Encapsulation of microalgae in hydrogel polymer and high-density algal biomass

Abstract

Aspects of the present disclosure generally relate to a new class of compositions that include microalgae encapsulated within a polymerized hydrogel. Aspects of the present disclosure also generally relate to high density algal biomass and to new processes for growing algal biomass. In an aspect is provided a composition that includes an encapsulant comprising a polymerized hydrogel, and microalgae encapsulated within the encapsulant. In another aspect, a process for encapsulating algae is provided. The process includes: introducing, to a device, a stream of core fluid comprising microalgae and a hydrogel precursor; contacting the stream of core fluid with two streams of a focusing fluid to form a thinner stream of core fluid comprising the microalgae and the hydrogel precursors, the focusing fluid having a different viscosity than the core fluid; and exposing the thinner stream of the core fluid to UV light to form a composition described herein.

Description

Encapsulation of Microalgae in Hydrogel Polymer and High-Density Algal BiomassFIELD

[0001] Aspects of the present disclosure generally relate to a new class of compositions that include microalgae encapsulated within a polymerized hydrogel and processes for forming such compositions. Aspects of the present disclosure also generally relate to high density algal biomass and to new processes for growing algal biomass.DESCRIPTION OF RELATED ART

[0002] Climate change is a dire threat to ecological, economic, and political systems and is compounded by environmental pollution and degraded arable land. One method to combat climate change is using algae biomass to capture carbon dioxide. Conventional technologies, however, produce algae very slowly and in low density. Further, mass production of the low density algae produced by conventional algaculture techniques is limited by the volume of water available.

[0003] There is a need for new compositions that include microalgae and processes of forming such compositions. There is also a need for new processes for producing high density algal biomass.SUMMARY

[0004] Aspects of the present disclosure generally relate to a new class of compositions that include microalgae encapsulated within a polymerized hydrogel and to processes for forming such compositions. Aspects of the present disclosure also generally relate to high density algal biomass and new processes for growing algal biomass. Unlike conventional approaches that produce algae very slowly and in low density, aspects described herein produce dense algae clusters rapidly. In further contrast to conventional approaches for producing algae, aspects of the present disclosure do not require large amounts of water.

[0005] In an aspect is provided a composition that includes an encapsulant comprising a polymerized hydrogel; and microalgae encapsulated within the encapsulant.

[0006] In another aspect is provided a composition that includes microalgae encapsulated within a polymerized hydrogel matrix.

[0007] In another aspect, a soil amendment is provided. The soil amendment includes a composition described herein and an exogenous carbon source.

[0008] In another aspect, a carbon sequestration composition is provided. The carbon sequestration composition includes a composition described herein.

[0009] In another aspect, a composition for removing nitrates from a medium that includes a composition described herein.

[0010] In another aspect, a process for encapsulating algae is provided. The process includes introducing, to a fluidic channel of a microfluidic device, a stream of an aqueous-based core fluid comprising microalgae and one or more hydrogel precursors. The process further includes contacting the stream of aqueous-based core fluid with two streams of an aqueous-based focusing fluid to form a thinner stream of the core fluid comprising the microalgae and the one or more hydrogel precursors, the aqueousbased focusing fluid having a different viscosity than the aqueous-based core fluid. The process further includes exposing the thinner stream of the core fluid to light to form a polymerized hydrogel thread encapsulating the microalgae.

[0011] In another aspect, a process for encapsulating algae is provided. The process includes introducing, to a fluidic channel of a microfluidic device, a stream of an aqueous-based core fluid comprising microalgae and one or more UV light- photoreactive hydrogel precursors. The process further includes contacting the stream of aqueous-based core fluid with two streams of an aqueous-based focusing fluid to form a thinner stream of the core fluid comprising the microalgae and the one or more UV light-photoreactive hydrogel precursors, the aqueous-based focusing fluid having a different viscosity than the aqueous-based core fluid. The process further includes exposing the thinner stream of the core fluid to UV light to form a polymerized hydrogel thread encapsulating the microalgae.

[0012] In another aspect, a process for growing algal biomass is provided. The process includes exposing a composition described herein to growth conditions to form a microalgae cluster within the polymerized hydrogel.

[0013] In another aspect, a process for forming microalgae particles (for example, droplets) is provided. The process includes introducing, to a fluidic channel of a microfluidic device, a stream of an aqueous-based core fluid comprising microalgae and one or more hydrogel precursors. The process further includes contacting the stream of aqueous-based core fluid with one or more streams of an aqueous-based focusing fluid to form hydrogel particles comprising the microalgae and the one or more hydrogel precursors, the aqueous-based focusing fluid having a different viscosity than the aqueous-based core fluid. The process further includes exposing the hydrogel particles to light to form a polymerized hydrogel particles encapsulating the microalgae.

[0014] In another aspect, a process for encapsulating algae is provided. The process includes introducing, to a fluidic channel of a microfluidic device, a stream of an aqueous-based core fluid comprising microalgae and one or more UV light- photoreactive hydrogel precursors. The process further includes contacting the stream of aqueous-based core fluid with one or more streams of an aqueous-based focusing fluid to form hydrogel particles comprising microalgae and one or more UV light- photoreactive hydrogel precursors. The process further includes exposing the hydrogel particles to UV light to form polymerized hydrogel particles encapsulating the microalgae.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] So that the manner in which the above recited features of the present disclosure may be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to aspects, some of which are illustrated in the drawings. It is to be noted, however, that the appended drawings illustrate only exemplary aspects and are therefore not to be considered limiting of its scope, may admit to other equally effective aspects.

[0016] FIG. 1A shows an illustration of a thread pulling apparatus in operation where the hydrogel precursor solution is focused into a thread.

[0017] FIG. IB is an illustration of microalgae growing in hydrogel threads.

[0018] FIG. 2A is a schematic of an example apparatus for forming polymerized hydrogel threads or droplets encapsulating microalgae.

[0019] FIG. 2B is a pictorial representation of selected elements of the example apparatus shown in FIG. 2A according to at least one aspect of the present disclosure.

[0020] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements and features of one aspect may be beneficially incorporated in other aspects without further recitation.DETAILED DESCRIPTION

[0021] Aspects of the present disclosure generally relate to a new class of compositions that include microalgae encapsulated within a polymerized hydrogel and to processes for forming such compositions. As used herein, a “composition” may include component(s) of the composition, reaction product(s) of two or more components of the composition, a remainder balance of remaining starting component(s), or combinations thereof. Compositions of the present disclosure may be prepared by any suitable mixing process. Aspects of the present disclosure also generally relate to high density algal biomass and to new processes for growing algal biomass. Aspects of the present disclosure may be utilized in various applications, for example, agriculture, carbon capture, and nitrate runoff, among other applications.

[0022] As described above, climate change represents a dire threat to the world. Carbon dioxide capture using algae biomass has been put forth as a method to combat climate change. However, conventional technologies produce algae very slowly and in low density, and are limited by the volume of water available. Therefore, there is a need for new compositions and methods for growing algal biomass.

[0023] To this end, the inventors discovered a new class of compositions that facilitate rapid growth of encapsulated algae. Advantageously, aspects described herein produce algae significantly faster and more densely than conventional methods. For example, while conventional growth rates are about 1 g / L per unit time, aspects of the present disclosure may produce growth rates of about 300 g / L or more per the same unit of time. In addition, the inventors found a scalable process for forming the compositions. Advantageously, the process may be free of separation techniques used for immiscible solvent methods such as centrifugation, solvents, filtration, or otherprocesses because processes of the present disclosure produce a polymerized hydrogel- encapsulated microalgae that is more dense than water and is easily recoverable.

[0024] The use of headings is for purposes of convenience only and does not limit the scope of the present disclosure. Aspects described herein may be combined with other aspects.Compositions

[0025] Aspects of the present disclosure generally relate to a new class of compositions that may include microalgae encapsulated within and / or dispersed in a polymerized hydrogel. The polymerized hydrogel may be a matrix. Besides the encapsulated microalgae, the polymerized hydrogel may further encapsulate a hydrogel matrix, nutrients, or combinations thereof, among other components.

[0026] The polymerized hydrogel may be in the form of, for example, a thread or a particle / droplet that encapsulates the microalgae. As described herein, the polymerized hydrogel may be formed from a hydrogel precursor solution that may include one or more photoreactive monomers, one or more linkers (which may be photoreactive), one or more photoinitiators, or combinations thereof.

[0027] The polymerized hydrogel encapsulants may be a polymerized hydrogel matrix. The polymerized hydrogel encapsulants may be in the form of, for example, polymerized hydrogel threads or polymerized hydrogel droplets (or particles).

[0028] The polymerized hydrogel thread encapsulant may have any suitable shape. For example, the polymerized hydrogel thread may appear as a thin and / or elongated polymerized hydrogel. The polymerized hydrogel thread may have a cylindrical shape, substantially cylindrical shape, tubular shape, substantially tubular shape, rod shaped, or substantially rod shaped. The shape of the polymerized hydrogel thread may be a result of the fabrication process to form the polymerized hydrogel thread.

[0029] The polymerized hydrogel droplets may be in the form of spherical particles or substantially spherical particles. Additionally, or alternatively, the hydrogel droplets may be in the form of disc-shaped or substantially disc-shaped particles.

[0030] The polymerized hydrogel may serve as an encapsulant that encapsulates the microalgae (or microalgae cell). The size of polymerized hydrogel encapsulants may be 5 millimeters (mm) or less. Larger elements than 5 mm may introduce transportlimitations that prevent algae from growing either at all or to their potential maximum density. Because 5 mm is a relatively small size, significant amounts of hydrogel elements may be used to encapsulate sufficient quantities of microalgae.

[0031] The polymerized hydrogel encapsulant (e.g., threads or droplets) may have any suitable diameter, such as less than 5 mm, such as less than about 1 mm, such as in a range from about 1 pm to about 1,000 pm, such as from about 100 pm to about 900 pm, such as from about 200 pm to about 800 pm, such as from about 300 pm to about 700 pm, such as from about 400 pm to about 600 pm, such as about 500 pm. The polymerized hydrogel threads and polymerized hydrogel droplets may have a diameter less than 500 pm, such as less than 250 pm, such as in a range from about 1 pm to about 200 pm, such as in a range from about 1 pm to about 100 pm, such as from about 10 pm to about 90 pm, such as from about 20 pm to about 80 pm, such as from about 30 pm to about 70 pm, such as from about 40 pm to about 60 pm, such as about 50 pm, or from about 1 pm to about 9 pm, such as from about 2 pm to about 8 pm, such as from about 3 pm to about 7 pm, such as from about 4 pm to about 6 pm, such as about 5 pm.

[0032] The diameter of the polymerized hydrogel encapsulant (for example, a thread or droplet) may be controlled by, for example, the relative volumetric flow rates of the stream of core fluid, the first stream of focusing fluid, and / or the second stream of focusing fluid. That is, varying the ratio of the flow between the stream of focusing fluid and the stream of core fluid may be utilized to change the diameter of the polymerized hydrogel thread pulled or the polymerized hydrogel droplet made.

[0033] The microalgae encapsulated within or dispersed in the polymerized hydrogel may be in the form of algae cells. The algae may be any suitable algae strain, for example, such as terrestrial algae, aquatic algae (e.g., freshwater algae), or combinations thereof. Illustrative, but non-limiting, examples of algae that be utilized include Tetradesmus deserlicola. Chlorella vulgaris, Chlorococcum infiisiomim, a species of Aster ochloris, Trebouxia arboricola, or combinations thereof.

[0034] The inventors found that when the algae is incorporated into the matrix of the polymerized hydrogel, the polymerized hydrogel matrix may serve to stress algae in order to increase algae production. While not wishing to be bound by any theory, itis believed that when the algae is encapsulated within and / or dispersed in a polymerized hydrogel matrix as described herein, the polymerized hydrogel matrix may serve to mimic the algae’s native environment but may also serve to stress the algae in a manner that is not present in the algae’s native environment. In so doing, rapid and large production of algae biomass may be observed. Aspects of the present disclosure are scalable.

