Use of carbon dioxide nanobubbles in aqueous culture

Abstract

Provided are an aqueous growth medium containing CO2 nanobubbles and method of culturing a microbe using such medium. Advantageously, the present method may provide much higher CO2 delivery and utilization efficiency than the conventional sparging processes.

Description

USE OF CARBON DIOXIDE NANOBUBBLES IN AQUEOUS CULTURECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 603.332, filed November 28, 2023, the content of which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under DE-EE0008515 awarded by the Department of Energy. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] Photosynthetic microbes including microalgae are useful renewable sources of biofuels, bioactive products and food ingredients that they synthesize from light and CO2, and thus are promising contributors to the bioeconomy. However, CO2 delivery' by sparging leads to unacceptable losses of CO2 to the air as most of the sparged CO2 is not taken up into aqueous medium. Conventional CO2 sparging of microalgal / cyanobactenal ponds suffers from a limited mass transfer efficiency. Macrobubbles of CO2 (1-10 millimeters) generated by spargers rise quickly to the surface where the gas is lost to the atmosphere. This gas transfer inefficiency leads to increased operational costs (energy and CO2 sourcing) and release of greenhouse gas into the atmosphere as part of a microalgal production facility. Smaller bubbles have the potential to improve gas exchange. Microbubbles (1-100 micrometers) offer better gas mass transfer. They rise more slowly than macrobubbles but eventually burst in the bulk liquid. There remains a need for systems that effectively use smaller CO2 bubbles to improve gas transfer efficiency, thereby improving carbon utilization for production of photosynthetic microbes.SUMMARY OF THE INVENTION

[0004] In one aspect, the present disclosure provides a method of culturing a microbe. The method can comprise supplying CO2 nanobubbles to an aqueous growth medium to generate a CO2 enriched medium and growing the microbe in the CO2 enriched medium.

[0005] The nanobubbles can have a size of about 10 nm to about 500 nm and an average size of about 200 nm. The infused CO2 in the CO2 enriched medium can have an inorganic carbon concentration of about 300 mg / L to about 500 mg / L, such as about 400 mg / L.

[0006] In some embodiments, the present method is used for culturing a photosynthetic microbe. The photosynthetic microbe can include, for example, a cyanobacterium or an alga. In particular embodiments, the photosynthetic microbe comprises Synechocystis .

[0007] In another aspect, an aqueous growth medium is provided, which comprises CO2 nanobubbles and at least one nutrient for microbe culture, optionally wherein the aqueous growth medium has a microbe therein.BRIEF DESCRIPTION OF DRAWINGS

[0008] FIG. 1 depicts the impact of metal ions (Cu2+, Ni2+, Zn2+) on the grow th of the TE / \s7r / 6 / > strain that was grown in BG-11 supplemented with 34 mM bicarbonate with added Cu2+at 11.6 pM, Ni2+at 2.7 pM, Zn2+at 7 pM and in combination of two or three metal ions at the listed concentrations under continuous light at a light intensity of 100 pmole photon m 2- s 1- .

[0009] FIG. 2 depicts the impact of EDTA at various concentrations on the growth of TE / slr 1609 culture in Nano BG-11 under continuous light. CO2 nano-water generated with NG25 at a gas pressure of 25 psi contained approximately 1500 ppm CO2. Control BG-11 was supplemented with 34 mM bicarbonate.

[0010] FIG. 3 show s the growth of TE / slr 1609 cultures in Nano BG-11 in the presence of various concentrations of EDTA. BG-11 medium containing 29.7 pM EDTA was used as a control, which has about 10 times more EDTA than the regular BG-11 medium. OD is the average from triplicate cultures. Nano BG-11 was made with CO2 nano-water generated at a pressure of 25 psi with the NG25 instrument.

[0011] FIG. 4 show s the growth of wild-type Synechocystis sp. PCC 6803 in Nano BG-11 with or without additional EDTA under continuous light. BG-11 control refers to a control culture grown in regular rather than Nano BG-11 medium. Cultures (50 ml each) were grown in shake flasks at 150 rpm. Nano BG-11 w as made with CO2 nanobubble water generated by the NG25 instrument at 100 psi.

