Acinetobacter baumannii and application thereof in denitrification

Abstract

The invention discloses acinetobacter baumannii and application thereof.The acinetobacter baumannii is preserved in Guangdong Microbial Culture Collection Center on August 16, 2024, the preservation number is GDMCC No: 65015, the acinetobacter baumannii has a good denitrification effect, and the degradation rate of ammonia nitrogen reaches 94.93%; the degradation rate on nitrate reaches 99.53%; and the degradation rate of nitrite reaches 96.94%. The immobilized microbial agent is further prepared from the bacillus subtilis and pseudomonas aeruginosa with vibrio parahaemolyticus resistance, and the immobilized microbial agent has good effects in the aspects of denitrification and vibrio parahaemolyticus resistance, especially shows better denitrification effect and antibacterial effect in actual culture application, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically, to Acinetobacter baumannii and application thereof in denitrification. Background Art

[0002] Litopenaeus vannamei (Boone), also known as Penaeus vannamei, was introduced and promoted by the Chinese Academy of Sciences in 1988. Since the 1990s, Penaeus vannamei has gradually become the most important shrimp culture variety in my country.

[0003] At present, the farming modes of whiteleg shrimp are mainly divided into pond farming and factory farming. Pond farming includes traditional earth pond farming and high-level pond farming, and factory farming includes cement ground flowing water farming and recirculating water farming. In terms of farming density, except for traditional earth pond farming, which belongs to the original low-density farming mode, the rest belong to high-density farming mode.

[0004] One of the most serious problems caused by high-density shrimp farming is the deterioration of water quality. The rapid increase in ammonia nitrogen and nitrite content in the farming water will pose a serious threat to the survival of the shrimp, and the farming wastewater containing excessive nitrates will have a certain impact on the ecology.

[0005] Under the high-density farming model, the rapid reproduction of a large number of pathogens due to the deterioration of water quality and the resulting infection of shrimp groups are also one of the serious problems in the farming process.

[0006] Acute hepatopancreas necrosis syndrome (AHPNS) caused by Vibrio parahaemolyticus is one of the most serious diseases in the past two years. It is particularly difficult to treat because of its rapid progression and high mortality rate. The most typical feature of shrimp infected with AHPNS is the obvious atrophy and pale color of the hepatopancreas. The other features are whitening of the whole body, softening of the carapace and easy peeling, and severe jejunum. Once infected, if not treated in time, 100% of shrimp will die within 1 to 3 days after the onset of the disease.

[0007] Therefore, in the process of high-density aquaculture, water bodies need to be treated in a timely manner. Biological methods are a powerful and emerging means of managing aquaculture water bodies. With the development of aquaculture, not only can beneficial microorganisms be added to aquaculture water bodies, but also related products of beneficial microorganisms. These live bacteria and related products are collectively referred to as microecological preparations. On the one hand, microecological preparations can purify water quality and maintain water ecological balance. On the other hand, they can effectively prevent and treat diseases, enhance the digestive and immune characteristics of aquatic animals, and will not make animals resistant to drugs. Summary of the invention

[0008] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an Acinetobacter baumannii and its application in denitrification.

[0009] The first object of the present invention is Acinetobacter baumannii.

[0010] The second object of the present invention is the use of the Acinetobacter baumannii in denitrification and / or preparation of denitrification products.

[0011] A third object of the invention is a product for denitrification.

[0012] A fourth object of the present invention is a bacterial composition.

[0013] A fifth object of the present invention is the use of the bacterial composition in denitrification and / or preparation of denitrification products.

[0014] The sixth object of the present invention is the use of the bacterial composition in combating Vibrio parahaemolyticus and / or preparing anti-Vibrio parahaemolyticus products.

[0015] The seventh object of the present invention is a bacterial agent.

[0016] The eighth object of the present invention is a method for preparing an immobilized bacterial agent.

[0017] The ninth objective of the present invention is the immobilized bacterial agent prepared by the preparation method.

[0018] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0019] The present invention claims protection for an Acinetobacter baumannii D-4, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 16, 2024, with the deposit number GDMCC No: 65015.

[0020] It is further claimed that the use of the Acinetobacter baumannii in denitrification and / or preparation of denitrification products is protected, wherein the denitrification is the degradation of one or more of ammonia nitrogen, nitrate and nitrite.

[0021] And, a product for denitrification, containing the Acinetobacter baumannii, wherein the denitrification is the degradation of one or more of ammonia nitrogen, nitrate and nitrite.

[0022] It also claims protection for a bacterial composition containing Pseudomonas aeruginosa and Acinetobacter baumannii, wherein the Pseudomonas aeruginosa is Pseudomonas aeruginosa D-1, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 31, 2024, with a deposit number of GDMCC No: 64925; the Acinetobacter baumannii is the Acinetobacter baumannii, and the denitrification is the degradation of one or more of ammonia nitrogen, nitrate and nitrite.

[0023] Preferably, calculated based on the number of bacteria, the dosage of Pseudomonas aeruginosa and Acinetobacter baumannii is 1-2:1-2.

[0024] Most preferably, the dosage of Pseudomonas aeruginosa and Acinetobacter baumannii is 2:1 calculated based on the number of bacteria.

[0025] The following applications are also claimed:

[0026] The bacterial composition is used in denitrification and / or preparation of denitrification products, wherein the denitrification is the degradation of one or more of ammonia nitrogen, nitrate and nitrite.

[0027] Application of the bacterial composition in fighting against Vibrio parahaemolyticus and / or in preparing products against Vibrio parahaemolyticus.

[0028] Preferably, the product immobilizes bacterial agents.

[0029] Also claimed is a bacterial agent containing the Acinetobacter baumannii or the bacterial composition

[0030] And, a method for preparing an immobilized bacterial agent, comprising fully dissolving and uniformly mixing polyvinyl alcohol, sodium alginate and kaolin to obtain a mixed solution, fully mixing the mixed solution with a bacterial solution containing the bacterial composition, dropping the solution into a CaCl2 solution, and solidifying the solution.

[0031] Preferably, the bacterial solution is prepared by: inoculating the Pseudomonas aeruginosa and the Acinetobacter baumannii into LB liquid culture medium respectively, culturing with shaking, adjusting the OD600 of the culture solution to 0.95-1.05, and mixing them in a volume ratio of 1-2:1-2.

[0032] Most preferably, the bacterial solution is prepared by: inoculating the Pseudomonas aeruginosa and the Acinetobacter baumannii into LB liquid culture medium respectively, culturing with shaking, adjusting the OD600 of the culture solution to 0.95-1.05, and mixing them in a volume ratio of 2:1.

[0033] Preferably, the concentrations of polyvinyl alcohol, sodium alginate and kaolin in the mixed solution are 6-10 g / 100 ml, 2-3 g / 100 ml and 2-3 g / 100 ml respectively.

[0034] More preferably, the concentrations of polyvinyl alcohol, sodium alginate and kaolin in the mixed solution are 6.5-9.5 g / 100 ml, 2-3 g / 100 ml and 2-3 g / 100 ml respectively.

[0035] More preferably, the concentrations of polyvinyl alcohol, sodium alginate and kaolin in the mixed solution are 6.5-7.5 g / 100 ml, 2-3 g / 100 ml and 2-3 g / 100 ml respectively.

[0036] Most preferably, the concentrations of polyvinyl alcohol, sodium alginate and kaolin in the mixed solution are 7 g / 100 ml, 2.5 g / 100 ml and 2.5 g / 100 ml respectively.

[0037] It is further claimed that the immobilized bacterial agent prepared by the preparation method is protected.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention discloses an Acinetobacter baumannii and its application. The Acinetobacter baumannii was deposited in the Guangdong Provincial Microbial Culture Collection Center on August 16, 2024, with a deposit number of GDMCC No: 65015. It has a good denitrification effect, and the degradation rate of ammonia nitrogen reaches 94.93%; the degradation rate of nitrate reaches 99.53%; and the degradation rate of nitrite reaches 96.94%. It is further used with Pseudomonas aeruginosa that also has resistance to Vibrio parahaemolyticus to prepare an immobilized bacterial agent. The immobilized bacterial agent has good effects in denitrification and resistance to Vibrio parahaemolyticus, especially in actual breeding applications. It shows better denitrification effect and antibacterial effect, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The colony morphology of strains D-1 (a) and D-4 (b) are shown.

