Methods and systems for nanobubble generation and use

Abstract

Presented herein are systems and methods related to associating nanobubbles (NBs) with a composition (e.g., a liquid composition). NBs are ultrafine gas domains in liquid with nano-scale diameters that are, on average, smaller than 1000 nm diameter (e.g., about 200nm in diameter or less). NBs have remarkably large interfacial surface areas and have unique physicochemical properties due to their small dimensions. The small size (e.g., diameter) and higher surface tension of NBs increase the interfacial gas-liquid surface area, which in turn promotes mass transfer. In some embodiments, NBs (e.g., oxygen NBs) can be suspended in a composition (e.g., water) long periods of time and produce reactive oxygen species without added catalysts. However, NB applications in water treatment applications have not been widely explored.

Description

Attorney Docket No. 2010363-0390METHODS AND SYSTEMS FOR NANOBUBBLE GENERATION AND USE CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is claims priority to and benefit of U.S. Provisional Application No.63 / 492,415 filed on March 27, 2023, the disclosure of which is hereby incorporated by reference in its entirety. GOVERNMENT SUPPORT

[0002] This invention was made with government support under NASA EPSCoR Cooperative agreement number: EP-23-03 and Federal Award No: 80NSSC22M0168. The government has certain rights in the invention. FIELD OF THE INVENTION

[0003] The present disclosure relates generally to methods and systems for generating nanobubbles in a composition. In some embodiments, the disclosure relates to methods and systems for removal of target substances (e.g., pollutants) from a composition using nanobubbles and / or adding payload substances to a composition using nanobubbles. BACKGROUND OF THE INVENTION

[0004] Activated sludge process is the biotic conversion of organic matter of wastewater to CO2and biomass. The biological conversion of organic compounds in activated sludge relies on the efficient transfer of dissolved oxygen (DO). Oxygen mass transfer from the gas phase to the liquid phase and microbial oxygen consumption are critical for effectively removing chemical oxygen demand (COD), microbial growth, cell maintenance, sludge decomposition, and other metabolic processes. Therefore, sufficient aeration is imperative for the activated sludge process.

[0005] However, aeration is an energy-intensive process with poor mass transfer efficiencies achieving only 30% oxygen transfer efficiency in a typical 4.5 m deep activated sludge reactor. According to the Water Environment & Reuse Foundation (WERF), publicly- owned wastewater treatment plants consume 1–4% of total energy production, representing about 1% of total retail electricity sales in the United States. Within this energy requirement, 50–90% of the total energy consumption for wastewater treatment is destined for the aeration of the activated sludge processes. Moreover, foulant accumulation in the fine pore diffusers Page 1 of 63 11877856v1Attorney Docket No. 2010363-0390reduces the oxygen transfer efficiency over time and requires increased air flux to maintain the same oxygen supply. Biofouling and scaling on and within the air diffusers further increase the cost of aeration due to the high airflow resistance.

[0006] Therefore, there exists a need for improved systems and methods for treatment of wastewater. SUMMARY OF THE INVENTION

[0007] Presented herein are systems and methods related to associating nanobubbles (NBs) with a composition (e.g., a liquid composition). NBs are ultrafine gas domains in liquid with nano-scale diameters that are, on average, smaller than 1000 nm diameter (e.g., about 200nm in diameter or less). NBs have remarkably large interfacial surface areas and have unique physicochemical properties due to their small dimensions. The small size (e.g., diameter) and higher surface tension of NBs increase the interfacial gas–liquid surface area, which in turn promotes mass transfer. In some embodiments, NBs (e.g., oxygen NBs) can be suspended in a composition (e.g., water) long periods of time (e.g., several months) and produce reactive oxygen species without added catalysts. However, NB applications in water treatment applications have not been explored.

[0008] NBs have potential to become a key component of next generation biphasic fluids. NBs can be applied to address essential water supply needs in space transport, survival, colonization, and other applications. In addition, NB technology can be applied to conventional water treatment (e.g., for drinking water) and wastewater treatment systems on Earth. In some embodiments, NBs can be used to add a payload substance (e.g., a gas or other desirable compound) to a composition and / or remove a target substance from a composition.

[0009] In one aspect, the disclosure encompasses methods comprising, associating a plurality of nanobubbles (e.g., in a first solution) with a composition.

[0010] In some embodiments, provided methods comprise removing one or more target substances from a composition using a plurality of nanobubbles.

[0011] In some embodiments, provided methods comprise adding one or more target substances to a composition using a plurality of nanobubbles.

[0012] In some embodiments, provided methods comprise adding at least a portion of one or more payload substances into a plurality of nanobubbles (e.g., substantially within the interior of the bubble). In some embodiments, provided methods comprise adding at least a portion of one or more payload substances on a surface of a plurality of nanobubbles. Page 2 of 63 11877856v1Attorney Docket No. 2010363-0390

[0013] In some embodiments, one or more payload substances comprise a gas.

[0014] In some embodiments, a plurality of nanobubbles comprises an elemental gas (e.g., hydrogen, nitrogen, oxygen, a noble gas, etc.), ozone gas (e.g., substantially pure ozone gas), or a combination of gases (e.g., atmospheric air).

[0015] In some embodiments, provided methods comprise storing a plurality of nanobubbles in a storage solution (e.g., separate from a composition) for a period of time (e.g., prior to the use of said plurality of nanobubbles). In some embodiments, a plurality of nanobubbles in a storage solution are stable (e.g., are substantially the same in number, density, size, charge, or composition) for a period of time. In some embodiments, a period of time is at least 30 days (e.g., 35 days, 40 days, 45 days, 50 days, 60 days, 70 days, 80 days, 90 days, or more). In some embodiments, a period of time is sufficient for long range ground transportation, long field missions in battleground, remote terrestrial expeditions, space expedition missions, and the like.

[0016] In some embodiments, provided methods comprise generating a plurality of nanobubbles on a surface of a substrate.

[0017] In some embodiments, provided methods comprise contacting a plurality of nanobubbles with a substrate (e.g., such that a plurality of nanobubbles adsorb to the substrate) (e.g., in the solution).

[0018] In some embodiments, a substrate is a hydrophobic substrate.

[0019] In some embodiments, a substrate comprises graphene (e.g., graphene nanosheets) (graphene oxide).

[0020] In some embodiments, provided methods comprise generating a plurality of nanobubbles on a substrate using one or more of the following methods: water-solvent exchange, differential temperature, differential pressure, ultrasonication, mechanical forces and electrolytic evolution.

[0021] In some embodiments, provided methods comprise removing a plurality of nanobubbles from a surface of a substrate (e.g., thereby removing one or more target substance from the composition).

[0022] In some embodiments, provided methods comprise removing a plurality of nanobubbles from a surface of a substrate using sonication or salt-destabilization (e.g., thereby releasing a payload substance).

[0023] In some embodiments, provided methods comprise destruction of a plurality of nanobubbles at an interphase (e.g., between a substrate and a nanobubble)(e.g., between a nanobubble and a composition)(e.g., between a nanobubble and a gas). Page 3 of 63 11877856v1Attorney Docket No. 2010363-0390

[0024] In some embodiments, provided methods comprise release of a payload substance from the plurality of destroyed nanobubbles.

[0025] In some embodiments, a composition is a wastewater solution.

[0026] In some embodiments, a wastewater solution comprises sludge (e.g., activated sludge).

[0027] In some embodiments, one or more target substances are pollutants.

[0028] In some embodiments, one or more target substances comprise one or more of the following: an organic pollutant (e.g., synthetic organic pollutants), an organosilane, urea, a perfluorochemical, a microorganism, a short chain fatty acid, a sugar, a protein, a surfactant, a nanoparticle, a dye (e.g., a color-causing chemical), a colloid, Methyl-Isoborneol (MIB), Geosmin, a nanoplastic (NP), and oxytetracycline.

[0029] In some embodiments, one of one or more target substances comprises an organic pollutant. In some embodiments, an organic pollutant is or comprises one or more of the following compounds or an equivalent or derivative thereof: naphthalene, 1- chloronapthalene, 1-napthol, 1,4-dichloronaphtalene, 1,8-dichloronaphtalene, phenanthrene, anthracene, pyrene, biphenyl, thrichloroethylene, tetrachloroethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane, carbon tetrachloride, 1,1-dichloroethylene, 1,2-dichloropropane, methylene blue, bisphenol A, carbamazepine, phenol, benzoic acid, atrazine, perfluorooctanesulfonate (PFOS), phenanthrene, PFOS / Perfluorooctanoic Acid (PFOA), and trichloroethylene (TCE).

[0030] In some embodiments, one or more target substances comprises a nanoplastic (NP). In some embodiments, a size of a nanoplastic is about 10μm or less, about 1μm or less, about 100nm or less in size (e.g., in diameter).

[0031] In some embodiments, a nanoplastic is or comprises polystyrene, latex, polyethylene tetrephtalate (PET), or a combination thereof.

[0032] In some embodiments, at least one of the one or more target substances comprises a plurality of nanoparticles (e.g., nanoplastics).

[0033] In some embodiments, provided methods comprise floating the plurality of nanoparticles using the plurality of nanobubbles (e.g., to the surface of the composition) (e.g., in nanoparticle-nanobubble flocs).

[0034] In some embodiments, provided methods comprises causing the nanoparticles to rise to a surface of the composition. Page 4 of 63 11877856v1Attorney Docket No. 2010363-0390

[0035] In some embodiment, provided methods comprise reducing the pH of the composition to a pH of about 6 or lower, to a pH of about 5 or lower, to a pH of about 3 or lower (e.g., to enhance removal of the one or more nanoparticles).

[0036] In some embodiments, the composition has a pH of about 6 or lower, to a pH of about 5 or lower, to a pH of about 3 or lower (e.g., to enhance removal of the one or more nanoparticles).

[0037] In some embodiments, provided methods comprise adding one or more coagulants and / or surfactants to the composition. In some embodiments, a coagulant is an aluminum based coagulant (e.g., aluminum sulfate, aluminum chloride, sodium aluminate). In some embodiments or an iron based coagulant (e.g., ferric sulfate, ferric sulfate, ferric chloride, ferric chloride sulfate, polymeric ferric sulfates).

[0038] In some embodiments, provided methods comprise reducing and / or neutralizing the magnitude of the zeta potential of one or more target substances (e.g., nanoplastics) (e.g., wherein the zeta potential is about -40mV or less (in absolute value) (e.g., about -33.8mV or less, about -25mV or less, about -16.7mV or less).

[0039] In some embodiments, provided methods comprise decreasing an abundance (e.g., number, density) of filamentous bacteria, mycobacterium, viruses or floc forming bacteria (e.g., Corynebacterium, Pseudomonas, Zoogloea) in a composition [e.g., in a wastewater solution (e.g., as compared to coarse bubble treated wastewater)(e.g., as compared to wastewater not treated with nanobubbles)].

[0040] In some embodiments, a plurality of nanobubbles are characterized in that a payload substance of at least a subset of (e.g., up to substantially all of) the plurality of nanobubbles are accessible to microorganisms (e.g., gases of the nanobubbles are accessible to microorganisms) (e.g., aerobic microorganisms, e.g., aerobic bacteria).

[0041] In some embodiments, provided methods comprise generating a plurality of nanobubbles with hydrogen peroxide (e.g., for micro-gravity applications, for off-grid applications), nozzle decompression, ultrasonic generation, venturi bubble generation, spiral flow mixing, static and dynamic mixing, ceramic oxygen generator (COG) (e.g., a solid state COG) (e.g., under microgravity conditions) or hammer-rotation (e.g., magnetic hammer rotation).

[0042] In some embodiments, provided methods comprise use of advanced oxidation process(es). In some embodiments, advanced oxidation process(es) comprise exposing a plurality of nanobubbles to metallic catalysts (e.g., iron (II)), UV light irradiation (e.g., using hydrogen peroxide / UV), or ultrasound (e.g., advanced cavitation). In some embodiments, Page 5 of 63 11877856v1Attorney Docket No. 2010363-0390nanobubble implosions may generate extreme local temperatures and pressures causing generation of reactive oxygen species. The reactivity of these radicals can cause oxidative destruction of substrates in addition to their shuttling.

[0043] In some embodiments, provided methods comprise generating a reactive oxygen species using a plurality of nanobubbles. In some embodiments, provided methods further comprise disrupting (e.g., imploding) a plurality of nanobubbles.

[0044] In some embodiments, the diameter (e.g., average diameter) of a plurality of nanobubbles is about 1000 nm or less (e.g., about 650 nm or less, about 400 nm or less, about 300 nm or less, about 200 nm or less, about 100 nm or less) (e.g., as measured using a nano- particle tracking analyzer).

[0045] In some embodiments, the density of a plurality of nanobubbles (e.g., in the composition)(e.g., in a first solution) is about 3.5x107bubbles per milliliter (e.g., greater than .5 x 107bubbles per mL, greater than 1 x 107bubbles per mL, greater than 1.8 greater x 107bubbles per mL, greater than 2 x 107bubbles per mL, greater than 3 x 107bubbles per mL, greater than 4 x 107bubbles per mL, greater than 5 x 107bubbles per mL, greater than 11.2 x 1010bubbles per mL) (e.g., as measured using a nano-particle tracking analyzer). In some embodiments, the density of a plurality of nanobubbles in a composition is characterized in that it does not affect the stability of the nanobubbles in the composition.

[0046] In some embodiments, the provided methods comprise removing a plurality of nanobubbles from the composition using salt addition, filtration, sonication, a vacuum, dielectric heating, radio waves, or light (e.g., UV light, IR light).

[0047] In another aspect, the disclosure encompasses methods of aquaculture production comprising methods disclosed herein.

[0048] In another aspect, the disclosure encompasses methods of treating wastewater under microgravity conditions comprising methods disclosed herein.

[0049] In another aspect, the disclosure encompasses methods of treating a water column for use in aquaculture comprising methods disclosed herein.

[0050] In another aspect, the disclosure encompasses methods of oxygen bleaching (e.g., for use in papermaking) comprising methods disclosed herein.

[0051] In another aspect, the disclosure encompasses methods of delignification comprising methods disclosed herein.

[0052] In another aspect, the disclosure encompasses systems configured to perform methods disclosed herein. Page 6 of 63 11877856v1Attorney Docket No. 2010363-0390

[0053] In another aspect, the disclosure encompasses methods of removing a pollutant from a composition, comprising: associating a plurality of nanobubbles with a composition, wherein the plurality of nanobubbles are about 500nm or less in diameter; reducing the zeta potential of the pollutant to about -25mV or less; and removing the pollutant from the composition using the plurality of nanobubbles, wherein the pollutant comprises nanoplastics of about 1μm or less in size, wherein removing the one or more target substances comprises causing the pollutant to rise to the surface of the composition.

[0054] In another aspect, the disclosure encompasses methods of removing organic pollutants from a composition, the methods comprising: generating the plurality of nanobubbles on a surface of a hydrophobic substrate, wherein the plurality of nanobubbles are about 500nm or less in diameter; contacting the plurality of nanobubbles with a composition, wherein the composition comprises the organic pollutants, thereby associating the organic pollutants with the plurality of nanobubbles; and removing the plurality of nanobubbles from the surface of the substrate using the plurality of nanobubbles using sonication and / or salt-destabilization.

[0055] In another aspect, the disclosure encompasses methods of removing one or more target substances from a composition, the method comprising, associating a plurality of nanobubbles with the composition, wherein the composition comprises one or more target substances and wherein the plurality of nanobubbles are about 500nm or less in diameter; and removing the one or more target substances from the composition using the plurality of nanobubbles, wherein removing the one or more target substances comprises removing the plurality of nanobubbles from the composition using sonication, wherein at least one of the one or more target substances comprises an organic pollutant. BRIEF DESCRIPTION OF THE FIGURES

[0056] Drawings are presented herein for illustration purposes, not for limitation. The foregoing and other objects, aspects, features, and advantages of the disclosure will become more apparent and may be better understood by referring to the following description taken in conjunction with the accompanying drawings.

[0057] FIG.1 is an illustration of surface and bulk nanobubbles and exemplary generation methods, according to an illustrative embodiment. Page 7 of 63 11877856v1Attorney Docket No. 2010363-0390

[0058] FIG.2 is an illustrative embodiment of an experimental setup for NB aeration experiments. The same setup was used for control experiments, but the NB generator was bypassed.

