Methods and compositions for inactivation of cyanobacteria and degradation of microcystin

Abstract

The growth of algae can be controlled in water by exposing water containing algae to agitation, for example shear stress and an electrical field. The water may further be detoxified of the microcystins produced by algae by treating the water with a microcystin-degrading bacterium, such as Sphingomonas sp. together with an electric field, with the application of the electric field causing a major acceleration of the toxin's degradation.

Description

[0001] METHODS AND COMPOSITIONS FOR INACTIVATION OF CYANOBACTERIA AND DEGRADATION OF MICROCYSTIN

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U.S. Provisional Application 63 / 507,784, filed June 13, 2023, the contents of which are hereby incorporated in its entirety.

[0004] BACKGROUND

[0005] Harmful algal blooms (HABs), large growths of cyanobacteria, are found in many freshwater and marine ecosystems but can disrupt normal water usage, damage the environment, and threaten human health. Over time, HAB frequency and intensity has increased, as well as their geographic distribution due to climate change and increased eutrophication, particularly recently in Lake Erie. Previous work describes the widespread presence of cyanobacterial blooms across the United States, with 62% of counties exhibiting detectable blooms. Lake Erie in particular serves as one of the most commonly studied and monitored fresh water bodies in the Great Lakes region of the U.S. and across the world, due to the persistent HABs that are dominated by the cyanobacteria genus Microcystis.

[0006] HABs are seen as harmful when they become a threat to public health and the environment. Common cyanobacteria, including Microcystis, can produce cyanotoxins, toxic chemical metabolites that are known to have damaging effects on cells and tissues. Cyanotoxin exposure can be through ingestion, inhalation, or dermal contact for both animals and humans. Microcystins (MCs) are some of the most well-studied cyanotoxins that cause damage in liver cells, with MC-LR being the most toxic of the MC congeners. MCs are taken up in liver cells and become inhibitors of protein phosphatases (PPI and PP2), the root of toxicity. Acute human symptoms of short-term MC exposure include hay fever-like symptoms, skin rashes, respiratory and gastrointestinal distress, while chronic human symptoms can be liver and kidney damage.

[0007] Microcystis and other MC-producing cyanobacterial blooms exhibit concentrations of cyanotoxins in their surrounding w aters. Particularly in Lake Erie, MC concentrations associated ssiX Microcystis blooms have exceeded the World Health Organization (WHO) drinking water guideline for MC (1 pg / L) near drinking water treatment intake sites. Both light and temperature affect toxin production in cyanobacteria, and studies suggest that the changing climate and warmer waters may favor more toxin-producing strains of cyanobacteria in the future. Due to their relative stability in water, MC and other cyanotoxin persistence is cause for concern. During recent summer high bloom seasons in Lake Erie, seven MC congeners were detected, with MC-LR as the most abundant, especially near shore. Lake Erie and other similar water bodies have experienced increased nutrient influxes, likely attributed to increased anthropogenic activities, causing increased growth of HABs.

[0008] Lake Erie serves as a drinking water supply for over 12 million people in the region, is important for protecting the environment, and is key to a $13 billion tourism and fishing industry for the region. Both the U.S. Environmental Protection Agency (U.S. EP A) and the WHO have established recreational and drinking water guidelines and recommendations regarding cyanobacteria and cyanotoxins for protecting public health. Management of these water bodies is crucial for protecting public health in both recreational and drinking water sources, and short and long term measures are. Unfortunately, monitoring and ultimately protecting these waters can be economically challenging. Local communities may have to handle adverse economic impacts caused by these blooms, and in some instances may dedicate more than $100,000 per month for additional treatments in order to ensure public health safety.

[0009] With rising climate challenges and observed increases in toxic HABs, there is a continued need to manage and mitigate the harmful effects of these cyanobacteria and, more importantly, their accompanying cyanotoxins. Because various conventional water treatment processes for MC removal, such as ozonation and chlorination, can lead to harmful by-products, other methods have been explored in an attempt to find a more sustainable and environmentally-friendly solution, such as ultrasound, UV light, and cold plasma. Other opportunities for MC removal come from the natural environment itself. It is known that naturally occurring bacteria in the same aquatic or sediment environment as cyanobacterial blooms are capable of degrading MCs when toxin levels are high, and have been isolated around the world. The first isolated microcystin-degrading bacteria was identified as Sphingomonas sp. ACM3962, and was capable of degrading MCs into various non-toxic compounds.

[0010] For all of these solutions, although successful, they exhibit challenges when trying to scale up for larger impact. These solutions face challenges with penetration in water depths, spatial effectiveness, residue production, increased health risk and exposure for operators, as examples. Therefore, a toxin management practice that is sustainable, environmentally friendly, and scalable is desired. Ultimately, reducing cyanobacteria populations would therefore decrease toxin levels. However, mass collapse of HABs may lead to vast increases in toxin in surrounding waters and may overwhelm treatment systems, as was the case in Toledo, Ohio in August 2014. The city underwent a 3-day state of emergency due to the unsafe MC levels in the finished drinking water, likely attributed to the unexpectedly high toxin concentrations and high bloom spikes throughout the summer and the inability7of the treatment infrastructure to manage these high toxin levels safely and efficiently. Therefore, addressing solutions geared towards toxins is also an important step for reducing public health risks.

[0011] There remains a need for improved systems and methods for the treatment and purification of water. There remains a need for improved systems and methods for the reduction of algae growths in water. There remains a need for improved systems and methods for decreasing the concetration of toxins, including algae-produced toxins such as microcystins, in water supplies.

[0012] BRIEF DESCRIPTION OF THE FIGURES

[0013] Figure 1 shows a schematic illustration of (a) MEF unit; (b) MEF system set-up; and (c) a flowchart of experimental design for Microcystin-LR degradation experiments. Figure 1(d) shows a schematic illustration of a vertical cross-section of SS+MEF unit.

[0014] Figure 2. Changes in percentage of (a) cell density, (b) chlorophyll a, and (c) phycocyanin of AT. aeruginosa over 72 h incubation (25°C) after different SS+MEF treatments (40°C). Data represent the means ± SD of three experiments each in triplicate. Capital letters refer to comparisons between the same treatment at different time points; Lower case letters refer to comparisons between different treatments at the same time point. For both sets of comparisons, different letters indicate significant differences (p < 0.05).

[0015] Figure 3. Changes in appearance of AL. aeruginosa culture over 72 h incubation (25°C) after different SS+MEF treatments (40°C).

[0016] Figure 4. Changes in percentage of (a) cell density, (b) chlorophyll a, and (c) phycocyanin of AL. aeruginosa over 72 h incubation (25°C) after different SS+MEF treatments (30°C). Data represent the means ± SD of three experiments each in triplicate. Capital letters refer to comparisons between the same treatment at different time points: Lower case leters refer to comparisons between different treatments at the same time point. For both sets of comparisons, different leters indicate significant differences (p < 0.05).

[0017] Figure 5. Changes in appearance of AL. aeruginosa culture over 72 h incubation (25°C) after different SS+MEF treatments (30°C).

[0018] Figure 6. Changes in percentage of (a) cell density, (b) chlorophyll a, and (c) phycocyanin of AL. aeruginosa over 72 h incubation (25°C) after different SS+MEF treatments (20°C). Data represent the means ± SD of three experiments each in triplicate. Capital leters refer to comparisons between the same treatment at different time points; Lower case leters refer to comparisons between different treatments at the same time point. For both sets of comparisons, different leters indicate significant differences p < 0.05).

[0019] Figure 7. Changes in appearance of AL. aeruginosa culture over 72 h incubation (25°C) after different SS+MEF treatments (20°C).

[0020] Figure 8. Changes in percentage of (a) cell density, (b) chlorophyll a, and (c) phycocyanin of AL. aeruginosa over 72 h incubation (25°C) after different SS+MEF treatments (10°C). Data represent the means ± SD of three experiments each in triplicate. Capital leters refer to comparisons between the same treatment at different time points; Lower case leters refer to comparisons between different treatments at the same time point. For both sets of comparisons, different leters indicate significant differences (p < 0.05).

[0021] Figure 9. Changes in appearance of AL. aeruginosa culture over 72 h incubation (25°C) after different SS+MEF treatments (10°C).

[0022] Figure 10. Waveforms of discharge currents upon applied voltage (sine waveform with 60 Hz frequency; 135 V / cm) under different rotation speeds of rotor electrode in the fluid.

[0023] Figure 1 1 . Relative changes of total mcyE gene over 72 h incubation (25°C) after different SS+MEF treatments at (a) 40°C and (b) 20°C. Data represent the means ± SD of three experiments each in triplicate. Capital leters refer to comparisons between different treatments at the same time point; Lower case leters refer to comparisons between the same treatment at different time points. For both sets of comparisons, different leters indicate significant differences (p < 0.05).

[0024] Figure 12A shows For electro-bio-degradation of MC-LR, with three conditions tested. The results are obtained from ADDA ELISA (Polyclonal antibody prepared against the Adda moiety binds to MC - allowing the congener-independent determination).

[0025] Dark green bar: Negative control (i.e. no treatment) Green bar: We conducted the incubation with 106CFU / mL Sphingomonas sp. after MEF (15 V / cm at 30°C, as a pre-treatment). Treatments were conducted up to 24 hrs.

[0026] Blue bar: Data from weak MEF (wMEF) - the applied electric field strength was set to 4 V / cm, which presented the lowest electric current readings (~3 mA) from the current setting while MEF was applied. A 100 ppb MC-LR sample with sub-inoculated 106CFU / mL Sphingomonas sp. was incubated for 15 hours with continuous MEF and agitation at 30°C and then transferred to the same incubation system with the control group for the next 9 h (*, without MEF).

[0027] Orange bar: Positive control - same agitated incubation at a temperature of 30°C with 106CFU / mL Sphingomonas sp. up to 24 hours.

