Sterilization method
阅读说明：本技术 灭菌方法 (Sterilization method ) 是由 R·M·帕什利 A·G·桑切斯 巴里·尼哈姆 于 2018-11-28 设计创作，主要内容包括：本公开涉及用于灭活水溶液中的微生物的方法、系统和设备。该方法包括使气体的气泡通过水溶液,其中所述气体包含按体积计至少10％的CO<Sub>2</Sub>并具有至少18℃的温度。所述系统包括：气体供应装置,以供应包含至少10％的CO<Sub>2</Sub>且温度高于18℃的气体；气体输送装置,以接收气体并将其以气泡的形式输送至水溶液中。所述设备包括：透气性材料,其具有被配置为使得水溶液能够流过所述流动表面的流动表面；布置在所述透气性材料的相对侧的腔室,以提供灭活所述微生物的气体,使得所述气体能够穿过该透气性材料并作为气泡进入水溶液。(The present disclosure relates to methods, systems, and devices for inactivating microorganisms in aqueous solutions. The method comprises passing bubbles of a gas through an aqueous solution, wherein the gas comprises at least 10% CO by volume 2 And has a temperature of at least 18 ℃. The system comprises: a gas supply device for supplying a gas containing at least 10% of CO 2 And a gas at a temperature above 18 ℃; a gas delivery device to receive the gas and deliver it in the form of bubbles into the aqueous solution. The apparatus comprises: a gas permeable material having a flow surface configured to enable an aqueous solution to flow through the flow surface; is arranged atA chamber on the opposite side of the gas permeable material to provide a gas that inactivates the microorganisms such that the gas can pass through the gas permeable material and enter the aqueous solution as bubbles.)
1. A method of inactivating microorganisms in an aqueous solution, the method comprising:
passing bubbles of a gas through the aqueous solution, wherein when the gas first contacts the aqueous solution, the gas comprises at least 10% CO by volume2And a temperature of at least 18 ℃.
2. The method of claim 1, wherein the microorganism is an algae, protozoa, fungus, spore, virus, or bacterium.
3. The method of claim 1, wherein the microorganism is a virus or a bacterium.
4. The method of any one of claims 1 to 3, wherein the gas bubbles pass through the aqueous solution when the aqueous solution is exposed to atmospheric pressure.
5. The method of any one of claims 1 to 4, wherein the bubbles have a diameter of 0.1mm to 7 mm.
6. The method of any one of claims 1 to 5, wherein the gas comprises 50% to 100% CO by volume2。
7. The method of any one of claims 1 to 6, wherein the gas comprises 10% to 50% CO by volume2。
8. The method according to any one of claims 1 to 7, wherein the temperature of the gas exceeds 100 ℃.
9. The method of any one of claims 1 to 7, wherein the temperature of the gas is from 18 ℃ to 100 ℃.
10. The method of any one of claims 1 to 9, wherein the gas bubbles are formed by passing the gas through a porous material in contact with the aqueous solution.
11. The method of any one of claims 1 to 10, wherein the gas bubbles occupy 10% to 60% of the total volume of the combination of the aqueous solution and the bubbles as the bubbles pass through the aqueous solution.
12. The method of any one of claims 1 to 11, wherein the bulk temperature of the aqueous solution is from 18 ℃ to 80 ℃.
13. The method of any one of claims 1 to 11, wherein the bulk temperature of the aqueous solution is from 18 ℃ to 55 ℃, and wherein the temperature of the gas is higher than the bulk temperature of the aqueous solution.
14. A system for inactivating microorganisms present in an aqueous solution, the system comprising:
-a gas supply device configured to supply a gas comprising at least 10% by volume of CO2The gas of (a), the gas having a temperature of at least 18 ℃ or higher;
-a gas delivery device configured to receive a supply of gas and deliver the gas in the form of bubbles into the aqueous solution.
15. The system of claim 14, wherein the gas delivery device is a bubble column vaporizer.
16. A system according to claim 14 or 15, wherein the gas supply means comprises a heater to heat the gas to form a gas having a temperature of at least 18 ℃ or above.
17. The system of any one of claims 14 to 16, wherein the gas supply further comprises a gas source adapted to provide a gas comprising at least 10% CO by volume2The gas of (2).
18. An apparatus for inactivating microorganisms present in an aqueous solution, the apparatus comprising:
-a gas permeable material having a flow surface on one side thereof configured to enable the aqueous solution to flow through the flow surface,
-a chamber arranged on the opposite side of the gas permeable material to supply a gas inactivating microorganisms present in the aqueous solution such that the gas is able to flow through the gas permeable material and into the aqueous solution as bubbles over the flow surface.
19. The apparatus of claim 18, wherein the flow surface is in the form of a channel.
20. The apparatus of claim 18 or 19, wherein the flow surface is configured such that the gas bubbles pass through the aqueous solution in a direction transverse to the direction of flow of the aqueous solution as the bubbles flow over the flow surface.
21. The apparatus of any one of claims 18 to 20, wherein the gas permeable material is selected such that gas bubbles passing into the aqueous solution have a diameter of about 0.1mm to about 7mm when the aqueous solution is at a pressure of about 0.9 to 1.5 bar.
Technical Field
The present invention relates to methods, systems and apparatus for inactivating microorganisms in aqueous solutions. The method, system or apparatus may be used to disinfect an aqueous solution.
Background
In many cases, it may be desirable to inactivate microorganisms such as viruses and bacteria in aqueous solutions. For example, the presence of microorganisms in water used in food or pharmaceutical manufacturing can cause contamination of the food or pharmaceutical and therefore the water may need to be treated to inactivate said microorganisms prior to use. Similarly, wastewater from agricultural or industrial uses or water obtained from environmental flowing water may contain pathogenic microorganisms that need to be inactivated before the water is used for industrial or agricultural purposes or for drinking water.
The waste water produced by human activities usually contains human enteroviruses such as hepatitis and rotavirus as well as bacteria such as e. If the water is to be reused, for example in agriculture, it must be disinfected.
Various methods are known to inactivate viruses, bacteria and other microorganisms in water and aqueous solutions. Such methods include heating, treatment with chemicals (e.g., ozone), irradiation (e.g., ultraviolet treatment), high pressure treatment, and filtration (e.g., membrane filtration). Many of these processes, particularly thermal processes, are energy intensive. More energy efficient processing techniques are urgently needed.
The World Health Organization (WHO) compares the thermal inactivation rates of different types of bacteria and viruses in hot liquids in its drinking water quality guidelines. They concluded that temperatures above 60 ℃ are effective in inactivating viruses and bacteria. Bacterial inactivation is faster than viral inactivation when the temperature range is between 60 ℃ and 65 ℃. These studies show that at water temperatures of 60 ℃, coli takes 300 seconds to achieve a 1.5log reduction, while viruses such as enterovirus, echovirus 6, coxsackie B4, and B5 take 1800 seconds to achieve a 4log reduction.
It would be desirable to provide an alternative method of inactivating viruses, bacteria or other microorganisms in an aqueous solution. It would be advantageous to provide a method that does not require the use of high pressures, can be performed at relatively low cost, and/or does not consume energy.
Disclosure of Invention
In a first aspect, the present invention provides a method of inactivating microorganisms in an aqueous solution, the method comprising:
passing bubbles of a gas through the aqueous solution, wherein when the gas first contacts the aqueous solution, the gas comprises at least 10% CO by volume2And has a temperature of at least 18 ℃.
In one embodiment, the microorganism is an algae, protozoa, fungus, or spore. In one embodiment, the microorganism is a virus or a bacterium. In one embodiment, the aqueous solution comprises a combination of one or more viruses and one or more bacteria, and the method is for inactivating the viruses and bacteria in the aqueous solution.
Typically, the gas bubbles pass through the aqueous solution when the aqueous solution is exposed to atmospheric or about atmospheric pressure. The aqueous solution may, for example, be exposed to a pressure of about 0.9 to 1.5 bar.
In one embodiment, the bubbles have a diameter of from 0.1mm to 7mm, for example from 1mm to 3 mm.
In one embodiment, the gas comprises from 50 to 100% by volume of CO2. In another embodiment, the gas comprises 10% to 50% by volume of CO2。
In one embodiment, the temperature of the gas exceeds 100 ℃. In another embodiment, the temperature of the gas is from 18 ℃ to 100 ℃.
In one embodiment, gas bubbles are formed by passing a gas through a porous material in contact with an aqueous solution, thereby forming gas bubbles on the surface of the material in contact with the aqueous solution.
In one embodiment, the gas bubbles occupy 10% to 60% of the total volume of the combination of aqueous solution and gas bubbles when the gas bubbles pass through the aqueous solution.
In one embodiment, the bulk temperature of the aqueous solution is from 18 ℃ to 80 ℃, e.g., from 18 ℃ to 55 ℃ or from 18 ℃ to 50 ℃.
In one embodiment, the bulk temperature of the aqueous solution is from 18 ℃ to 55 ℃, and the temperature of the gas is higher than the bulk temperature of the aqueous solution.
In one embodiment, the aqueous solution comprises a bubble coalescence inhibitor. The bubble coalescence inhibitor may for example be selected from NaCl, sucrose, emulsifiers and surfactants.
In a second aspect, the present invention provides a system for inactivating microorganisms present in an aqueous solution, the system comprising:
-a gas supply device configured to supply a gas containing at least 10% CO by volume2The gas of (a), the gas having a temperature of at least 18 ℃ or higher;
-a gas delivery device configured to receive a supply of gas and deliver the gas in the form of bubbles into the aqueous solution.
In a third aspect, the present invention provides an apparatus for inactivating microorganisms present in an aqueous solution, the apparatus comprising:
-a gas permeable material, said material having a flow surface on one side thereof, the flow surface being configured to enable the aqueous solution to flow therethrough,
-a chamber arranged on the opposite side of the gas permeable material to supply a gas inactivating microorganisms present in the aqueous solution such that the gas is able to pass through the gas permeable material and enter the aqueous solution to flow as bubbles over the flow surface.
