Bioreactor systems, compositions, and methods, including moderating biofouling in bioreactors

Abstract

Compositions and / or methods for growing biological material are generally described. In certain embodiments, an emulsion having an aqueous fluid and a non-aqueous fluid is described. The non-aqueous fluid may have greater wettability than the aqueous fluid, which may be advantageous for preventing biofouling, for example. The aqueous fluid may comprise a biological material, and the non-aqueous domain may comprise a compound. The compound may be capable of exiting the non-aqueous fluid and entering the aqueous fluid, where the compound may be at least partially consumed by the biological material. The compound entering the aqueous fluid may benefit the biological material (e.g., increase growth rate).

Description

[0001] BIOREACTOR SYSTEMS, COMPOSITIONS, AND METHODS, INCLUDING MODERATING BIOFOULING IN BIOREACTORS RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 752,211, filed January 31, 2025, and entitled “Bioreactor Systems, Compositions, and Methods, Including Moderating Biofouling In Bioreactors ,” and to U.S. Provisional Patent Application No. 63 / 796,810, filed April 29, 2025, and entitled “Machine-Learning Enabled Droplet-B ioreactor Platform,” which are incorporated herein by reference in their entirety for all purposes.

[0003] TECHNICAL FIELD

[0004] Systems and / or methods for at least partially separating species in a fluid solution or mixture from a container wall or other surface, including growing biological material in an aqueous fluid in ways that moderate biofouling, and / or for transporting species to and / or from an active phase of a multi-phase system are generally described.

[0005] BACKGROUND

[0006] Bioreactors allow organic matter to be cultivated under controlled conditions and at different scales. To maximize the productivity of bioreactors and decrease associated costs, it is generally desired to minimize the maintenance and downtime of bioreactors. Bioreactors capable of cultivating organic matter may suffer from biofouling, which is the accumulation of organic matter within its components. Biofouling may affect the function and efficiency of bioreactors, and typically increases downtime. Thus, improvements are needed.

[0007] SUMMARY

[0008] The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0009] In one aspect of the present disclosure, compositions are provided. In some embodiments, the composition comprises a multi-phase fluid arrangement comprising an active fluid phase and a transport fluid phase, wherein the active phase comprises a reactive and / or growth species, and the transport fluid phase is selected for the ability to transport a reactant to

[0010] #14861445vlthe reactive and / or growth species in the active fluid phase, and / or to remove a product from the reactive and / or growth species in the active fluid phase.

[0011] In some embodiments, an emulsion comprising an aqueous domain and a non-aqueous domain, the aqueous domain comprising an aqueous fluid and the non-aqueous domain comprising a non-aqueous fluid, wherein: the aqueous fluid comprises biological material; the non-aqueous fluid comprises a compound capable of exiting the non-aqueous domain and entering the aqueous fluid of the aqueous domain; the non-aqueous fluid has greater wettability to surfaces of a bioreactor than the aqueous fluid such that at least some of the non-aqueous fluid is positioned between at least some of the aqueous fluid and surfaces of the bioreactor; and the compound is capable of being at least partially consumed by the biological material in the aqueous fluid.

[0012] In another aspect, systems, such as a bioreactor system, are provided. In some embodiments, a container configured to house a bioreaction, having an interior surface; a source of a multi-phase fluid arrangement connectable to the bioreactor, wherein the multi-phase fluid arrangement comprises an active fluid phase in which a bioreaction occurs and which, when in contact with the surface, can cause biofouling of the surface, and a separating fluid phase, wherein separating fluid phase has a greater affinity for the surface than the active fluid phase.

[0013] In some embodiments, a source of an aqueous fluid comprising biological material; a source of a non-aqueous fluid comprising a compound; a vessel configured to receive a first input comprising the aqueous fluid and a second input comprising the non-aqueous fluid, wherein: the vessel is configured to form and / or maintain an emulsion comprising an aqueous domain, the aqueous domain comprising droplets of the aqueous fluid, and a non-aqueous domain comprising the non-aqueous fluid, at least some of the compound is capable of exiting the non-aqueous domain and entering the droplets of the aqueous domain, and the non-aqueous fluid has greater wettability to at least one surface of the vessel than the aqueous fluid.

[0014] In some embodiments, a reaction vessel; an emulsion in the vessel comprising: a discontinuous domain comprising a biological material; continuous non-aqueous domain comprising a nutrient for the biological material, wherein at least some of the nutrient is capable of passing from the non-aqueous domain into the aqueous domain, and the non-aqueous domain has greater wettability to surfaces of the vessel than the aqueous domain.

[0015] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures. In cases where the present

[0016] #14861445vlspecification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale unless otherwise indicated. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. The figures illustrate various embodiments and / or examples that fall within this disclosure, but do not represent the full scope of the disclosure or claims. In the figures:

[0019] FIGS. 1A and IB shows aqueous fluid and non-aqueous fluid in a vessel and an emulsion comprising an aqueous domain and a non-aqueous domain.

[0020] FIG. 2 shows, algae growing in aqueous droplets within oil.

[0021] FIG. 3 shows an optical image of droplets containing algae.

[0022] FIG. 4 shows an example of steps for a harvesting process in a reactor.

[0023] FIG. 5 shows a transparency mask used to make a microfluidic chip.

[0024] FIG. 6 shows steps for one type of emulsification generation.

[0025] FIG. 7 shows a chip submerged in oil and droplets rising to a later at the top of the oil. FIG. 8 shows an optical image of a single layer of droplets.

[0026] FIG. 9 shows a histogram of diameters of droplets.

[0027] FIG. 10 shows a cuvette with emulsions where absorbance is linear throughout the depth of the cuvette and the transmittance decreases according to the Beer Lambert Law.

[0028] FIG. 11 shows a cuvette with emulsions.

[0029] FIG. 12 shows light penetration growth of algae in droplets over 7 days with light only entering from the top.

[0030] FIG. 13 shows optical density of algae over time in bulk growth.

[0031] FIG. 14 shows cell growth in droplets with varying starting optical densities.

[0032] FIG. 15 shows containers of droplets on day 0 seeded with varying starting densities.

[0033] #14861445vlFIG. 16 shows a schematic of a flow path for oil flowing from a syringe pump into the top of a sealed container of algae and being forced out due to volume conservation into a waste container.

[0034] FIG. 17 shows a set-up for flowing oil with different carbon dioxide levels.

[0035] FIG. 18 shows algae growth over a 10 day period with three different levels of carbon dioxide.

[0036] FIGS. 19A-19C shows optical images of droplets with different carbon dioxide levels. FIG. 20 shows a reactor schematic with a main flow loop is shown on the bottom and gas lines to add and remove carbon dioxide above and contacted with the oil flow loop.

[0037] FIG. 21 shows, number of algae cells per droplet grown in a reactor.

[0038] FIGS. 22A-22D shows optical images of droplets and reactors at day 0 and day 6.

[0039] FIGS. 23A-23C shows a flow focusing chip, according to some embodiments.

[0040] FIGS. 24A-24C shows a droplet imaging chip and images of droplets acquired using the imaging chip, according to some embodiments.

[0041] FIG. 25 is a plot depicting the density of algae and total amount of CO2 sequestered in reactor over 8 days, according to some embodiments.

[0042] FIG. 26 is a schematic diagram of a flat-panel bioreactor, according to some embodiments.

[0043] FIG. 27 is a schematic diagram of a flat-panel bioreactor system enabling recycling of buffer materials, according to some embodiments.

[0044] FIG. 28 is a schematic diagram of an illustrative implementation of a computer system that may be used in connection with some embodiments.

[0045] DETAILED DESCRIPTION

[0046] Bioreactors are emerging as an important biomanufacturing tool. Bioreactor applications have expanded beyond traditional biologic pharmaceuticals — such as monoclonal antibodies (mAbs), insulin, and later mRNA vaccines — to encompass a diverse array of industries, including cosmetics, lab-grown meat, and bio-polymers. Among the diverse organisms cultivated in bioreactors, algae stand out due to their resilience and ability to convert CO2 efficiently. Algae provide a desirable solution for cost-effective CO2 utilization, as they have numerous high-value applications, including but not limited to cosmetics, biofuels, dietary supplements, aquaculture feeds, and water treatment.

[0047] #14861445vlPhotobioreactors, specialized for algae cultivation, offer advantages over traditional open pond systems. These include higher productivity, reduced land and resource use, and better contamination control. Some designs enable photobioreactors to exhibit improved volumetric and areal productivity, driven by a favorable surface-area-to-volume ratio and reduced algae settling. Despite these benefits, the adoption of photobioreactors is still limited by significant operational challenges, including high costs associated with labor, power, and nutrients, which often outweigh the gains in productivity.

[0048] Photobioreactor designs — such as tubular, flat panel, stirred tanks, bubble columns, and / or membrane and nature-inspired reactors aim to address these limitations but continue to face significant challenges. Some challenges include biofouling, high energy demands for pumping, and limitations in gas transfer rates. Biofouling, caused by biofilms forming on reactor surfaces, obstructs light penetration, reduces efficiency, and necessitates cleaning cycles that lower productivity and shorten reactor lifespans. Similarly, the high pumping costs needed to prevent algae settling and ensure adequate mixing can account for 30-95% of a photobioreactor’s power consumption. Finally, closed bioreactor systems often face gas transfer limitations, as insufficient CO2 supply and oxygen removal slow algae growth and eventually taper productivity. These challenges make classical open ponds, despite their inefficiencies, the dominant choice for algae cultivation, accounting for 99% of cultivated algae on a per- volume basis.

[0049] Various strategies have been proposed to address the above referenced challenges.

[0050] Biofouling can be mitigated by high-speed pumping or the use of particles to scrub surfaces, though both approaches add operational complexity and cost. Advanced materials and surface coatings have also been explored, but transparent, stable, and biocompatible coatings remain elusive, requiring monthly maintenance cycles . Electro-active surfaces offer a promising alternative, however their scale-up remains challenging for large reactor surfaces. Reducing pumping costs typically involves scaling up reactor dimensions, which compromises the high productivity and low land use that closed systems are designed to achieve. Finally, enhancing gas transfer rates typically involves either increasing the sparging rate or prolonging bubble residence time. However, higher sparging rates can result in excessive mixing, which may damage shear-sensitive cells and significantly increase pumping and gas costs. On the other hand, extending bubble residence time often requires the installation of physical barriers within the reactor to slow bubble ascent. While effective, these barriers increase the reactor's capital costs and are susceptible to fouling over time.