[0028] Figure 12B shows microcystin toxicity test results. The quantitative measurement of PP2A-alpha protein (based on the phosphatase activity inhibition by microcystin) is shown. Samples containing microcystins inhibit the enzy me activity proportionally to the amount of toxin contained in the sample.

[0029] Figure 13 shows the table with ODeoo, which is the change in cell concentration (In this case, Sphingomonas sp.), while they are incubated or treated.

[0030] Figure 14A-B show the data of residual MC-LR (at 15 and 24 hours) quantified with LC-MS / MS method. The residual MC-LR concentrations were compared to the initial concentration (at 0 hours) under 3 treatment groups: preMEF+SP group (high intensity MEF (15 V / cm) for initial 40 min, additional 24-hr agitation, incubated with Sphingomonas (SP) at 30°C); wMEF+<SP group (weak intensity MEF (4 V / cm) to MC+Sphingomonas (SP) solution for 15 hrs and additional 9-hr agitation at 30°C); control group (MC solution with Sphingomonas only, 24-hr agitation at 30°C).

[0031] Figure 15 depicts an exemplary reactor for use in the methods disclosed herein.

[0032] Figure 16 depicts an exemplary reactor for use in the methods disclosed herein.

[0033] Figure 17 depicts exemplary' rotors for use in the methods disclosed herein.

[0034] Figure 18 depicts a cross-section of an exemplary reactor for use in the methods disclosed herein.

[0035] Figure 19A depicts a cross-section of an exemplary' reactor for use in the methods disclosed herein.

[0036] Figure 19B depicts a cross-section of an exemplary reactor for use in the methods disclosed herein. DETAILED DESCRIPTION

[0037] Fluids

[0038] The disclosed methods can be used to disinfect any fluids where high temperatures are not feasible or desirable. In some embodiments the fluid is from a naturally occurring source, such as a pond or a lake. In addition, the methods described herein can be used to purify drinking water or other liquids that may be used for enteral or parenteral introduction to humans and animals. In other aspects, the water can be from a waste treatment plant or water purification system. In effect, any liquid medium, for any use, that supports algal or bacterial grow th may be disinfected by this method.

[0039] Mechanical Shear

[0040] Fluid undergoes shear when one area of fluid travels with a different velocity relative to an adjacent area. A high-shear mixer uses a rotating impeller or high speed rotor, or a series of such impellers or inline rotors, usually powered by an electric motor, to "w ork" the fluid, creating flow and shear. The tip velocity, or speed of the fluid at the outside diameter of the rotor, will be higher than the velocity at the center of the rotor, and it is this velocity difference that creates shear.

[0041] A stationary7component may be used in combination with the rotor and is referred to as the stator. The stator creates a close-clearance gap between the rotor and itself and forms an extremely high-shear zone for the material as it exits the rotor. The rotor and stator combined together are often referred to as the mixing head, or generator. A large high-shear rotor-stator mixer may contain a number of generators.

[0042] In some embodiments, mechanical shear is produced using a Taylor-Couette Device. Taylor-Couette flow is the flow of a viscous fluid sheared in the gap between two rotating coaxial cylinders. The basic apparatus consists of two coaxial cylinders. The gap between them is filled with fluid and the inner cylinder is made to rotate by a motor. Fluid elements follow circular paths and the flow' field has no axial or azimuthal dependence. How ever, as the rotation rate of the inner cylinder is increased the flow suddenly undergoes an instability' and a cellular pattern of toroidal vortices called Taylor rolls emerges producing Taylor- Couette flow. As the rotation is increased further, the Taylor rolls themselves become unstable giving way to a progressively more complicated flow states, eventually leading to turbulence.

[0043] In some embodiments, the mechanical shear has a shear rate of at least, 150 s’1, 200 s’1, 250 s’1, 300 s’1, 350 s’1, 400 s’1, 450 s’1, 500 s’1, 550 s’1, 600 s’1, 650 s’1, 700 s’1, 750 s’1, 800 s’1, 850 s’1, 900 s’1, 950 s’1, 1,000 s’1. 1,050 s’1. 1,100 s’11,150 s’1. 1,200 s’1. 1,250 s’1,

[0044] 1.300 s’1, 1.350 s’1, 1.400 s’1. 1.450 s’1. 1.500 s’1. 1,550 s’1. 1,600 s’1. 1,650 s’1, 1,700 s’1,

[0045] 1.750 s’1, 1,800 s’1, 1,950 s’1, 2,000 s’1, 2,050 s’1, 2,100 s’1, 2,150 s’1, 2,200 s’1, 2,250 s’1,

[0046] 2.300 s’1, 2,350 s’1, 2,400 s’1, 2,450 s’1, 2,500 s’1, 2,550 s’1, 2,600 s’1, 2,650 s’1, 2,700 s’1,

[0047] 2.750 s’1, 2,800 s’1, 2,950 s’1, or 3,000 s’1.

[0048] The use of the rotating shear may be generated, for example, by using a motor, connected to concentric rotating cylinder systems. The methods described herein can create the shear rate with a defined intensity at the middle of the annulus between the inner and the outer cylinders by rotating inner, outer, or both (co-rotating or counter-rotating). The shear rate was generated in the present embodiment by an angular rotation of the motor with an intensity- as measured by the RPM (revolutions per minute) by a tachometer. Shear rate values may be calculated by the RPM and radii of inner and outer cylinders. A person of ordinary- skill in the art will recognize that the RPM referred to herein is the speed of the rotating body as measured at the center of rotor.

[0049] Alternatively, it is possible to create a high shear environment by pumping fluid between parallel plates at a high rate and using the parallel plates as electrodes. Other embodiments are also possible including electric fields parallel to, perpendicular to the primary flow (o shearing) plane; or any combination thereof.

[0050] The description of any such embodiments is not intended to be restrictive, rather any means of shearing combined with a moderate electric field is intended to be covered by this invention.

[0051] Moderate Electric Field

[0052] In some embodiments, the electric field is applied between the rotor and the stator. Therefore, in some embodiments, one electrode is located on the rotor, and one electrode is located on the stator. In other embodiments, both electrodes are placed on the stator (e.g. opposite ends of the stator). In some embodiments, shear is created by pumping fluid between static parallel plates, which may serve wholly or partially as electrodes. In some embodiments, the electric field is less than about 1000 V / cm. In some embodiments, the electric field is less than about 200 V / cm. In some embodiments, the electric field is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 V / cm.

[0053] In some embodiments, the electric field has an arbitrary wave form. As described elsewhere, the moderate electric field may be provided as a squared waveform. Common waveforms for electrical currents include the square wave, the sine wave, the ramp, the Sawtooth wave, and the triangular wave. In a squared waveform, the amplitude of the wave alternates at a steady frequency between fixed minimum and maximum values, with the same duration at minimum and maximum.

[0054] In addition to having a waveform, moderate electric field can be provided with a duty cycle (fraction of field on-time to total time) of about 0 to about 1. In some embodiments, the squared waveform can have a frequency of about 0 to about 100 kHz. In some embodiments the frequency is about 60 Hz or above.

[0055] Temperature and Pressure

[0056] In some embodiments, the fluid is treated at a temperature less than 100 °C, 90 °C, 80 °C. 70°C, 60 °C. 50 °C, 40 °C, 20 °C, 10 °C, or 5 °C. In some embodiments, the fluid is treated at a temperature of about 5 to 100 °C. including about 10 to 60 °C. In some aspects, the fluid may be treated to a temperature in the range of about 15 °C to about 40 °C. In some embodiments, the fluid is treated at room temperature. In some embodiments, the fluid is cooled while treated. In some embodiments the apparatus comprises elements to reduce the temperature in the chamber to a desired temperature, such as in the range of about 20°C to about 60 C. In some embodiments, the fluid is treated at atmospheric pressure. In some embodiments, the pressure may be less than 50 MPa, for example about 1 atm (101.3 kPa).

[0057] Also disclosed herein are methods for treating a volume of w ater disposed w ithin a of a reactor, w hich include the step of agitating the volume of water in the presence of an electrical field. In certain aspects, the water may be agitated using a rotating rotor disposed within the volume of w ater. The rotating rotor applies a shearing force to the w ater, thereby inducing a shear stress.

[0058] In some implementations, the shear stress has a shear rate of at least 150 s'1, at least 200 s'1, at least 250 s'1, at least 300 s'1, at least 350 s'1, at least 400 s'1, at least 450 s'1, at least 500 s’1, 550 s’1, 600 s’1, 650 s’1, at least 700 s’1, at least 750 s’1, at least 800 s’1, at least 850 s’1, at least 900 s’1, at least 950 s’1, at least 1,000 s’1, at least 1,050 s’1, at least 1,100 s’1, at least 1,150 s’1, at least 1,200 s’1, 1,250 s’1, at least 1,300 s’1, at least 1,350 s’1, at least

[0059] 1,400 s’1, at least 1,450 s’1, at least 1,500 s’1, at least 1,550 s’1, at least 1,600 s’1, at least

[0060] 1,650 s’1, at least 1,700 s’1, at least 1,750 s’1, at least 1,800 s’1, at least 1,950 s’1, at least

[0061] 2,000 s’1, at least 2.050 s’1, at least 2.100 s’1, at least 2.150 s’1, at least 2.200 s’1, 2.250 s’1, at least 2,300 s’1, at least 2,350 s’1, at least 2,400 s’1, at least 2,450 s’1, at least 2,500 s’1, at least 2,550 s’1, at least 2,600 s’1, at least 2,650 s’1, at least 2,700 s’1, at least 2,750 s’1, at least 2,800 s’1, at least 2,950 s’1, or at least 3,000 s’1.

