A deep-sea bacterium capable of heterotrophic aerobic growth and autotrophic sulfur oxidation and denitrification and its applications.

Abstract

This invention discloses a deep-sea bacterium capable of heterotrophic aerobic growth and autotrophic sulfur oxidation and denitrification, and its applications. The bacterium is *Lihuangiaoceani*, strain number D14, registered at the China General Microbiological Culture Collection Center (CGMCC) No. 1.13774. This *Lihuangiaoceani* possesses facultative sulfur oxidation and denitrification capabilities, enabling heterotrophic growth under aerobic conditions and denitrification using organic matter and / or sodium thiosulfate under anaerobic conditions. It can utilize nitrate as an electron acceptor for sulfur oxidation under anaerobic conditions and oxygen as an electron acceptor for sulfur oxidation under aerobic conditions. It can be used to remove nitrates and thiosulfates from polluted water bodies or sediments, or for wastewater treatment in landscape water, urban rivers, and aquaculture, as well as for the remediation of eutrophic rivers and lakes.

Description

Technical Field

[0001] This invention relates to a strain of *Flavorella vulgaris* and its uses, particularly to a deep-sea bacterium capable of heterotrophic aerobic growth and possessing autotrophic sulfur oxidation and denitrification functions, and its applications. Background Technology

[0002] Denitrification, also known as nitrogen removal, refers to the process by which microorganisms use nitrate as the final electron acceptor in respiration, converting nitrogen from nitrate through a series of intermediate products (NO2). - Nitrification is the biochemical process by which nitrogen (NO and N2O) is reduced to nitrogen gas. The microorganisms involved in this process are collectively called denitrifying bacteria. Denitrification is a crucial link in the nitrogen cycle in nature and a major biological process for nitrogen removal in eutrophic water bodies. Denitrifying bacteria are important functional bacteria in the process of nitrogen removal and purification in water bodies, playing a vital role in the treatment of (wastewater) from landscape water, urban rivers, and aquaculture, as well as in the management of eutrophic rivers and lakes.

[0003] Most known denitrifying bacteria are heterotrophic, using organic matter as a carbon and nitrogen source and energy source, reducing nitrates and removing nitrogen through anaerobic respiration. However, using these heterotrophic denitrifying bacteria for eutrophic water purification has several drawbacks: for example, the COD in black and odorous water bodies is already low, and conventional heterotrophic denitrifying microorganisms struggle to utilize the low concentrations of organic matter, leading to a continuous accumulation of nitrate nitrogen. Adding additional organic carbon sources can easily increase COD, sludge production, and operating costs. Therefore, in practical applications, there is a greater desire to obtain and use denitrifying bacteria that can grow autotrophically. Currently, there are very few types of microorganisms with autotrophic denitrification capabilities; they can utilize inorganic substances such as reduced sulfur compounds, H2, or Fe. 2+ As an electron donor, NO3 - As an electron acceptor, it obtains energy and fixes CO2 as a carbon source for autotrophic growth. No additional organic carbon source is needed during use, and the overall operating cost is more than 50% lower than heterotrophic denitrification. Therefore, this new strain with autotrophic denitrification capability is a unique microbial resource with enormous potential application value, providing strains and microbial preparations for polluted water remediation and nitrogen and sulfur wastewater treatment. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to remove nitrogen and / or sulfur from the environment, such as removing nitrogen and / or sulfur from water bodies, soil and / or water sediments.

[0005] To address the above technical problems, the present invention first provides a *Flavorella vulgaris* bacterium.

[0006] The *Lihuangia oceani* strain provided in this invention is strain D14, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 1.13774. It is hereinafter referred to as *Lihuangia oceani* D14.

[0007] The present invention also provides a culture of *Flavorholia serratifolia*, which is a substance obtained by culturing the aforementioned *Flavorholia serratifolia* in a microbial culture medium.

[0008] The culture contained Lihuangia oceani D14.

[0009] The culture mentioned above is a fermentation product obtained by culturing Lihuangia oceani D14 in a microbial culture medium.

[0010] The substances in the above-mentioned cultures include Lihuangia oceani D14 and its metabolites.

[0011] In the above-mentioned cultures, the microbial culture medium can be a solid culture medium or a liquid culture medium.

[0012] The term "culture" refers to a liquid or solid product (all substances within the culture vessel are fermentation products) that has grown a microbial community after artificial inoculation and cultivation. It is a product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, and / or other components produced during the cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

[0013] The present invention also provides a microbial agent containing the aforementioned *Flavorholia spp.* or / and the metabolites of the aforementioned *Flavorholia spp.* or / and the aforementioned culture.

[0014] In this article, the metabolites mentioned can be obtained from the fermentation broth of *Lihuangia oceani* D14. The metabolites of *Lihuangia oceani* D14 can be sterile metabolites of *Lihuangiaoceani* D14 or bacterial metabolites of *Lihuangia oceani* D14. Specifically, the sterile metabolites of *Lihuangia oceani* D14 (sterile fermentation filtrate) can be prepared by culturing *Lihuangia oceani* D14 in a liquid culture medium, and filtering to remove *Lihuangia oceani* D14 from the liquid culture (fermentation broth) to obtain the sterile metabolites of *Lihuangiaoceani* D14. The bacterial metabolites of Lihuangia oceani D14 can be prepared by the following method: Lihuangia oceani D14 is cultured in a liquid fermentation medium, and the fermentation broth (containing Lihuangia oceani D14 and substances secreted into the liquid medium) is collected. This fermentation broth is the bacterial metabolites of Lihuangia oceani D14.