[0035] Besides the encapsulated microalgae, optional components such as nutrients and / or buffers may be encapsulated within the polymerized hydrogel threads / droplets. These optional components are described herein.Processes for Forming Compositions

[0036] Aspects of the present disclosure also generally relate to processes for forming compositions described herein. Conventional hydrogel microfabrication production techniques are inadequate. For example, conventional hydrogel microfabrication techniques rely on immiscible fluids such an oil phase and an aqueous phase that enable separation of an aqueous hydrogel precursor fluid from an oil phase. Having multiple fluid phases increases device complexity, operation, and costs. For example, the immiscible oil must be removed from the hydrogels once the hydrogels have been polymerized into discreet elements which frustrates scale-up and increases costs.

[0037] To create the massive amounts of hydrogel needed for industrial algal applications, processes described herein offer various advantages over conventional hydrogel microfabrication techniques. For example, thread pulling processes of the present disclosure provide significant advantages in terms of, for example, device simplicity and operation as no immiscible fluids are used. In addition, recovery of the polymerized hydrogel encapsulating the microalgae may be accomplished by simply removing excess fluid as the polymerized hydrogel is denser than water and sinks to the bottom of the collection vessel.

[0038] In further contrast to conventional techniques, processes described herein may be free of centrifugation, solvents, filtration of immiscible fluids, or other processes involving two immiscible fluids such as an oil phase and water phase.However, centrifugation, solvents, filtration, or other suitable processes may be utilized.

[0039] Various suitable processes and devices may be utilized to form compositions described herein.

[0040] For example, a microfluidic device may be utilized with a thread pulling process to form compositions described herein. Thread pulling processes described herein may be advantageous as such processes do not involve immiscible fluids. Instead, viscosity-mismatched fluids may be utilized.

[0041] Generally, for example, thread pulling processes of the present disclosure involve utilization of a core fluid (having a first viscosity) and a focusing fluid (having a second viscosity that is different from the first viscosity). In the thread pulling process, hydrodynamic focusing may be utilized to control tapering of the core fluid into a thread. In hydrodynamic focusing, a sheath fluid (or focusing fluid) is utilized to force a core fluid into a smaller core stream. With the viscosity-mismatched fluids, necking of the core fluid by the focusing fluids results in a microalgae thread as a smaller core stream. The thread may be subsequently polymerized into a polymerized hydrogel thread by exposure to ultraviolet (UV) light. Following polymerization, microalgae is encapsulated within and / or dispersed in the polymerized hydrogel thread.

[0042] The thread pulling process may include introducing a stream of core fluid into an opening of a microfluidic device. The core fluid may be a higher- viscosity lower-flow core fluid. The core fluid may be an aqueous-based core fluid.

[0043] The thread pulling process may further include flowing one or more streams of a focusing fluid into one or more different openings of the microfluidic device. The focusing fluid may be a lower-viscosity higher-flow focusing fluid. The focusing fluid may be an aqueous-based core fluid. A hydrogel precursor solution, microalgae, or combinations thereof may be added to the core fluid before, after, or during introducing the core fluid to the microfluidic device.

[0044] During the thread pulling process, positive pressure may be applied to enable flow of the stream of core fluid and streams of focusing fluid. Flow rates for the stream of core fluid may include a flow rate of about 0.1 pL / min or more, about 150 pL / min or less, or combinations thereof, such as in a range from about 25 pL / min toabout 125 pL / min, such as from about 50 pL / min to about 100 pL / min, such as from about 80 pL / min to about 100 pL / min or in a range from about 0.1 pL / min to about 15 pL / min, such as from about 0.5 pL / min to about 10 pL / min, such as from about 1 pL / min to about 5 pL / min. Flow rates for the streams of focusing fluid may include a flow rate of about 0.1 pL / min or more, about 200 pL / min or less, or combinations thereof, such as in a range from about 1 pL / min to about 150 pL / min, such as from about 25 gL / min to about 125 gL / min, such as from about 50 gL / min to about 100 gL / min, such as from about 75 gL / min to about 100 gL / min or in a range from about 0.1 gL / min to about 15 gL / min, such as from about 0.5 gL / min to about 10 gL / min, such as from about 1 gL / min to about 5 gL / min. The flow rate of the streams of focusing fluid may be the same or different from the flow rate of the core fluid.

[0045] The thread pulling process may further include contacting the core fluid with the one or more streams of the focusing fluid to form a thinner stream of core fluid comprising the microalgae and the hydrogel precursor solution. The lower viscosity focusing fluid focuses the higher viscosity core fluid comprising the hydrogel precursor solution and microalgae into a thread (thinner stream of the core fluid).

[0046] The thread pulling process may further include exposing the thinner stream of core fluid to UV light to form a polymerized hydrogel thread encapsulating the microalgae. The UV light causes polymerization of one or more hydrogel precursors. Conditions suitable to form the polymerized hydrogel-encapsulated microalgae (or microalgae cells) may include a suitable wavelength, or wavelength range of light, an energy density of the light, a time of exposure to the ultraviolet, or combinations thereof. Such conditions are further described herein.

[0047] Following polymerization, microalgae is encapsulated within and / or dispersed in the polymerized hydrogel thread. Varying the ratio of flow between the focusing fluid and the core fluid may be utilized to change the diameter of the thread pulled. The polymerized hydrogel thread may be recovered by utilizing its density. For example, the polymerized thread may be denser than water and therefore sink to the bottom of a collection vessel. This recovery of the polymerized thread is advantageous over conventional methods as it does not require separation techniques observed for immiscible solvents such as centrifugation, solvents, filtration, or other processesinvolving two immiscible fluids. An illustrative, but non-limiting, example of the thread pulling process is described in the Examples Section. One or more operations of the thread pulling process may be performed at any suitable temperature, such as a temperature in a range from about 15°C to about 35°C, such as from about 15°C to about 25°C.

[0048] After the polymerized hydrogel thread encapsulating the microalgae is formed, the polymerized hydrogel thread encapsulating the microalgae may be placed in an environment permissible to the growth of the microalgae. The microalgae may then rapidly grow. Processes to grow algal biomass are described herein.

[0049] Another example of forming compositions described herein also involves use of a microfluidic device to form polymerized hydrogel particles (droplets). Similar to the polymerized hydrogel threads, the algae may be encapsulated within and / or dispersed in the polymerized hydrogel droplets. Varying the ratio of flow between the focusing fluid and the core fluid may be utilized to change the diameter of the droplet formed. The polymerized hydrogel droplets may be recovered by utilizing their density. For example, the polymerized droplets may be denser than water and therefore sink to the bottom of a collection vessel. This recovery of the polymerized droplets is advantageous over conventional methods as it does not require separation techniques of conventional hydrogel fabrication methods using immiscible solvents as described above. Once encapsulated within and / or dispersed in the polymerized hydrogel droplets, the microalgae may be placed in an environment permissible to their growth. The microalgae may then rapidly grow. An illustrative, but non-limiting, example of forming polymerized hydrogel droplets encapsulating microalgae is described in the Examples Section. One or more operations of the process may be performed at any suitable temperature, such as a temperature in a range from about 15°C to about 35°C, such as from about 15°C to about 25°C. After the polymerized hydrogel droplet encapsulating the microalgae is formed, the polymerized hydrogel droplet encapsulating the microalgae may be placed in an environment permissible to the growth of the microalgae as described herein. The microalgae may then rapidly grow inside the polymerized hydrogel droplet.

[0050] Additionally, or alternatively, compositions that include microalgae encapsulated and / or dispersed within a hydrogel may be formed without use of a microfluidic device. For example, a composition may be formed that includes microalgae (e.g., microalgae cells) encapsulated within and / or dispersed in a crosslinked hydrogel. This crosslinked hydrogel is not made using ultraviolet light. In this example, the hydrogel may be formed by adding microalgae to a suitable media comprising any suitable gelling agent. The gelling agent may include, for example, barium chloride (BaCh). The microalgae may be in the form of an alginate salt, for example, sodium alginate. The mixture that includes the microalgae and the gelling agent is stirred and left to crosslink for a suitable period to form microalgae encapsulated within and / or dispersed in a crosslinked hydrogel. An illustrative, but nonlimiting, example of forming crosslinked hydrogel encapsulating microalgae is described in the Examples Section. The crosslinked hydrogel encapsulating microalgae may be placed in an environment permissible to the growth of the microalgae as described herein.

[0051] Additionally, or alternatively, three-dimensional (3D) printing may be utilized to form compositions described herein. An illustrative, but non-limiting, example of 3D printing to form compositions of the present disclosure is described in the Examples Section. Following 3D printing of the compositions, the compositions may be placed in an environment permissible to the growth of the microalgae as described herein.Hydrogel Precursor Solution

[0052] As described herein, a hydrogel precursor solution is used to form the hydrogel. Upon irradiation with ultraviolet light, one or more hydrogel precursors present in the hydrogel precursor solution react to form a polymerized hydrogel. The hydrogel precursor solution may be any suitable photopolymerizable hydrogel. Components of the hydrogel precursor solution may include polyethylene glycol diacrylate, gelatin methacylate, polyethylene glycol norbomene, or combinations thereof. The hydrogel precursor solution may further include a dithiolated linker. Any suitable UV photofragmentable photoinititator may be used, such as LAP (Lithium 2,4,6 trimethylbenzoylphosphinate or Irgacure 2959).

[0053] Hydrogel precursors may include photoreactive monomers, linkers (which may be photoreactive), photoinitiators, or combinations thereof. The hydrogel precursor solution may include one or more photoreactive monomers, one or more linkers, one or more photoinitators, other component(s), solvent(s), or combinations thereof. The hydrogel precursor solution may include microalgae. The hydrogel precursor solution may include one or more of those optional components described herein, such as nutrients and / or buffers. As described herein, the hydrogel precursor solution may be added to a core fluid prior to introduction into an apparatus for forming compositions of the present disclosure. Additionally, or alternatively, the hydrogel precursor solution may be mixed with a core fluid after introducing the hydrogel precursor solution into the device.

[0054] The one or more photoreactive monomers may comprise photoreactive functional groups chemically attached to, e.g., polyethylene glycol (PEG). Illustrative, but non-limiting, examples of photoreactive functional groups may include alkenes, acrylates, thiols, acids, or combinations thereof. Upon irradiation, the photoreactive monomers (with or without co-reactants, such as linkers described herein) may react to form a polymerized hydrogel.

[0055] The one or more photoreactive monomers may be UV light-photoreactive hydrogel precursors. That is, the one or more photoreactive monomers may become reactive when exposed to ultraviolet (UV) light to form a polymerized hydrogel.

[0056] Non-limiting examples of photoreactive monomers may include, but are not limited to, polyethylene glycol norbornene (PEGNB), polyethylene glycol diacrylate (PEGDA), PEG methacrylate, polyethylene glycol di-photodegradable acrylate (PEGdiDPA), derivatives thereof, or combinations thereof. The photoreactive monomers may be branched (e.g., ~20k 4-arm PEGNB and ~40k 8-arm PEGNB) or unbranched. Other PEG-based derivatives having varied reactive functional groups are also contemplated.

[0057] Photoreactive monomers may include non-PEG-based monomers such as acrylates, acids (e.g., lactic acid, hyaluronic acid), gelatin, collagen, or combinations thereof. For example, polylactic acid (PL A), acrylated hyaluronic acid, gelatin methacrylate, derivatives thereof, and combinations thereof may be used. Blockcopolymers and triblock copolymers may be used such as triblock PLA and PLA-PEG- PLA.

[0058] Molecular conformation of the photoreactive monomers may be varied to, e.g., impart desired material properties to the hydrogel microenvironment. For example, 1-arm molecular structures to 12-arm molecular structures may be used, such as 4-arm, 8-arm, or 12-arm molecular structures, such as 4-arm PEGNB, 8-arm PEGNB, 12-arm PEGNB, or combinations thereof.

[0059] The molecular weight and shape (e.g., number of arms on PEGNB) of the one or more photoreactive monomers, among other characteristics, may be adjusted. Adjustment of the molecular weight and shape of the photoreactive monomers (as well as the linker) enable the tuning of various properties of the polymerized hydrogel structure, and may confer a range of traits to the system depending on the desired use and desired effect on the algae (or algae cells).

