Comamonas testosteroni DP38 as well as fungicide and application thereof

Abstract

The invention discloses comamonas testosteroni DP38 as well as a microbial agent and application thereof, and belongs to the technical field of microbial wastewater purification. According to the invention, a comamonas testosterone i DP38 capable of efficiently removing nitrogen and phosphorus is separated, the preservation number of the comamonas testosterone i DP38 is GDMCC No: 66845, and the comamonas testosterone i DP38 is further prepared into an immobilized bacterial agent. Experimental results show that the comamonas testosteroni DP38 disclosed by the invention has efficient denitrification effect and phosphorus accumulating capacity, and can be directly used for ectopic treatment of aquatic water. Compared with comamonas testosteroni DP38, the nitrogen and phosphorus removal performance of the immobilized microbial agent in culture tail water is further improved, nitrate and phosphate can be basically removed, and the total nitrogen removal rate reaches 91.19%. The invention provides a new biological material for treatment of aquaculture tail water, and has a wide prospect in the field of microbial purification of wastewater.

Description

Technical Field

[0001] This invention relates to the field of microbial wastewater purification technology, and in particular to a strain of Trichomonas testis DP38, its inoculum, and its application. Background Technology

[0002] With the rapid development of intensive aquaculture, eutrophication of water bodies has become increasingly serious, primarily due to the excessive discharge of unused nutrients such as nitrogen and phosphorus from aquatic animals. Traditional wastewater denitrification and phosphorus removal processes (such as A...) 2 The treatment technologies (such as sludge treatment plants, sludge treatment plants, and sludge treatment plants) have problems such as unstable treatment efficiency, large sludge production, and high operating costs. There is an urgent need to develop new treatment technologies that are efficient, low-consumption, and environmentally friendly.

[0003] Immobilized microbial technology, by immobilizing specific functional strains on carrier materials, can significantly improve the environmental tolerance, bioactivity, and reusability of the strains, becoming a research hotspot in the field of water pollution control. Among these, biochar, due to its porous structure, high specific surface area, excellent adsorption performance, and abundant surface functional groups, has become an ideal carrier for microbial immobilization. It can effectively adsorb pollutants and provide attachment sites and a protective microenvironment for microorganisms, and can be further developed into highly functional composite carriers. By immobilizing screened, highly efficient denitrifying polyphosphate-accumulating bacteria on composite carriers to construct a "carrier-strain" functionalized system, targeted denitrification, efficient phosphorus removal, and improved system stability can be achieved. This technology combines the advantages of adsorption, chemical precipitation, and biodegradation, providing a promising solution for aquaculture wastewater treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of *Comamonas testosteroni* DP38, its inoculum, and its applications, to address the problems existing in the prior art. This invention isolates a strain of *Comamonas testosteroni* DP38, which possesses highly efficient denitrification and phosphorus accumulation capabilities, and can be directly used for the ex-situ treatment of aquaculture water. Further development into an immobilized inoculum further enhances its nitrogen and phosphorus removal performance in aquaculture wastewater. This invention provides a new biological material for the treatment of aquaculture wastewater and has broad prospects in the field of microbial wastewater purification.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a strain of *Comamonas testosteroni* DP38, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No. 66845, deposited on August 15, 2025, at the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] This invention provides an application of the above-mentioned Trichomonas vaginalis DP38 in the preparation of immobilized bacterial agents.

[0008] This invention also provides a method for preparing an immobilized bacterial agent, comprising the following steps:

[0009] Agricultural waste is mixed with iron powder, calcined, cooled, and washed to obtain modified biochar.

[0010] The modified biochar was mixed with the bacterial suspension of Trichomonas vaginalis DP38 and an encapsulating agent was added to obtain a mixture.

[0011] The mixture is dripped into a crosslinking agent and crosslinked and cured to obtain the immobilized bacterial agent.

[0012] Furthermore, the agricultural waste is coconut shell;

[0013] The mass ratio of the coconut shell to the iron powder is 1:1;

[0014] The calcination temperature was 400℃ and the time was 3 hours.

