0.65 mg/L HaSeOs, 0.5 mg/L vitamin B12, 0.5 mg/L biotin and 100 mg/L thiamine hydrochloride.

Example 1 : Bead Manufacture

[0109] As a first step in preparation of the beads 5 mg CA, 5 mg GA, 0.5 mL 50 mM Tris buffer (pH 8.0), 0.12 g sodium alginate, and 0.5 g paraffin were mixed with purified water to 5g. Paraffin was selected as a density-modifying material since it has low density (0.86 g/mL), low volatility, is water-immiscible and not toxic to algae. The mixture was constantly stirred for 20 min until a homogeneous emulsion was formed. As shown in Figure 1 , the emulsion was then extruded through a 25G needle (Terumo, Japan) at the rate of 0.3 mL/min by an NE-4000 syringe pump (Adelab Scientific, Australia) into a 5 % CaCh solution. Beads containing CA formed as droplets of the emulsion interacted with the Ca²⁺. The beads were taken out after 4 h and stored in Tris buffer at 4 °C. The free CA concentration in the curing and storing solutions was measured by a Wilbur-Anderson assay (described below) to determine the CA loss during preparation and storage.

[0110] The size of 20 wet beads were measured by a digital micrometer (Mitutoyo, Japan) as 2.26±0.09 mm. The bead density was 0.91 g/mL, as calculated after measuring the weight and volume of 300 beads. The beads proved to maintain good buoyancy for a year.

Example 2: Characterisation of CA-

[0111] Wilbur-Anderson Assay (W-A assay). The activity of CA was evaluated based on the Wilbur-Anderson method (Wilbur, K. M.; Anderson, N. G., Electrometric and colorimetric determination of carbonic anhydrase. Journal of biological chemistry 1948, 776(1), 147-154). In brief, 1.4ml of sample was mixed with 12.6 ml of 50mM Tris buffer in an ice bath before 6ml of CO2 saturated water was added. A pH meter (S220 SevenCompact, Mettler Toledo, US) was used to monitor the pH and temperature of the mixture (around 4°C). A stopwatch was used to record the time. The pH drop from 8.3 to 7.3 was measured. The Wilbur-Anderson Unit (WAU) is defined as (Tc/T_s)-1, where Tc and T_s refer to the time required for the certain pH drop without and with the presence of CA. Due to the slow speed of natural CO2 hydration, Tc was measured every day when Ts was measured, and an average Tc was calculated as 270± 40 s based on 25 measurements. The W-A assay was so sensitive that a reaction with enzyme concentration higher than 0.5 mg/L finished in less than 15s. For this reason, the sample dilution with Tris buffer was used when the enzyme concentration was greater than 0.5 mg/L p-NPA activity assay

[0112] CA activity was also measured based on its esterase activity for hydrolysis of para-nitrophenyl acetate (p-NPA).²⁶100 pL of p-NPA in acetonitrile (2.5 mg/mL) was mixed with 4.9 mL of CA in Tris buffer immediately before the activity assay. The concentration of the 4-nitrophenol (p-NP) produced was quantified by a Cary 3E UV- Vis spectrophotometer (Varian, Palo Alto, US) at a wavelength of 348 nm, which is the isosbestic point of the products. The molar extinction coefficient at 348nm was 5.2 mM- ¹ cm-¹ according to calibration experiment completed by measuring the absorbance of p-NP solutions with the concentrations 0, 0.2, 0.4, 0.6 and 0.8 mM.

Example 3: Bead Characterisation

Confocal laser scanning microscopy (CLSM) -

[0113] The distribution of oil and protein was observed using a Nikon A1 R+ confocal laser scanning microscope (Tokyo, Japan) equipped with the software NIS-Elements AR. Before microscopy, the beads were stored in 50 mg/L fluorescein isothiocyanate (FITC) and 15mg/L Nile red solution overnight and rinsed with MilliQ water. The beads were then placed on a glass slide. An air immersion 20x objective was used. Nile red and FITCs were imaged at excitation wavelength 488 nm and 561 nm respectively, emission wavelength at 500-550 nm and 570-620 nm respectively. The two images were overlayed, where the FITC labelled CA appears green, the Nile red labelled paraffin appears red and other phases appear black.