[0060] A molecular weight of the one or more photoreactive monomers may be in a range from about 100 Da to about 75,000 Da, such as from about 250 Da to about 50,000 Da, such as from about 5,000 Da to about 50,000 Da, such as from about 10,000 Da to about 45,000 Da, such as from about 15,000 Da to about 40,000 Da, such as from about 20,000 Da to about 35,000 Da, such as from about 25,000 Da to about 30,000 Da. Illustrative, but non-limiting, examples of the molecular weight of the photoreactive monomer may be in a range from about 250 Da to about 10,000 Da, such as from about 500 Da to about 9,000 Da, such as from about 1,000 Da to about 8,000 Da, such as from about 2,000 Da to about 7,000 Da, such as from about 3,000 Da to about 6,000 Da, such as from about 4,000 Da to about 5,000 Da. The molecular weight of the one or more photoreactive monomers may be 30,000 Da or less. The molecular weight of the one or more photoreactive monomers may range from MWi to MW2 where each of MWi to MW2 (in Da) may be, independently, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000,about 14,500, about 15,000, about 15,500, about 16,000, about 16,500, about 17,000, about 17,500, about 18,000, about 18,500, about 19,000, about 19,500, about 20,000, about 20,500, about 21,000, about 21,500, about 22,000, about 22,500, about 23,000, about 23,500, about 24,000, about 24,500, about 25,000, about 25,500, about 26,000, about 26,500, about 27,000, about 27,500, about 28,000, about 28,500, about 29,000, about 29,500, about 30,000, about 30,500, about 31,000, about 31,500, about 32,000, about 32,500, about 33,000, about 33,500, about 34,000, about 34,500, about 35,000, about 35,500, about 36,000, about 36,500, about 37,000, about 37,500, about 38,000, about 38,500, about 39,000, about 39,500, about 40,000, about 40,500, about 41,000, about 41,500, about 42,000, about 42,500, about 43,000, about 43,500, about 44,000, about 44,500, about 45,000, about 45,500, about 46,000, about 46,500, about 47,000, about 47,500, about 48,000, about 48,500, about 49,000, about 49,500, or about 50,000, as long as MWi < MW2. Higher or lower molecular weights of the one or more photoreactive monomers are contemplated. The molecular weight of the photoreactive monomer refers to the number average molecular weight (Mn). The Mnis the Mnprovided by the manufacturer of the photoreactive monomer.

[0061] Any suitable aqueous and / or organic solvents may be utilized as a portion of the hydrogel precursor solution. Such aqueous and / or organic solvents may include water, saline, phosphate buffered saline, appropriate biologically compatible liquid, or combinations thereof.

[0062] A concentration of the one or more photoreactive monomers in the hydrogel precursor solution may be in a range from about 5 wt% to about 75 wt%, such as from about 10 wt% to about 70 wt%, such as from about 15 wt% to about 65 wt%, such as from about 20 wt% to about 60 wt%, such as from about 25 wt% to about 55 wt%, such as from about 30 wt% to about 50 wt%, such as from about 35 wt% to about 45 wt%, based on a total weight percent of the components of the hydrogel precursor solution (not to exceed 100 wt%). The concentration of the one or more photoreactive monomers in the hydrogel precursor solution may be in a range from about 5 wt% to about 35 wt%, such as from about 10 wt% to about 30 wt%, such as from about 15 wt% to about 25 wt%, based on the total weight percent of the components of the hydrogelprecursor solution (not to exceed 100 wt%). Higher or lower concentrations of the one or more photoreactive monomers may be used depending on application.

[0063] The hydrogel precursor solution may include one or more linkers (which may be photoreactive). The one or more linkers may be UV light-photoreactive hydrogel precursors. That is, the one or more one or more linkers may become reactive when exposed to ultraviolet (UV) light to form a polymerized hydrogel.

[0064] The one or more linkers may include any suitable linker. Suitable linkers may include a dithiol linker, such as a polyethylene glycol-dithiol (PEG-dithiol) linker, a derivative thereof, or combinations thereof. PEG-dithiol is a thiolated PEG having two thiol groups. The linker may be referred to as a thiol-containing monomer or dithiol linker unless the context indicates otherwise. When a dithiol linker is utilized, one or more photoreactive monomer(s), such as PEGDA may react with the thiol-containing monomer(s) via, e.g., a step-growth polymerization reaction occurring between the ene portion of the photoreactive monomer(s) and the thiol of the thiol-containing monomer.

[0065] A molecular weight of the one or more linkers (e.g., the PEG-dithiol linker) may be in a range from about 500 Da to about 10,000 Da, such as from about 1,000 Da to about 9,500 Da, such as from about 1,500 Da to about 9,000 Da, such as from about 2,000 Da to about 8,500 Da, such as from about 2,500 Da to about 8,000 Da, such as from about 3,000 Da to about 7,500 Da, such as from about 3,500 Da to about 7,000 Da, such as from about 4,000 Da to about 6,500 Da, such as from about 4,500 Da to about 6,000 Da, such as from about 5,000 Da to about 5,500 Da. The molecular weight of the one or more linkers may be about 6,000 Da or less, such as in a range from about 500 Da to about 6,000 Da, such as from about 1,000 Da to about 5,000 Da, such as from about 1,500 Da to about 4,500 Da, such as from about 2,000 Da to about 4,000 Da, such as from about 2,500 Da to about 3,500 Da. The molecular weight of the one or more linkers may be in a range from MW3 to MW4 where each of MW3 to MW4 (in Da) is, independently, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, or about 10,000, as long as MW3 < MW4. The molecular weight of the linker refers to the number average molecularweight (Mn). The Mnis the Mnprovided by the manufacturer of the linker. Higher or lower molecular weights of the one or more linkers are contemplated. Illustrative, but non-limiting, examples of PEG-dithiol linkers may include ~1.5k PEG-dithiol, 3.5k PEG-dithiol, and ~5k PEG-dithiol.

[0066] A concentration of the one or more linkers (e.g., PEG-dithiol) in the hydrogel precursor solution may be in a range from about 1 mM to about 50 mM, such as from about 5 mM to about 45 mM, such as from about 10 mM to about 40 mM, such as from about 15 mM to about 35 mM, such as from about 20 mM to about 30 mM, based on a total molar concentration of the components of the hydrogel precursor solution.

[0067] The hydrogel precursor solution may include one or more photoinitiators. Illustrative, but non-limiting, examples of photoinitiators may include lithium phenyl- 2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator, 2-hydroxy-2-methyl propiophenone (for example, Irgacure™ 1173, Darocur™ 1173), or combinations thereof. The photoinitiator induces polymerization when exposed to ultraviolet light. The use of LAP, due to its robust initiation properties at low concentrations, may minimize deleterious effects on cells.

[0068] A concentration of the one or more photoinitiators in the hydrogel precursor solution may be in a range from about 0.0001 wt% to about 1 wt%, such as from about 0.001 wt% to about 0.9 wt%, such as from about 0.01 wt% to about 0.5 wt%, such as from about 0.05 wt% to about 0.1 wt%, based on the total wt% of the components of the hydrogel precursor solution. Higher or lower concentrations of the one or more photoinitiators may be used depending on, for example, the application or desired results.

[0069] Using the components described above, various formulations may be used to form the polymerized hydrogel-encapsulated microalgae or compositions thereof.

[0070] A photoreactive monomer may be utilized with a linker. Here, a nonlimiting formulation useful for the hydrogel precursor solution may include: (a) from about 0.1 wt% to about 40 wt%, such as from about 1 wt% to about 40 wt%, such as from about 5 wt% to about 35 wt%, such as from about 10 wt% to about 20 wt% of one or more photoreactive monomers, such as a PEGNB, ranging in molecular weight fromabout 500 Da to about 50,000 Da, such as from about 3,000 Da to about 50,000 Da, such as from about 5,000 Da to about 20,000 Da, such as from about 10,000 Da to about 15,000 Da; (b) from about 1 mM to about 100 mM, such as from about 5 mM to about 50 mM of a linker, such as PEG dithiol, ranging in molecular weight from about 100 Da to about 10,000 Da; and / or (c) from about 0.0001 wt% to about 1 wt%, such as from about 0.01 wt% to about 0.1 wt% of LAP photoinitiator. Additional components may be used as desired.

[0071] A first photoreactive monomer, such as PEGNB, may be utilized with a second photoreactive monomer such as PEGDA, PL A, PLA-PEG-PLA, etc., and a linker. Here, a non-limiting formulation may include the aforementioned formulation with about 0.1 wt% to about 40 wt%, such as from about 1 wt% to about 40 wt%, such as from about 5 wt% to about 35 wt%, such as from about 10 wt% to about 20 wt% of the second photoreactive monomer (e.g., PEGDA, PLA, PLA-PEG-PLA, etc.) having a molecular weight from about 1,000 Da to about 30,000 Da, such as from about 5,000 Da to about 20,000 Da, such as from about 10,000 Da to about 15,000 Da. Additional components may be used as desired.

[0072] An illustrative, but non-limiting, formulation useful to form a PEGPLA / NB composite hydrogels may include: (a) from about 0.1 wt% to about 40 wt%, such as from about 1 wt% to about 40 wt%, such as from about 5 wt% to about 35 wt% such as from about 10 wt% to about 20 wt% of a first photoreactive monomer (e.g., PLA-PEG- PLA, etc.) having a molecular weight from about 1,000 Da to about 30,000 Da, such as from about 5,000 Da to about 20,000 Da, such as from about 10,000 Da to about 15,000 Da; (b) from about 0.1 wt% to about 40 wt%, such as from about 1 wt% to about 40 wt%, such as from about 5 wt% to about 35 wt%, such as from about 10 wt% to about 20 wt% of a second photoreactive monomer (e.g., PEGNB, such as 4-arm PEGNB, 8- arm PEGNB, or a combination thereof) ranging in molecular weight from about 500 Da to about 50,000 Da, such as from about 3,000 Da to about 50,000 Da, such as from about 5,000 Da to about 20,000 Da, such as from about 10,000 Da to about 15,000 Da; (c) from about 1 mM to about 100 mM, such as from about 5 mM to about 50 mM PEG dithiol ranging in molecular weight from about 100 Da to about 10,000 Da; and / or (d)from about 0.0001 wt% to about 1 wt%, such as from about 0.01 wt% to about 0.1 wt% of the LAP photoinitiator. Additional components may be used as desired.Processes for Growing Algal Biomass

[0073] Aspects described herein also generally relate to processes for growing algae clusters. The process may include those operations described herein for forming the composition. As described herein, the composition may include the polymerized hydrogel-encapsulated microalgae (or microalgae cells). The process may further include placing the composition under conditions sufficient for growing algae clusters.

[0074] For example, and as described herein, such conditions for growing algae clusters may include light and temperature.

[0075] Any suitable conditions (growth conditions) for microalgal growth into algae clusters may be utilized, such as light and temperature. The quality and intensity of light may be adjusted to facilitate microalgae growth into dense algae clusters. The light may contact the polymerized hydrogel threads / droplets from any suitable direction. Typically, the light may have a wavelength in a range from about 400 nm to about 700 nm. The light may be natural light (solar) or artificial light. Different artificial light sources may be used for microalgal cultivation, such as light-emitting diodes (LEDs), halogen lamps, fluorescent lamps, incandescent bulbs, or combinations thereof. The use of LEDs may be favorable for production denser algae clusters as LEDs typically have better control of light and the use of different wavelengths.

[0076] The temperature utilized for microalgal growth may be any suitable temperature, such as a temperature in a range from about 15°C to about 45°C, such as from about 20°C to about 40°C, such as from about 25°C to about 35°C.

[0077] The polymerized microalgae threads or polymerized microalgae droplets may be exposed to the light and / or temperature for any suitable period. Suitable periods may be about 1 hour or more, such as 10 hours or more, such as from about 1 day to about 10 days, such as from about 2 days to about 5 days.

