Acinetobacter johnsonii strain scuec19 capable of efficiently degrading algal toxin and application thereof

Abstract

The application discloses a high-efficiency algicidal Acinetobacter junii SCUEC19 strain and application thereof, and belongs to the technical field of microorganisms. Acinetobacter junii The application first separates the Acinetobacter junii SCUEC19 strain, which has a preservation number of CCTCC NO: M 20261017; the Acinetobacter junii SCUEC19 strain can efficiently degrade microcystins, and has a good degradation effect on high-concentration microcystins; the Acinetobacter junii SCUEC19 strain has good salt tolerance, acid and alkali tolerance and drought resistance; the Acinetobacter junii SCUEC19 strain has good nitrogen fixation and indole acetic acid production capacity; and the bacillus licheniformis fermentation supernatant has a promoting effect on the growth capacity of the Acinetobacter junii SCUEC19 strain, so that the strain has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a highly efficient Acinetobacter juncea SCUEC19 strain that degrades algal toxins and its applications. Background Technology

[0002] Microcystins (MCs) are a class of monocyclic heptacapeptide hepatotoxicants mainly produced and released into the aquatic environment by cyanobacteria such as *Microcystis aeruginosa* and *Anabaena*. These toxins are chemically stable, resistant to acids, alkalis, and high temperatures, and are difficult to remove effectively using conventional water treatment processes. MCs exhibit strong hepatotoxicity, primarily by inhibiting protein phosphatase activity and disrupting the cytoskeleton, leading to hepatocyte damage, necrosis, and even inducing liver cancer. MCs can also trigger oxidative stress, causing toxicity to the kidneys, intestines, and nervous system. They frequently occur in eutrophic lakes, reservoirs, and nearshore waters, disrupting the balance of aquatic ecosystems and directly threatening human and animal health through drinking water and the food chain (such as seafood). Their pollution has become a global environmental and public health safety issue.

[0003] Currently, physical adsorption and chemical oxidation methods for controlling and removing microorganisms (MCs) pollution suffer from high costs and the potential for secondary pollution. Biodegradation, due to its environmental friendliness, high efficiency, and specificity, is considered the most promising remediation approach. Reported MCs-degrading microorganisms mainly include bacteria such as *Sphingomonas*, *Sphingobacter*, *Achromobacter*, and *Pseudomonas*. Their degradation mechanism primarily relies on microcystin-degrading enzymes (such as MlrA, MlrB, and MlrC) secreted by these strains, which open the rings of MCs, hydrolyze them, and gradually convert them into low-toxicity or non-toxic products. However, MCs in natural water bodies have complex structures and morphologies, fluctuate greatly in concentration, and are subject to diverse environmental factors. Existing degrading strains suffer from unstable degradation efficiency, limited environmental adaptability and colonization capacity, and limited functionality in practical applications. Therefore, screening and discovering new bacterial strains with high efficiency, stable degradation capabilities, and potentially strong environmental adaptability and multifunctionality is of great significance for developing efficient and safe MCs bioremediation technologies.

[0004] However, there are currently few studies and patent reports on the efficient degradation of MCs by Acinetobacter. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient Acinetobacter juncea SCUEC19 strain that degrades microcystin and its applications. The Acinetobacter juncea SCUEC19 strain provided by this invention exhibits good salt, acid, alkali, and drought tolerance. Furthermore, this Acinetobacter juncea SCUEC19 strain demonstrates good nitrogen fixation and indoleacetic acid production capabilities. The fermentation supernatant of Bacillus licheniformis promotes the growth of the Acinetobacter juncea SCUEC19 strain. In addition, this Acinetobacter juncea SCUEC19 strain can efficiently degrade high concentrations of microcystin.

[0006] In a first aspect, the present invention provides a strain of Acinetobacter jumbo that efficiently degrades algal toxins (… Acinetobacter junii ) SCUEC19 strain, Acinetobacter jonesi ( Acinetobacter junii The preservation number of strain SCUEC19 is CCTCC NO:M 20261017.

[0007] In some implementation schemes, Acinetobacter jumbo ( Acinetobacter junii The colony morphology of SCUEC19 strain is pale yellow, round, moist, and smooth. It is a Gram-negative bacterium, appearing as short rods. The optimal growth pH is 5.0-11.0, for example, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, or other values ​​within this range. The highest tolerated NaCl concentration is 6.0% (w / v). Acinetobacter juncea (… Acinetobacter junii The nucleotide sequence of the 16S rDNA of strain SCUEC19 is shown in SEQ ID NO.1.

[0008] In this invention, the SCUEC19 strain was isolated from sediment samples from Meiliang Bay in Taihu Lake, Jiangsu Province. The SCUEC19 strain tested negative for indole, methyl red, voltaic, and citrate tests.