[0114] The results of this characterisation is shown in Figure 2. Under the optical microscope, the CA-GA beads were spherical (Figure 2a) with buoying droplets of paraffin visible beneath the exterior surface (Figure 2b). 100 droplets were captured from the image and the bead diameter was measured as 40±16 pm. The droplets within the beads, stained with Nile red, were confirmed as paraffin oil droplets (Figure 2d) under CLSM, while CA stained with FITC was found dispersed throughout the droplets (Figure 2c).

Scanning Electron Microscope (SEM)

[0115] The morphology of the inner and outer surface of the materials was imaged by a FlexSEM 1000 (HITACHI, Tokyo, Japan) at accelerating voltage 15 kV and spot number 80 The microscope was equipped with a secondary electron (SE) detector, a back-scattered electron (BSE) detector, and an energy-dispersive X-ray (EDX) detector for element identification and mapping. Energy-dispersive X-ray (EDX) spectroscopy was applied for element identification and mapping. For examination of the cross- section structure of the beads, they were frozen with liquid nitrogen and cut in half with a blade.

[0116] SEM images of the beads show the cross-section of the beads as a gelatinous texture with visible bulges dispersed (Figure 3a), consistent with the droplets observed beneath the surface (Figure 2a). Both BSE and EDX images (Figure 3b, c) provided information on atomic weight, where areas with heavier elements were brighter than those with lighter elements. As confirmation, element mapping showed a similar distribution of calcium (Figure 3e) to the bright area in Figure 3c. Oxygen atoms, mainly originated from alginate, were evenly distributed (Figure 3d). While carbon is expected in both paraffin and alginate phases, it was denser in paraffin droplets (Figure 3f) due to a higher carbon content (85 wt%). The map indicated that calcium alginate was evenly distributed as the framework of the beads, while paraffin droplets were successfully embedded to provide stable buoyancy.

Coomassie Blue dyeing

[0117] Coomassie Brilliant Blue G-250 (CBBG) is a dye that can form non-covalent bonds with proteins.²⁷ It was thus used for enzyme identification in the alginate beads. CA-GA beads and blank beads were prepared in the same manner except for the addition or absence of CA. Two CA-GA beads and blank beads were suspended in 3 mL 0.01 mg/mL CBBG solution respectively. After storage under room temperature for 10 days, the light absorption of the solution was measured by a Cary 3E UV-Vis spectrophotometer (Varian, Palo Alto, US).

Example 4: Effect of crosslinking on CA loss during bead preparation

[0118] A series of studies was carried out comparing cross linked beads to noncross linked beads over a number of assays.

[0119] CA loss into the curing solution was much higher without GA presence (Table 1). Consistently, the hydrase activity of CA beads without GA decreased significantly throughout 10 cycles of assay, indicating CA leaching out from the beads. When CA was cross-linked with GA before encapsulation, it remained stable throughout 14 assay cycles (Figure 4). These results showed that the CA-GA beads have a higher resistance to denaturation and enzyme leaching compared with CA beads. As can be seen the presence of GA dramatically reduced the CA loss from 88±3 % to 19±1 % during bead formation. CA loss during storage under quiescent conditions was much less significant. For both CA and CA-GA beads, the storing solution showed stable CA activity over a week, indicating no ongoing CA loss with time (data not shown), probably because a CA concentration equilibrium was reached between the bead surface and the storing solution.

Table 1 : The Influence of GA on CA loss during the preparation and storage process

[0120] Following repeated use, the hydrase activity of the CA beads without GA decreased significantly throughout cycles of the W-A assay, with only 16% left after 5 cycles (Figure 4), indicating CA leaching from the beads in the washing step. Comparing the retention of CA during quiescent bead storage with the loss of CA during washing indicates loose entrapment of some of the enzyme. By contrast, when CA was cross-linked with GA before encapsulation, the beads retained around 100% activity throughout 10 assay cycles. These results showed that the CA-GA beads had a higher resistance to enzyme leaching compared with CA beads. Therefore, later experiments were all performed with beads containing GA-CA aggregates.

