Artificial biofilm on-line monitoring method based on synchrotron radiation technology

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to an artificial biofilm on-line monitoring method based on a synchrotron radiation technology, which comprises the following steps: coating a liquid artificial biofilm non-crosslinking solution on a nickel net, and carrying out chemical crosslinking curing or photo-crosslinking curing to obtain an artificial biofilm sample; rotating the artificial biofilm sample, performing multi-angle shooting on the artificial biofilm sample by using soft X-rays generated by a synchrotron radiation light source in the process, and collecting a series of slice images; and reconstructing the series of collected slice images through Avizo software, and independently marking and imaging bacteria in the images to obtain a three-dimensional picture of the artificial biofilm sample. By adopting the method provided by the invention for online monitoring of the artificial biofilm, nondestructive imaging of the artificial biofilm can be realized, and information such as growth state and distribution condition of loaded bacteria can be obtained.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and in particular relates to an online monitoring method for artificial biofilm based on synchrotron radiation technology. Background Art

[0002] The activated sludge method is the mainstream process for wastewater treatment throughout the world, including my country. As a multicellular microbial community, naturally domesticated activated sludge has problems such as poor adaptability and high sludge production. Especially in the face of new and difficult-to-degrade pollutants in wastewater, such as antibiotics, microplastics, flame retardants, surfactants, and persistent organic pollutants, there are problems such as low degradation efficiency and slow speed. Innovation in wastewater treatment technology is an urgent need for the country.

[0003] The use of synthetic biology technology to develop composite artificial bacterial communities and construct artificial biofilms with strong adaptability, high degradation efficiency, fast speed and low sludge production is considered to be an effective way to solve the above-mentioned problems. However, there is currently a lack of online monitoring methods for artificial biofilms, and it is difficult to understand the growth status and distribution of bacteria loaded on the biofilms without loss of quality. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an online monitoring method for artificial biofilms based on synchrotron radiation technology. The online monitoring of artificial biofilms using the method provided by the present invention can achieve non-destructive imaging and obtain information such as the growth status and distribution of loaded bacteria.

[0005] The present invention provides an online monitoring method for artificial biofilm based on synchrotron radiation technology, comprising the following steps:

[0006] coating a liquid artificial biofilm uncrosslinked solution onto a nickel mesh, and performing chemical crosslinking or photocrosslinking curing to obtain an artificial biofilm sample;

[0007] The artificial biofilm sample is rotated, and during this process, the artificial biofilm sample is photographed at multiple angles using soft X-rays generated by a synchrotron radiation source to collect a series of slice images;

[0008] The series of slice images collected are reconstructed by Avizo software, and the bacteria in the images are individually marked and imaged to obtain a three-dimensional picture of the artificial biofilm sample.

[0009] Preferably, the nickel mesh is 70-150 meshes.

[0010] Preferably, the uncrosslinked solution of the liquid artificial biomembrane corresponding to the chemical crosslinking solidification contains sodium alginate, cellulose, acetylene black and bacteria;

[0011] The chemical cross-linking and curing method is: adding a calcium chloride solution dropwise onto a nickel mesh coated with a liquid artificial biofilm uncross-linked solution.

[0012] Preferably, the concentration of the calcium chloride solution is 0.2 mol / L to saturation concentration.

[0013] Preferably, the uncrosslinked solution of the liquid artificial biomembrane corresponding to the photo-crosslinking curing contains methacrylated gelatin, a blue light photoinitiator and bacteria;

[0014] The photo-crosslinking and curing method is: irradiating the nickel mesh coated with the liquid artificial biofilm uncrosslinked solution with blue light.

[0015] Preferably, the blue light photoinitiator is phenyl 2,4,6-trimethylbenzoyl lithium phosphate.

[0016] Preferably, the wavelength of the blue light source is 405nm and the light intensity is 25mW / cm 2 .

[0017] Preferably, the energy of the soft X-ray is 284-530 eV.

[0018] Preferably, the exposure time of the shooting is ≤2s.

[0019] Preferably, the rotation angle is from -52° to +52°, and a series of slice images taken at intervals of 1° are collected during the rotation.