[0015] The active ingredient of the above-mentioned microbial agent may be Lihuangia oceani D14 or / and the metabolites of Lihuangia oceani D14 or / and the above-mentioned culture. The active ingredient of the above-mentioned microbial agent may also contain other biological or non-biological components. Other active ingredients of the above-mentioned microbial agent can be determined by those skilled in the art based on the effect of the microbial agent.

[0016] The aforementioned microbial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0017] The solid carrier can be a mineral material or a biological material; the mineral material can be at least one of peat moss, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material can be at least one of various crop straws, pine shells, rice straw, peanut shells, corn flour, soybean flour, starch, peat moss, and animal manure; the liquid carrier can be water; in the bacterial agent, the metabolites of *Lihuangia oceani* D14 or / and *Lihuangiaoceani* D14 can exist in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate. The bacterial agent can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder, or water-dispersible granules.

[0018] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the bacterial agent.

[0019] Furthermore, the aforementioned microbial agents can perform sulfur oxidation, oxidizing low-valence sulfur compounds into sulfates.

[0020] Furthermore, the above-mentioned microbial agents can perform denitrification, reducing nitrogen in nitrates to nitrogen gas (N2) or nitrous oxide (N2O) for removal.

[0021] Specifically, this refers to the sulfur oxidation process of Lihuangia oceani under anaerobic conditions using nitrate as an electron acceptor or under aerobic conditions using oxygen as an electron acceptor.

[0022] The present invention also provides a nitrogen remover containing the aforementioned *Flavorholia spp.* or / and its metabolites or / and its culture or / and its bacterial agent. The nitrogen remover can simultaneously utilize organic matter and sodium thiosulfate for denitrification.

[0023] Furthermore, the nitrogen remover can be a bacterial agent that removes nitrogen in an anaerobic environment. The nitrogen remover can reduce nitrate to nitrogen gas through denitrification.

[0024] The present invention also provides the use of the aforementioned *Flavorholia serratifolia*, the aforementioned metabolites of *Flavorholia serratifolia*, or the aforementioned culture in the preparation of a bacterial agent for removing nitrogen and / or sulfur from the environment.

[0025] The environment may be a body of water, soil, and / or aquatic sediments. The body of water may be wastewater.

[0026] Furthermore, the polysaccharide high molecular weight polyhydroxyalkanoates (PHA) and their analogues produced by Lihuangia oceani during use can be applied to fields requiring polysaccharide high molecular weight polyhydroxyalkanoates, their cultures, metabolites, or bacterial agents containing the bacteria.

[0027] The present invention also provides an application, specifically providing any one of the following applications of the aforementioned *Flavobacterium oxysporum*, the aforementioned metabolites of *Flavobacterium oxysporum*, the aforementioned culture, and the aforementioned bacterial agent:

[0028] N1. Oxidize sulfur in wastewater from a low oxidation state to a high oxidation state or remove nitrogen from wastewater through denitrification;

[0029] N2, products that oxidize sulfur in wastewater from a low oxidation state to a high oxidation state or remove nitrogen from wastewater through denitrification;

[0030] N3, oxidizes sulfur in sediments from low oxidation state to high oxidation state or removes nitrogen in sediments through denitrification;

[0031] N4. To prepare products that oxidize sulfur in sediments from low oxidation state to high oxidation state or remove nitrogen in sediments through denitrification;

[0032] N5. Oxidizes sulfur in the soil from a low oxidation state to a high oxidation state or removes nitrogen in the soil through denitrification;

[0033] N6. Prepare products that oxidize sulfur in soil from a low oxidation state to a high oxidation state or remove nitrogen in soil through denitrification;

[0034] N7, remediation of nitrogen and / or sulfur contaminated deposits;

[0035] N8. Products for the remediation of nitrogen and / or sulfur contaminated deposits;

[0036] N9, remediation of nitrogen and / or sulfur contaminated soil;

[0037] N10, products for remediating nitrogen and / or sulfur contaminated soil;

[0038] N11, Preparation of polysaccharide-producing products;

[0039] N12, products for preparing polyhydroxy fatty acids.

[0040] In the above applications, the wastewater includes at least one of marine aquaculture wastewater and marine product processing wastewater, and the sediment includes sediment from marine aquaculture water bodies.

[0041] In the above applications, the soil includes, but is not limited to, coastal or intertidal soils.

[0042] The *Lihuangia oceani* D14 of this invention is a heterotrophic aerobic bacterium with autotrophic sulfur oxidation and denitrification capabilities. It possesses facultative sulfur oxidation and denitrification functions, capable of simultaneously utilizing organic matter and sodium thiosulfate for denitrification. It can utilize nitrate as an electron acceptor for sulfur oxidation under anaerobic conditions and also utilize oxygen as an electron acceptor for sulfur oxidation under aerobic conditions. Based on this, *Lihuangia oceani* D14 can be used to simultaneously remove nitrates and thiosulfates from polluted water bodies or sediments, and can be applied to the treatment of (sewage) water in landscape water, urban rivers, and aquaculture, as well as the remediation of eutrophic rivers and lakes.