[0051] #14861445vlIn accordance with some embodiments, a droplet based photobioreactor configured to contain algae within aqueous micro-droplets in oil is described. By encapsulating algae within aqueous micro-droplets suspended in oil, the bioreactor, in some embodiments, substantially prevents algae from interacting with reactor surfaces, reducing, or even eliminating, biofouling. In some instances, the droplet configuration also minimizes algae settling, reducing pumping energy requirements by over 55% while desirable growth rates and productivity. Gas transfer may also be obtained through the oil phase, which serves as a permanent interface for CO2 diffusion into the droplets. While algae serves as the model organism throughout the present disclosure, the droplet-based system is equally applicable to other biological cultures, including, but not limited to, mammalian cells, which face similar challenges of fouling, stirring, and gas transfer inefficiencies. The compartmentalized nature of the droplets also reduces contamination risks, making this system particularly suitable for sensitive biological applications.

[0052] This disclosure involves management of chemical, biological, or biochemical species in a way that moderates the effect of those species on other species and / or on a surface proximate the species (e.g., a surface proximate a fluid combination containing the species). In one set of embodiments, the species is a biological species that might otherwise cause fouling of surfaces, e.g., interior surfaces of containers and / or tubing / channels in which the species is contained or otherwise manipulated, including bioreactors in which bioreactions are carried out, and the disclosure involves management of the biological species in a way that reduces or minimizes biofouling.

[0053] The disclosure involves, in one set of embodiments, at least partially separating a fluid within which at least one species is contained, from the surface. This can involve utilizing a multi-phase combination, e.g. a multi-phase fluid combination, where one of the fluids dissolves and / or otherwise carries the species to a greater extent than at least one other fluid of the combination. In this way, the species, e.g., one that would otherwise cause biofouling of the surface, is carried to a greater extent in a first phase of the fluid combination than in a second phase of the combination.

[0054] Multi-phase fluid combinations are known. Some involve at least two fluids where one is essentially immiscible with another, “essentially immiscible,” as used herein, means possessing characteristics that cause the fluids to separate form one another spontaneously to form at least two different phases. These can be fluids such as aqueous / non-aqueous fluid combinations (e.g., water-in-oil or oil-in-water) or other fluids that separate. Aqueous / aqueous multi-phase solutions also are known and can be employed in this manner, even though many molecules of one phase #14861445vl-1-

[0055] may be miscible with molecules of another phase. I.e., in multi-phase combinations, the various phases, and species within them, are not necessarily entirely immiscible with other phases and components within them. Certain amounts or molecules of one phase can be found in the other, and vice versa. But as would be understood by those of ordinary skill, a multi-phase combination (sometimes called a multi-phase solution) is identifiable in that certain species preferentially, or almost entirely self-select to be present in one phase, while others are present in another. Many multi-phase fluid combinations are determinable by instrumentation or even visual inspection.

[0056] Multiple phases of combinations useful in this disclosure can exist in layers, flow components, for example in a laminar flowing system, or can exist in a continuous / discontinuous combination such as an emulsion. As will be understood from this disclosure, just by virtue of arranging a multi-phase system, a species that preferentially resides in one species will be at least partially separated from a surface (e.g., an interior of a container), as compared to an arrangement in which the species is present in a continuous fluid bordered by the surface, unless it is present in a phase of a multi-phase arrangement (e.g., transport fluid phase) that is preferentially in contact with the surface. In this disclosure, one phase of a multi-phase system typically contains a first species which is ideally at least partially separated from a surface, such as a biological species (e.g., algae) that would otherwise promote biofouling of the surface, and another phase can be used to separate some or much of the first species from the surface and optionally to transport nutrients to and / or remove reaction product or waste from the phase containing the first species.

[0057] “Proximate,” as used herein in the context of a species proximate a surface, means the species is arranged with respect to the surface, or vice versa, in a way such that without the multi-phase arrangements disclosed herein, the species could interact with the surface in a manner that would be ideally moderated or eliminated. For example, a biological molecule that would otherwise add to biofouling of a surface of a bioreactor (e.g., vessel of a bioreactor) or related tubing or other equipment (or interiors of wells, channels, etc., or other surfaces) is proximate the surface if, without the multi-phase arrangement described herein, more of the biological molecule(s) would interact with the surface to increase biofouling.

[0058] In much of the disclosure herein, a multi-phase arrangement is described in the context of an aqueous-in-nonaqueous emulsion, or other emulsion, where a discontinuous aqueous phase (e.g., droplets) is combined with a second phase. The second phase may be continuous, may be non-aqueous, and may surround some or all of the droplets of the aqueous phase. But wherever such an arrangement is described herein, it is to be understood that any and all embodiments can #14861445vlextend beyond that specific arrangement into any other multi-phase arrangement facilitating management of fluid-contained species as described in this disclosure and, with the benefit of the disclosure, such arrangements can be readily formulated by those ordinary skill in the art to carry out various purposes described herein.

[0059] “Aqueous” and “nonaqueous” are given their ordinary meaning and, in this disclosure, to include phases that are more water-based (but not necessarily exclusively) and less water-based (but not necessarily exclusively), respectively. Typically, aqueous and nonaqueous phases are essentially immiscible.

[0060] In any of the arrangements described to herein, phases of a multi-phase arrangement can be selected such that a phase that has greater affinity for and / or otherwise is selected to contain more of the species ideally shielded from the surface has a lower affinity for the surface than at least one other phase. That is, at least one other phase, which contains less or essentially none of the species desirably shielded from the surface, has a greater affinity for the surface and thereby adds to separation of the species from the surface. Selection of fluids and surfaces having a greater or lesser affinity for each other is within the level of ordinary skill in the art and can be readily implemented across a broad range of surfaces and fluids based on this disclosure.

[0061] Another set of embodiments will now be described which can be used separately from, or in combination with, the concept described above in which a particular species (e.g., algae in a bioreactor) is inhibited from contact with a surface such as the interior of a reactor. This set of embodiments involves utilizing one phase (a transport phase) of a multi-phase arrangement to transport one or more compounds to, and / or to remove one or more compounds from, another phase (a reaction or growth phase) of the arrangement. One or more compounds transported to the reaction or growth phase can be nutrients or other species to interact with species such as biologies or biomolecules present in the reaction or growth phase, and one or more compounds removed from the reaction or growth phase can be reaction products (e.g., carbon dioxide), waste, excess nutrient, metabolites, or anything else desirably transported away. In this arrangement, the reaction or growth phase can be arranged to have a greater affinity for, or otherwise greater ability to contain the reactive and / or growth species such as a biologic, and the transport phase can be arranged to have the ability to transport compounds to and from the first phase, while optionally maintaining the multi-phase arrangement for reasons disclosed herein, and / or other reasons. The first phase can be an active fluid phase, meaning one in which a chemical or biochemical reaction or growth can occur, such as algae growth. In some cases, the active fluid phase is referred herein as a source of aqueous fluid. By reducing or preventing the #14861445vlactive liquid phase from being in contact with the surface of the bioreactor, biofouling may be reduced or prevented.

[0062] More specific arrangements are now described, which can be implemented in more extensive ways as described above.

[0063] Cultivation of organic matter, typically in a vessel, may allow for the sustainable production of biofuels, bioplastics, feed, and pharmaceuticals. Bioreactors offer a promising solution to culture fluid-contained species such as organic matter at large scales under controlled conditions without exposing organic matter to contaminants. However, the costs associated with operating bioreactors can be relatively high due to labor and maintenance costs. Biofilms and other biofouling products can form on surfaces of the bioreactors. This can limit the amount of light that can enter the bioreactor in some arrangements, thereby impeding the growth of organic matter. Moreover, biofilms may foul sensors or other operation-sensitive surfaces which reduces the productivity of bioreactors, as production is often halted to remove the biofilms from the reactor.

[0064] The present disclosure generally describes systems and methods of growing biological materials in an aqueous fluid in a manner that reduces contact of the biological materials with the walls of vessels, tubing, wells, channels, or essentially any other surface proximate the material(s), e.g. walls, vessels, tubing, wells, channels, etc. with which a medium containing the materials is brought into contact. In one set of embodiments, the biological materials are contained in a discontinuous aqueous fluid that is part of a multi-phase solution, e.g., a discontinuous aqueous phase combined with another fluid with which is it essentially immiscible, e.g. a non aqueous, second phase. The second phase can be continuous. For example, an arrangement can involve biological materials in droplets of an aqueous fluid contained within and essentially immiscible non aqueous fluid such as an oil. The droplets can be at least partially surrounded, or fully surrounded by the non-aqueous fluid (e.g., oil). As noted, the non-aqueous fluid may be essentially immiscible with the aqueous fluid and may form an aqueous-in-nonaqueous emulsion. This combination of materials and fluids can then be processed in any of a number of ways, some of which are described herein. The second phase can be a transport phase selected for the ability to move species (e.g., compounds) to and from the first phase, which can be an active phase, as described elsewhere herein.

[0065] As one phase (such as an oil) separates the aqueous droplets from surfaces of the bioreactor, biofouling of surfaces within the bioreactor is substantially limited or prevented. To facilitate the growth of biological material within aqueous droplets, compounds (e.g., nutrients #14861445vlsuch as carbon dioxide) may be transported through the oil phase of the emulsion such that at least some of the compounds are transported into the aqueous droplets. With limited biofouling of the bioreactor components and abundant access to nutrients for the biological material, the systems and / or methods described herein may facilitate the growth of biological material at advantageous rates, yields, and / or with advantageously low operating cost.

