An apparatus for exposing microorganisms to gaseous pollutants

Abstract

The present application belongs to the field of environmental protection technology, and specifically relates to a device for exposing microorganisms to gaseous pollutants. The device comprises two decompression air inlet pipes, a T-shaped gas mixing pipe, a porous exposure disc, and a pressurized air outlet pipe. The two horn-shaped decompression air inlet pipes are screw-connected to the two ends of the horizontal pipe of the T-shaped gas mixing pipe. The horn-shaped pressurized air outlet pipe is screw-connected to the lower end of the vertical pipe section of the T-shaped gas mixing pipe. The porous exposure disc is arranged in the vertical pipe section of the T-shaped gas mixing pipe to form an exposure cabin. The hydrogel microspheres of microorganisms are supported and fixed by the branch nails in the holes of the exposure disc to provide a contact environment for the gaseous pollutants and the hydrogel microspheres of microorganisms. By using the device, the sensitivity of the acute toxicity (IC50) of microorganisms to the same kind of pollutants is improved by 3-5 orders of magnitude. The gas flow, flow rate and gaseous pollutant exposure amount in the multiple holes are uniform and stable, and the toxicity deviation of the hydrogel microspheres of microorganisms in each hole does not exceed 5%.

Description

Technical Field

[0001] This invention belongs to the field of environmental protection technology, specifically relating to a device for exposing microorganisms to gaseous pollutants. Background Technology

[0002] The health risks posed by air pollution remain one of the most pressing environmental issues globally. In 2021, the World Health Organization released its latest Global Air Quality Guidelines (AQG 2021), emphasizing the significant link between complex air pollution problems and a range of non-communicable diseases. These diseases include, but are not limited to, chronic respiratory diseases, neurological diseases, cardiovascular diseases, and immune system disorders. Therefore, it is imperative to rapidly and economically understand the potential toxicity of the air in our living environment.

[0003] Currently, most studies on the toxic effects of direct exposure to gaseous pollutants use mice as models. This assessment method is time-consuming and relies on complex and expensive analyses, such as metagenomics and metabolomics. Furthermore, it is difficult to eliminate the influence of individual mouse variability on the results. Some researchers have also used 3D cell model systems to study the toxic effects of gaseous pollutants. However, this technique still has several problems, including high cost, complex operation, strict exposure conditions, and unstable toxicity feedback. Therefore, the detection of the toxic effects of gaseous pollutants is currently still in the laboratory research stage and cannot be applied to health-effect-based air quality monitoring and commercial detection. However, using certain microorganisms with natural toxicity sensitivity, or recombinant microorganisms with constructed gene pathways for specific toxicity responses, offers advantages in toxicity detection, including low cost, rapid toxicity response, and stable toxicity feedback. But because these microorganisms cannot be directly exposed to air, they are not currently used in the toxicity detection of gaseous pollutants. Summary of the Invention

[0004] To address the aforementioned technological gaps, the present invention aims to provide a device for the exposure of microorganisms to gaseous pollutants, thereby enabling low-cost, rapid, and high-throughput detection of the toxicity of gaseous pollutants.

[0005] The device for exposing microorganisms to gaseous pollutants provided by this invention comprises: two depressurized inlet pipes, a T-shaped gas mixing pipe, a porous exposure plate, and a pressurized outlet pipe; wherein:

[0006] The two pressure-reducing inlet pipes are used to introduce gaseous pollutants. They are horn-shaped with the small end being the air inlet and the large end being threaded. The diameter of the large end of the two pressure-reducing inlet pipes matches the diameter of the left and right ends of the horizontal pipe in the T-shaped gas mixing pipe. The large ends of the two pressure-reducing inlet pipes are respectively connected to the left and right ends of the T-shaped gas mixing pipe through threads.

[0007] The exposure tray, located in the vertical section of a T-shaped gas mixing tube, is a disc with a plurality of uniformly distributed channels. Each channel contains four trapezoidal supports to support hydrogel microspheres (also known as microbial hydrogel microspheres) immobilized with microorganisms, suspending the microspheres within the channels. These channels provide a contact environment for gaseous pollutants with the hydrogel microspheres. Specifically, gaseous pollutants continuously flow in through two depressurized air inlets, entering the channels of the exposure tray and uniformly sweeping across the surface of the hydrogel microspheres. This causes the pollutants to adhere to, accumulate, and diffuse inwards on the surface of the microspheres, allowing the microorganisms immobilized within the microspheres to come into contact with the gaseous pollutants. Different types of hydrogel microspheres immobilized with microorganisms can be placed in the channels to form an array for simultaneous detection of multiple toxicity indicators. The vertical section of the T-shaped gas mixing tube and the corresponding exposure tray constitute an exposure chamber.