[0043] Preservation Instructions

[0044] Lihuangia oceani

[0045] Strain number: D14

[0046] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0047] Collection institution abbreviation: CGMCC

[0048] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0049] Deposit date: June 30, 2022

[0050] Registered with the China National Collection Center (CGMCC) No. 1.13774. Attached Figure Description

[0051] Figure 1 Transmission electron microscope and scanning electron microscope images of Lihuangia oceani D14;

[0052] Figure 2 Phylogenetic clustering tree of Lihuangia oceani D14;

[0053] Figure 3 The heterotrophic aerobic growth curve of Lihuangia oceani D14 was obtained.

[0054] Figure 4 To stabilize the isotope labeling experiment, it was demonstrated that Lihuangia oceani D14 bacterial cells can carry out denitrification under autotrophic anaerobic conditions;

[0055] Figure 5The N2O-N content of Lihuangia oceani D14 during autotrophic anaerobic sulfur oxidation and denitrification changes with autotrophic culture time.

[0056] Figure 6 The concentration changes of nitrate-nitrogen, thiosulfate-sulfur, and sulfate-sulfur during autotrophic anaerobic sulfur oxidation and denitrification in Lihuangia oceani D14.

[0057] Figure 7 The changes in the concentrations of nitrous oxide-nitrogen, nitrate-nitrogen, thiosulfate-sulfur, and sulfate-sulfur during sulfur oxidation and denitrification by Lihuangia oceani D14 under anaerobic conditions with sodium thiosulfate.

[0058] Figure 8 The changes in the concentrations of nitrous oxide-nitrogen, nitrate-nitrogen, and sulfate-sulfur during denitrification by Lihuangia oceani D14 under anaerobic conditions without sodium thiosulfate.

[0059] Figure 9 The concentration changes of thiosulfate-sulfur and sulfate-sulfur during sulfur oxidation by Lihuangia oceani D14 under heterotrophic aerobic conditions. Detailed Implementation

[0060] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0062] Composition, concentration, and preparation of 2216E liquid culture medium: 5 g / L peptone, 1 g / L yeast extract, 0.1 g / L ferric citrate, solvent is 30 g / L sea salt solution (solvent is water, solute is sea salt (Sigma, catalog number S9883-1KG), pH 7.6-7.8). 2216E solid culture medium: Add 15 g / L agar to the 2216E liquid culture medium. Autoclave at 121℃ for 20 min, and use after natural cooling.

[0063] Artificial seawater: Sodium chloride 23.477 g / L, sodium sulfate 3.917 g / L, magnesium chloride hexahydrate 4.981 g / L, calcium chloride 1.102 g / L, sodium bicarbonate 192 mg / L, potassium chloride 664 mg / L, boric acid 26 mg / L, strontium chloride 24 mg / L, sodium fluoride 3 mg / L, potassium bromide 6 mg / L, natural pH.

[0064] Example 1: Isolation and Identification of Strain D14

[0065] I. Isolation of strain D14

[0066] A water sample from the water-sediment interface at a depth of 4213 meters in the Northwest Indian Ocean (69°20′31.84″E 4°00′10.06″N) was used as the sample. This water sample was collected on June 21, 2013, by the Institute of Microbiology, Chinese Academy of Sciences, during the 28th oceanographic expedition. Microbial cells from the sample were separated using flow cytometry and placed in 96-well plates (100 μL / well) containing 2216E liquid medium. The plates were incubated at 25°C for approximately 40 days. Single-cell cultures showing growth were then spread onto 2216E solid medium and incubated for 20-30 days. Single colonies were picked and further purified by liquid medium and streak plating to obtain a pure culture strain, which was named strain D14.

[0067] II. Morphological and physiological-biochemical identification of strain D14

[0068] Strain D14 grows slowly on 2216E solid medium at 30°C. After 3-4 weeks of culture, colonies are approximately 0.5 mm in diameter, round, raised, smooth, translucent, white, and with regular edges. Figure 1 As shown in Figure A, the bacteria are rod-shaped, 1.0-2.0 μm long and 0.5-0.8 μm wide, with single flagella and extracellular polysaccharide.

[0069] Strain D14 grows rapidly in liquid 2216E medium at 30°C. Furthermore, strong light inhibits its growth; therefore, cultivation should be carried out in darkness.

[0070] Strain D14 can grow under both aerobic and anaerobic conditions. The growth temperature range is 15-40℃, with an optimal growth temperature of 30-35℃. The growth pH range is 5.5-9.5, with an optimal growth pH of 6.5-8.5. The growth salinity is 0.5-6.5%, with an optimal salinity of 2.5%.

[0071] Strain D14 was cultured in 2216E liquid medium at 30°C and 160 rpm for 3 days. After collection, the cells were washed three times with sterile artificial seawater and transferred to a culture medium (40 g / L sucrose, 0.4 g / L yeast extract, artificial seawater as solvent). The cells were then incubated statically for 3 days. After fixation and sectioning, the cells were examined under a transmission electron microscope. Figure 1 In the middle B cell, high-molecular-weight polyhydroxyalkanoates (PHA) were observed; under a scanning electron microscope, the bacterium exhibited a unique starfish-like arrangement. Figure 1 C and Figure 1 (D).