[0012] FIG. 5 shows the growth of the TE / slrl609 strain in Nano BG-11 generated at various pressures ranging from 10 to 100 psi supplemented with 27 pM additional EDTA (29.7 pM total). CO2 nanobubble w ater generated at each pressure was collected after disposing of about 10 gallons of CO2 nanobubble w ater.INCORPORATION BY REFERENCE

[0013] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference.DETAILED DESCRIPTION OF THE INVENTION

[0014] This disclosure relates to the use of nanobubbles for growth of photosynthetic microbes. Various embodiments show that CO2 nanobubbles support their grow th as well as, if not better than, traditional inorganic carbon delivery methods. Moreover, CO2 nanobubbles have the advantage of less CO2 loss to the atmosphere compared to sparging.

[0015] Carbon dioxide is a primary feedstock for photosynthetic microbes in aqueous cultures, and efficient utilization of carbon dioxide has become an important sustainability target. Supply of CO2 to photosynthetic microbes in aqueous media traditionally is done via sparging, but sparging is a very inefficient process with the great majority of the CO2 ending up in the atmosphere, unused. The present disclosure aims to remedy this unsatisfactory situation by greatly reducing the escape of CO2 from the aqueous medium into the atmosphere by means of CO2 delivery' through nanobubbles that do not escape from the medium. Nanobubbles are stable, gas-filled cavities that when generated create a fluid containing a nanogas dispersion. Nanobubbles (e.g.,10 nanometers - 1 micrometer) are shown to have the ability to provide higher mass transfer levels between the gas bubbles and the aqueous medium than larger bubbles, likely because the nanobubbles have no appreciable rise rate. Their negative surface charge also prevents coalescence and, as a result, nanobubbles have prolonged stability- in aqueous environments. Their increased interfacial surface area provides nanobubbles with the potential for a more efficient and controlled gas dissolution as the demand for carbon warrants. To date, CO2 nanobubbles had not been applied to provide CO2 to cultures of photosynthetic microbes.

[0016] In various embodiments, the present disclosure provides a novel way to supply CO2 to photosynthetic microbes: CO2 is infused into the medium in nanobubbles, and such bubbles are stable in the medium, in contrast to larger bubbles. Photosynthetic microbes (e.g., cyanobacteria or algae) can use this inorganic carbon for photosynthesis and for incorporation into valuable products at rates comparable to or somewhat better than those achieved with traditional inorganic carbon supply. In any case, CO2 nanobubbles improve carbon utilization efficiency and may also improve the overall process economics by significantly reducing the CO2 loss to the atmosphere that occurs during typical sparging processes. The levels of CO2 inaqueous medium achievable using the nanobubble technology described herein may be more than sufficient for supplying microalgae (more specifically, the cyanobacterium Synechocystis) with their inorganic carbon requirements to support vigorous growth.

[0017] In one aspect, the present disclosure provides method of culturing a microbe, the method comprising: supplying CO2 nanobubbles to an aqueous growth medium to generate a CO2 enriched medium; and growing the microbe in the CO2 enriched medium.

[0018] The CO2 nanobubbles can be generated, for example, by the system and method described in U.S. Patent Nos. US 9,586,186 and US 10,080,998 (both to Nano Gas Technologies, Inc.), which are incorporated by reference herein in their entirety. A representative nonlimiting process can include infusing CO2 gas into water under a saturation pressure to generate a water-CCh mixture and releasing the water-CCh mixture under atmospheric pressure, thereby producing w ater saturated with CO2 nanobubbles. The saturation pressure can be up to 300 psi, including, but not limited to, about 10 psi to about 200 psi, about 10 psi to about 150 psi, or about 10 psi to about 100 psi. This process may result in more CO2 in water than what dissolves at standard temperature and pressure, thus improving carbon delivery and utilization for commercial cultivation of microbes.

[0019] In some embodiments, supplying CO2 nanobubbles to the aqueous growth medium comprises (a) mixing w ater containing CO2 nanobubbles with the aqueous growth medium, (b) dissolving a solid growth medium with water containing CO2 nanobubbles, or (c) infusing CO2 nanobubbles into the aqueous growth medium. In some embodiments, the water in (a) or (b) is saturated with CO2.