[0059] FIG.3 is a graph of dissolved oxygen concentration change in deionized water with time under the following conditions: nanobubble (NB) aeration (■), coarse bubble (CB) aeration (●), and without aeration (▴).

[0060] FIG.4 is a graph of bubble size distribution and concentration profile of NB aerated water with respect to time as measured using a nanoparticle tracking analyzer (NTA).

[0061] FIG.5 is a graph of average bubble concentration as produced over the course of an experiment. Error bars indicate the standard deviation of three measurements, and * indicates statistically different values according to the one-way ANOVA test at 95% level of significance (p < 0.05).

[0062] FIG.6 is a graph of average bubble size as produced over the course of an experiment. Error bars indicate the standard deviation of three measurements, and * indicates statistically different values according to the one-way ANOVA test at 95% level of significance (p < 0.05).

[0063] FIG.7 is a graph showing sCOD removal percentage of activated sludge spiked with glucose reacted for 48-h with NB aeration (●) and CB aeration (▴). The lines are included as a guide. Error bars indicate the standard deviation of triplicate sCOD samples.

[0064] FIG.8 is a series of images showing settled sludge volume at 30 min (SSV30) before aeration with nanobubbles (I) and 48 h after of aeration with coarse bubbles (CB 48h) (II) and nanobubbles-NB (NB 48h) (III). Duplicate measurements are shown in the images.

[0065] FIG.9 is a chart showing the percent of microbial community abundance on a genera level of 35 predominant genera. Samples from which the microbial communities are measured are noted as follows: 0 h: Activated sludge sample before aeration, CB 48 h: coarse bubble aeration for 48 h, NB 48 h: nanobubble aeration for 48 h.

[0066] FIG.10 is a dual hierarchal dendrogram evaluation of the taxonomic classification data at the genera level. Samples from which the microbial communities are measured are noted as follows: 0 h: Activated sludge sample before aeration, CB 48 h: coarse bubble aeration for 48 h, NB 48 h: nanobubble aeration for 48 h.

[0067] FIG.11 is a graph showing a Shannon Diversity Boxplot and Kruskal-Wallis pairwise comparisons of Shannon Entropy of samples of an activated sludge sample before Page 8 of 63 11877856v1Attorney Docket No. 2010363-0390aeration (0 h), after coarse bubble aeration for 48h (CB 48 h), and after nanobubble aeration for 48 h (NB 48 h).

[0068] FIG.12 is a principal coordinate plot of weighted UniFrac data, which is used to show data related to beta diversity. Samples from the experimental conditions are noted as follows: 0 h (activated sludge sample before aeration), CB 48 h (coarse bubble aeration for 48 h), and NB 48 h (nanobubble aeration for 48 h).

[0069] FIG.13 is an exemplary NanoSightTMNS300 configuration (courtesy of Malvern Panalytical) for nanoparticle tracking analysis of nanobubbles in a sample of a solution, according to an illustrative embodiment.

[0070] FIG.14A is a graph of the number of bubbles in a fluid sample over a period of 40 days.

[0071] FIG.14B is a graph of the density of bubbles in a fluid sample over a period of 40 days.

[0072] FIG.15 is a set of data demonstrating the effect of nanobubbles on hydrolysis of biosolids based on the length of contact time between the NBs and the biosolids.

[0073] FIG.16A is graph showing the change in geosmin concentration in a nanobubble (NB) containing solution as measured by GC-MS after treatment with ultrasonication for 30 minutes (t=30), as compared to distilled and deionized water (DI) which has not been treated with NBs. t=0 is representative of the initial concentration of geosmin in a control water sample. The water sample initially contained 100ng / L of geosmin.

[0074] FIG.16B is graph showing the change in 2-methylisoborneol (MIB) concentration in a nanobubble (NB) containing solution as measured by GC-MS after treatment with ultrasonication for 30 minutes (t=30), as compared to distilled and deionized water (DI) which has not been treated with NBs. t=0 is representative of the initial concentration of geosmin in a control water sample. The water sample initially contained 100ng / L of MIB.

[0075] FIG.16C is graph showing the change in geosmin concentration in a nanobubble (NB) containing solution as measured by GC-MS after treatment with ultrasonication for 30 minutes (t=30), as compared to distilled and deionized water (DI) which has not been treated with NBs. t=0 is representative of the initial concentration of geosmin in a control water sample. The water sample initially contained 1000ng / L of geosmin.

[0076] FIG.16D is graph showing the change in 2-methylisoborneol (MIB) concentration in a nanobubble (NB) containing solution as measured by GC-MS after treatment Page 9 of 63 11877856v1Attorney Docket No. 2010363-0390with ultrasonication for 30 minutes (t=30), as compared to distilled and deionized water (DI) which has not been treated with NBs. t=0 is representative of the initial concentration of geosmin in a control water sample. The water sample initially contained 1000ng / L of MIB.

[0077] FIG.17A is a graph demonstrating the change in concentration of nanobubbles over time of a solution containing nanobubbles (NBs) subjected to sonication for 1hr.

[0078] FIG.17B is a graph showing the size of nanobubbles over time in a solution containing nanobubbles (NBs) subjected to sonication for 1hr.

[0079] FIG.18A is a graph demonstrating the change in concentration of nanobubbles over time of a solution which was not provided with nanobubbles and subjected to sonication for 1hr.

[0080] FIG.18B is a graph demonstrating the change in size of nanobubbles over time of a solution which was not provided with nanobubbles and subjected to sonication for 1hr.

[0081] FIG.19 shows a UV-vis calibration curve for nanoplastics in nanopure and nanobubble water.

[0082] FIG.20 is a graph of the mass concentration of NPs measured by UV-vis in the subnatant after 5 min stirring at different pH levels (Conditions: initial plastic concentration = 38 μg / L, stirring speed = 400rpm, stirring time = 5 min).

[0083] FIG.21 is a graph of the normalized number concentration of nanoplastics (NPs) in solutions with pHs ranging from about 3.0 to about 9.0.

[0084] FIG.22 is a series of graphs showing particle size distributions at pH 3.0. CT0and CT denote the initial and final number concentration, respectively.

[0085] FIG.23 shows a series of NTA micrographs of nano-entities in the bulk phase before and after stirring at pH 3 for 5 min and 400 RPM.

[0086] FIG.24 shows a bar graph the NTA size analysis of nano-entities in the subnantant after stirring at 400 rpm for 5 min.

[0087] FIG.25 is a graph changes in particle size of nanobubbles, nanoplastics, and nanoplastics with nanobubbles after 5 min stirring at various pHs (Conditions: initial nanoplastic concentration = 38 µg / L, stirring speed = 400 RPM, stirring time = 5 min).

[0088] FIG.26 shows zeta potential measurements for nanobubbles and nanoplastics as pH is changed.

[0089] FIG.27, panels A-C show (a) mass concentration from UV-vis spectroscopy, (b) normalized number concentration from NTA, and (c) normalized average size of nanoplastics from NTA distributed at the meniscus and bulk phase of DI water and Page 10 of 63 11877856v1Attorney Docket No. 2010363-0390nanobubble solution after stirring (stirring time = 5 min unless stated otherwise, stirring speed = 400 RPM unless stated otherwise, pH is unmodified unless stated otherwise).

[0090] Fig.28 shows the visible appearance of a thin film after stirring NPs with nanobubbles at pH 3.

[0091] The features and advantages of the present disclosure will become more apparent from the detailed description set forth below when taken in conjunction with the drawings, in which like reference characters identify corresponding elements throughout. In the drawings, like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements. DEFINITIONS

[0092] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0093] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of" (or which "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of" (or "consists of") the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step. Page 11 of 63 11877856v1Attorney Docket No. 2010363-0390

[0094] “Composition”: As used herein, the term “composition” refers to a collection of various elements to which a plurality of nanobubbles are to be applied to elicit some effect (e.g., adding or removing at least one substance to / from the composition, such as oxygen or a pollutant, respectively). In some embodiments, a composition may be or comprise at least one liquid. For example, in some embodiments, a composition may be or comprise water (e.g., wastewater). In some embodiments, a composition may be or comprise a liquid and a solid (e.g., a mixture of a liquid and a solid). In some embodiments, a composition may be or comprise homogeneous or inhomogeneous mixtures of a liquid and a solid. For example, in some embodiments, a composition may comprise sludge. In some embodiments, a composition may be, for example, an ocean, a lake, a pond or another large body of water. When discussing applying methods described herein to large bodies of water properties being described, for example, concentrations of nanobubbles, concentrations of target substances, and other properties (e.g., pH) are understood to be local to an area of application of the methods.

[0095] “Improve,” “increase”, “inhibit” or “reduce”: As used herein, the terms “improve”, “increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system under otherwise comparable conditions absent presence of (e.g., prior to and / or after) a treatment. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant treatment.

[0096] “Payload Substance”: In general, the term “payload substance”, as used herein, refers to an agent that may be delivered or transported by association with another entity. In some embodiments, a payload substance is associated with and / or transported by a nanobubble. In some embodiments, a nanobubble is comprised of a gas, which is a payload substance. In some embodiments, such association between a nanobubble and a payload substance may be or include non-covalent interaction(s) (e.g., based on a charge of a nanobubble, e.g., the zeta potential). In some embodiments, association may be direct; in some embodiments, association may be indirect. The term “payload substance” is not limited to a particular chemical identity or type; for example, in some embodiments, a payload substance may be or comprise, for example, an entity of any chemical class including, for example, a lipid (e.g., a short chain fatty acid), a metal, a nucleic acid, a polypeptide, a saccharide (e.g., a sugar, a polysaccharide), a small molecule, a micro- Page 12 of 63 11877856v1Attorney Docket No. 2010363-0390organism, an organosilane, a perfluorochemical, a dye (e.g., a color-causing chemical), a colloid, an organic pollutant (e.g., a synthetic organic pollutant), a gas (an elemental gas (e.g., hydrogen, nitrogen, oxygen, a noble gas, etc.), ozone gas (e.g., substantially pure ozone gas), a combination or mixture of gases (e.g., atmospheric air)) or a combination or complex thereof. In some embodiments, a payload substance may be or comprise a natural product in that it is found in and / or is obtained from nature; alternatively or additionally, in some embodiments, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and / or produced through action of the hand of man and / or is not found in nature. In some embodiments, a payload substance may be or comprise an agent in isolated or pure form; in some embodiments, such agent may be in crude form.

[0097] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological and chemical arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. DETAILED DESCRIPTION

[0098] It is contemplated that systems, architectures, devices, methods, and processes of the claimed invention encompass variations and adaptations developed using information from the embodiments described herein. Adaptation and / or modification of the systems, architectures, devices, methods, and processes described herein may be performed, as contemplated by this description.

[0099] Throughout the description, where articles, devices, systems, and architectures are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are articles, devices, systems, and architectures of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps. Page 13 of 63 11877856v1Attorney Docket No. 2010363-0390

[0100] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0101] The mention herein of any publication, for example, in the Background section, is not an admission that the publication serves as prior art with respect to any of the claims presented herein. The Background section is presented for purposes of clarity and is not meant as a description of prior art with respect to any claim.

[0102] Documents are incorporated herein by reference as noted. Where there is any discrepancy in the meaning of a particular term, the meaning provided in the Definition section above is controlling.

[0103] Headers are provided for the convenience of the reader – the presence and / or placement of a header is not intended to limit the scope of the subject matter described herein.

[0104] Decreasing in the bubble size (e.g., diameter) from the micrometer level to the nanoscale (e.g., 50–1000 nm in diameter) results in the prolonged existence of gas bubbles in solutions (e.g., from days to months). NBs are stable gas cavities with extraordinary characteristics in some embodiments including, but not limited to, prolonged stability, ability to produce reactive oxygen species (e.g., hydroxyl radicals), and higher mass transfer efficiency in aqueous solutions. Compared to microbubbles, NBs can significantly increase the efficiency of mass transfer of gases (e.g., oxygen) in these systems.

[0105] The use of nanobubbles over microbubbles in many applications leads to an in a minimization of loss of gases and saving energy. Systems starving for a gas either biologically or chemically can benefit from miniscule sizes and high surface areas of nanobubbles (NBs). Mass transfer is a surficial reaction, which means the higher surface area will promote movement of molecules between phases. Therefore, the high surface area of nanobubbles enhances transfer of gases between phases.

[0106] Additionally NBs can be rapidly generated by energy efficient fluidic oscillations or other low-cost hydraulic, physicochemical and electrochemical techniques (e.g., as shown in FIG.1). NBs are stable in water for long periods of times (e.g., hours, days, weeks, months) and coexist with reactive oxygen species. This modern and paradoxical discovery of stable NBs combined with their catalyst-free oxidation potential represents a paradigm shift for advanced water treatment technologies. For example, the outstanding properties of NBs allow them to be used to develop energy-efficient, small foot-print water treatment systems for life support subsystems in aerospace industry. In addition, NBs can improve conventional water and Page 14 of 63 11877856v1Attorney Docket No. 2010363-0390wastewater treatment technologies on Earth. For example, extremely large gas-liquid interfaces of NBs can lift practical restrictions of oxygen delivery; therefore, can revolutionize oxygen mass transfer in biotic (e.g., activated sludge) and abiotic (e.g., advanced oxidation) processes. Additionally, NBs that are attached to surfaces can increase adsorption of dissolved oleophobic compounds or can bridge suspended solids via hydrophobic interactions. Moreover, air, oxygen or ozone NBs can facilitate production of reactive oxygen species including short-lived and quasi-stable radicals for advanced oxidation applications. Nanobubble Stability and Assessment

[0107] In some embodiments, NBs are stable when present in a composition, though particular conditions can result in a loss of stability. Buoyancy forces on NBs are relatively insignificant because Brownian motion typically governs their movement in a composition. Because Brownian motion governs their movement, NBs do not typically rise (e.g., to the surface of a composition / solution) on short time-scales in a composition (e.g., in pure water). This phenomenon partly explains their stability in liquids. The low mass of NBs due to Pickering Emulsion mechanisms also prevents their interfacial transport.

[0108] In some embodiments, NB stability is affected by solution chemistry (e.g., the presence or absence of compounds or chemicals in a solution), gas type of NBs (e.g., oxygen, ozone, nitrogen, etc.), temperature, pressure, mechanical vibrations, and other conditions. For example, increasing the temperature of a composition containing NBs could accelerate NBs’ erratic motion and their chances of coalescence and subsequent rise to surface. Changes in ambient pressure could also influence bubble size thus overall stability.

[0109] Charges of NBs aid in preventing their coalescence. Charged nanobubbles can repel each other and, as a result, cause their stability in suspension. Without wishing to be bound to a particular theory, the greater the magnitude of the charge of NBs, the more stable the NBs are in a composition. In some embodiments, high ionic strength of a composition may cause double layer compression and, subsequently, increase chances of collision between NBs. Increases in collisions (or the chance of collisions) result in a decrease in the overall stability of NBs. In some embodiments, external impacts including, but not limited to, vibrations, turbulence, and mixing deteriorate NB stability. In some embodiments, charges of NBs (e.g., NBs and their payload substance(s)) can also play a role in shuttling oppositely charged payload substances while avoiding NBs having same-charge payload substances. In some embodiments, oppositely charged payload substances can also bridge two nanobubbles and cause their coalition / aggregation in a composition. Page 15 of 63 11877856v1Attorney Docket No. 2010363-0390

[0110] In some embodiments, nanobubbles (NBs) are stored in a storage solution for a period of time. As used herein, the term “storage solution” refers to a solution that is capable of supporting the existence of particular nanobubbles for a period of time, but it differs from a composition in that the nanobubbles (and any payload substance) is not intended for use in a storage solution, simply to be stored there and eventually used in a composition. In some embodiments, NBs are stored in a storage solution, e.g., a liquid storage solution, a storage solution comprising of a liquid and solid) for a period of time. In some embodiments, NBs are stored for a period of time prior to their use in an application. In some embodiments, NBs are stored for at least an hour, 12 hours, 18 hours, a day, 10 days, 20 days, 30 days, 40 days, 2 months, 3 months or more prior to use.