[0041] Figure 2 The scanning electron microscopic morphologies of strains D-1 (a) and D-4 (b) are shown.

[0042] Figure 3 Agarose gel electrophoresis of D-1 and D-4 16S rDNA PCR products.

[0043] Figure 4 Phylogenetic tree of strains D-1(a) and D-4(b) based on 16S rDNA gene

[0044] Figure 5 NH4 + -Denitrification characteristics of bacteria when N is the only nitrogen source.

[0045] Figure 6 The NO3 - -Denitrification characteristics of bacteria when N is the only nitrogen source.

[0046] Figure 7 The NO2 - -Denitrification characteristics of bacteria when N is the only nitrogen source.

[0047] Figure 8 This is the counting result of co-culture of strain D-1 and Vibrio parahaemolyticus.

[0048] Fig. 9 The colony characteristics of strain D-1 on CAS medium.

[0049] Fig.10 This is the biofilm of strain D-1 stained with crystal violet; left: biofilm formed by bacterial liquid; right: culture medium as control group.

[0050] Fig.11 Colony characteristics of strain D-1 on skimmed milk powder nutrient agar medium at 24h (a) and 48h (b)

[0051] Fig.12 The growth of the strain on the LB plate at a distance of 1.2 cm and 0.6 cm.

[0052] Fig.13 Antibiotic resistance testing for bacterial strains

[0053] Fig.14 The denitrification effect of strain D-1 and strain D-4 at different ratios

[0054] Fig.15 The results show the co-culture counts of strain D-1 and strain D-4 with Vibrio parahaemolyticus at different ratios.

[0055] Fig.16 The denitrification effects of the three groups of immobilized beads were analyzed by one-way Anova. The significant differences were annotated by Duncan method. Different letters indicate significant differences.

[0056] Fig.17 This is the denitrification effect of three cycles of immobilized bacteria.

[0057] Fig.18 Scanning electron microscopic morphology of the surface (a) and interior (b) of the immobilized beads without bacteria added, and the surface (c) and interior (d) of the immobilized bacteria agent

[0058] Fig.19 The scanning electron microscopic morphology of the immobilized bacteria after 3 cycles

[0059] Fig. 20 Changes in ammonia nitrogen (a), nitrate (b), nitrite (c) contents and shrimp survival rate (d) during shrimp farming; data were subjected to Anova one-way analysis of variance, and significant differences were annotated using the Duncan method. Different letters indicate significant differences.

[0060] Fig.21 The survival rate of shrimp (a) and the changes in the number of Vibrio parahaemolyticus (b) during shrimp farming. DETAILED DESCRIPTION

[0061] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0062] Trace element solution: EDTA 50g / L, ZnSO4·7H2O 3.92g / L, MnCl2·4H2O 5.06g / L, FeSO4·7H2O 5.0g / L, MgSO4·7H2O 0.2g / L, CoCl2·6H2O 1.61g / L, CaCl2 5.5g / L, (NH4)6Mo7O2·4H2O 1.1g / L and CuSO4·5H2O 1.57g / L.

[0063] LB medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, pH about 7.0.

[0064] R2A liquid culture medium: 0.25g / L tryptone, 0.5g / L acid hydrolyzed casein, 0.5g / L yeast extract powder, 0.5g / L soluble starch, 0.3g / L dipotassium hydrogen phosphate, 0.1g / L magnesium sulfate, 0.3g / L sodium pyruvate, 0.25g / L peptone and 0.5g / L glucose, pH is about 7.2; R2A solid culture medium is based on R2A liquid culture medium, and 1.5g / L agar is added.

[0065] Nitration (DM) liquid culture medium: K2HPO4 1.50g / L, MgSO4 7H2O 0.01g / L, sodium citrate 5.66g / L, Na2HPO4 7.90g / L, NaNO3 0.841g / L, NaNO2 0.362g / L and NH4Cl 0.192g / L solution pH is about 7.0; nitration (DM) solid culture medium is based on nitration (DM) liquid culture medium, and 1.5g / L agar is added.

[0066] Bromothymol blue (BTB) medium: K2HPO4 1.5g / L, MgSO4 7H2O 0.01g / L, sodium citrate 5.66g / L, Na2HPO4 7.9g / L, NaNO3 0.841g / L, NaNO2 0.362g / L, NH4Cl0.192g / L, trace element solution 2ml, BTB solution 1ml and agar 15g / L, pH is about 7.0.

[0067] Heterotrophic nitrification medium: (NH4)2SO4 0.47 g / L, sodium citrate 5.62 g / L and Wei's salt solution 50 mL.

[0068] Weiss salt solution: K2HPO4 5.0g / L, MgSO4·7H2O 2.5g / L, NaCl 2.5g / L, MnSO4·4H2O0.05g / L and FeSO4·7H2O 0.05g / L.

[0069] Gries reagent: Dissolve 0.5 g of p-aminobenzenesulfonic acid in 50 mL of 30% acetic acid and store in a dark place. Dissolve 0.4 g of α-naphthylamine in 100 mL of distilled water and boil. Mix the colorless supernatant with 6 mL of 80% acetic acid. Mix the two liquids in equal amounts before use.

[0070] MRS medium: peptone 10g / L, beef extract powder 8.0g / L, yeast extract powder 4.0g / L, glucose 20.0g / L, dipotassium hydrogen phosphate 2.0g / L, diammonium hydrogen citrate 2.0g / L, sodium acetate 5.0g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L and Tween 80 1.0g / L. The pH of the solution is about 6.5. MRS solid medium is supplemented with 1.5% agar.

[0071] CAS detection culture medium: chrome azuro blue S (CAS) 60.5 mg / L, hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg / L, ferric chloride hexahydrate 2.645 mg / L, sodium dihydrogen phosphate dihydrate 295.25 mg / L, sodium dihydrogen phosphate dodecahydrate 1213.5 mg / L, ammonium chloride 125 mg / L, potassium dihydrogen phosphate 37.5 mg / L, sodium chloride 62.5 mg / L and agar 9000 mg / L, the solution pH is about 8.6.

[0072] Domestication and breeding of white shrimp: Carry out in a 40cm×23cm×25cm water tank, put 20 shrimps of the same size in each water tank, and cover the water tank with a layer of gauze. Clean up feed residues, feces and dead shrimps in time every morning and evening, feed feeding amount is 7% of shrimp body weight, control daily water change volume to 1 / 3 of the total volume, carry out a major water change every 5 days, the water change volume is 2 / 3 of the total volume, keep the water temperature at 20℃, salinity at 20ppt, dissolved oxygen at 5mg / L, and continuously aerate until the shrimp adapt to the new environment.

[0073] Example 1 Screening of strains

[0074] 1. Bacterial Isolation and Purification

[0075] 1. Experimental methods

[0076] Take 1 ml of aquaculture water (L. vannamei aquaculture water in Shanwei area) and use LB liquid medium, R2A liquid medium and denitrification liquid medium for enrichment culture respectively. The enriched bacterial culture fluid is heated with PBS phosphate buffer for 10 -1 , 10 -2 Up to 10 -8 Perform gradient dilution, spread 200 μL of the diluted bacterial solution on nutrient agar medium, R2A solid medium and DM solid medium, make 3 parallels for each group, and use an equal amount of PBS as a blank control. Culture at 30°C for 1 to 3 days, select as many different single bacteria as possible based on the size, color, transparency, etc. of the colonies, and store in glycerol after 3 times of streak purification.

[0077] 2. Experimental results

[0078] The enriched bacterial culture fluid was diluted and spread on three solid culture media, and a total of 82 strains of bacteria were isolated, of which 30 strains were isolated from LB medium, 26 strains were isolated from R2A medium, and 26 strains were isolated from denitrification medium. The strains were numbered L / R / D-X, where L, R, and D represent LB, R2A, and DM culture media, respectively, and X represents a digital number.

[0079] 2. Preliminary screening of denitrifying strains

[0080] 1. Experimental methods

[0081] The purified single strain was inoculated into LB medium for activation. 10 μL of the activated bacterial liquid was dropped onto the BTB denitrification identification medium, and an equal amount of sterile LB medium was used as a blank control. The culture was incubated in an inverted position at 30 °C. Due to the action of nitrate reductase in denitrifying bacteria, nitrate will be reduced to nitrite, thereby increasing the pH of the medium. Under the action of the acid-base indicator BTB, the medium changes from green to blue, and then the denitrification ability of the bacteria can be preliminarily judged.