Brief Description of Drawings
The invention will be further described, by way of example only, with reference to the accompanying drawings, in which:
FIG. 1 is a graph showing the evaluation of the gas N in the example2，CO2Ar, air and O2Schematic of the system/apparatus of the effect of passing aqueous solution through a bubble column evaporator at different temperatures. According to the experiment, the gas was pumped through a heater (electric heater) that maintained the gas temperature at a specific temperature just above the sinter surface. In the examples described herein, the bottom of the bubble column evaporator was equipped with a 40-100 μm pore size glass sintered body (type 2) having a diameter of 135 mm. Thermocouples were used to measure the temperature of the gas in the gas heater and the temperature of the aqueous solution in the bubble column evaporator.
FIG. 2 shows the results of 1 × 10 in 0.17M NaCl at 25 ℃ using DLS6Coli size distribution (peak (mean/area) at 1601d.nm) of E.coli ATCC15597 at cell/ml.
Fig. 3 is a photograph of the apparatus used in the examples, in which combustion gas was used as the gas. The photograph shows the exhaust from the generator (Honda EM2200) connected to a valve that provides an exhaust flow of 27l/min through the bubble column vaporizer.
FIG. 4 shows photographs of double-layered plaque assay dishes of MS2 virus in samples obtained from BCE subjected to CO at an entry temperature of 200 ℃2NaCl 0.17M solution after 3 minutes of treatment (a)0, (b)0.75, (c)2.25 and (d), as described in the examples.
FIG. 5 is a graph of the log of virus survival factor MS2 (PFU \ PFU0) versus exposure time (min) for different sparged gases (air, oxygen, nitrogen, carbon dioxide or argon at an inlet temperature of 200 ℃ or combustion gases from a 60 ℃ generator) in 0.17M NaCl solution.
FIG. 6 is a graph of the E.coli survival factor log (PFU \ PFU0) versus exposure time (min) for different sparging gases (either air, oxygen, nitrogen, carbon dioxide or argon at a temperature of 150 ℃ inlet, or combustion gas from a 58 ℃ generator, oxygen at 20 ℃ or air at 20 ℃) in 0.17M NaCl solution.
FIG. 7 is the log of virus survival factor for MS2 (PFU \ PFU0) versus different CO in 0.17M NaCl solution2Graph of the exposure time (min) of the bubbled carbon dioxide at entry temperatures (205 ℃, 150 ℃, 100 ℃, 11 ℃ and 20 ℃).
FIG. 8 is a graph of the log of virus survival factor for MS2 (PFU \ PFU0) versus the exposure time (min) to bubbling carbon dioxide in a 0.17M NaCl solution at 22 ℃ at a flow rate of 27l/min or 20 l/min.
Figure 9 is a perspective view of a cross-section of a continuous flow reactor (an embodiment of the third aspect) for performing the process of the invention in a continuous manner as an aqueous solution flows through the flow reactor.
Detailed Description
In a first aspect, the present invention provides a method of inactivating microorganisms in an aqueous solution, the method comprising:
passing bubbles of a gas through the aqueous solution, wherein when the gas first contacts the aqueous solution, the gas comprises at least 10% by volume of CO at a temperature of at least 18 ℃2。
The method (sometimes referred to herein as the "method of the invention") is capable of inactivating water-borne viruses and bacteria in an aqueous solution without boiling the aqueous solution or raising the overall temperature of the aqueous solution to more than about 60 ℃.
As used herein, "inactivating a microorganism" refers to inhibiting or reducing the viability of the microorganism or reducing the number of microorganisms present in an aqueous solution. Typically, inactivation of the microorganisms kills the microorganisms such that the microorganisms no longer survive.
Bubbles of gas may be passed through the aqueous solution in a bubble column evaporator.
Bubble Column Evaporators (BCEs) are generally cylindrical vessels in which a gas introduced through a bottom perforated fritted glass generates an ascending stream of bubbles in the liquid phase [1 ]. They are used in many biochemical, chemical and petrochemical industries [2 ]. In the wastewater industry, they are used as reactors for chlorination, oxidation and fermentation [3 ].
BCE has several advantages over other systems for contacting gases and liquids. BCE provides a high level of contact for chemical reactions between gases and liquids and provides good heat and mass transfer between gases and liquids [1 ]. These advantages would increase with increasing effective interface area if bubble coalescence suppression could be induced to control bubble size. Many bubbles coalesce as they pass through the aqueous solution. The use of various strong electrolytes in BCE can suppress bubble coalescence and can produce high density bubbles (1-3 mm diameter) [4 ]. The phenomenon of inhibition of bubble coalescence at physiological concentrations (0.17M) and above by specific salts has been studied since 1993 [4 ]. So far not explained. Bubbles do not generally coalesce in sodium chloride concentrations greater than 0.17M, and practically all gas bubbles coalesce below this concentration (see reference [4] for detailed results). The addition of salt to 0.17M NaCl inhibited bubble coalescence and improved BCE performance by creating a higher gas-water interface area [4 ]. In the experiments reported in the examples, all studies were performed in 0.17M NaCl solution, which provided a similar degree of bubble coalescence for the various gases used.
Xue et al [8] recently described the inactivation of fecal coliform using BCE. In the method described in this document, the coliform bacteria are inactivated using hot bubbles of air and nitrogen (at 150 ℃). The BCE method is used to inactivate coliforms in solution while maintaining a low column temperature.
In at least preferred embodiments, the use of a gas comprising at least 10% carbon dioxide by volume using the process of the present invention provides unexpected advantages over the process described in the process of Xue et al. For example, in some embodiments, bubbles of gas comprising at least 10% by volume carbon dioxide can effectively inactivate microorganisms, including viruses and bacteria, in an aqueous solution even when the temperature of the gas is well below 150 ℃, and even at room temperature (about 22 ℃). Thus, the use of a gas comprising at least 10% carbon dioxide by volume provides another mechanism for inactivating microorganisms, which is different from the heat inactivation described by Xue et al. As a result, less gas heating may be required using this embodiment of the method of the invention. Further, in some embodiments, bubbles of gas comprising at least 10% by volume carbon dioxide coalesce less in aqueous solution than bubbles of air and many other gases, even in the absence of a bubble coalescence inhibitor. Thus, in some embodiments, it is not necessary to include a bubble coalescence inhibitor in the aqueous solution to inhibit coalescence of the bubbles. In contrast, when bubbles of other gases such as air or nitrogen are used in a bubble column evaporator, it is often desirable to include a bubble coalescence inhibitor in the aqueous solution to inhibit coalescence of the bubbles.
Carbon dioxide has previously been successfully used for bacterial and viral inactivation when used as a supercritical fluid or used at high pressures. Other studies have also compared the use of explosive pressure reduction systems with CO under different high pressure and high temperature conditions2，N2，N2The inactivation ratio of baker's yeast (Saccharomyces cerevisiae) when subjected to explosive decompression of O and Ar. These studies found that CO2And N2The deactivation rate of O is higher than other gases. This is due to their solubility in water and subsequent uptake by cells [17]. In contrast to the methods used in these studies [14-17]The present invention can effectively inactivate microorganisms without using high pressure. Without wishing to be bound by theory, it is believed that the effectiveness of the process of the present invention is due at least in part to the large CO generated by passing the bubbles through the aqueous solution2-a liquid contact surface. This increases the amount of carbon dioxide dissolved in the solution, even when the pressure is maintained at about 1atm in an aqueous solution exposed to atmospheric pressure, which can produce results similar to those obtained by increasing the pressure in the dense phase carbon dioxide process.
Aqueous solution
The term "aqueous solution" refers to a liquid in which water is the only solvent or at least 50% by weight of the total solvent in the liquid. The aqueous solution may be part of an emulsion or microemulsion, such as the aqueous component of an oil/water emulsion or microemulsion. The aqueous solution may comprise water and a co-solvent miscible with water, such as methanol or ethanol, provided that water comprises at least 50% by weight of the solvent present. In some embodiments, water comprises at least 80%, such as 80% to 100%, 90% to 100%, 98% to 100%, 99% to 100%, 80% to 99%, or 90% to 99% by weight of the solvent in the aqueous solution.
The aqueous solution may be, for example, water containing microorganisms that are to be treated to provide potable water. For example, the invention may be used to treat water from environmental streams such as rivers or lakes that contain potentially pathogenic microorganisms to reduce the number of microorganisms to a sufficiently low level to make the water suitable for use as drinking water for humans or for agricultural use.
In some embodiments, the aqueous solution is a wastewater containing viruses, bacteria, and/or other microorganisms from agricultural use and the water is treated to inactivate the viruses, bacteria, and/or other microorganisms before being reused for further agricultural use or before being released into the environment.
In other embodiments, the aqueous solution may be a culture medium from a fermentation or bioreactor comprising bacteria for producing proteins. One problem with conventional fermentation processes that use bacteria to produce proteins is how to both stop the growth of the bacteria and how to extract the desired protein. In some cases, for example, the extraction of a desired protein may be difficult for proteins that are completely or partially hydrophobic, which tend to be "held together" by "hydrophobic forces" within the cell and thus "aggregated". In some previous methods, proteins were isolated and extracted using single-chain cationic surfactants, which disrupt cell membranes by detergency. Thereafter, the cationic surfactant coats the hydrophobin and separates the clumps into the desired individual molecular units by electrostatic repulsion between them, now charged by their cationic surfactant coating. However, it is then necessary to remove the surfactant from the protein, for example by passing the surfactant and protein through an ion exchange column, which can be expensive. The method of the present invention provides an alternative method of killing bacteria. Furthermore, in the case of hydrophobins, the system can be degassed to isolate the hydrophobins.