[0072] Biochemical identification results showed that strain D14 was a Gram-negative bacterium with CoQ-10 (95.7%) and CoQ-8 (4.3%) as cellular respiratory quinones. The polar lipid composition (an important chemical classification indicator) of strain D14 cells included phosphatidylglycerol (PG), lecithin (PC), phosphatidylethanolamine (PE), one unknown phospholipid (PL), and two unknown lipids (L).

[0073] III. Molecular taxonomic identification of strain D14

[0074] Total DNA was extracted from strain D14 using standard methods. Using the total DNA as a template, PCR amplification was performed using universal primers 27F and 1492R for the bacterial 16S rRNA gene. Primer information is as follows:

[0075] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0076] 1492R: 5'-GGTTACCTTGTTACGACTT-3';

[0077] The PCR system and procedure were as follows: 2.5 μL 10×PCR buffer, 2.0 μL 10.0 mmol / L dNTPs, 1.0 μL 10.0 pmol / μL 27F primers, 1.0 μL 10.0 pmol / μL 1492R primers, 0.3 μL 2.5 U / μL Taq enzyme, and deionized water to a final volume of 25.0 μL. The reaction was pre-denatured at 95℃ for 5 min, followed by denaturation at 94℃ for 45 s, annealing at 55℃ for 45 s, and extension at 72℃ for 1.5 min, for 30 cycles. A final extension at 72℃ for 10 min followed. The PCR amplification products were sent to a sequencing company for sequencing, and the obtained sequences were submitted to [the relevant authority / organization]. http: / / www.ezbiocloud.net / identifySequence similarity analysis was performed. The results are shown in Table 1. D14 showed similarity between 93.53% and 93.85% with four different genera in the order Hyphomicrobiales (redefined in 2020 to include and replace the previous order Rhizobiales) (as shown in Table 1). According to convention, a 16S rRNA gene similarity of less than 95% is sufficient to classify a microorganism into a new genus (Tindall BJ., et al., 2010), indicating that this bacterium represents a new genus and species in the order Hyphomicrobiales.

[0078] Table 1. Analysis of the similarity (%) of rRNA gene / ANI / AAI of strain D1416S

[0079] Serial Number Most similar sequence (16S rRNA GenBank accession number) 16S(％) ANI (%) AAI(%) division* 1 Mesorhizobium carmichaelinearum MonP1N1(JQ963057) 93.85 70.89 51.73 Phy 2 Chelativorans intermedius CC-MHSW-5(EU564843) 93.76 70.96 54.87 Phy 3 Rhodobium orientis MB312(D30792) 93.68 71.81 55.64 Rho 4 Breoghania sp. L-A4 (CP031841) 93.53 71.50 56.42 Bre 5 Chelativorans composti Nis3(AB563785) 93.53 - - Phy

[0080] *Phy:Phyllobacteriaceae; Rho:Rhodobiaceae; Bre:Breoghaniaceae; -:No genomic data.

[0081] IV. Genomic determination of strain D14

[0082] Total DNA was extracted from strain D14 using the standard CTAB method. After purification using the MGIEAsy DNA purification magnetic bead kit, the DNA was assembled using Annoroad's PacBio Sequel II third-generation sequencing combined with Illumina PE150 second-generation sequencing to obtain the complete genome sequence of strain D14. The genome size of strain D14 was determined to be 3.53M, with a G+C percentage of 65.7%, and it contained a set of rRNA gene sequences (i.e., 16S-23S-5S rRNA). The complete 16S rRNA gene sequence was extracted, as shown in SEQ ID NO.1, as follows:

[0083]

[0084] Based on the similarity analysis results in Table 1, 16S rRNA gene sequences of related genera and species in the order Micromycetes were downloaded from the GenBank database. Phylogenetic cluster analysis was performed using MEGA 7.0 software, and the resulting phylogenetic tree is shown below. Figure 2 The figure shows "Strain D14" T "" indicates strain D14.

[0085] The results showed that strain D14 clustered with six genera of the family Parvibaculaceae in the order Pyromycetes, indicating that D14 is a new genus and species in this family. The 16S rRNA gene similarity among them (91.8-93.0%) was between the known similarity values ​​among genera (90.2-94.7%) (Table 2).

[0086] The average nucleotide similarity (ANI) of strain D14 with similar genera and species was calculated on the website http: / / jspecies.ribohost.com / jspeciesws / #analyse. The results showed that the ANI values ​​of strain D14 and related strains were all less than 72%, lower than the ANI values ​​of strains from different species within the same genus (>73%) (Tables 1 and 3). Since the ANI value is less than 75%, the average amino acid similarity (AAI) is needed to determine the relationship between species. AAI is generally considered useful for classifying genera, with a threshold of approximately 65-72%. Therefore, the average amino acid similarity (AAI) was calculated, and the AAI values ​​of strain D14 and related strains were further analyzed. The results showed that the AAI values ​​of strain D14 with microorganisms from different genera of the order Mycomycetes were all less than 57%, significantly lower than the maximum AAI value of 62.48% for strains from different genera, and far lower than the AAI values ​​of strains from different species within the same genus (>73%) (Tables 1 and 4). Therefore, the results of ANI and AAI both support the view that strain D14 is a new genus and species of Hyphomicrobiales.

