A salt-tolerant hydrogel composite high-energy degrading bacterium derived from mangrove sediment and a preparation method and application thereof

Abstract

The application discloses a salt-tolerant hydrogel composite high-energy degrading bacterium derived from mangrove sediment and a preparation method and application thereof. The bacterium is Marinobacterium mangrovicola S01 and Vibrio tritonius K07, which are respectively preserved in the Guangdong Microbial Culture Collection Center and have the keeping numbers of GDMCC No: 68772 and 68773. The two kinds of bacterium are compounded, dropped into balls, and then solidified at low temperature to form the microspheres in the form of solidified hydrogel, and the surface is modified to form a semi-permeable protective outer membrane on the surface of the microspheres. The salt-tolerant hydrogel composite high-energy degrading bacterium has the use of treating high-salinity wastewater.

Description

Technical Field

[0001] This invention relates to the fields of bio-environmental protection technology and environmental microbiology, and in particular to a salt-tolerant hydrogel composite high-energy degrading bacterium derived from mangrove sediments, its preparation method and application. Background Technology

[0002] With the rapid development of mariculture in coastal areas of my country, the resulting mariculture wastewater has become one of the major sources of water pollution in my country's nearshore waters. Mariculture wastewater (such as wastewater from shrimp ponds and factory-farmed fish) typically exhibits characteristics of high salinity, high organic matter concentration, high nitrogen and phosphorus content, and high antibiotic content. This type of wastewater contains large amounts of uneaten feed, feces, and excrement. If discharged directly without effective treatment, it will lead to eutrophication of surrounding waters, trigger ecological disasters such as red tides, and even induce outbreaks of diseases in farmed organisms. The implementation of the new Fisheries Law has further raised the standards for the treatment and discharge of mariculture wastewater.

[0003] Currently, biological methods (microbial degradation) are the most economical and effective way to treat this type of wastewater. However, in practical engineering applications, traditional microbial agents often face the following technical bottlenecks: 1. Poor environmental resistance (difficult to tolerate high salinity and high toxicity): Most commercially available wastewater treatment microbial agents originate from freshwater or ordinary terrestrial environments. In high-salinity environments (especially the 1.5% to 3.5% salinity commonly found in mariculture), their cells will dehydrate, lyse, or become inactive due to a sudden increase in osmotic pressure. This results in extremely low colonization rates (less than 1%) of traditional microbial agents in mariculture wastewater, leading to low degradation efficiency.

[0004] 2. Weak synergistic effect and limited function of bacterial strains: Nitrogen sources in marine aquaculture wastewater mainly exist in the form of highly toxic ammonia nitrogen and nitrite nitrogen. Existing compound bacterial agents are mostly simple mixtures, lacking highly efficient ammonia-oxidizing and denitrifying bacteria that exhibit strong synergistic effects under salinity conditions. Their functions are limited, making it difficult to form a stable dominant bacterial community in aquaculture effluent.

[0005] 3. Insufficient stability of compound formulations: Traditional liquid or room-temperature solid bacterial agents are prone to a sharp drop in viable bacteria count during storage and transportation due to environmental fluctuations. When directly added to large-volume seawater aquaculture ponds, they are easily diluted by water flow or displaced by native wild bacteria. Many commercially available bacterial agents only show effects for less than a week, or even just one day. They cannot achieve sustained high-energy degradation.

[0006] Therefore, discovering highly efficient and special functional strains, constructing a synergistic metabolic network of composite strains, and combining hydrogel encapsulation technology to prepare a composite bacterial agent that can tolerate high salt and high ammonia nitrogen and efficiently degrade pollutants in pig farming and marine aquaculture wastewater is a technical problem that urgently needs to be solved in the field of bio-environmental protection. Summary of the Invention

[0007] The purpose of this invention is to provide a salt-tolerant hydrogel composite high-energy degrading bacteria.