[0121] Based on the assay results, the CA-GA beads exhibited a CO2 hydration rate of 0.079 μmol cm^-2 s"¹. Assuming a target microalgae growth rate of 25 g m^-2 d"¹ and dry cell carbon content of 50%, only 0.015 m² of valid bead area is needed per m² of algae culture, i.e. culture surface occupation rate is 0.77% - low enough to ignore light shadowing.

Example 5: Algae cultivation

[0122] A marine strain of Nannochloropsis sp. was obtained from the University of Melbourne Culture Collection [25], At the start of each cultivation period, the algae culture was diluted to 0.05 g/L with MF medium [25], 200 mL algae culture was put in a Schott bottle, magnetically stirred at 100 rpm. Since large-scale outdoor microalgae production is commonly performed in a raceway pond, a 10 L raceway pond detailed in our previous work( was also used as a further demonstration. In both cases, the algae culture was diluted to 0.05 g/L with the MF medium at the start of each cultivation period. The algae were grown at room temperature (26±1 °C) and illuminated by T5 Aquarium florescent globes from the top at 130±3 μmol m^{- 2} s^-1. An AquaOne air pump was used to facilitate ventilation within the headspace of the bottles and raceway chambers, to ensure the replacement of CO2-depleted air. Purified water was added on a daily basis to compensate for evaporation.

[0123] During microalgae growth, the culture pH and temperature were measured by the pH meter described above. Optical density (OD) was measured by the spectrophotometer at wavelength 750nm. OD can be converted to biomass concentration using the calibration formula: Biomass(gZL) = 0.2273OD (R²=0.9992). Culture samples were filtered, acidified and the nitrate concentration measured based on the absorbance at 275nm and 220 nm (Ultraviolet Spectrophotometric Screening Method).²⁸ The total inorganic carbon (TIC) concentration was measured by a CM5015 CO2 coulometer (UIC Inc., IL, US). 5.1 : Free CA

[0124] Initial experiments were performed to assess the activity of different concentrations of free CA and their effect on the growth of Nannochloropsis sp. Error! Reference source not found.(a) showed that the hydrase activity of free CA was linearly dependent on its concentration up to 0.4 mg/L. This trend did not continue at higher concentrations where the enzyme was more than needed. Within a 95% confidence level, the hydrase activity of CA was calculated to be 2800±300 WAU/mg CA, and the catalysed hydration rate was in the order of 10⁵ mol CO₂ mol CA^-1 s^-1, consistent with literature values 10⁴ - 106 s ¹.²⁹ These results were used to quantify active CA concentration in a solution.

[0125] Similarly, p-NPA hydrolysis rate (Figure 5(b)) showed a distinct linear correlation to CA concentration. In contrast to the highly sensitive W-A assay, p-NPA assay featured a much wider optimal enzyme range which was 1 -10 mg/L, allowing characterisation of enzyme activity beyond the linear range of the W-A method. Moreover, the catalysed hydrolysis rate was calculated as 0.14 mol p-NPA mol CA^-1 s ¹ , 10⁶ times slower than the CO2 hydration rate.

[0126] The growth rate of the microalgae increased slightly as free CA concentration increased (Figure 6). Biomass accumulated steadily over time, while the growth rate increased with increasing CA concentration. This was due to the enrichment of inorganic carbon by CA activity. Typically, 40 mg/L of CA improved the growth of algae by 87%. However, supply and continual replacement of this amount of free CA is not economical, which emphasizes the importance of the surface immobilization technique. A lower pH for higher CA concentration during the early days of growth is reasonable as CO2 hydration releases H+.

