Paracoccus pantotrophus and application thereof

Abstract

The invention relates to the technical field of aquaculture, and particularly discloses paracoccus pantotrophus P.p-O8 and application of the paracoccus pantotrophus P.p-O8, the paracoccus pantotrophus P.p-O8 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the paracoccus pantotrophus P.p-O8 is CGMCC NO.26522. The invention relates to a microbial inoculum prepared from paracoccus pantotrophus P.p-O8. The method has a huge application prospect in improving the quality of aquaculture water and repairing the polluted sediments in the cage culture area, improves the water environment quality and the survival conditions of aquaculture organisms, repairs the eutrophicated polluted ecological environment, and has high environmental protection value and economic benefit.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aquaculture, and particularly relates to a Paracoccus versutus for extensive culture and application thereof. BACKGROUND

[0002] Net cage culture is a high-density culture mode with high yield and high benefit. In order to meet the increasing demand of people for aquatic products and increase the global fish yield, the net cage culture has been widely promoted. However, the feeding, feces and metabolic waste in the culture activities cause a load to the sediments under the net cage. The serious eutrophication leads to the hypoxia of seabed, and the hydrogen sulfide and ammonia are accumulated in the sediments and pore water. In the areas with insufficient or lack of dissolved oxygen, the denitrifying bacteria hinder the process of completely converting nitrogen into nitrate, leading to the accumulation of ammonia nitrogen and nitrite, thereby affecting the health of organisms. At the same time, in the anoxic area, the incomplete biodegradation of sulfur-containing organic matter further promotes the formation of H2S. H2S is the end product of sulfate reduction, which can interfere with the metabolism of plants and animals and affect the health of plants and animals, which in turn can lead to large-scale death of aquatic organisms, and even cause damage to the local ecosystem. The negative effects on the ecosystem and the local environment also include the emission of greenhouse gases and the bottom-up negative feedback effect on the tertiary industry (such as tourism).

[0003] The methods such as Ulva, biomass combined diatoms and sodium alginate immobilized microalgae can effectively repair artificial pollution. Sodium alginate is a natural polysaccharide extracted from brown algae, and the cross-linking process is mild and causes less damage to cells. The immobilization of microorganisms with sodium alginate is more likely to maintain microbial activity, and at the same time has certain mechanical strength and permeability. At the same time, in the environmental ecological restoration, the indigenous bacterial flora is more suitable for the local environment than the exogenous bacterial flora. Therefore, the indigenous microorganisms from marine sediments, immobilized by sodium alginate, are more suitable for repairing the marine culture environment.

[0004] This study isolated a heterotrophic nitrifying aerobic denitrifying bacterium, O8, from sediments in the waters off Daqin Island. O8 efficiently removes sulfides, ammonia nitrogen, nitrite nitrogen, and COD from wastewater. Physiological, biochemical, and 16S rRNA gene identification confirmed that O8 is *Paracoccus pantotrophus*. Notably, *Paracoccus pantotrophus* possesses a complete sulfur oxidation gene cluster (sox), which can mediate sulfide oxidation. Microorganisms involved in the S and N cycles play a crucial role in the coupled biogeochemical cycle due to their metabolic adaptability and responsiveness to sediment nutrients, electron acceptors, donors, and environmental conditions. Therefore, we hypothesize that O8 may be a potential candidate species for ecological restoration in cage aquaculture areas. It could significantly impact nitrogen and sulfur cycles and the microbial system in the sediments of cage aquaculture areas, causing changes in the overall functional diversity, functional composition, and interactions of the sedimentary microbial community, thus achieving ecological restoration. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide a strain of Paracoccus pantrophicus and its application. The Paracoccus pantrophicus provided by this invention has functions such as efficient denitrification and desulfurization, and can play a role in the ecological restoration of cage aquaculture areas.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a strain of Paracoccus pantotrophus Pp-O8, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.26522.

[0008] This strain was obtained from sediments in the waters around Daqin Island. After enrichment, separation and purification, it was named Pp-O8 and deposited at the China General Microbiological Culture Collection Center on February 7, 2023.

[0009] Furthermore, the aforementioned pantrophic paracoccus Pp-O8 has the following colony morphology characteristics: the colony size is 1-2 mm, it is milky white, with neat edges, slightly raised, smooth and moist surface, opaque, and can be stretched into filaments when picked up. Under a microscope, the bacterial morphology is similar to short rods, and under a scanning electron microscope, it is paraspherical, with a bacterial size of 0.5-1 μm.

[0010] Furthermore, the pantrophic paracoccus Pp-O8 was identified as a Gram-negative bacterium by Gram staining.

[0011] Secondly, this invention provides the application of pantrophic paracoccus Pp-O8 in water environment purification and ecological restoration.

[0012] Furthermore, this includes the application of the aforementioned Paracoccus pleurotus Pp-O8 in reducing the content of ammonia nitrogen, nitrite nitrogen, and organic matter in aquaculture wastewater.

[0013] Furthermore, this includes the application of the aforementioned Paracoccus pleurotus Pp-O8 in reducing the content of ammonia nitrogen, nitrite nitrogen, organic matter, and sulfides in sediments of net cage aquaculture areas.

[0014] Furthermore, this includes the application of the pantrophic paracoccus Pp-O8 in adjusting the abundance of nitrogen and sulfur cycle-related microorganisms and the diversity of microbial communities in the sediment.

