Bacterial strain combination, biological ink, biological membrane and application thereof
Through artificial composite bacteria combination, activated sludge is strengthened, and the problem of low degradation efficiency of activated sludge on new pollutants is solved, efficient degradation of tetrabromobisphenol A is achieved, and the performance of wastewater treatment is improved.
Patent Information
- Application Number
- CN202411794719.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-05
- Publication Date
- 2025-01-10
AI Technical Summary
The activated sludge method has poor adaptability and low degradation efficiency in wastewater treatment, especially for new and difficult-to-degrade pollutants such as the flame retardant tetrabromobenzophenol A, which is difficult to meet the increasingly stringent environmental protection requirements.
Using artificial complex bacterial flora combinations, including Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa CDT, biofilms were formed by 3D bioprinting and added to the activated sludge system to enhance its degradation performance.
It significantly improves the degradation effect of unattached activated sludge on tetrabromobisphenol A and improves the efficiency and quality of wastewater treatment.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biodegradation, and in particular to a strain combination, bio-ink, biofilm and applications thereof. Background Art
[0002] As the mainstream process for wastewater treatment in the world, including my country, the activated sludge method has always played an important role. However, it should be noted that as a multicellular group composed of microorganisms, activated sludge has some problems that cannot be ignored in the process of natural domestication. For example, its adaptability is relatively poor, and it is difficult to quickly adapt to changes in wastewater quality; at the same time, the sludge output is high, which not only increases the difficulty and cost of treatment, but also may bring the risk of secondary pollution, especially in the face of new and difficult-to-degrade pollutants in wastewater such as antibiotics, microplastics, flame retardants, surfactants, and persistent organic pollutants. The activated sludge method often shows problems such as low degradation efficiency and slow speed, and it is difficult to meet increasingly stringent environmental protection requirements. Therefore, the innovation of wastewater treatment technology has become an urgent need for the country.
[0003] In order to solve these problems, the use of synthetic biology technology to develop composite artificial bacterial communities for addition is considered to be an effective approach. By specifically enhancing the characteristics of the pollutants, these composite artificial bacterial communities can more efficiently degrade new and difficult-to-degrade pollutants, thereby improving the efficiency and quality of wastewater treatment. This method provides new ideas and methods for solving the challenges faced in wastewater treatment, and provides rich reference examples for activated sludge intensification technology, which is expected to promote the further development and improvement of wastewater treatment technology. Summary of the invention
[0004] In view of this, the present invention provides a strain combination, a bio-ink, a biofilm and its application. The present invention provides a method for enhancing the degradation performance of activated sludge pollutants based on an artificial composite bacterial community. The method provided by the present invention can significantly improve the degradation effect of untamed activated sludge on flame retardants (tetrabromobisphenol A, etc.).
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] The invention provides a strain combination, comprising: Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa CDT.
[0007] In some embodiments of the present invention, in the above strain combination, the Pseudomonas aeruginosa PAO1 is derived from China Center for Type Culture Collection CCTCC KB 20082366.
[0008] In some embodiments of the present invention, in the above-mentioned strain combination, the Pseudomonas aeruginosa CDT is derived from the research group of Professor Ji Rong of Nanjing University (Effects of Cu 2+ and humic acids on degradation and fate of TBBPA in pure culture of Pseudomonas sp. strain CDT).
[0009] In some embodiments of the present invention, in the above-mentioned strain combination, the OD of the Pseudomonas aeruginosa PAO1 and the Pseudomonas aeruginosa CDT is 600 The ratio is: (1~3): (1~3).
[0010] In some embodiments of the present invention, in the above-mentioned strain combination, the OD of the Pseudomonas aeruginosa PAO1 and the Pseudomonas aeruginosa CDT is 600 The ratio is: 1:2.
[0011] The present invention also provides a microbial agent, characterized in that it comprises: the above-mentioned strain combination.
[0012] The present invention also provides biological ink, comprising: the above-mentioned strain combination and / or the above-mentioned microbial agent and liquid culture medium.