5.2: Comparison of free CA versus beads versus control

[0127] Evidence of successful enzyme loading into the beads was given by CBBG dyeing. After incubating the colourless beads in the CBBG solution(see para 120) for 10d, both the CA-GA beads and blank beads were stained blue (Figure 7a). However, the CBBG concentration in the CA-GA bead vial, as given by the absorbance at 555nm, was only 34% of that in the blank bead vial (Figure 7b), indicating binding of the dye with protein (i.e. GA) contained within the beads.

[0128] Figure 8 showed the productivity of the algae with GA. Free GA enhanced the productivity by 61% while CA-GA beads achieved more than 75%. After the microalgae cultivation process, free GA cannot be recycled. Most microscopic immobilization techniques require centrifugation or filtration to recycle the enzyme. However, micro- or nano- material supported GA cannot be easily separated from the algae cells whose size lies in the range of micrometres. By contrast, at the end of cultivation, the alginate beads with diameters 2-3 mm can be taken out directly from the culture surface for a second-cycle use, which dramatically simplifies the separation process and therefore reduces the cost.

5.3: Multi- Day Algae Growth Trial

[0129] Nannochloropsis sp. was cultivated under the following conditions: 1) a positive control group with air sparging at a flow rate of 280 mL/min, providing more than sufficient CO2 to ensure light-limited growth; 2) a negative control group limited to absorbing CO2 from the atmosphere representing carbon-limited growth; 3) with 2 g or 4 g of CA-GA beads facilitating CO2 absorbance from the atmosphere; 4) free GA, with an equivalent mass of GA content as in 2 g of CA-GA beads, facilitating CO2 absorbance from the atmosphere. In all the cases, CO2 was utilized by algae via the pathway shown in Eq. 2.

E q. 2

[0130] By consuming HCO3 via photosynthesis, the algae can maintain a concentration gradient of CO2/HCO3 to allow sustained uptake of CO2 from the atmosphere. Biomass accumulated during the cultivation period (Figure 9a), and a higher growth rate also corresponds to more rapid consumption of nitrate (Figure 9b), which is a nutrient for the algae cells. [0131] As CO2 solubility in the water phase is low, the supply of atmospheric CO2 is insufficient for concentrated algae growth in the natural environment, as was the case for the negative control group, which showed the lowest biomass growth rate 22.7±0.5 mg L^-1 d^-1 and nitrate consumption rate 1.38±0.03 mg L^-1 d^-1 (Figure 9). By contrast, air sparging marks the upper limit of the growth rate 100±3 mg L^-1 d^-1, with the nitrate in the culture depleted in 4 days. Air bubbles increase the gas-liquid contacting area, generate turbulence and narrow the mass transfer boundary layer, thus accelerating the CO2 dissolution reaction (Eq. 2, Step 1 ).

[0132] The presence of either free or immobilized CA enhanced algae growth to a similar extent: free CA improved the growth rate to 37±3 mg L^-1 d^-1, while 2 g and 4 g of CA-GA beads achieved 40±1 mg L^-1 d^-1 and 43±1 mg L^-1 d^-1 respectively (Figure 9a). This was due to the use of CA accelerating the CO2 hydration reaction (Eq. 2, Step 2). Importantly, the use of immobilized CA-GA beads shows not only comparable algae growth enhancement factor to the free CA, but also a much greater potential for industrial implementation. After microalgae cultivation, the free CA cannot be recycled via centrifugation or filtration from the algal cells whose sizes lie in the range of micrometres. By contrast, the buoyant alginate CA-GA beads with diameters around 2 mm can either be retained during continuous cultivation and harvesting or skimmed directly from the culture surface for second-cycle re-use. This dramatically simplifies the separation process and increases their useful lifetime, therefore reducing the cost.

[0133] The total inorganic carbon (TIC) concentration in the culture of the control group decreased as the algae grew, indicating that the demand for CO2/HCO3 exceeded the supply (Figure 10). While the CA-GA beads did not provide a TIC concentration as high as freshly added (day 1 ) free CA (89±10 ppm versus 102±2 ppm), the TIC concentration was more stable for the CA-GA beads, with 80% TIC remaining after 7 days. Given the algal growth was comparable for both cases, the higher TIC concentration may reflect less enzyme degradation with time.