[0015] Thirdly, the present invention provides a microbial agent for aquaculture or water environment purification and ecological restoration, the raw material of which includes the pantrophic paracoccus Pp-O8.

[0016] Furthermore, the microbial agent is in the form of fermentation broth or solidified microspheres.

[0017] Compared with the prior art, the advantages of the present invention are as follows:

[0018] This invention utilizes the fermentation broth of Paracoccus Pp-O8 to significantly reduce the content of ammonia nitrogen, nitrite nitrogen, and organic matter in aquaculture wastewater. It can also significantly reduce the content of ammonia nitrogen, nitrite nitrogen, organic matter, and sulfides in the sediment of cage aquaculture areas. Its immobilized microspheres can be used for ecological restoration of cage aquaculture areas. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of Paracoccus Pp-O8 used in this invention.

[0020] Figure 2 This is a morphological diagram of the paracoccus Pp-O8 strain used in this invention.

[0021] Figure 3 This is a scanning electron microscope image of Paracoccus Pp-O8 used in this invention.

[0022] Figure 4 The results show the removal of NO2--N, NH4+-N and COD by the paracoccus Pp-O8 strain used in this invention.

[0023] Figure 5 shows the screening results of carbon source (a), nitrogen source (b), and inorganic salt (c) of Paracoccus Pp-O8 used in this invention.

[0024] Figure 6 shows the optimized fermentation conditions of Paracoccus Pp-O8 used in this invention. (a) Temperature; (b) Aeration rate; (c) pH adjustment.

[0025] Figure 7The changes in bacterial (a) and archaea (b) species richness after in vitro remediation using the Paracoccus Pp-O8 strain employed in this invention.

[0026] Figure 8 This paper presents the changes in β-diversity of the microbial community after indoor remediation with Paracoccus Pp-O8 used in this invention. a) Principal coordinate analysis (PCoA) based on the weighted_unifrac distance algorithm; b) Non-metric multidimensional scaling analysis (NMDS) based on the unweighted_unifrac distance algorithm.

[0027] Figure 9 This invention utilizes the species-informed functional prediction (FAPROTAX) method following in vitro remediation of Paracoccus Pp-O8.

[0028] Figure 10 This is a CCA analysis of the microbial community and environmental factors after the indoor remediation of Paracoccus Pp-O8 used in this invention.

[0029] Figure 11 The changes in species richness of TOP35 (phyla) after in situ remediation using Paracoccus Pp-O8 as employed in this invention.

[0030] Figure 12 The UPGMA clustering tree based on Weighted Unifrac distance is used in this invention after in-situ remediation of Paracoccus Pp-O8.

[0031] Figure 13 The PCoA analysis diagram after in situ remediation of Paracoccus Pp-O8 used in this invention (left: based on Weighted Unifrac distance; right: based on Unweighted Unifrac distance).

[0032] Figure 14 The relative abundance clustering analysis of Tax4Fun2 functional annotations after in situ retrieval of Paracoccus Pp-O8 used in this invention. Detailed Implementation

[0033] 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.

[0034] The heterotrophic nitrifying-aerobic denitrifying Paracoccus strain used in the following examples is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 26522 and named Paracoccus Pp-O8.

[0035] The culture medium formulations involved in the examples are as follows:

[0036] Heterotrophic nitrification medium (NM): 0.5 g L⁻¹ (NH₄)₂SO₄, 0.36 g L⁻¹ KNO₃, 4.0 g L⁻¹ sodium citrate, 0.05% (v / v) trace element solution.

[0037] Trace element solution: 6.5 g L⁻¹ K₂HPO₄·3H₂O, 2.5 g L⁻¹ MgSO₄·7H₂O, 2.5 g L⁻¹ NaCl, 0.05 g L⁻¹ FeSO₄·7H₂O, 0.04 g L⁻¹ MnSO₄·H₂O. pH 7.0.

[0038] Aerobic denitrification medium (DM): 0.36 g L⁻¹ KNO₃, 10.55 g L⁻¹ Na₂HPO₄·12H₂O, 1.5 g L⁻¹ KH₂PO₄, 0.1 g L⁻¹ MgSO₄·7H₂O, 4.0 g L⁻¹ sodium citrate, 0.2% (v / v) trace element solution.

[0039] Trace element solution: 50.0 g L⁻¹ EDTA-Na₂, 2.2 g L⁻¹ ZnSO₄, 5.5 g L⁻¹ CaCl₂, 5.06 g L⁻¹ MnCl₂·4H₂O, 5.0 g L⁻¹ FeSO₄·7H₂O, 1.57 g L⁻¹ CuSO₄·5H₂O, 1.61 g L⁻¹ CoCl₂·6H₂O. pH 7.0.

[0040] Colorimetric medium (GN): 1.0 g L⁻¹ KNO₃, 8.5 g L⁻¹ sodium citrate, 1.0 g L⁻¹ L-asparagine, 1.0 g L⁻¹ KH₂PO₄, 1.0 g L⁻¹ MgSO₄·7H₂O, 0.2 g L⁻¹ CaCl₂·6H₂O, 0.05 g L⁻¹ FeCl₃·6H₂O, 0.1% (v / v) of 1% (w / w) bromothymol blue (BTB), adjusted to pH 7.0.