[0015] Furthermore, the mass-to-volume ratio of the modified biochar to the bacterial suspension is 10 g: 1 L.

[0016] Furthermore, the encapsulating agent comprises 1% sodium alginate by mass and 10% polyvinyl alcohol by mass;

[0017] The volume ratio of the bacterial suspension to the encapsulating agent is 1:1.

[0018] Furthermore, the crosslinking agent comprises 4% by mass of calcium chloride and 1% by mass of boric acid.

[0019] The present invention also provides an immobilized bacterial agent obtained according to the above preparation method.

[0020] The present invention also provides the application of the above-mentioned Trichomonas vaginalis DP38 or the above-mentioned immobilized bacterial agent in the purification of wastewater.

[0021] Furthermore, the wastewater is aquaculture wastewater with high nitrate and phosphorus content.

[0022] The present invention also provides a method for purifying wastewater, comprising the step of applying the above-mentioned Trichomonas vaginalis DP38 or the above-mentioned immobilized bacterial agent to the wastewater.

[0023] Furthermore, the wastewater is aquaculture wastewater with high nitrate and phosphorus content;

[0024] The amount of the immobilized bacterial agent applied is 80 g / L.

[0025] The present invention discloses the following technical effects:

[0026] This invention isolated a highly efficient nitrogen and phosphorus removal strain of *Comamonas testosteroni* DP38, with the preservation number GDMCC No: 66845, and further prepared it into an immobilized bacterial agent. Experimental results show that *Comamonas testosteroni* DP38 of this invention possesses highly efficient denitrification and phosphorus accumulation capabilities, simultaneously removing nitrate nitrogen, ammonia nitrogen, and phosphate generated during aquaculture. Furthermore, it accumulates very little nitrite during denitrification, resulting in no secondary pollution, and can be directly used for ex-situ treatment of aquaculture water. Compared to the bacterial suspension of *Comamonas testosteroni* DP38, the immobilized bacterial agent combines *Comamonas testosteroni* DP38 with biochar, increasing pore size and attachment sites for the strain. Iron can enhance the denitrification and phosphorus accumulation performance of *Comamonas testosteroni* DP38. In summary, the immobilized bacterial agent further improves the nitrogen and phosphorus removal performance in aquaculture wastewater, effectively removing nitrate and phosphate, with a total nitrogen removal rate of 91.19%. This invention provides new biological materials for the treatment of aquaculture wastewater and has broad prospects in the field of microbial wastewater purification. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The growth rate of Trichomonas testis DP38 in denitrified medium is shown in the figure.

[0029] Figure 2 Transmission electron microscopy image of Trichomonas testis DP38;

[0030] Figure 3 Phylogenetic tree of Trichomonas testis DP38;

[0031] Figure 4 Preliminary results of the denitrification capacity determination of Trichomonas vaginalis DP38;

[0032] Figure 5 The image shows the denitrification effect of biochar-immobilized strains in denitrification medium.

[0033] Figure 6 Examples of immobilized spheres with different combinations of embedding materials. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Iron-based materials (such as zero-valent iron and iron oxides) play a unique role in nitrogen and phosphorus removal processes: iron ions can directly remove phosphorus through chemical precipitation and promote denitrification processes (such as Fe) through redox reactions. 0 →Fe 2+ (Release electrons to drive nitrate reduction). Iron powder (Fe...) 0 Combining iron powder with biochar as a functionalized carrier can fully leverage their synergistic effect: biochar-loaded iron powder forms a porous conductive network, enhancing the carrier's mechanical strength and electron transport capacity; the iron powder continuously releases Fe... 2+ / Fe 3+ It enhances chemical phosphorus removal and acts as an electron donor to promote denitrification; the composite carrier improves the simultaneous removal efficiency of nitrogen and phosphorus through an adsorption-biodegradation coupling mechanism.

[0040] The molecular formula of bromothymol blue (BTB) is C2. 27 H 28 O5SBr2, CAS number 76-59-5.