[0008] To achieve the above objectives, the present invention provides a method for preparing salt-tolerant hydrogel composite high-energy degrading bacteria, characterized by comprising the following steps: S1. Preparation of hydrogel matrix solution: The gel matrix is ​​dissolved, then subjected to high pressure treatment at 550-600 MPa, preferably for 10 minutes, and then cooled to 25℃-40℃; the gel matrix is ​​composed of 8.0 w / v %–12.0 w / v % polyvinyl alcohol, 1.0 w / v %–2.0 w / v % sodium alginate and 0.1 w / v %–0.3 w / v % polyoxypropylene triol. S2. Preparation of bacterial solution: Marinobacterium mangrovicola S01 and Vibrio tritonius K07 were mixed to form a mixed bacterial solution, and then the mixed bacterial solution was thoroughly mixed with the above hydrogel matrix solution to obtain a bacterial mixture. S3. Immobilization of highly resistant hydrogel composite bacterial agents: S3.1. Droplet formation: The above-mentioned bacterial mixture is dropped into the crosslinking solution at a constant rate. Under the action of a magnetic stirrer, the droplets of the mixture fully contact and solidify into perfectly spherical microspheres. The crosslinking solution is a mixed aqueous solution containing 3.0 w / v %–6.0 w / v % boric acid and 1.0 w / v %–3.0 w / v % calcium chloride, with a pH of 6.0–6.5. S3.2. Low-temperature fixation: The microspheres generated above are further immersed and cured in a cross-linking solution at 2℃-6℃ for 2-6 hours to ensure complete internal cross-linking; to ensure that the hardness of the microspheres meets the standard; S4. Surface modification of high-resistance hydrogel composite bacterial agent: The solidified hydrogel-like microspheres were retrieved and washed with sterile physiological saline until neutral. The microspheres were then immersed in a chitosan solution for modification to form a semi-permeable protective outer membrane on the surface of the microspheres. After the modified microspheres were dried at a low temperature of 25-35℃, salt-resistant hydrogel composite high-energy degrading bacteria were obtained.

[0009] Furthermore, in step S1, the gel matrix is ​​heated and stirred in a water bath at 90℃-95℃ until completely dissolved.

[0010] Furthermore, in step S2, the cell abundance ratio of Marinobacterium mangrovicola S01 and Vibrio tritonius K07 in the mixed bacterial solution is 1:1; Optionally, in step S2, the volume ratio of the mixed bacterial solution to the hydrogel matrix solution is 1:5.

[0011] Furthermore, in step S3.2, the soaking and curing time is 2-6 hours.

[0012] Furthermore, in step S4, the concentration of the chitosan solution is 0.2 w / v %-0.5 w / v %; Optionally, the modification time in step S4 is 20-30 minutes.

[0013] The present invention also provides a salt-tolerant hydrogel composite high-energy degrading bacteria prepared by the preparation method described above.

[0014] The present invention also provides the use of the salt-tolerant hydrogel composite high-energy degrading bacteria in treating high-salinity wastewater.

[0015] Furthermore, the treatment of high-salinity wastewater refers to reducing the content of any one of the following in the high-salinity wastewater: COD, ammonia nitrogen, Nitrite content.

[0016] Furthermore, the high-salinity wastewater refers to aquaculture wastewater; Preferably, aquaculture wastewater refers to wastewater from aquaculture of seafood or livestock that has been fermented.

[0017] The salt-tolerant hydrogel composite high-energy degrading bacteria and its preparation method of the present invention have the following advantages compared with existing microbial degrading agent preparation technologies: 1. Suitable for high-salinity wastewater treatment: Most existing wastewater treatment bacterial agents are derived from freshwater or ordinary terrestrial environments, and cannot be used for high-salinity wastewater treatment. The bacterial strains of this invention are derived from mangrove wetland sediments used for long-term purification of mariculture tailwater. They are naturally tolerant to high salinity, osmotic pressure fluctuations and complex water stress environments. The bacterial agent has a high colonization rate and is not easily inactivated. Compared with commercially available bacterial agents, the colonization survival time can be extended by at least 3 months, and it can play a stable role in the long term.

[0018] 2. Comprehensive degradation efficiency and simultaneous removal of multiple pollutants: The complex bacterial community in the salt-tolerant hydrogel composite high-energy degrading bacteria has complementary functions and can synergistically degrade a variety of typical pollutants in water, such as organic matter, ammonia nitrogen, and nitrite. It has a high purification capacity for marine aquaculture wastewater, a wide range of applications, and strong treatment capabilities.

[0019] 3. Excellent compatibility with native microbial communities and low ecological risk: The selected wetland native microorganisms are highly compatible with the marine aquaculture habitat and will not cause the invasion of alien organisms. After application, they can be integrated into the original aquatic micro-ecosystem and help restore the aquatic ecosystem.