[0041] LB liquid medium: 10 g L⁻¹ tryptone, 5 g L⁻¹ yeast extract, 10 g L⁻¹ NaCl, pH 7.0.

[0042] Note: All liquid culture media were sterilized at 121°C for 20 min before use. Solid culture media were prepared by adding 1.5-2% agar to the above liquid culture media.

[0043] Example 1: Isolation, purification and identification of heterotrophic nitrifying-aerobic denitrifying pantotrophic Paracoccus pantotrophus Pp-O8

[0044] 1. Strains isolation and screening:

[0045] The *Paracoccus* heterotrophic nitrifying-aerobic denitrifying bacteria in this embodiment were mainly obtained through screening using the following methods:

[0046] Primary bacterial screening – screening of heterotrophic nitrifying microorganisms: enrichment and screening were carried out in a heterotrophic nitrification medium with ammonium sulfate as the sole nitrogen source. Collected water samples and sediments were inoculated into 250 mL of sterile NM at a 10% inoculum (volume or mass ratio); cultured at 30°C and 150 rpm min⁻¹ on a shaker; fresh NM was replaced every 48 hours at a 10% volume ratio; continuous culture for 30 days.

[0047] Initial bacterial screening:

[0048] Screening of aerobic denitrifying microorganisms: The domesticated microorganisms were inoculated into DM medium for further screening. DM used potassium nitrate as the sole nitrogen source. After culturing in NM for 30 days, 25 mL of NM supernatant was discarded every 24 h while 25 mL of fresh sterile DM medium was added. The mixture was then cultured in a shaker at 30℃ and 150 rpm min⁻¹ for 14 days.

[0049] Table 1. Physiological and biochemical identification results of the strains

[0050]

[0051] Bacterial rescreening:

[0052] Spreading culture: Take 1 mL of the bacterial suspension from the pre-screened water sample and spread it evenly on solid GN chromogenic medium; take 30 mL of the pre-screened biofilm sample (a mixture of bacterial suspension and biofilm) into a 50 mL sterile centrifuge tube, add sterile glass beads, and vortex to separate the bacteria attached to the carrier from the biofilm. After mixing thoroughly, take 1 mL of the bacterial suspension from the biofilm sample and spread it on solid GN chromogenic medium; the spreading procedure for the sediment sample is the same as that for the biofilm sample. Place the spread plates in a 30℃ incubator for incubation.

[0053] Screening of bacterial strains: Select a single blue colony from the solid chromogenic medium and inoculate it into 3 mL of sterile liquid GN chromogenic medium. Incubate at 30℃ and 150 rpm min-1 on a shaker. Observe the color change of the medium and select the strain that can turn the GN medium from green to blue as the secondary screening strain.

[0054] The screened strains were streaked and purified on prepared agar plates. The highest abundance colonies were selected and purified at least three times until a single colony was obtained. The colonies were examined under a microscope and stored at -80°C using the glycerol preservation method. The obtained strain was named O8 in the laboratory.

[0055] 2. Strain identification

[0056] The identification process and results of the Pp-O8 strain in this embodiment are as follows:

[0057] Morphological identification:

[0058] Microbial morphology and structure were observed using colony observation, optical microscopy, and scanning electron microscopy.

[0059] The colony morphology of strain Pp-O8 on LB medium is as follows: Figure 1 As shown: Colonies are 1-2 mm in size, milky white, with neat, slightly raised edges, smooth and moist surface, opaque, and can be stretched into strings when picked up. O8 Gram staining identifies them as Gram-negative bacteria. Figure 2 The bacteria have a short rod-like morphology. The O8 scanning electron microscope image is shown below. Figure 3 As shown: the bacterial cells are paraspherical and the cell size is 0.5-1 μm.

[0060] Physiological and biochemical identification:

[0061] The physiological and biochemical characteristics of the strains in this experiment were determined with reference to the literature and the Bergermin Manual of Bacterial Identification.

[0062] The physiological and biochemical characteristics of strain O8 were determined, and the results are shown in Table 1. O8 showed negative results in all sugar fermentation tests, indicating no acid or gas production; however, it showed positive results in both catalase and oxidase tests, meaning it can utilize sucrose and maltose. Based on morphological observations, it was preliminarily determined that the physiological and biochemical characteristics of O8 are consistent with those of Paracoccus versutus.

[0063] Molecular biological identification:

[0064] PCR amplification was performed using universal 16S rDNA primers 27F and 1429R. The PCR products were purified and sequenced by Beijing Qingke Biotechnology Co., Ltd. Homology comparison of the sequencing results with the NCBI database revealed that the target strain shared over 99% homology with multiple Paracoccus sp. strains, identifying strain O8 as *Paracoccus pantotrophus*. Therefore, the strain was renamed Pp-O8.

[0065] Strain preservation:

[0066] The heterotrophic nitrifying-aerobic denitrifying pantotrophic paracoccus (Paracoccus pantotrophus) Pp-O8, screened in the laboratory, has been deposited at the China General Microbiological Culture Collection Center (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC NO. 26522.

[0067] SEQ ID NO. 1

[0068]

[0069] Example 2:

[0070] The *P. p. O8* strain of *Paracoccus* in this embodiment exhibits excellent heterotrophic nitrification-aerobic denitrification performance and can be used for denitrification of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in aquaculture wastewater. Application includes the following steps:

[0071] The strain was inoculated into LB liquid medium and cultured in a constant temperature shaker at 30°C for 24 h to achieve an OD600 of 0.6–0.8, thus obtaining the fermentation broth.

