Morgan morganella and application thereof
Morganella morganii JHT-1, obtained through screening and purification, was used to prepare a microecological preparation, which solved the problem of excessive sulfide concentration in marine aquaculture areas, achieving efficient removal of sulfides, improving water quality, and reducing secondary pollution.
Patent Information
- Application Number
- CN202511228042.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-29
- Publication Date
- 2025-12-05
AI Technical Summary
The "aging" phenomenon caused by excessively high sulfide concentrations in marine aquaculture areas is easily caused by existing physical and chemical treatment methods, which are prone to secondary pollution and whose effects are difficult to sustain. Microbial immobilization technology has failed to effectively solve the problem of sulfide removal.
A strain of Morganella morganii JHT-1 was provided, which was obtained through enrichment, separation and purification. It has the ability to remove sulfides efficiently and can be made into a solid powder microecological preparation for use in aquaculture and domestic sewage.
It achieves efficient removal of sulfides from water and sewage, improves water quality, reduces secondary pollution, and has significant application value and broad application prospects.
Smart Images
Figure CN121065013A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial strain technology, and particularly relates to a strain of Morganella morganii and its applications. Background Technology
[0002] my country is a major producer of marine aquaculture. In nearshore aquaculture areas, to increase yields, feed is often added to the water, easily leading to severe organic loads and black, foul-smelling sediments. At the sediment-water interface in nearshore aquaculture areas, the aerobic degradation of organic matter results in a significant depletion of dissolved oxygen, causing a decrease in the water's oxidation-reduction potential (Eh). Under these conditions, sulfate-reducing bacteria (SRB) in the sediment produce large amounts of sulfides through sulfate respiration. These sulfides accumulate at the sediment-water interface and diffuse upwards. The toxic effects of high concentrations of sulfides severely threaten the aquaculture environment, especially benthic organisms, leading to sediment "aging" and hindering the healthy development of the aquaculture industry.
[0003] Currently, to address the "aging" phenomenon in marine aquaculture areas, especially the problem of excessively high sulfide concentrations, physical (filtration, sieving) or chemical (strong oxidants) methods are mostly used for treatment. However, these methods are prone to causing secondary pollution, and the treatment effect is difficult to sustain, easily leading to rebound. In the biological removal of sulfides, microorganisms play a crucial role in sulfide removal. To make the reaction more stable and prevent microorganisms from being lost, they are usually immobilized. These microorganisms, compared to the autotrophic microorganisms previously used for biological sulfide removal, are characterized by rapid growth and large biomass. Summary of the Invention
[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a strain of Morganella morganii and its application. The Morganella morganii provided by this invention has the function of efficiently removing sulfides and can improve aquaculture water and domestic sewage.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0006] Firstly, a strain of *Morganella morganii* JHT-1, deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.34498, was provided.
[0007] This strain was obtained from black and foul-smelling sediments in nearshore aquaculture areas. After enrichment, isolation, and purification, it was confirmed to be non-toxic using blood agar plate testing. Through morphological observation and molecular biological analysis, the strain was identified as *Morganella morganii*, named JHT-1 in the laboratory, and deposited at the China General Microbiological Culture Collection Center. This strain exhibits a strong ability to remove sulfides from water bodies and can be used to remove sulfides from aquaculture water and domestic sewage.
[0008] Furthermore, the Morganella morganii JHT-1 described above has the following colony morphology characteristics: colony size 10-25 μm, colonies are round, raised, moist, milky white, and the bacterial morphology is rod-shaped under scanning electron microscopy.
[0009] Furthermore, the Morganella morganii JHT-1 was identified as a Gram-negative bacterium by Gram staining.
[0010] Secondly, this invention provides the application of Morganella morganii JHT-1 in the removal of sulfides from aquaculture water and domestic sewage.
[0011] Thirdly, the present invention provides a microbial agent for removing sulfides, the raw material of which includes the aforementioned Morganella morganii JHT-18.
[0012] Furthermore, the bacterial agent is in the form of a solid powder.
[0013] Furthermore, the aforementioned microbial agent is used to remove sulfides from aquaculture water bodies and domestic sewage.
[0014] Compared with the prior art, the advantages of the present invention are as follows:
[0015] The Morganella morganii JHT-1 strain provided by this invention grows rapidly and has the ability to efficiently remove sulfides from water. The microecological preparations made from this strain have a high removal rate of sulfides from water and have important application value for improving the quality of aquaculture water and domestic sewage. Attached Figure Description
[0016] Figure 1 This is a colony morphology diagram of Morganella morganii JHT-1 used in this invention.
[0017] Figure 2 for Figure 1 A magnified view of a portion of the image.
[0018] Figure 3 This is a Gram staining image of Morganella morganii JHT-1 used in this invention.
[0019] Figure 4This is a scanning electron microscope image of Morganella morganii JHT-1 used in this invention.