[0041] The formulations of the various culture media used in the embodiments of this invention are shown in Table 1:

[0042] Table 1. Formulations of various culture media

[0043]

[0044] Example 1: Isolation, identification, and preservation of aerobic denitrifying bacteria

[0045] Comamonas testosteroni DP38 was isolated from a largemouth bass pond in Guangzhou, Guangdong Province. Details are as follows:

[0046] 1. Isolation and purification of strains

[0047] Take 10 g of activated sludge from the collected sediment sample and place it in an Erlenmeyer flask containing glass beads and 90 mL of sterile water. After thorough shaking, let it stand for 30 min. Take 5 mL of the supernatant and incubate it in LB medium at 30°C for 2 days for enrichment. After enrichment, take another 5 mL of the bacterial solution and incubate it in denitrification acclimatization medium at 30°C for 3 days. After acclimatization, take 1 mL of the bacterial solution and mix it thoroughly in 9 mL of sterile water, and repeat this process 10 times. -3 -10 -8 Serial dilutions were performed, with 0.2 mL of each dilution spread onto 1% BTB-denitrification medium in triplicate. The cultures were incubated at 30°C for 2 days. Blue-showing strains (a single colony with a blue halo) were used as the initial screening strains. Single colonies were picked and purified using the three-zone streak method, yielding 13 blue-showing strains. These strains were inoculated onto LB medium and incubated at 30°C for 48 hours. The cells were collected by centrifugation at 8000 r / min for 5 min, washed twice with sterile water, resuspended, and the OD of the culture was adjusted. 600 It is 0.8.

[0048] The bacterial culture was inoculated at a 2% inoculum onto denitrification medium in triplicate. The cultures were incubated at 30°C for 48 h. Samples were taken after centrifugation at 8000 r / min for 5 min, and the supernatant was collected. The phosphate (PO4) content was determined using ammonium molybdate spectrophotometry, ultraviolet spectrophotometry, and Nessler's reagent spectrophotometry, respectively. 3- ), nitrates (NO3) - ), nitrite (NO2) - ) and ammonia nitrogen (NH4) +The nitrogen and phosphorus removal rate of the strains (>80%) was calculated based on the nitrogen (N) content to screen for strains with denitrification and phosphorus accumulation functions. A strain with optimal denitrification and phosphorus accumulation functions was obtained and named strain DP38.

[0049] 2. Identification and preservation of strains

[0050] Strains DP38 were cultured on LB solid medium, colony morphology was observed, Gram staining was performed, and microscopic examination was conducted.

[0051] The biological characteristics of strain DP38 are shown in Table 2.

[0052] Table 2 Biological characteristics of strain DP38

[0053]

[0054] Strain strain DP38 was inoculated into denitrification medium and cultured, with samples taken periodically to measure OD. 600 Biomass was statistically analyzed. The results are as follows: Figure 1 As shown, strain DP38 continued to grow in the culture medium, reaching its maximum biomass after 24 h, and then entered a stable growth phase.

[0055] The cell morphology of strain DP38 was observed by transmission electron microscopy, and the results are as follows: Figure 2 As shown, it has single-ended flagella, no spores, no capsule, and the cells are rod-shaped and easily aggregate.

[0056] The genome of strain DP38 was extracted using a DNA extraction kit. Using the DP38 genome as a template, PCR amplification of the 16S rRNA gene was performed. Alignment of the obtained 16S rRNA gene sequence with NCBI revealed a high similarity (>99.9%) between strain DP38 and *Comamonas testosteroni*. Based on the 16S rRNA sequence, 20 strains most closely related at the species level were selected, and a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with MEGA 6.0 software. Figure 3 As shown. Based on the phylogenetic analysis of the 16S rRNA gene and its physiological and biochemical characteristics, strain DP38 was identified as a new strain of *Comamonas testosteroni*, and named *Comamonas testosteroni* DP38.

[0057] Commonas testosteroni DP38 was deposited on August 15, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66845.

