Rapid determination method for adhesive power of aerogenic microorganism aggregate

Abstract

The invention relates to the field of microbial aggregate detection, in particular to a method for rapidly measuring the adhesive force of aerogenic microbial aggregates, which comprises the following steps: sample preparation: taking a proper amount of microbial aggregates to be measured, and cleaning to remove suspended solids; meanwhile, taking inlet water of the reaction device, and filtering to remove suspended matters in the inlet water; sample culture: feeding water into the microbial aggregate without the suspended matters and the reaction device, and performing sample culture; observing and imaging through a microscope: observing the bubble separation condition of the cultured sample microorganism aggregate under the microscope, collecting a bubble image through a CCD camera, and obtaining the bubble particle size in the bubble image through image processing software; and adhesive force calculation: calculating the maximum adhesive force between the microbial aggregate and the bubbles according to the critical value of the diameter of the maximum bubble staying on the microbial aggregate. According to the invention, the adhesive force between the bubbles and the aggregate can be conveniently and accurately evaluated, and the measured adhesive force is close to a real environment value.

Description

Technical Field

[0001] The invention relates to the field of microbial aggregate detection, and in particular to a method for quickly determining the adhesion of gas-producing microbial aggregates. Background Art

[0002] Gas-producing microbial aggregates play a key role in sewage treatment, bioenergy production and bioremediation, including anaerobic ammonia oxidation, anaerobic methanogenesis, anaerobic hydrogen production, algal granular sludge, biofilm, etc. Gas-producing microbial aggregates enhance the ecological function and environmental adaptability of microorganisms by forming a complex community structure, which is beneficial to improving sewage treatment efficiency and biogas production. In addition, since gas-producing microbial aggregates have diverse metabolic pathways, exploring their behavior is also helpful for food fermentation and environmental biodegradation. Therefore, the research and application of gas-producing microbial aggregates has important scientific significance and broad development prospects.

[0003] In the fields of sewage treatment and bioenergy production, when the bubbles produced by gas-producing microbial aggregates detach from the aggregate surface, their adhesion will affect the mass transfer efficiency, gas production and gas production rate, thereby affecting the stable operation of the reactor. Therefore, analyzing the interaction between the gas phase and the solid phase, especially the interaction between a single bubble and the aggregate, is of great significance for guiding the optimization of the aggregate surface properties, increasing gas production and adjusting the reactor.

[0004] Currently in sewage treatment research, impact jet technology and CFD technology can both describe the fluid dynamics in the reactor, but it is still difficult to directly observe the interaction force between bubbles and aggregates at the single particle level. Some studies have also used bubble probe atomic force microscopy technology with different approach speeds to measure the interaction between bubbles and aggregates, but this method is complicated to operate and requires high experimental accuracy. To evaluate the adhesion between bubbles and aggregates, it is necessary to explore simpler and faster methods. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for quickly determining the adhesion of gas-producing microbial aggregates, which is used to conveniently and accurately evaluate the adhesion between bubbles and aggregates.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows: A method for rapidly determining the adhesion of gas-producing microbial aggregates comprises the following steps:

[0007] Sample preparation: Take an appropriate amount of microbial aggregates to be measured and wash them to remove suspended solids; at the same time, take the influent water of the reaction device and filter to remove suspended solids in the influent water;

[0008] Sample culture: the microbial aggregates and the reaction device from which the suspended matter has been removed are filled with water for sample culture;

[0009] Microscope observation and imaging: observe the bubble detachment of the cultured sample microbial aggregates under a microscope, collect bubble images with a CCD camera, and obtain the bubble particle size in the bubble image with image processing software;

[0010] Adhesion force calculation: The maximum adhesion force between microbial aggregates and bubbles is calculated based on the critical value of the maximum bubble diameter that stays on the microbial aggregates.

[0011] Furthermore, the washing of the microbial aggregates was performed using a sterile 1× PBS buffer, and the microbial aggregates were rinsed more than three times using a sterile 1× PBS buffer;

[0012] Filtration to remove suspended matter from the influent water is performed through a 0.1-1 μm filter membrane.