[0009] Furthermore, the 16S rDNA of the SCUEC19 strain was sequenced, and its 16S rDNA sequence was BLASTed in NCBI (https: / / www.ncbi.nlm.nih.gov / ). A phylogenetic tree was constructed using the NJ method in MEGA 11.0 software. The results showed that the SCUEC19 strain is related to... Acinetobacter junii Based on the highest similarity to ATCC 17908 and considering morphological, physiological and biochemical characteristics and molecular biological identification results, strain SCUEC19 was named Acinetobacter jung. Acinetobacter junii ) SCUEC19 strain.

[0010] In some implementation schemes, Acinetobacter jumbo ( Acinetobacter juniiSCUEC19 strain was able to completely degrade MC-LR at a concentration of 15.0 μg / mL under the conditions of pH 7.0, culture temperature of 30℃ and culture time of 12h.

[0011] In this invention, under specific conditions, the SCUEC19 strain can achieve efficient degradation of MC-LR.

[0012] In some implementation schemes, Acinetobacter jumbo ( Acinetobacter junii The highest concentration at which the SCUEC19 strain degraded MC-LR was at least 45.0 μg / mL, and the degradation rate of MC-LR by Acinetobacter jungiella SCUEC19 strain at a concentration of 45.0 μg / mL was 50.15% after 12 hours.

[0013] In this invention, the SCUEC19 strain can achieve efficient degradation of high concentrations of MC-LR (e.g., 45 μg / mL).

[0014] In a second aspect, the present invention provides Acinetobacter jumborum as described above ( Acinetobacter junii Applications of SCUEC19 strain in any of the following: 1) Salt tolerance; 2) Acid and alkali tolerance; 3) Drought tolerance; 4) Nitrogen fixation; 5) Indoleacetic acid production.

[0015] In this invention, the SCUEC19 strain exhibits good salt tolerance, acid and alkali tolerance, drought tolerance, nitrogen fixation, and indoleacetic acid production capabilities, thus showing promising application prospects.

[0016] In a third aspect, the present invention provides a biological agent comprising any of the above-mentioned Acinetobacter jumbo ( Acinetobacter junii ) SCUEC19 strain.

[0017] In some embodiments, the biological agent includes at least one of liquid and solid agents; wherein, the liquid agent contains Acinetobacter juncea (… Acinetobacter junii The concentration of SCUEC19 strain was 1.0 × 10⁻⁶. 10 cfu / mL -1.0×10 12 cfu / mL, for example, can be 1.0 × 10⁻⁶. 10 cfu / mL, 5.0×10 10 cfu / mL, 1.0×10 11 cfu / mL, 5.0×10 11 cfu / mL, 1.0×10 12 cfu / mL or other values ​​within that range; Acinetobacter junceta in solid bacterial agents ( Acinetobacter junii The concentration of SCUEC19 strain was 1.0 × 10⁻⁶. 10 cfu / g -1.0×10 12cfu / g, for example, can be 1.0 × 10⁻⁶. 10 cfu / g, 5.0×10 10 cfu / g, 1.0×10 11 cfu / g, 5.0×10 11 cfu / g, 1.0×10 12 cfu / g or other values ​​within that range.

[0018] It is understandable that Acinetobacter juncea ( ) is present in both liquid and solid bacterial agents. Acinetobacter junii The content of SCUEC19 strain can be adjusted according to actual needs, as long as it meets the requirements of the application. For example, in this invention, the liquid bacterial agent contains Acinetobacter juncea (… Acinetobacter junii The optimal content of SCUEC19 strain is 1.0 × 10⁻⁶. 10 cfu / mL -1.0×10 12 cfu / mL, Acinetobacter junceta in solid bacterial agent ( Acinetobacter junii The optimal content of SCUEC19 strain is 1.0 × 10⁻⁶. 10 cfu / g -1.0×10 12 cfu / g.

[0019] In some embodiments, the liquid bacterial agent is Acinetobacter jumbo (… Acinetobacter junii The SCUEC19 strain was obtained by inoculating it into a culture medium.

[0020] Understandably, the culture medium can be any common culture medium in the field, as long as it meets the requirements of Acinetobacter juncea (…). Acinetobacter junii The normal growth of SCUEC19 strain is sufficient. In this invention, the preferred culture medium is LB medium.

[0021] In some implementations, the solid microbial agent is obtained by drying the aforementioned liquid microbial agent.

[0022] In some preferred embodiments, the drying is spray drying, and the spray drying process conditions include: using corn starch as a carrier, controlling the material concentration to be 10-25% (W / V), for example, 10%, 15%, 20%, 25% or other values ​​within this range; the inlet air temperature to be 100-200°C, for example, 100°C, 120°C, 140°C, 160°C, 180°C, 200°C or other values ​​within this range; and the feed rate to be 10-30 mL / min, for example, 10 mL / min, 15 mL / min, 20 mL / min, 25 mL / min, 30 mL / min or other values ​​within this range.

[0023] In some preferred embodiments, the spray drying process conditions include: using corn starch as a carrier, controlling the material concentration at 16% (W / V), the inlet air temperature at 165°C, and the feed rate at 25 mL / min.