[0058] Example 2 Preliminary Denitrification and Phosphorus Removal Capacity Determination of Trichomonas vaginalis DP38

[0059] The purified *Trichomonas vaginalis* DP38 was inoculated into LB medium and incubated at 30°C and 160 r / min for 24 h. The activated bacterial suspension was centrifuged at 8000×g, the supernatant was discarded, and the suspension was washed twice with 0.75% sterile physiological saline. The suspension was resuspended, and the OD value of the bacterial suspension was calculated. 600 Adjust the pH to approximately 0.8, and inoculate 2 mL of the culture into 100 mL of denitrification medium containing KNO3 as the sole nitrogen source. Incubate at 30°C and 160 r / min for 48 h. During this period, take 10 mL samples of the culture multiple times to determine the nitrogen and phosphorus concentrations. Centrifuge at 8000×g and take the supernatant to determine the NO3 content in the culture medium. - -N, NH4 + -N, NO2 - -N and PO4 3- The concentration of -P was used to calculate the nitrogen and phosphorus removal rate.

[0060] Nitrogen and phosphorus removal effects are as follows Figure 4 As shown, after 24 h of culture, *Trichomonas testis* DP38 completely removed nitrate nitrogen and phosphate. From 0 to 16 h, a certain amount of nitrite nitrogen was generated due to denitrification, which was completely removed after 24 h. Phosphate levels showed a gradual decreasing trend from 0 to 24 h and were completely removed after 24 h. Furthermore, ammonia nitrogen content did not increase significantly from 0 to 24 h.

[0061] Example 3: Screening of methods for preparing immobilized particles

[0062] 1. Preparation of embedding agent

[0063] Weigh 10 g of polyvinyl alcohol (PVA) and 90 mL of water, add them to a beaker, and heat in a water bath at 90°C to dissolve them, thus obtaining a 10% polyvinyl alcohol solution; weigh 1 g of sodium alginate (SA) and 99 mL of water, add them to a beaker, and heat in a water bath at 90°C to dissolve them, and seal the beaker after complete dissolution to obtain a 1% sodium alginate solution.

[0064] The procedure for preparing sodium alginate solutions or polyvinyl alcohol solutions of different concentrations is the same as above, using polyvinyl alcohol solutions, sodium alginate solutions, or a mixture of the two at different concentrations as embedding agents.

[0065] 2. Preparation of crosslinking agent

[0066] Dissolve 1 g of boric acid in 100 mL of water. Once completely dissolved, add 4 g of calcium chloride, stir to dissolve, and adjust the pH to neutral for later use as a crosslinking agent.

[0067] 3. Preparation of microspheres

[0068] First, add 100 mL of crosslinking agent to a large beaker, insert a magnetic stir bar, place it on a stirrer, and adjust the speed to keep the solution in a rotating state. Then, use a pipette to add 10 mL of bacterial suspension to the beaker containing 10 mL of embedding agent, and stir well to obtain a mixture. Use a sterile syringe to drop the mixture dropwise into the crosslinking agent. After the dropwise addition is complete, remove the stir bar with tweezers, cover with plastic wrap, and place in a 4°C refrigerator for crosslinking. After the crosslinking and curing are complete, remove from the refrigerator, pour off the crosslinking agent, wash with sterile physiological saline, repeat the rinsing 2-3 times, and store at 4°C for later use.

[0069] The spherical morphology is shown in Table 3 and Figure 6 As shown, the immobilization method using a mixture of 1% sodium alginate and 10% polyvinyl alcohol for crosslinking was selected.

[0070] Table 3 Performance of immobilized microspheres under different embedding material combinations

[0071] Preparation materials sphericity strength Reusability 0.5% SA good Low Low 1% SA excellent middle excellent 1.5% SA Low middle middle 0.5% SA + 5% PVA Low Low Low 0.5% SA + 10% PVA Low Low Low 0.5% SA + 15% PVA Low middle Low 1% SA + 5% PVA Low Low Low 1% SA + 10% PVA good excellent excellent 1% SA + 15% PVA Low middle Low 1.5% SA + 5% PVA Low good middle 1.5% SA + 10% PVA Low good Low 1.5% SA + 15% PVA Low good Low

[0072] Example 4: Preparation of modified biochar-immobilized bacterial particles and their application in denitrification media.