[0111] In some embodiments, nanobubbles are stored in a storage solution for a period of time and are stable in that storage solution. Stability of nanobubbles (e.g., in a storage solution) can be assessed through a number of properties of the NBs in the storage solution. For example, stability can be assessed by parameters including, but not limited to, a number of NBs, density of NBs, size of NBs (e.g., an average size), charge of NBs (e.g., zeta potential), payload substance of the NBs, or makeup of NBs (e.g., gas(es) of NBs). In some embodiments, one or more properties of NBs in solution remains substantially unchanged for a period of time prior to use. In some embodiments, a NB property substantially remaining unchanged in a storage solution for a period of time is an indicator of stability in a storage solution.

[0112] In some embodiments, one or more NB properties (e.g., in a composition or a storage solution) can be assessed using a method as understood by a person of skill in the art based on the present disclosure including, but not limited to, microscopy (e.g., light microscopy), nano tracking analysis, electrophoretic light scattering (ELS), dynamic light scattering (DLS), or a combination of methods. Based on the present disclosure, a person of skill in the art would understand which techniques and methods are appropriate for determining a given NB property in a storage solution.

[0113] In some embodiments, stability of NBs is affected by their density (e.g., in a storage solution or a composition). If the density of NBs exceeds a threshold value, NBs may coalesce or become unstable. In some embodiments, the density of NBs is related to the duration over which the NBs are produced. In some embodiments, the density of a plurality of nanobubbles is about 3.5x107bubbles per milliliter. In some embodiments, the density of nanobubbles is greater than 0.5 x 107bubbles per mL, greater than 1 x 107bubbles per mL, greater than 2 x 107bubbles per mL, greater than 3 x 107bubbles per mL, greater than 4 x 107Page 16 of 63 11877856v1Attorney Docket No. 2010363-0390bubbles per mL, greater than 5 x 107bubbles per mL. In some embodiments, the density of nanobubbles is less than 5 x 107bubbles per mL. In some embodiments, the density of nanobubbles is measured using a nano particle tracking analyzer, DLS, or another suitable technique (e.g., as described herein).

[0114] In some embodiments, NB stability can be affected by properties of a composition or a storage solution in which they are present (e.g., as discussed herein). Nanobubble Composition and Payload Substance

[0115] In some embodiments described herein, NBs may be used to add or deliver a payload substance to a composition. NBs can be used to deliver any suitable payload substance to a composition or as part of a composition.

[0116] In some embodiments, a payload substance can be carried on a surface of a NB (e.g., adhered to or adsorbed to the NB surface). In some embodiments, a payload substance can be inserted into a nanobubble (e.g., within the nanobubble). NBs have a high surface area per volume, which makes them useful for carrying one or more payload substance on or close to their surface. In some embodiments, a payload substance is adhered to or adsorbed to the surface of the NBs. In some embodiments, a payload substance is a gas carried by NBs.

[0117] In some embodiments, a payload substance can be carried by multiple nanobubbles (e.g., through attachment of multiple nanobubbles to a surface). For example, with nanoplastics, attachment of multiple nanoparticles to surface(s) of an object may help to float / raise the nanoparticles to the surface of a composition (e.g., water).

[0118] In some embodiments, NBs have a charge (e.g., as measured by a zeta potential). In some embodiments, the charge of NBs can be changed based on the composition in which the NBs are present as described herein. In some embodiments, altering a composition of the NBs (e.g., the gas composition, e.g., the percentage and / or ratios of gases) alters the NB charge.

[0119] In some embodiments, NBs are destabilized or disrupted in order to release a payload substance. For example, destroying NBs (e.g., through sonication, salt- destabilization, or other mechanical or chemical techniques) releases their payload substance(s). In some embodiments, NBs can be destroyed at an interphase (e.g., to release a payload substance).

[0120] As discussed herein, NBs are ultrafine gas domains in liquid with nano-scale diameters that are, on average, smaller than 1000 nm diameter (e.g., about 900nm or less, Page 17 of 63 11877856v1Attorney Docket No. 2010363-0390about 800 nm or less, about 700 nm or less, about 600nm or less, about 500nm or less, about 400nm or less, about 300 nm or less, about 200nm or less, about 100 nm or less, about 50nm or less). In some embodiments, the longer NBs are produced in a composition (e.g., a liquid), the smaller the generated NBs become over time.

[0121] NBs can be generated or formed using any suitable gas or mixture of gases. The makeup of the NB gas depends on the application of the NBs and requirements of the NBs. In certain embodiments, for example, NBs comprising CO2increase CO2gas availability and delivery rates with notably higher buffering capacity. In other embodiments, NBs may be used to generate reactive oxygen species (ROS) and therefore contain oxygen and / or ozone. In some embodiments, the NBs may be inert (e.g., nitrogen nanobubbles).

[0122] In some embodiments, NBs comprise a single, substantially pure gas (e.g., an elemental gas or substantially a single gaseous compound). In some embodiments, NBs comprise an elemental gas (e.g., hydrogen, nitrogen, oxygen, a noble gas, etc.). In some embodiments, NBs comprise ozone or carbon dioxide (CO2). Nanobubble Generation

[0123] Nanobubbles can be generated using any suitable method to obtain NBs of an appropriate size, makeup (e.g., of gases), and density in a composition (e.g., as shown in FIG. 1). In some embodiments, multiple populations of NBs (e.g., having different diameters, sizes, gas compositions) can be generated for use in a particular application. In certain embodiments and without limitation, NBs are generated using hydrogen peroxide (e.g., for micro-gravity applications, for off-grid applications), nozzle decompression, ultrasonic generation, venturi bubble generation, spiral flow mixing, static and dynamic mixing, ceramic oxygen generator (COG) (e.g., a solid state COG) (e.g., under microgravity conditions) or hammer-rotation (e.g., magnetic hammer rotation). Devices and systems are described herein for generating

[0124] A person of skill in the art will be familiar with NB generation techniques useful in the methods and systems provided herein based on the current disclosure. Nanobubbles on Surfaces

[0125] In some embodiments, NBs are produced and associated with a surface of a substrate. For example, NBs that are attached to a surface can increase adsorption of dissolved oleophobic compounds or can bridge suspended solids via hydrophobic interactions. Additionally, advanced oxidation processes (AOPs) are often coupled with Page 18 of 63 11877856v1Attorney Docket No. 2010363-0390adsorption. Adsorbent surfaces can provide scaffolding for catalysts, amphoteric intermolecular electron shuttling, and transformation product trapping benefits.

[0126] In some embodiments, NBs can be produced (e.g., in a first solution) and then contacted with a surface of a substrate.

[0127] In some embodiments, NBs can be generated on a surface of a substrate using any suitable method (e.g., as disclosed herein). For example, as shown in FIG.1, NBs can be produced such that they adhere or adsorb to a surface of a substrate. In FIG.1, water is used as an exemplary composition, however the composition may be any suitable liquid(s) for forming and / or retaining NBs. In some embodiments, NBs can be generated on a surface of a substrate using one or more of the following methods: water-solvent exchange, differential temperature, differential pressure, ultrasonication, mechanical forces, and electrolytic evolution.

[0128] In some embodiments, a substrate with which NBs are associated with is a hydrophobic substrate. In some embodiments, a hydrophobic substrate is an activated carbon substrate or graphene (e.g., graphene oxide) (e.g., a sheet of graphene, e.g., a graphene nanosheet). Binding NBs to graphenes can change their aggregation behavior (graphene-NB sandwich structure) and influence their dynamic bundle formation in water resulting in targeted adsorptive ability in complex solutions by controlled molecular size-straining.

[0129] In addition, without wishing to be bound to any particular theory, NBs can attach to graphene surfaces and remain intact because they have low mass and their movement is dominated by Brownian motion that may not overcome the surface tension at interfaces. In some embodiments, NB-attached graphitic adsorbents improve adsorption of synthetic organic compounds via intermolecular interactions at the adsorbent-bubble-liquid interfaces.

[0130] For example, a significant increase in amphiphilic perfluorooctanesulfonate (PFOS, an organic pollutant) removal by carbon-based adsorbents was found when air bubbles formed on a surface of the carbon-based adsorbent. Without wishing to be bound to any particular theory, the polar sulfonic head of PFOS orients toward the water phase while the polarizable fluorinated alkene chain aligned toward air-NBs on the hydrophobic carbon surfaces.

[0131] In some embodiments, NB-coated graphene substrates are used to adsorb organic compounds to a surface. NB formation on a graphene substrate is governed by the interactions between the π-electrons of a graphitic surface, H-bond accepting / donating polar functional groups, and the negative charge of NBs. Without wishing to be bound to a Page 19 of 63 11877856v1Attorney Docket No. 2010363-0390particular theory, the π-electron density of graphitic surfaces promote induced polar interactions between the available electrons of gas molecules (e.g., from oxygen) in NBs. Similarly, permanent dipole moment of oxygen-containing functional groups may promote cluster formation around them via Lewis acid / base interactions.

[0132] In some embodiments, advanced electron microscopy, Raman spectroscopy, or FT-IR (Fourier-transform infrared) spectroscopy is used to assess structures and molecular interactions on a substrate (e.g., a graphene surface, e.g., a pristine or exhausted graphene surface). Considering the heterogeneity of graphene substrates (e.g., due to folds, wrinkles, and / or defects), multiple, different molecular interactions with a substrate can govern NB formation.

[0133] In some embodiments, NBs are removed from a surface of a substrate (e.g., to remove a payload substance associated with the NBs). In some embodiments, NBs on a surface of a substrate become associated with (e.g., bind to) target substance(s) (e.g., pollutants) in a composition. A target substance can be removed along with NBs from a surface of a substrate by disrupting a bond between the substrate and NBs. For example, NBs can be removed or released from a surface of a substrate using sonication or salt- destabilization. In some embodiments, NBs are destroyed, thereby releasing a target substance or a payload substance (e.g., from the substrate). Nanobubble Water / Waste Fluid Treatment

[0134] In some embodiments, NBs can be used to treat a composition (e.g., an effluvent, water, e.g., wastewater) containing one or more target substance. As used herein, “target substance” refers to any molecule, entity, or other discrete substance that a user wishes to add to or remove from a composition.

[0135] In some embodiments, NBs are used to treat wastewater. In general, wastewater is any liquid or liquid-solid mixture which has been used in a process which introduces one or more target substances (e.g., pollutants). In some embodiments, NBs can be used to treat any suitable composition which contains a target substance. In some embodiments, wastewater comprises sludge. Sludge is a residual, semi-solid material that is produced as a byproduct of sewage treatment. In some embodiments, sludge comprises inorganic and / or organic matter. In some embodiments, sludge comprises microorganisms (e.g., bacteria).

[0136] In some embodiments, NBs are used to treat a composition containing one or more target substances. For example, target substances can include, but are not limited to, an Page 20 of 63 11877856v1Attorney Docket No. 2010363-0390organic pollutant (e.g., synthetic organic pollutants), an organosilane, urea, a perfluorochemical, a microorganism, a short chain fatty acid, a sugar, a protein, a surfactant, a nanoparticle (e.g., plastic particles, e.g., nanoplastics), a dye (e.g., a color-causing chemical), a colloid, Methyl-Isoborneol (MIB), and Geosmin. In some embodiments, an organic pollutant is one or more of the following compounds or an equivalent or derivative thereof: naphthalene, 1-chloronapthalene, 1-napthol, 1,4-dichloronaphtalene, 1,8- dichloronaphtalene, phenanthrene, anthracene, pyrene, biphenyl, thrichloroethylene, tetrachloroethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane, carbon tetrachloride, 1,1- dichloroethylene, 1,2-dichloropropane, methylene blue, bisphenol A, carbamazepine, phenol, benzoic acid, atrazine, perfluorooctanesulfonate (PFOS), phenanthrene, PFOS / PFOA, and TCE.

[0137] In some embodiments, NBs are used to treat compositions containing nanoparticles. In some embodiments, nanoparticles may include plastics (e.g., nanoplastics) including, for example, polystyrene, a latex, polyethylene tetrephtalate (PET), or another type of plastic and combinations thereof. In certain embodiments, nanoparticles are less than 1μm in size (e.g., 100nm or less) in size (e.g., diameter).

[0138] In some embodiments, NBs may be used to float particles (e.g., nanoparticles) to the surface of a solution (e.g., a wastewater solution) through, for example, aggregation of the particles and bubbles (e.g., heteroaggregation).

[0139] In some embodiments, certain properties of a solution can have an effect on NB generation, stability, and / or the ability of nanobubbles to remove target substances from a solution. Parameters including, but not limited to, pH and distribution of nanobubbles in the composition may alter removal of target substances. In certain embodiments, coagulants (e.g., alum), pH altering substances, and surfactants may be added to a solution to enhance removal of target substances. In certain embodiments, the pH of the composition can be reduced (e.g., to a pH of about 9 or lower, to a pH of about 6 or lower, to a pH of about 3) to enhance removal of a target substance. In certain embodiments, the magnitude of a zeta potential of target substances can be reduced and / or neutralized to, for example, enhance removal of target substances. For example, in certain embodiments, the zeta potential is about -40mV or less (in absolute value) about -33.8mV or less, about -25mV or less, about -16.7mV or less. Coagulants, pH altering substances, and surfactants can be used to reduce the zeta potential of the target substances including, for example, nanoparticles (e.g., nanoparticles) to enhance removal. For example, coagulants can be an aluminum based coagulant (e.g., aluminum Page 21 of 63 11877856v1Attorney Docket No. 2010363-0390sulfate, aluminum chloride, sodium aluminate) or an iron based coagulant (e.g., ferric sulfate, ferric sulfate, ferric chloride, ferric chloride sulfate, polymeric ferric sulfates).

[0140] In some embodiments, NBs are used to alter the composition of bacteria or biofilm. In some embodiments, NBs decrease an abundance (e.g., number, density) of filamentous bacteria, mycobacterium, or floc forming bacteria (e.g., Corynebacterium, Pseudomonas, Zoogloea) in the wastewater solution (e.g., as compared to coarse bubble treated wastewater)(e.g., as compared to wastewater not treated with nanobubbles).

[0141] In oxygen deficient environments, filamentous bacteria gain an advantage over non-filamentous bacteria because their tentacles can reach more oxygen. When filamentous bacteria is predominant, wastewater treatment fails because the sludge does not settle down in time. If nanobubbles are applied and oxygen deficiency is overcome, then the filamentous bacteria growth is suppressed. In certain embodiments, NBs improve sludge settling ability by suppression of filamentous bacteria (e.g., by increasing access to oxygen for other populations of bacteria). In certain embodiments, NBs alter formation of microbial flocs, presence of individual species of microbes, and disposition of microbes in a floc (e.g., aerobic outer shell vs. anaerobic interior or core of a floc). Reactive Oxygen Species Generation

[0142] The metastable character of NBs results in highly reactive nano-interfaces of gas that can react with water molecules generating different ROS including short-lived radical species. In some embodiments, the stability of NBs can sustain a continuous and / or controlled yield of ROS.

[0143] In some embodiments, NBs are used to generate reactive oxygen species (ROS). In some embodiments, ROS are identified and / or quantified by electron parametric resonance (EPR). In some embodiments, samples containing generated NBs are added to a solution containing 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) as spin-trapping molecule that reacts with radicals and stabilizes the radicals. In some embodiments, samples are transferred to a 2 mm thin wall precision quartz EPR sample tube and analyzed under continuous wave EPR in a cylindrical mode resonator with a magnetic field modulation frequency 100 kHz, field modulation amplitude of 0.1 mT peak-to-peak, and microwave power of about 4 mW and a frequency of about 9.74 GHz. In some embodiments, other probe compounds of known reactivity with ROS and defined kinetic rate constants are used. In some embodiments, NBs comprising essentially nitrogen will not produce a ROS. Page 22 of 63 11877856v1Attorney Docket No. 2010363-0390

[0144] In some embodiments, quick scavenger testing can be used to detect the formation of ROS. In some embodiments an agent that is reactive with ROS (e.g., terephthalic acid, coumarin, p-chlorobenzoic acid) is used to aid in and / or verify the presence of radical ROS. In some embodiments, a nanobubble generator is (e.g., as described herein, e.g., an Acniti bench-top model) is used for quick scavenger testing. In some embodiments, chromatography is used to determine formation of hydroxylated by-products and / or disappearance of a parent compound. In some embodiments, phase-reverse C-18 columns coupled to a photodiode array detector are used to detect the formation of ROS.