[0020] In some embodiments, the method comprises mixing water containing CO2 nanobubbles with the aqueous growth medium, thus supplying CO2 nanobubbles to the aqueous growth medium. The volume ratio between water containing CO2 nanobubbles and the aqueous medium can be about 1: 1 to about 1000: 1. Suitable volume ratio includes, but is not limited to about 5:1, about 10: 1, about 100: 1, or about 1000: 1. In some embodiments, the volume ratio between water containing CO2 nanobubbles and the aqueous medium is about 10: 1 to about 1000: 1, such as about 50: 1, about 100: 1, about 200: 1, about 400: 1. about 600: 1, about 800: 1, or about 1000: 1. In some embodiments, the volume ratio is about 100: 1.

[0021] The nanobubbles can be a size (diameter) of about 10 nmto about 500 nm, including, but not limited to, about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, and about 500 nm. The nanobubbles can have an average size (diameter) of about 100 nm to about 300 nm, including, but not limited to, about 100 nm, about 150 nm. about 200 nm, about250 nm, and about 300 nm. In some embodiments, the CO2 nanobubbles in the CO2 enriched medium have an average size of about 200 nm.

[0022] The concentrations of the infused CO2 in water as described herein can be up to 1500 ppm (e.g., between 500 and 1500 ppm). The content of the infused CO2 can also be measured by inorganic carbon concentration. In some embodiments, the infused CO2 in the CO2 enriched medium has an inorganic carbon concentration of about 200 mg / L to about 500 mg / L, such as about 250 mg / L to about 500 mg / L or about 300 mg / L to about 500 mg / L. In some embodiments, the infused CO2 in the CO2 enriched medium has an inorganic carbon concentration of about 300 mg / L, about 350 mg / L, about 400 mg / L, or about 450 mg / L. In some embodiments, the infused CO2 in the CO2 enriched medium has an inorganic carbon concentration of about 400 mg / L.

[0023] In some embodiments, the method further comprising adding a metal chelator to the CO2 enriched medium. The metal chelators can form complexes with divalent cations to alleviate their negative impact on microbe culture. Suitable metal chelators include known compounds, such as ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA). hydroxyethylethylenediaminetriacetic acid (HEDTA). and nitrilotnacetic acid (NTA). In some embodiments, the metal chelator is EDTA. In some embodiments, the metal chelator can be added to the water containing CO2 nanobubbles prior to mixing the water with the aqueous medium or dissolving the solid growth medium in the water. The metal chelator in the CO2 enriched medium can have a concentration of about 1 pM to about 150 pM. such as about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 80 pM, about 100 pM, or about 120 pM.

[0024] The CO2 enriched medium can have an alkaline pH. In some embodiments, the CO2 enriched medium has a pH of about 8.0 to about 9.5, such as about 8.0, about 8.5, about, 9.0, or about 9.5. Since water with dissolved CO2 is acidic (e.g., pH 3.4-5), the pH of the water containing CO2 nanobubbles may be adjusted before being used for the present method. In some embodiments, the method further comprising adjusting a pH of the water containing CO2 nanobubbles to about 8 to about 11. For example, the pH can be adjusted to about 8.5, about 9.0, about 9.5, about 10.0, or about 10.5.

[0025] The CO2 nanobubbles in the CO2 enriched medium can be stable for a sufficient duration under the conditions for culturing a microbe. Stability of the CO2 nanobubbles as disclosed herein includes the ability to maintain their average size, surface charge, internal pressure, neutral buoyancy, and / or resistance to coalescence. The stable duration of the CO2nanobubbles as disclosed herein can be, for example, multiple days at ambient temperature (e.g.. 20-35 °C). In some embodiments, the CO2 nanobubbles in the CO2 enriched medium are stable from 1 to 14 days at a temperature of 25-35 °C. For example, the stable duration can be about 3 days at 25 °C, about 5 days at 25 °C, about 7 days at 25 °C. about 10 days at 25 °C, about 12 days at 25 °C, about 14 days at 25 °C, about 5 days at 30 °C, about 7 days at 30 °C, about 10 days at 30 °C, or about 14 days at 30 °C.