[0008] In this invention, a horn-shaped pressure-reducing inlet pipe is used, which can reduce the airflow velocity and pressure while keeping the flow rate constant, thereby assisting the T-shaped gas mixing pipe in evenly distributing the airflow throughout the entire pipe. Furthermore, after the gas enters from both ends, it collides with each other in the middle of the T-shaped gas mixing pipe to form turbulence and change the airflow direction, ensuring that the gas velocity and pressure are the same at any position on any cross section of the vertical pipe section of the T-shaped gas mixing pipe, thereby ensuring that the gas velocity and pressure through each hole on the porous exposure plate are consistent.

[0009] The pressurized outlet pipe is used to discharge gaseous pollutants. Its structure is also trumpet-shaped, and the diameter of its large end matches the diameter of the vertical section of the T-shaped gas mixing pipe. The two are connected by threads. The pressurized outlet pipe has the opposite function to the depressurized inlet pipe. The diameter of the pressurized outlet pipe at the end of the vertical section of the T-shaped gas mixing pipe gradually decreases from large to small. In addition to ensuring that the pipe diameter is consistent with the pipe diameter of other equipment, a certain pressure is formed at the outlet to ensure the uniformity and stability of the airflow at the lower end of the exposed plate.

[0010] Furthermore, to ensure a secure connection between the two pressure-reducing inlet pipes and the two ends of the T-shaped gas mixing pipe, the connection parts of the two pressure-reducing inlet pipes are designed with positive and negative threads, respectively.

[0011] Furthermore, the number of channels can be 10-30, and the channel diameter is 6-10 mm. The diameter of the microbial hydrogel microspheres is smaller than the channel diameter, for example, 4-8 mm.

[0012] Furthermore, there can be multiple exposure trays, threadedly connected vertically. These trays can be interconnected to form a multi-layered exposure array, which can multiply the number of hydrogel balls placed, thereby achieving high-throughput gaseous microbial exposure. The specific number of these trays can be determined according to actual needs, such as 2-4.

[0013] In an embodiment of the present invention, the diameter of the horizontal end of the T-shaped gas mixing pipe is 30 mm; the small end diameter of the inlet of the horn-shaped depressurizing inlet pipe is 6 mm, which is gradually enlarged to a large end diameter of 30 mm; the vertical section diameter of the T-shaped gas mixing pipe is 30 mm; and the large end diameter of the horn-shaped pressurizing inlet pipe is 30 mm, which is gradually reduced to a diameter of 6 mm.

[0014] In an embodiment of the present invention, there are 17 pores with a diameter of 8 mm. The diameter of the microbial hydrogel microspheres is 6 mm.

[0015] The main components of the device designed in this invention can be made of stainless steel that has undergone mirror polishing.

[0016] The method of using the device provided by this invention includes the following specific steps:

[0017] (1) Preparation of microbial hydrogel microspheres: Microorganisms were cultured in a medium containing 1.5% sodium alginate for 3-4 hours until the OD600 was greater than 0.1. Then, the microorganisms were uniformly added to a 1.5% calcium chloride solution at a rate of 1.5 ml / min using a peristaltic pump to obtain microbial hydrogel microspheres. The microorganisms were then placed in a culture medium for later use.

[0018] (2) According to the detection requirements, hydrogel microspheres immobilized with different types of microorganisms are sequentially placed in a porous exposure tray marked with channel numbers;

[0019] (3) Connect the porous exposure plate to the lower end of the vertical section of the T-shaped gas mixing pipe via threads, and connect it to the pressurized gas outlet pipe. Install silicone sealing rings at all threaded connections to ensure the airtightness of the entire exposure chamber;

[0020] (4) Install the exposure chamber into a high-throughput VOCs in-situ exposure toxicity detection system or a high-throughput atmospheric particulate matter in-situ exposure toxicity detection system. Gaseous pollutants enter the exposure chamber from the depressurization inlet pipes at both ends at a flow rate of 0.3-2.0 L / min.

[0021] (5) Continuously introduce gaseous pollutants for 15-60 minutes, then introduce clean air for 8-10 minutes to completely remove residual gaseous pollutants from the system; remove the multi-well exposure plate, transfer the hydrogel microbeads into the 96-well plate in sequence, and use an enzyme-linked immunosorbent assay (ELISA) reader to read the changes in the fluorescence pattern of the microbial hydrogel microbeads in each well.

[0022] Both the depressurization inlet pipe and the increase inlet pipe of the present invention are connected to other components of the high-throughput VOCs in-situ exposure toxicity detection system or the high-throughput atmospheric particulate matter in-situ exposure toxicity detection system via 10mm metal pipes.

[0023] In this invention, the flow rate of gaseous pollutants is preferably 0.5 L / min and should not exceed 2.0 L / min, so as to avoid the microbial hydrogel spheres losing water rapidly under the purging of high-velocity airflow, which would lead to the death of fixed microorganisms.