[0145] Different advanced oxidative processes (AOPs) require activation. For example, AOPs can be activated by metallic catalysts (e.g., iron (II) for Fenton processes), UV light irradiation (e.g., H2O2 / UV) or ultrasound (e.g., advanced cavitation). Accordingly, in some embodiments, an activator is used to alter production of ROS using NBs (e.g., oxygen NBs, ozone NBs). In some embodiments, NBs do not require the use of AOPs to generate ROS. Exemplary Uses of Nanobubbles in Microgravity Conditions

[0146] In some embodiments described herein, nanobubbles (NBs) can be used to remove one or more target substances from a composition. For example, the Water Processing Assembly (WPA) of International Space Station (ISS) utilizes oxygen gas to remove organic pollutants and disinfect the cabin’s drinking water. However, in a microgravity environment, separation of bubbles after their use is a tedious step. Nanobubbles (NB) have the potential to increase the gas delivery potential while eliminating the need for subsequent gas removal due to their minuscule size. In other words, NB solutions can be dramatically more reactive than biphasic solutions (e.g., oxygen microbubbles in water). Additionally, in some embodiments, compositions comprising NBs (e.g., NB solutions) can be pumped, piped and stored as single-phase incompressible fluids.

[0147] NBs are ultrafine domains of gas within liquid. In some embodiments, NBs are bubbles smaller than 1000 nm in diameter (e.g., 800nm or less, 700nm or less, 600nm or less, 500nm or less, 400nm or less, 300nm or less, 200nm or less). In some embodiments, the NBs are about 100 nm in diameter. In some embodiments, NBs can be generated by energy efficient fluidic oscillations or other low-cost hydraulic, physicochemical and electrochemical techniques as shown in, for example, FIG.1.

[0148] In some embodiments, NBs and coexist with reactive oxygen species. In some embodiments, stable NBs combined with their catalyst-free oxidation potential can be an inflection point of a paradigm shift for advanced water treatment technologies. Outstanding Page 23 of 63 11877856v1Attorney Docket No. 2010363-0390properties of NBs (e.g., high surface area, stability, and tunability) can be used to develop energy-efficient, small foot-print water treatment systems without the need for gas separation seemly for life support subsystems. Without limitation, these subsystems are useful in, for example, the aerospace industry and can improve conventional water treatment (e.g., drinking water) and wastewater treatment technologies on Earth.

[0149] Without wishing to be bound to any particular theory or limiting other notable benefits, NBs have a number properties that are desirable. For example, extremely large gas- liquid interfaces of NBs can lift practical restrictions of oxygen delivery by super-saturating the NB solution. The use of NBs can revolutionize oxygen mass transfer in biotic (e.g., activated sludge) and abiotic (e.g., advanced oxidation) processes. Additionally, NBs that are attached to surfaces can increase adsorption of compounds (e.g., dissolved oleophobic compounds). In some embodiments, NBs can bridge suspended solids via hydrophobic interactions. In some embodiments, the composition or payload substance of NBs can be adjusted. In some embodiments, air, oxygen, or ozone NBs can facilitate production of reactive oxygen species (ROSs). Without limitation, ROSs include short-lived and quasi- stable radicals for advanced oxidation applications.

[0150] The NB generation conditions influence NB properties. For example, without limitation, dissolved oxygen concentration, pH, viscosity, and behavior (mass transport, adsorption, reactivity) of NBs are affected by conditions under which NBs are generated and / or stored. In some embodiments, a NB generator is used to generate NBs in a composition or a storage solution. For example, an Acniti bench-top model (e.g., MicroStar FS-302AC-SW1 Nanobubble Generator). In some embodiments, NBs are produced via hammer-rotation technique (e.g., using an Acniti bench-top NB generator). The MicroStar FS-302AC-SW1 Nanobubble Generator is rated up to 29 psi gas pressure for rapid screening experiments suitable for rapid shifts in gas source and small-scale solution chemistry parametric testing. In some embodiments, a Moleaer customizable NB generator is used for NB generation. The Moleaer (25 XTB Nanobubble Generator) is capable of generating NB solutions at meso-scale 25 gal / min liquid flowrate. In some embodiments, a NB generator is engineered to operate at gas pressures up to 140 psi. In some embodiments, a ceramic oxygen generator (COG) is coupled to a NB generator.

[0151] In some embodiments, a COG is a solid-state COG. Solid state COGs can sustain safe and high-purity (e.g., greater than 99.9% purity) oxygen production. In some embodiments, a COG facilitates oxygen storage at pressures ranging 100psi to 200 psi. In some embodiments, a COG is an off-grid (e.g., in environments without ready-access to) Page 24 of 63 11877856v1Attorney Docket No. 2010363-0390source of continuous oxygen production. In some embodiments, a COG is a part of a life support system (e.g., on a space station, on a space shuttle).

[0152] In some embodiments, dynamic light scattering (DLS) is used to detect the zeta potential of NBs. Without wishing to be bound to any particular theory, the zeta potential is important to evaluate electrostatic-driven NB interactions. In some embodiments, without limitation, product solution characteristics such as pH, temperature, viscosity, oxidation- reduction potential, zeta potential, dissolved oxygen, adsorption, and reactive oxygen species generation are correlated to NB generation parameters. In some embodiments, NBs can be generated using varying gas pressures (e.g., 10 psi -140 psi) and generation durations (e.g., seconds, minutes, hours, or longer).

[0153] In some embodiments, advanced oxidation processes are coupled with adsorption. In some embodiments, adsorbent surfaces can provide scaffolding for catalysts, amphoteric intermolecular electron shuttling, and transformation product trapping benefits.

[0154] NBs can attach to graphene surfaces and remain intact because they have low mass and their movement is dominated by Brownian motion that may not overcome the surface tension at interfaces. NB-attached graphitic adsorbents can improve adsorption of synthetic organic compounds via intermolecular interactions at the adsorbent-bubble-liquid interfaces. The polar sulfonic head of PFOS was speculated to orient toward the water phase while the polarizable fluorinated alkene chain aligned toward air-NBs on the hydrophobic carbon surfaces. Non-destructive pollutant-transport interactions in the presence of NBs.

[0155] In some embodiments, target substances or payload substances include highly volatile compounds. Highly volatile compounds can accumulate inside NBs. In certain embodiments, and be removed after destabilization of the NBs. In certain embodiments, ultrasonic and salt driven destabilization are used to remove NBs from a composition. In certain embodiments, NBs can promote surface bridging, and bridging can change adsorption of pollutants by substrates (e.g., structured graphene nanosheets).

[0156] In some embodiments, graphene nanosheets are used as a substrate. Graphenes have stacked-sheet-like 2D structures with densely conjugated basal plane and dynamic “house-of-cards” bundle structure in water. Binding NBs to graphenes can also change their aggregation behavior (graphene-NB sandwich structure) and influence their dynamic bundle formation in water resulting in targeted adsorptive ability in complex solutions by controlled molecular size-straining. Therefore, the aggregation behavior of NB attached graphenes can be monitored by DLS as a model nano-adsorbent. Page 25 of 63 11877856v1Attorney Docket No. 2010363-0390Exemplary Uses of Nanobubbles

[0157] Nanobubbles can be used to enhance any process in which delivering a gas to a liquid more efficiently would improve a process. Additionally, NBs are useful in processes which require delivery of a payload substance. Understanding that NBs can be efficiently produced (e.g., as described herein) such that they are more energy and resource efficient, NBs are useful in many different applications. NBs can be useful in a multiple fields including, but not limited to, military, space / microgravity, aquaculture, water treatment (e.g., wastewater treatment), delignification, and paper production, among other applications.

[0158] In aquaculture there are several barriers that prevent its sustainable implementation and further global expansion. These obstacles are due to four main factors: (i) continuous and increasing production supply; (ii) insufficient biosecurity measures to control waterborne animal diseases; (iii) the exacerbated use of water resources; and (iv) the high environmental impact of effluents. Without wishing to limit other notable benefits, NBs may be an effective solution to solve these challenges. As discussed herein, NBs can remain suspended for days to months, enabling more efficient gas delivery and can produce hydroxyl radicals and other reactive oxygen species (ROS), among other benefits

[0159] For example, in aquaculture, NBs can enhance delivery of gases to water columns. In some embodiments, NBs can contribute to effective and sustainable oxygen delivery to dense biotic systems (e.g., those containing fish, seaweed, mussels, oysters, or other aquatic organisms). In some embodiments, effects NBs delivery to biotic systems can yield measurable effects on them including, but not limited to, higher productivity, increased growth rate (of organisms, e.g., fish, seaweed, mussels, oysters, etc.), increase in survival rate (of organisms), and increased oxygen consumption (e.g., in fish and shrimp farming). In some embodiments, animal weight gain (kg), differential size measurements (m), total tank harvest (kg), productivity per unit surface area (kg·m-2), specific growth rate (% d-1), higher survival rate (%), lower oxygen consumption (mg·kg-1product), economic feasibility, and erythrocyte and leukocyte count in animal blood may be used to evaluate and / or measure the effectiveness of NB delivery on organisms. In some embodiments, NBs may be used to reduce pH (e.g., to a pH of about 7). Without wishing to be bound to any particular theory, neutral (e.g., a pH of about 7) or moderately low water pH could improve fish growth compared to alkaline conditions by shifting fish metabolites to less toxic substances. A high pH can increase the amount of available free ammonia, which can be toxic to many aquatic organisms. A low pH can reduce the toxic effects of ammonia, as well as the rate of bacterial nitrification. In some embodiments, NBs may have a positive effect on probiotics in animal Page 26 of 63 11877856v1Attorney Docket No. 2010363-0390guts. For example, NBs can induce beneficial effects on shrimp gut by increasing the proportion of probiotics (e.g., probiotic organisms)(e.g., Rhodobacter, Oscillospira, and Faecalibacterium) and / or inhibiting harmful pathogenic bacteria (e.g., Vibrio parahaemolyticus). Without wishing to be bound to any particular theory, probiotics promote better digestion, inhibit harmful pathogenic bacteria (e.g., Vibrio parahaemolyticus), promote better digestion, and improve immune functions, which could also result in a lower frequency of disease and / or antibiotic use. Additionally, NBs may be used to treat shrimp for diseases such as, for example, acute hepatopancreatic necrosis. In some embodiments, NBs can be used in addition to antibiotic treatments.

[0160] In some embodiments, NBs can be used to aid in recovery of spent (e.g., waste) waters (e.g., in aquaculture) and / or using advanced oxidation. For example, pollutants may be degraded using air and / or ozone containing NBs. In some embodiments, NBs are used with recirculating aquaculture systems. In some embodiments, NBs are used to react with drugs (e.g., antibiotics) used in aquaculture applications to degrade and / or inactivate (e.g., via advanced oxidation processes) drugs in the systems prior to release into an environment. For example, certain drugs including, for example, oxytetracycline can react with and / or be degraded by NBs (or byproducts of NBs). In some embodiments, NBs are used to treat (e.g., disinfect, clean) water (e.g., wastewater) from aquaculture prior to release into an environment. In certain embodiments, NBs can be used to remove unpleasant taste and odor compounds including, but not limited to, 2-methylisoborneol and geosmin, which can make food unpalatable and inedible. In some embodiments, NBs can be used to decompose hydrogen sulfide. In some embodiments, NBs can be used to disinfect water from, for example, harmful bacteria (e.g., Vibrio parahaemolyticus, Aeromonas hydrophila, A. hydrophila BT14, mycobacteria, e,g, Mycobacterium chelonae) and / or viruses (e.g., Spring viremia of carp virus (SVCV)).

[0161] In some embodiments, NBs are used to clean membranes (e.g., used in aquaculture). For example, NBs can be used to decrease membrane fouling by degrading fouling substances that collect on the membranes and / or prevent fouling substances from adhering to membranes in order to improve membrane permeability, decrease rejection of antibiotics, and improve antifouling performance. In certain embodiments, the bulk NBs moving through a channel (e.g., a feed channel) induce turbulent flow, producing a uniform flow circulation and increasing the shear forces at the membrane surface. Shear forces due to NBs can hinder the accumulation and promote the disloging of fouling substances. Without wishing to be bound to any particular theory, pyrolytic decomposition that occurs due to Page 27 of 63 11877856v1Attorney Docket No. 2010363-0390‘bubble collapse’ may lead to formation of ROS, localized temperature increase, and detachment of adsorbed foulants on membranes, which increases the lifetime of membranes.

[0162] In some embodiments, NBs can be used to disinfect water (e.g., in aquaculture, for drinking, or other uses). For example, the high mass transfer efficiency of particles can be used to generate hydroxyl radicals and other reactive oxygen species (ROS) as described herein, which are useful in disinfecting water. In some embodiments, NBs containing oxygen and / or ozone may be used to control bacteria (e.g., Vibrio parahaemolyticus, Aeromonas hydrophila, A. hydrophila BT14, mycobacteria, e,g, Mycobacterium chelonae) and viruses (e.g., Spring viremia of carp virus (SVCV) (e.g., thereby increasing the survival rate of organisms including fish and shrimp).

[0163] In some embodiments, NBs alter and / or effect animal health (e.g., for aquatic animals) by, for example, modulating immune responses. In certain embodiments, NBs modulate (e.g., activate) immune response of animals. Innate immunity is a critical systemic response that inhibits pathogens and maintains homeostatic interactions. This is the first line of defense against infection, and therefore, gene expression of immune-related genes are important indicators of the animal’s response following a pathogenic infection. For example, in some embodiments, ozone NB exposure, results in an up-regulation of pro-inflammatory cytokines and lysozyme genes.

[0164] In another example, NBs are useful in paper production and processes. For example, oxygen bleaching is an environmentally sustainable method for whitening wood pulp prior to papermaking. The low solubility of oxygen in water is a key technical hurdle, which NBs can be used to overcome. In certain embodiments, NBs are used to

[0165] In another example, the ability to store NBs for a long period of time (e.g., in a storage solution) is useful for off-grid conditions or locations where access to resources is limited. For example, NBs can be stored in a storage solution for long periods of time for use in, without limitation, long range ground transportation, field missions (e.g., in a battleground), remote terrestrial expeditions, space expedition missions, and the like.

[0166] In another example, NBs are useful in cleaning surfaces and / or filters. For example, NBs can be generated to clean surfaces and / or filters of bathtubs, jacuzzies, and hot tubs. In some embodiments, NBs can be used to clean difficult to access places (e.g., within plumbing) within plumbed appliances. Page 28 of 63 11877856v1Attorney Docket No. 2010363-0390Experimental Example 1: Nanobubbles for Wastewater Treatment

[0167] In the present example and embodiments discussed herein, the disclosed technologies relate to treatment of wastewater and aeration of activated sludge. Findings presented herein show that NBs have the potential to curtail the loss of oxygen during activated sludge aeration due to their extensive surface areas and lack of buoyance in solution. In this study, NB aeration is explored as an approach to enhance aerobic activated sludge treatment and benchmarked against coarse bubble aeration at the lab scale. NB aerated activated sludge reactors achieved greater dissolved oxygen levels at faster rates. Additionally, soluble chemical oxygen demand was decreased by 10% when compared to coarse bubble (CB) aeration.

[0168] Aeration of activated sludge through pore diffusers provide air in the form of macrobubbles (e.g., ~ 5 mm in diameter) to supply oxygen for aerobic microorganisms. Smaller bubble diameters at the micrometric size (e.g., microbubbles) can increase the contact surface area and mass transfer coefficient, leading to a faster rise in the dissolved oxygen concentration. However, these microbubbles suffer from buoyancy forces and short contact times in comparison to nanobubbles (NBs).

[0169] Higher oxygen availability can accelerate the metabolic rates of aerobic microorganisms. A recent study reported DO (dissolved oxygen) concentrations as high as 40 mg L-1, with O2NBs, greatly surpassing the air saturation concentration of 8.26 mg L-1at 25°C and 1 atm. High DO levels maintained in solution for 24 h can augment biomass production, increase the aerobic fraction of biofilms, and substantially change the microbiome composition in an activated sludge reactor. On the other hand, an increase in DO can reduce waste activated sludge production by deeper oxygen penetration into the floc mass that can accelerate the aerobic decomposition of the biomass and ultimately decrease the sludge yield. Also, at high DO concentrations (higher than 4.0 mg L-1), anoxic zones of the flocs can collapse due to higher biological lysis rates and eventually cause enhanced digestion rates during subsequent sludge digestion steps. Therefore, a deeper understanding of the impact of NB aeration was necessary to understand its immediate and long-term effects on wastewater treatment process.