[0013] Furthermore, the filter membrane is made of cellulose acetate membrane, polyvinylidene fluoride membrane, polyethersulfone membrane, mixed cellulose ester microporous membrane or nylon membrane.

[0014] Furthermore, the sample culture includes: transferring the microbial aggregates and the reaction device after the water inflow into a well plate made of virgin polystyrene or polypropylene material, and carrying out sample culture in a closed reaction tank.

[0015] Furthermore, the reaction tank was sealed with a plastic wrap and statically cultured at 25-37° C. for 15 min-1 h.

[0016] Furthermore, the microscope is a common optical microscope or a fluorescence microscope.

[0017] Furthermore, the CCD camera is a high-sensitivity and high-shutter-speed camera, ensuring that there is no smear of bubbles in the bubble image, and the acquisition frame rate is 30-300fps.

[0018] Furthermore, the maximum adhesion force between microbial aggregates and bubbles was calculated using the following formula:

[0019] F a =(d 3 ρgπ) / 6

[0020] Where d is the maximum diameter of the bubble detaching from the microbial aggregate when the bubble stays on the surface above the center point of the microbial aggregate, ρ is the density of the liquid environment, that is, the water density of the reaction device, and g is a constant, which is the ratio of gravity to mass, and is taken as g = 9.8N / kg.

[0021] Furthermore, the step of determining the largest bubble includes: setting a movable light source outside the reaction device, and the CCD camera collects bubble images while the light source moves around a set circuit outside the reaction device; the CCD camera collects images of the bubbles to be tested when several groups of light sources are in different positions, and the contour of each bubble in the bubble image to be tested is identified, and the size value of each bubble in different bubble images to be tested is obtained based on the contour of the bubble, and the average size of each bubble in different bubble images to be tested is obtained, and the bubble with the largest average size is defined as the largest bubble.

[0022] Furthermore, the above-mentioned rapid determination method for the adhesion of gas-producing microbial aggregates is applicable to gas-producing microbial aggregates including anaerobic ammonia oxidation, anaerobic methanogenesis, anaerobic hydrogen production, bacterial and algal granular sludge and biofilm.

[0023] The above scheme has the following beneficial effects:

[0024] 1. The present invention is based on the analysis of the stress conditions of bubbles. Compared with the prior art, the measured index is only the bubble diameter, which can be obtained conveniently and efficiently by taking a camera microscope lens combined with image processing software. The measurement process does not contain chemical reactions, does not interfere with the bubbles and aggregates themselves and their adhesion, and the measured value is close to the real environment value.

[0025] 2. Compared with the prior art, the present invention provides a convenient and efficient measurement by using a camera, image processing software and a calculation formula when measuring the adhesion between bubbles and aggregates. The measurement operation is simple and the measurement efficiency is high.

[0026] 3. The present invention reflects the mass transfer efficiency, gas production and gas production rate of aggregates by measuring the adhesion between bubbles and aggregates, which is then used to guide the optimization of aggregate surface properties and reaction aggregate bed permeability, which is beneficial to increase gas production and adjust the stable reactor.

[0027] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of principle analysis of an embodiment of the present invention;

[0029] Figure 2 is a flow chart of a method according to an embodiment of the present invention;

[0030] Figure 3 A partial schematic diagram of bubbles produced by microbial aggregates collected in an embodiment of the present invention;

[0031] Figure 4Schematic diagram of the adhesion characteristics between bubbles and microbial aggregates in anaerobic ammonium oxidation granular sludge under different loads measured in an embodiment of the present invention;

[0032] Figure 5 Schematic diagram of the adhesion characteristics between methane bubbles and aggregates in anaerobic methanogenic granular sludge under different loads measured in an embodiment of the present invention. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The following is further described in detail through specific implementation methods:

[0035] Embodiment 1, as attached Figure 1 As shown: A method for rapidly determining the adhesion of gas-producing microbial aggregates, applicable gas-producing microbial aggregates mainly include anaerobic ammonia oxidation, anaerobic methanogenesis, anaerobic hydrogen production, bacterial algae granular sludge and biofilm, etc. It specifically includes the following steps:

[0036] S1. Sample preparation: Take an appropriate amount of microbial aggregates to be measured and wash them to remove suspended solids. The microbial aggregates are washed with sterile 1× PBS buffer by rinsing for more than three times.

