Artificial biofilm on-line monitoring method based on X-ray CT technology

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to an artificial biofilm on-line monitoring method based on an X-ray CT (Computed Tomography) technology, which comprises the following steps: cutting and sampling an artificial biofilm, and then fixing a sampled sample on an objective table; rotating the objective table, and performing multi-angle tomography on the artificial biofilm sample on the objective table by using X rays in the process to obtain a series of tomographic images; and carrying out noise reduction, reconstruction and rendering on the series of cross-sectional images through Dragonfly Pro software to obtain a three-dimensional reconstructed X-ray CT image 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 X-ray CT technology. Background Art

[0002] The rapid development of synthetic biology has brought new breakthroughs in wastewater treatment technology. By designing and constructing composite artificial bacterial communities and artificial biofilms, the problems of poor adaptability and low degradation efficiency in the traditional activated sludge method can be effectively solved.

[0003] The design of artificial biofilms not only considers the diversity and functional optimization of microbial communities, but also aims to improve their ability to treat complex pollutants and reduce sludge production. However, despite the broad prospects of this technology, the current research on artificial biofilms still faces challenges, especially the lack of effective online monitoring methods, which cannot monitor the status of artificial biofilms in real time. To achieve a comprehensive understanding of the growth status, spatial distribution and interaction of microorganisms in the biofilm with the matrix, it is necessary to rely on advanced imaging and analysis technologies. 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 X-ray CT 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 X-ray CT technology, comprising the following steps:

[0006] The artificial biofilm is cut and sampled, and then the sample is fixed on a stage;

[0007] The stage is rotated, and during this process, the artificial biofilm sample on the stage is subjected to multi-angle tomographic scanning using X-rays to obtain a series of tomographic images;

[0008] A series of the tomographic images were subjected to noise reduction, reconstruction and rendering by using DragonflyPro software to obtain three-dimensional reconstructed X-ray CT images of the artificial biofilm samples.

[0009] Preferably, the steps of preparing the artificial biofilm include:

[0010] The uncrosslinked solution of the liquid artificial biomembrane is chemically crosslinked or photocrosslinked to obtain the artificial biomembrane.

[0011] Preferably, the uncrosslinked solution of the liquid artificial biomembrane corresponding to the chemical crosslinking solidification contains sodium alginate and bacteria;

[0012] The chemical cross-linking and curing method is: adding calcium chloride solution to the uncross-linked solution of the liquid artificial biomembrane.

[0013] Preferably, the concentration of the calcium chloride solution is 0.5 mol / L to saturation concentration.

[0014] Preferably, the uncrosslinked solution of the liquid artificial biomembrane corresponding to the photo-crosslinking curing contains methacrylated gelatin, a blue light photoinitiator and bacteria;

[0015] The photo-crosslinking and curing method is: irradiating the uncrosslinked solution of the liquid artificial biomembrane with blue light.

[0016] Preferably, the blue light photoinitiator is phenyl 2,4,6-trimethylbenzoyl lithium phosphate.

[0017] Preferably, the wavelength of the blue light source is 405nm and the light intensity is 25mW / cm 2 .

[0018] Preferably, the thickness of the sample is 1 to 2 mm.

[0019] Preferably, the rotation is performed at a constant speed of 360°.

[0020] Preferably, the scanning voltage of the tomographic scanning is 300-350 kV, and the scanning current is 90-110 uA.

[0021] Compared with the prior art, the present invention provides an online monitoring method for artificial biofilm based on X-ray CT technology, comprising the following steps: cutting and sampling the artificial biofilm, and then fixing the sampled sample on a stage; the stage is rotated, and in this process, the artificial biofilm sample on the stage is subjected to multi-angle tomographic scanning by X-rays to obtain a series of tomographic images; a series of tomographic images are subjected to denoising, reconstruction and rendering by Dragonfly Pro software to obtain a three-dimensional reconstructed X-ray CT image of the artificial biofilm sample. The technical solution provided by the present invention can directly realize three-dimensional online imaging of artificial biofilm with the help of X-ray CT (X-ray computed tomography) technology, based on which the instantaneous three-dimensional structure slice of the artificial biofilm can be given, and the slice can be reconstructed by the integrated image analysis software DragonflyPro, so as to realize online monitoring of the growth status, pore distribution and other information of the artificial biofilm, and further explain the application of artificial biofilm in activated sludge enhancement. Compared with traditional characterization methods such as scanning electron microscopy and transmission electron microscopy, this method does not require complex pre-treatment steps such as microbial immobilization and gradient dehydration, which can minimize the damage to the artificial biofilm to be tested and provide a complete and non-invasive online monitoring method for artificial biofilms. The experimental results show that the method provided by the present invention does not require complex sample preparation, but only requires cutting and sampling of the artificial biofilm and fixing it on the stage. By rotating the stage, three-dimensional slices of artificial biofilms at various angles can be photographed, and after reconstruction, the instantaneous bacterial community distribution, pore conditions and other information of the artificial biofilm can be obtained without damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 This is a physical picture of the artificial organism provided in Example 2 of the present invention after being cut and divided into pieces;

[0024] Figure 2 is one of the tomographic images provided in Embodiment 2 of the present invention;

[0025] Figure 3 This is the artificial biomembrane reconstruction diagram provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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.