[0066] According to some embodiments, the systems and / or methods disclosed herein comprise a source of an aqueous fluid. The aqueous fluid may be a single fluid, in accordance with some embodiments. For example, the aqueous fluid comprises water, in accordance with certain embodiments. In certain embodiments, the aqueous fluid comprises a biological material. The biological material, according to certain embodiments, may comprise algae, as will be further discussed below. The aqueous fluid may have any of a variety of components dissolved and / or suspended therewith. For example, some examples components within the aqueous fluid include but are not limited to nutrients, ions, fertilizers, or the like. Certain components in the aqueous fluid may benefit the biological material. For example, the presence of nutrients (e.g., nitrogencontaining nutrients) may benefit the biological material (e.g., algae). As another example, the presence of certain gases (e.g., carbon dioxide) dissolved within the non-aqueous fluid may be transported to the aqueous fluid and thereby become advantageous for the biological material.

[0067] In certain embodiments, a source of non-aqueous fluid is included within the system and / or methods disclosed. According to some embodiments, the aqueous liquid and the nonaqueous liquid are not miscible with each other. According to some embodiments, the source of non-aqueous fluid may be an organic solvent, a non-polar liquid, or any fluid that does not form a homogenous mixture or solution with the aqueous fluid. In some embodiments, different components are dissolved and / or suspended within the non-aqueous fluid, as will be described in more detail below. As illustrated in FIG. 1A, system 100 comprises aqueous fluid 101 and nonaqueous fluid 102. In certain cases, as also illustrated in FIG. 1A, aqueous fluid 101 may comprise biological material 105 and non-aqueous fluid 102 may comprise compound 106.

[0068] In some embodiments, the systems and / or methods disclosed comprise a vessel. The vessel (e.g., bioreactor) may be configured to receive a first input and a second input. In certain embodiments, the first input comprises an aqueous fluid. In certain embodiments, the second input comprises a non-aqueous fluid. Referring back to FIG. 1A, system 100 comprises vessel 103 having surface 104. The surface of the vessel can be an internal surface of the vessel, in some embodiments. The vessel is also referred herein as a container having an interior surface configured to house a bioreaction. According to some embodiments, the system and / or methods #14861445vldescribed may prevent or reduce biofouling on the surface of the vessel, as described later herein. The vessel (e.g., vessel of a bioreactor) can have a source of a multi-phase fluid arrangement connected thereto, in such way that one or more phases (e.g, an active fluid phase and a transport fluid phase) can be introduced to the vessel.

[0069] The transport fluid phase may be selected for its ability to transport certain species to the active phase. Certain species may completely or partially dissolve in the transport fluid phase. As it may be appreciated by one skilled in the art, the solubility of the species within the transport fluid phase can be determined through known methods in the art, such as a preliminary solvent screening test to determine solubility, for example. In embodiments in which it may be desirable to transport species (e.g., a metabolite) from the active fluid phase to the transport fluid phase, a partial or complete dissolution of the species can be achieved.

[0070] In some embodiments, the compositions described herein comprise an emulsion. The aqueous fluid and non-aqueous fluid received by the vessel (e.g., a bioreactor) may allow the system to form and / or maintain an emulsion, according to some embodiments. As also described herein, the active fluid phase, transport fluid phase, and or separating phase may be used to form and / or maintain emulsions. The emulsion may be also known in the art as a colloid, a liquid-liquid dispersion, or the like. In some embodiments, the emulsion comprises a water-in-oil emulsion. In certain embodiments, the emulsion within the vessel comprises an aqueous domain and / or a non-aqueous domain. As illustrated in FIG. IB, emulsion 110 may comprise aqueous domain 111 and non-aqueous domain 112. In accordance with certain embodiments and also illustrated in FIG. IB, aqueous domain comprises biological material 105 and non-aqueous domain comprises compound 106.

[0071] In some embodiments, the aqueous domain of the emulsion comprises an aqueous fluid. The aqueous domain may have different components (e.g., an aqueous fluid and biological material) and / or phases (e.g., a liquid and a solid). In certain embodiments, the aqueous domain may have one or more components (e.g., biological material, compound). The aqueous fluid may comprise droplets of the aqueous fluid, as will be explained in more detail below, according to certain embodiments. According to some embodiments, the aqueous domain having droplets of the aqueous fluid may be beneficial for the systems and methods described herein. Without wishing to be bound to any theory, it is believed that the formation of droplets of the aqueous fluid dispersed in the non-aqueous fluid may prevent or reduce the aggregation of components (e.g., biological material) in the vessel.

[0072] #14861445vlIn some embodiments, the aqueous fluid can be in droplets. The aqueous domain comprising droplets of the aqueous fluids may be formed and / or dispersed in a variety of ways. For example, the droplets of the aqueous fluid may be monodisperse droplets (i.e., similar shape and / or size) or poly disperse droplets (i.e., different shape and / or size).

[0073] According to some embodiments, the aqueous domain can be non-continuous. The non-continuous aqueous domain within the vessel can be separated by other components (e.g., nonaqueous domain) and therefore be non-continuous. For example, the aqueous domain of an emulsion comprising droplets of the aqueous fluid may be separated by non-aqueous fluid (e.g., oil). A non-continuous aqueous domain may be beneficial to the systems and / or methods disclosed for different reasons (e.g., allow the migration of nutrients to the aqueous fluid). In certain cases, the aqueous domain may be continuous (e.g., droplets may be aggregated together locally within the vessel and thereby be continuous).

[0074] According to certain embodiments, the non-aqueous domain comprises a non-aqueous fluid. The non-aqueous domain may also comprise other components (e.g., nutrients, gases, liquids, solids, etc.). In some embodiments, the non-aqueous domain is continuous. For example, a nonaqueous domain comprising non-aqueous liquid (e.g., oil) may be continuous (e.g., a continuous phase) within the vessel.

[0075] In some embodiments, the non-aqueous fluid has greater wettability to surfaces of the vessel than the aqueous fluid. A greater wettability of the non-aqueous fluid compared to the aqueous fluid may be beneficial for the systems and / or methods described herein for various reasons. For example, in an emulsion comprising an aqueous domain and a non-aqueous domain, a greater wettability may allow the non-aqueous fluid (e.g., oil) to be in contact with the surface of the vessel, which, in some cases, may limit the aqueous fluid from being in contact with the surface of the vessel. In embodiments where the non-aqueous fluid has greater wettability than the aqueous fluid to surfaces of the vessel, certain components (e.g., biological material) dissolved or dispersed in the aqueous fluid may not accumulate on the surface of the vessel (e.g., bioreactor). A greater wettability of the non-aqueous fluid may allow at least some of the non-aqueous fluid to be positioned between at least some of the aqueous fluid and surfaces of the vessel, in accordance with certain embodiments. The non-aqueous fluid with greater wettability to the surface of the vessel is capable of separating the active phase from the at least one surface of the vessel. The non-aqueous fluid, in some cases, may be referred herein as a separating fluid phase.

[0076] #14861445vlIn certain embodiments in which an emulsion is formed and / or maintained, the discontinuous domain comprises a biological material. In some cases, the emulsion may have a continuous non-aqueous domain comprising a nutrient for the biological material, wherein at least some of the nutrient is capable of passing from the non-aqueous domain into the aqueous domain, and wherein the non-aqueous domain has greater wettability to surfaces of the vessel than the aqueous domain.

[0077] According to certain embodiments, a compound is capable of exiting the non-aqueous domain and entering the aqueous fluid of the aqueous domain. As schematically illustrated in FIG. IB, compound 106 exits non-aqueous domain 112 and enters aqueous domain 111 of emulsion 110. Also illustrated in FIG. IB, relocation 113 may allow compound 106 to be in the presence of biological material 105 in aqueous domain 111. The compound entering the aqueous fluid (e.g., droplets of aqueous fluid) may benefit the system and / or methods described in various ways. For example, some compounds (e.g., nutrients) may exit the non-aqueous domain and enter the aqueous domain (e.g., droplets of the aqueous domain) such that it can consumed by other components (e.g., biological material) within the aqueous domain. It is believed that the ability of at least some compounds from exiting the non-aqueous domain and entering the aqueous domain may benefit the growth rate, among other properties, of the biological material, as described herein. The compound is capable of reacting with another species (e.g., reactive and / or growth species) after transporting from the transport fluid phase to the active fluid phase. In such cases, the compound may be a reactant.

[0078] In some embodiments, the compound is a nutrient capable of being absorbed and / or metabolized by the biological material in the aqueous fluid. The nutrient may be capable of being absorbed and / or metabolized by the biological material (e.g., algae). In some cases, the nutrient may be molecules comprising carbon, nitrogen, phosphorus, silicon, calcium, magnesium, sodium, potassium, sulfur, manganese, copper, zinc, cobalt, molybdenum, among others. The nutrients may comprise vitamins (e.g., folic acid, niacin, panthotenic acid), carothenoids, polyphenols, or the like. Some examples of nutrients include but are not limited to ammonia, nitrate, nitrite, and / or osthophosphate.

[0079] In certain embodiments, the nutrient may comprise a gas, such as carbon dioxide, for example. In some embodiments in which the nutrient comprises carbon dioxide, the biological material (e.g., algae) may metabolize the nutrient (e.g., during photosynthesis). According to some embodiments, carbon dioxide present in the non-aqueous fluid can have a concentration. In some embodiments, the concentration of carbon dioxide present in the non-aqueous fluid is #14861445vlgreater than or equal to 0.04%, greater than or equal to 0.1 %, greater than or equal to 0.5 %, greater than or equal to 1 %, greater than or equal to 2 %, greater than or equal to 4 %, greater than or equal to 6 %, greater than or equal to 8 %, or greater than or equal to 10 %. In some embodiments, the concentration of carbon dioxide present in the non-aqueous fluid is less than or equal to 10 %, less than or equal to 8%, less than or equal to 6%, less than or equal to 4%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.1 %, or less than or equal to 0.04%. Combinations of the carbon dioxide concentration are possible (e.g., greater than or equal to 0.1% and greater than or equal to 8%).

[0080] In some embodiments, the system comprises a contactor capable of contacting gaseous carbon dioxide with the non-aqueous liquid. The non-aqueous fluid may be configured to enter the vessel. According to some embodiments, the non-aqueous fluid entering the vessel can have carbon dioxide concentration. In some embodiments, the concentration of carbon dioxide present in the non-aqueous fluid entering the vessel is greater than or equal to 0.04%, greater than or equal to 0.1 %, greater than or equal to 0.5 %, greater than or equal to 1 %, greater than or equal to 2 %, greater than or equal to 4 %, greater than or equal to 6 %, greater than or equal to 8 %, or greater than or equal to 10 %. In some embodiments, the concentration of carbon dioxide [resemt om the non-aqueous fluid entering the vessel is less than or equal to 10 %, less than or equal to 8%, less than or equal to 6%, less than or equal to 4%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.1 %, or less than or equal to 0.04%.