[0037] At the same time, the influent water of the reaction device is taken and filtered to remove suspended matter in the influent water. In this embodiment, the filtration is performed through a 0.1-1 μm filter membrane, and specifically, the filter membrane material can be cellulose acetate membrane, polyvinylidene fluoride membrane, polyethersulfone filter membrane, mixed cellulose ester microporous filter membrane or nylon filter membrane.

[0038] S2. Sample culture: The microbial aggregates and the reaction device after the suspended matter is removed are introduced into water for sample culture. The sample culture of this embodiment is specifically as follows: the microbial aggregates and the reaction device after the water is introduced are transferred into a well plate made of virgin polystyrene or polypropylene material, sealed with a cling film as a closed reaction tank, and statically cultured at 25-37°C for 15min-1h.

[0039] S3. Microscope observation and imaging: observe the bubble detachment of the cultured sample microbial aggregates under a microscope; collect bubble images through a CCD camera, and obtain the bubble particle size in the bubble image through image processing software. The microscope can be an ordinary optical microscope or a fluorescence microscope, and the CCD camera is a high-sensitivity and high-shutter speed camera. It is necessary to ensure that there is no smear in the bubble image collected, and the acquisition frame rate can be set to 30-300fps.

[0040] S4. Calculation of adhesion force: The maximum adhesion force between microbial aggregates and bubbles is calculated based on the critical value of the maximum bubble diameter remaining on the microbial aggregates.

[0041] Specifically, in combination with Figure 2 As shown in the figure, the adhesion force between the bubbles produced by the microbial aggregates and the aggregates is calculated by analyzing the force of the bubbles. When the bubbles stay on the surface above the center of the aggregate, the buoyancy of the bubbles themselves Fb and the adhesion force between the aggregates F a Equal, that is, F b =F a Therefore, the maximum adhesion force between the aggregate and the bubble can be calculated based on the critical value of the maximum bubble diameter staying on the aggregate, that is, the adhesion force is equal to the buoyancy of the bubble. The bubble adhesion force calculation formula is as follows:

[0042] F a =F b =(d 3 ρgπ) / 6

[0043] Where d is the maximum diameter of the bubble detaching from the microbial aggregate when the bubble stays on the surface above the center point of the microbial aggregate, π is the density of the liquid environment, that is, the water density of the reaction device, and g is a constant, which is the ratio of gravity to mass, and is taken as g = 9.8N / kg.

[0044] Example 2, the determination method of Example 1 is used to carry out the following example to further illustrate it:

[0045] Example 1: Determination of the adhesion characteristics between bubbles and aggregates produced by anaerobic ammonium oxidation granular sludge in an expanded granular sludge bed reactor at different nitrogen loading rates.

[0046] (1) Preliminary preparation: Different nitrogen loading rates (R1-R6: 0.88, 1.32, 2.64, 3.96, 5.28 and 7.92 kg N / (m 3After the six groups of anaerobic ammonium oxidation expanded granular sludge bed reactors in d) were running stably, about 5g of wet granular sludge was taken from each group and the following operations were performed: the single sludge particles were washed three times with sterile 1×PBS buffer, and the influent of the reactor was filtered through a 0.1μm PVDF membrane syringe-driven filter, and then the aggregates with suspended solids removed were divided into 96-well plates, and then degassed artificial wastewater with suspended solids removed was added according to the sludge concentration of the original reactor, and the plates were sealed with a fresh-keeping film and incubated at 30°C for 1h. The total nitrogen concentration was controlled at 200-240mg / L, and NH 4 Cl and NaNO 2 The fixed molar ratio is 1:1.2, and three groups are set up in parallel.

[0047] (2) Bubble size determination: The single bubbles generated by the granular sludge were observed under an inverted fluorescence microscope (Nikon Ti-E, Nikon Instruments, Japan) equipped with a Nikon CFI Apochromat total internal reflection fluorescence (TIRF) 60× oil (1.49NA) objective lens, and a high-speed CCD camera (Pike F-032) was used to image the single bubbles. The image was imported into the image processing software imageJ to adjust its gray value and background value to obtain the following: Figure 3 The large number of bubble images shown are then processed by software to count their size changes.