[0072] The above fermentation broth was inoculated into the aquaculture wastewater at a concentration of 1%, the culture bottle was sealed with sealing film, and placed at room temperature to stand. The contents of NH4+-N, NO2-N and COD in the wastewater were measured at 12 h, 24 h and 48 h.

[0073] The results showed that the removal rates of NH4+-N and NO2-N in the wastewater treated by Pp-O8 were almost 100% within 24 h. After 48 h, NH4+-N, NO2-N and COD in the aquaculture wastewater were almost completely removed.

[0074] Example 3:

[0075] In this embodiment, the large-scale fermentation of *P. p. O8* requires determining the fermentation medium and culture conditions. After fermentation, the culture medium is coated and immobilized, and the mixture can be used for sediment remediation, including the following steps:

[0076] Activation of Pp-O8:

[0077] Paracoccus pantotrophus Pp-O8, frozen at -80℃, was activated by streaking. Single colonies were picked and transferred to LB nutrient liquid medium, and cultured at 30℃ with shaking for 12 h for secondary activation, which is the seed culture.

[0078] Screening of fermentation media for Pp-O8:

[0079] The carbon source in the basal medium (5 g glucose, 10 g tryptone, 5 g NaCl, 1 L sterile water) was replaced by sucrose, rice bran, wheat bran, and molasses; the nitrogen source was replaced by peanut meal, fish meal, corn flour, and soybean meal; and the inorganic salts in the basal medium were replaced by NaCl, K2HPO4, KH2PO4, NaCl + K2HPO4 (4:1), NaCl + KH2PO4 (4:1), and K2HPO4 + K2HPO4 (2:1). The seed culture was inoculated at a rate of 1% into 100 mL of medium and cultured at 30℃ and 220 rpm for 24 h. OD600 was measured every 6 h. The results of the Pp-O8 medium selection are shown in Figures 5(a)-(c). Referring to Figure 5(a), among the screened carbon sources, glucose showed the best effect, superior to rice bran and wheat bran, while molasses and sucrose showed the worst results. Referring to Figure 5(b), among the screened nitrogen sources, tryptone showed the best effect, while soybean meal was similar in effect to tryptone and superior to the other three nitrogen sources. Referring to Figure 5(c), among the screened inorganic salts, K2HPO4 showed the best effect. However, it is superior to tryptone due to its large demand and high price; therefore, soybean meal, which is relatively inexpensive, was used as the fermentation nitrogen source.

[0080] Optimization of fermentation conditions for Pp-O8:

[0081] The screened carbon source, nitrogen source, and inorganic salt were validated in a 25 L fermenter. During fermentation, a constant rotation speed of 150 rpm min⁻¹ was maintained, while aeration rate, temperature, and pH were adjusted. OD600 was measured after 24 h of fermentation. The optimized fermentation conditions for Pp-O8 are shown in Figures 6(a)-(c). Referring to Figure 6(a), the results show that Pp-O8 exhibits the best growth performance at 32℃, slightly better than at 30℃. Meanwhile, referring to Figure 6(b), the growth rate and final concentration of the strain were similar at aeration rates of 0.75 and 1 vvm; therefore, an aeration rate of 0.75 vvm was used to cultivate the strain to save energy. Referring to Figure 6(c), the addition of glucose and K₂HPO₄ during cultivation not only effectively controlled the pH of the fermentation broth but also increased the final concentration of the fermentation broth.

[0082] Microbial fermentation:

[0083] Pp-O8 was inoculated into LB liquid medium and cultured at 30°C with shaking at 220 rpm min⁻¹ for 24 h to obtain a seed culture. The seed culture was then inoculated at a 1% inoculation rate into fermentation medium (5 g L⁻¹ glucose, 10 g L⁻¹ soybean meal, 5 g L⁻¹ K₂HPO₄) and fermented in a 25 L fermenter. The culture volume was set at 17 L, the shaking speed at 150 rpm min⁻¹, the temperature at 32°C, the aeration rate at 0.75 vvm, and the pH at 7.2. During the culture, glucose and K₂HPO₄ were added to adjust the pH. After 24 hours of fermentation, a Pp-O8 fermentation broth with a concentration of 33 billion CFU / mL was obtained.

[0084] Preparation of sustained-release granular bacterial agents:

[0085] A 6% sodium alginate-2% diatomaceous earth solution (w / v) was used as a carrier and mixed evenly with an equal volume of Pp-O8 fermentation broth. The mixture was then added dropwise to a 0.1M CaCl2 solution and immobilized for 12 hours to form granular mycelial cores.

[0086] The compound Bacillus bacterial suspension was added to sterile water at a ratio of 1:9, and 3-5% of H-type mordenite zeolite was added. Biofilm formation was allowed for 3-7 days. The biofilm zeolite and slow-release carbon source were mixed evenly at a mass ratio of 2:170. The Pp-O8 bacterial core and the Bacillus biofilm zeolite slow-release carbon source were fed into a roller granulator. Using the Pp-O8 bacterial core as the core, the Bacillus biofilm zeolite slow-release carbon source was used to form composite biological agent particles with a diameter of 0.3-2.5 cm. The thickness ratio of the bacterial core to the carbon source layer was set to 2:13, resulting in composite biological agent particles.