[0024] It is understood that the drying method can be conventionally selected according to actual use, and spray drying is preferred in this invention; and the carrier for spray drying can also be conventionally selected according to actual use, and corn starch is preferred in this invention.

[0025] In a fourth aspect, the present invention provides a combined bacterial agent comprising any of the above-mentioned Acinetobacter jumbo ( Acinetobacter junii SCUEC19 strain and Bacillus licheniformis fermentation supernatant; among which, the Bacillus licheniformis fermentation supernatant was effective against Acinetobacter jung. Acinetobacter junii The growth ability of the SCUEC19 strain is promoted.

[0026] In some implementations, the Bacillus licheniformis fermentation supernatant is obtained by inoculating the aforementioned Bacillus licheniformis into a culture medium and then culturing and isolating it.

[0027] It is understood that the culture medium can be any common culture medium in the art, as long as it can meet the normal growth requirements of the Bacillus licheniformis; the separation can be carried out using conventional separation methods in the art. For example, in this invention, the culture medium is preferably LB medium, and the separation is preferably centrifugation.

[0028] In a fifth aspect, the present invention provides Acinetobacter jumborum as described above ( Acinetobacter junii The application of SCUEC19 strain, any one of the above-mentioned biological agents, or the combination of the above-mentioned agents in the degradation of microcystin.

[0029] In some implementations, the microcystin includes at least one of MC-LR, MC-RR, and MC-YR.

[0030] The beneficial effects of this invention are: unlike the prior art, this invention is the first to isolate a strain of Acinetobacter juncus (…). Acinetobacter junii The SCUEC19 strain of Acinetobacter juncea exhibits efficient degradation of microcystin, showing good degradation effects even on high concentrations of microcystin. It also demonstrates good salt, acid, alkali, and drought tolerance, along with good nitrogen fixation and indoleacetic acid production capabilities. Furthermore, the fermentation supernatant of Bacillus licheniformis promotes the growth of the SCUEC19 strain. Therefore, this strain shows promising application prospects. Attached Figure Description

[0031] Figure 1The image shows a high-performance liquid chromatogram of the SCUEC19 strain degrading MC-LR in Example 1 of this invention. In the image, A is a chromatogram of 15.0 μg / mL MC-LR standard, B is a chromatogram of the SCUEC19 strain after 0 h of culture in medium containing 15.0 μg / mL MC-LR, and C is a chromatogram of the SCUEC19 strain after 24 h of culture in medium containing 15.0 μg / mL MC-LR. Figure 2 This is a Gram staining image of the SCUEC19 strain in Example 1 of the present invention; Figure 3 This is a phylogenetic tree diagram of the SCUEC19 strain in Example 1 of the present invention; Figure 4 This is a graph showing the effect of different culture times on the degradation of MC-LR and growth of the SCUEC19 strain in Example 2 of the present invention; Figure 5 This is a graph showing the effect of different initial MC-LR concentrations on the degradation of MC-LR by the SCUEC19 strain in Example 3 of the present invention; Figure 6 This is a graph showing the effect of different culture temperatures on the degradation of MC-LR by the SCUEC19 strain in Example 4 of this invention; Figure 7 This is a graph showing the effect of different pH values ​​on the degradation of MC-LR by the SCUEC19 strain in Example 5 of the present invention; Figure 8 This is a graph showing the growth results of SCUEC19 strain under different NaCl concentrations in Example 6 of the present invention; Figure 9 This is a graph showing the growth results of SCUEC19 strain under different pH conditions in Example 7 of the present invention; Figure 10 Different concentrations of PEG in Example 8 of this invention 6000 Figure showing the growth results of SCUEC19 strain under the specified conditions; Figure 11 The image shows the qualitative detection results of indoleacetic acid production by the SCUEC19 strain in Example 9 of this invention. In the image, A is LB medium without inoculation, and B is the supernatant of fermentation broth inoculated with the SCUEC19 strain. Figure 12 This is a graph showing the detection results of indoleacetic acid content produced by the SCUEC19 strain at different culture times in Example 9 of the present invention; Figure 13 The figure shows the growth of SCUEC19 strain on Ashby solid medium in Example 10 of the present invention. In this figure, A and C are negative control strains without nitrogen fixation ability, and B and D are Acinetobacter jung SCUEC19 strains.

[0032] Figure 14 This is a graph showing the effect of Bacillus fermentation supernatant on the growth of SCUEC19 strain in Example 11 of this invention; Figure 15 This is a degradation curve of MC-LR by Acinetobacter juni SCUEC19 strain in Example 12 of the present invention; Biological Preservation The strain provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20261017, deposited on May 20, 2026, and classified as Acinetobacter junceus SCUEC19. Acinetobacter junii SCUEC19). Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0035] The culture medium used in this invention is as follows: Inorganic salt basic liquid culture medium (MM liquid medium): Na2HPO4 1.6g, KH2PO4 1.0g, (NH4)2SO4 0.5g, MgSO4·7H2O 0.5g, CaCl2 0.0215g, NaNO3 0.5g, add distilled water to a final volume of 1L, pH 7.0.