[0170] Additionally, the activated sludge produced compact sludge yielding easier waste sludge for subsequent sludge handling. The samples showed fewer filamentous bacteria with a lower relative abundance of floc forming Corynebacterium, Pseudomonas, and Zoogloea in the sludge. The microbiome of the nanobubbletreated activated sludge showed Page 29 of 63 11877856v1Attorney Docket No. 2010363-0390significant shifts in the abundance of community members at the genus level and significantly lower alpha and beta diversities. Materials and Methods

[0171] A 7.5-gallon (28.4 L) cylindrical linear low-density polyethylene (LLDPE) biological tank reactor was filled with nanopure water (18.2 MΩ cm) and aerated for 1 h with NBs. An Acniti Microstar Nanobubble Generator (Model-FS302AC-SW1, 300 W) generated NBs in situ, using the reactor assembly shown in FIG.2. Power consumption was measured by the P3 International Kill A Watt™ electricity usage monitor (Model P4400). The NB generator was operated at 50 Hz speed, delivering airflow at a rate of 376 mL s-1in the 22°C- 27°C temperature range. Samples (60 mL) were withdrawn at 0, 2, 5, 8, 10, 30, and 60 min time intervals for analysis. Dissolved oxygen was measured simultaneously at the sampling times using a YSI 5100 DO meter (Probe: YSI 5905 gold / silver electrode). The oxygen saturation levels were computed using USGS DOTABLES online calculator (United States Geological Survey, 2008). Based on the two-film theory as a model for mass transfer between phases, O2 mass transfer in nanopure water can be estimated assuming a non-reactive system. The volumetric oxygen transfer coefficient (^^^) is expressed in the below equation: ln[(^^^^− ^^) / (^^^^− ^^)] = ^^^^

[0172] where Csat is the O2 saturation concentration at a given temperature, CL is the O2concentration in the bulk solution at time t, C0is the initial concentration of O2, and t is the aeration time. Media pH was monitored and measured using YSI Multilab probe 4110. Control aeration was performed in parallel with an identical setup, bypassing the NB generator with the same gas loading by bubbling air via coarse bubbles (CB). Both NB and CB aeration provided similar turbulent mixing regimes within the reactor. Air for the experiments was provided by the compressor located at the University of Maine, Orono, ME, USA (Dayton Electric Mfg. Co., Model: 4B237, capacity: 136.4 L, distribution network: 1′′ copper pipe regulated to 45 psi).

[0173] The NB size distribution and concentrations were measured for samples aerated up to 6h. Collected aliquots in borosilicate bottles stoppered without bottle headspace at 0, 2, 10, 30, 60, 180, and 360 min time intervals were analyzed for NB size distribution and concentration. The data corresponding to each time were evaluated using a Nanoparticle Tracking Analyzer (NTA) from Malvern Panalytical NanoSight NS300. Bubble diameters were calculated using the Stokes-Einstein equation as shown in the below equation: Page 30 of 63 11877856v1Attorney Docket No. 2010363-0390Δ^^(^)^^^4^= ^ =6^^

[0174] where kB is the × 10-23J K-1), T is the temperature in K (Kelvin), η is thes m-2, and r is the nanobubble radius in m. Based on the Brownian motion of the particle, the diffusion coefficient (D) was estimated using the mean square displacement of the particle measured by the NTA at a given time (t in seconds), as shown in the below equation: ΔR^= |r$(t) − r$(0)^| Effect of the nanobubble aeration on activated sludge

[0175] The effect of NB on activated sludge was assessed using fresh activated sludge samples obtained locally from the secondary activated sludge wastewater treatment plant located in Orono, Maine, USA. The activated sludge samples were mixed before the experiments, and measurements were started on the same day within 2 h in 22.7 L (5-gal) LLDPE reactors set up as illustrated in FIG.2. A difference between NB aeration and control reactors in the preliminary experiments was not observed because both initial soluble chemical oxygen demand (sCOD) values (<100 mg L-1) dropped to zero within the first sampling period. Therefore, to observe the impact of NB aeration, the activated sludge samples were spiked with 88.8 g of glucose (Sigma D (+) Glucose, 99.5%) to achieve an expected sCOD value of 5000 mg L-1prior to aeration. Theoretical oxygen demand for glucose mineralization was used to estimate the amount of glucose addition. A blank NB aeration experiment was performed by adding the same amount of glucose to nanopure water with no activated sludge to confirm that there was no change in glucose concentration. Aeration for the CB control was achieved by flowing air through silicone tubing (1 / 4´´ ID × 5 / 16´´ OD) into a glucose-spiked activated sludge sample. Airflow rates were constant throughout the NB and CB aeration experiment (376 mL s-1). Dependent variables such as sCOD, total solids (TS), 30 min settled sludge volume (SSV30), and SVI were measured to assess the efficiency of NB aeration according to the standard methods. For determining sCOD, approximately 10 mL aliquots (triplicate samples) from both NB aerated and CB- aerated solutions were taken at 0, 2, 4, 6, 8, 12, 18, 24, 30, 36, 42, and 48 h time intervals and filtered using glass fiber filters (Whatman; GF / F) to remove suspended solids. sCOD was determined following the standard method (Method 5220; Hach COD system, Hach company Loveland CO) using Hach 45,600 COD reactor and Hach DR 890 colorimeter. Page 31 of 63 11877856v1Attorney Docket No. 2010363-0390

[0176] In a separate set of experiments, the DO concentrations of the activated sludge systems were measured at 0, 2, 10, 30, 60, 120, 180, and 240 min time intervals under both NB and CB aeration without adding glucose using YSI 5100 DO meter. Analysis of sludge characteristics and microbial communities

[0177] Activated sludge samples from bioreactors aerated with NB and CBs were collected at predetermined time intervals (0, 24, and 48 h) and examined through a Nikon Eclipse E 400 phase-contrast microscope equipped with a Nikon Eclipse and Excelis video camera. The number of filamentous bacteria was counted in 5 distinct fields for each sample, and the average filamentous bacteria count across all fields was recorded. ImageJ software was used to count bacteria filaments. The analytical approach for counting the filamentous structures is described in the Supplementary Materials of Yaparante, 2022, Effect of air nanobubbles on oxygen transfer, oxygen uptake, and diversity of aerobic microbial consortium in activated sludge reactors, Bioresource Technology, 351, https: / / doi.org / 10.1016 / j.biortech.2022.127090, which is incorporated by reference with respect to the Supplementary Materials.

[0178] To investigate the effect of different aeration techniques on the microbial community, 10 mL of activated sludge samples were collected at t = 0 h (before aerating) and after 48 h aerating with both NBs and CBs. The samples were sent to Molecular Research LP (https: / / www.mrdnalab.com Shallowater Texas, USA) for microbial community analysis based on 16S rRNA. Genomic DNA was isolated from each water sample using DNeasy Power soil pro kit (Qiagen) according to the manufacturer’s instructions. A total of 3 replicates from the 0 h sample, 6 replicates from 48 h nanobubbled, and 6 replicates from coarse bubbled samples were processed. Amplicon sequencing was performed using next- generation sequencing technology (bTEFAP®) described by Dowd et al. (Dowd et al., 2008a; Dowd et al., 2008b). The V4 region of the 16 s rRNA was amplified using polymerase chain reaction (PCR) primer pair (i.e., 515F 5′-GTGYCAGCMGCCGCGGTAA-3′ (SEQ ID NO.1) and 806R 5′-GGACTACNVGGGTWTCTAAT-3′ (SEQ ID NO.2) ) in 30–35 cycle PCR in HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA). The Q25 sequence data were processed using MR DNA ribosomal and functional gene analysis pipeline (https: / / www.mrdnalab.com Shallowater Texas, USA). Zero radius operational taxonomic units (zOTU) were taxonomically classified using BLASTn (nucleotide Basic Local Alignment Tool) against a curated database derived from NCBI National Center for Biotechnology Information. The identified zOTUs were compiled into designated taxonomic Page 32 of 63 11877856v1Attorney Docket No. 2010363-0390levels with a relative percentage of sequences within each sample. Statistical analysis was performed by Molecular Research LP (https: / / www.mrdnalab.com Shallowater Texas, USA) using XLstat, NCSS 2007, “R” and NCSS 2010. Alpha and beta diversity were determined as described in previously published work using Qiime2 (Bolyen et al., 2018), which is incorporated by reference with respect to the description of alpha beta diversity analysis. Statistical significance reported for data analysis is defined at 95% level of significance. Results and Discussion Effect of nanobubble aeration on mass transfer efficiency

[0179] The levels of DO in nanopure water were monitored during aeration of the two tested conditions (NB and CB aeration) and compared against a passive system (no mixing and no aeration) open to the atmosphere but without active aeration through air bubbling. The no-aeration control showed no DO fluctuation over the 1 h experiment. FIG.3 shows that all three samples initially had similar DO levels within the range of 5.8–6.3 mg L-1. Under the experimental conditions (i.e., temperature: 26.3°C and atmospheric pressure: 760 mm Hg), this DO level corresponds to 72–78% oxygen saturation. Within 2 min, the DO level of CB aerated nanopure water increased from 6.3 ± 0.3 to 6.5 ± 0.1 mg L-1(i.e., 0.2 mg L-1increase) while NB-aerated nanopure water DO was increased from 5.9 ± 0.5 to 7.1 ± 0.3 mg L-1(i.e., 1.2 mg L-1increase). The NB aeration infused 1 mg L-1more oxygen into the nanopure water than aeration with CBs which shows 6 times faster DO elevation by NBs within the first 2 min of aeration. The DO level increase in the NB aerated water within 2 min was significant according to the one-way ANOVA test at 95% level of significance (p < 0.05), where DO increase in the CB aerated sample did not show statistical significance. These results indicate more effective gas transfer using NBs. Furthermore, following 10 min, both NB and CB aeration showed a linear increase in the DO levels. At 30 min, the DO levels for both tested conditions started to level off. After 1 h aeration, the DO level of NB water reached 7.9 ± 0.3 mg L-1while the DO level of coarse air bubbled water increased to 7.6 ± 0.1 mg L-1. Energy consumption measured for a 1 h NB generation experiment in nanopure water was 0.03 kW h. A 3.2-fold higher KLa value for the NB aerated (9.6 h-1) nanopure water was observed compared to CB aerated (3 h-1) samples. The DO level changes over time and oxygen transfer coefficients indicate that bubbling air NBs into the water can rapidly increase the DO level in less time than aerating with CBs. However, it should be noted that the NBs are gas pockets and not completely dissolved in water. Therefore, the DO measurements do not necessarily capture the actual benefit of oxygen Page 33 of 63 11877856v1Attorney Docket No. 2010363-0390present in the water in the form of NBs. These stable gas pockets may provide a relatively continuous supply of oxygen for systems biotically or abiotically starving for oxygen. Further investigation is necessary to quantify the impact of NB aeration by measuring total oxygen levels by distinguishing the utilization of dissolved gases vs. packed gases in NBs.

[0180] The pH of the nanopure water after 1 h for each of the three experimental scenarios is shown next to DO levels in FIG.3. Both NB- and CB-aerated water showed a lower pH value than non-aerated water. This low value can be attributed to the dissolution of CO2 along with oxygen due to the stirring of water during aeration, which might have altered the buffering capacity of the nanopure water. However, the difference in pH was not significant enough because both CB and NB reactors were mixing similarly and possibly dissolving CO2at similar rates. To confirm the presence of NBs in the DO experiments, an NTA was utilized, and NB size distribution and concentrations were obtained. The presence of complex sludge elements limits the analyses of NB content by nanoparticle tracking analysis, which must be conducted under particle-free clean solutions, such as nanopure water. However, results observed in nanopure water samples can provide an acceptable excess threshold approximation for the available air NBs in solution.

[0181] FIG.4 shows the concentration and size distribution of NBs produced over time. During generation, there was an increase in the concentration and a decrease in the average size of the NBs over time. The concentration of NB increases over time, as illustrated in FIG.5. Corresponding NB concentration and size values are also reported (see Supplementary Materials Section of Yaparante, 2022). It is important to note the 7-fold increase in average bubble concentration after a 360 min aeration with the nanobubbler, which showed a significant difference from the concentration at 0 min according to the one way ANOVA test at 95% level of significance (p < 0.05). The rapid elevation of DO level observed in FIG.3 can be attributed to this prompt generation of NBs, where the high surface area of these minuscule gas domains allowed the rapid transfer of gas into the solution until saturation was reached.

[0182] FIG.6 indicates the average diameter of NBs associated with the size distributions reported in FIG.4. An initial shrinkage in the average bubble diameter from 402 nm to 239 nm after 2 min was observed in nanopure water. After 10 min of NB generation, size distribution showed smaller NBs with approximately 125 nm and about 50 nm in diameter. At 30 min, peaks at approximately 100 nm and 150 nm were observed in the size distribution. A slight increase in the NB size may be due to the coalescence of smaller bubbles and Ostwald ripening. NB size distribution after 30 min showed more polydispersed Page 34 of 63 11877856v1Attorney Docket No. 2010363-0390characteristics (FIG.4) but a consistent size distribution in terms of the average diameter. NB aeration of nanopure water for 6 h exhibited the smallest average particle size and highest concentration among all the samples. The experiments were kept at a maximum of 6 h to mimic practical hydraulic retention times for activated sludge reactors, but an increase in aeration time can further increase the concentration of these NBs. These findings indicate that a longer NB generation time can produce more NB concentrations with smaller diameters, enhancing gas transfer into the liquid phase in activated sludge reactors. Effect of nanobubble aeration on the activated sludge process

[0183] Understanding the impact of NB aeration on activated sludge is important to elucidate mechanisms and possible applications in wastewater treatment. TS percentage, SSV30, and SVI values showed clear differences between NB and CB aerated samples. TS percentages were increased from 0.29 to 0.41% at the end of 48 h NB aeration vs.0.59% at the end of 48-hr CB aeration. This increase can be attributed to the rise of microbial community due to the added glucose at the point of aeration and remaining organic / inorganic matter. The slightly lower TS percentage for NB-aerated samples (0.41%) compared to CB aerated samples (0.59%) can be attributed to effective organic matter degradation by NB aeration. According to the one-way ANOVA test at 95% level of significance, this lower percent TS in the NB-aerated sample was statistically significant (p < 0.05) compared to CB- aerated samples. Increased DO concentration leads to higher cell mass lysis for activated sludge aeration with NB under aerobic processes. Therefore, the lower percent TS suggests that aerobic bacteria grow faster and enter the autolysis phase more rapidly due to effective NB oxygen transferability under NB aeration compared to CB aeration.

[0184] The percent removal of sCOD over 48 h is shown in FIG.7 for both aeration methods. Activated sludge aerated with NBs achieved 28.5% sCOD removal percentage within 2 h, while CB aeration only removed 9.7% sCOD at the same time interval. The efficiency of sCOD removal increased over time for both aeration methods, possibly due to greater oxygen delivery, especially at the relevant 4–8 h hydraulic retention time frames. After 12 h, sCOD removal efficiencies were consistently higher for NB-aerated samples. 10% higher sCOD removal was achieved with NB aeration of the activated sludge than CB aeration, after a 48 h period. This higher sCOD removal efficiency can be attributed to the NB-induced higher degradation of organic matter in the activated sludge. The results obtained from the blank experiment performed under NB aeration without activated sludge (glucose spiked nano pure water) showed that the sCOD level remained with no significant changes for 12 h period (see Page 35 of 63 11877856v1Attorney Docket No.2010363-0390 Supplementary Materials of Yaparante 2022). This finding indicates that NBs can rapidly increase the DO, and the DO is available for aerobic microorganisms to degrade dissolved organic matter in the sludge. The rapid DO elevation and higher oxygen retention ability in activated sludge (samples without added glucose) were also observed. DO changes for these samples are shown in the Supplementary Materials of Yaparante 2022.

[0185] During the first 2 min period, NB aerated activated sludge sample DO concentration was increased from 0.26 to 5.51 mg L-1while CB aerated samples only showed DO fluctuation from 0.23 to 0.21 mg L-1(26 times higher DO in NB aerated samples than CB aerated samples within 2 min). NB-aerated and CB-aerated sludge DO concentrations started to level off at 30 min. After 30 min, activated sludge aerated with NBs retained a DO level around 1.6–1.3 times higher than the CB aerated activated sludge consistently for the remainder of the experiment.