[0087] Sodium alginate and polyvinyl alcohol formed a gel in a 90°C constant-temperature water bath. Alkali-modified diatomaceous earth and subsequently added composite biological agent particles were added to the gel sequentially, ensuring the encapsulating carrier gel fully encapsulated the composite biological agent particles. The particles were then added to a boric acid (CaCl2) solution for crosslinking for 2-3 hours, followed by addition to a NaSO4 solution for another 1-2 hours of crosslinking. After washing, the sustained-release Pp-O8 granular bacterial agent was finally obtained. The total bacterial count of the encapsulated and immobilized sustained-release bacterial agent reached 10 billion CFU g⁻¹ as determined by the plate coating method.

[0088] Example 4:

[0089] The *P. p. O8* strain of *Paracoccus* in this embodiment exhibits excellent heterotrophic nitrification-aerobic denitrification performance and can be used for sediment remediation in net cage aquaculture. For indoor application, the following steps are included:

[0090] Microscopic experimental setup:

[0091] A 30×20×20 cm aquarium was used as the experimental container. Approximately 4000 g of sediment was added to the aquarium, followed by the slow addition of 4 L of filtered seawater along the edge of the aquarium to avoid disturbing the sediment. Pp-O8 was added to the overlying water at concentrations of 10³ CFU L⁻¹ (PL), 10⁴ CFU L⁻¹ (PM), and 10⁵ CFU L⁻¹ (PH). Mixed surface sediment (0–1 cm) was collected and thoroughly mixed on days 0 (C. 0d), 4 (PL. 4d, PM. 4d, PH. 4d), and 7 (PL. 7d, PM. 7d, PH. 7d). A portion of the sediment was frozen at -80°C for DNA extraction and nitrogen nutrient analysis, while the remaining samples were dried for TOC and AVS analysis.

[0092] Physicochemical property analysis:

[0093] Pore ​​water was extracted from sediments by centrifugation, and the concentrations of NH4+-N and NO2−-N in the pore water were determined using an automated continuous flow analysis system (AutoAnalyzer III, Seal, Germany). TOC (%) and AVS (mg Kg-1) in the sediment samples were analyzed according to the methods of the Chinese National Standard (GB 17378.5-2007). After Pp-O8 bioremediation, TOC decreased from 0.61% to 0.44% (Table 2), significantly lower than the control group (P < 0.05). Furthermore, TOC gradually decreased with increasing remediation time and bacterial concentration. NH4+-N and NO3−-N in the microscopic system were consumed, especially in the high-concentration treatment group (Table 2), indicating active nitrification and denitrification during the remediation process. Pp-O8 treatment can accelerate the coupling process of nitrification and denitrification, promoting nitrogen removal. In this study, after 7 days of repair, the AVS concentrations in the PL group, PM group and PH group decreased by 28.79%, 40.09% and 53.85%, respectively, which were significantly lower than those on day 4 and the control group (Table 2). At the same time, the high concentration group was significantly better than the low concentration group (Table 2).

[0094] Table 2. Changes in the physicochemical properties of the sediment after indoor remediation.

[0095]

[0096] Microbial community analysis includes the following steps:

[0097] DNA extraction, PCR amplification, and sequencing:

[0098] Genomic DNA was extracted from approximately 0.5 g of sampled sediment using a magnetic soil DNA extraction kit (TianGen, catalog number DP712). The V4-V5 region of the 16S rRNA gene was amplified using primers 515F (5'-GTGYCAGCMGCCGCGGTA-3') and 926R (5'-CCGYCAATTYMTTTRAGTTT-3'). The reaction mixture consisted of 15 μL of Phusion® High Fidelity PCR Master Mix (New England Biolabs), 2 μM of forward and reverse primers, and 10 ng of template DNA. The PCR program was as follows: denaturation at 98°C for 1 min; denaturation at 98°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, repeated 30 times; and a final extension at 72°C for 5 min. PCR products were detected by electrophoresis on a 2% agarose gel. Qualified PCR products were purified using magnetic beads and quantified using enzyme-linked immunosorbent assay (ELISA). Equal volumes of PCR products were mixed thoroughly and then detected by 2% agarose gel electrophoresis. The target band was recovered using a universal DNA purification and recovery kit (TianGen, catalog number DP210). Library construction was then performed using the NEB Next® Ultra™ II FS DNA PCR-free Library Prep Kit (New England Biolabs, catalog number E7410L). The constructed libraries were quantified using Qubit and Q-PCR. Once the libraries were deemed acceptable, they were sequenced using a NovaSeq 6000 at PE 250.

[0099] Sequence analysis:

[0100] First, the original data was merged using FLASH (V1.2.11). The original sequences were then analyzed according to specific filtering criteria of QIIME (V1.9.1, http: / / qiime.org / scripts / split_libraries_fastq.html) to obtain high-quality clean labels. The sequences were then analyzed using UPRASE software (Uparse v7.0.1001, http: / / drive5.com / uparse / ) with at least 97% similarity, and the UCHIME algorithm was used to detect and remove chimeric sequences. Each representative sequence was then annotated using the Silva database (http: / / www.arb-silva.de / ).