[0082] 2. Experimental results

[0083] Among the 82 isolated single strains, 13 strains had denitrification ability. The screening results are shown in Table 1. Among them, the media of 3 strains (L-8, D-1, and D-4) quickly turned blue within 24 h after inoculation, and the color was relatively deep. It was preliminarily determined that these 3 strains had the strongest denitrification ability; the media of 5 strains turned blue within 48 h after inoculation, with varying degrees of color depth, and the denitrification ability was relatively strong; the media of 5 strains also changed color after 48 h of inoculation, having denitrification ability, but the ability was weak.

[0084] Table 1 Preliminary screening of denitrifying strains

[0085]

[0086] Note: 1) “+++” represents strong, “++” represents relatively strong, and “+” represents weak

[0087] 2) When the BTB medium turns blue within < 1 day, the denitrification ability is strong “+++”; when 1 day < the BTB medium turns blue time < 2 days, the denitrification ability is relatively strong “++”; when 2 days < the BTB medium turns blue time, the denitrification ability is weak “+”.

[0088] III. Preliminary screening of nitrifying strains

[0089] 1. Experimental method

[0090] The single strains with denitrification function preliminarily screened were inoculated into LB medium for activation. 100 μL of the activated bacterial liquid was inoculated into 100 mL of heterotrophic nitrification medium, and the culture was shaken at 30 °C and 160 r / min for 48 h. 500 μL of the culture solution was taken into a white disk, and an equal amount of sterile medium was used as a blank control, and 2 drops of Griess reagent were added.

[0091] If the medium is red or brown, it is preliminarily judged that there is nitrite nitrogen in the medium. If the medium does not show color, a small amount of zinc powder is added after 5 min. If the medium turns red, it indicates the presence of nitrate nitrogen. The nitrification ability of the bacteria can be preliminarily judged according to the color reaction.

[0092] 2. Experimental results

[0093] The results are shown in Table 2. Among the 13 strains of bacteria with denitrification ability, 8 strains had no detected nitrification function, 5 strains had nitrification ability, among which 3 strains had the strongest nitrification function and 2 strains had relatively strong nitrification ability.

[0094] Combined with the initial screening results of denitrification function, strains D-1 and D-4 have both nitrification and denitrification functions.

[0095] Table 2 Preliminary screening of nitrifying strains

[0096]

[0097] Note: 1) “++” means strong, “+” means relatively strong, and “-” means no

[0098] 2) If the culture medium is pink, the nitrification capacity is strong "++"; if the culture medium is light pink, the nitrification capacity is relatively strong "+"; if the culture medium is colorless, the nitrification capacity is no "-".

[0099] Example 2 Antagonistic Effects of Strain D-1 and Strain D-4 on Vibrio parahaemolyticus

[0100] 1. Experimental Methods

[0101] Vibrio parahaemolyticus was inoculated into LB medium for activation, cultured at 30°C and 160 rpm for 24 h, and diluted to 10 5 Take 1mL of Vibrio parahaemolyticus dilution and mix it with DM agar medium which has been sterilized and cooled to about 40℃. Pour the mixture evenly into a plate as the identification medium for antagonistic bacteria of Vibrio parahaemolyticus.

[0102] The strains D-1 and D-4 were activated in LB medium, and the culture solution was applied to DM medium. The sterile LB culture solution was used as a control. Three groups of parallels were made for each strain. The plate was inverted and cultured at 30℃ for 24 hours. If a transparent inhibition zone appeared on the strain, it was preliminarily determined that the strain was an antagonist of Vibrio parahaemolyticus. The diameter of the inhibition zone was measured with a caliper.

[0103] 2. Experimental Results

[0104] Both strain D-1 and strain D-4 produced inhibition zones, and the diameters of the inhibition zones in DM agar medium were 37.15±0.49 mm and 18.7±1.27 mm, respectively.

[0105] Example 3 Genetic stability test of strains D-1 and D-4

[0106] 1. Experimental Methods

[0107] The strains D-1 and D-4 were subcultured five times on nutrient agar, and each time the strains were seeded on the identification medium of antagonistic bacteria to Vibrio parahaemolyticus, and the diameter of the inhibition zone was recorded. If the inhibition zone appeared in five subcultures and the size was stable, it was determined that the bacteria had the ability to antagonize Vibrio parahaemolyticus in a stably inherited manner.

[0108] 2. Experimental Results

[0109] The results are shown in Table 3. The D-1 strain has a stable and inherited ability to antagonize Vibrio parahaemolyticus, while the antagonistic properties of D-4 are lost during the passage.

[0110] Table 3 Stable genetic inheritance of the ability of D-1 and strains to antagonize Vibrio parahaemolyticus

[0111]

[0112] Example 4 Identification of strains D-1 and D-4

[0113] 1. Colony morphology

[0114] The appearance of strains D-1 and D-4 is as follows Figure 1 As shown: As shown in Figure (a), the single colony of the D-1 strain is light yellow, slightly transparent, round in shape, with neat edges, moist and without bulges; as shown in Figure (b), the single colony of the D-4 strain is beige, opaque, round in shape, with neat edges, moist and with bulges.

[0115] 2. Electron microscopic morphology observation

[0116] 1. Experimental methods

[0117] The bacterial suspension of strains D-1 and D-4 was rinsed twice with physiological saline, 1 mL of 2.5% glutaraldehyde fixative was added and mixed thoroughly, and fixed by standing for 4 h (the time should be extended as much as possible or overnight); after washing with physiological saline for 3 times, gradient ethanol shrinkage was performed, that is, 30%, 50%, 70%, 80%, and 90% ethanol were added once, and 100% ethanol was added twice. After standing for 15 min each time, the supernatant was removed by centrifugation (after dehydration with 70% ethanol, overnight, and gradient dehydration was continued the next day); the samples were sent for drying and then observed by scanning electron microscopy.

[0118] 2. Experimental results

[0119] The morphology of strains D-1 and D-4 under scanning electron microscopy is shown in Figure 2 As shown:

[0120] As shown in Figure (a), the D-1 strain is rod-shaped, has no flagella, no spores, and is (1.5~3.0)μm×(0.5~0.8)μm in size; as shown in Figure (b), the D-4 strain is rod-shaped, has no flagella, no spores, and is (1.0~1.6)μm×(0.6~1.0)μm in size.

[0121] 3. 16S rDNA Identification

[0122] 1. Experimental methods

[0123] After strain D-1 and strain D-4 were activated, the genomic DNA of the target strain was extracted using a bacterial DNA extraction kit and used as a template. The primers selected were bacterial 16S rDNA universal primers 27F (5′-AGAGTTTGATC(A / C)TGGCTCAG-3′) and 1492R (5′-TACGG(C / T)TACCTTGTTACGACTT-3′) for amplification.

[0124] The PCR reaction system is: 2×Taq PCR Mix 12.5μL, Forward Primer (27F) 1μL, Reverse Primer (1492R) 1μL, Template DNA 1μL and ddH2O 9.5μL

[0125] The PCR amplification products were detected by agarose gel electrophoresis and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing after the detection was correct. The sequencing results were submitted to the GenBank database.

[0126] 2. Experimental results

[0127] Results Figure 3 , there was a bright band near 1500bp, and there were no other non-specific amplification bands. The strains D-1 and D-4 were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing to obtain the 16S rDNA of strains D-1 and D-4. The sequencing results showed that the nucleotide sequence of the 16S rDNA of strain D-1 was as shown in SEQ ID NO:1, and the nucleotide sequence of the 16S rDNA of strain D-4 was as shown in SEQ ID NO:2.

[0128] (II) Evolutionary tree analysis

[0129] 1. Experimental methods

[0130] The 16S rDNA P sequences of D-1 and D-4 were analyzed by BLAST alignment on NCBI, and other sequences with higher homology were selected to construct a phylogenetic tree using the Neighbor-Joining method in MEGA 5.2 software.

[0131] 2. Experimental results

[0132] Results Figure 4 The 16S rRNA sequence of strain D-1 has high homology with many bacteria of the genus Pseudomonas. The similarity between D-1 and Pseudomonas aeruginosa strain DSM 50071 (NR117678.1), and between D-4 and Acinetobacter baumannii strain DSM 30007 (NR117677.1) are all over 99%. It is determined that strain D-1 is Pseudomonas aeruginosa and D-4 is Acinetobacter baumannii.