[0048] (3) Calculation of adhesion: Substitute the obtained bubble diameter into the formula (d 3 The adhesion force is calculated by ρgπ) / 6 and plotted into a graph, which can more intuitively show the distribution and characteristics of the adhesion force between the bubbles and aggregates produced by the anaerobic ammonium oxidation granular sludge in the six groups of reactors.

[0049] (4) Result analysis: Figure 4 As shown in the figure, with the increase of nitrogen load, the adhesion between bubbles and aggregates gradually increased from 5.26 mN in R1 to 11.34 mN in R4, and then gradually decreased to 7.68 mN in R6. Under medium load, the adhesion between bubbles and aggregates is the largest, making it easier for bubbles to stay in the gaps between aggregates in the aggregate bed, which may also be an important reason why the average particle size of micron bubbles in the R4 reactor is larger than that in other reactors.

[0050] Example 2: The adhesion characteristics between methane bubbles and aggregates produced by anaerobic methanogenic granular sludge in an upflow anaerobic sludge blanket reactor under different organic loading rates were determined.

[0051] (1) Preliminary preparation: A laboratory-scale upflow anaerobic sludge blanket reactor was set up at an organic loading rate of 10 kg COD / (m 3d) and cultured under the conditions of 5% sludge and 1% PBS. After stable operation, the biomass in the reactor was reduced by discharging sludge to cause load shock. Then, the organic load rate was slowly increased by increasing the influent COD concentration. The adhesion force was measured every 5 days. About 5 g of wet granular sludge was taken each time and rinsed three times with sterile 1× PBS buffer. The influent of the corresponding reactor was filtered through a 0.1 μm PVDF membrane syringe-driven filter and then divided into orifice plates made of polypropylene (PP) material. After sealing with a fresh-keeping film, the plates were cultured at 37°C for 1 hour in three parallel groups.

[0052] (2) Bubble size determination: Observe under Fei F20 electron microscope (Tridiem Gatan 200kV), and use Gatan UltraScan 1000FT CCD camera to image the single bubbles generated by granular sludge. Import the image into imageJ image processing software to adjust its gray value and background value, and then process it with software to count the size changes.

[0053] (3) Calculation of adhesion: Substitute the maximum particle size of the bubbles obtained in each measurement into the formula (d 3 ρgπ) / 6. Since the difference between the three parallel groups is small, the average value can be directly taken to get the adhesion force, which is plotted into a graph to more intuitively reflect the change of the adhesion force between the methane bubbles and aggregates produced by the anaerobic methanogenic granular sludge in the reactor with the organic loading rate.

[0054] (4) Result analysis: Figure 5 As shown, before the reactor was impacted, the organic loading rate was 12 kg COD / (m 3 d), the adhesion force is the largest, reaching 10.2 mN. After the reactor is impacted, the adhesion force drops to 3.6 mN, and then increases with the increase of organic loading rate. Finally, when the organic loading rate increases to 11 kg COD / (m 3 d), the adhesion force reached 9.4 mN. Compared with Example 1, the adhesion characteristics between the methane bubbles produced by the anaerobic methanogenic granular sludge and the aggregates are generally smaller than the adhesion between the anaerobic ammonium oxidation granular sludge and the bubbles.

[0055] Example 3: The difference compared to Example 1 is that a method for determining the maximum bubble is provided, and the specific steps include: first, a movable light source is set outside the reaction device, and the CCD camera collects the bubble image while the light source moves around a set circuit outside the reaction device; the light source can be an LED lamp or a spotlight, and when it is located at different positions to irradiate the bubbles, the boundary contours of the bubbles in the bubble images collected by the CCD camera are different, which matches the dynamic situation after the bubble is generated, ensuring that the CCD camera can obtain the size of the bubble at multiple different angles at one angle, avoiding the situation where the obtained bubble size is single and the judgment of the maximum bubble is inaccurate.