[0101] Data Analysis:

[0102] Before performing statistical tests, the normality of variance was checked using the Shapiro-Wilk test. One-way ANOVA and t-tests were used to determine statistical differences between treatments. If the variances did not meet the hypotheses after data transformation, the Kruskal-Wallis rank-sum test was applied. Statistical analysis was performed using Microsoft Excel 2016, IBM SPSS Statistics 25, or R software (version 3.5.3). A p-value < 0.05 was considered statistically significant. The number of observed species was calculated using QIIME2 software (version QIIME2-202006), Good's_coverage was used to check sequencing depth, and Observed_features, Chao1, Shannon, Simpson, and Pielou_e analyses were performed to analyze community diversity, richness, and consistency. To assess the complexity of bacterial and archaea community composition and compare differences between treatments, nonmetric multidimensional scaling analysis (NMDS) and principal coordinate analysis (PCoA) based on the unifrac distance algorithm were used, and the results were displayed using the vegan package in R software (version 3.5.3). Furthermore, to investigate the potential functions of the community, functional annotation analysis was performed using FAPROTAX software (version 1.2.6) or Tax4Fun2. Canonical correspondence analysis between environmental factors and the microbial community was conducted using the Geomicrobial website tools (https: / / www.omicshare.com / tools / ).

[0103] Results of indoor microbial community experiments:

[0104] Changes in α-diversity of the microbial community after Pp O8 repair by Paracoccus:

[0105] Among the alpha diversity indices of the microbial community, Chao and Observed features reflect the richness of the microbial community, while Good's coverage reflects the coverage of each sample (clone) library. The Simpson and Shannon indices reflect the diversity of the microbial community, and the Pielou e index reflects the evenness of the community. The changes in the microbial alpha diversity indices of samples at different time periods are shown in Table 3. On days 4 and 7, the Goods coverage, Observed features, Pielou e, Shannon, and Simpson indices of the experimental group were all lower than those of the control group, indicating that the richness and diversity of the bacterial community decreased in both treatments. Compared to the initial sediment, the added *P. pantotrophus* represents a more restricted niche, which ultimately led to a more even and specific bacterial community.

[0106] Table 3. Changes in α-diversity of sediment microbial community after indoor remediation.

[0107]

[0108] Changes in bacterial and archaeal diversity after Pp O8 repair of Paracoccus:

[0109] After processing the high-throughput sequencing data obtained from 9 sediment samples, a total of 40,558 primer sequence variants (ASVs) were obtained. Most of the top 30 phyla were suppressed after Pp O8 retrieval. Proteobacteria, Bacteroidetes, Cyanobacteria, and Firmicutes were the four most abundant phyla in the sediment bacterial community, accounting for >80% ( Figure 9 Compared to the control group, Bacteroidetes were stimulated in the experimental group, while the abundance of Proteobacteria, Firmicutes, Chloroflexi, Fusobacteria, Actinobacteria, Spirochaetes, Nitrospirae, Gemmatimonadetes, Deferribacteres, and Verrucomicrobia decreased. Figure 7 (a)). The abundance of Proteobacteria, to which Pp O8 belongs, showed a trend of first decreasing and then gradually stabilizing, but was highest in the highest concentration treatment. Proteobacteria, Firmicutes, and Nitrospirae contain many bacteria associated with global carbon, nitrogen, and sulfur cycles. The abundance of Gemmatimonadetes is closely related to the content of organic carbon, and Actinobacteria have the ability to degrade organic and inorganic pollutants. The decrease in their abundance may be due to the weakening of carbon, nitrogen, and sulfur cycle transformation caused by the large-scale degradation of organic matter.

[0110] In terms of archaea, unclassified type 2 accounted for over 75% ( Figure 7(b) may be a new phylum requiring further investigation. On day 7 of the remediation process, the abundance of Thaumarchaeota and Crenarchaeota decreased significantly. Thaumarchaeota are a type of ammonia-oxidizing archaea and are considered important members of the global nitrogen and carbon biogeochemical cycle; Crenarchaeota are key groups mediating nitrification. These changes indicate the succession of various functions within the microbial community, promoting nitrogen and sulfur cycling processes.

[0111] Changes in β-diversity of the microbial community after Pp O8 repair by Paracoccus:

[0112] We performed principal coordinate analysis (PCoA) based on weighted Unifrac distance and selected the principal coordinate combination with the highest contribution rate for graphical display. PCoA results showed that C.0d and C.4d clustered in the fourth quadrant, and the bacterial community treated with *P. pantotrophus* was significantly separated from the CK community. Figure 8 (a)). NMDS analysis can better reflect the nonlinear structure of the samples. NMDS results also showed that the distance between the control groups was relatively large at different stages of repair. Except for the highest concentration treatment on day seven, the distances between the other treatment groups were relatively close ( Figure 8 (b)). MRPP analysis is often used in conjunction with dimensionality reduction techniques such as PCoA and NMDS to determine whether the differences in microbial community structure between different groups are statistically significant. Based on the MRPP analysis results, there were no significant differences between PH.4d and C.4d and PM.4d, while significant differences existed between other groups (Table 4). Adding an appropriate amount of PpO8 to the water significantly improved the stability of the microbial composition in the sediment, and the stable microbial network facilitated the removal of nitrogen and sulfur.