[0013] The present invention also provides a method for preparing the above-mentioned biological ink, wherein the Pseudomonas aeruginosa PAO1 and the Pseudomonas aeruginosa CDT are respectively mixed with sodium alginate, and the biological ink is obtained after cross-linking.
[0014] The present invention also provides a biofilm, comprising: the above-mentioned bio-ink and / or the bio-ink obtained by the above-mentioned preparation method.
[0015] The present invention also provides a method for preparing the above-mentioned biofilm, wherein the bio-ink is 3D printed and cross-linked to obtain the biofilm.
[0016] In some embodiments of the present invention, in the above preparation method, the cross-linking uses a 0.5M calcium chloride solution.
[0017] The present invention also provides the use of the above-mentioned strain combination, the above-mentioned microbial agent, the above-mentioned biological ink, the biological ink obtained by the above-mentioned preparation method, the above-mentioned biofilm and / or the biofilm obtained by the above-mentioned preparation method in degrading tetrabromobisphenol A.
[0018] The invention also provides a method for degrading tetrabromobisphenol A, wherein the biofilm is mixed with pollutants.
[0019] The present invention provides a method for artificially constructing a composite bacterial community to enhance the performance of activated sludge, mainly by artificially cultivating a functional bacterial community, constructing it into a community, and adding it to an activated sludge system. The method provided by the present invention can significantly improve the degradation effect of untamed activated sludge on flame retardants (tetrabromobisphenol A, etc.). BRIEF DESCRIPTION OF THE DRAWINGS
[0020] 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 are briefly introduced below.
[0021] Figure 1 The volume-enhancing effect diagram of rhamnolipid provided in Example 1 of the present invention is shown;
[0022] Figure 2 The graph shows the degradation ability of Pseudomonas aeruginosa PAO1 to tetrabromobisphenol A;
[0023] Figure 3 A schematic diagram showing the effect of the composite bacterial community with the best ratio provided in Example 1 of the present invention;
[0024] Figure 4 FIG. 3 is a flowchart of machine learning provided by Embodiment 3 of the present invention;
[0025] Figure 5 FIG. 3 is a schematic diagram of a particle swarm algorithm provided in Example 3 of the present invention;
[0026] Figure 6 Schematic diagram showing degradation of the microbial hydrogel material under the optimal printing conditions provided in Example 3 of the present invention;
[0027] Figure 7Schematic diagram of the inner and outer layer configuration printed under the optimal printing conditions provided by Example 4 of the present invention; wherein: A shows a top view of the inner PAO1 outer CDT structure; B shows a side view of the inner PAO1 outer CDT structure; C shows a top view of the inner CDT outer PAO1 structure; D shows a side view of the inner CDT outer PAO1 structure;
[0028] Figure 8 The figure shows the strengthening effect of tetrabromobisphenol A provided in Example 4 of the present invention. DETAILED DESCRIPTION
[0029] The invention discloses a strain combination, biological ink, a biofilm and applications thereof.
[0030] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.
[0031] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.
[0032] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.
[0033] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.
[0034] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.
[0035] The invention relates to a method for enhancing the degradation performance of activated sludge pollutants based on artificial composite bacterial flora.
[0036] The present invention provides a method for enhancing the degradation performance of activated sludge on specific pollutants by artificial composite bacterial flora, and the specific steps are as follows: using LB culture medium to culture target microorganisms, selecting different microorganisms for use or compounding according to different pollutants, performing 3D bioprinting and cross-linking immobilization according to their characteristics, and finally putting the prepared engineering living material into the activated sludge system.
[0037] The culture method, degradation function and pathway of the bacterial flora have been scientifically verified and explained. The culture of the bacterial flora is mainly carried out by culturing it in a culture medium for several hours until its logarithmic growth phase, which is highly feasible for most units.
[0038] Preferably, the culture medium for culturing the bacteria uses the commonly used LB medium.
[0039] The hydrogel matrix and configuration, in this case, there is a survival competition between the two bacteria, so the optimal compound ratio is explored, and the inner and outer layer structures are formed by 3D printing to provide the best growth environment for both bacteria.