[0020] 4. Hydrogel matrix and semi-permeable membrane modification technology enhance the degradation ability and colonization time of the composite degrading bacterial agent. This invention uses polypropylene triol (PPT) as a modifier, which plays an excellent role in defoaming and structural fine-tuning during preparation in a 90℃-95℃ water bath. The prepared hydrogel microspheres have a highly uniform internal network, ensuring that nutrients in the wastewater can smoothly penetrate into the microspheres for bacterial degradation, while also endowing the microspheres with extremely high resistance to mechanical shearing and salting-out swelling, enabling them to withstand long-term aeration and scouring by seawater. Furthermore, the polyelectrolyte semi-permeable outer membrane formed by the reaction of chitosan and sodium alginate (SA) effectively slows down the penetration and diffusion rate of toxic substances such as heavy metals and residual antibiotics into the microspheres, providing valuable buffer time for the core bacterial community. Simultaneously, this semi-permeable membrane locks in the high-density core bacterial community inside the microspheres, preventing disordered loss during large-volume scouring, ensuring the long-term colonization and efficient proliferation of the composite bacterial agent in the treatment of marine aquaculture wastewater.

[0021] 5. Low operating and maintenance costs: No need for complex physical and chemical treatment equipment, can be added in situ, and is easy to operate; the bacterial community has a long-lasting effect and the frequency of addition is low. Compared with traditional treatment processes and ordinary bacterial agents, the overall operating cost is lower.

[0022] 6. Compared with similar products on the market, the present invention is not a simple “carrier + bacteria” in terms of structure, but a microscopic “multi-layer defense and tiered reactor” constructed through multi-component composite and surface modification, as shown in Table 1.

[0023] Table 1. Performance Comparison of Similar Products

[0024] Note: Traditional encapsulation products include industrial microbial agents for wastewater treatment, nitrifying bacteria, denitrifying bacteria, and activated sludge encapsulation pellets. Attached Figure Description

[0025] Figure 1 This invention is a mangrove marine bacterium ( Marinobacterium mangrovicola Phylogenetic tree diagram of S01 based on the 16S rRNA gene sequence; Figure 2 This invention is Vibrio tectorum ( Vibrio tritonius Phylogenetic tree diagram of K07 based on the 16S rRNA gene sequence; Figure 3 This is an electron microscope schematic diagram of the internal structure of a salt-tolerant hydrogel composite high-energy degrading bacteria. Detailed Implementation

[0026] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0027] In the following embodiments: COD and ammonia nitrogen were determined using the alkaline potassium permanganate method and the indophenol blue spectrophotometric method, respectively, as specified in "Marine Monitoring Standard Part 4: Seawater Analysis" (GB17378.4 / 39.1-2007).

[0028] Nitrite was determined using the continuous flow colorimetric method as specified in the Technical Specifications for Environmental Monitoring of Nearshore Marine Areas, Part III: Water Quality Monitoring of Nearshore Marine Areas (HJ442.3-2020).

[0029] Total nitrogen was measured using HJ 636. 2012 Alkaline potassium persulfate digestion-ultraviolet spectrophotometry.

[0030] Total phosphorus shall conform to GB 11893 1989 Ammonium molybdate spectrophotometric method.

[0031] A method for preparing a salt-tolerant hydrogel composite high-energy degrading bacteria, characterized by comprising the following steps: S1. Preparation of hydrogel matrix solution: The gel matrix is ​​dissolved, then subjected to high pressure treatment at 550-600 MPa for 10 min, and then cooled to 25℃-40℃; the gel matrix is ​​composed of polyvinyl alcohol with a mass fraction of 8.0 w / v %–12.0 w / v %, sodium alginate with a mass fraction of 1.0 w / v %–2.0 w / v %, and polyoxypropylene triol with a mass fraction of 0.1 w / v %–0.3 w / v %. S2. Preparation of bacterial solution: Marinobacterium mangrovicola S01 and Vibrio tritonius K07 were mixed to form a mixed bacterial solution, and then the mixed bacterial solution was thoroughly mixed with the above hydrogel matrix solution to obtain a bacterial mixture. S3. Immobilization of highly resistant hydrogel composite bacterial agents: S3.1. Droplet formation: The above-mentioned bacterial mixture is dropped into the crosslinking solution at a constant rate. Under the action of a magnetic stirrer, the droplets of the mixture fully contact and solidify into perfectly spherical microspheres. The crosslinking solution is a mixed aqueous solution containing 3.0 w / v %–6.0 w / v % boric acid and 1.0 w / v %–3.0 w / v % calcium chloride, with a pH of 6.0–6.5. S3.2. Low-temperature fixation: The microspheres generated above are further immersed and cured in a cross-linking solution at 2℃-6℃ for 2-6 hours to ensure complete internal cross-linking and that the hardness of the microspheres meets the standard; S4. Surface modification of high-resistance hydrogel composite bacterial agent: The solidified hydrogel-like microspheres were retrieved and washed with sterile physiological saline until neutral. The microspheres were then immersed in a chitosan solution for modification to form a semi-permeable protective outer membrane on the surface of the microspheres. After the modified microspheres were dried at low temperature, the salt-tolerant hydrogel composite high-energy degrading bacteria were obtained.