[0027] The present invention provides an online monitoring method for artificial biofilm based on X-ray CT technology, comprising the following steps:

[0028] The artificial biofilm is cut and sampled, and then the sample is fixed on a stage;

[0029] The stage is rotated, and during this process, the artificial biofilm sample on the stage is subjected to multi-angle tomographic scanning using X-rays to obtain a series of tomographic images;

[0030] A series of the tomographic images were subjected to noise reduction, reconstruction and rendering by using DragonflyPro software to obtain three-dimensional reconstructed X-ray CT images of the artificial biofilm samples.

[0031] In the online monitoring method provided by the present invention, the preparation step of the artificial biofilm preferably includes:

[0032] The uncrosslinked solution of the liquid artificial biomembrane is chemically crosslinked or photocrosslinked to obtain the artificial biomembrane.

[0033] In the above-mentioned artificial biofilm preparation step provided by the present invention, the OD of the uncrosslinked solution of the liquid artificial biofilm is 600 It is preferably 1 to 5, and more preferably 3.

[0034] In the above-mentioned artificial biofilm preparation steps provided by the present invention, the liquid artificial biofilm uncrosslinked solution corresponding to the chemical crosslinking curing preferably contains sodium alginate and bacteria; wherein the bacteria include but are not limited to Shewanella oneidensis MR-1; the concentration of the sodium alginate in the liquid artificial biofilm uncrosslinked 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). In the present invention, the preparation process of the liquid artificial biofilm uncrosslinked solution corresponding to the chemical crosslinking curing preferably includes: mixing the sodium alginate and the bacterial culture solution evenly to obtain the liquid artificial biofilm uncrosslinked solution.

[0035] In the artificial biomembrane preparation steps provided by the present invention, the chemical crosslinking and curing method is preferably: adding calcium chloride solution to the liquid artificial biomembrane uncrosslinked solution; wherein the concentration of the calcium chloride solution is preferably 0.5 mol / L to saturation concentration.

[0036] In the above-mentioned artificial biomembrane preparation step provided by the present invention, the liquid artificial biomembrane uncrosslinked solution corresponding to the photo-crosslinking curing contains methacryloyl gelatin, 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 biomembrane uncrosslinked solution is preferably 3-10% (w / v), specifically 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), 8% (w / v), 8.5% (w / v), 9% (w / v), 9. 5% (w / v) or 10% (w / v); the concentration of the blue light photoinitiator in the liquid artificial biomembrane uncross-linked 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 biofilm uncross-linked solution corresponding to the photo-cross-linking curing preferably includes: mixing methacrylated gelatin, blue light photoinitiator and liquid culture medium, heating, cooling, and then mixing evenly with bacterial culture fluid to obtain a liquid artificial biofilm uncross-linked 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 above-mentioned artificial biomembrane preparation steps provided by the present invention, the photo-crosslinking curing method is preferably: irradiating the liquid artificial biomembrane 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 thickness of the sampling sample is preferably 1 to 2 mm, specifically 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm or 2 mm.

[0039] In the online monitoring method provided by the present invention, the fixing method is preferably fixing with photo-crosslinking glue.

[0040] In the online monitoring method provided by the present invention, the rotation method is preferably a uniform rotation of 360°.

[0041] In the online monitoring method provided by the present invention, the scanning voltage of the tomography is preferably 300-350kV, specifically 300kV, 305kV, 310kV, 315kV, 320kV, 325kV, 330kV, 335kV, 340kV, 345kV or 350kV; the scanning current of the tomography is preferably 90-110uA, specifically 90uA, 91uA, 92uA, 93uA, 94uA, 95uA, 96uA, 97uA, 98uA, 99uA, 100uA, 101uA, 102uA, 103uA, 104uA, 105uA, 106uA, 107uA, 108uA, 109uA or 110uA.

[0042] In the online monitoring method provided by the present invention, the number of the tomographic images is preferably 800 to 1200, specifically 800, 850, 900, 950, 1000, 1050, 1100, 1150 or 1200.