Water has a high heat of vaporization. The heat of vaporization (also known as the enthalpy of vaporization or heat of vaporization) is the change in enthalpy required to convert a quantity of a substance from a liquid to a gas at a given pressure. For liquids with a high heat of evaporation, more heat is required to evaporate a given amount of liquid than for liquids with a lower heat of evaporation. Due to the high heat of evaporation of water, the passage of gases through an aqueous solution (particularly when the humidity of the gas used is low) generally results in a reduced heating effect of the overall liquid (due to the vapor trapped by the bubbles) compared to liquids with lower heat of evaporation.
In some embodiments, the bulk temperature of the aqueous solution is 18 ℃ to 80 ℃, e.g., 18 ℃ to 60 ℃, 18 ℃ to 55 ℃, 18 ℃ to 50 ℃, 20 ℃ to 80 ℃, 20 ℃ to 60 ℃, 20 ℃ to 55 ℃, or 20 ℃ to 50 ℃ as the bubbles of gas pass through the aqueous solution. The bulk temperature of a liquid refers to the temperature of the liquid away from a surface, such as the surface of a container holding the liquid or the surface of a bubble passing through the liquid. In the method of the invention, the bulk temperature of the aqueous solution may be determined by measuring the temperature of the aqueous solution at a point remote from the surface. Since the gas bubbles passing through the aqueous solution cause rapid mixing of the aqueous solution in the method of the present invention, the bulk temperature of the aqueous solution can generally be determined by measuring the temperature of the aqueous solution alone using a conventional thermometer or other device for measuring the temperature of the liquid. One skilled in the art will be able to select an appropriate method to determine the bulk temperature of the aqueous solution taking into account factors such as, for example, the method used to pass gas bubbles through the aqueous solution and the container used to hold the aqueous solution.
In some embodiments, the bulk temperature of the aqueous solution prior to passing the gas bubbles through the aqueous solution is from 10 ℃ to 80 ℃, e.g., from 10 ℃ to 30 ℃, from 10 ℃ to 50 ℃, from 18 ℃ to 80 ℃, from 18 ℃ to 60 ℃, from 18 ℃ to 55 ℃, or from 18 ℃ to 50 ℃. In some embodiments, the overall temperature of the aqueous solution may change (either increasing or decreasing depending on the particular gas or gas mixture and the relative temperatures of the gas and aqueous solution) as bubbles of gas pass through the aqueous solution.
The aqueous solution may contain a bubble coalescence inhibitor. Thus, in some embodiments, the aqueous solution comprises a bubble coalescence inhibitor. In some embodiments, the method comprises the step of adding a bubble coalescence inhibitor to the aqueous solution prior to the step of passing the bubbles through the aqueous solution. In other embodiments, the aqueous solution does not comprise a bubble coalescence inhibitor, does not comprise an added bubble coalescence inhibitor, or does not comprise a substantial amount of a bubble coalescence inhibitor or an added bubble coalescence inhibitor.
As used herein, the term "bubble coalescence inhibitor" refers to any substance that inhibits coalescence of bubbles in an aqueous solution when present in the aqueous solution beyond a certain concentration. One skilled in the art can readily determine whether the substance is a bubble coalescence inhibitor. For example, one skilled in the art can determine whether the substance is a bubble coalescence inhibitor by adding different concentrations of the substance to a sample of the aqueous solution and visually observing the effect of the substance on the coalescence of bubbles, e.g., air, through the aqueous solution. Examples of bubble coalescence inhibitors include certain salts, such as MgCl2，MgSO4，NaCl，NaBr，NaNO3，Na2SO4，CaCl2，Ca(NO3)2，KCl，KBr，KNO3，NH4Br，NH4NO3，CsBr，LiCl，LiNO3，LiSO4And various sugars, such as sucrose. Other bubble coalescence inhibitors include emulsifiers and surfactants. In some embodiments, the aqueous solution is or comprises wastewater, which may already comprise one or more bubble coalescence inhibitors. In some embodiments, the wastewater comprises lipids, surfactants, and/or biopolymers.
Gas bubbles containing at least 10% carbon dioxide by volume have less coalescence in aqueous solution than bubbles of air, nitrogen and many other gases. Therefore, in the method of the present invention, it is not necessary to include a bubble coalescence inhibitor in the aqueous solution. However, in some embodiments, a bubble coalescence inhibitor may be added to the aqueous solution to inhibit coalescence of the bubbles. In such embodiments, the bubble coalescence inhibitor is typically included in the aqueous solution in an amount effective to inhibit coalescence of gas bubbles in the aqueous solution.
In some embodiments, the bubble coalescence inhibitor is a surfactant or an emulsifier. The surfactant may be, for example, a nonionic surfactant, a cationic surfactant, an anionic surfactant (e.g., common soap), or a zwitterionic surfactant. The nonionic surfactant includes monododecyl octaglycol. The cationic surfactant comprises cetylpyridinium chloride. Anionic surfactants include sodium lauryl sulfate. Examples of the emulsifier include lipids, proteins, and fats serving as emulsifiers. Some polymers also act as emulsifiers, such as sodium carboxymethyl cellulose, methyl cellulose and polyoxyethylene stearate.
Gas bubbles
The method of the invention comprises passing bubbles of gas through the aqueous solution. The gas comprises at least 10% carbon dioxide by volume. Thus, the gas comprises 10 to 100% by volume of carbon dioxide.
As will be appreciated by those skilled in the art, the relative amounts of the gas components in the bubbles may vary as they pass through the aqueous solution. For example, when the gas comprises a mixture of gases, one or more of the gases is more readily soluble in the aqueous solution than the other gases. As another example, water or other components of the aqueous solution may be vaporized and incorporated into the gas in the bubbles as the bubbles pass through the aqueous solution. Unless otherwise indicated, reference herein to the amount of a gas component (e.g., CO in a gas)2Volume percent) refers to the content of a component in the gas when the gas is first contacted with an aqueous solution. Bubbles are formed in the aqueous solution. The gas that first comes into contact with the aqueous solution is sometimes referred to herein as the feed gas (inlet gas).
As will also be appreciated by those skilled in the art, the temperature of the gas in the bubbles may change as the bubbles pass through the aqueous solution. Unless otherwise indicated, reference herein to gas temperature refers to the temperature of the gas when it first comes into contact with an aqueous solution, i.e., the inlet gas temperature.
In some embodiments, the gas comprises from 50% to 100%, for example from 50% to 100%, by volume. 80% to 99% carbon dioxide.
In some embodiments, the gas comprises 10% to 98%, for example 10% to 80%, 10% to 90%, 10% to 80%, 10% to 50% or 10% to 20% carbon dioxide by volume. In some embodiments, the gas comprises from 15% to 100%, or from 20% to 100%, by volume, carbon dioxide.
Carbon dioxide, which is a greenhouse gas, is considered to be responsible for global warming. Many industries, such as landfill sites, biogas plants and coal-fired power plants, emit large amounts of carbon dioxide. The present invention advantageously provides a method of using CO-containing products produced by such industries2A method of producing a gas.
The gas temperature at which the gas first contacts the aqueous solution is at least 18 ℃.
In some embodiments, the temperature of the gas when it first contacts the aqueous solution is higher than the bulk temperature of the aqueous solution. In some embodiments, the bulk temperature of the aqueous solution is from 18 ℃ to 55 ℃, and the temperature of the gas is higher than the bulk temperature of the aqueous solution.
In some embodiments, the temperature of the gas is at least 50 ℃, e.g., at least 55 ℃, at least 60 ℃, or at least 100 ℃. In some embodiments, the temperature of the gas is from 50 ℃ to 1000 ℃, from 50 ℃ to 500 ℃, from 50 ℃ to 400 ℃, from 50 ℃ to 300 ℃, from 50 ℃ to 200 ℃. From 50 ℃ to 150 ℃, from 55 ℃ to 1000 ℃, from 55 ℃ to 500 ℃, from 55 ℃ to 400 ℃, from 55 ℃ to 300 ℃, from 55 ℃ to 200 ℃, from 55 ℃ to 150 ℃, from 60 ℃ to 1000 ℃, from 60 ℃ to 500 ℃, from 60 ℃ to 400 ℃, from 60 ℃ to 300 ℃, from 60 ℃ to 200 ℃ or from 60 ℃ to 150 ℃. In some embodiments, the temperature of the gas is at least 100 ℃, e.g., 100 ℃ to 1000 ℃, 100 ℃ to 500 ℃, 100 ℃ to 400 ℃, 100 ℃ to 300 ℃, 100 ℃ to 200 ℃, 150 ℃ to 1000 ℃, 150 ℃ to 500 ℃, 150 ℃ to 400 ℃, 150 ℃ to 300 ℃, 150 ℃ to 200 ℃, or 100 ℃ to 150 ℃.
When bubbles of gas at a temperature above the bulk temperature of the aqueous solution are introduced and passed through the aqueous solution, a transient hot surface layer is created around each bubble. For example, when gas bubbles with a temperature above 100 ℃ are introduced and passed through an aqueous solution with an overall temperature below 100 ℃, a transient hot surface layer is created around each bubble. The instantaneous hot surface layer has a temperature higher than the bulk temperature of the aqueous solution. Without being bound by theory, it is believed that the interaction of the microorganisms with the transient hot surface layer and the heated bubbles themselves leads to microorganism inactivation when the temperature of the gas is 100 ℃ or higher, even if the overall temperature of the aqueous solution is below the temperature at which microorganisms are inactivated.