[0032] Furthermore, in step S1, the gel matrix is ​​heated and stirred in a water bath at 90℃-95℃ until completely dissolved.

[0033] Furthermore, in step S1, the high-pressure treatment involves placing the dissolved gel matrix into an ultra-high pressure device at 550-600 MPa for 10 minutes.

[0034] Furthermore, in step S2, the cell abundance ratio of Marinobacterium mangrovicola S01 and Vibrio tritonius K07 in the mixed bacterial solution is 1:1; Optionally, in step S2, the volume ratio of the mixed bacterial solution to the hydrogel matrix solution is 1:5.

[0035] Furthermore, in step S3.2, the soaking and curing time is 2-6 hours.

[0036] Furthermore, in step S4, the concentration of the chitosan solution is 0.2 w / v %-0.5 w / v %; Optionally, the modification time in step S4 is 20-30 minutes.

[0037] Example 1: Preparation of salt-tolerant hydrogel composite high-energy degrading bacteria Step 1: Screening and synergistic domestication of salt-tolerant and heavy metal-degrading bacteria from mangrove sediments Take 5-10g of shallow surface sediment (10-15cm depth) from the mangrove wetland in Longhai District, Zhangzhou City, Fujian Province (used for purifying mariculture wastewater) and add it to a conical flask containing 100mL of double-distilled water. Seal the flask with sealing film and shake it at 25-30℃ for 24 hours at 150-180 rpm to obtain a mangrove sediment suspension. Transfer 1mL of the mangrove sediment suspension to 100mL of 2216E liquid medium containing NaCl. Use a gradient salting method (increase the NaCl mass fraction from 1.0% to 3.5% in 0.5% increments) for cross-subculturing. After acclimatization, the strain is then subjected to nitrite stress (NO2). - -N concentration is gradually increased from 5 mg / L to 50 mg / L in increments of 5 mg / L) or ammonia nitrogen stress method (NH4+). + The -N concentration was gradually increased from 5 mg / L to 50 mg / L in increments of 5 mg / L. Multiple generations of alternating acclimatization were carried out (each generation was cultured for 24-48 hours, and high-concentration transfer was performed every 24-48 hours). Salt-tolerant and highly efficient denitrifying / ammonia-oxidizing bacteria were screened out, and 11 strains were obtained, which were named 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9#, 10#, and 11# in sequence.

[0038] Step 2: Verification of strain degradation ability and study of degradation mechanism Single-strain degradation capacity verification: The 11 selected strains were activated to the logarithmic growth phase and then inoculated into high-salt, high-nitrogen marine aquaculture wastewater of Litopenaeus vannamei at an inoculation rate of 5% (v / v). They were then cultured in a constant-temperature shaker (conditions: temperature 25-30℃, rotation speed 150-180 rpm, culture period 3 days). Samples were taken every 12 hours to measure the concentrations of COD, ammonia nitrogen, and nitrite in the wastewater, and the degradation rates of COD, ammonia nitrogen, and nitrite by each strain were calculated (results are shown in Table 2).

[0039] Table 2. Degradation results of COD, ammonia nitrogen, and nitrite by each strain.

[0040] Note: The control group consisted of marine aquaculture wastewater from Litopenaeus vannamei, without the addition of any microbial strains.

[0041] As shown in Table 2, #1 and #7 exhibited the best effects in degrading COD, ammonia nitrogen, and nitrite. After degradation by #1, the COD concentration decreased to 31 mg / L, ammonia nitrogen to 1.22 mg / L, and nitrite to 0.45 mg / L. After degradation by #7, the COD concentration decreased to 27 mg / L, ammonia nitrogen to 1.51 mg / L, and nitrite to 0.35 mg / L.