[0043] In the online monitoring method provided by the present invention, the three-dimensional reconstructed X-ray CT image of the artificial biofilm sample contains the required information such as the growth status of the artificial biofilm and the distribution of bacteria.

[0044] The technical solution provided by the present invention can directly realize three-dimensional online imaging of artificial biofilms with the help of X-ray CT (X-ray computed tomography) technology, based on which the instantaneous three-dimensional structure slices of artificial biofilms can be given, and the slices can be reconstructed with the integrated image analysis software DragonflyPro, so that the growth status, pore distribution and other information of artificial biofilms can be monitored online, and the application of artificial biofilms in activated sludge enhancement can be further explained. Compared with traditional characterization methods such as scanning electron microscopy and transmission electron microscopy, this method does not require complex pre-treatment steps such as microbial immobilization and gradient dehydration, which can minimize the damage to the artificial biofilm to be tested, and provide a complete and non-invasive online monitoring method for artificial biofilms.

[0045] For the purpose of greater clarity, the invention is described in detail through the following examples.

[0046] Example 1

[0047] Take 1.0g of sodium alginate and place it in a centrifuge tube. Add 10mL of OD 600 3.0 Shewanella oneidensis MR-1 culture fluid is stirred evenly to obtain a liquid artificial biofilm solution with a sodium alginate concentration of 10% (w / v) that needs to be chemically cross-linked and formed.

[0048] 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 placed in a 37°C water bath for 10 minutes after being bathed at 60°C for 30 minutes. Then, the culture medium of Shewanella oneidensis MR-1 was added to the mixture to raise the OD to 1. 600 The concentration of methacryloyl gelatin is 7% (w / v) and the concentration of blue light photoinitiator is 0.25% (w / v), and the liquid artificial biomembrane solution to be photocrosslinked is obtained by shaking evenly.

[0049] Example 2

[0050] A 1 mL pipette was used to draw the uncrosslinked solution of the artificial biofilm and drip it into the culture dish, and then the sample was solidified by chemical or photo-crosslinking method; the chemical crosslinking method was to use 0.5 mol / L calcium chloride solution to crosslink the artificial biofilm, and the photo-crosslinking method was to use a blue light source (wavelength 405 nm, light intensity 25 mW / cm 2 )Irradiate the biofilm.

[0051] Taking photo-crosslinking curing as an example, the cured artificial biofilm is placed in a culture dish and cut into pieces with a knife to obtain pieces with a thickness of 1 mm. Figure 1 Then, it was fixed on the stage using photo-crosslinking glue; the stage was rotated 360° at a constant speed under X-rays by the control system, and during the rotation, the artificial biofilm sample on the stage was scanned at multiple angles using X-rays to obtain 1,000 tomographic images, one of which is shown in Figure 2 shown.

[0052] The 1000 tomographic images were de-noised, reconstructed and rendered using DragonflyPro software, and finally a three-dimensional reconstructed X-ray CT image of the artificial biofilm sample was obtained. Figure 3 shown.

[0053] 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 X-ray CT technology, characterized in that: The following steps are involved: The artificial biofilm is cut and sampled, and then the sample is fixed on a stage; The stage is rotated, and during this process, the artificial biofilm sample on the stage is subjected to multi-angle tomographic scanning using X-rays to obtain a series of tomographic images; A series of the tomographic images were subjected to noise reduction, reconstruction and rendering by using Dragonfly Pro software to obtain three-dimensional reconstructed X-ray CT images of the artificial biofilm samples.

2. The method for online monitoring of artificial biofilm according to claim 1, characterized in that: The preparation steps of the artificial biofilm include: The uncrosslinked solution of the liquid artificial biomembrane is chemically crosslinked or photocrosslinked to obtain the artificial biomembrane.

3. The method for online monitoring of artificial biofilm according to claim 2, characterized in that: The uncrosslinked solution of the liquid artificial biomembrane corresponding to the chemical crosslinking curing contains sodium alginate and bacteria; The chemical cross-linking and curing method is: adding calcium chloride solution to the uncross-linked solution of the liquid artificial biomembrane.

4. The method for online monitoring of artificial biofilm according to claim 3, characterized in that: The concentration of the calcium chloride solution is 0.5 mol / L to saturation concentration.

5. The method for online monitoring of artificial biofilm according to claim 2, 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 uncrosslinked solution of the liquid artificial biomembrane 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 thickness of the sample is 1 to 2 mm.

9. The method for online monitoring of artificial biofilm according to claim 1, characterized in that: The rotation method is to rotate 360° at a constant speed.

10. The method for online monitoring of artificial biofilm according to claim 1, wherein the scanning voltage of the tomography is 300-350 kV, and the scanning current is 90-110 uA.

Images