[0056] Then, the CCD camera is used to collect several groups of bubble images to be tested when the light source is at different positions, and the contour of each bubble in the bubble image to be tested is identified. The size value of each bubble in different bubble images to be tested is obtained based on the bubble contour, and the size mean of each bubble in different bubble images to be tested is obtained, and the bubble with the largest size mean is defined as the largest bubble. When the light source is irradiated on the bubble from different angles, the bubble contours in the bubble image to be tested collected by the CCD camera are different due to the light source at different angles irradiating the bubble. The bubble contours are obtained from multiple angles to accurately reflect the real size of the bubble, and then the largest bubble is accurately obtained.

[0057] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for rapidly determining the adhesion of gas-producing microbial aggregates, characterized in that: The steps include: Sample preparation: Take an appropriate amount of microbial aggregates to be measured and wash them to remove suspended solids; at the same time, take the influent water of the reaction device and filter to remove suspended solids in the influent water; Sample culture: the microbial aggregates and the reaction device from which the suspended matter has been removed are filled with water for sample culture; Microscope observation and imaging: observe the bubble detachment of the cultured sample microbial aggregates under a microscope, collect bubble images with a CCD camera, and obtain the bubble particle size in the bubble image with image processing software; Adhesion force calculation: The maximum adhesion force between microbial aggregates and bubbles is calculated based on the critical value of the maximum bubble diameter that stays on the microbial aggregates.

2. The method for rapid determination of adhesion of gas-producing microbial aggregates according to claim 1, characterized in that: The washing of microbial aggregates was carried out with sterile 1× PBS buffer, and the microbial aggregates were rinsed more than three times with sterile 1× PBS buffer; Filtration to remove suspended matter from the influent water is performed through a 0.1-1 μm filter membrane.

3. The method for rapidly determining the adhesion of gas-producing microbial aggregates according to claim 2, characterized in that: The filter membrane material is cellulose acetate membrane, polyvinylidene fluoride membrane, polyethersulfone membrane, mixed cellulose ester microporous membrane or nylon membrane.

4. The method for rapidly determining the adhesion of gas-producing microbial aggregates according to claim 1, characterized in that: The sample culture includes: transferring the microbial aggregates and the reaction device after entering the water into the orifice plate made of virgin polystyrene or polypropylene material, and carrying out sample culture in the closed reaction tank.

5. The method for rapid determination of adhesion of gas-producing microbial aggregates according to claim 4, characterized in that: Seal with plastic wrap as a closed reaction tank and culture statically at 25-37°C for 15min-1h.

6. The method for rapid determination of adhesion of gas-producing microbial aggregates according to claim 1, characterized in that: The microscope is a common optical microscope or a fluorescence microscope.

7. The method for rapid determination of adhesion of gas-producing microbial aggregates according to claim 6, characterized in that: The CCD camera is a high-sensitivity and high-shutter-speed camera, ensuring that there is no smear of bubbles in the bubble image, and the acquisition frame rate is 30-300fps.

8. The method for rapid determination of adhesion of gas-producing microbial aggregates according to claim 7, characterized in that: The maximum adhesion force between microbial aggregates and bubbles was calculated using the following formula: F a =(d 3 ρgπ) / 6 Where d is the maximum diameter of the bubble detaching from the microbial aggregate when the bubble stays on the surface above the center point of the microbial aggregate, ρ is the density of the liquid environment, that is, the water density of the reaction device, and g is a constant, which is the ratio of gravity to mass, and is taken as g = 9.8N / kg.

9. The method for rapid determination of adhesion of gas-producing microbial aggregates according to claim 1, characterized in that: The steps for determining the largest bubble include: setting a movable light source outside the reaction device, collecting bubble images with a CCD camera while the light source moves around a set route outside the reaction device; the CCD camera collects images of the bubbles to be tested when several groups of light sources are at different positions, identifying the contours of each bubble in the bubble images to be tested, obtaining the size value of each bubble in different bubble images to be tested based on the contours of the bubbles, obtaining the average size of each bubble in different bubble images to be tested, and defining the bubble with the largest average size as the largest bubble.

10. The method for rapidly determining the adhesion of gas-producing microbial aggregates according to any one of claims 1 to 9, characterized in that: Applicable gas-producing microbial aggregates include anaerobic ammonia oxidation, anaerobic methanogenesis, anaerobic hydrogen production, bacterial-algal granular sludge and biofilm.

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