[0133]

[0134] The nucleotide sequence of the 16S rDNA of strain D-4 is shown in SEQ ID NO:2.

[0135] CGGGCTTTACACATGCAGTCGAGCGGGGGAAGGTAGCTTGCTACCGGACCTAGCGGCG

[0136] GACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATCTCGAAAGGG

[0137] ATGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTGCGCTAA

[0138] TAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGA

[0139] TCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGAC

[0140] TCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGCCA

[0141] TGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGGCT

[0142] ACTTTAGTTAATACCTAGAGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTAACTC

[0143] TGTGCCAGCAGCCGCGGTAATACAGAGGGTGCGAGCGTTAATCGGATTTACTGGGCGT

[0144] AAAGCGTGCGTAGGCGGCTTATTAAGTCGGATGTGAAATCCCCGAGCTTAACTTGGGA

[0145] ATTGCATTCGATACTGGTGAGCTAGAGTATGGGAGAGGATGGTAGAATTCCAGGTGTAG

[0146] CGGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCCATCTGGCCTA

[0147] ATACTGACGCTGAGGTACGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGT

[0148] CCATGCCGTAAACGATGTCTACTAGCCGTTGGGGCCTTTGAGGCTTTAGTGGCGCAGCT

[0149] AACGCGATAAGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAATGAATT

[0150] GACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAC

[0151] CCTTACCTGGCCTTGACATACTAGGAAACTTTCCAGAGATGGATTGGTGCCTTCGGGAA

[0152] TCTAGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTGGGTTAAGTC

[0153] CGCACGAGCGCACCCTTTCCTTACTTGCCAGCATTCCGGATGGGAACTTAAGGATACTGCCATGACAACTGGAGGAGGCGGGGACGACGTCAAGTCATC.

[0154] The strains D-1 and D-4 were preserved separately. The strain D-1 (Pseudomonas aeruginosa D-1) was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 31, 2024, with the deposit number GDMCC No: 64925, and its taxonomic name was Pseudomonas aeruginosa; the strain D-4 (Acinetobacter baumannii D-4) was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 16, 2024, with the deposit number GDMCC No: 65015, and its taxonomic name was Acinetobacter baumannii.

[0155] Example 5 Nitrification-denitrification functional activity of strains D-1 and D-4

[0156] 1. Experimental Methods

[0157] The activated strains D-1 and D-4 were inoculated into 100 mL of heterotrophic nitrification medium and DM medium with nitrate and nitrite as the only nitrogen sources, respectively, and cultured at 30°C and 160 r / min for 5 days. The removal rates of ammonia nitrogen, nitrate nitrogen and nitrite were measured.

[0158] 2. Experimental Results

[0159] The results showed that the two strains D-1 and D-4 had high degradation rates for ammonia nitrogen, nitrate and nitrite, with the degradation rate for ammonia nitrogen reaching 96.90% and 94.93%; the degradation rate for nitrate reaching 97.18% and 99.53%; the degradation rate for nitrite reaching 93.84% and 96.94%, respectively, showing high heterotrophic nitrification-aerobic denitrification functional activity.

[0160] Example 6 Nitrogen degradation characteristics of strains D-1 and D-4

[0161] 1. Experimental Methods

[0162] The strains D-1 and D-4 were inoculated into LB medium for activation. The activated bacterial solution was centrifuged to collect the bacterial precipitate, washed three times with PBS and resuspended, and inoculated into 100 mL of DM medium with NaNO3, NaNO2 and (NH4)2SO4 as the only nitrogen source, respectively. The initial OD 600 0.1, 30℃, 160r / min shaking culture for 5 days. NO3 was measured every 24h - -N, NO2 - -N、NH4 + -N content.

[0163] 2. Experimental Results

[0164] 1. Degradation characteristics of ammonia nitrogen

[0165] The results are as follows Figure 5 As shown, strains D-1 and D-4 can convert the initial 274 mg / L NH4 + -N degradation was 0.57mg / L and 0.183mg / L, and the removal rates were 2.27mg / L / h and 2.28mg / L / h, respectively. The degradation rate peaked on the second day, at 4.49mg / L / h and 4.19mg / L / h, respectively. During the entire ammonia nitrogen consumption process, a small amount of intermediate products, nitrate and nitrite, were generated. NO3 - -N can reach up to 12.70mg / L and 11.319mg / L, NO2 --N can reach up to 1.264mg / L and 0.438mg / L, and the maximum amount of nitrite generated does not exceed 1.5mg / L, and its accumulation increases with NH4 + -N degradation also gradually decreased.

[0166] 2. Nitrate degradation characteristics

[0167] result Figure 6 As shown, strains D-1 and D-4 can convert the initial 273 mg / L NO3 - -N was degraded to 7.26mg / L and 10.10mg / L, with removal rates of 2.22mg / L / h and 2.19mg / L / h, respectively. The rate of nitrate degradation by D-1 was the highest on the third day, at 3.72mg / L / h. The rate of nitrate degradation by D-4 was the highest on the second day, at 4.35mg / L / h. In the later stage of degradation, the degradation rate slowed down due to the consumption of nutrients in the culture medium and the generation of bacterial metabolites. During the entire nitrate consumption process, a small amount of intermediate product nitrite was generated, but the maximum cumulative amount was very low, at 0.13mg / L and 1.39mg / L, respectively. Ammonia nitrogen was also generated in small amounts during the entire process, with the maximum generation amounts being 0.8mg / L and 0.67mg / L, respectively. It is possible that in the later stage of fermentation, the autolysis of the bacteria caused the release of intracellular ammonia nitrogen, or that the organic nitrogen of some dying cells was converted into ammonia nitrogen, resulting in the detection of a small amount of NH4 in the culture medium. + -N.

[0168] 3. Degradation characteristics of nitrite

[0169] result Figure 7 As shown, both strains were able to effectively degrade nitrite, and the nitrite degradation rates of D-1 and D-4 were 1.77 mg / L / h and 2.27 mg / L / h, respectively.

[0170] On the first and second days, the D-1 strain did not begin to degrade nitrite quickly compared to ammonia nitrogen and nitrate nitrogen. The possible reason was that the high concentration of nitrite stressed the growth of the strain, and the strain did not grow and reproduce rapidly during the adaptation period. However, after two days, the D-1 strain gradually regained its activity and began to degrade nitrite. On the fifth day, the degradation rate of the D-1 strain reached 3.91 mg / L / h.

[0171] D-4 did not show similar phenomena as D-1 in the early degradation process, and the maximum degradation rate reached 5.41 mg / L / h, indicating that the D-4 strain can tolerate high-concentration nitrite environment well and show strong denitrification performance. Ammonia nitrogen was also detected in small amounts throughout the process, with the highest cumulative amounts being 0.071 mg / L and 0.125 mg / L, respectively, which may be due to the autolysis of the bacteria and the conversion of organic nitrogen in the dying cells into ammonia nitrogen.

[0172] Antagonistic effects of the fermentation broth, supernatant and resuspended precipitate of Example 7D-1 against Vibrio parahaemolyticus

[0173] 1. Experimental Methods

[0174] The strain D-1 was inoculated into 100 mL of LB medium, and cultured at 30°C and 160 r / min with shaking for 24 h. 50 mL of the culture medium was taken for storage, and the remaining 50 mL of the culture medium was centrifuged to obtain the supernatant. The supernatant was filtered through a 0.22 μm bacterial filter to obtain a sterile supernatant. The precipitate was washed with sterile PBS and resuspended to obtain a resuspended precipitate. The sterile supernatant and resuspended precipitate were stored at 4°C.

[0175] Spread a 6mm sterile filter paper in the center of the identification medium for the antagonist of Vibrio parahaemolyticus. Take 20μL of culture medium, sterile supernatant and resuspended precipitate and drop them on the filter paper of the identification medium. An equal amount of sterile culture medium is used as a control. Each strain is made in triplicate. The plate is inverted and cultured at 30℃ for 24h, and the diameters of the inhibition zones of the fermentation broth, sterile supernatant and resuspended precipitate are compared.