[0036] Inorganic salt basic solid medium (MM solid medium): Add 15-20 g / L of agar to inorganic salt basic liquid medium (MM liquid medium).

[0037] LB medium: NaCl 10.0g, tryptone 10.0g, yeast extract 5.0g, deionized water 1.0L, pH 7.0.

[0038] LA medium: Add 15-20 g / L of agar to LB medium.

[0039] Ashby solid medium: sucrose 10.0g, NaCl 0.12g, MgSO4·7H2O 0.2g, CaCO3 1.0g, KH2PO4 0.5g, agar 15-20g, deionized water 1.0L, pH 7.0.

[0040] DLB medium: NaCl 1.0g, tryptone 1.0g, yeast extract 0.5g, deionized water 1.0L, pH 7.0.

[0041] In this invention, the Salkowski colorimetric solution is: 31 mL of 0.5 mol / L FeCl, 30 mL of H2SO4, and 50 mL of deionized water.

[0042] Example 1: Isolation, purification, and identification of SCUEC19 strain 1.1 Targeted enrichment and isolation of SCUEC19 strain The sediment samples from Meiliang Bay in Taihu Lake, Jiangsu Province, were acclimatized and cultured using an inorganic salt-based liquid medium (MM liquid medium) with microcystin-LR (MC-LR) as the sole carbon source. Colonies of different morphologies were selected based on their morphology, color, and size, and pure strains were obtained through multiple streak purifications. A total of 33 strains with the ability to degrade MC-LR were screened.

[0043] Ten microbial strains were tested in MM liquid medium with 15 μg / mL MC-LR as the sole carbon source. After culturing at 30℃ and 180 r / min for 24 h in a shaker, the remaining MC-LR content was determined by HPLC to assess the strains' ability to degrade MC-LR. Seven strains showed high microcystin-LR degradation capabilities, with degradation efficiencies all exceeding 70%. One strain achieved the highest microcystin-LR degradation rate of 100%, and was named SCUEC19 (see [link to article]). Figure 1 ).

[0044] from Figure 1 As can be seen, after 24 hours of culture, the SCUEC19 strain achieved a 100% degradation rate of MC-LR (no corresponding MC-LR chromatographic peak was observed in the chromatogram), indicating that the SCUEC19 strain has a good degradation ability for MC-LR.

[0045] 1.2 Identification of SCUEC19 strain Morphological identification: The colonies of SCUEC19 on LA medium were pale yellow, round, moist, and smooth.

[0046] After Gram staining, the SCUEC19 strain was observed under a light microscope, and the results were as follows: Figure 2 As shown.

[0047] from Figure 2 As can be seen from the data, the SCUEC19 strain is Gram-negative, exhibiting a short rod-like shape when Gram-stained.

[0048] Physiological and biochemical identification: The physiological and biochemical characteristics of SCUEC19 strain were determined using a bacterial biochemical identification kit. The results are shown in Table 1.

[0049] Table 1 Physiological and biochemical characteristics of SCUEC19 strain

[0050] Note: "-" indicates a negative result.

[0051] As can be seen from the results in Table 1, the indole test, methyl red test, voltaic test and citrate test of SCUEC19 strain were all negative.

[0052] Molecular biological identification: Genomic DNA was extracted from SCUEC19 strain, and using total DNA from SCUEC19 strain as a template, the 16S rDNA sequence was amplified using primers 16S rDNA-F and 16S rDNA-R. The nucleotide sequences of 16S rDNA-F and 16S rDNA-R are shown below: 16S rDNA-F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 2); 16S rDNA-R:5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.3); The amplified product was sent to the company for sequencing. Its 16S rDNA sequence is shown in SEQ ID NO.1, with a length of 1402 bp. This sequence was BLASTed using NCBI (https: / / www.ncbi.nlm.nih.gov / ), and a phylogenetic tree was constructed using the NJ method in MEGA 11.0 software. The results are as follows. Figure 3 As shown.

[0053] from Figure 3 It can be seen from this that the SCUEC19 strain is related to Acinetobacter junii Based on the highest similarity to ATCC 17908 and considering morphological, physiological and biochemical characteristics and molecular biological identification results, strain SCUEC19 was named Acinetobacter jung. Acinetobacter junii ) SCUEC19 strain.

[0054] Example 2: Determination of microcystin degradation characteristics of Acinetobacter jungi SCUEC19 strain Acinetobacter jumbo strain SCUEC19 was inoculated into MM liquid medium containing 15.0 μg / mL microcystin-LR (MC-LR) and cultured in a shaker at 30℃ and 180 r / min. Samples were taken at different culture times (0, 2, 4, 6, 8, 12, 16, 24 h), and the degradation ability of the strain on MC-LR within 24 h was determined by HPLC. The results are as follows. Figure 4 As shown.