[0020] Figure 5 This is a blood plate test diagram of Morganella morganii JHT-1 used in this invention. Detailed Implementation
[0021] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.
[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. The reagents and raw materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods or product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.
[0023] Example 1: Isolation, purification and identification of Morganella morganii JHT-1
[0024] 1. Enrichment and isolation of bacterial strains:
[0025] One g of foul-smelling sediment from the nearshore aquaculture area of Yantai was inoculated into 100 mL of LB solid medium, with glucose added as an organic matter source (50 mg / L). The medium was incubated at 30°C for 7 days, with 0.01 mg / L sodium sulfide added daily for continuous enrichment. The enriched culture was then serially diluted and plated onto LB solid medium, and incubated at 30°C. After incubation, different purified single colonies on the plates were named and selected, and their concentrations were determined using a sulfide assay kit. The results of the bacterial assays in the enriched culture are shown in Table 1 below.
[0026] Table 1. Results of sulfide removal rate determination of bacterial strains in enrichment culture medium
[0027] code name Sulfide removal rate (24h) Sulfide removal rate (48h) S-1 25.2±3.6% 20.36±3.8% S-2 1.3±0.18% 2.1±0.27% S-3 0.3±0.08% 0.31±0.01% S-4 10.6±1.98% 11.2±0.72% S-5 8.6±0.75% 6.6±0.65% S-6 32.5±5.1% 41.6±4.95% S-7 85±2.3% 95±1.9% S-8 65.6±7.8% 75.3±8.2% control group 0 0
[0028] Based on the sulfide determination results, strain S-7, which had the highest sulfide removal rate, was screened and purified. The strain was isolated using the plate spread method. Once the colonies reached a suitable size, they were repeatedly streaked until single strains with uniform colony morphology were obtained. Single colonies were picked and inoculated into LB broth and cultured with shaking at 30℃ and 120 r / min for 16–24 h. After cultivation, the bacterial cells were collected, and 30% glycerol was added as a cryoprotectant. After mixing, the cells were stored at -80℃ for subsequent experiments.
[0029] 2. Strain identification
[0030] The identification process and results of strain S-7 selected in this embodiment are as follows:
[0031] 2.1 Observation of strain characteristics:
[0032] Microbial morphology and structure were observed using colony observation, optical microscopy, and scanning electron microscopy.
[0033] Strain S-7, stored at -80℃, was spread onto LB solid medium, and its colony characteristics were observed. After activation, strain S-7 was stained with crystal violet and examined under a microscope. The microscopic surface morphology of the strain was observed using scanning electron microscopy. The specific steps are as follows:
[0034] a) Take the test strain in the logarithmic growth phase, centrifuge at 5000 r / min for 5 min and remove the supernatant;
[0035] b) Wash three times with PBS and gently suspend;
[0036] c) After washing with PBS, fix with 2.5% glutaraldehyde for 3 h; rinse with PBS 2-3 times, 10 min each time, and then rinse twice with pure water.
[0037] d) Dehydrate the sample with a gradient of 30%, 50%, 70%, 80%, and 90% alcohol for 10 min each; then dehydrate it twice with anhydrous ethanol for 10 min each time; finally, place the sample in a 1:1 mixture of ethanol and tert-butanol for 15 min; after centrifugation at each dehydration step, gently suspend the sample.
[0038] e) Place the prepared sample on a glass slide, let it dry, coat it, and observe it under a scanning electron microscope.
[0039] Morphological identification results:
[0040] The colony morphology of strain S-7 on LB medium is as follows: Figure 1 and Figure 2 As shown: Colonies are 10-25 μm in size, round, raised, moist, and milky white. See also Figure 3Gram staining identified it as a Gram-negative bacterium. See also Figure 4 It appears as a rod shape under a scanning electron microscope.
[0041] 2.2 Molecular biological identification:
[0042] PCR amplification was performed using universal 16S rDNA primers 27F and 1429R. The PCR products were purified and sequenced by Shanghai Sangon Biotech Co., Ltd. The sequencing results were compared with the NCBI database for homology, identifying strain S-7 as *Morganella morganii*, and the strain was renamed JHT-1.
[0043] 2.3 Safety analysis of the strain
[0044] The safety of bacterial strains was determined using the rabbit blood agar streak method. Strains with a clear zone and a greenish-brown zone around the colony exhibited α-hemolysis (severe) and β-hemolysis (mild), respectively, indicating toxicity. Strains without changes around the colony were considered non-hemolytic, indicating safety and non-toxicity. (See also...) Figure 5 The JH-1 strain showed no hemolytic changes around its colonies, indicating that it is a non-toxic strain according to safety analysis.
[0045] Example 2: Analysis of the fermentation, immobilization and sulfide removal capabilities of strain JHT-1
[0046] The JHT-1 strain at -80℃ was spread onto LB solid medium and activated. Then, strain JHT-1 was inoculated at a ratio of 1% into 200 mL of LB medium and cultured at 120 r / min and 30℃ with constant temperature shaking for 16-24 h, adjusting the bacterial concentration to 10-100 billion CFU / mL. 100 mL of the above bacterial solution was sprayed onto 200-1000 g of wheat bran (200 mesh size) and air-dried at room temperature to prepare a solid microecological preparation.