[0042] Based on the results, strains #1 and #7, which have the highest degradation capacity for COD, ammonia nitrogen, or nitrite, were finally selected to form a composite high-energy degrading bacteria.

[0043] The two degrading bacteria strains (1# and 7#) isolated above were subjected to 16S rDNA high-throughput sequencing and transcriptome functional analysis. The obtained gene sequences were sequenced (as shown in SEQ ID NO:1 and SEQ ID NO:2) and compared to determine the species of the degrading bacteria. (See [link to relevant documentation]). Figure 1-2 After quality control of the obtained transcriptome data, mapping was performed with the genome obtained from NCBI for this strain to determine the expression level of each gene. DESeq and other software were then used to identify significantly differentially expressed genes under different treatments. KEGG annotation and pathway enrichment analysis were performed on the identified differentially expressed genes, and the metabolic pathways were visualized using iPath 2.0. RT-qPCR was used to validate the significantly differentially expressed genes in the transcriptome, and the up- and down-regulated expression levels of these genes were calculated to determine the main function and metabolic mechanism of this degrading bacterium.

[0044] The sequence of Marinobacterium mangrovicola S01 is as follows:

[0045] >Vibrio tritonius K07

[0046] The two strains obtained from the above screening and their main functions are as follows: ①1# is *Bacillus mangroveensis* (… Marinobacterium mangrovicola S01): Naturally adapted to high-salt environments, it has a high adaptability to the environment, and due to its diverse metabolism, it can degrade a variety of complex and recalcitrant organic pollutants. Its preservation information is as follows: Strain name: Mangrove marine bacillus ( Marinobacterium mangrovicola S01); Preservation institution: Guangdong Provincial Microbial Culture Collection Center, GDMCC; Accession number: GDMCCNo: 68772; Deposit date: August 4, 2026; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0047] ②7# is Vibrio tectorum ( Vibrio tritonius K07: It has extremely strong salt tolerance and is mainly responsible for heterotrophic denitrification, while also decomposing nitrogen-containing organic compounds in the environment.

[0048] Its preservation information is as follows: Strain name: Vibrio tectorum ( Vibrio tritonius K07); Preservation institution: Guangdong Provincial Microbial Culture Collection Center, GDMCC; Accession number: GDMCCNo: 68773; Deposit date: August 4, 2026; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0049] Study on the ratio of compound high-energy degrading bacteria: The two bacteria mentioned above were activated to the logarithmic growth phase, and then *Bacillus mangroveensis* and *Vibrio tectorum* were mixed at different cell abundance ratios. After mixing, the mixture was transferred at a 5% (v / v) inoculation rate into high-salt, high-nitrogen marine aquaculture wastewater from *Litopenaeus vannamei*, and cultured in a constant-temperature shaker (conditions: temperature 25-30℃, rotation speed 150-180 rpm, culture period 3 days). The degradation capabilities of different treatment groups for COD, ammonia nitrogen, and nitrite are shown in Table 3.

[0050] Table 3. Degradation results of COD, ammonia nitrogen, and nitrite in different treatment groups.

[0051] Note: The control group consisted of marine aquaculture wastewater from Litopenaeus vannamei, without the addition of any microbial strains.

[0052] Table 3 shows that when the abundance ratio of *Bacillus mangroves* to *Vibrio tectorum* is (2-3):1, the degradation effects on COD, ammonia nitrogen, and nitrite are all very good, with COD decreasing to 11-12 mg / L, ammonia nitrogen decreasing to 0.52-0.95 mg / L, and nitrite decreasing to 0.25-0.57 mg / L. After comprehensive consideration, the optimal 2:1 ratio of high-energy degrading bacteria was selected.

[0053] Step 3: Immobilization of highly resistant hydrogel composite bacterial agent Preparation of the hydrogel matrix solution: Dissolve the gel matrix, which consists of 8.0%–12.0% polyvinyl alcohol (PVA), 1.0%–2.0% sodium alginate (SA), and 0.1%–0.3% polypropylene triol (PPT) by mass. Heat and stir in a water bath at 90℃–95℃ until completely dissolved. To ensure a finer dispersion and more uniform gel pores, treat the dissolved solution in an ultra-high pressure apparatus (550–600 MPa, 10 min). Then cool to 25℃–40℃ (too high a temperature will kill bacteria, too low a temperature will cause PVA to solidify prematurely).