[0055] from Figure 4 As can be seen, with the gradual degradation of MC-LR, the OD of Acinetobacter jumbo strain SCUEC19 decreased. 600 The value gradually increased, indicating that Acinetobacter juniorii SCUEC19 strain can grow using MC-LR as the sole carbon source. Furthermore, the concentration of MC-LR decreased slowly from 0 to 4 hours and rapidly from 4 to 8 hours. At 8 hours of culture, the concentration of MC-LR was approximately 2.0 μg / mL. When the culture time was extended to 12 hours, the concentration of MC-LR was 0.0 μg / mL, indicating that MC-LR had been completely degraded by Acinetobacter juniorii SCUEC19 strain. These results demonstrate that Acinetobacter juniorii SCUEC19 strain possesses a highly efficient ability to degrade MC-LR.

[0056] Example 3: Characterization of Acinetobacter jumboschneideri SCUEC19 strain in degrading high concentrations of MC-LR Acinetobacter jumbo strain SCUEC19 was inoculated into MM liquid medium containing MC-LR at final concentrations of 15.0, 25.0, 35.0, and 45.0 μg / mL (initial OD of SCUEC19 strain in the medium). 600 The sample (value 0.306) was cultured at 30℃ and 180 r / min in a constant temperature shaker for 12 h. The remaining MC-LR content was detected by HPLC, and the results are as follows: Figure 5 As shown.

[0057] from Figure 5 As can be seen, after culturing Acinetobacter jungiformis strain SCUEC19 in MM liquid medium with final MC-LR concentrations of 15.0, 25.0, 35.0, and 45.0 μg / mL for 12 h, the degradation rates were 100.00%, 89.28%, 76.56%, and 50.15%, respectively. The results indicate that Acinetobacter jungiformis strain SCUEC19 possesses the ability to degrade high concentrations of MC-LR.

[0058] Example 4: Effect of temperature on the degradation of MC-LR by Acinetobacter jungi SCUEC19 strain Acinetobacter jumbo strain SCUEC19 was inoculated into MM liquid medium containing MC-LR at a final concentration of 15.0 μg / mL (initial OD of SCUEC19 strain in the medium). 600 The MC-LR content was determined by HPLC after incubation at 180 rpm for 12 h in shakers at 15, 25, 30, 37, and 45 °C (value 0.306). The remaining MC-LR content was then analyzed. The results are as follows: Figure 6 As shown.

[0059] from Figure 6 As can be seen, at a temperature of 30℃, the degradation rate of MC-LR by Acinetobacter juniorhynchus strain SCUEC19 was 100.00% within 12 hours. At temperatures of 15℃ and 25℃ with a culture time of 12 hours, the degradation rates reached 72.64% and 92.83%, respectively. At temperatures of 37℃ and 45℃ with a culture time of 12 hours, the degradation rates reached 90.69% and 61.09%, respectively. These results indicate that Acinetobacter juniorhynchus strain SCUEC19 can efficiently degrade MC-LR over a wide temperature range.

[0060] Example 5: Determination of the effect of pH on the degradation of MC-LR by Acinetobacter juncea SCUEC19 Acinetobacter jumbo strain SCUEC19 was inoculated into MM liquid medium containing MC-LR at pH values ​​of 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 with a final concentration of 15.0 μg / mL. The initial OD of strain SCUEC19 in the medium was measured. 600 The value was 0.306. After incubation at 30℃ and 180 r / min for 12 h, the remaining MC-LR content was determined by HPLC. The results are as follows: Figure 7 As shown.

[0061] from Figure 7 The data shows that the degradation rate was highest at pH 7.0 (100.00%), while the degradation rates at pH 5.0 and 6.0 were 62.59% and 87.74%, respectively. At pH 8.0, 9.0, and 10.0, the degradation rates were 92.33%, 79.48%, and 67.55%, respectively. These results indicate that the optimal pH for Acinetobacter jumboschneidere SCUEC19 to degrade MC-LR is 7.0.

[0062] Example 6: Determination of salt tolerance of Acinetobacter jungi SCUEC19 strain Using LB medium as the basal medium, certain proportions of NaCl were added to it at concentrations of 0.1%, 0.5%, 1.0%, 3.0%, 6.0%, 9.0%, and 12.0% (w / v). Acinetobacter juncea SCUEC19 strain was inoculated into these media respectively, allowing the strain to reach its initial OD. 600 The value was 0.1, each concentration was repeated three times, and the cells were incubated at 30℃ and 180 rpm for 24 h, and the OD was measured. 600 Value, result as Figure 8 As shown.

[0063] from Figure 8 As can be seen, when the NaCl concentration is 0.1-6.0%, the OD of Acinetobacter jungi SCUEC19 strain... 600 When the concentration of NaCl increased to between 2.073 and 1.747, and the concentration of NaCl increased to between 9.0% and 12.0%, the growth of Acinetobacter juncea SCUEC19 strain was inhibited. The results indicate that Acinetobacter juncea SCUEC19 strain exhibits good salt tolerance.