[0087] Table 2. 16S rRNA gene similarity matrix (%) between strain D14 and type species of each genera in the Parvibaculaceae family.

[0088] Strain D14 100 A. baltica BA141 92.6 100 R. appendicifer ATCC BAA-2115 92.8 91.9 100 K.mangrovi R1DC25 93.0 91.7 94.7 100 Par.lavamentivorans DS-1 91.8 90.2 92.4 92.1 100 T. marinus MA2 92.2 91.2 92.0 91.3 93.6 100 Pyr.mobilis GYP-11 92.6 91.7 92.2 92.4 92.1 92.5 100

[0089] Table 3. ANI matrix (%) between strain D14 and type species of each genera in the Parvibaculaceae family.

[0090]

[0091] Table 4. AAI matrix (%) between strain D14 and type species of each genera in the Parvibaculaceae family.

[0092]

[0093] Further analysis of the genome of strain D14 revealed that it contains two copies of the key enzyme for PHA synthesis, namely the PHA synthase gene (SEQ ID NO.2, SEQ ID NO.3). The genome also encodes genes (clusters) for three pathways: carbon dioxide fixation, denitrification, and sulfur oxidation (SOx), including the key denitrification enzymes nitric oxide reductase (NorB) (SEQ ID NO.4) and nitrous oxide reductase (nosZ) (SEQ ID NO.5). This suggests that the bacterium possesses autotrophic sulfur oxidation and denitrification functions, capable of converting nitrate (NO3) into nitrogen oxides. - It is completely reduced to nitrogen (N2).

[0094] SEQ ID NO.2:

[0095]

[0096] SEQ ID NO.3:

[0097]

[0098] SEQ ID NO.4:

[0099]

[0100] SEQ ID NO.5:

[0101]

[0102] Based on morphological, physiological, biochemical, and molecular taxonomic identification results, strain D14 was identified as a new species belonging to the genus *Lihuangia* of the family Parvibaculaceae in the order Hyphomicrobiales, and named *Lihuangia oceani*. This strain was deposited on June 30, 2022, at the China General Microbiological Culture Collection Center (CGMCC; address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; postcode: 100101), with accession number CGMCC No. 1.13774, and will henceforth be referred to as *Lihuangia oceani* D14 or strain D14.

[0103] Example 2: Characteristics of Lihuangia oceani D14 growth under heterotrophic aerobic conditions

[0104] Strain D14 was activated on 2216E solid medium, inoculated into 2216E liquid medium, and cultured in a ZWYC-290C precision cell culture shaker (online OD detection type) at 30℃ and 160rpm for 5 days. A growth curve was plotted with culture time on the x-axis and the corresponding OD600 nm at each detection time point on the y-axis. The results are shown below. Figure 3 As shown, strain D14 has a logarithmic growth phase of 24-48 hours (1-2 days) under heterotrophic aerobic conditions, and its OD600nm reaches a maximum of 1.73 at 72 hours (3 days).

[0105] Example 3: Lihuangia oceani D14 exhibits sulfur oxidation and denitrification functions under anaerobic and autotrophic conditions.

[0106] I. Lihuangia oceani D14 bacterial cells possess denitrification function under anaerobic and autotrophic conditions.

[0107] The culture medium used in this experiment is as follows:

[0108] Contains 1g / L 15 N-stable isotope-labeled NaNO3 (where N is...) 15 N) and 1 g / L unlabeled NaNO3 (where N is N) and 14 Preparation of autotrophic culture medium containing sodium thiosulfate (N): NaNO3 (where N is N) 15 N) 1g / L, NaNO3 (where N is N) 1g / L 14The ingredients are: N 1 g / L, sodium bicarbonate 2 g / L, potassium nitrate 2 g / L, sodium thiosulfate 2.5 g / L, in a 30 g / L sea salt solution with pH 7.6-7.8. Sodium bicarbonate and sodium thiosulfate are sterilized by filtration through a 0.22 μm membrane. Other components are autoclaved at 121°C for 20 min and allowed to cool naturally before use.

[0109] The experiment was repeated three times, with two treatments each time: an experimental group and a control group.

[0110] 1. Experimental group: Strain D14 was cultured on 2216E liquid medium until the OD600nm reached 0.6 (using 2216E liquid medium as a blank control). 10 ml of bacterial cells were collected, washed three times with sterilized artificial seawater, and then a solution containing 1 g / L of [unspecified substance] was added. 15 N-stable isotope-labeled NaNO3 (where N is...) 15 N) and 1 g / L unlabeled NaNO3 (where N is N) and 14 The headspace gas was analyzed by gas chromatography-mass spectrometry (GC-MS) of headspace gas after incubation in an autotrophic medium containing sodium thiosulfate (N) at 30°C in the dark for 1 day (i.e., denitrification reaction of bacterial cells).