[0026] In some embodiments, the microbe comprises a photosynthetic microbe. In some embodiments, the photosynthetic microbe comprises a cyanobacterium or an alga. Suitable cyanobacteria include photosynthetic prokaryotes, such as freshwater unicellular species (e.g., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942), marine unicellular species (e.g.. Synechococcus sp PCC 7002), and nitrogen fixing filamentous species (e.g., Nos toe sp PCC 7120). Suitable algae include photosynthetic eukaryotes, such as unicellular green alga (e.g., Chlamydomonas reinhardtii) and diatom (e.g., Phaeodactylum tricornutum). In some embodiments, the photosynthetic microbe comprises Synechocystis.

[0027] The photosynthetic microbe can be cultured under known conditions (e.g., temperature, nutrients, and duration). Depending on the species of the microbe, the culturing conditions can be determined to achieve a desired yield or rate of production. In some embodiments, the photosynthetic microbe (e.g., Synechocystis)' is grown at a temperature of about 30 °C for about 7 days. In some embodiments, the photosynthetic microbe comprises Synechocystis, which is grown at a temperature of about 30 °C for about 5 days, about 7 days, about 10 days, or about 14 days.

[0028] In another aspect, the present disclosure provides an aqueous growth medium comprising CO2 nanobubbles and at least one nutrient for microbe culture. For example, the nutrient can be nitrate, phosphate, sulfate, chloride, carbonate, citrate, or a combination thereof. Suitable nutrients can include, for example, NaNCh, K.2HPO4, MgSO-i. CaCh, citric acid, ferric ammonium citrate, and Na2CCh. The grow th medium may also include other ingredients, such as one or more additional ingredients selected from H3BO3, MnCh, ZnSO4, Na2MoC>4, CuSC>4, and CO(NO3)2. In some embodiments, the aqueous grow th medium may optionally have a microbe therein.

[0029] The aqueous growth medium containing CO2 nanobubbles can be a CO2 enriched medium as described herein. In some embodiments, the nanobubbles can be a size (diameter) of about 10 nm to about 500 nm. The nanobubbles can have an average size (diameter) of about 100 nm to about 300 nm. including, but not limited to, about 100 nm, about 150 nm,about 200 nm, about 250 nm, and about 300 nm. In some embodiments, the CO2 nanobubbles have an average size of about 200 nm.

[0030] The CO2 nanobubbles in the aqueous growth medium can be stable for a sufficient duration under the conditions for culturing a microbe (e.g., 20-35 °C). In some embodiments, the CO2 nanobubbles in the aqueous growth medium are stable from 1 to 14 days at a temperature of 25-35 °C. For example, the stable duration can be about 5 days at 25 °C, about 7 days at 25 °C, about 14 days at 25 °C, about 5 days at 30 °C, about 7 days at 30 °C, or about 14 days at 30 °C.

[0031] In some embodiments, the CO2 nanobubbles have an average size of about 200 nm, the CO2 nanobubbles are stable from 1 to 14 days at a temperature of 25-35 °C, the CO2 in the CO2 nanobubbles has an inorganic carbon concentration of about 300 mg / L to about 500 mg / L, or any combination thereof.

[0032] In some embodiments, the aqueous growth medium has a pH of about 8.0 to about 9.5, such as about 8.0, about 8.5, about, 9.0, or about 9.5.

[0033] In some embodiments, the infused CO2 in the aqueous grow th medium as described herein has an inorganic carbon concentration of about 200 mg / L to about 500 mg / L, such as about 250 mg / L to about 500 mg / L or about 300 mg / L to about 500 mg / L. For example, the CO2 in the CO2 nanobubbles in the aqueous growth medium as described herein can have an inorganic carbon concentration of about 300 mg / L, about 350 mg / L, about 400 mg / L, or about 450 mg / L. In some embodiments, the inorganic carbon concentration is about 400 mg / L.

[0034] In some embodiments, the aqueous growth medium further comprises a metal chelator.

[0035] The CO2 nanobubble populations in aqueous medium as described herein can have an average size of about 200 nm and can be both stable and long lasting due to their neutral buoyancy and generally resistant to coalescence due to their negative surface charge. Moreover, the present CO2 nanobubble can have high internal pressures and provide high gas transfer efficiency. In specific embodiments, these nanobubble properties are used to efficiently deliver CO2 to cultures of photosynthetic microbes, specifically cyanobacteria, via CO2 nanobubbles. CO2 levels delivered this way can be orders of magnitude above air saturation and provide the photosynthetic microbes with an excellent source of inorganic carbon. The ability to generate large volumes of CCh-containing nanobubbles and their demonstrated use to support vigorous growlh of cyanobacterial cultures indicate that the technology has clearadvantages over CO2 delivery by traditional means (i.e., sparging), which is very inefficient and uneconomical in its CO2 utilization efficiency.