[0040] The hydrogel printing condition selection is characterized in that a variety of different printing conditions (hydrogel matrix concentration, pressure, speed, needle diameter, etc.) are learned through a machine learning method, and the optimal printing condition combination is returned through a particle swarm algorithm.
[0041] The crosslinking method is chemical crosslinking by calcium chloride solution or blue light induced photocrosslinking, the concentration of calcium chloride solution is (0.2 M ~ saturation), the wavelength of blue light is 405nm, and the light intensity is 25mW / cm 2 .
[0042] Preferably, the concentration of the calcium chloride solution used for the chemical crosslinking is 0.5 mol / L.
[0043] The target activated sludge for strengthening is mainly aerobic activated sludge or anaerobic granular sludge.
[0044] The present invention relates to a method for artificially constructing a composite bacterial community to enhance the performance of activated sludge, which is mainly carried out by artificially cultivating functional bacterial communities, constructing them into a community, and adding them to the activated sludge system. The activated sludge method is the mainstream process for wastewater treatment in the world, including my country. Natural domestication of activated sludge has problems such as poor adaptability and high sludge yield. The development of composite artificial bacterial communities and specific enhancement of pollutant characteristics are considered to be an effective way to solve the above problems. The bacteria used in the present invention are mainly two types of Pseudomonas, and the pollutants targeted are flame retardants (tetrabromobisphenol A, etc.). Bacteria that have been proven to have specific degradation functions are cultivated, and artificial biological living materials are formed by 3D bioprinting in an additive manufacturing manner, and are added to activated sludge to further promote the application of activated sludge in pollutant degradation.
[0045] In Examples 1 to 5 of the present invention, all the raw materials and reagents used can be purchased from the market.
[0046] The present invention will be further described below in conjunction with embodiments:
[0047] Example 1
[0048] The main biosurfactant produced by Pseudomonas aeruginosa PAO1 is rhamnolipid. Rhamnolipid was added to ultrapure water with 500 mg of tetrabromobisphenol A, and then the solution samples were taken at different time points for liquid chromatography detection. The detection mobile phase was 0.1% formic acid and methanol. Figure 1 As shown in Table 1 , over time, tetrabromobisphenol A, which was originally insoluble in water, gradually dissolved in water under the solubilization effect of rhamnolipid, confirming the solubilization potential of Pseudomonas aeruginosa PAO1.
[0049] Table 1
[0050]
[0051] Example 2 PAO1 bacteria does not have a degradation effect on TBBPA
[0052] The culture medium of Pseudomonas aeruginosa PAO1 was added to PBS buffer with 5 mg of tetrabromobisphenol A to make the final total volume 100 mL and the bacterial concentration OD 600 =2, then take sample points of the solution at different time points and use liquid phase for detection, the detection mobile phase is 0.1% formic acid and methanol. Figure 2As shown in Table 2, over time, the concentration of tetrabromobisphenol A fluctuated within a normal range, and part of tetrabromobisphenol A that was originally insoluble in water gradually dissolved in water under the solubilization effect of rhamnolipid produced by Pseudomonas aeruginosa PAO1, proving that Pseudomonas aeruginosa PAO1 could not significantly degrade tetrabromobisphenol A, and confirming the solubilization potential of Pseudomonas aeruginosa PAO1.
[0053] Table 2
[0054]
[0055] It can be seen from Examples 1 and 2 that Pseudomonas aeruginosa PAO1 does not have a degradation effect on TBBPA, but has a solubilization ability. In the art, solubilization means increasing the solubility of tetrabromobisphenol A by secreting biosurfactants, and no chemical reaction occurs during the solubilization process; degradation means dehalogenating tetrabromobisphenol A into other substances through biological enzymatic reactions.
[0056] Example 3
[0057] Different ratios (1:1, 1:2, 2:1, 1:3, 3:1) of free Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa CDT were added to the same bioreactor, which contained 10 mg / L tetrabromobisphenol A. After 24 hours, the solution samples were taken for liquid chromatography detection, and the detection mobile phase was 0.1% formic acid and methanol. Figure 3 As shown in Tables 3 and 4, the composite bacterial community with a ratio of 1:2 finally selected had the best degradation effect of tetrabromobisphenol A, far exceeding the single Pseudomonas aeruginosa CDT.