[0073] 1. Preparation method of composite biochar

[0074] 1.1 Preparation of *Trichomonas testis* DP38 bacterial suspension: 100 μL of *Trichomonas testis* DP38 was added to 100 mL of LB medium and cultured for 24 h. After centrifugation, the supernatant was discarded, and the culture was washed. Sterile physiological saline was then added to adjust the OD. 600 After adjusting the pH to 0.6-0.8, a bacterial suspension is obtained.

[0075] 1.2 The coconut shells were dried and ground, and large particles were removed by 100-mesh sieve to obtain solid powder. The solid powder and iron powder were mixed evenly at a mass ratio of 1:1. The solid powder was calcined at 400℃ for 3 hours and then cooled for 24 hours to obtain modified biochar.

[0076] 1.3 Mix 10 mL of bacterial suspension with 0.1 g of modified biochar until homogeneous, add 10 mL of encapsulating agent (sodium alginate and polyvinyl alcohol with mass fractions of 1% and 10%, respectively), stir until homogeneous, and obtain a mixed solution.

[0077] 1.4 Add 100 mL of crosslinking agent (4% calcium chloride and 1% boric acid solution) to a large beaker, add a magnetic stir bar, place it on a stirrer, and adjust the speed to keep the solution in a rotating state; use a sterile syringe to drop the mixture into the crosslinking agent drop by drop; after the dripping is complete, remove the stir bar with tweezers, cover with plastic wrap, and place in a 4℃ refrigerator for crosslinking. After the crosslinking and curing are complete, remove it from the refrigerator, pour off the crosslinking agent, wash with sterile physiological saline, repeat the rinsing 2-3 times, and store at 4℃ to obtain composite biochar immobilized bacterial particles (i.e., immobilized bacterial agent).

[0078] The above-mentioned composite biochar immobilized bacteria particles were inoculated into denitrification medium at an inoculation amount of 80 g / L. 10 mL of bacterial solution was taken at 0, 24 and 48 h of culture, centrifuged at 8000×g, and the nitrogen removal rate was calculated from the supernatant.

[0079] The denitrification and phosphorus removal effects of composite biochar-immobilized strains (without strict aseptic control) are as follows: Figure 5 As shown, after 48 hours, nitrate (100 mg / L) and phosphate (15 mg / L) were basically removed, and the total nitrogen removal rate reached 91.19±1.36%.

[0080] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of *Comamonas testosteroni* DP38, characterized in that, The *Trichomonas testis* DP38 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66845, deposited on August 15, 2025, at the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The use of *Trichomonas vaginalis* DP38 as described in claim 1 in the preparation of immobilized bacterial agents.

3. A method for preparing an immobilized bacterial agent, characterized in that, Includes the following steps: Agricultural waste is mixed with iron powder, calcined, cooled, and washed to obtain modified biochar. The modified biochar was mixed with the bacterial suspension of Trichomonas vaginalis DP38 as described in claim 1, and an encapsulating agent was added to obtain a mixture. The mixture is dripped into a crosslinking agent and crosslinked and cured to obtain the immobilized bacterial agent.

4. The preparation method according to claim 3, characterized in that, The agricultural waste is coconut shell; The mass ratio of the coconut shell to the iron powder is 1:1; The calcination temperature was 400℃ and the time was 3 hours.

5. The preparation method according to claim 3, characterized in that, The mass-to-volume ratio of the modified biochar to the bacterial suspension is 10 g: 1 L.

6. The preparation method according to claim 3, characterized in that, The embedding agent comprises 1% sodium alginate by mass and 10% polyvinyl alcohol by mass; The volume ratio of the bacterial suspension to the encapsulating agent is 1:

1.

7. The preparation method according to claim 3, characterized in that, The crosslinking agent comprises 4% calcium chloride and 1% boric acid by mass.

8. An immobilized bacterial agent obtained by the preparation method according to any one of claims 3-7.

9. The application of *Trichomonas testis* DP38 as described in claim 1 or the immobilized bacterial agent as described in claim 8 in the purification of wastewater.

10. A method for purifying wastewater, characterized in that, The method includes the step of applying the *Trichomonas vaginalis* DP38 of claim 1 or the immobilized bacterial agent of claim 8 to wastewater.

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