As the gas bubbles pass through the aqueous solution, the transient hot surface layer causes some vaporization of the aqueous solution, which is absorbed by the gas bubbles. This results in cooling of the bubbles as they pass through the aqueous solution, and the temperature and extent of the transient hot surface layer is reduced as the bubbles pass through the aqueous solution. The extent to which the bubbles are able to absorb the vaporized aqueous solution thus affects the extent to which the gas increases the overall temperature of the aqueous solution as the bubbles pass through the aqueous solution. When the gas has a low humidity, greater vaporization of the aqueous solution will occur than when the gas has a higher initial water content, resulting in more thermal energy being used for vaporization. The gas bubbles may pass through the aqueous solution without substantially increasing the overall temperature of the aqueous solution as a result of some of the aqueous solution vaporizing as the gas bubbles pass through the aqueous solution. Thus, the present invention provides a method useful for inactivating microorganisms in an aqueous solution that does not require heating the bulk solution to a temperature effective to inactivate the microorganisms.
In some embodiments, the gas has a relatively low humidity when it is first contacted with the aqueous solution (i.e., the feed gas). In some embodiments, the relative humidity of the gas is less than 50%, such as less than 40%, less than 25%, less than 20%, or less than 10%. Gases with relatively low humidity absorb water vapour more readily from the aqueous solution than gases with higher humidity and will therefore result in less heating of the bulk liquid.
In some embodiments, the aqueous solution comprises a surfactant or emulsifier. As described above, the surfactant may act as a bubble coalescence inhibitor. The presence of a surfactant or emulsifier in the aqueous solution may also increase the rate of inactivation of microorganisms, particularly when the temperature of the inlet air exceeds 100 ℃. The surfactant or emulsifier forms a coating around the gas bubbles. Since the surfactant or emulsifier is present in the surface layer of the bubbles, the boiling point of the surface layer of the bubbles is higher than the surface layer formed around the bubbles in the absence of the surfactant or emulsifier. As a result, the transient thermal surface layer of the bubbles can reach higher temperatures than bubbles formed in the absence of surfactants or emulsifiers. Exposing the microorganisms to a transient hot surface layer having a higher temperature may increase the inactivation rate of the microorganisms.
Surprisingly, the method of the present invention is still effective in inactivating one or more microorganisms in an aqueous solution when the gas temperature is below 100 ℃, for example at about 22 ℃ to 50 ℃. Without wishing to be bound by theory, it is believed that this is due to the large CO generated by passing the bubbles through the aqueous solution2-a liquid contact surface. This increases the amount of carbon dioxide dissolved in the solution even when the process of the invention is carried out in an aqueous solution exposed to water, and it is believed that a similar effect to that which can be achieved by increasing the pressure during dense phase carbon dioxide is produced, even if the pressure is maintained at around 1atm when the process of the invention is carried out in an aqueous solution exposed to atmospheric pressure. Thus, in some embodiments, the temperature of the gas is less than about 100 ℃, e.g., 18 ℃ to 100 ℃ or 22 ℃ to 100 ℃. In some embodiments, the temperature of the gas is from 18 ℃ to 50 ℃ or from 22 ℃ to 50 ℃.
In some embodiments, the intake air may be a combustion gas formed from the combustion of a carbon-containing fuel in air or another oxygen-containing atmosphere, such as the combustion of methane, natural gas, petroleum, coal, coke, charcoal, wood, biogas, biomass, or ethanol. In some embodiments, the combustion gas is an exhaust gas of an internal combustion engine. The combustion gas formed by combusting a fuel containing carbon in air comprises a mixture of gases. Gases formed by the combustion of carbonaceous fuels in air include carbon dioxide and other combustion products as well as nitrogen and argon (from air). The other combustion products typically include water, as well as small amounts of other gases, such as CO, H2，SO2And nitrogen oxides.
The combustion gas is used in the process of the present invention without further treatment. Advantageously, the combustion gas can be used immediately after combustion, while the gas has an elevated temperature from the combustion process.
The use of combustion gases in the process of the present invention has several advantages. First, the process of the present invention provides for the use of combustion gases that would otherwise normally be considered waste. The combustion gas may have an elevated temperature that may assist in the inactivation of microorganisms as the bubbles pass through the aqueous solution. In addition, trace gases present in the combustion gas, e.g. CO, H2，SO2And nitrogen oxides, which may themselves have a biocidal effect, contribute to the inactivation of microorganisms in aqueous solutions. Furthermore, the presence of water vapour in the combustion gas reduces the evaporative cooling effect of the bubble column, thereby causing the combustion gas to heat the aqueous solution to a greater extent than another gas having a lower amount of water vapour.
When the aqueous solution is exposed to atmospheric pressure, bubbles may be introduced into the aqueous solution, for example, using a gas inlet having a pressure in a pressure range just above atmospheric pressure, for example, in a range of 1 to 1.5 atmospheres.
In some embodiments, the gas bubbles are formed by passing a gas through a porous material, for example, through a porous material. And a porous glass sintered body which is brought into contact with the aqueous solution, thereby forming bubbles on the surface of the material which is brought into contact with the aqueous solution. As more gas passes through the porous material, bubbles are released from the surface of the material and rise through the aqueous solution. Advantageously, the temperature of the gas may be adjusted prior to the gas contacting the porous material, thereby providing a means of controlling the temperature of the gas as it contacts the aqueous solution. In some embodiments, the porous material is a sintered material (e.g., glass frit, or metal sinter, such as stainless steel sinter) or a porous ceramic (e.g., refractory ceramic). In some embodiments, the pore size of the porous material is in the range of 1 to 1000 μm (e.g., 10 to 500 μm, 20 to 200 μm, 40 to 100 μm, 50 to 80 μm). Especially 40 to 100 m. As will be appreciated, the size of the bubbles can be manipulated by one skilled in the art by varying various parameters, such as the pore size of the porous material, the viscosity of the aqueous solution, the surface tension of the aqueous solution, the gas flow rate and/or the gas pressure.
Typically, bubbles of gas are passed through an aqueous solution by passing the gas through a porous material at the bottom of a vessel or structure containing the aqueous solution to form bubbles of gas, which then rise through the aqueous solution.
Accordingly, in some embodiments, the present invention provides a method of inactivating a microorganism in an aqueous solution, the method comprising:
passing a gas through a porous material in contact with the aqueous solution to form bubbles of gas in the aqueous solution and causing the bubbles to rise through the aqueous solution,
wherein the gas comprises at least 10% CO by volume2And has a temperature of at least 18 ℃ when the gas first contacts the aqueous solution.
In some embodiments, the process is carried out in a bubble column evaporator (sometimes referred to as a bubble column reactor). Bubble column reactors typically consist of one or more vertically arranged cylindrical columns. The bubble column is configured such that gas in the form of bubbles is introduced into the lower part of the column and rises through the liquid phase. The gas escaping from the top surface of the liquid phase can be recaptured. The recovered gas may be recycled back to the bubble column reactor, reheated and reintroduced to the bottom of the column.
Preferably, the bubbles have a diameter of 0.1mm to 7mm, for example 0.1mm to 7mm, 0.1mm to 6mm, 0.1mm to 5mm, 0.1mm to 4mm, 0.1mm to 3mm, 0.1mm to 2mm, 0.1mm to 1mm, 0.5mm to 7mm, 0.5mm to 6mm, 0.5mm to 5mm, 0.5mm to 4mm, 0.5mm to 3mm, 0.5mm to 2mm, 0.5mm to 1mm, 1mm to 7mm, 1mm to 6mm, 1mm to 5mm, 1mm to 4mm, 1mm to 3mm, 1mm to 2mm, 2mm to 7mm, 2mm to 6mm, 2mm to 5mm, 2mm to 4mm or 2mm to 3 mm. The bubbles preferably pass through the aqueous solution at a high density. Generally, the aqueous solution becomes opaque due to the passage of high density bubbles. In some embodiments, the gas bubbles occupy 10% to 60% (e.g., 20% to 60%, 30% to 60%, 40% to 60%, 50% to 60%, 10% to 50%, 20% to 50%, 25% to 55%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to 40%, 30% to 40%, 10% to 30%, 20% to 30%, or 10% to 20%) of the total volume of the aqueous solution and gas bubbles as the gas bubbles pass through the aqueous solution.
When using BCE exposed to atmospheric pressure, the pressure of the gas bubbles in the column is typically about 1 atmosphere plus the hydrostatic pressure of the liquid in the column. As they leave the column, their pressure will drop to 1 atmosphere. The dry gas bubbles entering the bottom of the column will rapidly absorb a water vapor density corresponding to the temperature of the liquid in the column.
The gas bubbles may be passed through the aqueous solution, for example, in a continuous or intermittent manner. Preferably, the bubbles are passed through the aqueous solution in a continuous flow. In such embodiments, the gas bubbles typically occupy 10% to 60% of the total volume of the combination of aqueous solution and gas bubbles as the gas bubbles pass through the aqueous solution.
In some embodiments, the bubbles are passed through the aqueous solution in a continuous flow for a period of time greater than about 30 seconds, for example for a period of time greater than about 1 minute. In some embodiments, the bubbles are passed through the aqueous solution in a continuous flow for 30 seconds to 90 minutes, 1 minute to 30 minutes, 1 minute to 10 minutes, 2 minutes. From minutes to 30 minutes or from 5 minutes to 30 minutes.
In some embodiments, the bubbles are passed through the aqueous solution at a rate of greater than 0.1L of gas per L of aqueous solution per min, for example 0.1 to 1000, 1 to 1000, 10 to 1000, or 10 to 100L of gas per liter of aqueous solution per minute.
In a second aspect, the present invention provides a system for inactivating microorganisms present in an aqueous solution, the system comprising:
-a gas supply device configured to supply a gas containing at least 10% CO by volume2The gas of (a), the gas having a temperature of at least 18 ℃ or higher;
-a gas delivery device configured to receive a supply of gas and deliver the gas in the form of bubbles into the aqueous solution.
Features described in relation to the first aspect may also be applied to the second aspect.
In some specific embodiments, the gas delivery device is a bubble column evaporator or a bubble column reactor. In some specific embodiments, the gas delivery device is a bubble column evaporator or an array of bubble column reactors. In some specific embodiments, the array comprises 2 to 200 BCEs, for example 5-100 BCEs.