[0062] The rotor may be rotated a rate sufficient to induce Taylor rolls or Taylor-Couette flow in the volume of water. In certain implementations, the rotor rotates at a rate of at least 200 rpm, at least 300 rpm, at least 400 rpm, at least 500 rpm, at least 1,000 rpm, at least

[0063] 2.500 rpm, at least 5,000 rpm, at least 7,500 rpm, or at least 10,000 rpm.

[0064] In certain implementations, the rotor is a cylinder. In some implementations the rotor may include one or more paddles to increase the agitating force delivered by the rotor.

[0065] In certain implementations, the volume of water is disposed within a chamber configured to hold the water. In aspects, the chamber can be rotated. In certain implementations, the chamber rotates at a rate of at least 200 rpm, at least 300 rpm, at least 400 rpm, at least 500 rpm, at least 1,000 rpm, at least 2,500 rpm, at least 5,000 rpm. at least

[0066] 7.500 rpm, or at least 10,000 rpm. In still further aspects, a rotor can be disposed within the volume of water in the chamber, and both the rotor and the chamber rotated. In certain implementations, the chamber and rotor are rotated along the same axis. In the certain aspects, rotating chamber rotates at the same speed as the rotor, which may be in different directions, i.e., one of the chamber or rotor rotates clockwise and the other rotates counterclockwise.

[0067] In certain implementations the rotor rotates at a speed that is at least 110%, at least 125%. at least 150%, at least 200%, at least 250%, at least 300%, at least 500%. at least 1,000%. at least 2,000%, or at least 5,000% the speed of the rotating chamber. In certain implementations the chamber rotates at a speed that is at least 110%, at least 125%, at least 150%, at least 200%, at least 250%, at least 300%, at least 500%, at least 1,000%, at least 2,000%. or at least 5,000% the speed of the rotor.

[0068] In other implementations, the chamber is stationary, and the rotating rotor provides the agitating force. The electric field may be delivered through one or more electrodes. In certain implementations, the electric field may be provided across two electrodes. In certain implementations, both electrodes may be disposed within the volume of water. In some implementations, one electrode is disposed in the volume of water, and the other is included in either the rotor or chamber. In further implementations, one electrode may be provided in the rotor, and the other in the chamber.

[0069] The electrode may be provided in the rotor by placing the rotor in electrical communication with a power supply. In some aspects, the rotor itself is made of a conductive material, e.g., a metal, graphite, conductive polymer, etc. In other implementations, the rotor includes conductive material in combination with noncon ductive material. The electrode may be provided in the chamber using similar configurations.

[0070] Also disclosed herein are method for treating a volume of water, the method including the steps: a) providing a reactor, wherein the reactor includes: a first end wall, a second end axially opposite the first end wall, and a least one side wall extending between the first and second end walls, wherein said first end wall, second end wall, and side wall defines a chamber; and a rotor disposed within the chamber; wherein the chamber is in electrical communication with at least two electrodes; b) delivering the volume of water to the chamber; and c) agitating the volume of water in the presence of an electrical field.

[0071] In certain implementations, the rotor and at least one side wall are coaxial along a major axis. As used herein, the major axis extends along the longest linear length of the rotor. In some implementations, the at least one side wall forms a cylinder (e.g., the reactor includes a single side wall). In other implementations, the reactor includes three side walls, which together form a reactor having a triangular cross-section (taken perpendicular to the major axis). In further implementations, the reactor includes four side walls, which together form a reactor having a quadrilateral cross-section. In some implementations, the four side walls form a square.

[0072] In certain implementations, the rotor and the at least one side wall are concentric in that they share a center point along the same axis. In certain implementations, the rotor is a cylinder. In some implementations, the rotor can include one or more paddles. The paddles may be of any shape, and increase the surface area of water that is displaced by the rotating rotor. The rotor may be rotated a rate sufficient to induce Taylor rolls or Taylor-Couette flow in the volume of water. In certain implementations, the rotor rotates at a rate of at least 200 rpm, at least 300 rpm, at least 400 rpm, at least 500 rpm, at least 1,000 rpm, at least 2,500 rpm, at least 5,000 rpm, at least 7,500 rpm, or at least 10,000 rpm.

[0073] In certain implementations, the at least one side wall is stationary, while in other implementations the at least one side wall may be rotated along the same axis as the rotor, in the same or opposite direction as the rotor.

[0074] In certain implementations, the chamber includes a single rotor. In other implementations, the chamber includes a plurality of rotors. In some implementations, each rotor in the plurality of rotors is coaxial. In some implementations, each rotor in the plurality7of rotors rotates in the same directions. In other implementations, a first portion of the rotors rotates clockwise, and a second portion of the rotors rotates counterclockwise.

[0075] An exemplary reactor is depicted in Figure 15. The reactor includes chamber (1501) defined by at least one side wall (1502). A rotor (1503) may be rotated by a motor (1504). Water may be introduced to the chamber via inlet (1505), and water may exit the chamber via outlet (1506). In this example, both inlet and outlet define a portion of the at least one side wall. Two electrode (1507a, 1507b) may be disposed within the chamber, each in electrical communication with a power supply.

[0076] An exemplary reator is depicted in Figure 16 The reactor includes chamber (1601) defined by at least one side wall (1602). The reactor includes a first end wall (1608) and second end wall (1609) with the at least one side wall extending between them. A rotor (1603) may be disposed within the chamber along a major axis (1610). Side wall (1602) and rotor (1603) are coaxial with respect to the major axis. Water may be introduced to the chamber via inlet (1605), and water may exit the chamber via outlet (1606). In this example, inlet (1605) defines a portion of end wall (1609), and outlet (1606) defines a portion of end w all (1608).

[0077] Exemplary rotors are depicts in Figure 17, including cyclindrical rotor (1701) having one or more paddles (1702). Polygonal rotor (1703) may also include one or more paddles (not shown).

[0078] A partial cross-section of the Figure 15 reactor is depicted in Figure 18, showing chamber (1801), side wall (1802), rotor (1803), inlet (1805). outlet (1806), first and second electrodes (1807a, 1807b) disposed within the chamber, and power supply (1808) in electrical communication with the first and second electrodes. The rotor and side wall are concentric as they share the same center point.

[0079] A partial cross section of a separate reactor is depicted in Figure 19A, showing chamber (1901), side wall (1902), rotor (1903), inlet (1905), outlet (1906). In this example, side wall (1902) includes the first electrode and rotor (1903) includes the second electrode, both in electrical communication power supply (1908). The rotor and side wall are concentric as they share the same center point.

[0080] A partial cross section of a separate reactor is depicted in Figure 19B, showing chamber (1901), (1902), rotor (1903), inlet (1905), outlet (1906). In this example, the side wall includes a first portion (1902a) including the first electrode and a second portion (1902b) including the second electrode, both in electrical communication power supply (1908). The side wall includes insulating sections (1909a, 1909b) electrically isolating the first eletrodode from the section electrode. The rotor and side wall are concentric as they share the same center point.

[0081] A partial cross-section of a separate reactor is depicted in Figure 20, showing chamber (2001), four side walls (2002a, 2002b, 2002c, 2002d) defining a rectangular crosssection, and four rotors (2003a, 2003b, 2003c, 2003d) coaxially disposed in the chamber. Although Figure 20 depicts four rotors, the invention embraces any number of rotors disposed within the chamber. In some implementations, the reactor includes 2, 3. 4, 5, 6, 7. 8, 9, or 10 rotors. In some implementations, the reactor includes more than 10 rotors. The first and second electrode may be included in two or more of the side walls, show n here in opposite side walls 2002a and 2002d. Side w alls 2002b and 2002c may be made of insulating materials to isolate the electrodes. In some implementations, more than two electrodes may be disposed in the side walls, having a suitable number of isolating sections to prevent short circuits.

[0082] In certain implementations, the at least one side wall rotates at a rate of at least 200 rpm, at least 300 rpm. at least 400 rpm. at least 500 rpm, at least 1,000 rpm, at least 2.500 rpm, at least 5,000 rpm. at least 7,500 rpm. or at least 10,000 rpm. In the certain aspects, the at least one side wall rotates at the same speed as the rotor, which may be in different directions, i.e., one of the side w all(s) or rotor rotates clockwise and the other rotates counterclockwise.

[0083] In certain implementations the rotor rotates at a speed that is at least 110%, at least 125%, at least 150%, at least 200%, at least 250%, at least 300%, at least 500%, at least 1,000%. at least 2,000%, or at least 5,000% the speed of the side wall(s). In certain implementations the side wall(s) rotates at a speed that is at least 110%, at least 125%, at least 150%, at least 200%, at least 250%, at least 300%, at least 500%, at least 1,000%, at least 2,000%, or at least 5,000% the speed of the rotor.

[0084] The electric field may be delivered through one or more electrodes. In certain implementations, the electric field may be provided across two electrodes. In certain implementations, both electrodes may be disposed within the chamber. In some implementations, one electrode is disposed in the chamber, and the other is included in either the rotor or chamber. In further implementations, one electrode may be provided in the rotor, and the other in the chamber.

[0085] In various aspects, the volume of water is delivered to the reactor in a batch process or in a continuous process. The water may be provided to the chamber via an inlet, treated according to the methods disclosed herein, and the exited from the chamber via an outlet. The inlet and outlet may be spaced at opposite ends of the chamber, such that they are exposed to agitating force and electrical fields as they transit from inlet to outlet. In other implementation, a volume of water is delivered to the chamber, the volume is treated, and then exited the chamber. A second volume of water for treatment may then be introduced to the chamber.

[0086] In certain implementations, the water is treated at a temperature that is from 5-100 °C., form 5-60 °C., from 10-60 °C., from 15-60 °C., from 20-60 °C., from 20-50 °C., from 5-40 °C., from 5-25 °C., from 20-40 °C., from 20-30 °C., from 30-60 °C., from 30-50 °C., from 30-40 °C., from 40-60 °C., or from 50-60 °C.