[0020] Compared with the prior art, the present invention provides an online monitoring method for artificial biofilm based on synchrotron radiation technology, comprising the following steps: coating a liquid artificial biofilm uncrosslinked solution on a nickel mesh, performing chemical crosslinking or photocrosslinking to obtain an artificial biofilm sample; rotating the artificial biofilm sample, during which the artificial biofilm sample is photographed from multiple angles using soft X-rays generated by a synchrotron radiation light source, and a series of slice images are collected; reconstructing the collected series of slice images using Avizo software, and marking and imaging the bacteria in the image separately, to obtain a three-dimensional picture of the artificial biofilm sample. The technical solution provided by the present invention can directly realize high-contrast non-destructive three-dimensional imaging of artificial organisms with the help of soft X-rays generated by a synchrotron radiation light source, based on which an instantaneous three-dimensional structural slice of the artificial biofilm can be given, and the slice reconstruction is performed using the integrated image analysis software Avizo, which can realize online monitoring of its growth status and bacterial distribution information, and further promote the application of artificial biofilm in activated sludge enhancement. Compared with scanning electron microscopy, transmission electron microscopy and other characterization methods, this method does not require complicated pre-treatment steps such as immobilization and gradient dehydration, and can minimize the damage to the artificial biofilm to be tested. The experimental results show that the method provided by the present invention does not require complicated sample preparation. After loading and cross-linking, the three-dimensional slices of the artificial biofilm at various angles can be photographed by rotating the sample, and after reconstruction, the instantaneous bacterial community distribution and growth status of the artificial biofilm can be obtained without damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0022] Figure 1 This is a physical picture of the liquid artificial biomembrane provided in Example 1 of the present invention;

[0023] Figure 2 This is a physical picture of the artificial biomembrane after cross-linking provided in Example 2 of the present invention;

[0024] Figure 3 This is a schematic diagram of sample loading provided in Example 2 of the present invention;

[0025] Figure 4 It is one of the acquired image slices provided in Embodiment 2 of the present invention;

[0026] Figure 5 This is the artificial biomembrane reconstruction diagram provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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.

[0028] The present invention provides an online monitoring method for artificial biofilm based on synchrotron radiation technology, comprising the following steps:

[0029] coating a liquid artificial biofilm uncrosslinked solution onto a nickel mesh, and performing chemical crosslinking or photocrosslinking curing to obtain an artificial biofilm sample;

[0030] The artificial biofilm sample is rotated, and during this process, the artificial biofilm sample is photographed at multiple angles using soft X-rays generated by a synchrotron radiation source to collect a series of slice images;

[0031] The series of slice images collected are reconstructed by Avizo software, and the bacteria in the images are individually marked and imaged to obtain a three-dimensional picture of the artificial biofilm sample.

[0032] In the online monitoring method provided by the present invention, the nickel mesh is preferably 70-150 mesh, specifically 70 mesh, 80 mesh, 90 mesh, 100 mesh, 110 mesh, 120 mesh, 130 mesh, 140 mesh or 150 mesh.

[0033] In the online monitoring method provided by the present invention, the OD of the uncrosslinked solution of the artificial biofilm is 600 It is preferably 1 to 5, and more preferably 3.

[0034] In the online monitoring method provided by the present invention, the liquid artificial biomembrane uncross-linked solution corresponding to the chemical cross-linking curing preferably contains sodium alginate, cellulose, acetylene black and bacteria; wherein the bacteria include but are not limited to Shewanella oneidensis MR-1; the concentration of sodium alginate in the liquid artificial biomembrane uncross-linked solution is preferably 5-15% (w / v), specifically 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), 10% (w / v), 11% (w / v), 12% (w / v), 13% (w / v), 14% (w / v) or 15% (w / v); the concentration of cellulose in the liquid artificial biomembrane uncross-linked solution is preferably 10-20% (w / v), specifically 10% (w / v), 11% (w / v), 12% (w / v), 13% (w / v), 14% (w / v), 15% (w / v), 16% (w / v), 17% (w / v), 18% (w / v), 19% (w / v) or 20% (w / v); the concentration of acetylene black in the liquid artificial biofilm uncross-linked solution is preferably 2-8% (w / v), specifically 2% (w / v), 2.5% (w / v), 3% (w / v), 3.5% (w / v), 4% (w / v), 4.5% (w / v), 5% (w / v), 5.5% (w / v), 6% (w / v), 6.5% (w / v), 7% (w / v), 7.5% (w / v) or 8% (w / v). In the present invention, the preparation process of the liquid artificial biomembrane uncrosslinked solution corresponding to the chemical crosslinking curing preferably includes: uniformly mixing sodium alginate, cellulose, acetylene black and bacterial culture solution to obtain the liquid artificial biomembrane uncrosslinked solution.