In some embodiments, the gas supply includes a heater to heat the gas source or feed gas to form a gas having a temperature of at least 18 ℃ or greater.
In some embodiments, the gas supply further comprises a gas source. The gas source may be adapted to provide at least 10% CO by volume2The gas of (2). In some embodiments, the gas source may be a commercially available product, such as CO in a pressurized gas cylinder2(e.g., available from BOC Gas Australia). In some embodiments, the CO may be2With one or more other gases (e.g. N)2，O2Ar) to obtain CO in the desired ratio2(e.g., by volume or partial pressure). In some embodiments, CO2May be generated in situ, for example, by chemical reactions, including combustion. In some embodiments, the gas source is a combustion gas formed by combusting a fuel comprising carbon, particularly a fuel combusted in an internal combustion engine.
Fig. 1 is a schematic diagram depicting an embodiment of a system according to a second aspect of the invention. The
In a third aspect, the present invention provides an apparatus for inactivating microorganisms present in an aqueous solution, the apparatus comprising:
a gas permeable material having a flow surface on one side thereof, the flow surface being configured to enable an aqueous solution to flow through the flow surface,
-a chamber arranged on the opposite side of the gas permeable material to supply a gas inactivating microorganisms present in the aqueous solution, such that the gas is able to pass through the gas permeable material and enter as bubbles into the aqueous solution flowing through the flow surface.
The features described in relation to the first aspect may also be applied to the third aspect.
Typically, the gas inactivating the microorganisms present in the aqueous solution is a gas comprising at least 10% by volume of CO2And a gas having a temperature of at least 18 ℃.
In some embodiments, the flow of the aqueous solution is achieved by gravity. In some embodiments, the flow of the aqueous solution is performed by a pump.
In some embodiments, the apparatus includes a heating element positioned to heat the gas prior to contacting the gas permeable material. In some embodiments, the heating element is disposed in the chamber. In some embodiments, the heating element is disposed within or near the inlet of the chamber to heat the incoming gas as it enters the chamber. In some embodiments, the chamber wall is heated. In some embodiments, the heat exchanger is used to heat incoming gas.
In some embodiments, the flow surface is in the form of a channel. In some embodiments, the flow surface is a portion of a conduit or pipe. In some embodiments, the flow across the flow surface involves the flow of an aqueous solution from a first region of the surface to a second region of the surface.
In some embodiments, the flow surface is configured such that when the gas bubbles flow across the flow surface, the gas bubbles pass through the aqueous solution in a direction transverse to the direction of flow of the aqueous solution. In some embodiments, the gas bubbles rise in the aqueous solution, while the aqueous solution flows in a substantially horizontal direction (e.g., from about 0 ° to about 20 °, from about 0 ° to about 10 °, or from about 0 ° to about 5 °).
In some embodiments, the gas permeable material is selected such that the diameter of the gas bubbles entering the aqueous solution is from about 0.1mm to about 7mm when the aqueous solution is at a pressure of about 0.9 to 1.5 bar. As will be appreciated by those skilled in the art, this may be achieved by selecting a breathable material of appropriate porosity that operates at a particular pressure or pressure range.
In some embodiments, the apparatus comprises a cover configured to capture the gas as it exits the aqueous solution.
The process of the invention may be carried out in a continuous manner, for example using the apparatus of the third aspect, as the aqueous solution flows between the two locations (for example, as the aqueous solution moves through the channel). Figure 9 is a perspective view of a cross-section of a continuous flow reactor that may be used to perform the method of the present invention as an aqueous solution moves through the flow reactor. As shown generally in fig. 9, a gas (e.g., CO)2Gas or combustion gas) is introduced into the chamber through the inlet. Optionally, a heater in the chamber is used to heat the gas. The gas then flows through a porous structure, such as channels formed by a porous ceramic, such as a refractory ceramic, to form channels in the porous structure through which the aqueous solution flows. The bubbles then rise through the aqueous solution, inactivating microorganisms in the aqueous solution as the aqueous solution moves along the channel.
In more detail, FIG. 9 is a diagram depicting a third aspect according to the present inventionSchematic illustration of an embodiment of an apparatus. The
Microorganisms
Typically, the microorganism is a bacterium, such as e.coli or a virus. However, the method of the invention may also be used to inactivate other microorganisms, such as algae, protozoa, fungi or spores.
In some embodiments, the microorganism is a bacterium selected from the group consisting of: escherichia coli, Clostridium botulinum, Campylobacter jejuni, Vibrio cholerae, Vibrio vulnificus, Vibrio alginolyticus, Vibrio parahaemolyticus, Mycobacterium marinum, dysentery-causing species such as Shigella shigella, Legionella species such as Legionella pneumophila, Leptospira species, or Salmonella typhi.
In some embodiments, the microorganism is a virus selected from the group consisting of coronavirus, hepatitis a virus, poliovirus, or polyoma virus.
In some embodiments, the microorganism is a protozoan, such as cryptosporidium parvum or another pathogenic protozoan.
In some embodiments, the microbial inactivation in the aqueous solution is at least a 1log reduction, such as a 1 to 4log reduction, a 1 to 3log reduction, a 1 to 2log reduction, a 1 to 1.5log reduction, a 1.5 to 4log reduction, a 1.5 to 3log reduction, a 1.5 to 2log reduction, a2 to 4log reduction, a2 to 3log reduction. The reduction is relative to the initial microbial content in the initial solution (i.e., prior to contact with the gas). In some embodiments, the microbial inactivation is accomplished within 90 minutes, such as 30 seconds to 90 minutes, 30 seconds to 30 minutes, 30 seconds to 10 minutes, 30 seconds to 5 minutes, 1 minute to 90 minutes, 1 minute to 30 minutes, 1 minute to 10 minutes, 1 minute to 5 minutes, 2 minutes to 30 minutes, or 5 minutes to 30 minutes.
Examples
Various embodiments of the invention are described below with reference to the following non-limiting examples.
Model water treatment virus and bacteria system
MS2 virus
MS2 phage (ATCC 15597-B1) [12, 13] were selected as model viruses to evaluate the efficiency of the virus inactivation process. MS2 is generally quantified by counting infected units by a standard plaque assay commonly used to detect MS2[20] in treated drinking and waste water.
MS2 is used as a replacement for enterovirus because it is inactivated only at temperatures above 60 ℃, is resistant to high salinity, and is sensitive only to low pH. This means that it has a high resistance to environmental stress [21 ].
According to [22], MS2 is a member of a class of bacteriophages called bacteriophages. Its entire genome has been sequenced. It is a single-stranded RNA molecule with a positive sense of 3,569 nucleotides and has an icosahedral structure. The hydrodynamic radius of the virus is about 13nm [22 ].
Virus inactivation was analyzed using an optimized two-layer plaque assay technique described in Cormier et al [23], which detects only infectious viruses by using E.coli as the host.
Coliform bacteria
Escherichia coli is a gram-negative bacterium having a diameter of 1.1 to 1.5 μm and a length of 2.0 to 6.0. mu.m, and is in the form of a straight cylinder [24 ]. It is present in the gastrointestinal tract of animals and humans. Coli strains are harmless or pathogenic to the host. They can be found in water and soil due to fecal contamination. Thus, its presence in water is often used as an indicator to monitor water quality [25 ].
Coli C-3000(ATCC15597) was used as a representative model for bacteria in water for many studies [26,27 ]. Escherichia coli C-3000(ATCC15597) is an organism with
Spinks et al [29] demonstrated inactivation of pathogenic bacteria at temperatures ranging from 55 ℃ to 65 ℃. Other studies have found that E.coli shows first signs of heat inactivation at temperatures above 55 ℃ and reaches very high inactivation rates at 60 [30 ]. This is expected because membrane phospholipids have a phase transition and lose their order state at this temperature.
Materials and methods
Experimental solution
The electrolyte solution [31] was prepared and sterilized by autoclaving in Aesculap420 at 15psi and 121-.
Typically, the solution used contained 0.17M NaCl in 300ml of Milli-Q water (. gtoreq.99% purity, obtained from Sigma-Aldrich).
Tabor et al [32 ]]It was found that the coalescence of bubbles in water depends on the type of gas and the pH. In the Tabor et al article, CO was observed2The bubbles do not coalesce, while other gases (e.g. Ar, N)2And air) will coalesce. Therefore, a 0.17M NaCl solution was used in the experiments described below to ensure that bubble coalescence was similar for all gases studied, eliminating possible confounding variables. In the experiment, NaCl (0.17M) was used to reduce bubble size and inhibit coalescence, butWastewater generally contains lipids, surfactants and biopolymers, and can stabilize air bubbles and reduce the size of the air bubbles even at low levels.
The MS2 virus is resistant to high salinity and is stable in the presence of 1 to 2M NaCl [33 ]. Previous studies [5, 8 and 9] have shown that 0.17M NaCl solution does not inactivate E.coli. Therefore, the concentration of NaCl should not be a factor in viral or bacterial inactivation.
Preparation of culture Medium for Virus experiments
A specific optimized bilayer plaque assay technique can be used to assess the concentration of active MS2 virus as described in Cormier et al [23 ]. This plaque assay is commonly used to detect MS2 in treated drinking, waste and sea water. Water quality was assessed by the ability of the phage to kill host bacteria and allow the phage to multiply on bacterial host cell confluent regions immobilized on agar layers [23,28,34,35 ].
This medium is not commercially available, so 1.5L was prepared from both solutions (a and B) prior to each experiment. To prepare solution A, 15g of tryptone, 1.5g of yeast extract, 12 g of NaCl and 1425ml of Milli-Q water were used. The pH was 6.9 as measured with a Thermosscientific Orion Star A214 pH meter. The solution was aseptically dispensed into 3 containers containing different amounts of agar (1% bottom agar, 0.5% top agar, no agar in the medium) used in the experiment was of Sigma-Aldrich molecular biology grade. These solutions were heated to boiling to dissolve the agar and sterilized by autoclaving in Aesculap420 at 15psi, 121-.