[0024] In this invention, the minimum exposure time for gaseous pollutants should not be less than 15 minutes, and the maximum exposure time should not exceed 60 minutes, preferably 20-30 minutes.

[0025] The beneficial effects of this invention are as follows:

[0026] This invention presents a device for exposing microorganisms to gaseous pollutants. Microorganisms with natural susceptibility or recombinant microorganisms possessing specific toxicity response gene pathways are rapidly immobilized into hydrogel microspheres using calcium alginate hydrogel. These microspheres are then assembled into a porous exposure tray within an exposure chamber, where they remain semi-suspended within the sealed pores. After assembly, gaseous pollutants are uniformly introduced into each pore of the porous exposure tray, continuously purging the microspheres. The pollutants adhere to and accumulate on the surface of the microspheres, diffusing inwards, thus achieving simultaneous gaseous exposure of different types of microorganisms. Compared to traditional liquid exposure toxicity detection methods, the enhanced effect of the exposure chamber increases the acute toxicity (IC50) sensitivity of microorganisms to the same pollutant by 3-5 orders of magnitude. The porous design and multi-layered exposure trays allow the exposure chamber to simultaneously assemble multiple hydrogel spheres immobilized with different microorganisms, enabling high-throughput simultaneous exposure of different microorganisms to the same air pollutant. During high-throughput simultaneous exposure, the gas flow rate, velocity, and gaseous pollutant exposure levels in each exposure panel and pore are uniform and stable. The toxicity deviation exhibited by the microbial hydrogel spheres in each exposure panel and pore does not exceed 5%, and changes in flow rate and gaseous pollutant concentration do not affect the system's stability. This makes it possible to monitor air quality through in-situ gaseous toxicity responses and can be widely applied to environmental risk assessments based on health effects. Attached Figure Description

[0027] Figure 1 This is a structural diagram of the microbial air pollutant exposure device of the present invention.

[0028] Figure 2 These are structural diagrams of a porous exposure plate in a microbial air pollutant exposure device; (a) is a schematic diagram of the overall structure, (b) is a front view of the exposure plate, and (c) is a top view of the exposure plate.

[0029] The following labels are used in the diagram: 1 is a pressure-reducing inlet pipe, 2 is another pressure-reducing inlet pipe, 3 is a T-shaped gas mixing pipe, 4 is a porous exposed plate, 5 is a trapezoidal support, and 6 is an internally threaded pressurized outlet pipe. Detailed Implementation

[0030] Seven different VOCs with nine concentration gradients were precisely generated using a commercial VOCs generator, involving aldehydes (formaldehyde and acetaldehyde), pyridines (pyridine), alcohols (n-butanol), acyls and ketones (acetylacetone), benzenes, and phenols (phenol). Using Vibrio fischeri, a naturally luminescent bacterium, as the immobilization target, controlled self-assembly microbial gas-liquid interface exposure passive colonization hydrogel microspheres were used for immobilization and assembled into an exposure chamber. VOCs were sequentially introduced into the gaseous exposure chamber at a flow rate of 0.5 L / min. After exposure for 15 min, 30 min, 45 min, and 60 min, the microspheres were removed, and the degree of luminescence intensity reduction was measured using a microplate reader. Analysis software was used to fit complete gaseous pollutant exposure concentration-acute toxicity curves for different VOCs and mixed VOCs, obtaining the acute toxicity IC50 values ​​of the gaseous pollutants. Following the above procedures and operations, acute toxicity detection of VOCs was achieved. Compared with corresponding acute toxicity exposure experiments on liquid VOCs, Vibrio fischeri showed a 565.2-14261.5-fold increase in response to gaseous pollutants during gaseous exposure. The most significant increases were observed in common VOCs formaldehyde and phenol, with formaldehyde having a detection limit of 1.3 mg / m³. 3 The limit of detection for phenol is 20.31 mg / m³. 3 The limit of detection for mixed pollutants is 13.21 mg / m³. 3 The levels are close to the upper limit of air quality standards. In actual air toxicity testing of gaseous pollutants, the use of a fully automated atmospheric pre-concentrator can easily achieve the detection of trace amounts of gaseous pollutants. See Table 1 for details.

[0031] Table 1 shows the differences in acute toxicity sensitivity of six VOCs in gaseous and liquid states.