5.4: Multi- Day Bead Replenishment Algae Growth Trial

[0134] Fresh beads were repeatedly used in three rounds of 7-day cultivation of Nannochloropsis sp. The growth enhancement at this 7-day point was quite stable at around 40% (Figure 11 a), proving the CA was stable when immobilized in the calcium alginate beads. It was also found that water evaporation was slightly reduced (20% on average) due to the beads covering part of the culture surface (Figure 11 b), which was beneficial for maintaining a stable culture environment. According to the measured bead density, 11% of the bead volume would be exposed to the air, and the theoretical surface coverage of 2g beads to the algae culture was thus estimated as 33%. However, the water loss of only 20% indicated that around 1/3 of the beads were being temporarily immersed in the medium by the vortex of the stirrer. This problem can be readily avoided at a large-scale algal pond with a calmer water surface, which promises a higher growth enhancement factor for the beads.

5.5: Algae growth In a raceway pond.

[0135] In the mini raceway pond (Figure 12a), the growth of Nannochloropsis sp. was again improved upon the addition of these beads, to 4.4±0.1 g rrr^z d'¹, corresponding to a CO2 fixation rate of 8.0±0.2 g nr² d'¹ (Figure 12b). The growth enhancement in the raceways was 16%, compared to 75% in the smaller scale set up (Figure 9a). Similar to the previous discussion about air sparging, this was possibly because the paddle wheel, which is disproportionately large compared to the paddle wheel in a full-scale algal pond, provides significant aeration (and CO2 delivery) to the control group. Also, many of the beads became attached to the stirrer blades over the growth period and did not float freely, which is a problem that could be easily avoided or managed on a large scale (in which the paddle wheels occupy a much smaller proportion of the raceways).

[0136] As can be seen from the examples the CA-GA beads retained virtually all hydrase activity throughout 10 assay cycles. Compared with a natural growth rate of 22.7±0.5 mg L"¹ d"¹, free CA and CA-GA beads increased the productivity of Nannochloropsis salina to 37±3 mg L^-1 d^-1 and 40±1 mg L^-1 d^-1, respectively. The CA- GA beads further provided a stable growth enhancement for three rounds of microalgae cultivation, confirming that these buoyant beads can be readily recovered and re-used, which is promising for industrial biomass production.

Economic Calculations [0137] Based on the CA-catalysed CO2 hydration rate measured in the W-A assay, 1 cm² of the bead surface area exhibited a CO2 hydration rate of 5.2 μmol min"¹. Assuming the carbon content is 50% of the dry cell mass, to achieve a target microalgae biomass productivity of 25 g m"² d"¹ in a traditional raceway pond, 5.0 g beads are needed per square metre of algae culture, corresponding to only 0.35% of the pond surface coverage, which is low enough to ignore the side effect of light shadowing.

[0138] At lab-scale, CA accounts for over 98% of the material costs of bead production. Based on the previous assumptions, Table 2 outlines some projected cost scenarios as a function of scale and CA lifetime. Assuming a CA lifetime of a fortnight as a conservative estimate, the lab-scale cost for the beads is around $50 per tonne of dry biomass. However, a greater number of lifecycles can be possible, particularly if a CA enzyme can be developed that is more resistant to seawater and other impurities (e.g., if a CA from a marine microalga could be produced at scale); or if a freshwater algae species is used. Further, the cost of the enzyme itself is expected to reduce significantly if produced on a large scale. Under these conditions, the cost is thus projected to fall, possibly to around $0.2 per tonne of dry biomass.

Table 2: Estimated cost of CA-GA beads needed to produce algal biomass In different scenarios, In USD per tonne off dry biomass.

Note: t Assuming CA is purchased from the supplier Sigma-Aldrich with a price of $1822/g. t Assuming CA is produced economically at a large scale with the price of $50/g.