[0111] 2. Control group: containing 1g / L 15 N stable isotope labeling 1 NaNO3 (where N is...) 15 N) and 1 g / L unlabeled NaNO3 (where N is N) and 14 The culture medium (without strain D14) containing sodium thiosulfate was incubated at 30°C in the dark for 1 day, and then analyzed for the presence of N2O using an Agilent 7890A / 5975C GC / MS.

[0112] The abundance of N2O in the headspace of the experimental and control groups was detected by GC / MS. The GC / MS detection conditions were as follows: a GS-Carbon Plot (30m×0.32mm×3.0μm, Agilent, USA) analytical column was used, with a sample loading volume of 20μL headspace gas (5190-1503 Agilent 50μL gas-tight needle sampling); the injection temperature was 160℃, the split ratio was 5:1, the initial temperature was 35℃, held for 5 min, and then increased from 140℃ at 20℃ / min, held for 2 min. The carrier gas was helium, and the flow rate was 1.3 ml / min.

[0113] The results are as follows Figure 4 As shown, Figure 4The upper figure shows the peak of N2O with a molecular weight of 44 detected by gas chromatography-mass spectrometry, and the lower figure shows the peak of N2O with a molecular weight of 45 detected by gas chromatography-mass spectrometry. The horizontal axis represents retention time, and the vertical axis represents the response signal of gas chromatography-mass spectrometry. It can be seen that the experimental group can clearly detect N2O with stable isotope labeling, while the control group cannot detect N2O.

[0114] To determine whether the N2O generated during denitrification could be further reduced to N2, headspace gas from the above experimental group was detected using a Thermo Fisher Trace GC / Delta VAdvantage IRMS (Thermo Fisher Scientific, USA). The detection method was performed according to the reference (Ai GM., et al., 2011), specifically: 5 μL of headspace gas was collected using a 10 μL gas-tight syringe, and the δ¹⁴ Ω·cm of N2 was analyzed by GC-IRMS. 15 The analytical conditions for N were as follows: GS-Carbon Plot column (30m × 0.32mm × 3.0μm); injection temperature 160℃, split ratio 30:1, column initial temperature 35℃, held for 6 min, then increased to 140℃ at 20℃ / min, held for 2 min; carrier gas was He, flow rate 1 mL / min. IRMS was set to nitrogen analysis mode, detecting ions at m / z 28 and 29. The detection results confirmed the presence of N. 15 The presence of N-labeled nitrogen indicates that strain D14 can reduce nitrate to N2O and further to N2, thereby achieving complete nitrate conversion into nitrogen gas for denitrification.

[0115] II. Lihuangia oceani D14 possesses sulfur oxidation and denitrification functions under anaerobic and autotrophic conditions.

[0116] The culture medium used in this experiment was prepared as follows:

[0117] Composition and preparation of autotrophic sulfur oxidation-denitrification medium: Sodium bicarbonate 2 g / L, potassium nitrate 2 g / L, sodium thiosulfate 2.5 g / L, solvent is 30 g / L sea salt solution (solvent is water, solute is sea salt (Sigma, product number S9883-1KG), pH 7.6-7.8). Sodium bicarbonate and sodium thiosulfate are sterilized by filtration through a 0.22 μm filter membrane. Other components are autoclaved at 121℃ for 20 min and used after natural cooling.

[0118] Composition and preparation of sodium thiosulfate-free culture medium: 2 g / L sodium bicarbonate, 2 g / L potassium nitrate, solvent is 30 g / L sea salt solution, pH 7.6-7.8. Sodium bicarbonate is sterilized by filtration through a 0.22 μm filter membrane, and other components are autoclaved at 121℃ for 20 min and used after natural cooling.

[0119] The experiment was repeated three times, with three treatments each time: the experimental group, the control group without sodium thiosulfate, and the blank control group without inoculation.

[0120] 1. Experimental Group: Strain D14 was activated on 2216E liquid medium. Cells with an OD600nm of 0.6 (using 2216E liquid medium as a blank control) were collected, washed three times with sterilized artificial seawater, and inoculated at a 2% inoculum into sterilized headspace vials (100 mL volume). Each headspace vial contained 50 mL of autotrophic sulfur oxidation-denitrification medium, with three replicates. The headspace vials were purged with nitrogen, capped, and placed in an anaerobic incubator at 30°C for anaerobic incubation. The culture was shaken every 24 hours. Headspace gas samples were collected on days 0, 14, 21, and 28, and N2O was measured according to the method described in step one.

[0121] 2. Sodium thiosulfate-free control group: Strain D14 was activated on 2216E liquid medium. Cells with an OD600nm of 0.6 (using 2216E liquid medium as a blank control) were collected, washed three times with sterilized artificial seawater, and inoculated at a 2% inoculum into sterilized headspace vials (100 mL volume). Each headspace vial contained 50 mL of sodium thiosulfate-free medium, with three replicates. The headspace vials were purged with nitrogen, capped, and placed in an anaerobic incubator at 30°C for anaerobic incubation. The culture was shaken every 24 hours. Headspace gas samples were collected on days 0, 14, 21, and 28, and N2O was measured according to the method described in step one.