[0035] In the online monitoring method provided by the present invention, the chemical cross-linking and curing method is preferably: adding a calcium chloride solution to a nickel mesh coated with a liquid artificial biofilm uncross-linked solution; wherein the concentration of the calcium chloride solution is preferably 0.2 mol / L to saturation concentration, more preferably 0.5 mol / L.

[0036] In the online monitoring method provided by the present invention, the liquid artificial biofilm uncrosslinked solution corresponding to the photo-crosslinking curing contains methacryloyl gelatin, a blue light photoinitiator and bacteria; wherein the blue light photoinitiator is preferably phenyl 2,4,6-trimethylbenzoyl lithium phosphate (LAP); the bacteria include but are not limited to Shewanella oneidensis MR-1; the concentration of the methacryloyl gelatin in the liquid artificial biofilm uncrosslinked solution is preferably 2-15% (w / v), specifically 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), 10% (w / v), 11% (w / v), 12% (w / v), 13% (w / v), 14% (w / v) or 15% (w / v). 5% (w / v); the concentration of the blue light photoinitiator in the liquid artificial biomembrane uncrosslinked solution is preferably 0.05-0.5% (w / v), specifically 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), 0.25% (w / v), 0.3% (w / v), 0.35% (w / v), 0.4% (w / v), 0.45% (w / v) or 0.5% (w / v). In the present invention, the preparation process of the liquid artificial biomembrane uncrosslinked solution corresponding to the photocrosslinking curing preferably includes: mixing methacrylated gelatin, blue light photoinitiator and liquid culture medium, heating, cooling, and then mixing evenly with bacterial culture medium to obtain a liquid artificial biomembrane uncrosslinked solution; wherein the heating temperature is preferably 50-70°C, more preferably 60°C; the cooling temperature is preferably 35-40°C, more preferably 37°C.

[0037] In the online monitoring method provided by the present invention, the photo-crosslinking curing method is preferably: irradiating the nickel mesh coated with the liquid artificial biofilm uncrosslinked solution with blue light; wherein the blue light irradiation light source wavelength is 405nm, and the light intensity is 25mW / cm 2 .

[0038] In the online monitoring method provided by the present invention, the energy of the soft X-ray is preferably 284-530eV, specifically 284eV, 290eV, 300eV, 320eV, 350eV, 370eV, 400eV, 420eV, 450eV, 470eV, 500eV, 520eV or 530eV.

[0039] In the online monitoring method provided by the present invention, the exposure time of the shooting is preferably ≤2s, as a long exposure time may cause damage to the bacteria.

[0040] In the online monitoring method provided by the present invention, the rotation angle is preferably from -52° to +52°, and during the rotation process, a series of slice images taken at intervals of 1° are preferably collected.

[0041] In the online monitoring method provided by the present invention, the three-dimensional image of the artificial biofilm sample contains the required information such as the growth status of the artificial biofilm and the distribution of bacteria.

[0042] The technical solution provided by the present invention can directly realize high-contrast non-destructive three-dimensional imaging of artificial organisms with the help of soft X-rays generated by synchrotron radiation light sources. Based on this, the instantaneous three-dimensional structure slices of artificial biofilms can be given, and the slices can be reconstructed with the integrated image analysis software Avizo, which can realize online monitoring of its growth status and bacterial distribution information, and further promote the application of artificial biofilms in activated sludge enhancement. Compared with scanning electron microscopy, transmission electron microscopy and other characterization methods, this method does not require complex pre-treatment steps such as immobilization and gradient dehydration, and can minimize the damage to the artificial biofilm to be tested.

[0043] For the purpose of greater clarity, the invention is described in detail through the following embodiments.

[0044] Example 1

[0045] Mix 1.0 g of sodium alginate, 1.5 g of cellulose, and 400 mg of acetylene black in a centrifuge tube, and add 10 mL of OD 600 3.0 Shewanella oneidensis MR-1 culture fluid is stirred evenly to obtain a liquid artificial biomembrane solution that needs to be chemically cross-linked and formed. The sodium alginate concentration in the liquid artificial biomembrane solution is 10% (w / v), the acetylene black concentration is 4% (w / v), and the cellulose concentration is 15% (w / v).