[0113] Table 4 Statistical Analysis of MRPP

[0114]

[0115]

[0116] Note: A smaller Observe Delta value indicates smaller within-group differences, while a larger Expect delta value indicates larger between-group differences. An A value greater than 0 indicates that between-group differences are greater than within-group differences, and an A value less than 0 indicates that within-group differences are greater than between-group differences. A Significance value less than 0.05 indicates a significant difference.

[0117] Predicted nitrogen and sulfur cycle-related functions after Pp O8 repair by Paracoccus:

[0118] FAPROTAX was used to predict the nitrogen and sulfur cycling functions in sediments, and the T-test was used to examine the differences between the treatment and control groups. Compared with the control group, after 4 days, PpO8 treatment significantly reduced the relative abundance of nitrogen cycling (nitrogen fixation, nitrogen respiration, nitrate respiration, chemoheterotrophy, agro-chemoheterotrophy, nitrate reduction) and increased the relative abundance of sulfur cycling (sulfur respiration, sulfur compound respiration, sulfate respiration, etc.). Figure 9 This indicates that PpO8 causes changes in the metabolic adaptation of sedimentary S and N cycling microorganisms, leading to different responses to sediment nutrients, electron acceptors and donors, and environmental conditions, thus altering the coupled biogeochemical cycling process.

[0119] Correlation analysis between the microbial community and environmental factors after Pp O8 remediation by Paracoccus:

[0120] CCA stands for Canonical Correspondence Analysis, a direct ordination analysis method based on a unimodal model. Points represent different samples, and arrows emanating from the origin represent different environmental factors. The length of the arrow represents the strength of the environmental factor's influence on community change; the longer the arrow, the greater the influence. The angle between the arrow and the coordinate axis represents the correlation between the environmental factor and the axis; the smaller the angle, the higher the correlation. The angle between a species and an environmental factor represents the positive or negative correlation between the two (acute angle: positive correlation; obtuse angle: negative correlation; right angle: no correlation). Drawing perpendicular lines from different samples to each environmental factor, the closer the projection points, the more similar the environmental factor's attribute values ​​among the samples, indicating that the environmental factor's influence on the samples is comparable. Figure 10 As shown, AVS and TOC in sediments have a slightly greater impact on the microbial community than NH4+-N and NO2-N. The concentrations of NH4+-N, NO2-N, AVS, and TOC are negatively correlated with the abundance of Proteobacteria and Fusobacteria, and positively correlated with Bacteroidetes, Chloroflexi, and Firmicutes. Higher abundances of Proteobacteria and Fusobacteria result in more significant removal of NH4+-N, NO2-N, AVS, and TOC from sediments.

[0121] Example 5:

[0122] The *Paracoccus* Pp-O8 immobilized microspheres of this embodiment exhibit excellent heterotrophic nitrification-aerobic denitrification performance and can be used for sediment remediation in net cage aquaculture. In-situ remediation includes the following steps:

[0123] Paracoccus Pp-O8 immobilized microspheres were deployed at a final concentration of 10⁴ CFU L⁻¹ into the non-cultured zone (NonAc), cultured zone (Ac), and remediation zone (AcRe). Sediment samples were collected at different time points after deployment, and the microbial community and physicochemical factors in the sediments were analyzed. The specific steps were the same as in Case Study 4.

[0124] Analysis of the physical and chemical properties of sediments:

[0125] After in-situ remediation of the cage aquaculture area, sediment samples were collected to detect AVS and TOC, and the results are shown in Table 5. AVS and TOC levels were low in the non-aquaculture area, while they slightly decreased in both the aquaculture and aquaculture-remediation areas after remediation. However, due to continuous aquaculture activities, AVS and TOC levels in the aquaculture area remained significantly higher than in the non-aquaculture and aquaculture-remediation areas. This may be because the removal rate of pollutants by Pp-O8 is primarily determined by the metabolic state of the bacteria in the soil, rather than the inoculum size. In this study, the metabolic pathways in the sediment and overlying water may have changed after coating. Alternatively, the contact area ratio between the coated Pp-O8 and the sediment and overlying water may have changed, resulting in a slightly less effective in-situ treatment compared to laboratory results.

[0126] Table 5 Changes in the physicochemical properties of sediments after in-situ remediation

[0127]

[0128]

[0129] Sediment microbial community analysis:

[0130] Changes in α-diversity of microbial communities after in-situ remediation with immobilized Pp-O8:

[0131] 16S analysis of the microbial community revealed that the non-cultured area had the highest Chao_1, Observed features, Pielou e, and Shannon indices, indicating that the introduction of immobilized Pp-O8 had no effect on the microbial α-diversity in the non-cultured area (Table 6). In the cultured area, in-situ remediation with Pp-O8 increased the Chao_1 index, while other α-diversity indicators showed no significant changes. This may be related to the continuous input of uneaten feed and feces from culture, and the limited impact of Pp-O8 on sediment in the cultured area. However, in the culture remediation area, all α-diversity indices showed a decreasing trend, consistent with the results of the microscopic experiments. In the microscopic experiments, there was no continuous input of uneaten feed and feces, which is closer to the usage scenario of the culture remediation area. *P. pantotrophus* occupies a more restricted ecological niche relative to the sediment in the culture remediation area, ultimately leading to a more homogeneous and specific bacterial community.