[0176] 2. Experimental Results

[0177] The results are shown in Table 5. The fermentation broth, fermentation supernatant and fermentation precipitate of D-1 strain all formed inhibition zones, and the sizes of the inhibition zones were almost the same, all larger than 15 mm, indicating that it can antagonize Vibrio parahaemolyticus. The ability of the supernatant to produce inhibition zones indicates that the antibacterial substances produced by the strain can be secreted outside the cell.

[0178] Table 5 Inhibition zones formed by fermentation broth, supernatant and resuspended precipitate of D-1

[0179]

[0180] Example 8 Co-culture of D-1 strain and Vibrio parahaemolyticus

[0181] 1. Experimental Methods

[0182] D-1 strain and Vibrio parahaemolyticus were inoculated into 100mL LB medium, shaken and cultured at 30℃ and 160r / min for 24h as seed solution, and 200μL of D-1 strain and Vibrio parahaemolyticus seed solution were inoculated into 100mL LB medium for co-culture, with Vibrio parahaemolyticus cultured alone as control. After shaking and cultured at 30℃ and 160r / min for 48h, gradient dilution was performed and spread on TCBS solid medium, with three groups of parallels for each concentration, and the plate was inverted and cultured at 30℃ for 24h before counting. Since Vibrio parahaemolyticus is blue-green on TCBS medium, blue-green circular colonies were selected for counting.

[0183] 2. Experimental Results

[0184] Results Figure 8 The result of single culture count of Vibrio parahaemolyticus was 26.16×10 10 CFU / mL, the co-culture count of D-1 and Vibrio parahaemolyticus was 11.83×10 10 CFU / mL, the co-culture results were significantly lower than those of Vibrio parahaemolyticus alone, indicating that the D-1 strain can effectively inhibit the reproduction of Vibrio parahaemolyticus.

[0185] Example 9 Effect of environmental factors on strain antagonism

[0186] 1. Effect of temperature on the antagonistic ability of strains

[0187] 1. Experimental methods

[0188] The culture fluid, sterile supernatant and resuspended precipitate of the D-1 strain prepared in Example 7 were taken, and the three samples were treated at 30, 60, 80, 100 and 121°C for 30 min, and immediately returned to room temperature. The 30°C treatment was used as a control, and the diameter of the inhibition zone was determined by the method of Example 7, and each group was repeated three times.

[0189] 2. Experimental results

[0190] The culture medium of the D-1 strain contains extracellular antibacterial substances and bacteria, the sterile supernatant contains extracellular antibacterial substances, and the resuspended precipitate is the resuspension of the bacteria. During the plate culture process, the bacteria in the culture medium and the resuspended precipitate will grow rapidly and produce antibacterial active substances.

[0191] The results are shown in Table 6. At 30 and 60 °C, the sizes of the inhibition zones of the three samples all decreased with the increase in temperature, but the size of the inhibition zone was still above 20 mm, indicating that although the treatment at 60 °C reduced the antibacterial activity, the impact was relatively small. At 80 and 100 °C, all three still had inhibition zones, but their sizes decreased to below 20 mm. After autoclaving at 121 °C, the fermentation broth and the supernatant had inhibition zones below 10 mm, and the precipitate had no antibacterial effect, indicating that the bacteria in the fermentation broth and the precipitate could not grow after being inactivated by high temperature and could not produce antibacterial substances, while the extracellular antibacterial substances in the culture medium and the sterile supernatant could still exert antibacterial effects, indicating that the extracellular antibacterial substances of the strain had good thermal stability.

[0192] Under the conditions of 100 °C pasteurization and 121 °C high-pressure steam sterilization, the extracellular antibacterial substances of strain D-1 could maintain antibacterial activity and had good thermal stability.

[0193] Table 6 Effect of temperature on the antagonistic ability of the strain

[0194]

[0195] II. Effect of pH on the antagonistic ability of the strain

[0196] 1. Experimental method

[0197] Take the culture solution, sterile supernatant and resuspended precipitate of strain D-1 prepared in Example 7. Adjust the pH of the three samples to 3, 5, 7, 9, 11 and 13 with HCl solution and NaOH solution respectively, and adjust the volume of the samples to the same with sterile PBS. Treat for 30 min, then water bath at 37 °C for 1 h, and then adjust the pH value to 7. Measure the diameter of the inhibition zone by the method of Example 7, and repeat each group three times.

[0198] 2. Experimental results

[0199] The results are shown in Table 7. When pH = 3, none of the three samples produced inhibition zones, indicating that both the bacteria and the extracellular antibacterial substances were inhibited by strong acid and had no antagonistic ability against Vibrio parahaemolyticus. When 5 < pH < 9, the sizes of the inhibition zones of the three samples were all greater than 20 mm, and the antibacterial activity of the extracellular antibacterial substances produced by the strain was relatively high. When pH = 11, the antibacterial activity decreased slightly, and when pH = 13, the extracellular antibacterial substances still had antibacterial activity. The results showed that the extracellular antibacterial substances of the strain had good acid-base stability.

[0200] Table 7 Effect of pH on the antagonistic ability of the strain

[0201]

[0202] III. Effect of ultraviolet light on the antagonistic ability of the strain

[0203] 1. Experimental methods

[0204] The culture fluid, sterile supernatant and resuspended precipitate of the D-1 strain prepared in Example 7 were taken, and the three samples were irradiated with ultraviolet light for 30, 60, 90 and 120 min. The untreated samples were used as controls, and the diameter of the inhibition zone was determined by the method of Example 7. Each group was repeated three times.

[0205] 2. Experimental results

[0206] The results are shown in Table 8: After 30 and 60 minutes of ultraviolet irradiation, there was no significant difference in the size of the inhibition zone of the three samples, all of which were around 30 mm, indicating that the activity of the antibacterial substance did not change significantly. After 90 minutes of ultraviolet irradiation, the diameter of the precipitated inhibition zone was 0 mm, indicating that the bacteria were inhibited and could not grow, but the diameter of the inhibition zone of the fermentation broth and the supernatant was around 20 mm, indicating that the extracellular antibacterial substances were still tolerable. After 120 minutes of irradiation, both the bacteria and the extracellular antibacterial substances were inactive. The results show that the antibacterial substances produced by the strain are ultraviolet stable to a certain extent.

[0207] Table 8 Effect of ultraviolet light on the antagonistic ability of strains

[0208]

[0209]

[0210] Example 10D-1 Iron uptake and biofilm production ability of strain

[0211] 1. Iron uptake capacity of D-1 strain

[0212] 1. Experimental methods

[0213] The D-1 strain was inoculated into CAS agar medium and cultured at 30°C for 48 h to observe whether there was any obvious color change around the colonies.

[0214] 2. Experimental results

[0215] The results are as follows Fig. 9 As shown, a distinct orange-yellow halo appeared around the D-1 colony, indicating that the strain can produce iron carriers to chelate iron in the culture medium and has an iron uptake system.

[0216] 2. Biofilm production ability of D-1 strain

[0217] 1. Experimental methods

[0218] After the D-1 strain was activated, it was inoculated into LB medium, and the culture solution was diluted to OD600 = 0.02. 150 μL of the culture solution was aspirated into a 96-well cell culture plate, and incubated at 30°C for 48 hours. The culture solution was removed, and the plate was rinsed with PBS three times, and then dried at 30°C. 1% crystal violet solution was added and incubated for 20 minutes, rinsed and dried, and 150 μL of dimethyl sulfoxide was added to the wells, incubated for 20 minutes, and the absorbance at 590 nm was measured. If D590 nm ≥ 1, the strain was considered to have good biofilm formation ability.

[0219] 2. Experimental results

[0220] The results are as follows Fig.10 As shown, OD590 nm = 3.65 ± 0.03, indicating that the D-1 strain has a strong ability to produce biofilm. The strong ability to produce biofilm can promote the colonization of the D-1 strain in the intestine of aquatic products, adhere to the epithelial surface, interfere with the adhesion of pathogens to the intestinal mucosa, and play a role in regulating the balance of intestinal flora and resisting the invasion of pathogens.

[0221] Example 11 Enzyme production capacity of D-1 strain

[0222] 1. Experimental Methods

[0223] Agar diffusion method was used to determine the enzyme production capacity:

[0224] Add skim milk powder to the nutrient agar medium to detect the protease activity of the D-1 strain. After the identification medium cools down, drop the same volume of bacterial liquid in the center of the plate and culture for 24 hours. The presence of a transparent proteolysis zone around the colony indicates that the strain has protease activity.

