A biofouling mitigation method of synergistically degrading extracellular polymeric substances by multiple enzymes

Abstract

This invention discloses a method for alleviating bioclogging through the synergistic degradation of extracellular polymers by multiple enzymes, comprising: selecting freeze-dried *Pseudomonas aeruginosa* and reviving and culturing it to obtain bacterial solution a; expanding the reviving bacterial solution a and mixing it to obtain bacterial solution b; re-inoculating bacterial solution b into a new culture medium until the logarithmic growth phase to obtain bacterial solution c; centrifuging bacterial solution c in the logarithmic growth phase and adding MOPS buffer solution to the precipitate to obtain a bacterial suspension; preparing a compound enzyme agent and adding the prepared compound enzyme agent to the bacterial suspension to react and decompose the extracellular polymers secreted by the microorganisms, thereby solving the clogging problem. This invention can alleviate the formation of bioclogging without affecting the water quality of reinjected water. Compared with single enzymes or combinations of two enzymes, it has advantages such as lower dosage, lower cost, better effect, and faster results.

Description

Technical Field

[0001] This invention belongs to the field of artificial reinjection microbial blockage and relates to a biological blockage relief method for multi-enzyme synergistic degradation of extracellular polymeric substances (EPS), which is used to prevent the formation of microbial films and organic blockages during mine water reinjection and helps to improve reinjection efficiency. Background Technology

[0002] Mine water reinjection is a passive treatment method to reduce mine water discharge by reinjecting pre-treated mine water into the aquifer to achieve "transfer and storage" of mine water. However, blockage will directly lead to the reinjection volume being lower than the water extraction volume. This not only wastes a large amount of groundwater resources because they cannot be injected, but also causes the regional groundwater level to drop in the long term, which in turn triggers a series of environmental and geological problems, forming a "chain reaction" of negative effects.

[0003] Mine water contains a large number of bacteria and other microorganisms, which can multiply rapidly under suitable nutrient conditions. During reinjection, these microorganisms and their metabolic products easily occupy the pore channels of the aquifer, leading to a decrease in reinjection efficiency; this process is called microbial blockage. Once microbial blockage forms, the permeability of the medium is often more difficult to restore. Therefore, bioblockage is a key factor restricting the widespread application of mine water reinjection technology. Bioblockage is mainly caused by algal growth and the accumulation of microbial cells and their related metabolic products (such as extracellular polymeric substances, EPS) in the medium. However, reducing the generation of aquifer microbial EPS during reinjection is key to mitigating bioblockage. Current solutions mainly rely on chemical sterilization (such as ozone, chlorine-containing disinfectants, etc.) and physical cleaning, but chemical sterilization easily causes secondary pollution, while physical cleaning cannot prevent blockage. Therefore, a new preventive measure is needed that can both prevent blockage and eliminate secondary pollution. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for alleviating bioclogging by multi-enzyme synergistic degradation of extracellular polymers, which can alleviate the formation of bioclogging without affecting the water quality of reinjected water.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A method for alleviating bioblocking through multi-enzyme synergistic degradation of extracellular polymers includes the following steps: Step 1: Select freeze-dried Pseudomonas aeruginosa and revive and activate it on LB solid medium to obtain bacterial solution a; Step 2: Inoculate the revived bacterial culture a into a new LB medium for expansion culture, and mix well to form bacterial culture b; Step 3: Inoculate bacterial solution b into a new culture medium again until the logarithmic phase, to obtain bacterial solution c; Step 4: Centrifuge the bacterial culture c in the logarithmic phase to obtain a precipitate, and then add MOPS buffer solution to the precipitate to obtain a bacterial suspension. Step 5: Prepare a compound enzyme agent. Add the prepared compound enzyme agent to the bacterial suspension to react and decompose the extracellular polymers secreted by microorganisms, thus solving the blockage problem. The compound enzyme agent includes protease, α-amylase and cellulase.

[0007] The present invention also includes the following technical features: Specifically, in step 1, the resuscitation and activation culture includes: using a pipette to aspirate Pseudomonas aeruginosa and inoculating it onto a sterilized LB solid medium plate; completely dissolving the lyophilized Pseudomonas aeruginosa bacteria; transferring the dissolved bacterial solution to a centrifuge tube containing culture medium and mixing it thoroughly for culture.

[0008] Specifically, in step 1, the culture temperature and time are as follows: culture at 37℃ for 24-48 hours to obtain bacterial solution a.