[0186] Table 1 shown below shows the average number of filaments counted by observing sludge samples from the microscope per 1 drop of activated sludge at 5 pre- determined sites of observation under the microscope. Table 1. Average number of filaments.times under CB aeration and NB aeration. Filamentous bacteria serve as the backbone of floc formation, providing a structure for floc formation and assisting solid–liquid separation. Therefore, activated sludge settling is most efficient when there is a moderate number of filamentous bacteria. When there are too many filaments, floc cannot settle, compacting poorly (bulking). When there are too few filaments, floc cannot be formed. Filamentous sludge bulking deteriorates the effluent quality and leads to sludge washout with the treated effluent, disturbing the overall treatment system. In the 12 and 48 h observation intervals, more filamentous bacteria were detected in CB-aerated samples than the NB treated sludge. Without wishing to be bound to any particular theory, the lower abundance of filamentous bacteria in NB-aerated samples is attributed to more efficient DO delivery into the activated sludge than CB aeration. Tentacle-like filaments lose their advantage in the floc if DO is abundant and maintaining this morphology becomes burdensome; thus, well-aerated systems Page 36 of 63 11877856v1Attorney Docket No.2010363-0390 have less filamentous structures in their floc micro-assemblies. According to statistical analysis (two-tailed, homoscedastic t-test), the tests showed the average number of filaments for CB aeration vs. NB aeration are significantly different from each aeration method at 12 h and 48 h (p < 0.05). The growth of filamentous bacteria is favored by oxygen deficiency. Therefore, low DO has been identified as the primary cause of filamentous growth in activated sludge processes.

[0188] Furthermore, activated sludge flocs’ settleability, compressibility, and dewaterability are also affected by floc forming bacteria. If the growth rate of the floc forming species is too fast, dispersed, non-settleable floc formation occurs. Corynebacterium, Pseudomonas, and Zoogloea are considered a few of the most important floc forming bacteria identified in activated sludge. DNA analysis of the activated sludge samples before and after aeration confirmed that the percent relative abundance of these three flocforming bacteria diminished upon NB treatment. As shown in Table 2 below, the Corynebacterium, Pseudomonas, and Zoogloea percent relative abundance under NB treatment showed 0.018%, 0.07%, and 0.49% decrease, respectively, compared to the CB treated samples. This decrease for all three bacteria was significant according to the one-way ANOVA test at 95% level of significance (p < 0.05). Table 2. The genera percent relative abundance before aeration and after CB and NB aeration. Standard deviations are calculated based on analysis of three samples.

[0189] Without wishing to be bound to any particular theory, the floc forming bacteria abundance decrease in the case of NB aeration might be due to the impact of the NBs and shearing effects during the aeration with the nanobubble generator. Also, sludge settleability is affected by the competition between floc-formers and filamentous Page 37 of 63 11877856v1Attorney Docket No. 2010363-0390microorganisms. Therefore, it is important to compare the impact of DO supply through NB vs. CB on floc forming and filamentous microorganisms separately. Microbial community variation under NB aeration

[0190] Based on DNA sequence analysis, the relative abundances of the dominant phyla before aerating (0 h) were: 47.8% proteobacteria, 21.9% actinobacteria, 16.5% bacteroidetes, 3.4% firmicutes, 3.2%, planctomycetes, 2.9% chloroflexi, 1.6% nitrospirae and 0.5% chlamydiae. Among the zOTUs in the activated sludge samples, 356 genera were identified. Among those, 35 predominant genera were selected according to the higher relative abundance of each genus, as shown in FIG.9. During the 48 h aeration period, the relative abundance of some of the bacterial genera showed a decrease, and some were increased with time compared to the initial activated sludge sample (0 h). Among the changes, Aciditerrimonas, Acidovorax, and Zoogloea decreased with aeration, whether with NB or CB; however, the decreases were greater for NBs (i.e., NB 48 h). The genera Acidovorax and Zoogloea proved to be the most dominant in the oxygen limited enrichment. In aerobic conditions, the relative abundance of these bacteria were reduced. Mycobacterium increased with CB aeration and decreased with exposure to NBs. The growth of Mycobacterium species declined under anaerobic conditions due to the lack of metabolic oxygen. Furthermore, a decrease in Mycobacterium growth under high oxygen levels can be found due to oxidative stress.

[0191] Cytophaga, Devosia, Rhodobacter, and Tabrizicola each exhibited an apparent increase in the relative abundance in the aerated samples as compared to their initial concentration. Additionally, these were more abundant in the NB samples than CB samples. The increase can be due to the aerobic activity of those microorganisms. These differential changes in the microbial communities demonstrate that NBs generate different conditions in the floc and create different selective pressures in comparison to conventional aeration. These changes can be attributed to the elevated DO, reduced COD, BOD, zeta potential, localized high temperature, pressure, and hydroxyl radicals generated in NB-treated water. To the best of the inventors’ knowledge, existing NB aeration studies have not yet reported the impact of NBs on different microorganism species to evaluate the activated sludge quality under NB aeration.

[0192] A visual overview of relative abundance changes of the 35 predominant genera for all the trials of 0 h, CB 48 h, and NB 48 hr are shown in the double dendrogram of FIG.10. The x-axis shows the 3 trials of activated sludge samples before aeration, 6 trials of samples aerated with coarse air bubbles for 48 h (CB 48 h), and 6 trials of samples aerated Page 38 of 63 11877856v1Attorney Docket No. 2010363-0390with nanobubbles for 48 h (NB 48 h). The heat map represents the relative percentage of each genus, and these genera are shown along the right y axis. Clustering shown in FIG.10 clearly indicates the microbial communities resulting from the different aeration methods (CB 48 h vs. NB 48 h) are distinct from each other. Also, the microbial communities in both NB and CB samples differed from the 0 h samples.

[0193] Alpha diversity provides a measure of the diversity of microorganisms within a specific ecosystem and is usually expressed by the species richness. Statistical comparison of observed amplicon zOTUs and the Shannon diversity indices of the microbial diversity (alpha diversity) for each treatment group (before aeration and 48-h after NB and CB aeration) were conducted using Kruskal-Wallis pairwise comparisons. Shannon diversity boxplots are shown in FIG.11. According to the Kruskal Wallis pairwise comparisons, microbial diversity within the NB 48 h samples is significantly less than both 0 h and CB 48 h groups (p < 0.05). There was no significant difference in microbial diversity revealed between 0 h and CB 48 h groups (p > 0.05). Beta diversity provides a comparison of microbial community structure in different environments by creating individual phylogenetic trees without considering the taxonomy. This beta diversity analysis was done using a weighted UniFrac distance matrix. Principal coordinate analysis plots were used to show the data for beta diversity, as shown in FIG.12. A clear distinction between the three groups showed a significant difference between 0 h, CB 48 h, and NB 48 h samples (p < 0.05), in agreement with the clustering shown in FIG.10.

[0194] The significant difference in the microbial community and diversity demonstrated according to the taxonomic classification data (FIGs.9 and 10) and alpha and beta diversity analysis (FIGs.11 and 12) suggests that NB aeration has a considerable influence on the activated sludge microbiome activity. Furthermore, DO elevation, variations in TS, sCOD removal percentage, filamentous and floc forming bacteria abundance, and sludge settleability further confirm NBs’ ability to change the physicochemical characteristics in aqueous systems compared to CB aeration. The decline in filamentous and some of the floc forming bacteria and cloudier effluent in NB-aerated activated sludge samples implies that sludge settleability can be adversely impacted if NBs are used without a proper assessment of sludge quality parameters for extended periods. Conclusion

[0195] In situ NB aeration showed effective and rapid oxygenation of activated sludge. NBs reduced the abundance of filamentous bacteria and resulted in a lower settled Page 39 of 63 11877856v1Attorney Docket No. 2010363-0390sludge volume compared to CB aeration, potentially due to efficient oxygen supply into the activated sludge. A 10% higher sCOD was consumed within 48 h under NB aeration compared to CB aeration. Floc forming bacterial abundance was notably decreased, influencing the sludge settleability. Significant differences between NB and CB aerated samples’ microbiome were observed, indicating different selective pressures on the microbial consortium. Microbial diversity was significantly decreased in the NB aerated activated sludge. Experimental Example 2: Nanobubble Stability

[0196] The present example shows an embodiment which relates to determining the stability of nanobubbles (NBs) over a period of time.

[0197] FIG.13 is a diagram of a NanoSightTMNS300 configuration (courtesy of Malvern Panalytical). The NanoSightTMNS300 is used to analyze nanoparticles through nanoparticle tracking analysis. The NanoSightTMNS300 can be used to analyze the size distribution and / or concentration of nanobubbles in a sample. A microscope is positioned above a solution containing nanobubbles. A 50μm laser beam passes through the sample and nanobubbles. An example of the visualized nanobubbles through which the laser beam passes is shown in the top right panel of FIG.13. Under the sample, a metalized surface contacts a glass support.

[0198] FIG.14 is a series of graphs depicting NB stability in a solution over time. In panel A of FIG.14, the graph shows the number of nanobubbles (NBs) in solution as the number of bubbles per milliliter of fluid. The plot shows the change in the number of bubbles and bubble diameter over a 40 day period. In panel B of FIG.14, the bubble number density (bubbles ∙ mL-1) versus time (in days) is shown. In the experiment, bubbles remain in solution for a period of at least 40 days. The data in both plots were obtained using the NanoSightTMNS300 using a 0.5μm green laser beam under a microscope. Experimental Example 3: Evaluating generation capability of reactive oxygen species

[0199] Among other things, the present example relates to the effect of reactive oxygen species on organic matter, according to an embodiment.

[0200] The metastable character of NBs results in highly reactive nano-interfaces of gas that can react with water molecules generating different ROS including short-lived radical species. Among ROS, hydroxyl radical (●OH) and superoxide (O2● -), are strong oxidants that have high standard reduction potentials of 2.80 V vs standard H2electrode Page 40 of 63 11877856v1Attorney Docket No. 2010363-0390(SHE) and 1.65 V vs SHE, respectively. These radical oxidants are non-selective and because of their high reactivity, they are capable of degrading and mineralizing organic compounds and inactivating bacteria.

[0201] To demonstrate oxygen NB reactivity, hydrolization of organic matter (indicated by changes of soluble chemical oxygen demand in biosolids) in degassed ultrapure water was compared to a NB solution. A statistically significant (p < 0.05) 97% increase in soluble organic content after 5-minute stirring in oxygen NBs solution (FIG.15).

[0202] ROS species can be generated using NBs comprised of air. Without wishing to be bound to any particular theory, the generation of ROS is mostly associated to the oxygen gas interfacing with the aqueous solution since an O2 source is required to generate reactive oxygen species. Oxygen NBs can form ROS with no chemical addition and with significantly reduced energy input in comparison to energy intensive light-driven processes. Due to the stability of NBs, NBs can sustain a continuous and controlled yield of ROS. In some embodiments, ROS generation via nanobubbles is applied to advanced oxidation process (AOPs) for water treatment and sanitation. Experimental Example 4: 2-methylisoborneol (MIB) and Geosmin (GSM) Removal

[0203] The current example relates to the removal of MIB and GSM from a solution by using ultrasound on a composition containing NBs. MIB and GSM are naturally occurring compounds that impact taste and odor of water.

[0204] In the present example, the effect of NBs on the removal of GSM and MIB was tested. Samples were spiked with either 1000ng / L or 100 ng / L of GSM or MIB. Samples were taken at t=0 to determine the initial, measurable concentration of GSM and MIB in solution. Nanobubbles were generated in solution by an Acniti Nanobubble Generator. Mild ultrasonication was applied for 30 minutes (t=30) at isothermal conditions to NB solutions (e.g., substantially constant temperatures) using a Branson bath sonicator to remove NBs from NB solutions. The same ultrasonic treatment conditions were applied to no-NB control bottles containing distilled and deionized water (DI). The concentration of MIB and GSM in solution was measured by GC-MS (gas chromatograph – mass spectrometry).

[0205] FIGs.16A, 16B, 16C, and 16D show the effect of sonication and NB treatment on the removal of MIB and GSM from water. In FIG.16A and 16C, sonication of distilled, deionized water for 30 minutes (t=30) resulted in the removal of 16.9% of 100ng / mL GSM and 30.4% of 1000ng / mL GSM as compared to the initial sample taken at Page 41 of 63 11877856v1Attorney Docket No. 2010363-0390t=0. With NBs, 30 minutes of sonication of the NB solution results in removal of 48.9% of 100ng / mL GSM and 43.6% of 1000ng / mL GSM as compared to the initial sample. These results show that with GSM, NBs enhance removal.

[0206] In FIG.16B and 16D, sonication of distilled, deionized water for 30 minutes resulted in the removal of 30.3% of 100ng / mL MIB and 37.3% of 1000ng / mL MIB as compared to the initial sample taken at t=0. With NBs, 30 minutes of sonication of the NB solution (t=30) results in removal of 62.6% of 100ng / mL MIB and 50.5% of 1000ng / mL MIB as compared to the initial sample. These results show that with MIB, NBs enhance removal. Experimental Example 5: Effect of Ultrasonication on Solutions

[0207] The present example relates to the effect of ultrasonic treatment of solutions containing NBs and solutions without NBs.

[0208] In FIGs.17A and 17B, the effect of ultrasonic treatment of a solution containing NBs is shown. NBs were generated in a solution Acniti Nanobubble Generator. The samples containing NBs were subjected to treatment with sonication via a Branson bath sonicator. The NB concentration and diameter were measured using a Malvern Nanosight NTA (nanoparticle tracking analyzer).

[0209] In FIG.17A, the experiment shows that the NB concentration initially decreases when the sample containing NBs is subjected to sonication. The concentration of NBs decreases within the first few minutes of treatment, indicating that sonication contributes to the removal of NBs. However, the concentration of NBs does eventually increase, which shows that sonication itself does produce NBs. FIG.17B shows the size of the NBs in the NB solution remains relatively stable over the first 10 minutes of treatment, with the average bubble size being about 100nm.

[0210] FIGs.18A and 18B show the effect of sonication on Nanopure water in which no NB treatment was applied. The same sonication treatment as applied to the samples containing NBs was applied to the non-nanobubble containing solution. Initially, a low concentration of NBs is present in the solution. Sonication alone caused the concentration of NBs to significantly increase in the solution. FIG.18B shows the average size of bubbles present in the NB solution during the course of the experiment. Experimental Example 6: Nanoplastics

[0211] Nanoplastics (NPs, typically described as polymeric particulates <1,000 nm) form in the environment by the weathering of plastic debris due to environmental stressors Page 42 of 63 11877856v1Attorney Docket No. 2010363-0390such as ultraviolet radiation, physical forces, chemical oxidants, and biological degradation. NPs are ubiquitous in bodies of water in nature and their minuscule size and surface charge make them difficult to eliminate.

[0212] At low trophic levels, NPs can bind to algae or can be directly ingested by aquatic organisms. Unlike larger plastics, the small size of nanoplastics enables them to permeate biological membranes resulting in reproductive, developmental, neurological, endocrinal, and intestinal toxicity. For example, in Daphnia, polystyrene NPs stimulate reactive oxygen species (ROS) generation and decrease antioxidant enzyme activity. Without wishing to be bound to any specific theory, this leads to a retardation of the growth rate and reproductive ability and shortens the lifespan of zooplanktons. Polystyrene NPs also interfere with the embryonic development of zebrafish, oysters, sea urchins, and mussels causing hormonal disorders, gonadal damage, inflammation, oxidative stress, and imbalances in energy metabolism. At higher trophic levels, ingesting NPs is also problematic because they can reduce the viability of HepG2 liver cells and destroy antioxidant capabilities. NPs have also been detected in human cells and are shown to be cytotoxic to the human reproductive, digestive, and nervous systems by inducing oxidative stress, causing inflammation, and metabolic disorders. In addition, ingestion of NPs may indirectly result in contaminant uptake by leaching toxic monomers, oligomers, additives, and / or adsorbed micropollutants (e.g., the Trojan horse mechanism).

[0213] These implications of NP pollution necessitate water treatment strategies as they pose ecological and human health risks. Nanobubble-based water treatment can enable NP removal. The removal of NPs via flotation has not been explored yet. Removing ultrafine NPs by coarse bubble flotation is challenging because of the low collision probability between the short-lived coarse bubbles and fine particulates. Nanobubbles, on the other hand, are on the same scale as NPs, and they have greater retention times in water thus, they present an opportunity for NP flotation. Suspended nanobubbles can attach to the surface of NPs, improve dissolved air concentration, and result in higher froth flotation efficiencies. In addition, smaller bubbles adhere more strongly to surfaces than coarser bubbles and hence are less likely to detach from surfaces during flotation. Enhanced flotation recoveries with nanobubbles may also extend to coarser materials by multiple bubble attachments.