[0081] Combinations of the carbon dioxide concentration in non-aqueous fluid entering the vessel are possible (e.g., greater than or equal to 0.04% and greater than or equal to 10%).

[0082] In some embodiments, the system comprises a source of the compound. The source of the compound may be fluidly connectable to the vessel. The source of the compound may be introduced into the non-aqueous domain.

[0083] In certain embodiments, the system comprises an outlet configured to remove, from the non-aqueous domain, a reaction product of the biological material. The reaction product can be a product of a bioreaction in the active phase, for example. The reaction product configured to be removed from the non-aqueous domain may be a reaction byproduct, a metabolite, or the like. For example, the biological material (e.g., a plant) may form a metabolite (e.g., oxygen) during photosynthesis, which can be removed. In some embodiments, the outlet (e.g., hose, pipe, tube) connected to the vessel may allow the removal of a reaction product while operating the system.

[0084] According to certain embodiments, the system is capable of removing the non-aqueous fluid from the vessel when the concentration of the compound is below a threshold

[0085] #14861445vlconcentration. For example, it may be advantageous to remove at least some of the non-aqueous fluid (e.g., oil) when the concentration of the compound (e.g., carbon dioxide) is below a threshold concentration (e.g., any value below 0.04%). In some embodiments, the non-aqueous fluid removed from the vessel may be replaced by non-aqueous fluid, where the non-aqueous fluid being added may have a concentration of the compound that may benefit the system. In some embodiments, the removed non-aqueous fluid comprises byproducts generated by the biological material. In some embodiments, the removed non-aqueous fluid (e.g., oil) may be reused in the system.

[0086] In some embodiments, the non-aqueous fluid may move within the vessel at a flow rate. According to some embodiments, the flow rate of the non-aqueous fluid may have a flow rate of less than or equal to 2 ml / min, less than or equal to 1 ml / min, less than or equal to 0.5 ml / min, less than or equal to 0.1 ml / min, less than or equal to 0.05 ml / min, less than or equal to 0.01 ml / min, less than or equal to 0.005 ml / min, less than or equal to 0.001 ml / min, or less than or equal to 0.0001 ml / min. In some embodiments, the flow rate of the non-aqueous fluid may be greater than or equal to 0.0001 ml / min, greater than or equal to 0.001 ml / min, greater than or equal to 0.005 ml / min, greater than or equal to 0.01 ml / min, greater than or equal to 0.05 ml / min, or greater than or equal to 0.1 ml / min. Combination of the recirculated flow rate are possible, for example, less than or equal to 2 ml / min and greater than or equal to 0.0001 ml / min.

[0087] In some embodiments, the vessel may have any of a variety of suitable internal volumes. In some embodiments, the vessel has an interior volume suitable for laboratory and / or bench scale processes. In some embodiments, the vessel has an interior volume suitable for industrial scale processes. In some embodiments, the vessel has an interior volume greater than or equal to 1 cm3, greater than or equal to 10 cm3, greater than or equal to 50 cm3, greater than or equal to 100 cm3, greater than or equal to 500 cm3, greater than or equal to 1000 cm3, greater than or equal to 2000 cm3, greater than or equal to 5000 cm3, greater than or equal to 10000 cm3, greater than or equal to 100000 cm3, or greater.

[0088] In some embodiments, the aqueous fluid comprises a growth medium. The growth medium may comprise polysaccharides (e.g., agar), vitamins (e.g., thianine), buffering agents, water, trace elements (e.g., magnesium, zinc, copper), among other components of a growth medium known in the art.

[0089] According to certain embodiments, the non-aqueous fluid comprises oil. Some nonlimiting examples of oil include biocompatible oils, vegetable oils, mineral oils, silicone oils, seed oils, fruit oils, animal fats, waxes, triglycerides, or the like. For example, in some

[0090] #14861445vlembodiments, the oil comprises 2-(trifluoromethyl)-3-ethoxydodecafluorohexane, also named HFE-75003M.

[0091] In some embodiments, the droplets comprise at least one dimension of a maximum size. Some droplets (e.g., droplets of aqueous fluid) may have a particular dimension (e.g., diameter) of a certain size. In some embodiments, the maximum dimension of the droplets is greater than or equal to 400 microns, greater than or equal to 350 microns, greater than or equal to 300 microns, greater than or equal to 250 microns, greater than or equal to 200 microns, greater than or equal to 150 microns, greater than or equal to 100 microns, or greater than or equal to 50 microns. In some embodiments, the maximum dimension of the droplets is less than or equal to 50 microns, less than or equal to 100 microns, less than or equal to 150 microns, less than or equal to 200 microns, less than or equal to 250 microns, less than or equal to 300 microns, less than or equal to 350 microns, or less than or equal to 400 microns. Combination of the dimensions are possible (e.g., less than or equal to 400 microns and greater than or equal to 50 microns).

[0092] The droplets of the aqueous fluid in the non-aqueous fluid may have a packing fraction, according to certain embodiments. In some embodiments, the droplets of the aqueous fluid in the non-aqueous fluid can have a packing fraction that is greater than or equal to 0.95, greater than or equal to 0.9, greater than or equal to 0.8, greater than or equal to 0.7, greater than or equal to 0.6, greater than or equal to 0.5, or greater than or equal to 0.4. In some embodiments, the droplets of the aqueous fluid in the non-aqueous fluid can have a packing fraction that is less than or equal to 0.4, less than or equal to 0.5, less than or equal to 0.6, less than or equal to 0.7, less than or equal to 0.8, less than or equal to 0.9, or less than or equal to 0.95. Combination are possible (e.g., less than or equal to 0.7 and greater than or equal to 0.5).

[0093] In some embodiments, the system has a growth rate of growing biological material. According to some embodiments, the growth rate of the biological material is greater than or equal to 0.13 g / L / day, greater than or equal to 0.1 g / L / day, greater than or equal to 0.5 g / L / day, greater than or equal to 0.01 g / L / day, greater than or equal to 0.001 g / L / day, or greater than or equal to 0.0001 g / L / day.

[0094] In some embodiments, the biological material may be at least one type of biological material. In some embodiments, the biological material comprises algae. The type of algae may include but are not limited to Chlorella Vulgaris, Chlorophyta, Rhodophyta, Phaeophyta, Chrysophyta, Xanthophyte, Pyrrophyta, and / or Euglenophta. In some cases, the biological

[0095] #14861445vlmaterial may not need to be an algae but may be microorganisms, animal cells, plant cells, stems cells, and or a viruses.

[0096] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0097] EXAMPLE 1

[0098] This example describes a droplet-based reactor and methods of using the same. The inoculum or algae used to seed the reactor were turned into droplets in oil with a membrane or similar droplet generating technique. The algae and oil were then cultivated within a reactor (FIG. 2), where the algae are not able to foul the surface of the reactor because the oil preferentially wets the surfaces rather than the water within the droplets. Once the droplets have remained in the reactor for a set period, the algae were separated from the oil (FIG. 3). This can be done in an emulsion separation process, such as a gravitational method, a mechanical method, electrocoalescence, or more. Once the algae and oil are separated, the algae was dewatered and dried just as is currently done with bioreactors. An example of an algae harvesting procedure is shown in FIG. 4. The droplet-based photobioreactor described herein involves droplet generation and separation. The oil can then be reused to generate more droplets.

[0099] Instead of bulk algae growth, the system may be designed for droplets. This includes generating emulsions that are stable and investigating the growth of algae within the droplets. Significant parameters such as light, addition of nutrients, mass transfer, temperature, light intensity, pH, and more are commonly known to impact growth and may change with the switch from bulk growth to droplets. Initial optimizations and growth in droplets on a larger scale are shown.

[0100] Historically, algae have been grown in open ponds, recently cited to be around 99%. However, there are benefits of switching to a closed photobioreactor, including preventing culture contamination, better control of the growth conditions, and higher productivity. Both higher volumetric productivity and higher areal productivity can be obtained, largely linked to higher surface area to volume of the reactor and limited settling within the reactor compared to open ponds.

[0101] In this example, chlorella vulgaris was used. It is a common freshwater microalga that can be used for a variety of low, medium, and high value products. The oil used was HFE 7500 with 2% weight of 008-FluorSurfactant, purchased from RAN Biotechnologies, as it is a biocompatible oil with surfactant added. The medium was Modified Bold 3N Medium

[0102] #14861445vlpurchased premixed from UTEX. All experiments growing algae were done in a Percival I30BLL incubator with a 12 hour on / 12 hour off light cycle at 100 pmol / m2 / sec irradiation to maintain growth conditions. Cells were counted by compressing the droplets in microchannels and processing the image in MATLAB. Cell counts were confirmed to reach an average value by 30 droplets, and 40 droplets were counted in all cases. To inform the reactor design, initial experiments were conducted to show droplet generation and optimize certain parameters, including packing fraction, light penetration, starting concentration, and CO2 addition.

[0103] In order to create droplets at scale, a technique different from microfluidics may be used. One possibility is step emulsification (FIG. 6), which allows for parallelizing the outlet channels. Besides a dependency on the flow rates for the shear force, the size of the droplets in this case may depend on the geometry of the outlet. This allows for any number of channels and can be scaled to larger volumes. Emulsion generation will likely be scaled further using either a mesh or a membrane with holes in the specified length scales to generate droplets on the order of 100 pm. For example, droplets on this length scale can be generated by impinging droplets on a mesh, resulting in the droplets breaking up into smaller droplets on the length scale of the mesh. For the purpose of the bench scale reactor, a step emulsification device was made using soft lithography.