[0009] Specifically, in step 2, the expanded culture includes: placing bacterial culture a in LB liquid medium and culturing to obtain bacterial culture b in the stable phase.

[0010] Specifically, in step 2, the culture temperature and time are: 37℃, and the culture time is 16-20h until the growth is stable, to obtain the stable bacterial solution b.

[0011] Specifically, in step 2, the extraction volume of bacterial solution a is 0.5-1 ml, and the inoculation volume ratio is 0.5-1 ml of bacterial solution to 6-12 ml of culture medium.

[0012] Specifically, in step 3, the inoculation volume ratio is bacterial solution b 0.5-1 : culture medium 4-8.

[0013] Specifically, in step 4, the centrifugation speed is 3000-4000 rpm and the centrifugation time is 3-5 min.

[0014] Specifically, in the compound enzyme agent, protease, α-amylase and cellulase are prepared in a volume ratio of 3-5:2-4:2-3.

[0015] Compared with the prior art, the present invention has the following technical effects: This invention selects Pseudomonas aeruginosa as the model microorganism to prepare bacterial extracellular polymeric substances (EPS). It also selects a biological enzyme method to degrade the EPS that causes bioclogging, which not only alleviates the formation of bioclogging but also does not affect the water quality of reinjected water. Compared with single enzymes or combinations of two enzymes, it has the advantages of lower dosage, lower cost, better effect, and faster results. Attached Figure Description

[0016] Figure 1 This is a detailed flowchart of an embodiment of the present invention.

[0017] Figure 2 A comparison chart showing the total EPS before and after adding different proportions of compound microbial agents. Detailed Implementation

[0018] This invention provides a method for alleviating bioclogging by multi-enzyme synergistic degradation of extracellular polymeric substances (EPS), which can alleviate the formation of bioclogging without affecting the water quality of reinjected water, and includes the following steps: Freeze-dried Pseudomonas aeruginosa was selected and revived and activated on LB solid medium to obtain bacterial solution a. The revival and activation culture included: using a pipette to take 0.5 mL of Pseudomonas aeruginosa and inoculating it onto a sterilized LB solid medium plate, completely dissolving the freeze-dried bacteria of the genus Pseudomonas aeruginosa, transferring the dissolved bacterial solution to a centrifuge tube containing 5 mL of LB medium, mixing well, and incubating at 37℃ for 24-48 h to obtain bacterial solution a.

[0019] The revived bacterial culture a was inoculated into a new LB medium for expansion culture, and mixed well to become bacterial culture b. The expansion culture included: placing bacterial culture a in LB liquid medium and controlling the culture temperature at 37℃ for 16-20 hours until the growth stationary phase, to obtain bacterial culture b in the stationary phase; wherein the extraction volume of bacterial culture a was 0.5-1 ml, and the inoculation volume ratio was bacterial culture 0.5-1: medium 6-12.

[0020] Bacterial solution b was re-inoculated into a new culture medium at a certain ratio until the logarithmic phase, to obtain bacterial solution c; wherein the inoculation volume ratio was bacterial solution b 0.5-1 : culture medium 4-8.

[0021] The bacterial culture c in the logarithmic phase was centrifuged to obtain a precipitate. Then, 3-morpholinopropanesulfonic acid (MOPS) buffer solution was added to the precipitate to obtain a bacterial suspension for subsequent EPS testing. The centrifugation speed was 3000-4000 rpm and the centrifugation time was 3-5 min.

[0022] EPS test was performed on the initially cultured bacterial suspension to obtain its initial concentration.

[0023] The compound enzyme agent is prepared by mixing protease, α-amylase and cellulase in a volume ratio of 3-5:2-4:2-3. The prepared compound enzyme agent is added to the bacterial suspension to react and decompose the extracellular polymers secreted by microorganisms, thus solving the clogging problem.

[0024] EPS tests were performed on the bacterial suspension after the reaction to obtain its final concentration and analyze its degradation efficiency.