Solution B improved the visibility of the virus. The solution was prepared by mixing 1.5g glucose, 0.441 g CaCl2And 0.015g thiamine were added to 75ml of Milli-Q water and filtered through a 0.22 μm filter for sterilization, and then added aseptically to each of the 3 solutions A separately as it was cooled to 50 ℃.
The bottom agar was poured into 100mmx15mm petri dishes and dried on Bunsen burner to maintain local environmental sterility until the agar was not too dry or too moist [34 ].
Preparation of culture Medium for Escherichia coli experiments
Plaque assay is commonly used to detect E.coli in treated drinking water, wastewater and seawater. Water quality was assessed by the ability of bacteria to multiply in the agar layer [26,34 ].
For each experiment, 1L was prepared from two solutions (a and B). To prepare solution A, 13g of tryptone, 1g of yeast extract, 6 g of NaC and 1000ml of Milli-Q water were used. The pH was 6.9 as measured with a thermo Scientific Orion Star A214 pH meter. The solution was aseptically dispensed into two containers containing different amounts of agar (1.41% agar medium and no agar medium) used in the experiment as Sigma-Aldrich molecular biology grade. These solutions were heated to boiling to dissolve the agar and sterilized by autoclaving in Aesculap420 at 15psi and 121-.
Solution B improved the growth of bacteria. The solution was prepared as follows: sterilization was performed by adding 1g of glucose and 0.010g of thiamine to 50ml of Milli-Q water, and filtering through a 0.22 μm filter, which was then aseptically added to 2 solutions A, respectively, when cooled to 50 ℃.
The 1.41% agar solution was poured into a 100mmx15mm petri dish and dried next to the bunsen burner to maintain local environment sterility until the agar was either not too dry or too moist [34 ].
Bacterial strains
Coli C-3000(ATCC15597) was used as a representative model for aquatic bacteria [26,27], for E.coli inactivation experiments and for viral experiments as MS2 viral hosts [28 ]. Coli size and Zeta potential were measured in 0.17M NaCl solution with different bubbles using a Malvern Zetasizer nano-series instrument.
For successful plaque assays, E.coli C-3000(ATCC15597) must be in exponential growth phase. This can be achieved by culturing two different bacterial cultures: overnight cultures and log phase cultures [28,31,35 ]. The overnight cultures were grown in 10ml agar-free medium at 37 ℃ for 18-20 h in Labtech digital incubator LIB-030M with shaking at 110rpm using a PSU-10i orbital shaker. Overnight culture resulted in a large number of bacteria in the culture and was used as a reference standard.
To start the logarithmic phase of E.coli cultures, 1ml of overnight culture was transferred to 25-30ml agar-free broth and incubated at 37 ℃ for 3h with gentle shaking at 110 rpm. To prevent cell loss of F-pill, it was then rapidly cooled in a refrigerator at 5 ℃. The Optical Density (OD) of the log phase E.coli culture was then measured using a UV-VIS spectrometer UVmini-1240. The OD reading at 520nm was between 0.8 and 1.1, indicating that this culture can be used in plaque assays for viral experiments and as a standard for E.coli experiments.
Viral strains
Lyophilized MS2 phage vials were obtained from the American type culture Collection. Bacteriophage MS2(ATCC 15597-B1) was replicated using E.coli C-3000(ATCC15597) according to International Standard ISO10705-1[31] and the U.S. environmental protection agency's Manual of ultraviolet Disinfection guide [36 ]. Zeta potentials of MS2 virus in 0.17M NaCl solution with different air bubbles were measured using a Malvern Zetasizer nano series instrument.
Viral and bacterial dilution
The concentrations of MS2 phage and E.coli were calculated as follows: 1.0ml agar-free medium was added to the vial and 12 serial 10-fold dilutions [35 ] were made by placing 0.50ml phage and E.coli (in another experiment) into a test tube containing 4.50ml agar-free medium]. Mixing 0.1
After overnight incubation, at 37 ℃ for 18-24h, individual plaques can be counted and the concentrations of MS2 phage and E.coli calculated using the following formula:
PFU/mL ═ of undiluted peak suspension (PFU1+ PFU2 … PFUn)/(V1+ V2 … Vn)
Here, PFU is the number of plaque forming units in the plate, Vn is the volume (mL) of each undiluted sample added to the plate containing countable plaques, and n is the number of countable plaques.
Bubble column evaporator process
FIG. 1 shows a diagram for evaluating a gas N at different temperatures2，CO2Ar, air and O2Schematic of the system/apparatus of the effect of passing the aqueous solution in the bubble column evaporator. Depending on the experiment, the gas was pumped through an electric heater that maintained the bubble temperature directly above the sinter surface at the bottom of the bubble column evaporator. The bottom of the bubble column evaporator was fitted with a sintered glass (type 2) of 40-100 μm pore size with a diameter of 135 mm.
After pouring the aqueous solution into the column, the temperature of the solution was measured at the center of the column solution with a thermocouple. The gas was then passed through sintered glass into 300ml of solution to inactivate the MS2 virus or e.coli (in separate experiments).
When combustion gas was used, a bubble column evaporator similar to that used in the other experiments was used and the generator (Honda EM2200) vent line was connected to a valve that provided a 27l/min vent gas flow through the bubble column (as shown in FIG. 3.)
Disinfection experiment
The experiment was carried out using a 0.17M NaCl solution (initial temperature 22.5 ℃ C.) with CO as the feed gas2Argon, nitrogen, air, oxygen and combustion gas mixtures, for e2Argon, nitrogen, air and oxygen at 150 deg.C, combustion gas at 58 deg.C, oxygen and air at 20 deg.C, CO for MS2 virus experiment2Argon, nitrogen, air and oxygen at 200 ℃ and combustion gas at 60 ℃, the experiments described were carried out in BCE and as a comparison in a water bath at 22.5 ℃ and pH 4.2. Further experiments were also carried out in BCE using MS2 virus in 0.17M NaCl solution (initial temperature approximately 22 ℃) using CO with different entry temperatures (205 ℃, 150 ℃, 100 ℃, 11 ℃ and 20 ℃)2Gas and CO with different flow rates (27l/min and 20l/min) at an inlet temperature of 22 ℃2A gas.
The counting of phage and E.coli activity was performed by plaque assay [23,34,35 ].
Once solutions with known concentrations of coliphage and E.coli were prepared, experiments were conducted to investigate the process of inactivating coliphage at different gases and inlet air temperatures. For the virus experiments, 1.3ml samples were collected every 6 minutes from 10 to 15mm above the sintered central region over 36 minutes, and for the E.coli experiments, 1.3ml samples were collected every 2 min. 0.07ml of each sample was spotted in triplicate according to the two-layer plaque assay technique [36 ].
To evaluate the effect of solution pH on MS2 phage inactivation and e.coli experiments, comparative tests were performed in a water bath in another beaker using a 0.17M NaCl solution at pH 4.1. In these experiments, samples were periodically removed from the beaker and analyzed as before.
Data analysis
Plaque counts were performed on all 18-21 plates per experiment. Mean and standard deviation for each triplicate sample virus or bacterial survival factors were used: log10(PFU/PFU0) In which PFU0Is the initial number of PFUs per sample, PFUs is the PFU per sample after exposure time (in min). When plotted, a second order polynomial distribution was applied to fit the survival curve [21]]。
Results and discussion
Some common results are shown in FIG. 4, which shows CO at 200 ℃ in BCE2Photographs of the petri dishes used in the MS2 virus-inactivated double-layer plaque assay technique in NaCl 0.17M aqueous solution after entering temperature treatments (a)0, (b)0.75, (c)2.25 and (d)3 min. Complete inactivation of 295PFU per plate per sample was achieved using a total heat input of only 0.02kJ/ml (46.5 ℃ solution temperature at the end of the experiment) and only 3.5 min.
1. Effect of pH on viral and bacterial inactivation when bubbling CO2 in aqueous solution
When using CO in a bubble column2The pH of the water dropped from 5.9 to 4.2 in less than 45 seconds with gas.
When CO is present2When dissolved in water, 99% remain as dissolved molecular gas and less than 1% become carbonic acid (H)2CO3). This lowers the pH of the water to about 4 (equation 2). Decomposition of carbonic acid into bicarbonate ionsAnd carbonate ion(see
CO2(g)→CO2(aq) (1)
To determine whether the decrease in pH due to dissolved carbon dioxide (
Carbonic acid (H) was generated by bubbling 27l/min2CO3). Pure carbon dioxide was injected through a 0.17M NaCl solution at 22 ℃ over 10 min. After bubbling, the solution was continuously stirred in a beaker, sampled every 3min at a pH of 4.2, and only a 0.002 log reduction in virus was observed. For E.coli, only 0.08 log removal was observed, indicating that low pH had a slight inactivation of E.coli but no effect on the virus. Some authors describe a small, low pH inactivation of microbial cells, as the membrane prevents proton penetration, but due to chemical modification of the phospholipid bilayer of the membrane, the low pH renders the membrane resistant to other substances such as CO2Is more permeable [38,39 ]]. Cheng et al [14]It was observed that there was little change in inactivation for 3 different viruses (MS2, Q β and φ X174) at different pH conditions (
Therefore, when CO is introduced2The pH drop was considered to be independent of the high viral and bacterial inactivation observed when bubbled into the BCE.
2. Effect of different gases on Virus and bacterial inactivation
In these experiments, the deactivation rates of MS2 virus (ATCC 15597-B1) and Escherichia coli (ATCC15597) were determined when bubbling inlet gas through a 0.17M NaCl solution at 200 deg.C (for virus) and 150 deg.C (for Escherichia coli) in a bubble column evaporator. For different gases (nitrogen, oxygen, CO) present in the air2And argon) were performed. 99% of the air is a mixture of nitrogen and oxygen, and the other 1% of the gas comprises argon and CO2(among other gases).