[0054] Bacterial suspension mixing: Mix the bacterial suspension of the composite high-energy degrading bacteria obtained in step two (cell concentration > 10). 8 The CFU / mL solution was thoroughly mixed with the cooled gel matrix at a volume ratio of 1:5, and stirred slowly to ensure uniformity, avoiding the generation of a large number of air bubbles, to obtain a bacterial mixture.

[0055] Preparation of crosslinking solution: Prepare a mixed aqueous solution containing 3.0%–6.0% boric acid (saturated) and 1.0%–3.0% calcium chloride, and adjust the pH to 6.0–6.5 using NaHCO3.

[0056] Droplet addition to form microspheres: Using a peristaltic pump connected to a sizing nozzle (with an inner diameter of 0.8-1.2 mm), the bacterial mixture is dripped into the crosslinking solution at a constant rate (e.g., 0.5–2.0 mL / min). Under the action of a magnetic stirrer, the droplets of the mixture come into full contact with the crosslinking solution and solidify into perfectly round microspheres.

[0057] Low-temperature fixation: The microspheres generated above are further immersed and cured in a cross-linking solution at 2℃-6℃ for 2-6 hours to ensure complete internal cross-linking and that the hardness of the microspheres meets the standard.

[0058] The resulting perfectly spherical microspheres are called microspheres S.

[0059] Step 4: Surface modification of high-resistance hydrogel composite bacterial agent The solidified hydrogel-like microspheres are retrieved and washed with sterile physiological saline until neutral. The microspheres are then immersed in a 0.2%-0.5% (w / w) chitosan solution for 20-30 minutes (preferably 0.4% (w / w) chitosan solution for 20 minutes) to form a semi-permeable protective outer membrane on the microsphere surface. This membrane effectively prevents the disordered loss of the internal core bacterial species and slows down the penetration and diffusion of external heavy metals, antibiotics, and other toxic substances into the interior. After drying the modified microspheres at a low temperature (e.g., 25-35°C, preferably 30°C), the salt-tolerant hydrogel composite high-energy degrading bacteria are obtained.

[0060] An electron micrograph of the internal structure of the salt-tolerant hydrogel composite high-energy degrading bacteria is shown below. Figure 3 ,from Figure 3 It can be seen that the salt-tolerant hydrogel composite high-energy degrading bacteria has a porous three-dimensional network structure with uniform internal pore height, forming a PVA-SA-PPT ternary interpenetrating network framework. This structure exhibits resistance to mechanical shear forces and salt precipitation swelling, and can withstand long-term aeration and scouring of seawater. The specific surface area is 1.7268 m². 2 / kg, average pore size is 1.352mm, total pore volume is 0.065cm³. 3 / kg provides more effective contact and attachment surface area for microorganisms. This salt-tolerant hydrogel composite high-energy degrading bacteria has an optimized mass transfer radial spatial gradient of "high oxygen-micro oxygen-anaerobic". Because of the different structures and pores, oxygen is present inside the hydrogel. Different structures and oxygen concentrations in the air can cause different reactions in the microbial community.

[0061] Example 2: Application in seahorse farming wastewater Take 100g of the above-mentioned salt-tolerant hydrogel composite high-energy degrading bacteria and add 1m 3 In the aeration tank of the seahorse farming wastewater treatment plant (salinity 25-32‰), samples were taken every 24 hours for 30 consecutive days, then every 15 days thereafter, continuing for 90 days. The COD, ammonia nitrogen, and nitrite levels in the collected water samples were determined. The results are shown in Table 4.

[0062] Table 4. Application results of salt-tolerant hydrogel composite high-energy degrading bacteria in seahorse aquaculture wastewater

[0063] Table 4 shows that the salt-tolerant hydrogel composite high-energy degrading bacteria of this invention have a very good dynamic treatment effect on seahorse farming wastewater for 90 consecutive days. After adding the salt-tolerant hydrogel composite high-energy degrading bacteria of this invention, the water quality on the second day met the requirements for wastewater reuse and could continue to be used for seahorse farming. This example tested for 90 days, and the results in Table 4 show that the salt-tolerant hydrogel composite high-energy degrading bacteria of this invention have a high colonization rate and are not easily inactivated. The colonization survival time can be extended by at least 3 months, and it plays a stable role in the long term.