[0225] Soluble starch in nutrient agar medium was used to detect the amylase activity of the D-1 strain. A 1% iodine solution was dropped on the surface of the culture medium plate. A visible decolorization halo appeared around the colony, indicating that the strain had amylase activity.

[0226] 1% Tween 80 was added to the nutrient agar medium to detect the lipase activity of the D-1 strain. A fatty acid precipitation ring was formed around the colony, indicating that the strain had lipase activity.

[0227] 2. Experimental Results

[0228] The digestive enzyme activity of D-1 strain Fig.11 As shown: on the nutrient agar medium supplemented with skimmed milk powder, there was a transparent proteolysis zone around the colonies, and after the plate was cultured for 1 day, all the white skimmed milk powder was completely degraded, indicating that the strain had a high protease activity; and no amylase activity and lipase activity of the strain were detected.

[0229] Example 12 Antagonism between D-1 and D-4 strains

[0230] 1. Experimental Methods

[0231] D-1 strain and D-4 strain were inoculated into 50 mL LB liquid culture medium, respectively, and cultured at 30°C, 160 r / min shaking for 24 h. The OD600 of the bacterial culture solution was adjusted to 1.0 with sterile PBS. 1 μL of bacterial culture solution was taken from each strain and dropped on the surface of LB solid culture medium. The positions of the two strains were 1.2 cm and 0.6 cm apart, respectively. The plate was inverted and cultured at 30°C for 24 h. The antagonism of the two strains was observed and recorded.

[0232] 2. Experimental Results

[0233] The results are as follows Fig.12 As shown, the two strains grew normally when they were 1.2 cm apart, and the colony morphology of the two strains did not change when they were 0.6 cm apart, indicating that the growth of the two strains was not affected by each other, there was no antagonistic relationship between the two strains, and the bacterial flora could be compounded.

[0234] Example 13 Ecological safety of strains D-1 and D-4

[0235] 1. Experimental Methods

[0236] The paper diffusion method was used to conduct an ecological safety test on the two strains of bacteria. The antibiotics selected were TC (tetracycline), CM (chloramphenicol), OTC (oxytetracycline), HYG (hygromycin), EM (erythromycin), GM (gentamicin), and TOB (tobramycin). The ecological safety to be tested was inoculated into LB medium for activation, cultured at 30°C and 160r / min for 24h, and diluted to 10 with PBS. 5 cfu / mL. Take 1mL of bacterial culture dilution and mix it with liquid nutrient agar at about 40℃. Pour the mixture evenly into a plate. After the plate solidifies, spread a 6mm sterile filter paper in the center. Take 50μL of antibiotic solution and drop it on the filter paper. An equal amount of antibiotic solvent is used as a control. Three groups of parallels are made for each antibiotic. The plate is inverted and cultured at 30℃ for 24h, and then the transparent circle is observed.

[0237] 2. Experimental Results

[0238] The results of antibiotic resistance test of the strains are as follows Fig.13 Compared with the CK group treated with sterile water, inhibition zones appeared around the filter papers in all antibiotic-treated experimental groups, indicating that the strains were sensitive to all seven antibiotics tested and had high ecological safety.

[0239] Example 14 Safety of D-1 and D-4 strains

[0240] 1. Experimental Methods

[0241] Ten healthy shrimps were randomly selected from each tank and fed for 24 hours for safety experiments. A total of four treatment groups were set up, namely: CK group, D-1 group, D-4 group, and mixed bacteria group (bacteria amount D-1:D-4=1:1), with three replicates in each group. 8 cfu / m 3 The concentration is sprayed once every 3 days.

[0242] Clean up feed residues and feces in time every morning and evening, control the water change volume to 1 / 3 of the total volume, control the water temperature at around 20℃, and aerate continuously. After 6 days, count the survival status of each treatment group.

[0243] Survival rate: SR (%) = (N f / N i )×100%.

[0244] N f N is the number of shrimps at the end of the experiment. i is the number of shrimps stocked at the beginning of the experiment.

[0245] 2. Experimental Results

[0246] The survival rate of shrimp in the CK group was 72.73%, the survival rate of shrimp in the D-1 group was 84.85%, the survival rate of shrimp in the D-4 group was 75.76%, and the survival rate of shrimp in the D-1 and D-4 mixed bacteria group was 78.79%. The survival rates of shrimp in the three treatment groups were all higher than those in the control group without any treatment, but there was no significant difference, indicating that the strain had no obvious toxic effect on shrimp and was relatively safe and could be used in subsequent breeding experiments. On the other hand, the survival rate of shrimp in the D-1 group and the mixed bacteria group was higher than that in the control group, indicating that strain D-1 had a growth-promoting effect on shrimp.

[0247] Example 15 Effect of the ratio of strain D-1 to strain D-4 on denitrification and antibacterial activity

[0248] 1. Experimental Methods

[0249] Single bacteria of D-1 and D-4 were inoculated into 50 mL LB liquid culture medium, respectively, and cultured at 30°C and 160 r / min with shaking for 24 h. The OD600 of the bacterial culture medium was adjusted to 1.0 with sterile PBS.

[0250] (1) Effect of the ratio of D-1 strain to D-4 strain on denitrification

[0251] The single bacterial culture solutions (OD600 to 1.0) of the D-1 strain and the D-4 strain were mixed at volume ratios of 1:1, 1:2 and 2:1, respectively, to obtain mixed bacterial solutions. The mixed bacterial solutions were inoculated into 100 mL of DM medium and cultured at 30°C and 160 r / min for 3 days. The removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen were detected respectively.

[0252] (2) Effect of the ratio of D-1 to D-4 strains on antibacterial activity

[0253] The single bacterial culture liquid (OD600 to 1.0) of D-1 strain and D-4 strain was mixed at a volume ratio of 1:1, 1:2 and 2:1 to obtain a mixed bacterial liquid. 200 μL of the mixed bacterial liquid and the seed liquid of Vibrio parahaemolyticus were co-cultured in 100 mL LB medium, and the single culture of Vibrio parahaemolyticus was used as a control. After shaking culture at 30°C and 160r / min for 48 hours, the mixture was diluted stepwise and spread on LB solid medium. Three groups of parallels were made for each concentration, and the plates were inverted and cultured at 30°C for 24 hours before counting.

[0254] 2. Experimental Results

[0255] The results are as follows Fig.14 As shown: the removal rates of ammonia nitrogen with the ratio of D-1:D-4 of 1:1, 1:2 and 2:1 were 97.28%, 94.96% and 94.98% respectively; the removal rates of nitrate were 96.93%, 97.09% and 96.53% respectively; the removal rates of nitrite were 96.57%, 97.21% and 94.81% respectively. There was no significant difference in the denitrification effect of the three ratios of bacteria.

[0256] The results are as follows Fig.15 As shown: The number of Vibrio in the CK group was 7.21×10 7 The CFU / mL reached a peak of 10.7×10 7 CFU / mL, and then gradually decreased to 2.23×10 7 CFU / mL. The number of Vibrio at the D-1:D-4 ratio of 1:1, 1:2 and 2:1 was 1.65×10 7 CFU / mL, 2.11×10 7 CFU / mL, 1.21×10 7 CFU / mL, and then continued to decrease, reaching 0.02×10 7 CFU / mL, 0.12×10 7 CFU / mL, 0.03×10 7 CFU / mL, the number of Vibrio in the three experimental groups was significantly different from that in the CK group, and all had obvious antibacterial effects, while there was no significant difference among the three experimental groups.

[0257] In summary, there is no significant difference in the denitrification effect and antibacterial effect of the three ratios of bacterial flora. However, the early growth of the Vibrio parahaemolyticus antagonist D-1 will be inhibited in the presence of high concentrations of nitrite. In order to ensure that the antibacterial effect can be exerted in the presence of high concentrations of nitrite, the composite ratio is selected after comprehensive consideration: D-1: D-4 = 2:1.

[0258] Example 16 Effect of immobilization materials on the performance of immobilized beads

[0259] 1. Experimental Methods

[0260] The single bacteria of D-1 strain and D-4 strain were inoculated into 100 mL LB liquid culture medium, respectively, and cultured at 30°C and 160 r / min with shaking for 24 h. The OD600 of the bacterial culture solution was adjusted to 1.0 with sterile PBS to obtain a bacterial suspension.