[0078] A variety of other culture parameters such as pH, salinity, nutrients availability, CO2, dissolved oxygen concentration, or combinations thereof, may be utilized to grow the algal biomass as described herein. The culture parameters, as optional growth conditions, are discussed with respect to, at least, the OptionalComponents section. The process for growing algal biomass results in dense algae clusters encapsulated in the polymerized hydrogel.

[0079] The polymerization process and / or encapsulation process described herein improves microalgae cell viability and facilitates growth of dense microalgae clusters relative to conventional techniques. For example, it is believed that the polymerization and / or encapsulation described herein may mitigate reactive oxygen species (ROS) through active participation in the cross-linking mechanism of, e.g., PEGNB, contributing to the polymerization of the network rather than removing electrons from cellular membranes and destabilizing them, which can kill cells or contribute to cell death. In polymerizations with PEGDA, ROS may be mitigated by purging oxygen from the microenvironment via a non-reactive or inert gas which is free or substantially free of oxygen may be used, such as nitrogen and / or noble gases (e.g., argon). For polymerizations using mixtures of PEGDA and PEGNB, ROS may be mitigated by the addition of PEGNB and its above properties, but may be further mitigated through purging of the microenvironment with inert gas.

[0080] The polymerized hydrogel-encapsulated microalgae (or microalgae cells) formed by aspects described herein maintain cell viability longer than unencapsulated counterparts. In addition, the polymerized hydrogel-encapsulated microalgae (or microalgae cells) formed by aspects described herein show better growth than their unencapsulated counterparts. For example, polymerized hydrogel-encapsulated microalgae (or microalgae cells) formed by aspects described herein exhibit an increase in algae growth density relative to a control composition comprising an unencapsulated microalgae under the same conditions. Under the same conditions, the relative increase in algae growth density using the encapsulated microalgae may be about 1.5x or more than that of the unencapsulated microalgae, such as about 3* or more, such as about 5 or more, such as about 10x or more, such as about 20 x or more, such as about 30x or more, such as about 100x or more, such as about 300x or more. For example, the inventors found that aspects described herein may produce algae significantly faster and more densely than conventional methods. For example, while conventional growth rates for unencapsulated microalgae are about 1 g / L per unit time, aspects of the presentdisclosure may produce growth rates of about 300 g / L per the same unit time. Algae growth density refers to the population of algae within a given area.

[0081] Compositions comprising dense algae clusters of microalgae entrapped in hydrogels may be placed in various locations (e.g., the ground, canals, areas upstream of potable water, etc.) for use as, e.g., a soil amendment, a carbon (CO2) sequestration composition, or a composition for removing nitrates.Optional Components

[0082] As described herein, one or more optional components may be encapsulated within the polymerized hydrogel threads or polymerized hydrogel droplets, such as nutrients to stabilize the microalgae and / or components to assist the microalgae in growing. Additionally, or alternatively, the compositions comprising the microalgae encapsulated within the polymerized hydrogel threads or droplets may be placed in a culture (or medium) comprising the one or more optional components.

[0083] Here, besides light and temperature, microalgal growth may be influenced by a variety of other culture parameters, such as nutrients, CO2, dissolved oxygen concentration, pH, salinity, or combinations thereof.

[0084] Nutrients may include carbon, phosphorous, nitrogen, or combinations thereof, among others. The nutrients may be added to the hydrogel precursor solution prior to encapsulation. Additionally, or alternatively, the polymerized hydrogel threads or droplets containing the microalgae may be placed in a solution comprising the nutrients.

[0085] Various carbon sources may be utilized to provide carbon nutrients. Carbon sources may include a bicarbonate salt (HCO3 salt), a sugar (such as glucose), an acid, an alcohol, or combinations thereof. Various nitrogen sources may be utilized to provide nitrogen nutrients. Nitrogen nutrients may include urea, nitrite, nitrate, or combinations thereof. Various phosphorous sources may be utilized to provide phosphorous nutrients. Phosphorous nutrients may include polyphosphate, pyrophosphate, orthophosphate, metaphosphate, or combinations thereof.

[0086] Other nutrients may include micronutrients such as Mg, S, Se, K, Na, Cl, Ca, Mo, Fe, Mn, Zn, Cu, B, Co, or combinations thereof. These micronutrients may befound in both wastewater and seawater. Fertilizers and salts may be used as sources of these micronutrients.

[0087] A growth medium may be utilized to provide nutrients. The growth medium may be encapsulated within the polymerized hydrogel encapsulating the microalgae. Additionally, or alternatively, the polymerized hydrogel encapsulating the microalgae may be placed in a growth medium. A non-limiting example of a growth medium, and non-limiting recipe, is shown in Table 1.Table 1

[0088] Carbon dioxide (CO2) is also involved in the growth of microalgae. The CO2 may be supplied to the microalgae in the form of salts, such as a bicarbonate salt, and / or by injection of CCh-rich air into the culture (growth medium).

[0089] Salinity and pH are also involved in the growth of microalgae. The salinity may be any suitable salinity, such as in a range of ± 20% of the dissolved solids shown in Table 1. The pH may be any suitable pH, such as in a range from about 3 to about 11. Buffers may be utilized to control the pH and or salinity.

[0090] A fertilizer rich agricultural runoff may be used in addition to, or instead of, the growth medium to grow the dense algae clusters while encapsulated in the polymerized hydrogel.

[0091] Aspects of the present disclosure may be further understood by the following non-limiting examples. The following non-limiting examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use aspects of the present disclosure, and are not intended to limit the scope of aspects of the present disclosure.Examples

[0092] Aspects described herein enable extremely high-density production of algal biomass that may be achieved by algae encapsulation and growth within a confining matrix, such as a polymerized hydrogel matrix. While not wishing to be bound by any theory, it is believed that the increased rate of biomass accumulation may be due to the use of a soil-derived algae, Tetradesmus deserticola.

[0093] Conventional algae bioprocesses utilize aquatic species. In contrast, aspects described herein may utilize a terrestrial algae, though aquatic species of algae are contemplated.

[0094] The terrestrial algae grows in suspension culture at rates commensurate with aquatic species. In contrast, when encapsulated in a polymerized hydrogel as described herein, microalgae from the genus Tetradesmus deserticola divide more rapidly than when grown in liquid culture, with doubling times reducing to approximately 18 hours or less compared to 26-30 hours in liquid culture. The encapsulation does not affect cell viability and Tetradesmus sp. grows to at least 10-fold higher density compared to algae grown in liquid culture on the same timeframe.1. Example Fabrication of Microalgae Encapsulated within a Polymerized Hydrogel

[0095] Physical properties of fluids and / or microfluidic techniques may be taken advantage of to form a polymerized hydrogel thread. For example, viscosity- mismatched fluids may be utilized. In some examples, a core fluid (having a first viscosity) and a focusing fluid (having a second viscosity that is different from the firstviscosity) may be utilized. Microfluidic techniques, such as hydrodynamic focusing, may be utilized to control tapering of the core fluid.

[0096] In some examples, and as shown in FIG. 1 A, two side streams of focusing fluid are introduced at a junction immediately prior to the control region and used to pinch the stream of core fluid such that the final width and position of the core fluid may be a function of the relative volumetric flow rates of the core fluid and the two side streams of focusing fluid. With the viscosity -mismatched fluids, necking of the core fluid results in a microalgae thread.

[0097] FIG. 1 A shows an illustration of a thread pulling apparatus 100 in operation according to at least one aspect of the present disclosure. The thread pulling apparatus 100 may be a microfluidic device. Dimensions and characteristics of apparatus 200 of FIG. 2A may be used for thread pulling apparatus 100. The thread pulling apparatus 100 includes a fluidic channel 103. The thread pulling apparatus 100 may further include an inlet 101a for introducing a core fluid 131a. The thread pulling apparatus 100 may further include inlets 101b, 101c for introducing two streams of a focusing fluid 132. The thread pulling apparatus 100 may further include a junction 105. At junction 105, the three streams meet, where the three streams include a stream of the core fluid 131a, a first stream of the focusing fluid 132, and a second stream of the focusing fluid 132.

[0098] The core fluid 131a and the focusing fluid 132 may both be aqueous-based fluids. The core fluid 13 la and the focusing fluid 132 have mismatched viscosities. That is, the core fluid 131a and the focusing fluid 132 have different viscosities. The core fluid 13 la may be a higher viscosity, lower-flow fluid. The core fluid 13 la may include a hydrogel precursor solution. The core fluid 131a may further include microalgae 134, such as Tetradesmus deserticola algae. The focusing fluid 132 may be a lower viscosity, higher-flow fluid.

[0099] The microalgae may be encapsulated and entrapped into a hydrogel matrix (e.g., a polymerized hydrogel thread) by a flow-focusing microfluidic device (e.g., thread pulling apparatus 100 or apparatus 200).

[0100] The core fluid 131a comprising the hydrogel precursor solution and the microalgae may be focused into a thinner stream of core fluid (also referred to as ahydrogel thread 131b) by the lower viscosity focusing fluid. This thinner stream of core fluid (the hydrogel thread 131b) has a diameter that is smaller than a diameter of the stream of core fluid 131a.

[0101] At this stage, the hydrogel thread 131b is a thinner stream of the core fluid and includes the hydrogel precursor solution and the microalgae. The hydrogel thread 131b may then be exposed to ultraviolet light from a source 106 of ultraviolet light. The source 106 of ultraviolet light may be optically coupled to a portion of the thread pulling apparatus 100 that is downstream of the junction 105. Upon exposure to the ultraviolet light, under suitable conditions, the hydrogel thread 131b forms a polymerized hydrogel thread 133 encapsulating the microalgae 134. Because the polymerized hydrogel thread 133 is more dense than water, the polymerized hydrogel thread 133 sinks to the bottom of a collection vessel (not shown) coupled to the thread pulling apparatus 100. The water may then be removed, by, for example, pouring the water from the collection vessel.

[0102] Once encapsulated in the polymerized hydrogel thread, the microalgae may be placed in a bright environment permissible to their growth. The algae rapidly grow to densities unachievable with liquid cultures.

[0103] It is noted that microscale laminar flow prevents the higher- viscosity core fluid from mixing with the lower-viscosity focusing fluid throughout the thread pulling process. In addition, varying the ratio of flow between the focusing fluid and the core fluid may be utilized to change the diameter of the thread pulled.

[0104] To confirm the operation of the thread pulling apparatus, a fluorescently labeled hydrogel precursor solution was added to the core fluid. A stream of the higher- viscosity core fluid 131a and two side streams of the lower- viscosity focusing fluid 132 were introduced into the junction 105 through inlet 101a (inlet for the core fluid), inlet 101b (inlet for the first stream of focusing fluid), and inlet 101c (inlet for the second stream of focusing fluid). The focusing fluid pinched the core fluid stream such that the final width and position of the hydrogel thread 131b. The pinching, or focusing, of the core fluid 13 la by the two side streams of focusing fluid 132 leads to a thinner stream of core fluid (also called a hydrogel thread 131b). As described above, the thinner stream of core fluid (the hydrogel thread 131b) comprising the hydrogel precursorsolution and microalgae becomes a polymerized hydrogel thread 133 upon exposure to a suitable wavelength(s) of ultraviolet light.

[0105] For confirmation, fluorescence was used to detect the core fluid 131a and the thinner stream of core fluid (the hydrogel thread 131b). Upon excitation of the fluorescently labeled components present in the core fluid, emitted light showed that the core fluid 131a formed into a thinner stream (or hydrogel thread 131b) as it was guided into the thread by the focusing fluid 132. This confirmed the operation of the thread pulling apparatus whereby the fluorescently labeled hydrogel precursor solution being focused into a thread.

[0106] Experiments were also performed to confirm that different sizes of the polymerized hydrogel thread may be formed using aspects described herein. The diameter of the polymerized hydrogel thread may be controlled by, for example, the relative volumetric flow rates of the stream of core fluid, the first stream of focusing fluid, and the second stream of core fluid. That is, varying the ratio of the flow between the focusing fluid and the core fluid may be utilized to change the diameter of the thread pulled. As confirmed by microscope, polymerized hydrogel threads of approximately 5 pm in diameter, approximately 50 pm in diameter, and approximately 100 pm in diameter were formed, indicating that aspects of the present disclosure may be used to generate polymerized hydrogel threads encapsulating microalgae of any suitable size.