[0047] The JHT-1 bacterial agent prepared by the above method was applied at a concentration of 1.0 × 10⁻⁶. 6 CFU / mL or 1.0×10 8 The working concentration of CFU / mL was applied to laboratory-prepared sodium sulfide (0.02 mg / L) wastewater and allowed to stand at room temperature. The removal capacity of JHT-1 for sulfides in wastewater was analyzed at 0 h, 12 h, 24 h, and 48 h. The removal rate of sodium sulfide in laboratory sodium sulfide wastewater is shown in Table 2 below.
[0048] Table 2. Removal rate of sodium sulfide from laboratory sodium sulfide wastewater by strain JHT-1
[0049] Time (h) <![CDATA[Sulfide removal rate (1.0×10 6 CFU / mL)]]> <![CDATA[Sulfide removal rate (1.0×10 8 CFU / mL)]]> 0 0 0 12 89.5±5.3% 95.6±10.3% 24 92.3±7.8% 98.6±2.6% 48 95.6±8.9% 99.2±3.5%
[0050] Example 3: Verification of the wastewater treatment capacity of JHT-1 microecological preparation
[0051] Case 1: Wastewater from a shrimp farm in Muping District, Yantai City, was tested and found to contain 0.02 mg / L of sulfide. The JHT-1 microecological preparation (1.0 × 10⁻⁶ mg / L) prepared using the method described in Example 2 was then used. 11 When CFU / g was added to the aquaculture water at a concentration of 100g / mu*m, the sulfide content of the aquaculture water was measured to be (1±0.1)×10 after 24 hours. -6 mg / L, with a removal rate of nearly 100%, and effectively improved shrimp vitality and feed intake, while reducing odor in the greenhouse air.
[0052] Case 2: Odorous domestic sewage was collected from Yantai City, and JHT-1 microecological preparation (1.0 × 10⁻⁶ g / mu*m³ water) prepared according to the method in Example 2 was added at a concentration of 80 g / mu*m³ water. 12 The sulfide content in the domestic sewage was 0.4 mg / L (CFU / g). After 48 h, the sulfide content in the wastewater was measured to be 0.01 mg / L, with a removal rate of approximately 97.5%, and the deodorization effect was significant.
[0053] Case 3: JHT-1 probiotic preparation (1.0 × 10⁻⁶) prepared using the method in Example 2. 11 The CFU / g solution was added to grass carp ponds in Wuhan, Hubei Province, at a concentration of 100g / mu*m. The sulfide concentration in the aquaculture water was 0.02 mg / L. After 12 hours of application, the sulfide concentration in the water was measured to be 0.001 mg / L, indicating a good removal rate and significant effect.
[0054] Example 4: Preservation of microbial strains
[0055] As demonstrated in the preceding examples, the JHT-1 strain obtained by this invention exhibits a strong ability to remove sulfides from aquaculture water and domestic sewage, making it a safe, multifunctional, and highly efficient water treatment strain. Based on these characteristics, the cultivation and immobilization of this strain, and its application in the removal of sulfides from aquaculture water and urban sewage, demonstrates significant application value and broad prospects.
[0056] The safe and efficient sulfide removal strain JHT-1 obtained by this invention has been deposited at the China General Microbiological Culture Collection Center (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC NO.34498 and deposit date of May 9, 2025.
[0057] In summary, the strain JHT-1 screened in this invention can significantly reduce sulfides in aquaculture water and urban sewage, thereby improving water quality. Theoretically, it should also be effective against aquaculture wastewater, aquaculture pond sludge, and polluted soil from waste treatment plants.
[0058] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.
Claims
1. A strain of Morganella morganii JHT-1, characterized in that, It is preserved in China General Microbiological Culture Collection Center, and its preservation number is CGMCC NO.34498.
2. The Morganella morganii JHT-1 according to claim 1, characterized in that, The colony morphology characteristics are as follows: colony size is 10-25 μm, colony is round, raised, surface is wet, and is milky white, and under scanning electron microscope, the bacterial morphology is rod-shaped.
3. The Morganella morganii JHT-1 according to claim 1, characterized by that, Gram staining identification is gram-negative bacteria.
4. The application of Morganella morganii JHT-1 in removing sulfides in aquaculture water and domestic sewage according to claim 1.
5. A microbial agent for removing sulfides, characterized by, The raw material comprises the Morganella morganii JHT-18 according to claim 1.
6. The bacterial agent of claim 6, wherein The bacterial agent is in the form of solid powder.
7. The bacterial agent of claim 5 or 6, wherein It is used for removing sulfides in aquaculture water and domestic sewage. It is used for removing sulfides in aquaculture water and domestic sewage.