[0036] More specifically, the advantages of the present method utilizing CO2 nanobubbles over the conventional sparging processes include, but are not limited to, (1) smaller bubble size and consequently a greater interfacial surface area and higher gas mass transfer; (2) less waste of CO2 due to the neutral buoyancy of nanobubbles; (3) better retention of inorganic carbon i.e., CO2) in cultivation systems for photosynthetic microbes; (4) competitive production rates of photosynthetic microbes; and (5) higher carbon utilization efficiency.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present disclosure, including definitions, will control. Suitable methods and materials are described herein, although methods and materials similar or equivalent to those described herein can be used in practice. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety'. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0038] The terms "comprise(s)," "include(s)." "having," "has." "can," "contain(s)." and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility7of additional acts or structures. The singular forms "a," "an" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.

[0039] The modifier "about" used in connection with a quantity7is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier "about" should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression "from about 2 to about 4" also discloses the range "from 2 to 4." The term "about" may refer to plus or minus 10% of the indicated number. For example, " about 10%" may indicate a range of 9% to 11 %. and " about 1" may mean from 0.9- 1.1. Other meanings of "about" may be apparent from the context, such as rounding off, so, for example "about 1" may also mean from 0.5 to 1.4.

[0040] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2. 6.3, 6.4, 6.5, 6.6. 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.EXAMPLES

[0041] Nanobubble Production

[0042] CO2 nanobubbles were produced using lab-scale nanobubble generating unit supplied by Nano Gas Technologies (Houston. TX). such as model NG25. Typically, these units use a pressure vessel that includes a gas nozzle and a liquid atomizer for addition of CO2 and water, respectively, to the interior volume of the pressure vessel. The components and operation of a representative nanobubble generating unit are described, for example, in U.S. Patent Nos. US 9,586,186 and US 10,080,998. The nanogas dispersion was then collected in an appropriate container for further analysis and use.

[0043] Nano Gas Unit Operation

[0044] For the study, the nanobubble generating unit was connected directly to a tap water source using a simple garden hose. During the study the unit was also tested with higher purity water sources, such as lab-derived deionized or distilled water, the water was pumped from a 5-gallon bucket directly into the nanobubble unit using a simple sump-pump set-up using the appropriate water connections. A cylinder containing pressurized CO2 was used as the gas source for the nanobubble generating unit.

[0045] Analysis of Nanobubble Solutions:

[0046] During the test, the unit and the pressure used during operation varied, with the nanogas dispersion collected from the unit's nozzle coming out at a flow rate of about 1-3 gallons per minute. This nanogas dispersion contained a fairly high amount of soluble carbon dioxide along with CO2 nanobubbles. A rapid way of determining the dissolved CO2 concentration in water is with a test kit available from CHEMetrics (Midland, VA). CHEMetrics’ Carbon Dioxide (dissolved) Test Kits employ a sodium hydroxide titrant and phenolphthalein indicator. Results are expressed as parts per million (ppm) or milligrams / liter (mg / L) of CO2. These kits are able to determine the CO2 concentration in water from between 10 and 2500 ppm. Depending on the Nano Gas Technologies nanobubble generator used, soluble CO2 concentrations ranged from between 500 and 1500 ppm. CO2 nanobubble concentrations can be determined using a Nanoparticle Tracking Analysis (NTA) instrument such as the NanoSight 300 (Malvern Panalytical, UK). Total (dissolved) Carbon (TC) was analyzed in samples in a sealed vial without headspace using a Shimadzu TOC-V instrument. These samples were injected directly onto a combustion tube without exposure to air. Allcarbon was converted to CO2 and measured via NDIR (non-dispersive infrared) detection. Samples for Inorganic Carbon (carbonate & bicarbonate) analysis were collected as for total carbon (TC) and were analyzed in the same Shimadzu instrument but were acidified with 2 N HC1 to 5% acid within a sealed syringe and were then purged with carrier gas. The resultant CO2 is transferred via the instrument plumbing to the NDIR detector for measurement.