[0064] Example 7: Determination of acid and alkali resistance of Acinetobacter jungi SCUEC19 strain Using LB medium as the basal medium, different pH values ​​(2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0) of culture media were prepared. Acinetobacter juncea SCUEC19 strain was inoculated into these media respectively, allowing the strain to reach its initial OD value. 600 The value was 0.1, each gradient was repeated 3 times, and the cells were incubated at 30℃ and 180 rpm for 24 h, and the OD was measured. 600 Value, result as Figure 9 As shown.

[0065] from Figure 9 As can be seen, the OD of Acinetobacter jumboschneideri SCUEC19 strain was [not specified] when the pH of the culture medium was between 5.0 and 11.0. 600 The value is 1.965-1.858, and the OD value is 2.0. 600 The value was 0.248, and the pH of the culture medium was 12.0. OD 600 The value was 0.242, indicating that Acinetobacter jumboschneideri SCUEC19 strain can grow normally in a culture medium with a pH between 5.0 and 11.0, and has good acid and alkali resistance.

[0066] Example 8: Determination of drought resistance of Acinetobacter jungi SCUEC19 strain Using LB medium as the basal medium, different polyethylene glycol (PEG) formulations were prepared. 6000Acinetobacter juncea SCUEC19 strain was inoculated into culture media with concentrations of 0.0%, 10.0%, 20.0%, 25.0%, and 30.0% (w / v) respectively, allowing the strain to reach its initial OD. 600 The value was 0.1, with three replicates for each concentration, in PEG-free samples. 6000 LB medium was used as a control, and the cells were incubated at 30℃ and 180 rpm for 24 h. OD was then measured. 600 Value, result as Figure 10 As shown.

[0067] from Figure 10 As can be seen from this, with PEG 6000 With increasing concentration, the absorbance of Acinetobacter jumbo strain SCUEC19 gradually decreased at 600 nm in PEG. 6000 At a concentration of 0.0%, OD 600 The value is 2.164; PEG 6000 At a concentration of 10.0%, OD 600 The value is 1.530; when the PEG concentration is 20.0%, the OD... 600 The value was 1.205; when the PEG concentration was 25.0% and 30.0%, the growth of Acinetobacter jungi SCUEC19 decreased to 0.774 and 0.595, respectively, indicating that Acinetobacter jungi SCUEC19 has good drought resistance.

[0068] Example 9: Determination of Indoleacetic Acid Production Performance of Acinetobacter juncea SCUEC19 Strain Acinetobacter jumbo strain SCUEC19 was inoculated into LB medium containing 100 mg / L L-tryptophan, and the initial OD was... 600 The value was 0.1. The culture was incubated at 30℃ and 180 rpm for 24 h on a shaker. The bacterial culture was centrifuged at 8000 rpm for 10 min, and 1 mL of the supernatant was placed in a six-well plate. An equal volume of Salkowski colorimetric reagent was added and mixed thoroughly. Uninoculated culture medium was used as a negative control. After a dark reaction at room temperature for 30 min, the color change was observed. The results are as follows: Figure 11 As shown.

[0069] from Figure 11 As can be seen, the color of the mixture did not change when the culture medium was not inoculated with the strain. Figure 11 A); after inoculating the supernatant of the fermentation broth of SCUEC19 strain, the mixture turned red ( Figure 11 (B) The results showed that Acinetobacter jumbo strain SCUEC19 had the ability to produce indoleacetic acid.

[0070] Furthermore, indoleacetic acid standard solutions of 0, 5, 10, 20, 25, 30, 40, and 50 mg / L were prepared, and OD values ​​were measured after mixing with the colorimetric reagent. 530 To determine the initial OD value, a standard curve of indoleacetic acid was plotted. Acinetobacter jumbo strain SCUEC19 was inoculated into LB medium containing 100 mg / L L-tryptophan. 600 The value was 0.1, and each concentration was repeated three times. The ability to produce indoleacetic acid was determined by incubation at 30℃ and 180 r / min. 3 mL of the fermentation broth of the test strain was taken at 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h, respectively. After centrifugation at 12000 r / min for 2 min, the supernatant was collected, and an equal volume of Salkowski chromogenic reagent was added and mixed well. After a dark reaction at room temperature for 30 min, the OD was measured. 530 The indoleacetic acid (IAA) content in the bacterial culture was calculated based on the IAA standard curve, and the results are as follows: Figure 12 As shown.

[0071] from Figure 12 As can be seen, the indoleacetic acid (IAA) content in the culture medium of Acinetobacter juncea SCUEC19 strain increased rapidly within 0-72 h, reaching 25.65 mg / L at 72 h. Between 72-144 h, the IAA content increased slowly, reaching 29.09 mg / L at 144 h, and then decreased to 28.55 mg / L at 168 h. These results indicate that Acinetobacter juncea SCUEC19 strain has a good capacity for producing IAA.