[0087] In various aspects, the electric field is provided at a rate from 10-200 V / cm, from 25-200 V / cm, from 50-200 V / cm, from 100-200 V / cm, from 100-150 V / cm, from 150-200 V / cm, from 10-100 V / cm, from 10-50 V / cm, from 10-25 V / cm, from 25-100 V / cm, from 25-50 V / cm, from 25-75 V / cm, from 50-75 V / cm, from 0.5-5 V / cm, from 1-2.5 V / cm, from 1-5 V / cm. from 2.5-5 V / cm, from 1-10 V / cm, from 5-10 V / cm, or from 5-15 V / cm.

[0088] The electric field can be applied to the water for a penod of from 1-180 minutes, from 10-120 minutes, from 10-90 minutes, from 10-60 minutes, from 10-30 minutes, from 20-90 minutes, from 20-40 minutes, from 30-90 minutes, from 30-40 minutes, from 40-90 minutes, from 40-50 minutes, from 50-90 minutes, from 50-60 minutes, or from 60-90 minutes. In certain implementations, the electric field is applied as a square wave, a sine wave, a ramp, a sawtooth wave, a triangular wave, or a combination thereof. In some implementations, the electric field is applied as sinusoidal waveform, for example as a sinusoidal waveform having a frequency from 5-100 Hz, from 5-75 Hz, from 5-50 Hz, from 5-25 Hz, from 10-25 Hz, from 10-100 Hz, from 25-50 Hz, from 25-100 Hz, from 40-100 Hz, from 40-75 Hz. from 50-75 Hz, or from 75-100 Hz.

[0089] The water may be treated according to the methods disclosed herein to reduce a number of different contaminants. In certain implementations, the water includes an algae, which may be reduced, killed, inactivated, inhibited, or otherwise inhibited from growth / multiplication using the methods disclosed herein. In certain implementations, the growth rate of the algae is reduced by 50% or more. In certain implementations, the algae includes Microcystis aeruginosa.

[0090] In certain implementations, the water includes a toxin, for example an oligopeptide toxin produced by harmful algae. In certain implematations. the water includes a microcystin-type toxin. The water may be combined with a microcystin-degrading organism and treated according to the methods disclosed herein.

[0091] In some implementations, the water includes a microcystin-degrading bacteria, for example aAcidovorax sp. , Acinelobacler sp.. Aeromonas sp. , Alcaligenes sp., Arlhrobacler sp., Bacillus sp., Bifidobacterium sp.. Bordetella sp.. Bosea asp., Brevibacillus sp., Brevibacterium sp., Brevundimonas sp., Burkholderia sp., Chryseobacterium sp., Cupriavidus sp., Enterobacter sp., Hyphomicrobium sp., Klebsiella sp., Lactobacillu sp., Lysinibacillus sp.. Mesorhizobium sp., Methylobacillus sp., Methylotenera sp., Morganella sp., Nitrosococcu sp., Novosphingobium sp., Ochrobactrum sp., Paucibacter sp., Polaromonas sp.. Pseudomonas sp., Pseudoxanthomonas sp., Polynucleobacter sp., Ralstonia sp., Rhizobium sp., Rhodococcus sp., Sandaracinobacter sp., Sphingomonas sp., Sphingopyxis sp., Stenotrophomonas sp., Steroidobacter sp., Variovorax sp., or a combination thereof. Various microcystin toxins and species of microcystin-degrading bacteria are disclosed in Toxins (2020) 12, 268, which is hereby incorporated in its entirety

[0092] In certain implementations the microcystin-degrading bacteria comprises Sphingomonas sp.

[0093] In certain implementations the microcystin-degrading bacteria includes one or more of, two or more of, three or more of, four or more of. five or more of, six or more of, or seven of the following bacteria: Acinetobacter sp., Hyphomicrobium aestuarii. Pseudoxanthomonas sp., Rhizobium sp., Sphingobium sp., Sphingomonas sp. and Steroidobacter sp.

[0094] In certain implementations the microcystin-degrading bacteria includes one or more of, two or more of, three or more of, four or more of, five or more of, six or more of, seven or more, eight or more, nine or more, or ten of the following bacteria: Acinetobacter sp., Aeromonas sp., Novosphingobium sp., Ochrobactrum sp., Pseudomonas sp., Rhodococcus sp., Sphingomonas sp., Sphingopyxis sp., Stenotrophomonas sp. and Steroidobacter sp.

[0095] Water from any source may be treated according to the methods disclosed herein. For example, the water can be from a natural water source such as a pond or lake. In other implementations, the methods and systems disclosed herein can be included in a water purification system, for example in-line with one or more other systems for water purification.

[0096] EXAMPLE

[0097] EXAMPLE 1: Impact of combined shear stress and moderate electric field (MEF) on inactivation of Microcystis aeruginosa, a bloom-forming cyanobacterium

[0098] The aim of this work was to determine the effectiveness of SS+MEF in controlling M. aeruginosa. For this, cultures of AT aeruginosa were subject to SS+MEF at varying intensities and temperatures during 72-hour period and then evaluated for growth and overall culture health. The findings provide support for real-time application for algal bloom control with low energy use, volumetric efficacy, and technical scalability to improve water quality while not generating chemical byproducts.

[0099] Materials and Methods

[0100] Cyanobacteria host maintenance and monitoring

[0101] Cyanobacteria cultures of Microcystis aeruginosa PCC 7806 (Institut Pasteur, Paris, France) were grown in CT media at 24°C under 12-hour light cycles at 2-week intervals (Jiang et al., 2017). Each subsequent experiment utilized fresh cyanobacteria cultures grown for 7 days in fresh CT media. As a proxy for cyanobacterial growth, chlorophyll a (Chi a) and phycocyanin (PC) levels were monitored using both an AquaFluor Handheld Fluorometer (8000-010 Turner Designs, Sunnyvale, CA, USA) and a 96-well plate Absorbance Reader (ELx808, BioTek, Winooski, VT, USA). Excitation / emission wavelengths for both pigments were 450 / 665 nm and 595 / 670 nm. respectively.

[0102] Cyanobacteria were grown to an approximate concentration of 107cells / mL prior to each experimental trial.

[0103] SS+MEF setup and treatment conditions

[0104] For all SS+MEF treatments, the experimental unit shown in Fig. 1 was used. Before and after each treatment, the entire inoculum-contact part of the apparatus was rinsed thoroughly with ethanol followed by autoclaving (at 110°C for 15 min) for sterilization. The MEF treatment applied in this investigation was sinusoidal waveform AC at 135 V / cm with a frequency of 60 Hz. Two rotational speeds were used in this research: 4000 and 9500 rpm. The SS+MEF treatment was continuously applied during the entire experiment. The treatment intensity varied by treatment time as 10, 20, and 40 minutes at test temperatures in the range of 10 to 40°C. This temperature range was chosen since it represents seasonal variation in the temperate climate zone, reflective of true temperatures in common water bodies containing HABs.

[0105] Following such SS+MEF treatment, each sample was transferred into sterile 250 mL Erlenmeyer flasks and incubated at the same conditions described above. Samples were taken and measured immediately before and after SS+MEF, as well as 72 hours after SS+MEF treatments. The sample before SS+MEF was the negative control. A positive control was also included for systematic comparisons (no SS+MEF treatment; but exposed to the same treatment times and test temperatures as the SS+MEF treatment).

[0106] For mechanical fluid shear, consider a concentric cylindrical system where an inner cylinder of radius A is rotating at a constant speed (rad / s) in an outer cylinder or cup of radius A, ss . The shear rate (y) for the Newtonian fluid can be calculated from Eq. (1): where + is rotation speed of the inner cylinder (rpm), operating between 500-9500 rpm in present studies (500, 1500, 5500, and 9500 rpm were calculated to shear rates of 151.5, 454.6, 1666.8, and 2879 s’1, respectively).

[0107] The treatment under 0 V / cm with the slowest angular rotation at 151.5 s’1was assumed to be equivalent to conventional treatment with gentle stirring. Constant temperature was maintained by circulating cooling water, with agitation being increased by the rotating inner electrode.

[0108] Sample collection and visual analyses

[0109] Samples were taken periodically and analyzed immediately before and after treatments, as well as at the 72 hours after treatment (post-incubation) period. For quantifying cyanobacteria grow th, cell-counting using a hemocytometer was initially utilized (Cat. No. 0267110, Fisher Scientific, Hampton, NH, USA). The small volume required for hemocytometer counting (10 pL) did not seem representative of the total culture volume (35 mL). Therefore, as a proxy for cyanobacteria densities, samples (250 pL) were immediately subject to fluorescence readings using a 96-well plate Absorbance Reader (BioTek). Two photosynthetic pigments known to have a positive association to cyanobacteria and Microcystis cell density. Chi a and PC, were tested in triplicate and subsequently averaged. For tracking visual changes in culture color, photos of each sample culture were taken at time 0 (post-treatment) and time 72 hours (post-incubation period). The change in culture appearance from blue-green to white indicates a loss in photosynthetic pigments and, ultimately, cell death.