[0261] Dissolve polyvinyl alcohol, sodium alginate and kaolin in 80℃ distilled water according to the concentrations in Table 9 and mix them evenly. When cooled to room temperature, add the pre-made bacterial suspension and stir for 15 minutes to ensure the uniformity of the entire solution. Use a syringe to drop the mixture into a 4g / ml CaCl2 solution and let it stand overnight. The formed immobilized beads were cultured in the culture medium for 48 hours, washed three times with PBS and stored at 4℃.

[0262] Table 9 Amount of immobilized materials

[0263]

[0264] Performance evaluation methods:

[0265] Ball-forming property: When the immobilized spheres are dropped into the CaCl2 solution, are they uniform in size, round and smooth, are there any tails, and are the spheres formed quickly?

[0266] Adhesion: When the immobilized beads are dropped into the CaCl2 solution, do the beads stick to each other? When stirred with a glass rod, are the beads evenly dispersed?

[0267] Stability: Weigh 2 g of uniformly sized immobilized beads into a flask containing 100 mL of distilled water, shake at 30°C, 160 r / min for 24 h, and observe the breakage of the beads.

[0268] Mechanical strength: Weigh 2 g of uniformly sized immobilized spheres and place them on filter paper to absorb moisture. Use a 200 g weight to apply pressure on the spheres and observe the deformation of the spheres.

[0269] Water content: Weigh 2 g of uniformly sized beads, dry them on filter paper, and then fix them in an oven at 50°C to constant weight. Calculate the water absorption rate of the beads.

[0270] Nitrogen removal performance: Weigh 2 g of uniformly sized beads fixed on filter paper after absorbing water, and inoculate them into 100 mL of DM culture medium with NaNO3, NaNO2 and (NH4)2SO4 as the only nitrogen source, respectively. Culture at 30℃ and 160r / min with shaking for 5 days, and measure the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen.

[0271] 2. Experimental Results

[0272] The results are shown in Table 10: The immobilized beads of groups 1, 3, 4 and 5 did not stick together when the CaCl2 solution was added, and were evenly dispersed. The immobilized beads of groups 1, 4 and 5 were round and smooth, without tailing when forming beads, and were relatively stable; the immobilized beads of groups 4 and 5 had high mechanical strength; the water content of the immobilized beads of group 5 reached 75% or more. In summary, immobilized beads 1, 4 and 5 were selected for the next step of denitrification performance evaluation.

[0273] Table 10 Physical properties of immobilized beads

[0274]

[0275]

[0276] The results are as follows Fig.16 , the ammonia nitrogen removal rates of No. 1, No. 4, and No. 5 were 90.94%, 95.54%, and 94.17% respectively; the nitrate removal rates were 92.02%, 96.44%, and 95.76% respectively; the nitrite removal rates were 92.20%, 96.82%, and 97.36%. The removal rates of the three groups of immobilized beads for the three nitrogen sources were all above 90%. The removal rates of the immobilized beads No. 4 and No. 5 were significantly higher than those of the No. 1 bead, and there was no significant difference between No. 4 and No. 5. The only difference between the immobilized materials No. 4 and No. 5 is the different content of polyvinyl alcohol, which is not easy to dissolve. The difficulty of preparation increases with the increase of concentration. After comprehensive consideration, the No. 4 ratio was selected: 2.5g / ml kaolin + 2.5g / ml sodium alginate + 7g / ml polyvinyl alcohol.

[0277] Example 17 A method for preparing an immobilized bacterial agent for denitrification of water bodies

[0278] Single bacteria of D-1 strain and D-4 strain were inoculated into 50 mL LB liquid culture medium, respectively, and cultured at 30°C, 160 r / min with shaking for 24 h, and the OD600 of the bacterial culture solution was adjusted to 1.0 with sterile PBS. The single bacterial culture solutions of D-1 strain and D-4 strain (OD600 were both 1.0) were mixed in a volume ratio of 2:1 to obtain a 2:1 mixed bacterial solution.

[0279] Polyvinyl alcohol, sodium alginate and kaolin were dissolved in 80°C distilled water and then mixed evenly. When cooled to room temperature, the mixed bacterial solution was added and stirred for 15 minutes to ensure the uniformity of the entire solution to obtain the immobilized system, wherein the concentrations of polyvinyl alcohol, sodium alginate and kaolin in the immobilized system were 7g / 100ml, 2.5g / 100ml and 2.5g / 100ml, respectively, and the amount of the mixed bacterial solution was 6ml / 100ml.

[0280] The mixture was added dropwise into a 4 g / ml CaCl2 solution using a syringe and allowed to stand overnight.

[0281] At the same time, immobilized beads without added bacteria were prepared, and the preparation method was the same as above, except that the mixed bacterial solution was replaced with pure water.

[0282] Example 18 Repeatability of Immobilized Bacterial Agent for Water Denitrification

[0283] 1. Experimental Methods

[0284] Weigh 2 g of the immobilized bacterial agent prepared in Example 17, absorb the water, and inoculate it into 100 mL of DM medium with NaNO3, NaNO2 and (NH4)2SO4 as the sole nitrogen source, respectively, at 30°C, 160 r / min shaking culture for 5 days, and measure the removal rates of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen. One cycle is 7 days, the culture medium is replaced every seven days, and the above experiment is repeated three times.

[0285] 2. Experimental Results

[0286] Repeatability Fig.17 As shown in the figure, the removal rates of ammonia nitrogen in the three cycles were 98.28%, 96.30%, and 93.64% respectively; the removal rates of nitrate were 96.93%, 94.09%, and 87.86% respectively; and the removal rates of nitrite were 97.90%, 95.88%, and 93.81%. The denitrification effect of the immobilized bacteria in each cycle was good. The removal rates of ammonia nitrogen, nitrate, and nitrite in the third cycle were significantly different from those in the second and first cycles, but they were still above 85%, indicating that the prepared immobilized beads had a good reuse effect. However, after each cycle, the mass of the beads gradually decreased. After the fourth cycle, the beads gradually disintegrated. Although the material will gradually dissolve in high concentrations of ammonia nitrogen and nitrate, in the actual application of aquaculture water, after one application cycle, the beads gradually dissipated and would not block the pipeline with the discharge of aquaculture water. There was no need to recycle the discarded beads, which reduced the cost of later aquaculture wastewater treatment.

[0287] Example 19 Characterization of immobilized bacterial agent

[0288] 1. Experimental Methods

[0289] The immobilized bacterial agent prepared in Example 17, the immobilized beads without bacteria added prepared in Example 17, and the immobilized bacterial agent after three cycles of Example 18 were dried, 1 g was weighed, washed three times with sterile saline, fixed overnight with 1 mL of 2.5% glutaraldehyde fixative, and continued to wash three times with saline. Ethanol was added for gradient dehydration, dehydrated once with 30%, 50%, 70%, 80%, and 90% ethanol in sequence, and dehydrated twice with 100% ethanol, each time standing for 15 min; after dehydration, the sample was sent for drying and observed under a scanning electron microscope.

[0290] 2. Experimental Results

[0291] Results Fig.18 Under a scanning electron microscope, the surface of the immobilized beads without added bacteria is rich in wrinkles and protrusions, providing a larger area for microbial attachment. The internal pores of the unimmobilized composite carrier are large and abundant, with a three-dimensional tubular structure, and the surface area for microbial attachment is also larger. At the same time, it can improve the pressure resistance of microorganisms in water.

[0292] A large number of bacteria are attached to the surface of the immobilized bacterial agent prepared in Example 17, and many strains are also fused inside the carrier, indicating that the composite material is more conducive to the attachment and reproduction and growth of microorganisms. The fusion of strains and materials can also improve the survival rate and pressure resistance of microorganisms.

[0293] After three cycles, the results are shown in Fig.19 The immobilized bacteria gradually dissolved, the surface of the immobilized beads changed, the carrier became loose, and the number of microorganisms attached decreased, but the dissolved carrier could still adsorb some microorganisms.