[0072] Example 10: Determination of nitrogen fixation capacity of Acinetobacter jungi SCUEC19 strain Acinetobacter jumbo strain SCUEC19 was inoculated onto LA medium plates and activated for 12 h. It was then streaked onto Ashby agar plates with Escherichia coli as a blank control. The culture was carried out at 30°C for 24 h. The strain continued to grow even after three consecutive subculturings. Results are as follows: Figure 13 As shown.

[0073] from Figure 13 As can be seen, the negative control strains (A and C) had no nitrogen-fixing ability and showed no obvious growth on Ashby solid medium. The Acinetobacter jungi SCUEC19 strains (B and D) formed clear and robust colonies on Ashby solid medium and showed good growth. The results indicate that Acinetobacter jungi SCUEC19 strain has a good nitrogen-fixing ability.

[0074] Example 11: Determination of the growth-promoting effect of Bacillus licheniformis fermentation supernatant on Acinetobacter juncea SCUEC19 strain Acinetobacter jumboschneidera sinensis strain SCUEC19 and Bacillus licheniformis strain (ATCC 14580) preserved in glycerol were streaked onto LA medium for activation. Single colonies were picked and inoculated into LB medium and cultured at 30°C and 180 rpm for 12 h with shaking to obtain seed culture. The cells were collected by centrifugation at 4°C and 8000 rpm for 5 min, washed twice with sterile water, and transferred to 50 mL of DLB ​​medium to allow initial OD to develop. 600 The concentration was set at 0.05, and different volumes of supernatant from Bacillus licheniformis culture (cultured for 12 h) and DLB medium were added to make a total added volume of 5000 μL. The culture was incubated at 30℃ with shaking at 180 r / min, and samples were taken at 0, 1, 2, 3, 5, 7, 9, and 12 h to determine the OD of the bacterial culture. 600 For each sample, three parallel experiments were set up, and the results are as follows: Figure 14 As shown.

[0075] from Figure 14 As can be seen, the SCUEC19 strain with added Bacillus licheniformis culture supernatant showed significantly faster growth compared to the strain without the supernatant. In 50 mL DLB medium, the growth rate of SCUEC19 strain was 1.1 times that without the supernatant when 2.5 mL of Bacillus licheniformis culture supernatant was added, while the growth rate was 1.3 times that without the supernatant when 5.0 mL of supernatant was added. These results indicate that the Bacillus licheniformis fermentation supernatant has the characteristic of promoting the growth of the SCUEC19 strain.

[0076] Example 12: Application test of Acinetobacter jungi SCUEC19 strain in degrading aqueous solutions containing algal toxins. Acinetobacter jung SCUEC19 strain was inoculated into LB medium and cultured at 30°C and 180 rpm for 12 h in a constant temperature shaker. After culture, the culture was centrifuged at 3000 rpm for 8 min at 4°C, the supernatant was discarded, and the bacterial pellet was retained. The bacterial pellet was washed once with sterile water, and then washed twice with MM liquid medium. The washed bacterial pellet was resuspended in MM medium to OD. 600 The concentration was set at 0.5, and MC-LR was added to bring the final concentration to 15.0 μg / mL. The mixture was incubated at 30℃ and 180 rpm in a constant temperature shaker. The control treatment did not include any added microorganisms. Samples were taken at 0, 2, 4, 6, 8, 14, 16, and 24 hours after incubation, and the remaining MC-LR content was determined by HPLC. Each experimental group was run in triplicate. Results are as follows: Figure 15 As shown.

[0077] from Figure 15As can be seen, the MC-LR concentration in the control group remained unchanged. However, in the treatment with Acinetobacter juncea SCUEC19, the MC-LR concentration decreased slowly within 0-4 hours, and rapidly within 4-8 hours. At 8 hours of incubation, the MC-LR concentration was approximately 2.0 μg / mL. When the incubation time was extended to 16 hours, the MC-LR concentration was 0.0 μg / mL, indicating that MC-LR had been completely degraded by Acinetobacter juncea SCUEC19. The results show that Acinetobacter juncea SCUEC19 can completely degrade algal toxins in an aqueous solution with a final concentration of 15.0 μg / mL MC-LR.

[0078] Example 13 Application test of Acinetobacter jungi SCUEC19 strain in algal solution containing algal toxins 13.1 Activation of Acinetobacter jumbo strain SCUEC19 The Acinetobacter jungi strain SCUEC19, preserved in glycerol, was activated and inoculated onto LA medium, then incubated at 30°C for 12 hours. The activated strain was then picked and inoculated onto LB medium, incubated at 30°C for 12-16 hours to obtain the culture medium. Further spray drying was performed to obtain a dry powder bacterial agent. The preferred spray drying conditions were: corn starch as the carrier, a material concentration of 16% (W / V), an inlet air temperature of 165°C, and a feed rate of 25 mL / min, resulting in a viable count of Acinetobacter jungi strain SCUEC19 of approximately 1.0 × 10⁻⁶. 10 cfu / g -1.0×10 12 cfu / g.