[0107] FIG. IB is an illustration of microalgae growing in polymerized hydrogel threads. The polymerized hydrogel thread has a diameter and a length. The length of the polymerized hydrogel thread may include turns in the thread as shown in FIG. IB. The diameter may be an average diameter over the whole length of the polymerized hydrogel thread. The diameter of the thread is the dimension smaller than the length of the thread.

[0108] The polymerized hydrogel thread 133 includes the microalgae as single microalgae 134 (e.g., a single microalgae cell). After a certain period (represented by arrow 151), e.g., 48 hours, the polymerized hydrogel thread includes the microalgae as dense algae clusters 152 (e.g., two or more microalgae cells, such as 3 or more microalgae cells, such as up to about 300 microalgae cells). Here, the single microalgae 134 grew into the dense algae clusters 152 in an environment permissible to theirgrowth. This growth into dense algae clusters was confirmed by microscope, showing that, at first, the single algae was present in the polymerized hydrogel thread, and then grew into the dense algae clusters.2. Example Fabrication Using Microfluidic Device

[0109] A non-limiting example of a microfluidic device is described in U.S. Patent Application Publication No. 2021 / 0171587, which is incorporated herein by reference in its entirety. FIG. 2A is a schematic of an apparatus 200 for forming polymerized hydrogel droplets encapsulating microalgae, according to at least one aspect. FIG. 2B is a pictorial representation of selected elements of the example apparatus shown in FIG. 2 A. Although the apparatus 200 of FIG. 2 A and 2B are illustrated with respect to forming polymerized hydrogel droplets encapsulating microalgae, the apparatus 200 may be utilized to form polymerized hydrogel threads encapsulating microalgae.

[0110] Apparatus 200 includes a microfluidic device 201. The microfluidic device may include an introduction area 205, a contacting area 210, and a mixing area 212. Briefly, core fluid, microalgae, and hydrogel precursor solution may be introduced to the microfluidic device 201 in the introduction area 205 to form a core fluid mixture; the focusing fluid may contact the core fluid mixture in the contacting area 210; and after contact, the core fluid mixture comprising the hydrogel precursor solution and the microalgae forms droplets in the focusing fluid and travels through mixing area 212. As described herein, the core fluid mixture may further include one or more optional components such as nutrients and / or buffers.[OHl] The microfluidic device 201 may include a fluidic channel 203. The fluidic channel 203 may have a diameter of micrometers (pm) to millimeters (mm). For example, the fluidic channel 203 has a diameter from about 1 pm to about 2 mm and / or a depth of about 1 pm to about 2 mm. The microfluidic device 201 may have an opening 202 for introducing microalgae in a solution, an opening 204 for introducing a fluid, and an opening 206 for introducing a different fluid. Any one of these openings may be used for introducing hydrogel precursors. Any of these openings may optionally be utilized for introducing the one or more optional components such as nutrients and / or buffers.

[0112] A channel 215 may fluidly couple the opening 202 to the fluidic channel 203 such that microalgae in a solution may be introduced to the fluidic channel 203. Similarly, channel 216 may fluidly couple the opening 204 to the fluidic channel 203 and channel 217 may fluidly couple the opening 206 to the fluidic channel 203 such that various fluids and / or hydrogel precursors may be introduced to the fluidic channel 203. The channels 215, 216, and 217 may, independently, have the same or different dimensions. For example, the channel 215 may have a larger or smaller diameter than that of channel 216 and / or channel 217. Channels 215, 216, and 217 may, independently, have a diameter that is larger than, smaller than, or the same dimensions as fluidic channel 203.

[0113] The microalgae and various fluids (fluid comprising hydrogel precursor solution, fluid comprising one or more optional components) may meet at a junction 209 and flow through the fluidic channel 203 in a co-stream. As shown in FIG. 2B, 209a is a pictorial representation of this co-streaming channel where the streams of microalgae 222, hydrogel precursor solution 221, and optional fluid 220 (comprising one or more optional components such as nutrients and / or buffers) flow through the fluidic channel 203.

[0114] The microalgae may be introduced to the device through the same opening as, and together with, the hydrogel precursor solution. Additionally, or alternatively, the microalgae may be introduced to the device through a separate opening. The microalgae may be in an aqueous buffer such as Dulbecco’s Modified Eagle’s Medium (DMEM), phosphate buffered saline or combinations thereof. The microalgae in media may be part of the hydrogel precursor solution.

[0115] The microfluidic device 201 may include another channel 213 for introducing the focusing fluid (the fluid of different viscosity than the core fluid). The focusing fluid forces encapsulation of the cargo (e.g., microalgae, hydrogel precursor solution, among other components) in a hydrogel droplet or a hydrogel thread. The focusing fluid may travel through channel 213 and pinch off the core fluid mixture to form hydrogel droplets 224 or a hydrogel thread (for example, hydrogel thread 131b) at a contacting area 210. Channel 213 may have the same or different dimensions as channels 215, 216, and 217. Channel 213 may have a larger or smaller diameter thanthat of channels 215, 216, and 217, independently, and may have a diameter that is larger than, smaller than, or the same dimensions as fluidic channel 203.

[0116] Tubings, or other suitable apparatus, may be coupled to the individual openings 202, 204, 206, to enable introduction of the microalgae and various fluids to the fluidic channel 203. Tubings, or other suitable apparatus may be coupled to each end of channel 213 to enable introduction of the focusing fluid to the fluidic channel 203. The focusing fluid may be introduced at one or both ends of the channel 213.

[0117] The fluidic channel 203 includes the contacting area 210. The contacting area includes a junction where the fluid of differing viscosity (e.g., the focusing fluid) may pinch off the core fluid (comprising the hydrogel precursor solution and microalgae) to form hydrogel droplets or a thread (for example, hydrogel thread 131b).

[0118] Illustration 210a of FIG. 2B is a pictorial representation of contacting area 210 showing hydrogel droplets 224 being pinched off by one or more streams of focusing fluid 232 at or near the junction. The hydrogel droplets 224 include the core fluid, hydrogel precursor solution, microalgae. The hydrogel droplets 224 may optionally include optional components such as nutrients and / or buffers. As shown in illustration 210a, the focusing fluid 232, may travel through channel 213, while the core fluid (that includes the hydrogel precursor solution and the microalgae) may travel through the fluidic channel 203. A dispersion may be formed when the core fluid and the focusing fluid 232 contact to form droplets. Alternatively, the hydrogel thread 131b may be formed when the core fluid and the focusing fluid 232 contact.

[0119] After the hydrogel droplets 224 are formed, the dispersion of the droplets may be caused to flow (by, e.g., positive pressure) through the mixing area 212 of the fluidic channel 203 and towards fluidic channel exit 214. Here, the hydrogel droplets 224 may travel along the mixing area 212 such that the hydrogel droplets become sufficiently mixed to form hydrogel droplets 225. The fluidic channel 203 of the mixing area 212 may have a serpentine shape / design, the length of which enables, e.g., sufficient mixing of the components. 212a of FIG. 2B is a pictorial representation of a portion of mixing area 212 where components within hydrogel droplets 224 sufficiently mix, while traveling through mixing area 212, to form hydrogel droplets 225.

[0120] After sufficient mixing, the dispersion of the core fluid (in the form of microalgae-containing droplets / particles 224) in the focusing fluid move toward the fluidic channel exit 214 where they may be collected for further processing and / or analysis. Other materials (fluids, excess components, etc.) may exit the fluidic channel exit 214 along with the droplets / particles. Upon exit, the droplets / particles may be purified, or otherwise isolated, from the other materials exiting the apparatus 200.

[0121] The apparatus 200 further includes a source of ultraviolet light optically coupled to the microfluidic device 201. The source of ultraviolet light (not shown in FIG. 2 A) may be optically coupled to a portion of the mixing area 212, a portion of the fluidic channel 203 between the mixing area 212 and the fluidic channel exit 214, and / or the fluidic channel exit 214. Upon exposure of the droplets to ultraviolet light, the polymerized hydrogel droplets encapsulating the microalgae may be formed. Conditions for exposing the hydrogel droplets to UV light are described herein.

[0122] The polymerized hydrogel droplets may be recovered based on density as the polymerized hydrogel droplets are denser than water (fluids present in the microfluidic device) and sink to the bottom of the collection vessel (coupled to fluidic channel exit 214). After forming the composition (the microalgae encapsulated in the polymerized hydrogel droplet), the composition is placed in an environment permissible to microalgae growth into algae clusters inside the polymerized hydrogel droplets as described herein.

[0123] Movement of the various materials from the one or more openings 202, 204, 206, and the ends of channel 213 to the fluidic channel exit 214 may be controlled by, e.g., a pumping mechanism, a pressure regulation system (such as those available from Fluigent), and / or electrodes. Such elements controlling the movement may be placed at opposing ends of or along various regions along a length of the fluidic channel 203. Design parameters to effectively form droplets / particles at desired concentrations include the length and width of the channels, the spacing between the channels and / or openings, and the flow rates of fluid flowing through the microfluidic device.

[0124] With respect to the hydrogel threads 131b, the hydrogel threads 131b may be caused to flow through the mixing area 212 of the fluidic channel 203 and towards fluidic channel exit 214 by the positive pressure of the focusing fluid and / or core fluid.Here, the hydrogel thread 131b may travel along the mixing area 212. The fluidic channel 203 of the mixing area 212 may have any suitable shape / design. The hydrogel thread 131b (comprising the hydrogel precursor solution and the microalgae), which is focused by the focusing fluid, moves toward the fluidic channel exit 214 where it may be collected for further processing. As the hydrogel thread 131b moves toward the fluidic channel exit 214, the hydrogel thread 131b is polymerized with ultraviolet light using a source of ultraviolet light (for example, source 106). The source of ultraviolet light may be optically coupled to a portion of the mixing area 212, a portion of the fluidic channel 203 between the mixing area 212 and the fluidic channel exit 214, and / or the fluidic channel exit 214. Upon exposure of the hydrogel thread 131b to ultraviolet light, the polymerized hydrogel thread 133 encapsulating the microalgae 134 may be formed. Conditions for exposing the hydrogel thread to UV light are described herein.

[0125] The polymerized hydrogel thread 133 may be recovered based on density as the polymerized hydrogel thread 133 is denser than water and sinks to the bottom of the collection vessel (coupled to fluidic channel exit 214). For example, the fluids / components utilized to form the polymerized hydrogel thread 133 may be separated from the polymerized hydrogel thread 133. After forming the composition (the microalgae encapsulated in the polymerized hydrogel thread), the composition is placed in an environment permissible to microalgae growth into algae clusters inside the polymerized hydrogel thread as described herein.

[0126] The non-limiting microfluidic device is only one of many suitable ways to encapsulate the microalgae. It is also noted that microfluidic devices are not required to encapsulate microalgae. For example, and as described herein, the microalgae may be encapsulated by using a pipette. Here, a dispersion of the microalgae may be pipetted into a suitable media comprising, for example, barium chloride (BaCh) as a gelling agent, hydrogel precursors, or combinations thereof. Other suitable methods and apparatus may be utilized.

[0127] That is, the polymerized hydrogel droplets encapsulating the microalgae may be left to grow under conditions suitable for growth of algae clusters as described herein.3. Example Process for Forming the Composition

[0128] The composition may include microalgae (or microalgae cells) encapsulated in a polymerized hydrogel thread(s) or in a polymerized hydrogel droplet(s).

[0129] The process may include introducing a core fluid (for example, core fluid 131a) to a device. The device may be any suitable device such as thread pulling apparatus 100 or apparatus 200. The core fluid introduced to the device may include a core fluid mixture that includes core fluid, hydrogel precursor solution, microalgae (e.g., microalgae cells), or combinations thereof. Alternatively, one or more of the core fluid, microalgae, and hydrogel precursor solution may be introduced separately into the device. In the latter case, the core fluid, microalgae, and hydrogel precursor solution may meet at junction 209 and flow through the fluidic channel 203 in a co-stream. The microalgae may be introduced with a media such as a buffer, nutrients, or combinations thereof.