[0110] RNA extraction and digital droplet PCR (dd-PCR)

[0111] To quantify viable cell densify using genetic methods, RNA was extracted from the samples and tested for a microcystin-producing Microcystis gene concentrations. Cyanobacteria cultures were briefly mixed by hand (swirled) and samples (1 mL) were aseptically removed at the 0- and 72-hour time mark for all trials. Samples were placed in 2- mL glass tubes (Cat. No. 66010-562, VWR, Radnor, PA, USA) and either stored at -80°C for 3-5 days, or immediately processed. RNA was extracted using the RNeasy Mini Kit (Cat. No. 74104-50, QIAGEN, Germantown. MD. USA) following the manufacturer’s instructions. A DNase-treatment was completed following extraction using the RNase-free kit (Cat. No. 79254-50, QIAGEN) to remove any remaining DNA in the sample (to exclusively capture living cell genetic material, represented by the RNA). Total RNA (10 pl) was then reverse transcribed (Cat no. 4368814. High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems). Reverse transcription was performed by incubation at 25°C for 10 minutes, 50°C for 10 minutes, and 85°C for 5 minutes. The resulting cDNA was then used for cyanobacterial gene quantifications. For quantification of cyanobacteria, a microcystin-producing Microcystis gene was quantified using a droplet digital PCR (ddPCR) system (Bio-Rad, Hercules, CA, USA). More specifically, the microcystin synthetase gene (mcyE) that codes Microcystis-specific MC production was targeted to quantify living Microcystis aeruginosa in all samples. The znqyE ddPCR assay, including primer and probes, was utilized according to a previous study. Amplifications were conducted in 20 pL reaction mixtures containing dd-PCR supermix for probes (Bio-Rad), DNase- & RNase-free water (Fisher Scientific, Hampton, NH, USA), 250 nM of forward and reverse primers, 250 nM of probe, and cDNA templates.

[0112] After droplet generation using a QX200 Droplet Generator (Bio-Rad), target genes were amplified using a Bio-Rad C1000 touch PCR thermal cycler (Bio-Rad). The cycling conditions were as follows: an initial 95°C for 10 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing for 30 seconds, extension at 60°C for 30 seconds, and enzyme deactivation at 98°C for 10 minutes, followed by an infinite hold at 4°C. Following amplification, target gene concentrations were determined by a QX200 droplet reader (Bio-Rad) and QuantaSoft version 1.7 (Bio-Rad).

[0113] Statistical analysis

[0114] Three replications for each treatment were used for all microbiological and fluorescence measurements unless otherwise stated. The results were analyzed by ANOVA of SAS software 9.3 (SAS Institute, Cary, NC, USA). Differences among mean values (± standard error of the means) were determined by Student t-test for pairwise comparisons (at a = 0.05) to determine significant differences between controls and treated groups. For significance testing of fluorometry and gene quantification results, comparisons were drawn between and across treatment parameters.

[0115] Results and Discussion

[0116] After SS+MEF treatment at 40°C (Fig. 2), a sharp decrease was observed in the mean values of change in ODeso but thereafter was a slight gradual decrease in cell density (ODeso) with increasing treatment time. All changes in cell concentration were negative (i.e., decreased concentration) for both applied rotation speeds. With longer treatment time, the results from the higher rotation speed showed significantly greater inhibition of growth (up to 222 %, at 40 min treatment), compared to the positive controls. For the positive controls, after a slight initial increase in the changes in ODeso, samples gradually decreased over time during the incubation period. Notably, the changes were opposite to the SS+MEF treatments, indicating growth and not inactivation of cyanobacteria under identical temperature conditions when the electric field is absent. These differences clearly highlight the inactivation role of the electric field to cyanobacteria.

[0117] In the case of the positive controls at 40°C, the decrease in ODsso over time is likely due to over-exposure to a higher temperature than that for optimal growth of M. aeruginosa, typically ranging from 27.5-35°C. Particularly, with extended thermal exposure, the peroxidation of thylakoid lipids increases, and the contents of proline, indicating the protective response of A / . aeruginosa, decrease. In the case of SS+MEF, thylakoid membrane fluidity could be altered, while the phase transition of its bulk lipids from the liquid-crystalline to the gel state occurs by applied heat, which may cause increased susceptibility of. T aeruginosa and resulted in growth suppression (Fig 2(a)).

[0118] Similar patterns were shown in the changes of Chi a and PC (Fig. 2(b) and (c), respectively). It appears reasonable that the concentrations of pigments and their changes were in proportion to cell densities and their growth rates, supporting the idea that cell densities and these two pigments are positively associated (Marion et al., 2012). Especially, damaged cells can cause the release of organic materials, w hich result in a higher initial pigment concentration. Notably, SS+MEF at 40°C induced effects on Chi a and PC that were qualitatively similar to the effects on cell density. Interestingly, in the case of positive controls, increases in Chi a and PC after 10-minute exposure at 40°C were followed by gradual decreases as treatment time increased. This can be explained by heat shock response in AT. aeruginosa by up-regulation of expression in related proteins (i.e., HspA and HtpG) upon heat exposure. These proteins experience loss of function after prolonged heat shock and induced stress. A possible reason for these results was due to recombinant protein aggregation and hampering of metabolic activity. Taking all of this into account, it is important to note the role that temperature alone plays in influencing cyanobacterial growth, as seen in the decrease in positive change over increased exposure time across all measured parameters.

[0119] In the culture discoloration results at 40°C (Fig. 3), post-incubation discolorations after SS+MEF became apparent at both rotation speeds. Slight yellow -green translucent appearance ofM. aeruginosa cultures was observed with increasing SS+MEF treatment time at 0 h incubation, and samples became completely discolored (clearing of culture) after 72 hours of incubation. Both temperature and MEF seem to influence cell viability at these experimental conditions. In addition, the discoloration after 72 hours of incubation demonstrates accumulated inactivation effects of SS+MEF on cyanobacteria which do not appear right after the treatment. It is clear that photosynthesis was no longer occurring in these samples following MEF treatment and 72 hours incubation, and that M. aeruginosa had effectively been inactivated.

[0120] In Figs. 4 and 5. the SS+MEF at 30°C (the optimal growth temperature of M. aeruginosa), presented similar trends in ODeso measurements (negative values in percent change) and appearance (discoloration) as at the 40°C treatments. The degrees of change in cell density (Fig. 4(a)) were on a similar level and these differences were not more than 5% for 40 minutes SS+MEF at both rotation speeds. In the case of the shortest treatment time (10 min), there were small differences in cell density changes at 4000 rpm but overall, the differences were significantly higher than those at 9500 rpm (see Fig. 4(a), 10 minute data points, lower-case letter comparisons). However, as treatment progressed, the trend reversed (change in an optical density greater for 9500 rpm greater than for 4000 rpm; see Fig. 4(a), upper case comparisons at 40 mm.) In the Chi a and PC results, a similar nature of changes was observed; maximum values were found in the SS+MEF with 4000 rpm.

[0121] A possible factor that explains the above dependence on rotational speed is the electrical current levels as affected by fluid rotation. Unlike the stable electric currents with 60 Hz sine waveform at 4000 rpm, currents fluctuated periodically at 9500 rpm (Fig. 10). The measured values of the average electric current at 9500 rpm were approximately 49.7% lower than in the case of 4000 rpm. Periodic fluctuation in electric current was also observed at 9500 rpm, (with their maximum current 53.1% increase in comparison to the mean, and the minimum current shrinking to 51.6% of the mean); as opposed to stable currents when the fluid was rotated at 4000 rpm. This appears to be due to flow separation occurring due to increased centripetal acceleration at 9500 rpm. Furthermore, although their average current values dropped, SS+MEF at high rotation speed would show sharper and narrower waveforms at peak, somewhat similar to pulsations. This can explain the shortterm (10 minute) time point; however, the increased effectiveness of 9500 rpm at 40 minutes is likely due to the accumulation of synergistic mechanical shear plus MEF effects. Similar patterns in results were shown at a lower temperature (10 and 20°C; discussed herein). As with the appearance and discoloration observed at 40°C, those at 30°C became clear after 72 hours incubation, showing the efficacy of SS+MEF in inactivation at this temperature. However, unlike the 40°C treatments, there seem to be no strong visual differences directly after treatment (0 hours), displaying how this temperature may be less of a factor in immediate decreases in culture health compared to higher temperature treatments.

[0122] At below-optimal temperatures (20°C and 10°C, Figs 6-7 and Figs 8-9, respectively), the growth suppression of aeruginosa by SS+MEF also increased significantly with the increase of SS+MEF treatment time. The data show that most M. aeruginosa were inactive and viable cyanobacteria were reduced at low temperatures. Still, there appeared to be small positive changes in ODeso (indicating slight grow th, albeit greatly suppressed, see Fig. 6(a)) in most cases at 20°C, and in all cases (see Fig. 8(a)) at 10°C. In both 20°C and 10°C temperature groups, inactivation was observed at the longest treatment time (40 minutes) at 9500 rpm. SS+MEF of bacteria (i.e., Escherichia coh K12 an Listeria innocua) affects mostly viability, because the bacterial damage is mainly in the form of electrophysical disinfection. In a similar manner, it is possible that physical damage may be sufficient to prevent reproduction without the use of heat yet allow microorganisms to repair sublethal damage and perform some live algal functions. The changes in fluorescence intensities for Chi a and PC over 72 hours incubation also support these findings; both results reveal that during the first 10 minutes of SS+MEF the change in optical density at 9500 rpm was positive and less affected than at 4000 rpm. However, the percent change at 9500 rpm was largely negative after 40 minutes of SS+MEF, more so than the 4000 rpm treated group.