[0294] Example 20 Application of immobilized bacterial agent in denitrification of aquaculture water

[0295] 1. Experimental Methods

[0296] A total of 4 treatment groups were set up with whiteleg shrimp of the same size and healthy growth after domestication and breeding, namely: CK group, bacterial solution group (2:1 mixed bacterial solution prepared in Example 17), immobilized bacterial agent group (immobilized bacterial agent prepared in Example 17, Immobilization), and market microecological bacterial agent group (Tianyi EM original dew water adjustment special type, Microbialagent), with three replicates in each group.

[0297] On the first day of a cycle, the fungicide was sprayed (10 10 cfu / m 3 ), and then collect water samples from the treatment group every day to measure the nitrate (NO3 - -N), nitrite (NO2 - -N), ammonium salt (NH4+ -N) content, and determine the difference in shrimp survival rate, and control the temperature (T), dissolved oxygen (DO), pH and salinity (Sal) of the water body to maintain a stable level. In one cycle, no feeding, no feces cleaning, no water change, in order to simulate the natural aquaculture water environment.

[0298] 2. Experimental Results

[0299] The results are as follows Fig. 20 As shown, the results showed that the highest survival rate of shrimp was in the bacterial liquid group (D-1&D-4) at 96.67%, followed by the immobilized bacteria group (Immobilization) at 83.33%, the third was the market microecological bacteria group (Microbialagent) at 66.67%, and the lowest was the control group at 50.00%. There were obvious differences in the survival rates among the four groups.

[0300] The contents of ammonia nitrogen, nitrate and nitrite in the CK group gradually increased, with the highest cumulative amounts being 3.80 mg / L, 4.32 mg / L and 0.15 mg / L, respectively. The contents of nitrate and nitrite were within the tolerance range of shrimp, but the ammonia nitrogen content reached 1.53 mg / L on the second day, far exceeding the tolerance value of ammonia nitrogen for shrimp (0.60 mg / L), which had a serious impact on the survival of shrimp.

[0301] Under the action of different microbial agents, the contents of ammonia nitrogen, nitrate and nitrite in the three experimental groups remained relatively stable. The maximum accumulation of ammonia nitrogen, nitrate and nitrite in the immobilization group was 0.54 mg / L, 0.75 mg / L and 0.07 mg / L respectively from the second day to the seventh day; the maximum accumulation of ammonia nitrogen, nitrate and nitrite in the bacterial liquid group (D-1 & D-4) was 0.34 mg / L, 0.78 mg / L and 0.08 mg / L respectively from the second day to the seventh day; the maximum accumulation of ammonia nitrogen, nitrate and nitrite in the market microecological agent group was 0.39 mg / L, 0.22 mg / L and 0.05 mg / L respectively from the second day to the seventh day. The maximum accumulation of ammonia nitrogen, nitrate and nitrite in the three experimental groups did not exceed the tolerance value of shrimp, which is consistent with the phenomenon that the survival rate of shrimp is much higher than that of the CK group.

[0302] In summary, the non-immobilized bacterial group (D-1&D-4) and the immobilized bacterial group (Immobilization) had better denitrification effects and higher shrimp survival rates.

[0303] Example 21 Application of immobilized bacterial agent in antibacterial treatment of aquaculture water

[0304] 1. Experimental Methods

[0305] A total of 4 treatment groups were set up with whiteleg shrimp of the same size and healthy growth after domestication and breeding, namely: CK group, bacterial solution group (2:1 mixed bacterial solution prepared in Example 17), immobilized bacterial agent group (immobilized bacterial agent prepared in Example 17, Immobilization), and market microecological bacterial agent group (Tianyi EM original dew water adjustment special type, Microbialagent), with three replicates in each group.

[0306] During the whole experiment, each group was sprayed with water for the first two days, and then infected with Vibrio on the third day. 9 cfu / mL of Vibrio parahaemolyticus culture fluid, and Vibrio infection was carried out indoors for 24 hours.

[0307] After the Vibrio infection, the number of Vibrio in the water body is tested every day to reflect the antibacterial effect of the strain on the pathogen. In a cycle of Vibrio infection, water samples of the treatment group are collected every day, and the water samples are gradient diluted, and the appropriate concentration is selected to be coated on the Vibrio selective culture medium. Three groups of parallels are made for each concentration. The plates are inverted and cultured at 30°C for 24 hours and then counted; the survival rate of shrimp is measured, and the temperature (T), dissolved oxygen (DO), pH and salinity (Sal) of the water body are controlled to remain at a stable level. In one cycle, no feeding and no water change are done, but the feces of the shrimp need to be cleaned every day.

[0308] 2. Experimental Results

[0309] The antibacterial results were as follows Fig.21 As shown, the survival rates of shrimps in the three experimental groups were significantly different from those in the control group. The highest survival rate of shrimps was in the bacterial liquid group (D-1 & D-4) at 46.67%, followed by the immobilized bacteria group (Immobilization) at 36.67%, and the third was the commercial microbial agent group (Microbial agent) at 33.33%.

[0310] After Vibrio infection, the counts of Vibrio in the three experimental groups were significantly different from those in the control group. The number of Vibrio in the control group reached a peak of 8.36×10 on the third day. 4 CFU / mL, and later decreased to 6.97×104CFU / mL due to the reduction of nutrients in the water, but still maintained a high level, and the corresponding shrimp survival rate of the control group was also the lowest. The number of Vibrio in the immobilization group gradually decreased over time, from 4.15×10 4 CFU / mL decreased to 0.51×10 4 CFU / mL, the lowest in the bacterial liquid group (D-1 & D-4) was 0.43×10 4CFU / mL, the lowest in the microbial agent group on the market is 0.91×10 4 CFU / mL. The number of Vibrio in the bacterial liquid group (D-1&D-4) and the immobilized bacterial agent group (Immobilization) in the early stage was less than that in the market microecological bacterial agent group (Microbial agent), indicating that the antibacterial effect of these two groups was exerted earlier, the effect was stronger, and the overall effect was better than that of the market bacterial agent group. Compared with the bacterial liquid group (D-1&D-4) and the immobilized bacterial agent group (Immobilization), the number of Vibrio in the unimmobilized bacterial agent group increased in the later stage, while the number of Vibrio in the immobilized bacterial agent group remained relatively stable. It can be seen that the antibacterial effect of the immobilized bacterial agent group is more stable and long-lasting.

[0311] The antibacterial effect of the microbial agent is reflected in the survival rate of shrimp on the one hand, and in the count of Vibrio after infection on the other hand. In general, the antibacterial effect of the immobilized microbial agent is more lasting and stable.

Claims

1. An Acinetobacter baumannii D-4, characterized in that On August 16, 2024, it was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 65015.

2. The use of Acinetobacter baumannii D-4 according to claim 1 in denitrification and / or preparation of denitrification products, characterized in that: The denitrification is the degradation of one or more of ammonia nitrogen, nitrate and nitrite.

3. A product for denitrification, characterized in that: Containing the Acinetobacter baumannii D-4 according to claim 1, the denitrification is the degradation of one or more of ammonia nitrogen, nitrate and nitrite.

4. A bacterial composition, characterized in that It contains Pseudomonas aeruginosa and Acinetobacter baumannii, wherein the Pseudomonas aeruginosa is Pseudomonas aeruginosa D-1, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on July 31, 2024, with a deposit number of GDMCC No: 64925; the Acinetobacter baumannii is the Acinetobacter baumannii D-4 described in claim 1, and the denitrification is the degradation of one or more of ammonia nitrogen, nitrate and nitrite.

5. Use of the bacterial composition according to claim 4 in denitrification and / or preparation of denitrification products, characterized in that: The denitrification is the degradation of one or more of ammonia nitrogen, nitrate and nitrite.

6. Use of the bacterial composition according to claim 4 in combating Vibrio parahaemolyticus and / or in preparing anti-Vibrio parahaemolyticus products.

7. The use according to claim 5 or 6, characterized in that: The product is an immobilized bacterial agent.

8. A bacterial agent, characterized in that Contains the Acinetobacter baumannii D-4 according to claim 1 or the bacterial composition according to claim 4.

9. A method for preparing an immobilized bacterial agent, characterized in that: The polyvinyl alcohol, sodium alginate and kaolin are fully dissolved and mixed to obtain a mixed solution, the mixed solution is fully mixed with a bacterial solution containing the bacterial composition according to claim 3, and then dropped into a CaCl2 solution and solidified to obtain the obtained solution.

10. The immobilized bacterial agent prepared by the preparation method according to claim 9.

Images