[0046] 0.7 g of methacrylated gelatin was mixed with 10 mL of liquid culture medium containing 0.25% (w / v) blue light photoinitiator phenyl 2,4,6-trimethylbenzoyl phosphate lithium salt (LAP), and the mixture was placed in a 37°C water bath for 10 minutes after being heated at 60°C for 30 minutes. 10 mL of Shewanella oneidensis MR-1 culture medium was added to the mixture to make the OD 600 is 3.0, and shaken evenly to obtain a liquid artificial biofilm solution (such as Figure 1 As shown), the concentration of methacryloyl gelatin in the liquid artificial biomembrane solution is 7% (w / v), and the concentration of blue light photoinitiator is 0.25% (w / v).

[0047] Example 2

[0048] Use a 100 μL pipette to pipette a small amount of uncrosslinked artificial biofilm solution onto a 100-mesh nickel grid. After the grid is covered with the artificial biofilm solution, solidify the sample by chemical or photo-crosslinking. The chemical crosslinking method is to use a 0.5 mol / L calcium chloride solution to crosslink the artificial biofilm, and the photo-crosslinking method is to use a blue light source (wavelength 405 nm, light intensity 25 mW / cm 2 ) to irradiate the biofilm. Taking photo cross-linking as an example, the obtained artificial biofilm after immobilization is as follows Figure 2 shown.

[0049] Taking photo-crosslinking as an example, the cured artificial biofilm sample is placed in the sample chamber and observed under a microscope. Figure 3 As shown; the sample chamber then rotates from -52° to +52°; during the rotation, the artificial biofilm sample is photographed at multiple angles using soft X-rays (energy of 520 eV) generated by a synchrotron radiation source, and 105 slice images taken at 1° intervals are collected (one slice image is shown in FIG. Figure 4 The exposure time for each shot is 2 seconds.

[0050] The collected series of slice images are reconstructed by Avizo software, and the bacteria in the image are individually marked and imaged using the "OrthoSlice" function, and finally a three-dimensional image of the artificial biofilm sample is obtained, such as Figure 5 shown.

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An online monitoring method for artificial biofilm based on synchrotron radiation technology, characterized in that: The following steps are involved: coating a liquid artificial biofilm uncrosslinked solution onto a nickel mesh, and performing chemical crosslinking or photocrosslinking curing to obtain an artificial biofilm sample; The artificial biofilm sample is rotated, and during this process, the artificial biofilm sample is photographed at multiple angles using soft X-rays generated by a synchrotron radiation source to collect a series of slice images; The series of slice images collected are reconstructed by Avizo software, and the bacteria in the images are individually marked and imaged to obtain a three-dimensional picture of the artificial biofilm sample.

2. The method for online monitoring of artificial biofilm according to claim 1, characterized in that: The nickel mesh has a mesh size of 70 to 150.

3. The method for online monitoring of artificial biofilm according to claim 1, characterized in that: The uncrosslinked solution of the liquid artificial biomembrane corresponding to the chemical crosslinking curing contains sodium alginate, cellulose, acetylene black and bacteria; The chemical cross-linking and curing method is: adding a calcium chloride solution dropwise onto a nickel mesh coated with a liquid artificial biofilm uncross-linked solution.

4. The method for online monitoring of artificial biofilm according to claim 3, characterized in that: The concentration of the calcium chloride solution is 0.2 mol / L to saturation concentration.

5. The method for online monitoring of artificial biofilm according to claim 1, characterized in that: The liquid artificial biomembrane uncrosslinked solution corresponding to the photo-crosslinking curing contains methacrylated gelatin, blue light photoinitiator and bacteria; The photo-crosslinking and curing method is: irradiating the nickel mesh coated with the liquid artificial biofilm uncrosslinked solution with blue light.

6. The method for online monitoring of artificial biofilm according to claim 5, characterized in that: The blue light photoinitiator is phenyl 2,4,6-trimethylbenzoyl lithium phosphate.

7. The method for online monitoring of artificial biofilm according to claim 5, characterized in that: The blue light source has a wavelength of 405 nm and a light intensity of 25 mW / cm 2 .

8. The method for online monitoring of artificial biofilm according to claim 1, characterized in that: The energy of the soft X-ray is 284-530 eV. 9 . The method for online monitoring of artificial biofilm according to claim 1 , wherein the exposure time of the shooting is ≤ 2 s.

10. The method for online monitoring of artificial biofilm according to claim 1, characterized in that: The rotation angle is from -52° to +52°, and a series of slice images taken at intervals of 1° are collected during the rotation.

Images