[0047] CO2 Levels in Nanobubble Water:

[0048] Inorganic carbon levels obtained at different pressures of the Nano Gas instrument are provided in Table 1. Inorganic carbon levels ranged between about 300 and 400 mg / L, which is several orders of magnitude higher than in air-saturated water (~0.5 mg CO2 / L, and -0.15 mg C / L). This illustrates the power of CO2 delivery via nanobubbles.Table 1. Inorganic carbon in CO2 nanobubble water that was adjusted to pH 9 with NaOH after generation at various gas pressures

[0049] Leaching of Metal Ions:

[0050] CO2 nanobubble solutions are acidic (pH 3.4-5). Care must be taken regarding the composition of metal fittings and tubing in the Nano Gas Unit that are in contact with the CO2 nanobubble solution as metal ions may be leached that are toxic to cyanobacterial cultures. As indicated in Table 2, micromolar concentrations of potentially toxic metal ions including those of Cu, Ni, Zn, AL Fe and Pb were observed in at least one of several nanobubble samples tested. Indeed, micromolar concentrations of Cu and Ni ions were shown to have negative effects on cyanobacterial growth (FIG. 1). The issue was ameliorated to a large extent by raising the pH of the water before feeding into the nanobubble generator (Table 2). Moreover, addition of micromolar concentrations of EDTA, which complexes divalent cations, alleviated most of the negative impact of leached metal ions in BG-11 made with nanobubble water on culturing a strain of the cyanobacterium Synechocystis sp. PCC 6803 (FIGS. 2-4), thus providing amechanism to overcome any negative impacts of metal ions leached from the nanobubblegenerating instrument.Table 2. Metal ions detected in CO2 nanobubble water samples. Concentrations are listed in microM (pM). LOD: limit of detection.Note: Nano-H20 was first generated by Nano Unit from low pressure at 25 psi and then with increasing pressures. Dl-water in the Nano Unit from previous run was not drained off and therefore the acidic Nano-water generated from previous run had been sitting in Nano Unit until the current run that resulted in higher level of leached metals accumulated in the pressurevessel. Thus, Nano-f bO water generated first at 25 psi contained the much higher level of metals compared to the Nano-tbO generated at 50 psi and 100 psi.

[0051] Growth of Synechocystis Cultures with Nanobubbles:

[0052] As indicated in Tables 1 and 2, Nano BG-11 can be generated at different Nano Gas instrument pressures (10-100 psi) using the C25 and NG25 units (or up to an instrument pressure of 300 psi using the L6 unit - not shown), yielding different results regarding the amount of inorganic carbon in the medium. In the presence of sufficient EDTA to complex any toxic metal ions, cultures grown in Nano BG-11 generated at higher CO2 pressure (50-100 psi) grew somewhat better than the control (FIG. 5). However, Nano BG-11 generated at lower CO2 pressures did not grow as well after a day (FIG. 5), which could be due to the lower inorganic carbon compared to that generated at higher pressures. (Table 2). In any case, we demonstrate here that grow th of photosynthetic microbes with CO2 derived from nanobubbles readily competes with that under control conditions.

[0053] Synechocystis Growth Medium:

[0054] The standard medium used to grow Synechocystis cultures was BG-1 1. a standard mineral medium used for culturing cyanobacteria. BG-11 medium contains 17.6 mM NaNCh, 0.23 mM K2HPO4, 0.3 mM MgSCU, 0.24 mM CaCl2, 0.031 mM citric acid, 0.021 mM ferric ammonium citrate. 0.0027 mM Na2EDTA, 0.19 mM Na2COs, 46 pM H3BO3, 9 pM MnCh, 0.77 pM ZnSO4. 1.6 pM Na2MoO4, 0.3 pM CuSO4, and 0.17 pM Co(NO3)2. Synechocystis grows best at alkaline pH, between 7.5 and 1 1. On the other hand, CO2 nanobubble water is acidic (pH 3.4-5) due to carbonic acid (CO2 dissolved in water). Given the Synechocystis preference for more alkaline culture environments, the freshly generated CO2 nanobubble water was mixed immediately with 100-fold concentrated BG-11 and then the pH of the medium was adjusted to pH 9 with NaOH to produce a growth medium enriched with CO2 nanobubbles, which is referred to as Nano BG-11. For example, to make 1 L of Nano BG-11 medium, about 950 mL of freshly generated CO2 nanobubble water w as mixed immediately with 10 mL of 100-fold concentrated BG-11 medium. The pH was then adjusted to pH 9 and the volume of the medium was brought to 1 L by adding a small amount of CO2 nanobubble water. Control BG-1 1 cultures (i.e., no CO2 nanobubbles) had bicarbonate added as the carbon source. The amount of bicarbonate added to the control medium varied depending on the carbon content of the Nano BG-11 medium.