[0122] 3. Blank control group without inoculation: Sterilized headspace vials (100 mL volume) contained 50 mL of autotrophic sulfur oxidation-denitrification medium, with three replicates. The headspace vials were purged with nitrogen, capped, and placed in an anaerobic incubator at 30°C for anaerobic incubation. The culture was shaken every 24 hours, and headspace gas samples were collected on days 0, 14, 21, and 28 to determine N2O according to the method in step one.

[0123] The results are as follows Figure 5 As shown, the N2O content in the experimental group increased with the autotrophic culture time. When the culture medium did not contain sodium thiosulfate, N2O could not be detected in the sodium thiosulfate-free control group. At the same time, since strain D14 was not inoculated, N2O could not be detected in the blank control group without inoculation. This indicates that strain D14 requires sodium thiosulfate as an electron donor for denitrification in the absence of organic matter.

[0124] In addition, NO3 was measured by ion chromatography in the experimental group after 14 days of culture. - -N, SO4 2- -S、S2O3 2--S, Ion Chromatography Determination Method: Sample Pretreatment: After centrifuging the sample at 13000 r / min for 5 min, take the supernatant and dilute it 100-200 times with water, then filter it through a 0.22 μm microporous membrane. Chromatographic Conditions: Automatic online eluent: EGCIII KOHRFIC TM Column: Dionex IonPac TM AS19-4um IC analytical column; column temperature: 30℃; flow rate: 1mL / min; anion suppressor ADRS600: automatic regeneration suppression mode, suppression current 50mA; conductivity detector detection; injection volume 25μL.

[0125] The results are as follows Figure 6 As shown, NO3 after 14 days of cultivation - -N and S2O3 2- The amount of -S decreased slightly, while SO4 2- The amount of -S increased slightly. Overall, it can be seen that strain D14 can oxidize thiosulfate to sulfate and reduce nitrate to N2O and N2 under anaerobic conditions in the absence of organic matter.

[0126] Example 4: Denitrification function of Lihuangia oceani D14 under organic anaerobic conditions

[0127] The culture medium used in this experiment is as follows:

[0128] Composition and preparation of heterotrophic denitrification medium: 2216E liquid medium with 2 g / L potassium nitrate and 2.5 g / L sodium thiosulfate. Sodium thiosulfate was sterilized by filtration through a 0.22 μm filter membrane. Other components were autoclaved at 121℃ for 20 min and used after natural cooling.

[0129] Composition and preparation of heterotrophic denitrification medium without sodium thiosulfate: 2216E liquid medium with 2 g / L potassium nitrate, autoclaved at 121℃ for 20 min, and used after natural cooling.

[0130] The experiment was repeated three times, with three treatments each time: the experimental group, the control group without sodium thiosulfate, and the blank control group without inoculation.

[0131] 1. Experimental Group: Strain D14 was activated on 2216E liquid medium. Cells with an OD600nm of 0.6 were collected, washed three times with sterilized artificial seawater, and inoculated at a 2% inoculum into sterilized headspace bottles (100 mL volume) containing 50 mL of heterotrophic denitrification medium. After nitrogen purging, the bottles were capped and placed in an anaerobic incubator at 30°C for anaerobic cultivation. The culture was shaken every 24 hours. After 7 days of cultivation, samples were taken and the cell concentration was determined using a spectrophotometer.

[0132] 2. Control group without sodium thiosulfate: Strain D14 was activated on 2216E liquid medium. Cells with an OD600nm of 0.6 were collected, washed three times with sterilized artificial seawater, and inoculated at a 2% inoculation rate into a 100mL headspace vial containing 50mL of sodium thiosulfate-free heterotrophic denitrification medium. After nitrogen purging, the vial was capped and placed in an anaerobic incubator at 30℃ for anaerobic cultivation. The culture was shaken every 24 hours. After 7 days of cultivation, samples were taken and the cell concentration was determined using a spectrophotometer.

[0133] 3. Blank control group without inoculation: Sterilized headspace bottles (100 mL volume) contained 50 mL of heterotrophic denitrification medium, with three replicates. After nitrogen purging, the bottles were capped and placed in an anaerobic incubator at 30 °C for anaerobic incubation. The culture was shaken every 24 hours, and after 7 days of incubation, samples were taken and the bacterial cell concentration was determined using a spectrophotometer.

[0134] The results showed that the average OD600nm of the experimental group was 0.24±0.016, the average OD600nm of the control group without sodium thiosulfate was 0.12±0.023, and the OD600nm of the blank control group without inoculation showed no change. This indicates that *Lihuangia oceani* D14 can proliferate under organic anaerobic conditions.

[0135] Simultaneously, NO2-N headspace gas was detected using an Agilent 7890A / 5975C GC / MS according to the method in Example 3(1), and NO3 was determined using ion chromatography according to the method in Example 3(2). - -N, SO4 2- -S、S2O3 2- -S. Results of the experimental group are as follows Figure 7 As shown, thiosulfate (S2O3) could be detected after 7 days of cultivation under organic anaerobic conditions (experimental group). 2- The concentration of -S decreases, and the sulfate (SO4) concentration decreases. 2- The increased concentration of nitrate nitrogen (NO3-) indicates that thiosulfate is oxidized to sulfate; simultaneously, nitrate nitrogen (NO3-) increases. - The decrease in N⁻N concentration and the production of N₂O indicate the presence of denitrification in the sodium thiosulfate-containing experimental group; the results for the control group without sodium thiosulfate are as follows: Figure 8 As shown, in the control group without sodium thiosulfate, sulfate (SO4) 2- The concentration of -S remained unchanged, while the nitrate nitrogen (NO3) concentration remained unchanged. - The concentration of N2O decreased slightly, and a small amount of N2O was produced, but the change was significantly less than when sodium thiosulfate was present; the blank control group without inoculation showed no change. This indicates that strain D14 can perform both sulfur oxidation denitrification using thiosulfate and anaerobic denitrification in the presence of organic matter.