[0123] Some variations were observed in the degree of changes in cell densities and metabolic activities when compared with visual discoloration results at 20°C and 10°C (in Fig. 7 and 9, respectively). After 72 hour post-treatment incubation, changes in ODeso reoccurred with much slower growth rates than the untreated samples (in Fig. 7(a) excluding 40 minutes MEF at 9500 rpm; and Fig. 8(a)). However, Chi a and PC exhibited much bigger suppressions in the treated samples versus the positive controls, and showed substantial discoloration results (Figs. 7 and 9). More specifically, at 20°C for Chi a and PC, an experimental time of 20 and 40 minutes exhibit greater negative changes. At 10°C for all parameters and for both MEF treatments, an experimental time of 10 minutes exhibited high positive percent changes, while 20 and 40 minute treatment times exhibited either lower positive percent changes or relatively high negative changes. Besides reduced MEF efficacy at lower temperatures and w aveform changes by rotation speed, one of the possible reasons for a slight reduction in efficacy is the formation of dead algae aggregates which envelop live algae and shield them from the destructive effects of SS+MEF. It is noted that the resistance to inactivation observed in aggregated or colony-formed Al. aeruginosa, which is widely believed to be membrane destruction, is not transmitted to successive generations. Therefore, aggregation is considered as a cyanobacteria defense mechanism against physical damage and other forms of stress which can also result in aggregation (cluster formation). Also, at the lower temperatures, restricted photosynthetic activity of aeruginosa could have occurred after SS+MEF. Since low-temperature stress (chill stress) can cause alteration of the desaturase-meditated upregulation of membrane lipids of Al. aeruginosa, such a temperature-dependent response can affect the degree of fluidity and partially the structure of the thylakoid membrane; thus their degree of injury or response to SS+MEF can be varied. From Wang’s studies (2010), it was reported that the decrease in the fluidity of the thylakoid membrane can cause significant inhibition of the photosynthetic activity’ and growth rate of Al. aeruginosa. These effects can explain the observed results at different low-temperature conditions. Regardless of the above points, SS+MEF is successful at either inactivating or inhibiting the growth of Al aeruginosa at all temperatures. If low temperatures are used, longer treatment times are required to obtain similar results as at a higher experimental temperature and to ensure a negative percent change in cell densities parameters. Moreover, discoloration after 72 hours provides clear evidence of the efficacy of this process.

[0124] The effects of temperatures were also confirmed by the results from dd-PCR (Fig. 11). Expression of mcyE genes showed significant treatment-by -temperature interaction effects through data from 40°C (selected as representative of elevated temperature) and 20°C (selected for representative below-optimal temperature). As observed in ODsso at 40°C, no significant difference in relative change was found for mcyE expression over treatment time for SS+MEF at 4000 rpm, but did show a gradual decrease over time for SS+MEF at 9500 rpm. In the case of below-optimal temperature, their expression was varied regardless of rotation speeds and treatment time. Albeit significant, all fold change in mcyE expression was within 0.5 log compared to changes in the negative control. This roughly parallels the observed changes in ODeso at 20°C, indicating that there was still some growth of M. aeruginosa, albeit suppressed, and their incorporating synthesis of microcystin. Gas vesicles within cyanobacteria cells act as a crucial mechanism for natural buoyancy, allow ing them to stay afloat on the surface of water bodies and photosynthesize. To remove growth media to prevent any extraction interferences or inhibition, samples that were frozen at -80°C prior to RNA extraction and were centrifuged. Fluorometry and visual appearance showed positive changes.

[0125] Exploration of the effects of combined shear and MEF treatments reveals the enhancement of inactivation and grow th suppression of M. aeruginosa and their temperature-dependent changes in cell densities, pigments, and mcyE gene copies. Disclosed herein is a low energy, volumetric, and technically scalable solution for controlling cyanobacteria and protecting public health.

[0126] Conclusions

[0127] This work clearly shows that SS+MEF treatment is effective in the inactivation and / or highly suppressing growth of AT. aeruginosa over a range of temperatures from 10 to 40°C, within the range of variation of temperatures of water bodies in the temperate zone. Inactivation is more clearly observed at higher temperatures, and growth suppression is appeared at lower temperatures. In all cases, discoloration (loss of green color) of treated samples after 72 hours incubation indicated the efficacy of the treatment process. The effect of rotational speed depends on temperature - with the high-speed, higher temperatures being the most effective. At intermediate and lower temperatures (less than or equal to 30°C), lower agitation rates seem to improve results, at least partly due to changes in waveforms, which improves electrical efficacy under lower speeds. However, the higher speed resulted in greater efficacy over time, likely due to greater physical damage in cell integrity. These findings show that the SS+MEF treatment can be used year-round to gradually decrease the population of AT aerigunosa in water bodies, and works despite seasonal temperature variations.

[0128] Technical note

[0129] To specify the flow conditions in the rotating flow system at several flow rates, Taylor (Ta) number are used to characterize the flow regime for this system. The flow' regimes described by Ta numbers are defined by equations (2). Beyond a critical Ta number, flow evolves to toroidal Taylor vortex flow, in which case inertial forces cause a small axisymmetric cellular secondary motion. where ' and are the radii of inner cylinder and outer cylinder, respectively; is an angular velocity of the inner cylinder; and is the kinematic viscosity of the fluid. At a critical value of the Taylor number the action of centrifugal forces leads to an axisymmetric instability consisting of counter-rotating toroidal cells of uniform width stacked one above the other. The SS+MEF unit in present study is designed to batch system without propagation up or down the annulus. The effect of rotating fluid on current flow between electrodes was tested under various rotation speeds while alternating currents were applied in sinusoidal waveform with 60 Hz frequency in the SS+MEF unit (Fig. 10).

[0130] EXAMPLE 2: Application of moderate electric fields and mcirocystin-degrading bacteria for controlling microcystin and its toxicity in water

[0131] Methods and Materials

[0132] Microcystin crude extraction from M. aeruginosa

[0133] Cyanobacteria cultures of Microcystis aeruginosa PCC 7806 (Institut Pasteur, Paris, France) were grown in CT media at 24°C under 12-hour light cycles for two weeks (Jang et al., 2004; Jiang et al., 2017). As a proxy for cyanobacterial growth, chlorophyll-a and phycocyanin levels were monitored using both an AquaFluor Handheld Fluorometer (8000- 010 Turner Designs, Sunnyvale, CA, USA) and a 96-well plate Absorbance Reader (ELx808, BioTek, Winooski, VT, USA). Excitation, emission wavelengths for both pigments were 450 ± 20 nm, 665 nm and 595 ± 20 nm, 670 nm, respectively (Marion et al., 2012). Cyanobacteria was grown to an approximate concentration of 1 x 107cells / mL.

[0134] Once grown to the desired cell concentration, the culture was transferred to 50-mL glass amber vials (Fisher Scientific, Hampton, NH). Crude extraction of MC-LR was adapted from general low-cost extraction methods utilizing sonication and freeze-and-thaw . The cultures were placed on ice for 30 minutes, then sonicated at 0°C (five five-minute cycles with 30 seconds rest and brief mixing) using a % gallon benchtop sonicator (80 watts, model 2510, Branson Ultrasonics, Danbury, CT, USA) to rupture the healthy cyanobacteria cells to release intracellular toxins. The samples w ere then subject to four freeze-and-thaw cycles, transferred between -20°C and 23°C, with gentle inverting between each cycle. Following the final thaw cycle, the vials were centrifuged at 10,000 x g for 30 minutes for cyanobacterial cell debris removal. Final toxin concentrations were tested using the methods described below. Toxin concentrations used for spiking experiments were diluted to the desired experimental quantity described below.

[0135] Microcystin-degrading bacteria growth and maintenance

[0136] Sphingomonas sp. ACM3962 served as the primary’ microcystin-degrading bacteria used for these experiments, as it is a confirmed MC degrader. This bacteria strain was generously donated by Dr. Steven Wilhelm (University of Tennessee, Knoxville) for this work. Following multiple streaking steps to generate single colonies (grown on LB-Agar plates at 32°C), single colonies were outgrown in LB liquid media (10-mL) overnight in an Incu-Shaker™ Mini (28°C at 200 RPM, 10L, Benchmark Scientific. Edison, NJ, USA). Cell culture concentrations were determined via ODeoo (Genesys™ 20 Spectrophotometer, Thermo Spectronic Spectronic, Rochester, NY, USA), for a final concentration of 106CFU / mL. Once grown to the desired concentration, the bacteria cultures are briefly spun down for 2 minutes at 4.5 RPM (Thermo Scientific CL2 Low Speed Tabletop Centrifuge. 175 watts. Grand Island, NY, USA), the media is decanted, and the cells are washed using peptone w ater (1%). The culture is re-spun down and re-suspended in 10 mL peptone w ater (1%), to ensure the culture does not utilize nutrients from rich media. New' cultures were grown to the same cell concentration for each trial and experiment.

[0137] Moderate electric field experimental treatments

[0138] The MEF treatment was performed using a laboratory-scale cylindrical liquid- jacketed beaker, designed with an inlet and outlet port for circulating cold tempering fluid (Figure 1A).

[0139] The MEF unit consisted of a pair of curved platinized-titanium electrodes with an inter-electrode distance of 5 cm. Voltages were monitored using a digital oscilloscope (TBS1000C, Tektronix, Beaverton, OR, USA) with a sinusoidal wave at a 60 Hz frequency, connected to a variable transformer (Variac). The electric fields ranged from 4 to 15 V / cm, and the temperature w as monitored by a K-type thermocouple (precision temperature ± 1°C, Omega. 709, USA) placed in the geometric center of the sample volume and connected into a data-acquisition unit (34970A, Agilent Technologies, Inc., Palo Alto, CA, USA) and data logger softw are for data storage (Figure IB).

[0140] In a preliminary study, the effect of MEF on MC-LR degradation was investigated under various temperature settings. The MEF treatments on 100 pg / L (ppb) MC-LR solution (time 0) started when the temperature reached target ranges from 20-90°C. For the MEF system, as described above, the cell was connected to a refrigerated circulating bath to reach the desired temperature and maintain it during the treatment time of 40 min.