[0058] Table 3
[0059]
[0060] Table 4
[0061]
[0062] Example 4
[0063] The composite flora of Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa CDT was designed for 3D bioprinting with an inside-out configuration to separate the two and provide the best growth environment. The conditions for 3D bioprinting are complex, and different pressures, printing speeds, needle diameters, etc. correspond to different printing effects, such as Figure 4 , Figure 5 As shown in Figure 1, we use machine learning to analyze the printing effects under different printing conditions and return the best printing conditions through the particle swarm algorithm. In this example, the best printing condition combination is: concentration 7.34%, pressure 78.30 Pa, speed 6.06 mm / s, and needle diameter 0.43 mm. Figure 6 As shown in Tables 3 to 5, the degradation effect of the printed flora under this printing condition was further optimized.
[0064] Table 5
[0065]
[0066] Example 5
[0067] Weigh 2 portions of 0.7 g sodium alginate and divide them into two 50 mL centrifuge tubes, add Pseudomonas aeruginosa PAO1 culture medium (culture medium includes: 1×PBS, main components are Na2HPO4, KH2PO4, NaCl and KCl, pH is 7.4) and Pseudomonas aeruginosa CDT culture medium (culture medium includes: 1×PBS, main components are Na2HPO4, KH2PO4, NaCl and KCl, pH is 7.4) with OD600 of 2.0, stir evenly to obtain the bio-ink that needs to be chemically cross-linked. Print it into an inner and outer layer configuration according to the printing conditions selected in Example 3 by 3D bioprinting, such as Figure 7 As shown in the figure, the former is outside and the latter is inside. Then, a 0.5 M calcium chloride solution is used to cross-link them. After 10 minutes of cross-linking, an artificial biofilm is formed. It is put into the activated sludge system to degrade 10 mg / L of tetrabromobisphenol A. Figure 8 As shown in Tables 6 and 7, before the addition of the enhanced artificial biofilm, the activated sludge itself did not have the ability to degrade tetrabromobisphenol A. The enhanced biofilm achieved significant degradation optimization. Although the degradation rate was slow due to the intrinsic characteristics of tetrabromobisphenol A, it could reach about 60% in 48 hours.
[0068] Table 6
[0069]
[0070] Table 7
[0071]
[0072] 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. A strain combination, characterized in that: include: Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa CDT.
2. The bacterial strain combination according to claim 1, characterized in that OD of the Pseudomonas aeruginosa PAO1 and the Pseudomonas aeruginosa CDT 600 The value ratio is: (1~3): (1~3).
3. The bacterial strain combination according to claim 1 or 2, characterized in that: OD of the Pseudomonas aeruginosa PAO1 and the Pseudomonas aeruginosa CDT 600 The value ratio is: 1:
2.
4. A microbial agent, characterized in that: include: The strain combination according to any one of claims 1 to 3.
5. Bio-ink, characterized in that include: The strain combination according to any one of claims 1 to 3 and / or the microbial agent and liquid culture medium according to claim 4.
6. The method for preparing the biological ink according to claim 5, characterized in that: The Pseudomonas aeruginosa PAO1 and the Pseudomonas aeruginosa CDT are respectively mixed with sodium alginate, and after cross-linking, the bio-ink is obtained.
7. A biofilm, characterized in that include: The biological ink as claimed in claim 5 and / or the biological ink obtained by the preparation method as claimed in claim 6.
8. The method for preparing a biofilm according to claim 7, characterized in that: The bio-ink is 3D printed and cross-linked to obtain the biofilm.
9. Use of the strain combination according to any one of claims 1 to 3, the microbial agent according to claim 4, the biological ink according to claim 5, the biological ink obtained by the preparation method according to claim 6, the biofilm according to claim 7 and / or the biofilm obtained by the preparation method according to claim 8 in degrading tetrabromobisphenol A.
10. A method for degrading tetrabromobisphenol A, characterized in that: The biofilm is mixed with a pollutant.