Finally, the initial MS2 virus and the subsequent E.coli were inactivated with combustion gas from the generator at a temperature of about 60 deg.C (60 deg.C for MS2 virus experiments and 58 deg.C for E.coli experiments).
The results of these experiments are shown in figures 5 and 6.
The MS2 virus inactivation rates in 200 ℃ gas were found to be very similar, except for CO2And combustion gases. When Escherichia coli is inactivated using 150 ℃ gas, the inactivation rate of oxygen production is highest, followed by CO2And combustion gases.
Prior study [32][17]When CO is introduced2,Ar,N2And when air is bubbled through the water, the CO is generated2Soluble in water, which differs from the rest of the gas in terms of the change in the pH of the gas bubbles and the zeta potential.
Enomoto et al [17 ]]It was found that when using explosive decomposition systems, other gases which are not readily soluble in water (e.g. Ar or N) are used2) In contrast, gases highly soluble in water (e.g., CO)2And N2O) can realize higher inactivation rate of the beer yeast. Tabor et al [32 ]]Evidence of CO2The effect of preventing the coalescence of bubbles is much better than other gases. Bubble coalescence inhibition is a very important variable in BCE performance because it increases the gas/water interface area, which is why all experiments were performed in 0.17M NaCl solution [4]To ensureAll gases were kept at similar levels of inhibition of bubble coalescence in the column solution.
Carbon dioxide has a higher water solubility than other gases used in these experiments, produces lower Zeta potentials of coliform and virus, and lowers the pH (see table 1).
The solubility of the different gases used at 52 ℃ for the virus and 43 ℃ for E.coli was obtained from literature values (http:// www.engineeringtoolbox.com). The pH value was measured during the experiment and the Zeta potential value was measured during the experiment at a virus concentration of 10 per ml8The concentration of Escherichia coli is 10 per ml7。
Solubility of gas in water at 43 ℃ for E.coli and 52 ℃ for viruses. Grams of gas dissolved in 1Kg of water at 1atm
Table 1: solubility of gases in the study of viruses and bacteria, microbial Zeta potential, pH, heat capacity, water temperature at the end of the experiment, thickness of the heating layer around the bubbles, and average temperature of the layer.
The "water temperature" in Table 1 is the aqueous solution temperature at the end of the experiment (for E.coli experiments this means after 10min for all gases; for virus experiments after 37.5min for all gases except carbon dioxide and combustion gases and after 4min for carbon dioxide and combustion gases). For all experiments, except for viral experiments using carbon dioxide gas or combustion gas, the temperature at the end of the experiment was the equilibrium solution temperature (i.e., the temperature was kept constant with the temperature as further bubbles passed through the solution). In these experiments, the aqueous solution generally reached the equilibrium solution temperature after about 5 min. The average temperature of the heating layer around the bubbles reported in table 1 was calculated according to the water temperature reported in table 1 as follows.
In Table 1 (viral part), CO at the end of the experiment according to its heat capacity2Temperature ratio ofThe expected temperature under steady state conditions is low, only 46.5 ℃. This is because only the average temperature of the water after 4min is used, since this is due to the use of CO2The time required to achieve complete inactivation of the virus. For oxygen, nitrogen, argon and air, an average temperature after 37.5min was used, as this is the time required to achieve 1.5log viral inactivation. For bacteria, an average temperature was obtained after 10min, which is the time required to achieve an average of 1.5log of e.coli inactivation.
For the intake air temperature exceeding 100 ℃, the temperature and thickness of the hot water layer around the surface of the bubble having a diameter of 1mm can be roughly estimated using the following equations (5), (6) and (7).
The temperature of the hot water layer around the surface of a bubble of 1mm diameter can be roughly estimated by the following formula:
wherein T isl(in degrees C.) is the average (transient) temperature of the hot water layer around the bubble, and Tc(° c) is the temperature of the solution in BCE, assuming the hot bubble cools from the inlet air temperature to 100 ℃.
The thickness of the equilibrium transient heating layer can be estimated by balancing the heat provided by the cooling bubbles with the heat required to raise the film to this average temperature.
Thus, the volume V of the membrane is given by:
V＝4πr2z (6)
where V is r > > z the volume of the layer of thickness z around the bubble.
Thus, the thermal energy balance is given by:
CpΔTV＝Cwater (W)Δt4πr2ρwz (7)
Wherein C isp，CWater (W)Heat capacity of gas and water, respectively, pwIs the mass density of the liquid water, Δ T is the cooling of the bubbles, and Δ T is the transient temperature rise in the water layer.
In practice, it may be expected that about half of the heat provided by the cooling bubbles will beFor evaporating water to CO2In the air bubble, the thus calculated rough estimate of the film thickness will be halved.
Table 1 gives the common results of this calculation.
3. Inactivation of MS2 Virus with different gases at 200 deg.C
Mechanism of heat inactivation
When the gas entry temperature is 200 ℃, the collision between the transient thermal layer around the rising thermal bubble and the MS2 virus inactivates the virus. MS2 virus is rapidly inactivated at temperatures above 60 [21, 40], and therefore, if the temperature of the hot layer around the bubble is above 60 ℃, the hot bubble will effectively inactivate the virus. It has previously been demonstrated (Garrido et al [7]), that MS2 virus can be inactivated during collision in BCE by heat exchange with a hot bubble at 150 ℃. This mechanism appears to be related to how close the virus and bubble are at the time of collision. In earlier experiments, the MS2 virus survival factor was tested and correlated with the interaction surface forces between the virus and the air bubbles. The reported results show that electrolyte added at 150 ℃ can control the surface forces between the virus and the hot surface of the bubble, thereby affecting the inactivation rate.
The MS2 virus survival factors for several different gases at 200 ℃ and combustion gas at 60 ℃ were compared using the BCE method and the results are shown in FIG. 5. Air, oxygen, nitrogen and argon with an inlet temperature of 200 c were found to have very similar rates of viral inactivation, with 1.33 and 1.67-log reductions after 37.5 minutes of treatment. When hot bubbles form on the surface of the sintering furnace, a thin layer of hot water is formed briefly around the bubble surface and has a similar average temperature, ranging from 73 ℃ to 76.9 ℃, and thickness values of about 55 to 91nm for these four gases (see table 1). Without being bound by theory, it is believed that collisions between the transient thermal layer surrounding the rising thermal bubble and the MS2 virus are the basic mechanism by which these 4 gases inactivate the virus.
2Mechanism of CO inactivation
In contrast, when using CO at 200 ℃ and 60 ℃ respectively2(100％CO2) And combustion gas (according to textbook "basic knowledge of Combustion" chapter 2[ 41)]，CO212.5% to 14%), virus and hot CO in the experiment2Collisions between hot water layers around bubbles do not appear to be the only mechanism leading to rapid virus inactivation, and the results observed in these studies were 2.2log inactivation with combustion gases in as little as 3.5min, with pure CO2The deactivation rate was 2.7log (see FIGS. 4 and 5).
When combustion gases are used, there may be CO removal due to minor constituents and impurities in the fuel and different fuel/air ratios2，H2O and N2Other combustion products than oxygen. These gases typically include: carbon monoxide (CO), hydrogen (H)2) Sulfur Oxide (SO)2) And nitrogen oxides (NOx), such as NO and NO2[41]. The small presence of these gases may explain when using pure CO at 200 deg.C2And similar results were obtained using combustion gases only at an entry temperature of 60 c. For example, Richard F. et al [42 ]]Research into SO2And its high solubility in water.
Francis et al [43] studied the latent heat of vaporization using different brine solutions in BCE and estimated a dry nitrogen inlet temperature of 60 ℃ and an equilibrium temperature of 22 ℃. However, the combustion gas is saturated with water, and therefore does not use heat to evaporate the water vapor into bubbles. This explains why when the inlet temperature of the combustion gases is only 60 c, the equilibrium temperature of the water remains at a high temperature of 39.5 c instead of 22 c if the gases are dried. Thus, if the bulk of the heat in the hot gas bubbles is not used to vaporize the water, a feed gas temperature of 60℃ can also inactivate the virus by increasing the bulk temperature of the aqueous solution.
When CO is present2When the gas bubbles pass through the sintering zone, larger CO is continuously generated2The liquid contacts the surface. Even if the pressure in the bubble column is maintained at about 1atm, this increases the amount of CO dissolved in the solution2In an amount to produce an effect similar to that which can be achieved by increasing the pressure in a Dense Phase Carbon Dioxide (DPCD) process. Many authors have demonstrated the use of pressurized CO2Can inactivate microorganisms [14-16 ]]. Explaining the mechanism of this deactivation and its use in waterHigh solubility in (1).
Cheng et al [14]A mechanism for inactivation based on bacteriophages MS2 and Q β was proposed, which is based on penetration of CO2 inside the capsid under high pressure and expansion after decompression, thus destroying the capsid2Protein binding may also disrupt the capsid inactivating the virus.
4. Results of inactivation of Escherichia coli (ATCC15597) with gas at 150 deg.C
Mechanism of heat inactivation
When Escherichia coli (ATCC15597) was inactivated in BCE with air and nitrogen at an entry temperature of 150 ℃ with Shahid et al [ 9]]The reported inactivation results for E.coli were similar (FIG. 6), using a natural lake water sample. Collisions between transient hot layers surrounding rising bubbles and collisions of the hot bubbles themselves with coliform bacteria are considered as the basic mechanism of coliform inactivation [8]]. In the use of high-temperature gas N2The basic mechanism of coliform inactivation in current work also uses the above presumption, Ar and air. However, as the results reported herein show, when CO is used2Or combustion of gases, this is not the only mechanism of coliform inactivation.