[0139] It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

[0140] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

[0141] Future patent applications may be filed in Australia or overseas on the basis of the present application, for example by claiming priority from the present application, by claiming a divisional status and/or by claiming a continuation status. It is to be understood that the following provisional claims are provided by way of example only, and are not intended to limit the scope of what may be claimed in any such future application. Furthermore, the claims should not be considered to limit the understanding of (or exclude other understandings of) the invention inherent in the present disclosure. Features may be added to or omitted from the provisional claims at a later date, so as to further define the invention.

Claims

1. A hydrogel bead containing cross-linked carbonic anhydrase, wherein the (hydrated) bead has a density of less than 1.03 g/cm³ (at 20°C).

2. A hydrogel bead according to claim 1 wherein the (hydrated) bead has a density of less than 1.00 g/cm³ (at 20°C).

3. A hydrogel bead according to claim 1 or 2 wherein the bead comprises (a) one or more hydrophilic gel materials (b) cross-linked carbonic anhydrase and (c) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C.

4. A hydrogel bead according to claim 3 wherein the hydrophilic gel material is selected from the group consisting of polyethylene glycol (PEG) -thiol, PEG-acrylate, acrylamide, N, N'-bis (acryloyl) citamine (BACy), PEG, polypropylene oxide (PPO) , polyacrylic acid, poly (hydroxyethyl methacrylate) (PHEMA), poly (methyl methacrylate) (PMMA), poly (N-isopropylacrylamide) (PNIPAAm), poly (lactic acid) (PLA), poly (Lactic acid-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly (vinylsulfonic acid) (PVSA), poly (L-aspartic acid), poly (L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone , diethylene glycol diallyl ether, ethylene glycol acrylates, polymethylene glycol diacrylates, polyethylene glycol diacrylates, trimethylropropane trimethacrylate, ethoxylated trimethylol triacrylates or ethoxylated pentaerythritol tetraacrylates or combinations thereof

5. A hydrogel bead according to any one of claims 3 or 4 wherein the hydrophilic gel material is alginate.

6. A hydrogel bead according to any one of claims 3 to 5 wherein the hydrophilic gel material is crosslinked.

A hydrogel bead according to any one of claims 3 to 6 wherein the bead contains from 2wt% to 6wt% of the hydrophilic gel material.

8. A hydrogel bead according to any one of claims 3 to 7 wherein the density modifying material is a hydrophobic organic liquid.

9. A hydrogel bead according to claim 7 8 wherein the hydrophobic organic liquid is an oil.

10. A hydrogel bead according to any one of claims 8 to 9 wherein the hydrophobic organic liquid is selected from the group consisting of mineral oils and edible oils.

11. A hydrogel bead according to any one of claims 8 to 10 wherein the hydrophobic organic liquid is paraffin.

12. A hydrogel bead according to any one of claims 8 to 11 wherein the hydrophobic organic liquid is present in the hydrogel bead in an amount of from 5wt% to 50wt%.

13. A hydrogel bead according to any one of claims 1 to 12 wherein the bead contains from 0.01 wt% to 1 wt% of the cross-linked carbonic anhydrase.

14. A hydrogel bead according any one of claims 1 to 13 wherein the carbonic anhydrase is crosslinked with glutaraldehyde.

15. A method for producing a hydrogel bead comprising cross-linked carbonic anhydrase, wherein the (hydrated) bead has a density of less than 1 .03 g/cm³ (at 20°C), the method comprising:

(a) forming an aqueous emulsion comprising (i) one or more hydrophilic gel materials (ii) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C and either (iii) cross-linked carbonic anhydrase or (iiia) carbonic anhydrase and (iiib) a crosslinking agent for the carbonic anhydrase; and (iv) water;

(b) treating the emulsion to form the hydrogel bead.

16 A method according to claim 15 wherein the emulsion comprises (i) one or more hydrophilic gel materials (ii) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C, (iii) cross-linked carbonic anhydrase and (iv) water.

17. A method according to claim 15 wherein the emulsion comprises (i) one or more hydrophilic gel materials (ii) a density modifying material that is immiscible with the one or more hydrophilic gel materials, the density modifying material having a density of less than 0.98g/cm³ at 20°C (iiia) carbonic anhydrase and (iiib) a crosslinking agent for the carbonic anhydrase and (iv) water.