[0104] A transparency mask (FIG. 5) may be used to make a microfluidic chip. The one inlet pointed to, at the middle right of the chip, is for the aqueous medium and algae. The outlets are along the top and bottom of the mask. The chip is submerged in oil and the droplets rise vertically to a layer at the top of the oil FIG. 7. Single layer of droplets under 4x magnification are shown in FIG. 8. A histogram of the diameters of hundreds of droplets measured with a MATLAB image analysis script is shown in FIG. 9.

[0105] The packing fraction of the emulsion is important because the final overall algae density decreases as the volume of oil in the reactor increases. Because the droplets are liquid and relatively large emulsions, they deform and do not remain as spheres when there are many layers of droplets stacked on top of each other, which increases the packing fraction. Measuring the packing fraction is simple due to the density difference between oil and water. By measuring the volume and mass of total emulsion, the only two unknowns are the volume of water and the volume of oil, and thus the packing fraction, ff, can be determined:

[0106] total oil 3” Vwater (1)

[0107] ^total Poil^oil 3” Pou water (2)

[0108] #14861445vlf ^water total (3)

[0109] The packing fraction can vary when there is flow of oil and droplets. However, it remains near 0.9, which is larger than random sphere packing, ~0.6. Additionally, by creating larger droplets with more deformation or a variation of droplet sizes, this fraction could potentially be increased more.

[0110] Another major impact on algae cultivation is light penetration. With open pond reactors, light can only enter from the surface of the water and also gets absorbed by the water through the relatively large depth. Bioreactors allow for a larger surface area to volume ratio than an open pond and thus more access to light. However, adding interfaces between oil and water impacts the way the light interacts due to the different refractive indices. Thus, when droplets are introduced into the system, light penetration and algae growth may be impacted.

[0111] Adding oil interfaces between the water droplets impacts the way the light interacts due to the different refractive indices. Thus, when droplets are introduced into the system, light penetration and algae growth will be impacted. To understand the extent of this impact, the absorption of the emulsion was measured using a ThermoFisher NanoDrop One UV-Vis. The optical density, OD, or absorbance, across multiple wavelengths of a 1 cm x 1 cm cuvette was -3. The OD is linear across a homogeneous solution, starting at 0 and increasing to the measured value. Using the Beer Lambert Law:

[0112] OD = -log10T (4)

[0113] the transmittance, T, was determined. By a depth of 1 cm, the light transmitted is significantly lower (FIG. 10). Visually, it is very clear that light is not transmitted well through the emulsion, as seen in FIG. 11. Thus, creating a reactor at a larger length scale is important to examine growth rates and the feasibility of scaling this type of reactor. Adding a flow or mixing into the system would also help in allowing all algae to access the same amount of light. Note that this may be dependent on the droplet size distribution.

[0114] The light penetration was also explored by growing algae in a tube where light was allowed to enter from the top. The tube was 1 cm diameter, and the emulsions were filled to a depth of 4 cm (FIG. 12). The droplets were introduced into the tube with ambient conditions and a starting bulk optical density of 1, as described in the starting conditions experiments. The sides and bottoms of the tube were covered with tape to prevent light from entering the tube elsewhere. The algae were left for a week to grow, and results show a clear gradient where the #14861445vlgreenest droplets are at the top. While there is a gradient, the algae still grew throughout the whole depth of the tube, seen in FIG. 12, showing the possibility of scaling the reactor to the order of centimeters.

[0115] Additionally, to inform optimal initial conditions and understand the growth over time, experiments for starting algae density were executed. The optical density was measured for the bulk algae system with a NanoDrop One UV-Vis. First, algae were grown in bulk in an Erlenmeyer flask on a mixing plate, and the OD was measured every two days, seen in FIG. 13, using a wavelength of 750nm. These OD values can also be converted to cell density through standard cell counting techniques.

[0116] The next experiment varied starting densities, measured by the OD prior to generating the droplets. These values were determined based on the bulk growth case and chosen as 0.4, 0.8, 1.2, 1.6, and 2.0. First, five levels of algae with the correct OD were obtained, and then droplets were generated using these levels of algae. The five containers of algae in droplets are shown in FIG. 15. The number of cells per droplet was then measured on both day zero and day two.

[0117] The increase in cell number between the days can be seen in FIG. 14. Based on these results, a starting OD of 1 was chosen for the final reactor experiments to capture the exponential growth. Moving forward, this value could change significantly; for example, a high density may impact light penetration, or an optimal level may be impacted once CO2 levels are optimized.

[0118] A function of bioreactors is enhancing mass transport to the cells, which can be shown with insufficient solubility versus CO2 usage and diffusion length scales that are too small. This is one reason, for example, there is mixing with bubble columns or other flow induced mixing to increase growth. Common methods such as vigorous mixing and bubbling gas, however, may not work due to the stability of the emulsions and maintaining the algae within the droplets. It was shown that dissolving CO2 into the water droplets prior to the cultivation is insufficient to support them through their full growth cycle. Hence, CO2 may consistently be replenished in the droplets for the duration of the experiments. Furthermore, it is shown that a mass transfer limitation exists in CO2 diffusing from the oil into the droplets, leading to a mixing requirement to prevent a large gradient in CO2 concentration in the droplets and subsequently a large gradient in growth performance. To solve both problems, the system shown in FIG. 16, namely dripping oil saturated with CO2 at the top of the reactor, allowing it to flow to the bottom due to its higher density. Spent oil is then removed from the bottom. Removal of excess oil also acts as a

[0119] #14861445vlmechanism to remove O2 from the reactor. Small scale experiments were conducted to prove the viability of this idea (FIG. 17). By flowing oil with varying levels of CO2, an optimum level at the predicted 5% CO2 can be seen. At day zero, all the droplets start with the same density of cells. Over a period of 10 days, it is clear the 5% case increases in density much quicker than both the droplets with 10% and atmospheric CO2 levels. The droplets with the 10% CO2 level are still growing, but the level is likely too high, leading to an acidic environment. The droplets with atmospheric CO2 levels likely are not able to get enough CO2 to grow quickly. This can be seen in FIG. 18 and 19A-19C, where there is both quantitatively and qualitatively the most algae in the 5% case.

[0120] As algae are sensitive to the pH of the environment, a desirable level of CO2 dissolved in the oil is investigated. To achieve different levels of CO2, varying percentages of CO2 saturated oil and unsaturated oil were pulled directly into a syringe. The oil is saturated with CO2 by bubbling 99.9% CO2 for five minutes. Three levels of CO2 were tested: 0.04% (atmospheric), 5%, and 10%. Using syringe pumps, oil was flown at 2 pl / min through approximately 130 pl of droplets, or 0.015 ploii / min / pldK>Piet, which was chosen based on predicted CO2 consumption to maintain an infinite source of CO2. The experiment was run for 10 days, periodically counting the number of cells per droplet.

[0121] To convert the number of cells per droplet to cell density, the volume was determined with the average droplet diameter, 175 pm. This gave an average volume of 2.8xl0-6ml / droplet. The highest growth rate occured for 5% CO2 between day 4 and day 7, with an average increase of 184 cells / droplet each day. Using the average cell mass, this growth can be converted to 1.47 g / L / day in the droplets. Assuming a 0.9 packing fraction, a 1.32 g / L / day overall growth rate with minimal optimization overall was obtained.

[0122] Lab-scale droplet-based algae cultivation in oil flow-through reactor

[0123] Numerous factors have been investigated to inform the design of a reactor. For the oil and surfactant concentration utilized, droplets around 175 pm diameter were stable for months and grow algae well. The light path can be on a scale of centimeters. Finally, the CO2 level should be near 5%, and CO2can be added through oil to achieve this. In view of this, algae growth within a flow-through concept reactor was demonstrated. To scale the process from microliter to milliliter volumes, several modifications may be made . First, a scalable technique for droplet generation was implemented. Second, improved CO2 dissolution was implemented and precise mass control

[0124] #14861445vlusing a membrane contactor and a system of mass flow controllers (MFC) was established. Finally, these components into a continuous flow-loop, enabling the sustained circulation of algae-containing droplets.

[0125] Thus, a reactor was created on a larger scale, where the goal of this reactor was to show the successful scaling of cultivating algae within droplets (FIG. 20). One of the major factors determined by previous experiments was to maintain CO2 levels higher than atmospheric levels and lower than 10% by flowing oil through the reactor. To do this, there was a main flow loop for the oil as well as gas lines to add CO2 to the oil in this reactor.

[0126] Growth was done over an 8 day period (see FIG. 21 and FIGS. 22A-22B). From day 0 to day 8, there was clearly an increase in cell density when imaging the droplets within a chip. Between day 2 and day 4, the growth was 180 cells / droplet / day. This gives a density growth rate of 1.61 g / L / day in the droplets and 1.45 g / L / day overall within the reactor assuming a 0.9 packing fraction (FIG. 25). To add CO2 to the oil, 1 ml / minute of 12% CO2 and 88% air was flown through the membrane contactor. The oil was pumped by the peristaltic pump at -0.015 ploii / min / pldropiet. Faster oil flow rates over an order of magnitude higher were found to lead to cell death. The CO2 levels in the system were also measured in this reactor and have significant room for optimization to prevent any transients over the first days and periodic changes in the levels.

[0127] The number of cells in the droplets can be converted to cell density using the average volume. These droplets were created using the step emulsification device, meaning the average volume was 2.5xlO-6ml / droplet. Between day 2 and day 4, the growth was 180 cells / droplet each day, which is the highest growth rate. Based on the average cell mass, this gives a density growth rate of 1.61 g / L / day in the droplets and 1.45 g / L / day overall within the reactor assuming a 0.9 packing fraction.

[0128] The algae in the droplets can also be seen qualitatively. From day 0 to day 6, there is clearly an increase in cell density when imaging the droplets within a chip, seen in FIG. 22A and 22C for day 0 and FIG. 22B and 22D for day 6. Additionally, in these cases, the droplets consistently have algae growing, rather than some droplets with no algae. Visually, the reactor is much greener with algae on day 6 than day 0.

[0129] This example addresses challenges in algae photobioreactor design, such as biofouling, high pumping costs, and limited gas transfer rates, through the development of a droplet-based photobioreactor system. By encapsulating algae within aqueous micro-droplets suspended in oil, the system mitigates biofouling and minimizes the energy expenditure associated with pumping, #14861445vlachieving reductions in operational costs. Furthermore, enhanced gas exchange via CO2 diffusion through the oil phase and a large and stable water-oil interface ensures sustained growth rates comparable to traditional systems while avoiding the limitations of classical gas sparging methods.