[0141] The combined effect of MEF and biological agents for MC-LR degradation was investigated. Three different groups were tested: controls (positive and negative), pre-MEF (MEF was applied as a pre-treatment before biological agent inoculation), and w-MEF (low intensity [weak] MEF application) (Figure 1C). For the control group. 100 ppb MC-LR sample was sub-inoculated with Sphingomonas sp. (106CFU / mL) and incubated for 24 h at 30°C under 160 RPM agitation using the Incu-Shaker™ Mini (28°C at 200 RPM, 10L, Benchmark Scientific). For the pre-MEF treatment group, the applied electrical field strength was set to 15 V / cm, based on the electric current of MC-LR solution at 100 ppb (~85 mA) and working temperature controls (30°C). After MEF treatment of 40 min at 30°C, the Sphingomonas sp. (106CFU / mL) was inoculated into the MEF -treated MC-LR solution and incubated for 24 h at 30°C under 160 RPM agitation in the Incu-Shaker™

[0142] Mini. For the final treatment group of weak MEF (w-MEF), the applied electric field strength was set to 4 V / cm, which presented the lowest electric current readings (~3 mA). The 100 ppb MC-LR sample with sub-inoculated Sphingomonas sp. (106CFU / mL) was incubated for 15 h with continuous MEF and agitation at 30°C, and then transferred to the Incu-Shaker™ for the remaining 9 hours at 30°C.

[0143] For each experimental group, experiments were run and sampled in duplicate. Two sample volumes (1 rnL and 3 mL) were aseptically removed at times (hours) 0, 5, 10, 15, and 24, and stored in glass vials at -20°C until further processing. An additional sample set was taken for the pre-MEF group prior to MEF application. The different sample volumes were used for the two MC concentration methods described below.

[0144] Microcystin concentration quantification

[0145] As an initial test of total microcystin levels in the collected samples, an enzyme- linked immunosorbent assay (ELISA; Microcystins / Nodularins (ADDA) kit, Abraxis. Warminster, PA, USA) was employed. Following manufacturer instructions, pre-coated pipettes were used to transfer 50 pL of sample from the 1 mL aliquot into the 96-well plate. Each sample was tested in duplicate. To test for toxicity of present MC-LR, another ELISA kit (Microcystins / Nodularins PP2A (EPA ETV), Abraxis) was employed in a similar manner following manufacturer instructions. Each sample was tested in duplicate. For both tests, a final plate reading for sample detection was completed using the 96-well plate Absorbance Reader (ELx808, BioTek).

[0146] Although widely used and most common for water testing in the region, there exists some uncertainty7associated with ELISA kit methods for measuring total MC concentrations in complex matrices. Therefore, a subset of samples was prepared and tested for MC using liquid chromatography tandem-mass spectrometry (LC-MS / MS) through the Nutrient & Phytochemical Analytic Shared Resource (The Ohio State University, Columbus, OH, USA) (Manubolu et al., 2018). Briefly, samples were subject to equal parts methanol and bath sonicated to lyse any bacterial cells, followed by a brief centrifugation and filtration through 0.2 pm PTFE membrane filters (Waters. MA, USA). Sample (5 pL) was then injected into a Cl 8 UPLC chromatograph that was paired to a Triple Quadrupole mass spectrometer (QTrap 5500, ABSciex, Concord, Canada). Samples provided were 3 mL in total volume. Due to cost limitations, only a subset of samples (w-MEF treatment group) was run in duplicate. Quantifications were provided in units of pg / L (ppb) of detected MC- LR.

[0147] Statistical analyses

[0148] Data was visualized and analyzed using R Studio for Mac OSX, Version x (citation). Packages used include x (citation).

[0149] Results

[0150] The combined effects between MEF and MC -degrading Sphingomonas (SP) were tested via overnight treatment (Figure 12-14). In Figure 12A, the preMEF+NF group showed more than 50% reduction of MC after 24 h incubation. Compared to results from control group (SP only), MC reduction levels after MEF pretreatment were enhanced right after 10 h of incubation. In the case of wMEF+SF, 75.7-83.3% reduction of MC was observed after 10-15 h continous low intensity of MEF treatment. The control group, which was only treated with Sphingomonas, shows the lowest reduction of MC (24.5%), showing only incremental trends over 24 h incubation. Similar results were seen in the PP2A method, which has known as direct indication of the toxicity of MC (Figure 12B). The preMEF+SF group shows 53.7% reduction of MC-LR after 10-15 h incubation, compared to the initial MC level. The wMEF+SF group shows higher reduction of MC-LR after 5 h MEF treatment: their reduction reaching a maximum of 87. 1%. The control group shows 45.2% reduction of MC confirming that overnight incubation with Sphingomonas has a positive effect for toxicity control. From Figure 13, the changes in Sphingomonas concentrations during this study can explain the possible mechanisms of combination of MEF and MC-derading bactiera on MC degradation. For example, the larger changes in optical density with wMEF may indicate either the enhancement of MC -degrading cell grow th under MEF. Concomitant with preMEF results, MEF may have some other effects with residual metabolites from MC compounds that boost the cell growth or MC- degradation activity.

[0151] The MC concentrations were also quantified with LC-MS / MS (Figure 14A) for further validation of the MC concentration measurements. While the control group showed a gradual decrease during 24 h incubation, both preMEF+5'P and wMEF+SP presented steeper reductions up to 87.9% and nondetactable level, respectively. This agrees with our findings from ELISA tests. Collectively, MEF-based techniology can be utilized for improvement of the MC-LR degrading efficiency with the presence of Sphingomonas sp. and their syniergistic combination effects can be used for cyanobacterial toxin controls.

[0152] ADDITIONAL EMBODIMENTS

[0153] 1. A method for controlling growth of algae in water, the method comprising exposing water containing algae to shear stress and a moderate electrical field, thereby reducing the growth of the algae in the water.

[0154] 2. The method of embodiment 1, wherein the algae is Microcystis aeruginosa

[0155] 3. The method of embodiment 1 or 2, wherein the algae growth is reduced by 50% or more.

[0156] 4. The method of any one of embodiments 1-3, wherein the algae is inactivated.

[0157] 5. The method of any one of embodiments 1-4, wherein the algae is inhibited.

[0158] 6. The method of any one of embodiments 1-5, wherein microcystin is subsequently reduced.

[0159] 7. The method of any one of embodiments 1-6, w herein the water is from a natural w ater source such as a pond or lake.

[0160] 8. The method of any one of embodiments 1-7, wherein the w ater is from water punfication center.

[0161] 9. The method of any one of embodiments 1-8, wherein length of time of exposure to shear stress and moderate electrical field is inversely proportional to temperature of the water.

[0162] 10. The method of any one of embodiments 1-9, wherein the algae is exposed to shear stress and moderate electrical field and simultaneously. 11. The method of any one of embodiments 1-10, wherein the algae is further treated with an algae-reducing substance.

[0163] 12. A device for controlling grow th of algae in water, wherein the device provides shear stress and moderate electrical field simultaneously.

[0164] 13. The device pictured in Figure 1.

[0165] 14. A method of detoxifying water by exposing the water to a microcystin-degrading bacteria.

[0166] 15. The method of embodiment 14, wherein the bacteria is Sphingomonas sp..

[0167] 16. The method of embodiment 14 or 15, wherein the water is first exposed to shear stress and a moderate electrical field.

[0168] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the follow ing claims.

Claims

CLAIMSWhat is claimed is:

1. A method for treating a volume of water disposed within a reactor, comprising agitating the volume of water in the presence of an electrical field.

2. The method according to claim 1. wherein the agitating comprises rotating a rotor contacting the volume the water.

3. The method according to claim 1, wherein the agitating comprises shearing the water.

4. The method according to claim 1. wherein the agitating comprises inducing a shear stress in the water.

5. The method according to claim 4, wherein the shear stress has a shear rate of at least 150 s’1, at least 200 s’1, at least 250 s’1, at least 300 s’1, at least 350 s’1, at least 400 s’ at least 450 s’1, at least 500 s’1, 550 s’1, 600 s’1, 650 s’1, at least 700 s’1, at least 750 s’1, at least 800 s’1, at least 850 s’1, at least 900 s’1, at least 950 s’1, at least 1,000 s’1, at least 1,050 s’1, at least 1,100 s’1, at least 1,150 s’1, at least 1,200 s’1, 1,250 s’1, at least 1,300 s’1, at least 1,350 s’1, at least 1,400 s’1, at least 1,450 s’1, at least 1,500 s’1, at least 1,550 s’1, at least 1,600 s’1, at least 1,650 s’1, at least 1,700 s’1, at least 1,750 s’1, at least 1,800 s’1, at least 1,950 s’1, at least 2,000 s’1, at least 2,050 s’1, at least 2,100 s’1, at least 2,150 s’1, at least 2,200 s’1, 2,250 s’1, at least 2,300 s’1, at least 2,350 s’1, at least 2,400 s’1, at least 2,450 s’1, at least 2,500 s’1, at least 2,550 s’1, at least 2,600 s’1, at least 2,650 s’1, at least 2,700 s’1, at least 2,750 s’1, at least 2,800 s’1, at least 2,950 s’1, or at least 3,000 s’1.

6. The method according to claim 4. wherein the shear stress is induced by contacting the volume of water with a rotating rotor.

7. The method according to claim 6, wherein the rotor rotates at a rate of at least 200 rpm, at least 300 rpm, at least 400 rpm, at least 500 rpm, at least 1,000 rpm, at least 2,500 rpm, at least 5,000 rpm, at least 7,500 rpm. or at least 10,000 rpm.

8. The method according to claim 6. wherein the rotating rotor and volume of water are disposed within a chamber.

9. The method according to claim 6, wherein the rotating rotor and volume of water are disposed within a stationary chamber.

10. The method according to claim 6. wherein the rotating rotor and volume of water are disposed within a rotating chamber.

11. The method according to claim 6. wherein the rotating rotor and volume of water are disposed within a rotating chamber, wherein the rotating chamber rotates along the same axis as the rotor, in the same or opposite direction.

12. The method of claim 12, wherein the rotating chamber rotates at the same speed as the rotor, but in the opposite direction.

13. The method of claim 12, wherein the rotating rotor ace rotates at a speed that is at least 110%, at least 125%, at least 150%, at least 200%, at least 250%, at least 300%, at least 500%, at least 1,000%, at least 2,000%, or at least 5,000% the speed of the rotating chamber.