[0079] 13.2 Acinetobacter jungi strain SCUEC19 was used to treat algal solutions containing algal toxins. The method of using Acinetobacter jungi SCUEC19 strain in algal solutions: 0.5% Acinetobacter jungi SCUEC19 strain spray-dried granules were inoculated into the algal solutions, stirred evenly, and treated at room temperature for 15 days and 30 days. Each treatment group was set up with 5 replicates. The results are shown in Table 2 below.

[0080] Table 2. Microcystin content in algal broth of Acinetobacter juncea SCUEC19 strain at different fermentation times.

[0081] Note: For liquid chromatography, the detection limit for algal toxins is 0.06 μg / L.

[0082] Table 2 shows that, compared with treatment day 0, the contents of microcystin-LR, microcystin-RR, and microcystin-YR in the algal solution treated with Acinetobacter juncea SCUEC19 strain after 15 and 30 days of treatment were 0.43 μg / L, 0.35 μg / L, 0.36 μg / L and not detected, 0.08 μg / L, and 0.09 μg / L, respectively. The results indicate that Acinetobacter juncea SCUEC19 strain has good degradation performance for microcystin-LR, microcystin-RR, and microcystin-YR in the algal solution.

[0083] In summary, the Acinetobacter jungi strain SCUEC19 provided by this invention exhibits good salt, acid, alkali, and drought resistance. Furthermore, this Acinetobacter jungi strain SCUEC19 demonstrates good nitrogen fixation and indoleacetic acid production capabilities. The fermentation supernatant of Bacillus licheniformis promotes the growth of Acinetobacter jungi strain SCUEC19. In addition, this Acinetobacter jungi strain SCUEC19 can efficiently degrade microcystin.

[0084] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0085] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

[0086] The nucleotide sequence of the 16S rDNA of strain SCUEC19 (SEQ ID NO.1)

Claims

1. A highly efficient Acinetobacter jungi strain SCUEC19 that degrades algal toxins, characterized in that, The preservation number of the Acinetobacter juni SCUEC19 strain is CCTCC NO:M 20261017.

2. The Acinetobacter jumbo strain SCUEC19 according to claim 1, characterized in that, The colony morphology of Acinetobacter jungiformis strain SCUEC19 is pale yellow, round, moist, and smooth. It is a Gram-negative bacterium, short rod-shaped, with an optimal growth pH of 5.0-11.0 and a maximum tolerance to NaCl concentration of 6.0%. The nucleotide sequence of the 16S rDNA of Acinetobacter jungiformis strain SCUEC19 is shown in SEQ ID NO.

1.

3. The Acinetobacter jungi SCUEC19 strain according to claim 1, characterized in that, The Acinetobacter jungii SCUEC19 strain was able to completely degrade MC-LR at a concentration of 15.0 μg / mL under the conditions of pH 7.0, culture temperature of 30℃ and culture time of 12h.

4. The Acinetobacter jungi strain SCUEC19 according to claim 1, characterized in that, The highest concentration of MC-LR that the Acinetobacter juniorhynchus SCUEC19 strain degraded was at least 45.0 μg / mL, and the degradation rate of MC-LR at a concentration of 45.0 μg / mL by the Acinetobacter juniorhynchus SCUEC19 strain was 50.15% after 12 hours.

5. The use of Acinetobacter jumbo strain SCUEC19 as described in any one of claims 1-4 in any of the following: 1) Salt resistant; 2) Acid and alkali resistant; 3) Drought resistant; 4) Nitrogen fixation; 5) Production of indoleacetic acid.

6. A biological agent, characterized in that, Includes the Acinetobacter jumbo strain SCUEC19 as described in any one of claims 1-4.

7. The biological agent according to claim 6, characterized in that, The biological agent includes at least one of liquid and solid agents; The liquid bacterial agent contains 1.0 × 10⁻⁶ Acinetobacter von Willebrandi SCUEC19 strain. 10 cfu / mL -1.0×10 12 The concentration of cfu / mL in the solid bacterial agent is 1.0 × 10⁻⁶ CFU / mL, and the content of Acinetobacter juncea SCUEC19 strain is 1.0 × 10⁻⁶ CFU / mL. 10 cfu / g -1.0×10 12 cfu / g.

8. A combined microbial agent, characterized in that, Includes Acinetobacter jungi SCUEC19 strain and Bacillus licheniformis fermentation supernatant as described in any one of claims 1-4; The fermentation supernatant of Bacillus licheniformis promotes the growth of Acinetobacter jung SCUEC19 strain.

9. The use of Acinetobacter jumbo strain SCUEC19 as described in any one of claims 1-4, the biological agent as described in any one of claims 6-7, or the combined bacterial agent as described in claim 8 in the degradation of microcystin.

10. The application according to claim 9, characterized in that, The microcystin includes at least one of MC-LR, MC-RR, and MC-YR.

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