[0025] Reaction Principle: Proteins in EPS form a cross-linked network structure through covalent bonds, ionic bonds, and hydrophobic interactions. Proteases "cleave" these long-chain proteins into short peptides or individual amino acids. EPS contains a wide variety of polysaccharides, many of which contain fragments or similar structures composed of α-1,4-glycosidic bonds. α-Amylase can effectively degrade these polysaccharides, disrupting the polysaccharide network in EPS that serves as a "filler matrix" and carbon source reserve. This hollows out the EPS matrix, reduces viscosity, alters hydration capacity, and destabilizes the overall structure. Microbially produced EPS contains cellulose or similar β-glucan structures. Cellulase can specifically degrade these structural polysaccharides with high crystallinity and strength. It specifically breaks down the structural fibers in EPS composed of β-linked polysaccharides, further disrupting the integrity of EPS. EPS is a complex mixture composed of proteins, polysaccharides, nucleic acids, lipids, etc. Using a compound enzyme preparation (i.e., containing multiple enzymes mentioned above), the three enzymes work synergistically to attack the EPS network structure simultaneously from different angles. Proteases disrupt the protein backbone, while α-amylase and cellulase degrade different types of polysaccharide chains, respectively. This multi-pronged approach breaks down EPS and biofilms more quickly and thoroughly than using a single enzyme.

[0026] The following are specific embodiments of the present invention. It should be noted that the present invention is not limited to the following specific embodiments. All equivalent modifications made based on the technical solutions of this application fall within the protection scope of the present invention.

[0027] Example 1: This embodiment provides a method for alleviating bioblocking by multi-enzyme synergistic degradation of extracellular polymeric substances (EPS), such as... Figure 1 As shown, it includes the following steps: (1) Select freeze-dried Pseudomonas aeruginosa and activate it on LB solid medium. The activation culture steps are as follows: use a pipette to take 0.5 mL of Pseudomonas aeruginosa and inoculate it on a sterilized LB solid medium plate. Transfer the dissolved bacterial solution to a centrifuge tube containing 5 mL of LB medium and mix well. Incubate at 37°C for 24 h.

[0028] After successful activation, *Pseudomonas aeruginosa* was inoculated into LB liquid medium for expansion culture to obtain bacterial growth culture. The expansion culture steps are as follows: *Pseudomonas aeruginosa*, after activation, was placed in LB liquid medium at a ratio of 1:14, and the culture temperature was controlled at 37℃ for 20 hours until the stable growth phase; thus, the stable phase bacterial culture b was obtained. (2) The stable bacterial culture b was inoculated into a new culture medium at a ratio of 1:10 and cultured for 4 hours to the logarithmic phase to obtain the logarithmic phase bacterial culture c; the bacterial culture c was centrifuged, and the supernatant was discarded to obtain the bacterial precipitate. The centrifugation speed was 3000 rpm and the time was 3 min. (3) Add the collected bacterial precipitate to MOPS buffer to obtain a bacterial suspension containing EPS, and test its EPS content; (4) The compound enzyme agent is prepared by mixing protease, α-amylase and cellulase in a ratio of 5:4:3. The prepared compound enzyme agent is added to the bacterial suspension to react and degrade EPS in the suspension. The EPS content after the reaction is tested.

[0029] Example 2: (1) Select freeze-dried Pseudomonas aeruginosa and activate it on LB solid medium. The activation culture steps are as follows: use a pipette to take 0.5 mL of Pseudomonas aeruginosa and inoculate it on a sterilized LB solid medium plate. Transfer the dissolved bacterial solution to a centrifuge tube containing 5 mL of LB medium and mix well. Incubate at 37°C for 40 h.

[0030] After successful activation, *Pseudomonas aeruginosa* was inoculated into LB liquid medium for expansion culture to obtain bacterial growth culture. The expansion culture steps are as follows: *Pseudomonas aeruginosa*, after activation, was placed in LB liquid medium at a ratio of 1:10, and the culture temperature was controlled at 37℃ for 16 hours until the stable growth phase; thus, the stable phase bacterial culture b was obtained. (2) The stable bacterial culture b was inoculated into a new culture medium at a ratio of 1:8 and cultured for 3 hours to the logarithmic phase to obtain the logarithmic phase bacterial culture c; the bacterial culture c was centrifuged, and the supernatant was discarded to obtain the bacterial precipitate. The centrifugation speed was 4000 rpm and the time was 5 min. (3) Add the collected bacterial precipitate to MOPS buffer to obtain a bacterial suspension containing EPS, and test its EPS content; (4) The compound enzyme agent is prepared by mixing protease, α-amylase and cellulase in a ratio of 4:3:2. The prepared compound enzyme agent is added to the bacterial suspension to react and degrade EPS in the suspension. The EPS content after the reaction is tested.