[0130] The operation of introducing the core fluid into the device may be performed at any suitable flow rate. Suitable flow rates for introducing the core fluid may include a flow rate of about 0.1 pL / min or more, about 150 pL / min or less, or combinations thereof, such as in a range from about 25 pL / min to about 125 pL / min, such as from about 50 pL / min to about 100 pL / min, such as from about 80 pL / min to about 100 pL / min or in a range from about 0.1 pL / min to about 15 pL / min, such as from about 0.5 pL / min to about 10 pL / min, such as from about 1 pL / min to about 5 pL / min.

[0131] As described above, the core fluid, hydrogel precursor solution, and the microalgae stream may be introduced at the same time or separate times to the same or different openings of the device. For example, a mixture comprising the core fluid, hydrogel precursor solution, and microalgae (or microalgae cells) may be introduced via inlet 101a of thread pulling apparatus 100. As another example, each of the core fluid, the hydrogel precursor solution, and the microalgae may be introduced through different openings of the device. For example, the core fluid may be introduced via opening 204 of apparatus 200, the hydrogel precursor solution may be introduced via opening 202 of apparatus 200, and the microalgae (or microalgae cells) in a medium may be introduced via opening 206 of apparatus 200. When one or more of the core fluid, hydrogel precursor solution, or microalgae (in a medium) are introduced to the device separately, these materials may be, independently, introduced to the device atthe same or different flow rates such as those flow rates described above, such as a flow rate of about 0.1 gL / min or more, about 150 gL / min or less, or combinations thereof, such as in a range from about 25 gL / min to about 125 gL / min, such as from about 50 gL / min to about 100 gL / min, such as from about 80 gL / min to about 100 gL / min or in a range from about 0.1 gL / min to about 15 gL / min, such as from about 0.5 gL / min to about 10 gL / min, such as from about 1 gL / min to about 5 gL / min.

[0132] The resulting core fluid mixture comprising the core fluid, hydrogel precursor solution, and microalgae (or microalgae cells) flows through a channel of the device (for example, fluidic channel 103 of thread pulling apparatus 100 or fluidic channel 203 of apparatus 200.

[0133] The process may further include introducing the focusing fluid (for example, focusing fluid 132) to the device. For example, the focusing fluid may be introduced via inlets 101b, 101c of thread pulling apparatus 100 or introduced into channel 213 of apparatus 200. The focusing fluid may be introduced at any suitable flow rate. Suitable flow rates of the focusing fluid may be 0.1 gL / min or more, about 200 gL / min or less, or combinations thereof, such as in a range from about 1 gL / min to about 150 gL / min, such as from about 25 gL / min to about 125 gL / min, such as from about 50 gL / min to about 100 gL / min, such as from about 75 gL / min to about 100 gL / min or in a range from about 0.1 gL / min to about 15 gL / min, such as from about 0.5 gL / min to about 10 gL / min, such as from about 1 gL / min to about 5 gL / min. The flow rate of the focusing fluid may be the same or different from the flow rates of other materials in the device.

[0134] The process may further include contacting the focusing fluid with the core fluid at a junction of the device (for example, junction 105 of thread pulling apparatus 100 or a junction within contacting area 210 of apparatus 200).

[0135] After contact, a hydrogel thread (for example, hydrogel thread 131b) or a hydrogel droplet (for example, hydrogel droplet 224) is formed. As described above, the focusing fluid has a different viscosity than the core fluid such that the core fluid and focusing fluid are viscosity mismatched. This results in the formation of the hydrogel thread or hydrogel droplet.

[0136] The process may further include exposing the hydrogel thread or hydrogel droplet to ultraviolet light under conditions sufficient to form a polymerized hydrogelthread (e.g., polymerized hydrogel thread 133) encapsulating microalgae or a polymerized hydrogel droplet encapsulating microalgae.

[0137] Exposing the hydrogel thread or hydrogel droplet to ultraviolet light causes reaction of components present in the hydrogel precursor solution which forms a portion of the core fluid. These components include one or more photoreactive monomers, one or more optional linkers (for example, dithiol linkers), optional photoinitiators, or combinations thereof. The reaction may take the form of “click” chemistry, polymerization, click polymerization, and / or curing such that components of the reaction mixture react. For example, the mixture may be polymerized by exposure to ultraviolet light, under polymerization conditions, to form the composition comprising the polymerized hydrogel-encapsulated microalgae (or microalgae cells). “Polymerized hydrogel-encapsulated microalgae (or microalgae cells)” refers to the polymerized hydrogel thread encapsulating microalgae (or microalgae cells) and to the polymerized hydrogel droplet encapsulating microalgae (or microalgae cells).

[0138] Conditions for the reaction to form the polymerized hydrogel-encapsulated microalgae (or microalgae cells) may include one or more of the following parameters:

[0139] (a) Exposing the hydrogel thread or hydrogel droplet to ultraviolet light at a desired wavelength or wavelength range, such as a wavelength or wavelength in a range from about 290 nm to about 500 nm, such as from about 320 nm to about 460 nm, such as from about 340 nm to about 440 nm, such as from about 360 nm to about 420 nm, such as from about 380 nm to about 400 nm or from about 400 nm to about 420 nm, such as about 365 nm or about 405 nm. The wavelength or wavelength range of light may be from about 290 nm to about 460 nm, such as from about 350 nm to about 450 nm, such as from about 375 nm to about 425 nm. The wavelength or wavelength range may be constant or varying during polymerization. It is contemplated that other wavelengths of light may be used with appropriate reacting photoinitiators.

[0140] (b) An energy density of the ultraviolet light may be in a range from about1 mW / cm2to about 10,000 mW / cm2, such as from about 10 mW / cm2to about 1,000 mW / cm2, such as from about 50 mW / cm2to about 500 mW / cm2, such as from about 75 mW / cm2to about 150 mW / cm2, such as from about 80 mW / cm2to about 120 mW / cm2.

[0141] (c) A duration of exposure to the ultraviolet light may be about 1 millisecond or more, 5 minutes or less, or a combination thereof, such as in a range from about 3 milliseconds to about 60 seconds, such as from about 5 milliseconds to about 50 seconds, such as from about 50 milliseconds to about 45 seconds, such as from about 100 milliseconds to about 40 seconds, such as from about 0.5 seconds to about 30 seconds, such as from about 1 second to about 20 seconds.

[0142] The process may further include removing the polymerized hydrogel- encapsulated microalgae (or microalgae cells) from the fluid. The polymerized hydrogel-encapsulated microalgae (or microalgae cells) may be recovered based on density as it is denser than the fluid surrounding the polymerized hydrogel-encapsulated microalgae (or microalgae cells). The fluid may include focusing fluid, core fluid, unreacted components, or combinations thereof, among other components.

[0143] If desired, the polymerized hydrogel-encapsulated microalgae (or microalgae cells) may be purified or otherwise isolated from the other materials exiting the thread pulling apparatus 100 or the apparatus 200.4. Example Applications4, A, Soil Amendment

[0144] Soil amendments are products added to the soil to improve the soil condition or its physical properties. Algae has previously been used as a “green fertilizer” dating back to the application of kelp and seaweed to fields, serving to improve soil quality through the addition of nutrients and organic carbon.

[0145] A soil amendment may include a composition described herein, such as a composition comprising a polymerized hydrogel encapsulating a dense algae cluster. Algal media described herein may be used to grow the dense algae clusters while encapsulated in the polymerized hydrogel. Additionally, or alternatively, fertilizer rich agricultural runoff may be used to grow the dense algae clusters while encapsulated in the polymerized hydrogel.

[0146] The soil amendment may further include an exogenous carbon source. As used herein, an exogenous carbon source refers to a carbon source added to the composition after the formation of the composition. Suitable exogenous carbon sourcesmay include pyrolyzed coal char, coal, coal char, or combinations thereof, such as pyrolyzed coal char.

[0147] The algae (for example, Tetradesmus deserticold) encapsulated within the polymerized hydrogel, and with or without a carbon source, may be applied to fields in a viable state where it proliferates in the soil. This may serve to, for example, amend the quality of the soil through the addition of organic carbon and nutrients from decaying algae, but in a self-renewing manner as the algae continues to grow. Because Tetradesmus deserticola also binds strongly to surfaces, it also aggregates loose soils, which prevents erosion, holds more water, and sequesters fertilizer, preventing runoff. 4,B, Terrestrial Alga: Carbon Sinks for Environmental Remediation

[0148] Proposed herein is a unique approach to carbon sequestration that simultaneously reduces agricultural pollution and restores the health of soils. The approach may include use of an algal species (for example, Tetradesmus deserticola) that has been isolated from desert soils known as biotic crusts. These algae behave similarly to aquatic or marine algae when grown in conventional bioreactors. However, when confined within soft hydrogel matrices as described herein, this species may grow more than ten times as fast and densely when unencapsulated in an aqueous suspension. This ten-fold increase in biomass production represents a generational step increase for algae bioprocesses and offers paradigm shifting possibilities for algae as a carbon sink.

[0149] While algae has been touted as a key cog in the production of carbon-neutral chemicals and fuels, its potential has ultimately been limited by an inability to scale to the needed production targets. The central challenge of this problem may be addressed by aspects described herein.

[0150] Soils are a powerful carbon sink, or a tremendous source of carbon emissions depending on soil health. Improving soil health by reintroducing native organisms, such as algae, sequesters carbon at all stages. A native algae encapsulated in a polymerized hydrogel may be utilized to capture carbon dioxide (CO2) directly from air.

[0151] A warmer, drier climate in the American West increases water stress in agriculture. This, warmer, drier, new normal necessitates novel approaches in both agricultural practices and carbon sequestration. The terrestrial green algae, Tetradesmusdeserlicola. may be added to soils of semi-arid agricultural croplands as a strategy that limits water loss, improves soil health, and sequesters carbon. Because Tetradesmus deserticola is typically found in deserts across the desert southwestern US and into the Baja peninsula, it thrives in a variety of soil conditions, with each algal division further sequestering carbon and increasing soil health. With millions of acres of semi-arid cropland in North America at risk of drought, significant quantities of algae are needed. Because Tetradesmus deserticola algae lives in the sandy, dry soils of the American West, referred to as an extremophile, it survives extreme temperatures, desiccation, high salinity, heavy metals, high pH soils, and high levels of ultraviolet light. In its native desert habitat, Tetradesmus deserticola is a major component of biotic soil crusts, producing polysaccharides and cellulosic debris that bind soil, and retain water and nutrients. Additionally, it possesses a unique motility system that enables its ability to disperse throughout the environment. The water and nutrient holding capacity of Tetradesmus deserticola is significant, as evidenced in test samples, where water loss is reduced by (70%), Nitrate loss by (90%) and Phosphate loss by (32%), compared to untreated soil. Pilot studies in Wyoming fields showed 15% increase in corn ear biomass from algal treated test plots. Tetradesmus" environmental insensitivity allows for novel growth strategies.

[0152] The inventors have developed a technology and a generational breakthrough that increases the CO2 capture capacity of this algae by more than 5 times that of conventional algaculture techniques. Mass production of the low density, planktonic algae produced by conventional algaculture techniques is limited by the volume of water available. Aspects described herein take advantage of the Tetradesmus rapid doubling when entrapped, encapsulating algae in biodegradable hydrogel beads to achieve significant increase over conventional algaculture over the same time fame. Facile recovery is possible with millimeter scale, dense algae laden beads compared to planktonic algae, removing an inefficient processing step.