[0064] Example 3: Application in wastewater from Litopenaeus vannamei farming Take 100g of the above-mentioned salt-tolerant hydrogel composite high-energy degrading bacteria and add 1m 3 In the aeration tank of Litopenaeus vannamei farm wastewater (salinity 22-30‰), samples were taken every 24 hours for 30 consecutive days. The COD, ammonia nitrogen, and nitrite in the water samples were measured. The results are shown in Table 5.

[0065] Table 5. Application results of salt-tolerant hydrogel composite high-energy degrading bacteria in seahorse aquaculture wastewater.

[0066] Table 5 shows that the salt-tolerant hydrogel composite high-energy degrading bacteria of this invention have a very good dynamic treatment effect on the wastewater from Litopenaeus vannamei farming for 30 consecutive days. After adding the salt-tolerant hydrogel composite high-energy degrading bacteria of this invention, the water quality on the second day met the requirements for wastewater reuse and could continue to be used for Litopenaeus vannamei farming. This example tested for 30 days, and the results in Table 4 show that the salt-tolerant hydrogel composite high-energy degrading bacteria of this invention have a high colonization rate and are not easily inactivated.

[0067] Example 4: Application in pig farming wastewater One kg of the salt-tolerant hydrogel composite high-energy degrading bacteria of this invention was added to the aerobic tank of pig farming wastewater (project scale: 800 tons / day, aerobic tank retention time: 4 hours) to enhance the treatment efficiency of the original treatment system. The treatment efficiency before and after 4 hours of addition is shown in Table 6. The pig farming wastewater is the total wastewater that needs to be treated after fermentation of pig manure and urine, with a salinity typically between 35-45‰.

[0068] Table 6 Comparison of water quality before and after adding salt-tolerant hydrogel composite high-energy degrading bacteria to pig farming wastewater

[0069] Note: Degradation rate = (Influent water quality - Effluent water quality) / Influent water quality 100%.

[0070] As shown in Table 6, the salt-tolerant hydrogel composite high-energy degrading bacteria of the present invention can effectively treat pig farming wastewater with high salt, high organic matter, and high ammonia nitrogen. After adding the salt-tolerant hydrogel composite high-energy degrading bacteria for 4 hours, the degradation efficiency of the main pollutants (COD, ammonia nitrogen, total nitrogen, and total phosphorus) in the biochemical section of pig farming tailwater reached 93.21%, 96.03%, 95.29%, and 91.26%, respectively.

[0071] Example 4: Stirring Shear Test To verify the extremely high dynamic mechanical scouring, shearing, and collision resistance of the salt-tolerant hydrogel composite high-energy degrading bacteria, 100 salt-tolerant hydrogel composite high-energy degrading bacteria were added to a 5L beaker, along with 2L of artificial seawater. Four control groups were set up: three groups were stirred at speeds of 100, 200, and 300 rpm, respectively, while the other group was aerated and stirred using an aeration device. The four groups were treated for 12, 24, 48, and 72 hours, respectively, and the integrity rate was recorded after treatment. The results are shown in Table 7.

[0072] Completeness rate: R=N t / N0×100%.

[0073] in: R is the completeness rate; N0 = Initial number of intact microspheres; N t = Number of intact microspheres after shearing.

[0074] Table 7. Integrity Rate of Salt-Tolerant Hydrogel Composite High-Energy Degrading Bacteria (%)

[0075] As can be seen from Table 7, the salt-tolerant hydrogel composite high-energy degrading bacteria of the present invention have resistance to mechanical shearing and resistance to salt precipitation swelling, and can withstand long-term aeration and scouring of seawater.

[0076] Example 5 Resistance Experiment Take 100g of the microspheres S1 obtained in Example 1 (without step four treatment) and 100g of the salt-tolerant hydrogel composite high-energy degrading bacteria obtained in Example 1 (after step four modification treatment), and add them to 1m 3 Sampling was conducted on artificial seawater (with increased organic matter and ammonia nitrogen content) and pig farming wastewater after 12 hours of aeration. The COD and ammonia nitrogen levels were then measured, and the results are shown in Table 8. The raw pig farming wastewater was found to have a total copper content of 12.3 mg / L, a total zinc content of 18.7 mg / L, and a total antibiotic content (including tetracyclines, sulfonamides, and macrolides) of 300 μg / L. This indicates that the pig farming wastewater is a high-salt wastewater contaminated with heavy metals and antibiotics, while the artificial seawater does not contain heavy metals or antibiotics.