By bubbling over a period of 10min, survival factors of e.coli were compared in the following cases: introducing into 150 deg.C air, oxygen, nitrogen, and CO2And argon, into combustion gas at a temperature of 58 c and into oxygen and air at a temperature of 20 c and the results are shown in figure 6. After 10min of treatment at 150 ℃, the air and nitrogen inactivation rates of E.coli were very similar, decreasing by 1.43 and 1.74-log, respectively. No inactivation was detected in air at 20 ℃, only a 0.03log reduction. The deactivation rate of argon was lower, only 0.40log reduction after 10min, probably due to its low heat capacity (only 0.52kJ/kgK), only half of that of nitrogen and air (Table 1). For these 3 gases, when hot bubbles are formed on the surface of the sintered body, a thin hot water layer is formed around the bubble surface, and the average temperature of the thin layer is similar, ranging from 69.3 ℃ to 72.4 ℃ (table 1). Coli rapidly inactivates at temperatures above 60 ℃ [29]]Thus, if the temperature of the thermal layer surrounding the bubble is above 60 ℃, the thermal bubble will haveColi was effectively inactivated, so when air was bubbled at 20 ℃, no e coli inactivation was observed (0.03 log reduction), supporting the thermal collision hypothesis and establishing a baseline.
Mechanism of CO2 inactivation
Carbon dioxide (100% CO) was used at 150 ℃ and 58 ℃ respectively2) And combustion gas (according to textbook "basic knowledge of Combustion" chapter 2[ 41)]12.5% to 14% CO2) Enterobacter and hot CO2The large collisions between bubbles does not appear to be the only mechanism explaining the higher deactivation rate, 2.63log for combustion gases after 10min and for pure CO2Is 2.84log (see FIG. 6).
Different mechanisms have been proposed to explain the antimicrobial action of dissolved carbon dioxide. In dense phase carbon dioxide (38)]In
When combustion gases are used, there may be CO removal due to minor constituents and impurities in the fuel and different fuel/air ratios2，H2O and N2Other combustion products than oxygen. These gases are typically: carbon monoxide (CO), hydrogen (H)2) Sulfur Oxide (SO)2) And nitrogen oxides (NOx), such as NO and NO2[41]. The presence of these gases in small amounts may also result in bacterial inactivation.
Oxidation/combustion mechanism
In BCE, the hot oxygen bubbles generated at 150 ℃ using 0.17M NaCl solution with e.coli produced the best inactivation, a 2.90log reduction after only 3.5min (fig. 6). The pure hot oxygen can be directly contacted or introducedThe reactive oxygen species generated at 150 ℃ oxidize and burn E.coli. As is well known, H2O2Can inactivate Escherichia coli, and the inactivation effect is accompanied by H2O2Increase in concentration [43]. Proteins, lipids and nucleic acids may also be respired by O2Is incompletely reduced and oxidized by reactive oxygen species [44]. The Escherichia coli is in the shape of straight cylindrical rod with diameter of 1.1-1.5 μm and length of 2.0-6.0 μm [24]]. Compared to the size of the bubbles, 1-3mm, and therefore these cells can easily penetrate the thin hot water layer around the oxygen bubbles (table 1) and directly contact the hot oxygen.
In contrast, the results obtained for E.coli using hot oxygen (at 150 ℃) as the injection gas were found to be much more efficient than the results for the MS2 virus (entry temperature of 200 ℃), which MS2 virus has a much smaller hydrodynamic radius of about [22] at 13 nm. Thus, the lower solubility of oxygen in water (table 1), the inactivation time of the virus (37min) compared to e.coli (10min) and a 0.65log reduction of e.coli (fig. 6) when oxygen is used at an entry temperature of 150 ℃, supports the hypothesis that e.coli oxidation/combustion by direct contact with the gas phase is unlikely to occur in much smaller viruses.
5. Comparison of the Heat inactivation mechanisms of E.coli and Virus
The World Health Organization (WHO) compared the thermal inactivation rates of different types of bacteria and viruses in hot liquids in its drinking water quality guidelines [40 ]. They concluded that temperatures above 60 ℃ are effective in inactivating viruses and bacteria. Bacterial inactivation is faster than viral inactivation when the temperature range is between 60 ℃ and 65 ℃. These studies show that at 60 ℃ water temperature, E.coli takes 300 seconds to achieve a 1.5log reduction, while viruses of the enterovirus, echovirus 6, coxsackievirus B4, coxsackievirus B5 take 1800 seconds to achieve a 4log reduction [40 ].
In our experiments, when bubbled in nitrogen or air at 150 ℃, E.coli achieved 1.5log inactivation within 10min (FIG. 6) (where the solution temperature was 43 ℃ for nitrogen and 45 ℃ for air in 10 minutes) and MS2 virus was 200 ℃. (FIG. 6)1 to 1.5log deactivation was achieved after approximately 15 to 37min in air, oxygen or nitrogen (solution temperature at 37.5min 52 ℃ for air, 50 ℃ for oxygen and 54 ℃ for nitrogen) (FIG. 5). However, when bubbling CO at 150 deg.C2Or 58 ℃ combustion gas, the E.coli reached 2.6log inactivation within 10min (FIG. 6) (where the solution temperature was for CO over 10min)243 ℃ for combustion gas, 40 ℃ for combustion gas and with CO at 200 ℃2Or 60 ℃ combustion gas, 2.0log inactivation of the MS2 virus was achieved in 5 minutes (4 minutes dissolution temperature for CO)2At 47 c, 30 c for combustion gas) (fig. 5). It is recommended that in this method a gas with an entry temperature above about 100 c is used, and that viruses and bacteria are inactivated when they collide with the heated gas bubble, whether a hot water layer around the bubble or hot gas inside the bubble. These results are consistent with WHO data that bacteria inactivate faster than viruses at similar water temperatures. However, when utilized, the catalyst has a composition containing at least 10% CO by volume2This phenomenon is greatly and surprisingly enhanced with the BCE of the gas.
6.CO2Effect of bubble temperature on Virus inactivation
Using the apparatus shown in FIG. 1, CO was used at an inlet temperature of 205 deg.C, 150 deg.C, 100 deg.C, 11 deg.C or 20 deg.C2The gas was subjected to further experiments. The results are shown in fig. 7. At the beginning of the process, the temperature of the aqueous solution was about room temperature (about 22 ℃), the solution temperature reported in fig. 7 being the temperature of the relevant gas used at the end of the experiment (for example, for CO with an entry temperature of 100 ℃)2After 6 minutes, the bulk temperature of the aqueous solution was 34 ℃).
For gases having an entry temperature of 100 c or higher, when hot bubbles are formed on the surface of the sintered body, a thin layer of hot water is formed around the surface of the bubbles. The thickness and temperature of such thin, transient layers may be important parameters for virus inactivation. Without wishing to be bound by theory, it is believed that collisions between these hot bubbles and coliform bacteria can inactivate the coliform bacteria. It is further believed that a similar mechanism is also effective for inactivating viruses when they are close enough to the surface of the air bubbles within the hot water layer.
When CO is generated at room temperature2With air bubbles, 1log of virus was reduced in only 10 minutes (fig. 7 and 8). At higher entry temperatures, viral inactivation also increased, with entry of CO at 150 ℃2The reduction was 2.3log after 7 minutes at temperature and 3log after 3 minutes at 200 ℃ (fig. 7).
Isenschmid et al, 1995[45 [)]It is believed that dissolved compressed CO is present when the solution temperature exceeds 18 deg.C2Concentration is a key parameter for cell death. This may explain why CO is at 9 deg.C2After 6.5 minutes at the column temperature, at an entry temperature of 11 ℃, there was no significant CO with a 0.1 log reduction2Inactivation effect (fig. 7). If the solution temperature rises above 18 ℃, either as a layer around the bubbles or as the bulk of the solution, CO2The permeability through the capsid will increase, resulting in CO2Effect on virus inactivation of
7. Effect of gas flow Rate on Virus inactivation Using CO2
FIG. 8 compares the use of 2 different COs within 22min2Effect on virus inactivation at flow rate run for 10 min. From these results, it is clear that although the first 27l/min of CO2Gas inactivation of the virus was more effective than 20l/min, but the rate of inactivation after 2 minutes was almost doubled, reducing 0.35log and 0.62log, with both rates of inactivation being almost the same, about 1log, at approximately 10 minutes.
Conclusion
At atmospheric pressure so as to contain at least 10% by volume CO2Bubbling through an aqueous liquid can be used to effectively inactivate common aquatic pathogens (including viruses and bacteria), which appear to pass through two different mechanisms depending on the inlet gas temperature.
The mechanism of thermal inactivation is based on heat transfer between the hot bubbles and the pathogens during collisions. This mechanism is considered effective when the gas temperature exceeds about 100 ℃.
By means of a gas mixture containing at least 10% CO by volume2The second inactivation mechanism seems to come into play, which seems to be based on the permeation of carbon dioxide molecules through the bacterial membrane and the viral capsid. The bubbles contain at least 10% CO by volume2Will produce high density CO at atmospheric pressure2The gas can effectively inactivate viruses and bacteria in the water, and other gases such as air, nitrogen and argon can only have limited inactivation effect. The pH reduction due to carbon dioxide sparging was found to be independent of the high viral and bacterial inactivation obtained using carbon dioxide sparging. Appears to contain at least 10% CO by volume2The gas bubbles through the aqueous solution produce a higher liquid-gas interface area and are due to CO2Solubility in water is high and therefore plays an important role in viral and bacterial inactivation. This mechanism works when the gas temperature is 18 ℃ or higher.
It will be understood that, if any prior art publication is referred to herein, this reference does not constitute an admission that the publication forms part of the common general knowledge in the art in australia or in any other country.
In the appended claims and the previous description of the invention, unless the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
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