[0130] As demonstrated, droplet-based cultivation offers not only technical feasibility but also economic advantages, paving the way for its scalability and broader adoption in industrial settings. The lab-scale experiments validated the system's ability to maintain high productivity levels with optimized light penetration, CO2 concentration, and droplet stability. Moreover, the modular nature of the droplet-based reactor suggests potential applications beyond microalgae, extending to other sensitive cell cultures where contamination and biofouling are significant concerns.

[0131] Methods

[0132] Flow Focusing Chip

[0133] The flow focusing technique can be seen in FIG. 23A, where the aqueous medium with algae is input in the middle and the oil is input from the outsides, pinching off droplets of algae contained in medium. Other methods such as coflowing or a T-junction could similarly be used. For this device, the size of the droplets depends on the flow rates of both the oil and the water. The microfluidic chip in this case was manufactured using soft lithography in a clean room. First, to make a mold, a 4-inch diameter silicon wafer was plasma cleaned for 5 minutes. Then a 100 pm thick layer of MicroChem SU8 2100 was added using a spin coater at 3000 rpm. The wafer was then prebaked, at 65°C for 5 minutes and 95°C for 25 minutes. Using a mask aligner, the wafer was then exposed with a transparency (FIG. 23B) mask and a hard contact setting. The white areas are the channels. The oil is input from the left most circle. The algae and medium are input from the middle circle. The chip outlet is the right circle. The mask was designed in CAD, as seen in FIG. 23B, and made by Artnet Pro Inc. The wafer was then post baked at the same temperatures for 5 minutes and 11 minutes, respectively. Finally, the wafer was developed on the spin coater using Propylene glycol monomethyl ether acetate (PGMEA) then cleaned with 100% isopropyl alcohol (IPA), leaving only the mold for the channels on the wafer. Once the mold was dried, it was silanized with Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane, purchased from Sigma Aldrich, by adding 5pl on to a glass slide in the same desiccator as the mold and pulling vacuum overnight. This hydrophobized the surface and helped prevent the PDMS from adhering in the next step.

[0134] #14861445vlPolydimethylsiloxane (PDMS), Sylgard 184 purchased from Dow, was prepared with 10 \N / \NC / C elastomer and curing agent and poured over the wafer. Once any air bubbles were removed by pulling vacuum, the mold with PDMS was baked near 60 °C until cured. It was then peeled off the mold and excess PDMS was trimmed off. The holes for the oil inlet, water inlet, and emulsion outlet were created using a blunt tip 17-gauge needle. Finally, the PDMS and a glass slide were plasma cleaned with O2 for one minute then pressed together to bond the PDMS to the slide and close the microchannels.

[0135] By using a constant pressure pump, 5.0xl03Pa and 6.0xl03Pa were applied to the algae and oil respectively to flow both through the chip, creating approximately 175pm diameter droplets at ~lml / hr. An illustration of the set-up can be found in FIG. 23C. There is significant room to optimize this size, but larger droplets -400 pm in diameter generated with a previous chip were found to be unstable.

[0136] Step Emulsification Chip

[0137] A very similar process for mold manufacturing using Microchem SU8 was used; only changes were made to baking temperature and spinning speed according to the SU8 user manual for a thinner layer of SU8. There are 80 total outlet channels on this chip with a final oulet height, h, of 42 pm, which depended on the thickness of the SU8, and a final outlet width of 300 pm, which was set based on the transparency mask, as seen in FIG. 5. For this chip, only one hole was punched, as pointed to for the inlet, which is for the aqueous medium and algae.

[0138] Once the mold was made, the PDMS was poured similarly, making sure to remove any bubbles at the outlet channels. When cutting the chip out of the circular piece of PDMS, careful attention was paid to the channels to make sure the chip was left with clean rectangular outlets. After plasma cleaning and adhering the PDMS to the glass slide, the chip was left to sit overnight to become hydrophobic. This was necessary because plasma cleaning the PDMS caused it to become temporarily hydrophilic, however the exact recovery time of hydrophobicity was not investigated.

[0139]

[0140] Quantifying the number of algae in each droplet is necessary to determine the growth rates and compare the results to existing literature and industry levels. There are a variety of techniques to count cells, including but not limited to an automated cell counter and manually counting with a hemocytometer. The challenge with quantifying the growth for this case is that

[0141] #14861445vlthe algae are trapped within the aqueous droplets. This is a 3D shape where the algae continuously move with any flow in the droplet. An image of a droplet containing algae is shown in FIG. 24A. Thus, a two-step process was developed to obtain an accurate cell count: compressing the droplets to image and using image processing to count.

[0142] First, the droplet is compressed so all the algae are spread out into a layer and within the focal plane of a microscope (Zeiss 1X73, 10X objective). To do this, microfluidic chips were created with varying channel heights to maximize compression of the droplets while still preventing coalescence. Once the droplets were added to the chip, the algae eventually were found to settle to the bottom of the compressed droplet. An image of a compressed droplet with algae is shown in FIG. 24B. A channel height of 50 pm ultimately allowed for optimal imaging.

[0143] The chips, shown in FIG. 24C, were manufactured by laser cutting acrylic top and bottom pieces as well as microfluidic tape of set thicknesses obtained from Nitto Denko Corporation. The top acrylic piece had two holes: one for adding the droplets and a second so the air displaced can flow out of the chip. They were then assembled with the holes on the top acrylic piece aligning with the ends of the channel created by the microfluidic tape. Droplets were introduced using a pipette and flowed into the microchannel due to capillary forces. This was done carefully at a constant rate because introducing the droplets too quickly led to more droplet break up as well as coalescence.

[0144] Lab-Scale Reactor

[0145] The oil in the flow loop is shown with a solid line in FIG. 20 and flowed counterclockwise. It was pumped with a Kamoer KXP-100 peristaltic pump. To obtain a similar flow rate to the small-scale CO2 experiments, the oil was pumped at 0.14 ml / min, or 0.6 rpm; the pump was calibrated over an hour. The previous small-scale CO2 experiments used a -30% lower normalized flow rate, but the resolution of the pump could not obtain this exact flow rate.

[0146] From the pump, the oil first flowed through a membrane contactor, 3M™ Liqui-Cel™ SP 0.5X1 Series Membrane Contactor G680, Polyolefin UP I purchased from Quantum Flow Technologies. This allowed for enhanced mass transport between the oil and the gas by using capillary membranes to increase the contact surface area. Furthermore, it minimizes the loss of undissolved CO2 and limits evaporative losses of the oil, advantages over the alternative of sparging CO2 through the oil. The oil was then dripped into the reactor at the top of a small headspace. Within the reactor, there was approximately 7ml of droplets in a ~lcm diameter clear

[0147] #14861445vlglass tube (FIGS. 22C-22D). This was chosen to show the algae can be grown within droplets in a reactor on the centimeter length scale.

[0148] Next, the oil flowed out the bottom of the reactor to a CO2 sensor. At this location, a 20% gas phase sensor from CChMeter, ExplorIR®-W-X 20% CO2 Sensor CM-0123, was used to measure the level of CO2 in the oil. To obtain this, the sensor opening was attached to a small headspace of gas above the liquid. This was done by 3D printing a sensor holder with a Formlabs Form 2 printer, which had an oil inlet and outlet as well as an opening to epoxy the sensor. The oil then finally flowed back to the peristaltic pump. Luer fittings were used throughout the loop to connect the tubes.

[0149] To add CO2 to the oil, the gas lines shown in FIG. 20 were used. The gas was first obtained from a CO2 cylinder of 99.9% food grade CO2 and a pressurized air-line. Masterflex® Mass Flowmeter Controllers, MFLX32907-53 and MFLX32907-57, were used to mix the CO2 and air at the correct concentrations. These have flow rate ranges of 0.50 to 50.0 ml / min and 0.05 to 5.00 ml / min, respectively. A total of 1 ml / minute composed of 12% CO2 and 88% air was used.

[0150] After mixing the gas by combining the CO2 and air, it was bubbled into a humidifying container with water and HFE 7500 to saturate the gas and prevent stripping of oil or water from the reactor and oil flow loop. The saturated gas was then contacted with the oil in the main flow loop using the membrane contactor. After contacting the gas and oil, the gas flowed to a 10% gas phase CO2 sensor from CO2Meter, 030-7-0007 K30 10% CO2 Sensor CM-0049, so the levels of CO2 in the gas both before and after contacting the oil were known. After the sensor, the gas was no longer needed, but because the flow rate was low (Iml / min), the gas flowed out of the sensor into a beaker with water to limit back diffusion to the sensor. The gas bubbled through the water and was allowed to exit the system.

[0151] When setting up the experiment, all tubes and surfaces were cleaned with 70% ethanol prior to any addition of oil or algae to the reactor. Both CO2 sensors were calibrated with N2 at their given flow rates and checked with a known 7.4% CO2 prior to use. The light ran in cycles of 12 hours on and 12 hours off at an irradiance level of 100 pmol / m2 / sec in a Percival I30BLL incubator.

[0152] EXAMPLE 2

[0153] #14861445vlThis example describes a reactor for introducing droplets into cultivation. This reactor can be compared to a helical tubular reactor, which is one of the photobioreactors in an extensive technoeconomic analysis done by the National Renewable Energy Laboratory (NREL). This includes both the productivity and the power requirements.

[0154] Compared to helical tubular reactor, the productivity of this reactor is similar: 1.45 g / L / day versus NREL’s 1.48 g / L / day. Open ponds are 0.13 g / L / day . With optimization, the productivity of this reactor can easily be increased. Regardless, because the oil preferentially wets the surface, there is no biofouling in the reactor. Wetting of oil over water can be obtained in both glass and plastic. The reactor can easily be emptied and left completely clean, opening the possibilities for different reactor geometries. This means that the reactor can recover any downtime necessary for cleaning, which can significantly increase the overall volumetric productivity as well as save in any cleaning costs including labor, chemicals, cleaning systems, and more.