18. A method according to any one of claims 15 to 17 wherein step (b) comprises the steps of:

(b1) subjecting the emulsion to gelling conditions to form a gel; and

(b2) extruding the gel to form a hydrogel bead.

19. A method according to any one of claims 15 to 17 wherein step (b) comprises injecting the emulsion into a gelling solution to form a hydrogel bead.

20. A method according to any one of claims 15 to 19 wherein the hydrophilic gel material is selected from the group consisting of polyethylene glycol (PEG) -thiol, PEG- acrylate, acrylamide, N, N'-bis (acryloyl) citamine (BACy), PEG, polypropylene oxide (PPO) , polyacrylic acid, poly (hydroxyethyl methacrylate) (PHEMA), poly (methyl methacrylate) (PMMA), poly (N-isopropylacrylamide) (PNIPAAm), poly (lactic acid) (PLA), poly (Lactic acid-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly (vinylsulfonic acid) (PVSA), poly (L-aspartic acid), poly (L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone , diethylene glycol diallyl ether, ethylene glycol acrylates, polymethylene glycol diacrylates, polyethylene glycol diacrylates, trimethylropropane trimethacrylate, ethoxylated trimethylol triacrylates or ethoxylated pentaerythritol tetraacrylates or combinations thereof

21. A method according to any one of claims claim 15 to 20 wherein the hydrophilic gel material is alginate.

22. A method according to any one of claims 15 to 21 wherein the emulsion contains from 2wt% to 3wt% of the hydrophilic gel material.

23. A method according to any one of claims 15 to 22 wherein the density modifying material is a hydrophobic organic liquid.

24. A method according to claim 23 wherein the hydrophobic organic liquid is an oil.

25. A method according to any one of claims 23 to 24 wherein the hydrophobic organic liquid is selected from the group consisting of mineral oils and edible oils.

26. A method according to any one of claims 23 to 25 wherein the hydrophobic organic liquid is paraffin.

27. A method according to any one of claims 23 to 25 wherein the hydrophobic organic liquid is present in the emulsion in an amount of from 5wt% to 50wt%.

28. A method according to any one of claims 15 to 27 wherein the emulsion contains from 0.01 wt% to 5wt% of the carbonic anhydrase.

29. A method according to claim 28 wherein the carbonic anhydrase is crosslinked.

30. A method according to claim 29 wherein the carbonic anhydrase is crosslinked with glutaraldehyde.

31. A method according to claim 19 wherein the gelling solution contains a multivalent metal ion.

32. A method according to any claim 31 wherein the multivalent metal ion is Ca²⁺.

33. A method of increasing the uptake of CO2 into an aqueous medium the method comprising applying a hydrogel bead according to any one of claims 1 to 14 to the surface of the aqueous medium.

34. A method according to claim 33 wherein the aqueous medium is at a temperature of from 5°C to 40°C.

35. A method according to claim 33 or 34 wherein the aqueous medium is at a temperature of from 20°C to 30°C.

36. A method according to claim 33 to 35 wherein the aqueous medium is at a temperature of from 25°C to 28°C.

37. A method according to any one of claims 33 to 36 wherein the beads cover from 10% to 50% of the surface of the aqueous medium.

38. A method according to any one of claims 33 to 37 wherein the aqueous medium is agitated.

39. A method of promoting algae growth in an aqueous medium, the method comprising applying a hydrogel bead according to any one of claims 1 to 14 to the surface of the aqueous medium.

40. A method according to claim 39 wherein the aqueous medium is at a temperature of from 5°C to 40°C.

41. A method according to claim 39 or 40 wherein the aqueous medium is at a temperature of from 20°C to 30°C.

42. A method according to claim 39 to 41 wherein the aqueous medium is at a temperature of from 25°C to 28°C.

43. A method according to any one of claims 39 to 42 wherein the beads cover from 10% to 50% of the surface of the aqueous medium.

44. A method according to any one of claims 39 to 43 wherein the aqueous medium is agitated.