[0214] In the present example, the inventors have used nanobubble-based flotation to remove NPs from water by nanobubble-NP hetero-aggregation. Among other things, the objectives of the present example were to: (i) investigate the removal of NPs using nanobubble flotation, (ii) understand the surface chemistry and underlining mechanism of Page 43 of 63 11877856v1Attorney Docket No. 2010363-0390nanobubble-NP attachment, and (iii) study the probability and rate of NP-nanobubble collision, attachment, and flotation. To achieve these objectives, the inventors experimentally tested the influence of pH and stirring speed as independent variables on nanoplastic- nanobubble attachment, rising velocity, and removal and provide corresponding computational results. MATERIALS AND METHODS Nanobubble-based flotation experimental procedure

[0215] Information about materials and reagents used in this study is presented in Text S1 in the supplementary information (SI) section. For flotation experiments, 10 µL of a solution comprising 100 nm polystyrene NPs was added to 40 mL of a solution with or a solution without of nanobubbles reaching. The final nanoplastic concentration of the solutions was 38 µg / L. The nanobubble solution was produced using a Moleaer 25 XTB nanobubble generator (e.g., as described herein) as detailed in Text S1. The water height of each suspension was 2.4 cm and the diameter of the beaker was 4.6 cm. Each sample was stirred at either 100 rpm (Re = 81) or 400 rpm (Re = 325) for 5 min using a Heidolph MR Hei-Tec magnetic stirrer. The stirring rod was cylindrical with 2.5 cm length and 0.7 cm diameter. After stirring, the suspension was observed for fluid motion, and samples were drawn from the stagnant float and subnatant of the suspension. A plastic syringe was placed at the meniscus of the suspension to collect the float and then at the midpoint-bottom to collect the subnatant. The influence of pH on nanoplastic flotation with nanobubble solution was investigated by adjusting the pH of the solution to 3.0, 5.0, and 9.0 using 0.1 M HCl or 0.1 M NaOH. All pH measurements for solutions are presented in Table 1 in Text S1. Control experiments were conducted for solutions containing nanobubbles only and NPs only in ultrapure distilled and deionized water (DDI, Barnstead NANOpure infinity ultrapure water system, >18.2 ΩM∙cm). The effect of velocity gradient on plastic-bubble attachment was examined by adjusting the stirring speed of NP in nanobubble solution at 100 rpm vs.400 rpm. Analytical procedures

[0216] The analytical method for determining the NP concentration using a Hach DR 6000 UV-vis spectrophotometer at λ=249 nm is described in Text S2 (including calibration curve in Fig.19) and Text S1 in the Supplementary Information (SI) section. The number, concentration and size of NPs and nanobubbles were determined by nanoparticle tracking Page 44 of 63 11877856v1Attorney Docket No. 2010363-0390analysis (NTA) using a NanoSight NS300. Solution pH was measured using MultiLab IDS 4010-3W, and zeta potential was quantified using Zetasizer Nano-ZS. Analytical procedures are described in detail in Text S2 of the SI section. The data points presented are averaged from triplicate experiments with standard error bars. A one-way ANOVA test at 95% level of significance was used to determine the statistical significance. RESULTS AND DISCUSSION Nanobubble-based flotation of nanoplastics

[0217] Nanobubble and NP solutions looked indistinguishable to the naked eye before and after the flotation experiments. The only visible difference was the appearance of a thin film resembling an immiscible oil layer on water after stirring NPs with nanobubbles at pH 3. FIG.28 shows a thin film after stirring NPs with nanobubbles at pH 3. The formation of this film was attributed to the migration of suspended NP-nanobubble flocs to the air-water interface. FIG.20 presents the mass concentration of NPs measured by UV-vis in the subnatant after 5 min stirring at different pH levels (Conditions: initial plastic concentration = 38 μg / L, stirring speed = 400rpm, stirring time = 5 min). The p-values show a statistically significant difference NP concentration in nanobubble solution vs. DI water sample at a 95% level of confidence. It should be noted that the detection of NPs by UV-vis was not influenced by the presence of nanobubbles as indicated by the calibration curves presented in FIG.19. FIG.19 shows a UV-vis calibration curve for nanoplastics in nanopure and nanobubble water. After stirring the nanobubble solutions with NPs, the concentration of NPs decreased as a function of pH, resulting in the removal of 60%, 9%, 6%, and 0% of NPs from the solutions at pHs 3.0, 5.0, 6.0, and 9.0, respectively. In contrast, little to no changes were noted when the same experiment was conducted in the absence of nanobubbles, resulting in the removal of 8%, 3%, 0%, and 0% of NPs from the solutions at pH 3.0, 5.0, 7.0, and 9.0, respectively.

[0218] NTA analysis was performed, and normalized number concentrations are found in FIG.21. The results were showed a consistent decrease in number concentration as the pH decreased to 3.0. The nanoscopic entity counts at pH 5.0, 6.0, and 9.0 remained relatively unchanged (within 20% of the initial concentrations). Specifically, at pH 3.0, a significant decrease of 61% in the initial NP concentration (p-value = 0.0025) was observed after stirring the NPs in the nanobubble mixture. This was attributed to the destabilization of nanobubbles and the removal of NPs. However, since NTA does not distinguish nanobubbles from NPs, control experiments with only nanobubbles and only NPs were also conducted at Page 45 of 63 11877856v1Attorney Docket No. 2010363-0390pH 3.0. These control experiments showed 58% and 19% decreases in number concentrations after stirring only nanobubbles and only NPs, respectively. This confirmed that the NPs were relatively stable in DDI water during stirring at pH 3.0 but they were destabilized when nanobubbles were in the system. Therefore, the primary mechanism of flotation was speculated to be hetero-aggregation of nanobubbles with NPs with subsequent rise of the bubble-particle flocs (i.e., upward sweeping) to the surface of the vessel.

[0219] In FIG.22, the size distribution of nanobubbles showed polydisperse “peaks and shoulders” ranging from 80 to 300 nm, with a noticeable decline in the intensity of the peaks especially at 80 to 200 nm range after 5 min of stirring at pH 3.0. The size distribution of NPs, on the other hand, was uniform around 100 nm, and it maintained monodispersity after stirring despite a slight reduction in the intensity. The curve representing the mixture of NP and nanobubbles exhibited both the characteristic 100 nm NP peak and the nanobubble “peaks and shoulders”. After stirring at pH 3.0, a notable decrease in the NP peak (at 100 nm intensity) was observed, resulting in a net loss of 106 million entities ∙ mL-1in number concentration. Considering the initial number concentration of nanobubble solution, the loss of 65 million entities ∙ mL-1was assumed to be removal of nanobubbles, yielding a loss of 41 million NPs∙ mL-1from water in 5 min. This corresponds to 51% NP removal, and it aligns well with the UV-vis observation, which was indicating 60% removal. The NTA micrographs in FIG.23 further illustrate the changes in number concentration at pH 3.0. FIG.23 shows NTA micrographs of nano-entities in the bulk phase before and after stirring at pH 3 for 5 min and 400 RPM. Mechanistic insights into bubble-plastic attachment and nanoplastic floatation

[0220] The changes in size after stirring in pH 3.0 are presented in FIG.24. The average increase in size by particle aggregation for all other pHs were negligible .i.e., <5% FIG.25. FIG.25 is a graph changes in particle size of nanobubbles, nanoplastics, and nanoplastics with nanobubbles after 5 min stirring at various pHs (Conditions: initial nanoplastic concentration = 38 µg / L, stirring speed = 400 RPM, stirring time = 5 min). Also at pH 3.0, the average size of NPs alone remained unchanged indicating that stirring at pH 3.0 does not cause homo-aggregation of NPs, confirming the findings from NTA and UV-vis spectroscopy. On the other hand, the size of nanobubbles without NPs increased by 14%, indicating that the bubbles were able to coalesce. The nanobubble solution with NPs showed 27% increase in size indicating not only coalescence of bubbles, but also floc forming interactions with NPs. However, it should be noted that the NTA measurements were Page 46 of 63 11877856v1Attorney Docket No. 2010363-0390conducted for the residual nanoscopic entities in suspension after the treatment. The increasing size of bubbles and NP-nanobubble flocs were attributed to the decrease of electrostatic repulsion between NPs and nanobubbles at pH 3.0. As shown in FIG.26, zeta potential diminished with decreasing pH and led to an almost complete charge neutralization for nanobubbles and notable compression of electric double layer for NPs. The decrease in electrostatic repulsion was attributed to interparticle bridging. At a pH of about 6, both NPs and nanobubbles were negatively charged with zeta potentials of -33.8 mV and -17.8 mV, respectively. The NPs remained negatively charged at pH 3.0 with zeta potential of -16.7 mV while nanobubbles had nearly no charge i.e., -1.6 mV at pH 3.0.

[0221] To gain mechanistic insights regarding the nanobubble-based NP flotation mechanism, the changes in density, concentration, and size as well as nanoplastic-nanobubble attachment efficiencies and rising velocities were computed using the equations in Text S3. Nanobubbles and NPs have average diameters of 109 and 137 nm, respectively. The densities of polystyrene NPs and air nanobubbles were assumed to be 1,070 and 409 kg∙m-3, respectively. Based on these assumptions, the minimum volume of gas (ϕg) required to float all NPs was computed as 6.56×10-6mL of gas∙L of solution-1. To determine whether enough gas volume in nanobubbles is present at pH 3.0, the critical diameter required for the nanobubbles, to overcome their Brownian motion and rise, was calculated to be 488 nm. This implies that at least 3.6 nanobubbles are needed to come together to reach the critical rising diameter. In addition, the number of nanobubbles that coalesced and traveled upwards was computed to be 18 million bubbles∙mL-1. The total volume occupied by 18 million nanobubbles∙mL-1is 1.11×10-3 mL∙L-1, which is three orders of magnitude greater than the minimum gas volume, ϕg, required for 100% NP removal. This implies that the small gas volume packed in nanobubbles is not limiting the success of NP flotation. For our case, the generation of at least 112 million nanobubbles∙L-1could ensure complete NP removal. Lastly, based on the assumption that 3.6 nanobubbles coalesced at pH 3.0 to form “the rising nanobubbles with critical size,” the results indicate that each “rising nanobubble” attaches to 2.3 NPs (assuming 100% collision-attachment efficiency).

[0222] The coalesced nanobubbles are computed to rise at a velocity of 2.97 µm min-1while NPs settled slowly at 0.03 µm∙min-1. However, when nanobubbles attach with NPs to form NP-nanobubble flocs, they are computed to rise at a speed of 2.93 µm∙min-1. This is two orders of magnitude greater than the settling rate of NPs, indicating that attaching nanobubbles to NPs will significantly accelerate NP mobility. Moreover, in a typical flotation cell, coarse bubbles provide additional rising velocity as they attach to the NP-nanobubble Page 47 of 63 11877856v1Attorney Docket No. 2010363-0390flocs. Nevertheless, this rising velocity of the NP-nanobubble flocs is still not enough to achieve flotation in a 2.4 cm suspension height within 5 min. This indicates that incidental vertical fluid mixing enhanced the rising of NP-nanobubble flocs leading to flotation in 5 min due to the vortex created during stirring. Control experiments under static conditions (no stirring for 5 min) confirmed the impact of stirring on NP-nanobubble attachment and flotation (FIG.27, panels a-c). There was no difference between the subnatant and float concentrations when there was no mixing, which signified that agglomeration of the nano- entities proceeds immediately after the electric double layer is overcome without stirring. However, the agglomerated particles could not rise to the surface in 5 min, hence no increase in the float number concentration occurred due to the lack of the external rising force created by stirring.

[0223] Similarly, the rise of NP-nanobubble flocs during stirring was confirmed by analyzing and comparing the concentration and size of NPs in the float and subnatant (i.e., bulk (FIG.27, panel a). In NP-only control, the mass and number concentrations (FIG.27, panels a and b), and size of NPs (FIG.27, panel c) in the float were the same as the NPs in the suspension after pH 3.0 and 6.0 treatment for 5 and 30 min. This indicates that without nanobubbles, NPs do not accumulate in the froth. On the contrary, in the nanobubble solution at pH 3.0, the mass concentration of NPs in the float after treatment was 123% more than the subnatant. This difference between the float and subnatant upholds the deductions that the NPs attach to nanobubbles at pH 3.0 and rise to the air-water interface.

[0224] The attachment of NP-nanobubble can occur via the formation of a three- phase wetting perimeter and a spontaneous rupture of a fluid film between the bubble and particle. This attachment is propelled by hydrophobic forces and affected by the contact angle between particles and bubbles, liquid surface tension, and particle surface properties like zeta potential and roughness (e.g., surface roughness of the particle). Alternatively, contactless flotation could take place, which is influenced by attractive interparticle forces overcoming repulsive forces. The former mechanism of nanobubble-NP attachment is assumed to have taken place here since the thickness of the electric double layer was the driver of NP- nanobubble attachment and flotation. The rate at which the NPs and nanobubbles collide and attach is also governed by the induced velocity gradient during mixing. This was studied experimentally by varying the stirring speeds (e.g., 400 rpm, 100 rpm). The equations for calculating the velocity gradient, collision frequency, and the rate of attachment are in Text S4 and the results are discussed in Text S5. The findings indicate the role of hydrodynamic shearing forces from agitation that may require optimization for the envisioned applications, Page 48 of 63 11877856v1Attorney Docket No. 2010363-0390NP characterization, and nanobubble properties as 100 rpm showed better hetero-aggregation and flotation than 400 rpm. This also demonstrates that NPs and nanobubbles can attach and float under minimal stirring indicating the potential low energy demand of the nanobubble- enabled flotation of NPs. Environmental and Engineering Implications

[0225] The findings from this work show that nanobubbles can attach to NPs once the electric double layer is minimized. Attachment of nanobubbles to NPs is important to NP flotation and a key step to NP removal from water. The technique demonstrated herein has the potential to work as a standalone treatment process for removing nanoplastics from hyperclean effluents. Moreover, NP-nanobubble attachment can serve as an ancillary step to the conventional coarse bubble flotation and reduce the amount of surfactant, frother, and collector that is used for flotation. Moreover, coagulants like alum (i.e., aluminum sulfate) are a potential alternative to the use of acid. SUPPLEMENTARY INFORMATION Supplementary Text – Section 1 (Text S1) Materials and reagents

[0226] 100 nm polystyrene latex indicator standard (0.01% weight / volume) was purchased from Malvern Panalytical and used as a model nanoplastic. Sodium hydroxide (analytical grade, 99%) and hydrochloric acid (ACS reagent grade, 37%) were procured from Acros Organics. Nanobubble generation

[0227] Air nanobubbles were generated in a 32-gallon nanopure water (NW) using a Moleaer XTB 25 generator. The system was made up of a gas pressure of 100 PSIG, a gas flow rate of 1 LPM, and 13 PSI water pumping pressure for 1 h. The generated nanobubbles were transferred to 1 L polypropylene bottles, ensuring it was filled to the brim with no headspace, and then stored in a refrigerator at 4 °C. The number concentration and size distribution of the bubble were analyzed using NanoSight NS 300. Page 49 of 63 11877856v1Attorney Docket No. 2010363-0390Supplementary Text – Section 2 (Text S2) UV-vis spectroscopy

[0228] The concentration of nanoplastics was analyzed using a Hach DR 6000 UV- vis spectrophotometer in a 1 cm plastic cuvette at a wavelength of 249 nm. To ensure data reliability for low concentration measurements, each experiments were performed in triplicates and each sample was measured at least five times with the UV-vis spectrophotometer, and the average of three values was selected. The concentrations of nanoplastics were determined using a linear equation generated from the calibration curve (Fig. S1) that relates the absorbance to concentration. The calibration curve in Fig. S1 shows that nanoplastics can be detected in both DI water and nanobubble solution without any interference by the nanobubbles. Nanoparticle Tracking Analysis (NTA)