14. The method of claim 1, wherein the electric field is applied between a first electrode and a second electrode.

15. The method of claim 14, wherein the first electrode is disposed within the volume of water.

16. The method of claim 14, wherein the first electrode and second electrode are disposed within the volume of water.

17. The method of claim 14, wherein the rotor comprises the first electrode and the second electrode is disposed within the volume of water.

18. The method of claim 14, wherein the chamber comprises the first electrode and the second electrode is disposed within the volume of water.

19. The method of claim 14, wherein the rotor comprises the first electrode and the chamber comprises the second electrode.

20. A method for treating a volume of water, the method comprising: a) providing a reactor, wherein the reactor comprises: a first end wall, a second end axially opposite the first end wall, and a least one side wall extending between the first and second end walls, wherein said first end wall, second end wall, and side wall defines a chamber; and a rotor disposed within the chamber; wherein the chamber is in electrical communication with at least two electrodes; b) delivering the volume of water to the chamber; c) agitating the volume of water in the presence of an electrical field.

21. The method of claim 20, wherein the rotor and at least one side wall are coaxial.

22. The method of claim 20, wherein the rotor is concentric with the outer wall.

23. The method of claim 20, wherein the rotor is a cylinder.

24. The method of claim 23, wherein the rotor comprises a paddle.

25. The method of claim 20, wherein the volume of water is agitated by rotating the rotor.

26. The method of claim 20, wherein the at least one side wall is stationary'.

27. The method of claim 20, wherein at least one side wall is rotated along the same axis at the rotor, in the same or opposite direction as the rotor.

28. The method of claim 23, wherein the rotor provides Taylor-Couette flow in the volume of water.

29. The method according to claim 25, wherein the volume of water has a shear rate of at least 150 s’1, at least 200 s’1, at least 250 s’1, at least 300 s’1, at least 350 s’1, at least 400 s’1, at least 450 s’1, at least 500 s’1, 550 s’1, 600 s’1, 650 s’1, at least 700 s’1, at least 750 s’1, at least 800 s’1, at least 850 s’1, at least 900 s’1, at least 950 s’1, at least 1,000 s’1, at least 1,050 s’1, at least 1,100 s’1, at least 1,150 s’1, at least 1,200 s’1, 1,250 s’1, at least 1,300 s’1, at least 1,350 s’1, at least 1,400 s’1, at least 1,450 s’1, at least 1,500 s’1, at least 1,550 s’1, at least 1,600 s’1, at least 1,650 s’1, at least 1,700 s’1, at least 1,750 s’1, at least 1,800 s’1, at least 1,950 s’1, at least 2,000 s’1, at least 2,050 s’1, at least 2,100 s’1, at least 2,150 s’1, at least 2,200 s’1, 2,250 s’1, at least 2,300 s’1, at least 2,350 s’1, at least 2,400 s’1, at least 2,450 s’1, at least 2,500 s’1, at least 2,550 s’1, at least 2,600 s’1, at least 2,650 s’1, at least 2,700 s’1, at least 2,750 s’1, at least 2,800 s’1, at least 2,950 s’1, or at least 3,000 s’1.

30. The method according to claim 20, wherein the at least one side wall rotates at a rate of at least 200 rpm, at least 300 rpm, at least 400 rpm, at least 500 rpm, at least 1,000 rpm, at least 2,500 rpm, at least 5,000 rpm. at least 7,500 rpm. or at least 10,000 rpm.

31. The method of claim 25, wherein the at least one side wall rotates at a speed that is the same or different as the rotor.

32. The method of claim 25, wherein the rotor rotates at a higher speed than the at least one side wall, or the rotor rotates at a slower speed than the at least one side wall.

33. The method of claim 31, wherein the rotor rotates at a speed that is at least 110%, at least 125%, at least 150%, at least 200%, at least 250%, at least 300%, at least 500%, at least 1,000%, at least 2,000%. or at least 5,000% the speed of the at least one side wall.

34. The method of claim 20, wherein the first electrode and second electrode are disposed within the chamber.

35. The method of claim 20, wherein the rotor comprises the first electrode and the second electrode is disposed within the chamber.

36. The method of claim 20, wherein the at least one side wall comprises the first electrode and the second electrode is disposed within the chamber.

37. The method of claim 20, wherein the rotor comprises the first electrode and the at least one side wall comprises the second electrode.

38. The method of claim 20, wherein the at least one side wall comprises a first portion comprising the first electrode, a second portion comprising the second electrode, and an insulating portion electrically isolating the first electrode from the second electrode.

39. The method of any of claims 1-38, wherein the volume of water is delivered to the reactor in a batch process or in a continuous process.

40. The method of any of claims 1-39, wherein the volume of water has a temperature that is from 5-100 °C., form 5-60 °C., from 10-60 °C., from 15-60 °C., from 20-60 °C., from 20-50 °C., from 5-40 °C., from 5-25 °C., from 20-40 °C., from 20-30 °C., from 30-60 °C., from 30-50 °C., from 30-40 °C., from 40-60 °C., or from 50-60 °C.

41. The method of any of claims 1-40, wherein the electric field is from 10-200 V / cm, from 25-200 V / cm, from 50-200 V / cm, from 100-200 V / cm, from 100-150 V / cm, from 150-200 V / cm, from 10-100 V / cm, from 10-50 V / cm, from 10-25 V / cm, from 25-100 V / cm, from 25-50 V / cm, from 25-75 V / cm, from 50-75 V / cm, from 1-10 V / cm, from 5-10 V / cm, or from 5-15 V / cm.

42. The method of any of claims 1-41, wherein the electric field is applied for a period of from 1-180 minutes, from 10-120 minutes, from 10-90 minutes, from 10-60 minutes, from 10-30 minutes, from 20-90 minutes, from 20-40 minutes, from 30-90 minutes, from 30-40 minutes, from 40-90 minutes, from 40-50 minutes, from 50-90 minutes, from 50-60 minutes, or from 60-90 minutes.

43. The method of any of claims 1-42, wherein the electric field is applied as a square wave, a sine wave, a ramp, aSawtooth wave, a triangular wave, or a combination thereof.

44. The method of any of claims 1-43, wherein the electric field is applied as sinusoidal waveform having a frequency from 5-100 Hz, from 5-75 Hz, from 5-50 Hz, from 5-25 Hz. from 10-25 Hz, from 10-100 Hz, from 25-50 Hz, from 25-100 Hz, from 40- 100 Hz, from 40-75 Hz, from 50-75 Hz, or from 75-100 Hz.

45. The method of any of claims 1-44, wherein the volume of water comprises an algae.

46. The method of any of claims 1-45, wherein the volume of water comprises Microcystis aeruginosa.

47. The method of any of claims 1-46, wherein the algae growth is reduced by 50% or more.

48. The method of any of claims 1-46, wherein the algae is inactivated.

49. The method of any of claims 1-46, wherein the algae is inhibited.

50. The method of any of claims 1-49, wherein the volume of water comprises a microcystin-degrading organism.

51. The method of claim 50, wherein the volume of water comprises a microcystindegrading bacteria.

52. The method of claim 51, wherein the microcystin-degrading bacteria comprises Acidovorax sp.,Acinetobacter sp., Aeromonas sp.,Alcaligenes sp., Arthrobacter sp., Bacillus sp., Bifidobacterium sp., Bordetella sp., Bosea asp., Brevibacillus sp., Brevibacterium sp., Brevundimonas sp., Burkholderia sp., Chryseobacterium sp., Cupriavidus sp.. Enterobacter sp., Hyphomicrobium sp.. Klebsiella sp., Lactobacillu sp., Lysinibacillus ^.. Mesorhizobium sp., Methylobacillus sp., Methylotenera sp., Morganella sp., Nitrosococcu sp., Novosphingobium sp., Ochrobactrum sp., Paucibacter sp., Polaromonas sp., Pseudomonas sp., Pseudoxanthomonas sp., Polynucleobacter sp., Ralstonia sp., Rhizobium sp., Rhodococcus sp., Sandaracinobacter sp.. Sphingomonas sp.. Sphingopyxis sp., Stenotrophomonas sp., Steroidobacter sp., Variovorax sp., or a combination thereof.

53. The method of claim 52, wherein the microcystin-degrading bacteria comprises Sphingomonas sp.

54. The method of claim 52, wherein the microcystin-degrading bacteria comprises one or more of, two or more of. three or more of. four or more of, five or more of, six or more of, or seven of the following bacteria: Acinetobacter sp., Hyphomicrobium aestuarii, Pseudoxanthomonas sp., Rhizobium sp., Sphingobium sp., Sphingomonas sp. and Steroidobacter sp.

55. The method of claim 52, wherein the microcystin-degrading bacteria comprises one or more of, two or more of, three or more of, four or more of, five or more of, six ormore of. seven or more, eight or more, nine or more, or ten of the following bacteria: Acinetobacter s\ . Aeromonas sp., Novosphingobium sp., Ochrobactrum sp., Pseudomonas sp., Rhodococcus sp., Sphingomonas sp., Sphingopyxis sp., Stenotrophomonas sp. and Steroidobacter sp.

56. The method of any of claims 1-55, wherein the concentration of microcystin in the volume of water is reduced.

57. The method of any of claims 1-56, wherein the water is from a natural water source such as a pond or lake.

58. The method of any of claims 1-57, wherein the water is from water purification center.

59. The method of any of claims 1-58, wherein length of time of exposure to shear stress and moderate electrical field is inversely proportional to temperature of the water.

60. The method of any of claims 1-59, wherein the algae is exposed to shear stress and moderate electrical field and simultaneously.

61. The method of any of claims 1-60, wherein the algae is further treated with an algae- reducing substance.

Images