[0031] Example 3: In this embodiment, there are no changes in steps (1)-(3) during the cultivation process. In step (4), the protease, α-amylase and cellulase are prepared in a ratio of 3.5:3:2 or 3:2:2 or 2:3:3, respectively. The prepared compound enzyme agent is added to the bacterial suspension to react, degrade the EPS in the suspension, and test the EPS content after the reaction.

[0032] Comparative Example 1: During the cultivation process, steps (1)-(3) remained unchanged. In step (4), only a single protease was added to the bacterial suspension to react with EPS, and the EPS content after the reaction was tested.

[0033] Comparative Example 2: During the cultivation process, there were no changes in steps (1) to (3). In step (4), protease and α-amylase were added to the bacterial suspension in a ratio of 3:2 to react with EPS, and the EPS content after the reaction was tested.

[0034] Comparative Example 3: During the cultivation process, there were no changes in steps (1) to (3). In step (4), α-amylase and cellulase were added to the bacterial suspension in a 1:1 ratio to react with EPS, and the EPS content after the reaction was tested.

[0035] The comparison chart of the total EPS before and after adding different proportions of compound microbial agent in the above embodiments and comparative examples is shown in the figure below. Figure 2 As shown.

Claims

1. A biofouling mitigation method for the synergistic degradation of extracellular polymeric substances by multiple enzymes, characterized in that, Includes the following steps: Step 1: Select freeze-dried Pseudomonas aeruginosa and revive and activate it on LB solid medium to obtain bacterial solution a; Step 2: Inoculate the revived bacterial culture a into a new LB medium for expansion culture, and mix well to form bacterial culture b; Step 3: Inoculate bacterial solution b into a new culture medium again until the logarithmic phase, to obtain bacterial solution c; Step 4: Centrifuge the bacterial culture c in the logarithmic phase to obtain a precipitate, and then add MOPS buffer solution to the precipitate to obtain a bacterial suspension. Step 5: Prepare a compound enzyme agent. Add the prepared compound enzyme agent to the bacterial suspension to react and decompose the extracellular polymers secreted by microorganisms, thus solving the blockage problem. The compound enzyme agent includes protease, α-amylase and cellulase.

2. The method for alleviating bioblocking by multi-enzyme synergistic degradation of extracellular polymers as described in claim 1, characterized in that, In step 1, the resuscitation and activation culture includes: using a pipette to aspirate Pseudomonas aeruginosa and inoculating it onto a sterilized LB solid medium plate; dissolving all the freeze-dried Pseudomonas aeruginosa bacteria; transferring the dissolved bacterial solution to a centrifuge tube containing culture medium and mixing it thoroughly for culture.

3. The method for alleviating bioblocking by multi-enzyme synergistic degradation of extracellular polymers as described in claim 2, characterized in that, In step 1, the culture temperature and time are as follows: culture at 37℃ for 24-48 hours to obtain bacterial solution a.

4. The method for alleviating bioblocking by multi-enzyme synergistic degradation of extracellular polymers as described in claim 1, characterized in that, In step 2, the expansion culture includes: placing bacterial culture a in LB liquid medium and culturing to obtain bacterial culture b in the stable phase.

5. The method for alleviating bioblocking by multi-enzyme synergistic degradation of extracellular polymers as described in claim 4, characterized in that, In step 2, the culture temperature and time are: 37℃, and the culture time is 16-20h until the growth is stable, to obtain the stable bacterial solution b.

6. The method for alleviating bioblocking by multi-enzyme synergistic degradation of extracellular polymers as described in claim 4, characterized in that, In step 2, the extraction volume of bacterial solution a is 0.5-1 ml, and the inoculation volume ratio is bacterial solution 0.5-1 : culture medium 6-12.

7. The method for alleviating bioblocking by multi-enzyme synergistic degradation of extracellular polymers as described in claim 1, characterized in that, In step 3, the inoculation volume ratio is bacterial solution b 0.5-1 : culture medium 4-8.

8. The method for alleviating bioblocking by multi-enzyme synergistic degradation of extracellular polymers as described in claim 1, characterized in that, In step 4, the centrifugation speed is 3000-4000 rpm and the centrifugation time is 3-5 min.

9. The method for alleviating bioblocking by multi-enzyme synergistic degradation of extracellular polymers as described in claim 1, characterized in that, In the compound enzyme agent, protease, α-amylase and cellulase are prepared in a volume ratio of 3-5:2-4:2-3.

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