[0155] Additionally, the total power requirement for the NREL reactor is 1.664 kWh / m2 / day, with pumping making up 0.4881 kWh / m2 / day not including aeration. These assume high flow rates to mix the system, which prevents settling and fouling of the reactor walls. Three systems will be analyzed to compare the power requirements of the droplet photobioreactor: pumping, emulsion generation, and emulsion separation.

[0156] Emulsion generation will likely be scaled using either a mesh or a membrane with holes in the specified length scales to generate droplets on the order of 100 pm. The minimum amount of energy required depends on the interfacial energy between the oil and the water. Assuming an interfacial energy of 20 mN / m, filling the reactor each day would require around 6x1 O’6kWh / m2 / day. However, the energy to generate emulsions will likely be higher due to the pressure drop to flow the liquid through channels and the resulting velocity of the droplets.

[0157] While the energy will be higher, it will still be substantially below the energy required for an atomizing nozzle. For example, liquid pressurized nozzles from Exair use a pressure around 17 bar. The power required is then pressure multiplied by the flow rate, and to fill the volume of the entire photobioreactor each day with this method, it would take 0.014 kWh / m2 / day, which is already much lower than the pumping energy.

[0158] Next, for pumping the system, pumping cost is based on the power and the pump efficiency. The pressure drop can be estimated for low Reynolds number flows by the Hagen-Poiseuille equation:

[0159] #14861445vl

[0160]

[0161] where p is the viscosity, L is the length of the tube, Q is the flow rate, and D is the diameter of the tube. Assuming a worst-case scenario, where oil is only added through one end of a photobioreactor, oil replacement matches the droplet-based bioreactor experimental rates, and the tube diameter is 4 cm, the total pumping energy would be 0.04 kWh / m2 / day. However, oil flow rate likely may need to be lower to limit mixing. More inlets and outlets for oil can be added throughout the reactor rather than at one end to achieve the desired decreased flow rate.

[0162] Finally for emulsion separation, it depends highly on the stability of the emulsion. This can be done with gravitational methods, mechanical methods, electrocoalescence, or more. For example, applying a high voltage to achieve corona discharge, yet using negligible current is sufficient to separate the emulsions. Overall, this power requirement is negligible.

[0163] Experimentally, this power requirement can be calculated with the 6 kV voltage used and an assumed 0.005 mA current due to a reading of 0.00 mA on an output with a resolution of 0.01 mA. On this small scale, the emulsion separation took approximately 10 min / ml. Scaling this to the volume of the reactor, if all the emulsions are separated in a day, it would take ~ 0.14 kWh / m2 / day. This is slightly larger than other energy requirements, but it is likely there is a more efficient method for separating the emulsion and much optimization to be done in the area.

[0164] Putting all the values together and assuming a 70% efficient pump for both the droplet generation and oil pumping, the maximum energy requirement would be -0.22 kWh / m2 / day, which is still lower than the energy requirements for pumping in the helical tubular reactor. Hence, there is likely a possibility for savings with power as well as enhanced productivity. This reactor has the ability to prevent all fouling, where there has yet to be a surface treatment that can do this.

[0165] The droplet based bioreactor shows great promise in preventing biofouling and creating a more economical reactor. The reactor can both increase productivity through preventing any need for down time and possibly save money through power savings and lower labor requirements. This work is not only limited to chlorella vulgaris, but any bioreactor with microorganisms that may foul. Due to the high capital costs of a bioreactor in general, it may be more feasible to apply this to medium and high value products, however this would require a more intensive analysis for a specific product. Examples of the products include pharmaceuticals, feed, food, cosmetics and more.

[0166] #14861445vlWhile algae have been the example used here, a known need for bioreactors is providing oxygen for any aerobic cells, and there has also been a range in reactors to optimize the mass transport. The droplet bioreactor has been shown to use oil replacement as a mechanism to enable mass transport; this mechanism could also be applied to other bioreactors to enhance oxygen transport. The flow rates could remain low here as well for these reactors, possibly reducing agitation in the reactor. Thus, there is potential to expand this technology as a broad cell culturing technique.

[0167] MACHINE LEARNING IMPLEMENTATION

[0168] An intelligent droplet-based flat panel bioreactor is described herein that eliminates and / or mitigates biofouling and separation problems. Briefly, the algae will be encapsulated in micro to millimetric sized droplets. The droplet packing is designed to reach high packing within a medium of algae oil itself. This has two-fold advantage in preventing biofouling as algae are confined to the droplets and don’t contact the wall and the extraction / separation processes are significantly simplified. When the algae in the droplets are ready to harvest, ultrasonication of the oil-droplet solution causes the algae to burst, releasing the oils into the droplets. The released oil will then quickly coalesce with the surrounding oil medium. Thus, the separation step may be avoided. Since the algae droplets are surrounded by oil, the algae do not contact the wall of the bioreactor, eliminating the wall fouling problem as well. In some embodiments, machinelearning is applied to improve droplet distribution and growth conditions to provide in- situ control and maximize extraction efficiency, as well as minimize labor operating cost.

[0169] Currently, 99% of algae production occurs in open pond reactors. Due to hydrodynamic constraints, open pond reactors have a lower water thickness limit of 0.2 m, limiting their volume of medium to area exposed to light ratio. Decreasing water thickness allows more light to penetrate the medium, increasing growth rate and algae concentration. As such, thin, flat panel photobioreactors have been proposed as an alternative to thick pool, open pond reactors. The biomass density in flat panel reactors has been found to be 7x the biomass density in open pond reactors in practice (0.3 g / L to 2.3 g / L).

[0170] Presently, one advantage of open pond reactors is that they are relatively easy to clean compared to flat panel reactors. Open pond reactor walls can be continuously brushed during operation and cultivation of the algae. Flat panel reactor geometry is not conducive to continuous cleaning. During the cultivation and growth of algae, algae tend to attach to bioreactor walls, decreasing the amount of light reaching the algae in the bioreactor over time. In #14861445vlfact, for two weeks of uptime, a one- week downtime is required to drain and clean the walls of a typical closed bioreactor. Although closed bioreactors have been found to produce algae more efficiently than open ponds while operational, the downtime and cleaning often make the open pond reactor more profitable in the long run. Thus, eliminating biofouling of algae on reactor walls is a current technical challenge and is a significant bottleneck.

[0171] After the algae has grown, the desired algal oil is separated from the algae and culture medium for processing. Since the algae concentration in open pond reactors is relatively low, large quantities of water are processed to attain the same amount of useful oil compared to a flat panel reactor. Typically, algae are separated from the culture medium using centrifugation, which is energy intensive and costly. Separation accounts for 20% of cost in open pond systems, due to lower algae concentration, while for only 10% in flat panel systems. Furthermore, growth parameters tend to be based on global sensor measurements leading to suboptimal performance.

[0172] Herein, we describe a Droplet-Photobioreactor (Droplet-PBR), as shown in FIGs. 26 and 27, that will eliminate and / or mitigate the need to clean the photobioreactor walls and separate the algae using costly and energy intensive centrifugation techniques, while taking advantage of the benefits and advances of flat panel reactors.

[0173] In some embodiments, the bioreactors described herein include three categories of technical improvements: (1) the generation of algae droplets in lab-scale droplet-reactor and optimizing their growth characteristics, (2) the design of an integrated flat panel system to flow and manipulate algae droplets, and (3) a machine-learning framework to optimize microalgae growth through droplet manipulation and system inputs. In this manner, the PBR systems described herein enable higher area to volume ratio algae panels combined with increased automation of algal oil extraction, which provides the most prudent path to improving the commercial viability for microalgae PBR systems.

[0174] In some embodiments, algae containing droplets 2612 are created and suspended in an oil phase 2614 within a bioreactor 2610, as can be seen in FIG. 26. Every droplet 2612 acts as a contained bio-environment which can be engineered for optimal algal growth. The oil-phase 2614 may also be engineered. Depending on the product, different oils can be used. For example, for an omega-3 producing facility, the oil phase can be omega-3 oil. To extract the algal oil, algae cells may be burst through ultrasonication, releasing the oils into the droplets. The oil will then quickly coalesce with the surrounding oil medium.

[0175] This process may be performed using an integrated method to flow and manipulate algae droplets in a system 2600 including a flat panel bioreactor 2610. As one non-limiting example, #14861445vlelectroosmotic forces may be used to cause algae droplets 2612 to flow through the bioreactor 2610. As another non-limiting example, dielectrophoretic forces may be used to cause algae droplets 2612 to flow through the bioreactor 2610. Due to the elimination of both algae settling and biofouling, a turbulent flow is not necessary. Instead, a low energy laminar flow can be used to sort and flow droplets when necessary.

[0176] In some embodiments, computer vision and machine learning may be used to characterize algae droplets 2612 according to their readiness for harvest. As one example, a camera 2620 may be used to capture images of algae droplets 2612 at a location of the bioreactor 2610 prior to a separation stage. The captured images may be provided to a machine learning model for classification of the algae droplets 2612 according to whether the droplets contain protein crystals that are unprepared or ready for harvesting. As one example, a U-Net-based machine learning model may be used to perform image segmentation of images acquired by camera 2620. Using this framework for algae cultivation, the machine learning system is able to track microalgae growth patterns and detect the optimal time to remove droplets from the photobioreactor and harvest the algae.

[0177] In some embodiments, because each droplet contains its own nutrient profile, a machine learning architecture can be trained to determine which droplets contain healthy and unhealthy algae. Thus, the growth conditions of the algae, such as light exposure, pH, and nutrient supply, can be monitored and optimized on a droplet-by- droplet basis. The machine learning architecture may further be trained to determine optimal nutrition and CO2 rates for algae growth.

[0178] In some embodiments, the bioreactor may enable recycling of the buffer supporting algae growth, as shown in the example of FIG. 27. In the system 2700 of FIG. 27, a flat-panel bioreactor 2710 is used to cultivate algae in droplets suspended in a suitable oil, after which a camera 2720 may be used to acquire images for use (e.g., as input to a machine learning model) to determine whether the algae droplets are ready for harvesting. For the algae droplets that are ready for harvesting, an ultrasound device 2730 may be used to rupture the algae droplets, allowing the algal oil to coalesce with the oil used for droplet suspension. The buffer fluids may remain as droplets suspended in the algal oil mixture, and may be returned to the flat-panel bioreactor 2710 for further algae cultivation.