[0153] A carbon (e.g., CO2) sequestration composition may include a composition described herein (e.g., a microalgae encapsulated within a polymerized hydrogel, a microalgae crosslinked in a hydrogel matrix, or a 3D-printed composition). Algal media described herein may be used to grow the dense algae clusters while encapsulated inthe polymerized hydrogel. Additionally, or alternatively, fertilizer rich agricultural runoff may be used to grow the dense algae clusters while encapsulated in the polymerized hydrogel. Bags of the dense algae clusters of Tetradesmus entrapped in hydrogels may be placed in polluted canals to soak up excess fertilizer while sequestering carbon (e.g., CO2), easily harvested and applied to fields to improve soil fertility. These small, single celled native algae pose no risk to humans, animals or to the environment.4.C. Nitrate Runoff

[0154] Nitrate runoff causes nitrate contamination of surface and ground water. Nitrate contamination is a risk to human health and contributes to eutrophication, which is the excess growth of aquatic plant life that kills marine animals due to lack of oxygen. Typically, nitrate runoff is caused by fertilizer.

[0155] Nitrate runoff is difficult and costly to filter from drinking water. Poor rural communities have the largest issues with nitrate pollution due to difficulty removing nitrates. The EPA limit of nitrate in drinking water is 10 mg / L.

[0156] A composition for removing nitrates may include a composition described herein, such as a composition comprising a polymerized hydrogel encapsulating a dense algae cluster. Algal media described herein may be used to grow the dense algae clusters while encapsulated in the polymerized hydrogel. Additionally, or alternatively, fertilizer rich agricultural runoff may be used to grow the dense algae clusters while encapsulated in the polymerized hydrogel.

[0157] Compositions described herein (e.g., encapsulated algae) have the ability in soil to reduce this by 90%, using, e.g., Tetradesmus deserticola algae upstream of a potable water source and may turn 10 mg / L nitrate undrinkable water into 1 mg / L potable water with the added benefit of carbon capture and soil fertility.

[0158] Compositions and processes described herein may be used to form, as a conservative estimate, 10 g / L of algae in beads per harvest cycle. With 1 liter of volume, there are 100 kg algae per harvest cycle. This may take the form of a strip of bags 100 meters long, 10 meters wide with 10 cm of bag depth. These bags may be added upstream of a potable water source to remove nitrates. Overall, this example shows thatadding encapsulated algae upstream of a potable water source may act as an “artificial riparian buffer”.5. Microalgae Biomass Generation in Crosslinked Hydrogel Matrices

[0159] A non-limiting example of microalgae biomass generation and encapsulation is described in this section.5.1, Cultivation of Tetradesmus Deserticola

[0160] Tetradesmus deserticola was cultivated in Erlenmeyer flasks in a Heracell 150 incubator kept at 30°C and atmospheric (1.3%) CO2. Parts of the culture were removed consistently for experiments and fresh medium was added to keep the algae in their growth phase. Growth medium, and the recipe, are shown in Table 1.5.2, Growth Platform Development

[0161] The platform and scheme is a high throughput, homogeneous growth platform and an encapsulation scheme that is scalable, with minimal requirements for equipment.

[0162] 5.2.1 Hydrogel Selection. Tetradesmus deserticola lends itself to encapsulation and seems to thrive in alginate-based hydrogels. In order to minimize the use of high technology equipment, such as 3D printers, and to simplify the production process, hydrogels from different concentrations of sodium alginate were chosen. To make a given concentration of hydrogel precursor, an appropriate amount of sodium alginate (Kimica International, grade I-1G-80) was weighed out using a high precision scale (Mettler Toledo, SXSR105DU) and added to Biopak Milli-Q water. The suspension was combined on a magnetic stirrer (Heidolph, Mix’n’Heat Core) at 50°C until fully dissolved. It was then kept in a fridge at 8°C at least overnight to settle.

[0163] 5.2.2 Fabrication of Sodium Alginate Beads. The given concentration of sodium-alginate precursor was prepared as described in 5.2.1. A high concentration, IM BaCh crosslinker solution was prepared using Barium chloride dihydrate (Sigma- Aldrich, B0750-500G) and 50mL of Mili-Q water. This solution was then further diluted to the desired concentration according to the experiment conditions using Milli- Q water. An electronic pipette (Rainin, E4XLS) was used to drop the sodium alginate precursor into a continuously stirred crosslinker bath. Unless stated otherwise, the formed beads were then left to stir in the bath for 10 minutes, after which they weremoved to 24-well plates (TPP Switzerland, 92024) filled with 2 mL crosslinker and left there for another 110 minutes, for a total crosslinking time of 2 hours. The pipette settings were as follows: aliquot size (25 pL); dispension speed (1); aspiration speed (1); rest time (1 second). For biotic beads, a sodium alginate suspension containing algae was used, as described in 5.2.3.

[0164] 5.2.3. Encapsulation. Part of the suspension described in 5.1 was taken from the Erlenmeyer flask and pipetted into a 2 mL Eppendorf tube or a 15 mL falcon tube and centrifuged for 5 minutes at 3400 relative centrifugal force (ref) and 5000 ref respectively using a centrifuge (Eppendorf, 5430 R). The supernatant was pipetted out and the pellets were resuspended with the sodium alginate precursor outlined in 5.2.1. This suspension was then shaken and mixed with a vortex (Scientific Industries, Vortex Genie 2) until a homogeneous liquid was achieved. The encapsulated cultures were diluted to match the optical density at 664 nm of the corresponding aqueous suspension.6. Microalgae Biomass Generation in 3D Printed Matrices

[0165] A non-limiting example of microalgae biomass generation and encapsulation is described in this section.6,1 Cultivation of Tetradesmus deserticola

[0166] Dried algae of the species Tetradesmus deserticola were rehydrated and cultured in medium in Erlenmeyer flasks, which were mounted on a shaker plate (150 rpm) inside an incubator (Heraeus, Heracell 150). The medium recipe is provided in Table 1. The temperature inside the incubator was kept at 30°C and the illumination was set on 12-hour on and off cycle at 180 pmol photons m2s ' . No gases were added in the incubator. When the culture started to look very green or form clumps, indicating high algae density, a part of it was centrifuged for 5 minutes at 3500 ref. The medium was then pipetted out and replaced with a larger amount of fresh medium to start the cultivation process again.6,2, Gels

[0167] 6.2.1. Pluronic F127 & F127-bis-urethane methacrylate (F127 & F127-BUM) hydrogel. To prepare 6 g of a 10 wt% F127 & 10 wt% F127-BUM hydrogel with 0.1 wt% LAP: 2.34 ml of medium was added to 0.6 g (20 wt%) of F 127 (Pluronic, P2443) and mixed on ice on a magnetic stirrer until fully dissolved. The same was donefor F127-BUM. Then, both solutions were added together. 114 pL (0.12 g; 0.1 wt%) of LAP (5 wt% suspension) was added. This was then left at 4°C for approximately 5 minutes and then mixed with a vortex mixer. Crosslinking of F127 & F127-BUM hydrogels was done with 405 nm light at an intensity of 10 mW / cm2 for durations between 1-3 minutes.

[0168] 6.2.2. Alginate-Methylcellulose (Alg-MC) hydrogel. To prepare 4 g of a1 wt% alg-MC hydrogel: 40 mg (1 wt%) of sodium alginate (KIMICA, L1G-80) was dissolved in 3.6 mL of Milli-Q water at 50°C for approximately 30 minutes on a magnetic stirring plate (300 rpm) and then left overnight at 4°C. Then, 360 mg (9 wt%) of methylcellulose (Sigma-Aldrich, M0512, viscosity: 4000cP) was added and mixed in with a metal spatula and left overnight at 4°C again to swell. Before use, the gel was centrifuged for 1 min at 7200 ref to remove bigger air bubbles that form during the mixing with methylcellulose. Crosslinking was done with 10-100 mM BaCh (Sigma- Aldrich, B0750) solutions for 15 minutes. The samples were washed with media once for 10 min before incubation to remove excess of the crosslinker.

[0169] 6.2.3. Alginate-Polymer-Nanoparticle (Alg-PNP) hydrogel. To prepare 4 g of a 1 wt. % alg-PNP hydrogel: 40 mg (1 wt%) sodium alginate was dissolved in 1.88 ml of Milli-Q water at 50 °C for approximately 30 minutes on a magnetic stirring plate (300 rpm). After it had dissolved, 80 mg (2 wt%) of 2-hydroxyethyl cellulose (Sigma- Aldrich, 434981, average Mv: 1300000) was added and mixed in using a stirring plate and, later, with a spatula. This was then left in the fridge at 4°C until it was visibly homogeneous after one to two days. Parallel to that, 0.5714 ml (800 mg) of colloidal silica (Ludox, 420778, TM-50 (50 wt% suspension in H2O)) was mixed with 1.2 ml of Milli-Q water to obtain a 20 wt% CSP solution. On the day of use both parts, equal in mass (2 g each), were mixed by hand for approximately 1 minute using two syringes connected with a Luer-Lock to obtain the final gel. Crosslinking was done with 10-100 mM BaC12 solutions for 15 minutes. The samples were washed with media once for 10 min before incubation to remove excess of the crosslinker.

[0170] 6.2.4. Polyethylene glycol diacrylate-Polymer-Nanoparticle (PEGDA-PNP) hydrogel. To prepare 4 g of a 5 wt% PEGDA-PNP hydrogel: 200 mg (5 wt%) PEGDA (Laysan Bio, MW: 3400) was dissolved in 1.2 ml of Milli-Q water forapproximately 30 minutes on a magnetic stirring plate (300 rpm). After it had dissolved, 80 mg (2 wt. %) of 2-hydroxyethyl cellulose was added and mixed in using a stirring plate. Parallel to that, 0.5714 ml (800 mg) of colloidal silica was mixed with 1.64 mL of Milli-Q water to obtain a 20 wt% CSP solution. On the day of use 76 pL (80 mg) of LAP (5 wt% suspension) was added to the PEGDA-HEC-solution and mixed thoroughly. Both parts were then mixed by hand for approximately 1 minute using two syringes connected with a Luer-Lock to obtain the final gel. Crosslinking of PEGDA- PNP hydrogels was done with 405 nm light at an intensity of 10 mW / cm2for 1 minute.6.3, Encapsulation Procedure

[0171] Up to 1 mL (in general: 10 % of hydrogel mass) of the liquid algae culture was transferred to an Eppendorf tube and then centrifuged for 5 minutes at 3500 ref. The liquid was then pipetted out, leaving only pallet of algae in the tube. Then, the hydrogel was added and mixed with the algae using a spatula (Alg-MC, Alg-PNP, PEGDA-PNP) or a vortex mixer (Fl 27 & F127-BUM).6.4, 3D Bioprinting

[0172] All 3D printing was done using an extrusion-based Cellink BioX printer. Hydrogel was transferred to a 3 ml printer cartridge which was then outfitted with a 22G conical nozzle tip (diameter = 0.41 mm). Alg-PNP was printed with a pressure of 40-60 kPa at speeds of 2-6 mm / s Alg-MC was printed with a pressure of 300-400 kPa. This pressure was achieved by connection of the 3D printing system to a compressed air line. Printing speed was between 1-5 mm / s.Aspects Listing

[0173] The present disclosure provides, among others, the following aspects, each of which may be considered as optionally including any alternate aspects:

[0174] Aspect 1. A composition comprising: an encapsulant comprising a polymerized hydrogel; and microalgae encapsulated within the encapsulant.

[0175] Aspect 2. The composition according to Aspect 1, wherein the encapsulant is in the form of a thread, droplet, or combinations thereof.

[0176] Aspect 3. The composition according to any one of Aspects 1-2, wherein the encapsulant has a diameter of less than 5 mm.

[0177] Aspect 4. The composition according to any one of Aspects 1-3, wherein the encapsulant has a diameter of less than 1 mm.

[0178] Aspect 5. The composition according to any one of Aspects 1-4, wherein the microalgae comprises terrestrial algae, aquatic algae, or combinations thereof.

[0179] Aspect 6. The composition according to Aspect 5, wherein the terrestrial algae comprises Tetradesmus deserticola.

[0180] Aspect 7. The composition according to any one of Aspects 1-6, wherein the encapsulated microalgae of the composition, when exposed to growth conditions, exhibits an algae growth density per unit time that is greater than a control composition comprising unencapsulated microalgae under the same growth conditions.