[0229] NanoSight NS300 (Malvern Panalytical, UK) was employed for the assessment of number concentration, average size, and size distribution of triplicate samples of nanoplastics, nanobubbles, and the mixture of nanoplastics and nanobubbles. The instrument characterizes nano-entities (particles and bubbles) based on the nanoparticle tracking analysis (NTA) principle as explained elsewhere. Our NanoSight NS300 is equipped with a green laser and a syringe pump. Each measurement entailed a 60-second video recording with a camera level of 13-15. The detection threshold was set at 5-20. The NTA software analyzed five consecutive video recordings for each NanoSight measurement, producing a number / size distribution, with the reported results representing averages from the five measurements. Zeta potential

[0230] The zeta potential of the nanobubbles and nanoplastics from triplicate experiments was measured using Zetasizer Nano-ZS. For each sample, the initial and final pH after the experiment, presented in Table 3, was measured using MultiLab IDS 4010-3W. Prior to the zeta potential measurement, the refractive index and absorption values of nanobubbles and nanoplastics were set as shown in Table 4. The dispersant was set to water with temperature = 25 °C, Viscosity = 0.8872 cP, refractive index = 1.330, and dielectric constant = 78.5. Samples were loaded in a DTS1060C-Clear zeta cell and the equilibration time was set to 120 s. The zeta potential was calculated using the Smoluchowski model and Page 50 of 63 11877856v1Attorney Docket No. 2010363-0390an F(Ka) value of 1.5. As a control measurement, the zeta potential of DI water was measured to be -0.75 ± 0.37. Table 3. Changes in pH before and after stirring Nanobubbles Nanoplastics in DI water pH before ± SE pH after ± SE pH before ± SE pH after ± SE 9.14 ± 0.15 7.32 ± 0.27 9.10 ± 0.04 7.38 ± 0.07 6.33 ± 0.18 6.27 ± 0.15 6.28 ± 0.22 6.11 ± 0.33 4.70 ± 0.14 4.56 ± 0.19 5.04 ± 0.17 5.02 ± 0.13 3.11 ± 0.01 3.09 ± 0.01 3.08 ± 0.01 3.05 ± 0.02 Table 4. Parameters of nanobubbles and nanoplastics used in zeta potential measurement Sample Material Refractive index Absorption Nanobubbles Air 1.00 0.00 Nanoplastics Polystyrene latex 1.59 0.01 Supplementary Text – Section 3 (Text S3) Rising and settling calculations Computations for settling velocity of nanoplastics

[0231] The following set of equations from a previous study was used to estimate the settling velocity of nanoplastics. Polystyrene latex are nanospheres, hence shape factor = 1, and diameter of the nanoplastic (d) = equivalent sphere diameter

[0232] The settling velocity was calculated using Stoke’s law in Eq. (1) assuming a laminar flow: ('()'*)+,(- vs =$. /

[0233] Where, vs= settling velocity at laminar flow (m / s)

[0234] ρp = density of nanoplastic (kg / m3)

[0235] ρw= density of water at 20 °C (998.2 kg / m3)

[0236] dp = diameter of nanoplastic (m)

[0237] g = acceleration due to gravity (9.81 m / s2)

[0238] μ= dynamic viscosity of water at 20 °C (1.002 × 10-3kg / m.s). Page 51 of 63 11877856v1Attorney Docket No. 2010363-0390

[0239] The validity of the vs was verified by confirming with the Reynolds number (Re) given by: Re =1+2'*3

[0240] Where ν = kinematic viscosity of water at 20 °C (1.003 × 10-6m2 / s), w = iterative settling velocity (m / s), w = vs in the first iteration ' w = 45+(()'*6789'*9 g where, CD= Drag coefficientCD =^5;<, for Re <0.5 Plastic-bubble aggregate rising velocity calculations

[0241] For flotation to occur, nanobubbles need to overcome Brownian motion and rise. The critical size required by bubbles (db) to overcome Brownian motion and rise can be calculated using Eq.5 shown below. $J I=>= ?216kTμ^^H

[0242] Where, k = Boltzmann’s constant (1.38 × 10−23 J / K)

[0243] T = absolute temperature (293 K)

[0244] t = time (s)

[0245] db = bubble diameter, m

[0246] According to Crittenden et al. (2012), the following expressions can be used to determine the theoretical rising velocity of a particle-bubble floc.

[0247] The formula for calculating the density of the plastic–bubble floc (ρpb, kg / m3) is given by: 6 =ρKd6K + NGρG

[0248] Where, ρb = air bubble density (kg / m3)

[0249] Nb = number of bubbles attached to floc particle

[0250] The equivalent spherical diameter of the plastic–bubble aggregate (dpd) was determined using Eq.6. 6$dK+= (d6K+ NGdG)6Page 52 of 63 11877856v1Attorney Docket No. 2010363-0390

[0251] The rising velocity of a plastic-bubble aggregate (vpb) is given by: $.RI$J$.^IvKG = Pg(ρ1)ρKG)dKG

[0252] The minimum is expressed by:W(='*Y'Z

[0253] Where, ϕg = minimum volume of gas needed for flotation (mL / L)

[0254] ϕp = volume of particles (mL / L) Supplementary Text – Section 4 (Text S4) Stirring speed and velocity gradient formulas

[0255] The overall plastic-bubble collision rate depends on the rates of macroscale flocculation, microscale flocculation and differential settling flocculation between nanoplastics and nanobubbles. Furthermore, the rate of plastic-bubble attachments (rpb) can be determined by the particle concentrations and a collision frequency function (βpb).

[0256] rpb= αβpbnpnb

[0257] Where, rpb = rate of attachment between nanoplastics and nanoplastics (

[0258] α = collision efficiency factor (attachments per collision). α = 1, assuming effective destabilization of nanoplastics and nanoplastics at a pH of 3.

[0259] βpb= overall collision frequency between nanoplastics and nanoplastics (m3 / s)

[0260] np = nanoplastics concentration

[0261] nb= nanoplastics concentration

[0262] βpb = βM + βμ + βDS

[0263] βM = macroscale collision frequency, =$ 6[ \̅(=^+ =>)

[0264] βμ= microscale collision frequency, =_^`ac ?$+$(=^+ =>)

[0265] βDS= differential settling collisionfg'(Y'*hi [g=6^ + =>h(=^− =>)] gradient (s−1)

[0267] G =4^jk

[0268] Where, P = power input of horizontal mixing (W = kg·m2 / s3)

[0269] V = volume of nanoplastic-nanobubble suspension, m3Page 53 of 63 11877856v1Attorney Docket No. 2010363-0390] P =l or [0270mn 2'pq^ coefficient on paddleof paddle (m2)

[0273] ρ = density of suspension = density of water (kg / m3)

[0274] vR = velocity of paddle relative to fluid (m / s)

[0275] CDP =^5st+5√st+ 0.4

[0276] Where, Re = Reynolds number

[0277] Re =v,wxj = diameter of impeller (0.009 m)= rotational speed (revolutions per second)

[0280] Ap = L×W

[0281] Where, L = length of paddle

[0282] W = width of paddle

[0283] vR= 2π(L / 2)N×0.75

[0284] 0.75 = relative velocity of paddle with respect to fluid

[0285] Elements of different implementations described herein may be combined to form other implementations not specifically set forth above. Elements may be left out of the processes, computer programs, databases, etc. described herein without adversely affecting their operation. In addition, the logic flows depicted in the figures do not require the particular order shown, or sequential order, to achieve desirable results. Various separate elements may be combined into one or more individual elements to perform the functions described herein.

[0286] Throughout the description, where apparatus and systems are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are apparatus, and systems of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps. Page 54 of 63 11877856v1Attorney Docket No. 2010363-0390

[0287] It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0288] While the invention has been particularly shown and described with reference to specific preferred embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Page 55 of 63 11877856v1Attorney Docket No. 2010363-0390Listing of Sequences GTGYCAGCMGCCGCGGTAA (SEQ ID NO.1) GGACTACNVGGGTWTCTAAT (SEQ ID NO.2) Page 56 of 63 11877856v1

Claims

Attorney Docket No. 2010363-0390What is claimed is:

1. A method comprising, associating a plurality of nanobubbles with a composition.

2. The method of claim 1 further comprising, removing one or more target substances from the composition using the plurality of nanobubbles.

3. The method of claims 1 or 2 further comprising, adding one or more payload substances to the composition using the plurality of nanobubbles.

4. The method of claim 3, wherein a portion of at least one of the one or more payload substances are on the surface at least one of the plurality of nanobubbles.

5. The method of claims 3 or 4, wherein a portion of at least one of the one or more payload substances are inside at least one of the plurality of nanobubbles.

6. The method of any one of claims 4 or 5, wherein the one or more payload substances comprise a gas.

7. The method of any one of the previous claims further comprising, storing the plurality of nanobubbles in a storage solution for a period of time.

8. The method of claim 6, wherein the plurality of nanobubbles in the storage solution are stable for the period of time.

9. The method of claims 7 or 8, wherein the period of time is at least 30 days.

10. The method of any one of the previous claims, wherein the nanobubble comprises an elemental gas, ozone gas, or a mixture of gases. Page 57 of 63 11877856v1Attorney Docket No. 2010363-039011. The method of any one of the previous claims, wherein the method comprises generating the plurality of nanobubbles on a surface of a substrate.

12. The method of any one of the previous claims, wherein the method comprises contacting the plurality of nanobubbles with a substrate.

13. The method of claims 11 or 12, wherein the substrate is a hydrophobic substrate.

14. The method of any one of claims 11-13, wherein the substrate comprises graphene.

15. The method of any one of claims 11-14, wherein the method comprises generating the plurality of nanobubbles on the substrate using of one or more of the following methods: water-solvent exchange, differential temperature, differential pressure, ultrasonication, mechanical forces, and electrolytic evolution.

16. The method of any one of the previous claims, wherein the method comprises removing the plurality of nanobubbles from a surface of a substrate.

17. The method of claim 16, wherein the method comprises removing the plurality of nanobubbles from the surface of the substrate using sonication or salt-destabilization.

18. The method of claims 16 or 17, wherein the method comprises destroying nanobubbles at an interphase.

19. The method of claims 4-18, wherein the method comprises releasing the one or more payload substances from the nanobubbles.

20. The method of any one of the previous claims, wherein the composition is a wastewater solution.

21. The method of claims 20, wherein the wastewater solution comprises sludge.

22. The method of any one of the previous claims, wherein the one or more target substances are pollutants. Page 58 of 63 11877856v1Attorney Docket No. 2010363-039023. The method of any one of claims 2-22, wherein the one or more target substances comprise one or more of the following: an organic pollutant, an organosilane, urea, a perfluorochemical, a microorganism, a short chain fatty acid, a sugar, a protein, a surfactant, a nanoparticle, a dye, a colloid, Methyl-Isoborneol (MIB), Geosmin, a nanoplastic (NP), and oxytetracycline.

24. The method of claim 23, wherein at least one of the one or more target substances comprises an organic pollutant, and the organic pollutant is one or more of the following compounds or an equivalent or derivative thereof: naphthalene, 1-chloronapthalene, 1- napthol, 1,4-dichloronaphtalene, 1,8-dichloronaphtalene, phenanthrene, anthracene, pyrene, biphenyl, thrichloroethylene, tetrachloroethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane, carbon tetrachloride, 1,1-dichloroethylene, 1,2-dichloropropane, methylene blue, bisphenol A, carbamazepine, phenol, benzoic acid, atrazine, perfluorooctanesulfonate (PFOS), phenanthrene, PFOS / PFOA, and TCE.

25. The method of any one of claims 2-24, wherein the one or more target substances comprises a nanoplastic (NP) 26. The method of claim 25, wherein the size of the nanoplastic is about 10μm or less, about 1μm or less, about 100nm or less in size.

27. The method of either claims 25 or 26, wherein the nanoplastic is or comprises polystyrene, latex, polyethylene tetrephtalate (PET), or a combination thereof.

28. The method of any one of claims 2-27, wherein at least one of the one or more target substances comprises a plurality of nanoparticles.

29. The method of claim 28, wherein the method comprises causing the nanoparticles to rise to a surface of the composition.

30. The method of any one of the previous claims, wherein the method comprises reducing the pH of the composition to a pH of about 6 or lower, to a pH of about 5 or lower, to a pH of about 3 or lower (e.g., to enhance removal of the one or more nanoparticles). Page 59 of 63 11877856v1Attorney Docket No. 2010363-039031. The method of any one of claims 1-29, wherein the composition has a pH of about 6 or lower, to a pH of about 5 or lower, to a pH of about 3 or lower (e.g., to enhance removal of the one or more nanoparticles).

32. The method of any one of the previous claims, wherein the method comprises adding one or more coagulants and / or surfactants to the composition.

33. The method of any one of the previous claims, wherein the method comprises agitating the composition (e.g., to distribute the nanobubbles).

34. The method of any one of claims 2 to 33, wherein the method comprises reducing and / or neutralizing the magnitude of the zeta potential of the one or more target substances (e.g., wherein the zeta potential is about -40mV or less (in absolute value) (e.g., about - 33.8mV or less, about -25mV or less, about -16.7mV or less).

35. The method of any one of the previous claims, wherein the method comprises decreasing an abundance of filamentous bacteria, mycobacterium, viruses, or floc forming bacteria in the composition (e.g., in a wastewater solution).

36. The method of any one of the previous claims, wherein the plurality of nanobubbles are characterized in that the payload substance of at least a subset of the plurality of nanobubbles are accessible to microorganisms.

37. The method of any one of the previous claims, wherein the method comprises generating the plurality of nanobubbles with hydrogen peroxide, nozzle decompression, ultrasonic generation, venturi bubble generation, spiral flow mixing, static and dynamic mixing, ceramic oxygen generator (COG).

38. The method of any one of the previous claims, wherein the method comprises use of advanced oxidation process(es). Page 60 of 63 11877856v1Attorney Docket No. 2010363-039039. The method of claim 38, wherein the advanced oxidation process(es) comprises exposing the plurality of nanobubbles to metallic catalysts, UV light irradiation, or ultrasound.

40. The method of any one of the previous claims, wherein the method comprises generating a reactive oxygen species using the plurality of nanobubbles.

41. The method of claim 40, wherein the method further comprises disrupting the plurality of nanobubbles.

42. The method of any one of the previous claims, wherein the diameter of the plurality of nanobubbles is about 1000 nm or less.

43. The method of any one of the previous claims, wherein the density of the plurality of nanobubbles is greater than 0.5 x 107bubbles per mL.

44. The method of any one of the previous claims, wherein the method comprises removing the plurality of nanobubbles from the composition using salt addition, filtration, sonication, a vacuum, dielectric heating, radio waves, or light.

45. A method of aquaculture production comprising the methods of any one of claims 1-44.

46. A method of treating wastewater under microgravity conditions comprising the methods of any one of claims 1-44.

47. A method of treating a water column for use in aquaculture comprising the methods of any one of claims 1-44.

48. A method of oxygen bleaching comprising the methods of any one of claims 1-44.

49. A method of delignification comprising the method of any one of claims 1-44.

50. A system configured to perform the method of any one of claims 1-49. Page 61 of 63 11877856v1Attorney Docket No. 2010363-039051. A method of removing a pollutant from a composition, the method comprising: associating a plurality of nanobubbles with a composition, wherein the plurality of nanobubbles are about 500nm or less in diameter; reducing the zeta potential of the pollutant to about -25mV or less; and removing the pollutant from the composition using the plurality of nanobubbles, wherein the pollutant comprises nanoplastics of about 1μm or less in size, wherein removing the one or more target substances comprises causing the pollutant to rise to the surface of the composition.

52. A method comprising removing organic pollutants from a composition, the method comprising: generating the plurality of nanobubbles on a surface of a hydrophobic substrate, wherein the plurality of nanobubbles are about 500nm or less in diameter; contacting the plurality of nanobubbles with a composition, wherein the composition comprises the organic pollutants, thereby associating the organic pollutants with the plurality of nanobubbles; and removing the plurality of nanobubbles from the surface of the substrate using the plurality of nanobubbles using sonication and / or salt-destabilization.

53. A method of removing one or more target substances from a composition, the method comprising, associating a plurality of nanobubbles with the composition, wherein the composition comprises one or more target substances and wherein the plurality of nanobubbles are about 500nm or less in diameter; and removing the one or more target substances from the composition using the plurality of nanobubbles, wherein removing the one or more target substances comprises removing the plurality of nanobubbles from the composition using sonication, wherein at least one of the one or more target substances comprises an organic pollutant. Page 62 of 63 11877856v1

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