[0055] Growth of Synechocystis'.

[0056] Synechocystis sp. PCC 6803 cultures (wild ty pe and strain TE / As7r7609 that has been modified genetically to produce and excrete free fatty’ acid laurate) are typically grown at laboratory scale (50 ml tubes or culture flasks) with either control BG-1 1 or Nano BG-11. A dodecane overlay may be used to limit gas exchange between air and the culture medium. Cultures in culture flasks were shaken slowly, and small magnetic stirrers were used to facilitate even mixing of cultures in tubes. Cultures were incubated at 30°C and the culture density was monitored over time by measuring optical density (OD) at 730 nm.

[0057] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary’ aspects. Various features and advantages of the invention are set forth in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method of culturing a microbe, the method comprising: supplying CO2 nanobubbles to an aqueous growth medium to generate a CO2 enriched medium; and growdng the microbe in the CO2 enriched medium.

2. The method of claim 1, wherein supplying CO2 nanobubbles to the aqueous growth medium comprises (a) mixing water containing CO2 nanobubbles with the aqueous growth medium, (b) dissolving a solid growth medium with water containing CO2 nanobubbles, or (c) infusing CO2 nanobubbles into the aqueous growth medium.

3. The method of claim 2, wherein the water in (a) or (b) is saturated with CO2.

4. The method of claim 2, comprising (a) mixing water containing CO2 nanobubbles with the aqueous growth medium, wherein the volume ratio between water containing CO2 nanobubbles and the aqueous medium is about 10: 1 to about 1000: 1.

5. The method of claim 1 , wherein the CO2 nanobubbles in the CO2 enriched medium have an average size of 200 nm.

6. The method of claim 1, wherein the infused CO2 in the CO2 enriched medium has an inorganic carbon concentration of about 300 mg / L to about 500 mg / L.

7. The method of claim 1, further comprising adding a metal chelator to the CO2 enriched medium.

8. The method of claim 2, further comprising adding a metal chelator to the water containing CO2 nanobubbles in (a) or (b), prior to mixing with the aqueous medium or dissolving the solid growth medium.

9. The method of claim 1, w herein the CO2 enriched medium has a pH of about 8.0 to about 9.5.

10. The method of claim 2, further comprising adjusting a pH of the water containing CO2 nanobubbles in (a) or (b) to about 8 to about 11.

11. The method of claim 1 , wherein the CO2 nanobubbles in the CO2 enriched medium are stable from 1 to 14 days at a temperature of 25-35 °C.

12. The method of claim 1, wherein the microbe comprises a photosynthetic microbe.

13. The method of claim 12, wherein the photosynthetic microbe comprises a cyanobacterium or an alga.

14. The method of claim 12, wherein the photosynthetic microbe comprises Synechocystis .

15. The method of claim 12, wherein the photosynthetic microbe is grown at a temperature of about 30 °C for about 7 days.

16. An aqueous grow th medium comprising CO2 nanobubbles and at least one nutrient for microbe culture, optionally wherein the aqueous growth medium has a microbe therein.

17. The aqueous growth medium of claim 16, wherein the nutrient is selected from the group consisting of nitrate, phosphate, sulfate, chloride, carbonate, citrate, and a combination thereof.

18. The aqueous growth medium of claim 16, wherein the CO2 nanobubbles have an average size of about 200 nm, wherein the CO2 nanobubbles are stable from 1 to 14 days at a temperature of 25-35 °C, wherein the CO2 in the CO2 nanobubbles has an inorganic carbon concentration of about 300 mg / L to about 500 mg / L, or any combination thereof.

19. The aqueous growth medium of claim 16, wherein the aqueous growth media has a pH of about 8.0 to about 9.5.

20. The aqueous growth medium of claim 16, further comprising a metal chelator.

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