[0181] Aspect 8. The composition according to any one of Aspects 1-7, wherein the encapsulated microalgae of the composition, when exposed to growth conditions, exhibits an increase in algae growth density per unit time that is about 10x or more than a control composition comprising unencapsulated microalgae under the same growth conditions.

[0182] Aspect 9. The composition according to any one of Aspects 1-8, wherein the microalgae is a single microalgae cell.

[0183] Aspect 10. The composition according to any one of Aspects 1-9, wherein the microalgae is an algae cluster comprising two or more microalgae cells, for example, an algae cluster described herein.

[0184] Aspect 11. A composition comprising: microalgae encapsulated within a polymerized hydrogel matrix.

[0185] Aspect 12. A soil amendment, comprising: a composition described herein (for example, the composition according to any one of Aspects 1-11); and an exogenous carbon source.

[0186] Aspect 13. The soil amendment according to Aspect 12, wherein the exogenous carbon source comprises pyrolyzed coal char, coal, coal char, or combinations thereof.

[0187] Aspect 14. A carbon (e.g., CO2) sequestration composition, comprising: a composition described herein (for example, the composition according to any one of Aspects 1-11).

[0188] Aspect 15. A composition for removing nitrates from a medium (for example, soil or water), the composition comprising: a composition described herein (for example, the composition according to any one of Aspects 1-11).

[0189] Aspect 16. A process for encapsulating algae, the process comprising: introducing, to a fluidic channel of a microfluidic device, a stream of an aqueous-based core fluid comprising microalgae and one or more hydrogel precursors; contacting the stream of aqueous-based core fluid with two streams of an aqueous-based focusing fluid to form a thinner stream of the core fluid comprising the microalgae and the one or more hydrogel precursors, the aqueous-based focusing fluid having a different viscosity than the aqueous-based core fluid; and exposing the thinner stream of the core fluid to light to form a composition described herein (for example, a polymerized hydrogel thread encapsulating the microalgae).

[0190] Aspect 17. A process for encapsulating algae, the process comprising: introducing, to a fluidic channel of a microfluidic device, a stream of an aqueous-based core fluid comprising microalgae and one or more UV light- photoreactive hydrogel precursors; contacting the stream of aqueous-based core fluid with two streams of an aqueous-based focusing fluid to form a thinner stream of the core fluid comprising the microalgae and the one or more UV light-photoreactive hydrogel precursors, the aqueous-based focusing fluid having a different viscosity than the aqueous-based core fluid; and exposing the thinner stream of the core fluid to UV light to form a composition described herein (for example, a polymerized hydrogel thread encapsulating the microalgae).

[0191] Aspect 18. The process according to any one of Aspects 16-17, wherein hydrodynamic focusing by the aqueous-based focusing fluid causes tapering of the aqueous-based core fluid to form the thinner stream of the core fluid.

[0192] Aspect 19. The process according to any one of Aspects 16-18, wherein the polymerized hydrogel thread has a diameter of less than 1 mm.

[0193] Aspect 20. The process according to any one of Aspects 16-19, wherein the microalgae comprises terrestrial algae, aquatic algae, or combinations thereof.

[0194] Aspect 21. The process according to any one of Aspects 16-20, wherein: the stream of the aqueous-based core fluid is introduced at a first end of the microfluidic device; and the process further comprises applying positive pressure to the microfluidic device to cause the thinner stream of the core fluid to flow towards a second end of the microfluidic device.

[0195] Aspect 22. The process according to any one of Aspects 16-21, wherein one or more operations of the process is performed at a temperature that is from about 15°C to about 25°C.

[0196] Aspect 23. The process according to any one of Aspects 16-22, further comprising isolating the composition by removing the water.

[0197] Aspect 24. A process for growing algal biomass, the process comprising: exposing a composition described herein (for example, the composition according to any one of Aspects 1-11, such as a microalgae encapsulated within a polymerized hydrogel) to growth conditions to form a microalgae cluster within the polymerized hydrogel.

[0198] Aspect 25. The process according to Aspect 24, wherein the microalgae comprises terrestrial algae, aquatic algae, or combinations thereof.

[0199] Aspect 26. A process for forming microalgae particles (for example, droplets), the process comprising: introducing, to a fluidic channel of a microfluidic device, a stream of an aqueous-based core fluid comprising microalgae and one or more hydrogel precursors; contacting the stream of aqueous-based core fluid with one or more streams of an aqueous-based focusing fluid to form hydrogel particles (or droplets) comprising the microalgae and the one or more hydrogel precursors, the aqueous-based focusing fluid having a different viscosity than the aqueous-based core fluid; and exposing the hydrogel particles (or droplets) to light to form a composition described herein (for example, polymerized hydrogel particles (or droplets) encapsulating the microalgae).

[0200] Aspect 27. A process for encapsulating algae, the process comprising: introducing, to a fluidic channel of a microfluidic device, a stream of an aqueous-based core fluid comprising microalgae and one or more UV light- photoreactive hydrogel precursors; contacting the stream of aqueous-based core fluid with one or more streams of an aqueous-based focusing fluid to form hydrogel particles (or droplets) comprising microalgae and one or more UV light-photoreactive hydrogel precursors; and exposing the hydrogel particles (or droplets) to UV light to form a composition described herein (for example, polymerized hydrogel particles (droplets) encapsulating the microalgae).

[0201] Aspect 28. The process according to any one of Aspects 26-27, wherein hydrodynamic focusing by the aqueous-based focusing fluid causes formation of the hydrogel particles (or droplets) of the core fluid.

[0202] Aspect 29. The process according to any one of Aspects 26-28, wherein the polymerized hydrogel particles (or droplets) have a diameter of less than 1 mm.

[0203] Aspect 30. The process according to any one of Aspects 26-29, wherein the microalgae comprises terrestrial algae, aquatic algae, or combinations thereof.

[0204] Aspect 31. The process according to any one of Aspects 26-30, wherein: the stream of the aqueous-based core fluid is introduced at a first end of the microfluidic device; and the process further comprises applying positive pressure to the microfluidic device to cause the hydrogel particles (or droplets) to flow towards a second end of the microfluidic device.

[0205] Aspect 32. The process according to any one of Aspects 26-31, wherein one or more operations of the process is performed at a temperature that is from about 15°C to about 25°C.

[0206] Aspect 33. The process according to any one of Aspects 26-32, further comprising isolating the composition (for example, polymerized hydrogel particles (droplets) encapsulating the microalgae) by removing the water.

[0207] All documents described herein are incorporated by reference herein, including any priority documents and / or testing procedures to the extent they are notinconsistent with this text. As is apparent from the foregoing general description and the specific aspects, while forms of the aspects have been illustrated and described, various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, it is not intended that the present disclosure be limited thereby. Likewise, the term “comprising” is considered synonymous with the term “including.” Likewise whenever a composition, an element, a group of elements, or a method is preceded with the transitional phrase “comprising,” it is understood that we also contemplate the same composition, method, or group of elements with transitional phrases “consisting essentially of,” “consisting of,” “selected from the group of consisting of,” or “Is” preceding the recitation of the composition, element, elements, or method, and vice versa, such as the terms “comprising,” “consisting essentially of,” “consisting of’ also include the product of the combinations of elements listed after the term.

[0208] In the foregoing, reference is made to aspects of the disclosure. However, it should be understood that the disclosure is not limited to specific described aspects. Instead, any combination of the following features and elements, whether related to different aspects or not, is contemplated to implement and practice the disclosure. Furthermore, although aspects of the disclosure may achieve advantages over other possible solutions and / or over the prior art, whether or not a particular advantage is achieved by a given aspect is not limiting of the disclosure. Thus, the foregoing aspects, features, embodiments, and advantages are merely illustrative and are not considered elements or limitations of the appended claims except where explicitly recited in a claim(s). Likewise, reference to “the disclosure” shall not be construed as a generalization of any inventive subject matter disclosed herein and shall not be considered to be an element or limitation of the appended claims except where explicitly recited in a claim(s).

[0209] For purposes of this present disclosure, and unless otherwise specified, all numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and consider experimental error and variations that would be expected by a person having ordinary skill in the art. For the sake of brevity, only certain ranges are explicitly disclosed herein. However, rangesfrom any lower limit may be combined with any upper limit to recite a range not explicitly recited, as well as, ranges from any lower limit may be combined with any other lower limit to recite a range not explicitly recited, in the same way, ranges from any upper limit may be combined with any other upper limit to recite a range not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the subranges 1 to 4, 1.5 to 4.5, 1 to 2, among other subranges. As another example, the recitation of the numerical ranges 1 to 5, such as 2 to 4, includes the subranges 1 to 4 and 2 to 5, among other subranges. Additionally, within a range includes every point or individual value between its end points even though not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the numbers 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, among other numbers. Thus, every point or individual value may serve as its own lower or upper limit combined with any other point or individual value or any other lower or upper limit, to recite a range not explicitly recited.

[0210] As used herein, the indefinite article “a” or “an” shall mean “at least one” unless specified to the contrary or the context clearly indicates otherwise. For example, aspects comprising a “droplet” include aspects comprising one, two, or more droplets, unless specified to the contrary or the context clearly indicates only one droplet is included.

[0211] While the foregoing is directed to aspects of the present disclosure, other and further aspects of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

ClaimsWhat is claimed is:

1. A composition comprising: an encapsulant comprising a polymerized hydrogel; and microalgae encapsulated within the encapsulant.

2. The composition according to claim 1, wherein the encapsulant is in the form of a thread, droplet, or combinations thereof.

3. The composition according to claim 1, wherein the encapsulant has a diameter of less than 5 mm.

4. The composition according to claim 1, wherein the encapsulant has a diameter of less than 1 mm.

5. The composition according to claim 1, wherein the microalgae comprises terrestrial algae, aquatic algae, or combinations thereof.

6. The composition according to claim 5, wherein the terrestrial algae comprises Tetradesmus deserticola.

7. The composition according to claim 1, wherein the encapsulated microalgae of the composition, when exposed to growth conditions, exhibits an algae growth density per unit time that is greater than a control composition comprising unencapsulated microalgae under the same growth conditions.

8. The composition according to claim 1, wherein the encapsulated microalgae of the composition, when exposed to growth conditions, exhibits an increase in algae growth density per unit time that is about 10x or more than a control composition comprising unencapsulated microalgae under the same growth conditions.

9. The composition according to claim 1, wherein the microalgae is a single microalgae cell.

10. The composition according to claim 1, wherein the microalgae is an algae cluster comprising two or more microalgae cells.

11. A soil amendment, comprising: the composition according to claim 1; and an exogenous carbon source.

12. The soil amendment according to claim 11, wherein the exogenous carbon source comprises pyrolyzed coal char, coal, coal char, or combinations thereof.

13. A carbon sequestration composition, comprising: the composition according to claim 1.

14. A composition for removing nitrates from soil or water, the composition comprising: the composition according to claim 1.

15. A process for encapsulating algae, the process comprising: introducing, to a fluidic channel of a microfluidic device, a stream of an aqueous-based core fluid comprising microalgae and one or more UV light- photoreactive hydrogel precursors; contacting the stream of aqueous-based core fluid with two streams of an aqueous-based focusing fluid to form a thinner stream of the core fluid comprising the microalgae and the one or more UV light-photoreactive hydrogel precursors, the aqueous-based focusing fluid having a different viscosity than the aqueous-based core fluid; and exposing the thinner stream of the core fluid to UV light to form a polymerized hydrogel thread encapsulating the microalgae.

16. The process according to claim 15, wherein hydrodynamic focusing by the aqueous-based focusing fluid causes tapering of the aqueous-based core fluid to form the thinner stream of the core fluid.

17. The process according to claim 15, wherein the polymerized hydrogel thread has a diameter of less than 1 mm.

18. The process according to claim 15, wherein the microalgae comprises terrestrial algae, aquatic algae, or combinations thereof.

19. A process for growing algal biomass, the process comprising: exposing microalgae encapsulated within a polymerized hydrogel to growth conditions to form a microalgae cluster within the polymerized hydrogel.

20. The process according to claim 19, wherein the microalgae comprises terrestrial algae, aquatic algae, or combinations thereof.

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