[0077] Table 8 Results of Resistance Test

[0078] As can be seen from Table 8, the salt-tolerant hydrogel composite high-energy degrading bacteria (after modification treatment in step four) obtained in Example 1 of the present invention can survive in water containing heavy metals and antibiotics, and greatly remove the COD and ammonia nitrogen content in the water.

[0079] This indicates that pig farming wastewater is high-salt wastewater contaminated with heavy metals and antibiotics, while artificial seawater does not contain heavy metals or antibiotics. This experiment demonstrates the important role of surface modification with salt-tolerant hydrogel composite high-energy degrading bacteria in resisting heavy metals and antibiotics, as shown in step four.

[0080] The experimental results above show that the modified salt-tolerant hydrogel composite high-energy degrading bacteria have a certain resistance to pig farming wastewater containing heavy metals and antibiotics, and have a better degradation effect on pollutants.

[0081] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A method for preparing a salt-tolerant hydrogel composite high-energy degrading bacteria, characterized in that, Includes the following steps: S1. Preparation of hydrogel matrix solution: The gel matrix is ​​dissolved, then subjected to high pressure treatment at 550-600 MPa, and then cooled to 25℃-40℃; the gel matrix is ​​composed of 8.0 w / v %–12.0 w / v % polyvinyl alcohol, 1.0 w / v %–2.0 w / v % sodium alginate and 0.1 w / v %–0.3 w / v % polyoxypropylene triol; S2. Preparation of bacterial solution: Marinobacterium mangrovicola S01 and Vibrio tritonius K07 were mixed to form a mixed bacterial solution, and then the mixed bacterial solution was thoroughly mixed with the above hydrogel matrix solution to obtain a bacterial mixture. S3. Immobilization of highly resistant hydrogel composite bacterial agents: S3.

1. Droplet addition to form microspheres: The above-mentioned bacterial mixture is added dropwise to the crosslinking solution at a constant rate. Under the action of a magnetic stirrer, the droplets of the mixture fully contact and solidify into perfectly spherical microspheres. The crosslinking solution is a mixed aqueous solution containing 3.0 w / v %–6.0 w / v % boric acid and 1.0 w / v %–3.0 w / v % calcium chloride, with a pH of 6.0–6.

5. S3.

2. Low-temperature fixation: The microspheres generated above are further immersed and cured in a cross-linking solution at 2℃-6℃ for 2-6 hours to ensure complete internal cross-linking; S4. Surface modification of high-resistance hydrogel composite bacterial agent: The solidified hydrogel-like microspheres were retrieved and washed with sterile physiological saline until neutral. The microspheres were then immersed in a chitosan solution for modification to form a semi-permeable protective outer membrane on the surface of the microspheres. The modified microspheres were dried at a low temperature of 25-35℃ to obtain salt-tolerant hydrogel composite high-energy degrading bacteria.

2. The preparation method according to claim 1, characterized in that, In step S1, the gel matrix is ​​heated and stirred in a water bath at 90℃-95℃ until it is completely dissolved.

3. The preparation method according to claim 1, characterized in that, In step S2, the cell abundance ratio of Marinobacterium mangrovicola S01 to Vibrio tritonius K07 in the mixed bacterial solution is 2:1; In step S2, the volume ratio of the mixed bacterial solution to the hydrogel matrix solution is 1:

5.

4. The preparation method according to claim 1, characterized in that, In step S4, the concentration of the chitosan solution is 0.2 w / v % - 0.5 w / v % %. In step S4, the modification time is 20-30 minutes.

5. A salt-tolerant hydrogel composite high-energy degrading bacterium prepared by any one of the preparation methods of claims 1-4.

6. The use of the salt-tolerant hydrogel composite high-energy degrading bacteria according to claim 5 in the treatment of high-salinity wastewater.

7. The use as described in claim 6, characterized in that, The treatment of high-salinity wastewater refers to reducing the content of any one of the following in the high-salinity wastewater: COD, ammonia nitrogen, Nitrite content.

8. The use as described in claim 6, characterized in that, The high-salinity wastewater refers to aquaculture wastewater.

9. The use as described in claim 8, characterized in that, The aforementioned aquaculture wastewater refers to wastewater from aquaculture of seafood or livestock, which is then fermented.

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