[0179] An illustrative implementation of a computer system 2800 that may be used in connection with any of the embodiments of the technology described herein (e.g., such as the method of FIGs. 26 and / or 27) is shown in FIG. 28. The computer system 2800 includes one or more processors 2810 and one or more articles of manufacture that comprise non-transitory #14861445vlcomputer-readable storage media (e.g., memory 2820 and one or more non-volatile storage media 2830). The processor 2810 may control writing data to and reading data from the memory 2820 and the non-volatile storage device 2830 in any suitable manner, as the aspects of the technology described herein are not limited to any particular techniques for writing or reading data. To perform any of the functionality described herein, the processor 2810 may execute one or more processor-executable instructions stored in one or more non-transitory computer-readable storage media (e.g., the memory 2820), which may serve as non-transitory computer-readable storage media storing processor-executable instructions for execution by the processor 2810.

[0180] Computing system 2800 may also include a network input / output (I / O) interface 2840 via which the computing device may communicate with other computing devices (e.g., over a network) and may also include one or more user I / O interfaces 2850, via which the computing device may provide output to and receive input from a user. The user I / O interfaces may include devices such as a keyboard, a mouse, a microphone, a display device (e.g., a monitor or touch screen), speakers, a camera, and / or various other types of I / O devices.

[0181] The above-described embodiments can be implemented in any of numerous ways. For example, the embodiments may be implemented using hardware, software, or a combination thereof. When implemented in software, the software code can be executed on any suitable processor (e.g., a microprocessor) or collection of processors, whether provided in a single computing device or distributed among multiple computing devices. It should be appreciated that any component or collection of components that perform the functions described above can be generically considered as one or more controllers that control the above-discussed functions. The one or more controllers can be implemented in numerous ways, such as with dedicated hardware, or with general purpose hardware (e.g., one or more processors) that is programmed using microcode or software to perform the functions recited above.

[0182] In this respect, it should be appreciated that one implementation of the embodiments described herein comprises at least one computer-readable storage medium (e.g., RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or other tangible, non-transitory computer-readable storage medium) encoded with a computer program (i.e., a plurality of executable instructions) that, when executed on one or more processors, performs the above-discussed functions of one or more embodiments. The computer-readable medium may be transportable such that the program #14861445vlstored thereon can be loaded onto any computing device to implement aspects of the techniques discussed herein. In addition, it should be appreciated that the reference to a computer program which, when executed, performs any of the above-discussed functions, is not limited to an application program running on a host computer. Rather, the terms computer program and software are used herein in a generic sense to reference any type of computer code (e.g., application software, firmware, microcode, or any other form of computer instruction) that can be employed to program one or more processors to implement aspects of the techniques discussed herein.

[0183] It will be apparent that example aspects, as described above, may be implemented in many different forms of software, firmware, and hardware in the implementations illustrated in the figures. Further, certain portions of the implementations may be implemented as a “module” that performs one or more functions. This module may include hardware, such as a processor, an application-specific integrated circuit (ASIC), or a field-programmable gate array (FPGA), or a combination of hardware and software.

[0184] The terms “program” or “software” are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that can be employed to program a computer or other processor to implement various aspects as described above. Additionally, it should be appreciated that according to one aspect, one or more computer programs that when executed perform methods of the present disclosure need not reside on a single computer or processor but may be distributed in a modular fashion among a number of different computers or processors to implement various aspects of the present disclosure.

[0185] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.

[0186] Also, data structures may be stored in computer-readable media in any suitable form. For simplicity of illustration, data structures may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a computer-readable medium that convey relationship between the fields. However, any suitable mechanism may be used to establish a relationship between information in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationship between data elements.

[0187] #14861445vlWhen implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers.

[0188] Further, it should be appreciated that a computer may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer, as non-limiting examples. Additionally, a computer may be embedded in a device not generally regarded as a computer but with suitable processing capabilities, including a Personal Digital Assistant (PDA), a smartphone, a tablet, or any other suitable portable or fixed electronic device.

[0189] Also, a computer may have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output.

[0190] Examples of input devices that can be used for a user interface include keyboards, and pointing devices, such as mice, touch pads, and digitizing tablets. As another example, a computer may receive input information through speech recognition or in other audible formats.

[0191] Such computers may be interconnected by one or more networks in any suitable form, including a local area network or a wide area network, such as an enterprise network, and intelligent network (IN) or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks or fiber optic networks.

[0192] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the #14861445vlappended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0193] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0194] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0195] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0196] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements #14861445vland not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0197] As used herein, “wt%” is an abbreviation of weight percentage. As used herein, “at%” is an abbreviation of atomic percentage.

[0198] Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way.

[0199] Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and / or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

[0200] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0201] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0202] #14861445vl

Claims

CLAIMSWhat is claimed is:

1. A composition, comprising:a multi-phase fluid arrangement comprising an active fluid phase and a transport fluid phase, wherein the active phase comprises a reactive and / or growth species, and the transport fluid phase is selected for the ability to transport a reactant to the reactive and / or growth species in the active fluid phase, and / or to remove a product from the reactive and / or growth species in the active fluid phase.

2. A bioreactor system, comprising:a container configured to house a bioreaction, having an interior surface;a source of a multi-phase fluid arrangement connectable to the bioreactor, wherein the multi-phase fluid arrangement comprises an active fluid phase in which a bioreaction occurs and which, when in contact with the surface, can cause biofouling of the surface, and a separating fluid phase, wherein separating fluid phase has a greater affinity for the surface than the active fluid phase.

3. A system, comprising:a source of an aqueous fluid comprising biological material;a source of a non-aqueous fluid comprising a compound;a vessel configured to receive a first input comprising the aqueous fluid and a second input comprising the non-aqueous fluid, wherein:the vessel is configured to form and / or maintain an emulsion comprising an aqueous domain, the aqueous domain comprising droplets of the aqueous fluid, and a non-aqueous domain comprising the non-aqueous fluid,at least some of the compound is capable of exiting the non-aqueous domain and entering the droplets of the aqueous domain, andthe non-aqueous fluid has greater wettability to at least one surface of the vessel than the aqueous fluid.#14861445vl4. A composition, comprising:an emulsion comprising an aqueous domain and a non-aqueous domain, the aqueous domain comprising an aqueous fluid and the non-aqueous domain comprising a non-aqueous fluid, wherein:the aqueous fluid comprises biological material;the non-aqueous fluid comprises a compound capable of exiting the nonaqueous domain and entering the aqueous fluid of the aqueous domain;the non-aqueous fluid has greater wettability to surfaces of a bioreactor than the aqueous fluid such that at least some of the non-aqueous fluid is positioned between at least some of the aqueous fluid and surfaces of the bioreactor; andthe compound is capable of being at least partially consumed by the biological material in the aqueous fluid.

5. A system, comprising:a reaction vessel;an emulsion in the vessel comprising:a discontinuous domain comprising a biological material; continuous non-aqueous domain comprising a nutrient for the biological material, wherein at least some of the nutrient is capable of passing from the nonaqueous domain into a aqueous domain, and the non-aqueous domain has greater wettability to surfaces of the vessel than the aqueous domain.

6. The system or composition of claim 5, wherein the non-aqueous domain is continuous, aqueous domain is non-continuous.

7. The system or composition of any one of claims 3-4, wherein at least some of the non-aqueous fluid is positioned between at least some of the aqueous fluid and surfaces of the vessel.#14861445vl8. The system or composition of any one of claims 3-7, wherein the compound is a nutrient capable of being absorbed and / or metabolized by the biological material in the aqueous fluid.

9. The system or composition of any one of claims 3-8, wherein the nutrient comprises a gas10. The system or composition of claim 9, wherein the gas comprises carbon dioxide.

11. The system or composition of claim 10, wherein the carbon dioxide is present in the non-aqueous fluid at a concentration of greater than or equal to 0.04%.

12. The system or composition of any one of claims 3-11, wherein the system further comprises a source of the compound fluidly connectable to the vessel, configured to introduce the compound into the non-aqueous domain.

13. The system or composition of any one of claims 3-12, further comprising an outlet configured to remove, from the non-aqueous domain, a reaction product of the biological material.

14. The system or composition of any one of claims 3-13, wherein the system further comprises a contactor capable of contacting gaseous carbon dioxide with the nonaqueous fluid such that the non-aqueous fluid entering the vessel has a concentration of carbon dioxide greater than or equal to 0.04% and less than or equal to 10%.

15. The system or composition of any one of claims 3-14, wherein the system is capable of removing the non-aqueous fluid such that the non-aqueous fluid is removed from the vessel when the concentration of the compound in the vessel is below a threshold concentration.#14861445vl16. The system or composition of claim 15, wherein the removed non-aqueous fluid comprises byproducts generated by the biological material.

17. The system or composition of any one of claims 3-16, wherein the non-aqueous fluid is recirculated at a flow rate of less than or equal to 2 ml / min.

18. The system or composition of any one of claims 3-17, wherein the non-aqueous fluid is positioned between the aqueous fluid and surface of the vessel such that contact between the biological material and surfaces of the vessel is inhibited.

19. The system or composition of any one of claims 1-18, wherein vessel has an interior volume greater than or equal to 1 cm3.

20. The system or composition of any one of claims 3-19, wherein the aqueous fluid comprises growth medium.

21. The system or composition of any one of claims 3-20, wherein the non-aqueous fluid comprises oil.

22. The system or composition of any one of claims 3-21, wherein the maximum dimension of the droplets is less than or equal to 400 microns and greater than or equal to 50 microns.

23. The system or composition of any one of claims 3-22, wherein the packing fraction of the droplets of the aqueous fluid in the non-aqueous fluid is greater than or equal to 0.6.

24. The system or composition of any one of claims 3-23, wherein the system is capable of growing the biological material has a growth rate of greater than or equal to 0.13 g / L / day.#14861445vl25. The system or composition of any one of claims 3-24, wherein the biological material comprises algae.

26. The system or composition of any one of claims 3-25, wherein the emulsion is a water-in-oil emulsion.#14861445vl

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