[0136] Example 5: Sulfur oxidation function of Lihuangia oceani D14 under heterotrophic aerobic conditions

[0137] The experiment was repeated three times, with three treatments each time: the experimental group and a blank control group without inoculation.

[0138] 1. Experimental Group: Strain D14 was activated in 2216E liquid medium. Cells with an OD600nm value of 0.6 were collected (using 2216E liquid medium as a blank control). The cells were washed three times with sterilized artificial seawater and inoculated at a 2% inoculation rate into sterilized Erlenmeyer flasks (250 mL volume). Each flask was pre-filled with 100 mL of 2216E liquid medium containing 5.0 g / L Na2S2O3·5H2O (i.e., 2216E liquid medium with added Na2S2O3·5H2O to a final concentration of 5.0 g / L). Three replicates were performed. The flasks were incubated at 30°C and shaken at 180 rpm. After 3 days of incubation, samples were taken to measure the OD600nm value and SO42- content of the cells. 2- -S、S2O3 2- -S concentration was determined using the same method as in Example 4.

[0139] 2. Blank control group without inoculation: The volume of the sterilized conical flask was 250 mL. 100 mL of 2216E liquid culture medium containing 5.0 g / L Na2S2O3·5H2O was added to the aforementioned 2216E liquid culture medium to a final concentration of 5.0 g / L. The flask was placed in a shaker at 180 rpm and incubated at 30 °C. After 3 days of incubation, the OD600 nm value of the liquid in the conical flask was measured.

[0140] The average OD600nm value after 7 days of culture was 0.75±0.015, indicating that *Lihuangiaoceani* D14 can proliferate under heterotrophic aerobic conditions. (SO4) 2- -S、S2O3 2- -S changes as follows Figure 9 As shown, sodium thiosulfate (S2O3) in the culture medium of strain D14 in the experimental group... 2- -S) decreased significantly, SO4 ions decreased significantly. 2- An increase in -S concentration results in the formation of sulfate (SO4). 2- -S), thus it can be seen that strain D14 can carry out sulfur oxidation under heterotrophic aerobic conditions.

[0141] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. *Flavobacterium flocculationii*, characterized by: The Flavobacterium oceanus is Lihuangia oceani with the strain number D14, and the registration number of China General Microbiological Culture Collection Center is CGMCC No. 1.13774.

2. A bacterial agent, characterized in that: The bacterial agent contains *Flavorholia oryzae* as described in claim 1.

3. The bacterial agent of claim 2, wherein: The bacterial agent can perform sulfur oxidation, oxidizing low-valence sulfur compounds into sulfates.

4. The bacterial agent of claim 2, wherein: The bacterial agent can perform denitrification, reducing nitrogen in nitrates to nitrogen gas or nitrous oxide and removing it from the environment.

5. Denitrification agent, characterized in that: The denitrifying agent contains *Flavobacterium tumefaciens* as described in claim 1 or / and the bacterial agent as described in claim 3.

6. The use of the *Flavobacterium flocculationii* as described in claim 1 in the preparation of a microbial agent for removing nitrogen from the environment.

7. The use of *Flavobacterium tumefaciens* as described in claim 1 in the preparation of sulfur-oxidizing agents.

8. Any one of the following applications of the *Flavobacterium flocculationii* of claim 1, the bacterial agent of claim 2, and the nitrogen remover of claim 5: N1. Oxidize sulfur in wastewater from a low oxidation state to a high oxidation state or remove nitrogen from wastewater through denitrification; N2, products that oxidize sulfur in wastewater from a low oxidation state to a high oxidation state or remove nitrogen from wastewater through denitrification; N3, oxidizes sulfur in sediments from low oxidation state to high oxidation state or removes nitrogen in sediments through denitrification; N4. To prepare products that oxidize sulfur in sediments from low oxidation state to high oxidation state or remove nitrogen in sediments through denitrification; N5. Oxidizes sulfur in the soil from a low oxidation state to a high oxidation state or removes nitrogen in the soil through denitrification; N6. Prepare products that oxidize sulfur in soil from a low oxidation state to a high oxidation state or remove nitrogen in soil through denitrification; N7, remediation of nitrogen and / or sulfur contaminated deposits; N8. Products for the remediation of nitrogen and / or sulfur contaminated deposits; N9, remediation of nitrogen and / or sulfur contaminated soil; N10, products for the remediation of nitrogen and / or sulfur contaminated soil.

9. The application according to claim 8, characterized in that: The wastewater includes at least one of marine aquaculture wastewater and marine product processing wastewater, and the sediment includes sediment from marine aquaculture water bodies.

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