[0032]

Claims

1. A device for exposing microorganisms to gaseous pollutants, characterized in that, include: Two pressure-reducing inlet pipes, one T-shaped gas mixing pipe, one porous exposure plate, and one pressurized outlet pipe; wherein: The two pressure-reducing inlet pipes are used to introduce gaseous pollutants. They are horn-shaped with the small end being the air inlet and the large end being threaded. The diameter of the large end of the two pressure-reducing inlet pipes matches the diameter of the left and right ends of the horizontal pipe in the T-shaped gas mixing pipe. The large ends of the two pressure-reducing inlet pipes are respectively connected to the left and right ends of the T-shaped gas mixing pipe through threads. The exposure tray, located in the vertical section of a T-shaped gas mixing tube, is a disc with a number of evenly distributed channels. Each channel contains four trapezoidal supports to support hydrogel microspheres immobilized with microorganisms, suspending the microspheres within the channels. These channels provide a contact environment for gaseous pollutants with the hydrogel microspheres. Specifically, gaseous pollutants continuously flow in through two depressurized air inlets, entering the channels of the exposure tray and evenly sweeping across the surface of the hydrogel microspheres. This causes the pollutants to adhere to and accumulate on the surface of the microspheres, diffusing inwards, allowing the microorganisms immobilized within the microspheres to come into contact with the gaseous pollutants. Hydrogel microspheres immobilizing different types of microorganisms are placed in the channels, forming an array for simultaneous detection of multiple toxicity indicators. The vertical section of the T-shaped gas mixing tube and the corresponding exposure tray constitute an exposure chamber. A horn-shaped pressure-reducing inlet pipe is used to ensure that the airflow velocity and pressure are reduced while the flow rate remains constant, so as to assist the T-shaped gas mixing pipe in evenly distributing the airflow throughout the entire pipe. Furthermore, after the gas enters from both ends, it collides with each other in the middle of the T-shaped gas mixing pipe to form turbulence and change the airflow direction, ensuring that the gas velocity and pressure are the same at any position on any cross section of the vertical pipe section of the T-shaped gas mixing pipe, thereby ensuring that the gas velocity and pressure are consistent through each hole on the porous exposure plate. The pressurized outlet pipe is used to discharge gaseous pollutants. Its structure is also trumpet-shaped, and the diameter of its large end matches the diameter of the vertical section of the T-shaped gas mixing pipe. The two are connected by threads. The pressurized outlet pipe has the opposite function to the depressurized inlet pipe. The pressurized outlet pipe at the end of the vertical section of the T-shaped gas mixing pipe, in addition to ensuring that the pipe diameter is the same as that of other equipment, forms pressure at the outlet to ensure the uniformity and stability of the airflow at the lower end of the exposed plate. The diameter of the horizontal pipe at both ends of the T-shaped gas mixing pipe is 30 mm; the diameter of the small end of the inlet of the horn-shaped depressurizing inlet pipe is 6 mm, which gradually increases to the large end diameter of 30 mm; the diameter of the vertical section of the T-shaped gas mixing pipe is 30 mm; the diameter of the large end of the horn-shaped pressurizing inlet pipe is 30 mm, which gradually decreases to the diameter of 6 mm.

2. The apparatus according to claim 1, characterized in that, The two pressure-reducing inlet pipes and the horizontal pipe of the T-shaped gas mixing pipe are designed with positive and negative threads at their respective ends.

3. The apparatus according to claim 1, characterized in that, The number of channels is 10-30, and the channel diameter is 6-10 mm; the diameter of the hydrogel microspheres is smaller than the channel diameter, which is 4-8 mm.

4. The apparatus according to claim 1, characterized in that, The exposure chambers are multiple and connected by threads in sequence. The multiple exposure chambers are interconnected to form a multi-layer exposure assembly, which increases the number of hydrogel microspheres placed and achieves high-throughput gaseous microbial exposure.

5. A method of using the apparatus for exposing microorganisms to gaseous pollutants as described in any one of claims 1-4, characterized in that, Using the naturally luminescent bacterium *Vibrio fischeri* as the immobilization target, controlled self-assembly microbial gas-liquid interface exposure passive colonization hydrogel microspheres were used for immobilization and assembled into an exposure chamber; the specific steps are as follows: (1) Preparation of hydrogel microspheres immobilized with microorganisms; (2) According to the detection requirements, hydrogel microspheres immobilized with different types of microorganisms are sequentially placed in a porous exposure tray marked with channel numbers; (3) Connect the porous exposure plate to the lower end of the vertical section of the T-shaped gas mixing pipe by thread, and connect it to the pressurized gas outlet pipe; install silicone sealing rings at all threaded connections to ensure the airtightness of the entire exposure chamber; (4) Install the exposure chamber into a high-throughput VOCs in-situ exposure toxicity detection system or a high-throughput atmospheric particulate matter in-situ exposure toxicity detection system. Gaseous pollutants enter the exposure chamber from the depressurization inlet pipes at both ends at a flow rate of 0.3-2.0 L / min. (5) Continuously introduce gaseous pollutants for 15-60 min, then introduce clean air for 8-10 min to completely remove residual gaseous pollutants from the system; Remove the multi-well exposure plate and transfer the hydrogel microspheres sequentially into the 96-well plate. Use a microplate reader to read the changes in fluorescence signal of the microbial hydrogel microspheres in each well.

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