[0132] Table 6. Changes in α-diversity of microbial community after in-situ remediation

[0133]

[0134] Changes in bacterial diversity after in situ remediation with immobilized Pp-O8:

[0135] The changes in species richness in different regions after the introduction of immobilized Pp-O8 are as follows: Figure 11 As shown. Twenty-six days after in-situ remediation, the abundance of Proteobacteria, Fusobacteria, Spirochaetes, and Firmicutes increased in both the aquaculture and remediation zones, while the abundance of Crenarchaeota, Chloroflexi, Candidatus, Omnitrophica, Planctomycetes, Candidatus Latescibacteria, and Nitrospirae decreased in the remediation zone. Some results are consistent with laboratory experiments, suggesting that in addition to strain coating factors, uncontrollable factors such as tides, waves, temperature, and water pressure in the field may have altered the Pp-O8 metabolic pathway, affecting species abundance in the sediment.

[0136] Changes in β-diversity of microbial communities after in-situ remediation with immobilized Pp-O8:

[0137] According to UPGMA clustering tree ( Figure 12 ) and PCoA analysis ( Figure 13 Before remediation, the samples from the aquaculture area were far apart and clustered relatively dispersed, indicating significant differences in community structure. After remediation, the sampling points in each area became relatively clustered, and the community structure became relatively stable. The sampling points in the aquaculture remediation area were closer together and gradually clustered with those in the non-aquaculture area, indicating that the community structure and stability of the aquaculture remediation area were gradually recovering to those of the non-aquaculture area as remediation progressed. Ac.6.28 and NonAc.6.2 clustered together, indicating that after 26 days of bioremediation, the microbial structure of the aquaculture area was similar to that of the non-aquaculture area before the introduction of Pp-O8, demonstrating a good remediation effect.

[0138] Predicted nitrogen and sulfur cycle-related functions after in-situ remediation with immobilized Pp-O8:

[0139] Predicting the function of sediments using Tax4Fun2 ( Figure 14Tax4Fun functional prediction was achieved using the nearest neighbor method based on minimum 16S rRNA sequence similarity. Specifically, 16S rRNA gene sequences from the whole genomes of prokaryotes in the KEGG database were extracted and aligned to the SILVA SSU Ref NR database (BLAST bitscore >1500) using the BLASTN algorithm to establish a correlation matrix. Functional information from the whole genomes of prokaryotes in the KEGG database, annotated using UProC and PAUDA methods, was mapped to the SILVA database to achieve SILVA database functional annotation. Sequencing samples were clustered into OTUs using SILVA database sequences as reference sequences to obtain functional annotation information. On day 11 of repair, metabolic and genetic information processing-related functions in the AcRe region were higher than before repair, while functions related to organismal systems, environmental information processing, and cellular processes were lower than before repair. This indicates that metabolic activities such as energy production and utilization, organic matter synthesis and degradation, and genetic information exchange and signal transduction activities were enhanced in the sediment microbial community at this time. Conversely, on day 26 of repair, the metabolic and genetic information processing functions in the Ac region were lower than before repair, but the functions related to organismal systems, environmental information processing, and cellular processes were higher than before repair, indicating that the microbial community's perception and response to external environmental information was enhanced at this time, and the microbial proliferation rate was fast.

[0140] In summary, this strain and its metabolites can significantly reduce the levels of ammonia nitrogen, nitrite nitrogen, organic matter, and sulfides in aquaculture wastewater and cage aquaculture areas, thereby improving aquaculture water quality and accelerating ecological restoration in cage aquaculture areas. Theoretically, it should also have an effect on aquaculture wastewater and soil contaminated by waste treatment plants.

[0141] 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 pantotrophic paracoccus (Paracoccus pantotrophus) Pp-O8, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.26522.

2. The pantrophic paracoccus Pp-O8 according to claim 1, characterized in that, The colony morphology is characterized by a size of 1-2 mm, a milky white color, neat and slightly raised edges, a smooth and moist surface, opacity, and the ability to be stretched into strings when picked up. Under a microscope, the bacterial cells appear as short rods, while under a scanning electron microscope they appear as paraspherical cells with a size of 0.5-1 μm.

3. The pantrophic paracoccus Pp-O8 according to claim 1, characterized in that, Gram staining identified it as a Gram-negative bacterium.

4. The application of the pantrophic paracoccus Pp-O8 as described in claim 1 in water environment purification and ecological restoration.

5. The application according to claim 4, characterized in that, This includes the application of the aforementioned pantrophic paracoccus Pp-O8 in reducing the content of ammonia nitrogen, nitrite nitrogen, and organic matter in aquaculture wastewater.

6. The application according to claim 4, characterized in that, This includes the application of the aforementioned pantrophic paracoccus Pp-O8 in reducing the content of ammonia nitrogen, nitrite nitrogen, organic matter and sulfides in sediments in net cage aquaculture areas.

7. The application according to claim 6, characterized in that, This includes the application of the pantrophic paracoccus Pp-O8 in adjusting the abundance of nitrogen and sulfur cycle-related microorganisms and the diversity of microbial communities in the sediment.

8. A microbial agent for aquaculture or water environment purification and ecological restoration, characterized in that, The raw material includes the pantrophic paracoccus Pp-O8 as described in claim 1.

9. The microbial agent according to claim 8, characterized in that, The inoculant is in the form of fermentation broth or solidified microspheres.

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