CN116004480A - Deep sea bacteria capable of heterotrophic aerobic growth and having autotrophic sulfur oxidation denitrification function and application thereof - Google Patents

Abstract

The invention discloses a deep sea bacterium which can grow heterotrophically and aerobically and has the function of autotrophic sulfur oxidation denitrification and application thereof. The deep sea bacteria are ocean Huang Lishi bacteria, lihuanginiaoceani, the strain number of the deep sea bacteria is D14, and the registration number of the deep sea bacteria in the China general microbiological culture Collection center (CGMCC) is CGMCC No.1.13774. The ocean Huang Lishi bacteria have facultative sulphur oxidation denitrification function, can perform heterotrophic growth under aerobic conditions, can perform denitrification by using organic matters and/or sodium thiosulfate under anaerobic conditions, can perform sulphur oxidation by using nitrate as an electron acceptor under anaerobic conditions, can perform sulphur oxidation by using oxygen as an electron acceptor under aerobic conditions, and can be used for removing nitrate and thiosulfate in polluted water or sediment, or treating sewage such as landscape water, urban rivers, aquaculture and the like and treating eutrophic rivers and lakes.

Description

Deep sea bacteria capable of heterotrophic aerobic growth and having autotrophic sulfur oxidation denitrification function and application thereof

Technical Field

The invention relates to a strain of ocean Huang Lishi bacteria and application thereof, in particular to a strain of deep sea bacteria which can grow heterotrophically and aerobically and has autotrophic sulfur oxidation denitrification function and application thereof.

Background

Denitrification, i.e. denitrification, refers to the use of nitrate as the final electron acceptor for respiration by microorganisms, passing nitrogen from the nitrate through a series of intermediates (NO ₂ ^- 、NO、N ₂ O) biochemical processes of reduction to nitrogen, the microorganisms involved in this process are collectively referred to as denitrifiers. Denitrification is a key ring in natural nitrogen circulation, is also a main biological process for removing nitrogen in eutrophic water, and is an important functional flora in the process of removing nitrogen and purifying water, thereby playing an important role in the treatment of landscape water, urban rivers, aquaculture and other (sewage) water and the treatment of eutrophic rivers and lakes.

Most denitrifying bacteria known at present are heterotrophic bacteria, which take organic matters as carbon and nitrogen sources and reduce and denitrify nitrate through anaerobic respiration; the heterotrophic denitrifying bacteria have a plurality of defects when being used for purifying the eutrophic water body: if the COD in the black and odorous water body is lower, the conventional heterotrophic denitrification microorganism is difficult to utilize organic matters with lower concentration in the water body, so that nitrate nitrogen in the water body is continuously accumulated, if an organic carbon source is additionally added, the COD in the water body is easily increased, the sludge yield is increased, the running cost is increased and the like are easily caused; so that in the practical application,it is further desirable to obtain and use denitrifying bacteria that are autotrophic. At present, few microorganisms with autotrophic denitrification function can utilize inorganic matters such as reduced sulfur compounds and H ₂ Or Fe (Fe) ²⁺ As electron donor, NO ₃ ^- Capturing energy as an electron acceptor and fixing CO ₂ When the carbon source is used for autotrophic growth, an organic carbon source is not required to be added, and the whole operation cost can be reduced by more than 50 percent compared with heterotrophic denitrification. Therefore, the new strain with autotrophic denitrification capability is a unique microorganism resource, has great potential application value, and can provide strains and microorganism preparations for polluted water body restoration and nitrogen-sulfur wastewater treatment.

Disclosure of Invention

The technical problem to be solved by the present invention is how to remove nitrogen and/or sulphur from the environment, such as from water, soil and/or water sediments.

In order to solve the technical problems, the invention firstly provides the ocean Huang Lishi strain.

The ocean Huang Lishi bacteria provided by the invention is Lihuangia oceani, the strain number of the ocean Huang Lishi bacteria is D14, and the registration number of the ocean Huang Lishi bacteria in the China general microbiological culture Collection center (CGMCC) is 1.13774. Hereinafter, this is abbreviated as "ocean Huang Lishi bacteria" (Lihuanginia oceani) D14.

The invention also provides a culture of the ocean Huang Lishi bacteria, which is obtained by culturing the ocean Huang Lishi bacteria in a microorganism culture medium.

The culture contained Huang Lishi strain (Lihuangia oceanii) D14.

Among the above cultures, the culture is a fermentation product obtained by culturing Huang Lishi strain (Lihuangia oceani) D14 in a microbial medium.

In the above culture, the substances include metabolites of the ocean Huang Lishi bacteria (Lihuangia oceani) D14 and the ocean Huang Lishi bacteria (Lihuangia oceani) D14.

In the above culture, the microorganism culture medium may be a solid medium or a liquid medium.

The term "culture" refers to a generic term for liquid or solid products (all substances in the culture vessel, i.e. fermentation products) grown with a population of microorganisms after artificial inoculation and cultivation. I.e. the product obtained by growing and/or amplifying the microorganism, which may be a biologically pure culture of the microorganism, or may contain a certain amount of medium, metabolites and/or other components produced during the cultivation. The term "culture" also includes subcultures obtained by passaging microorganisms, which may be a culture of a certain generation or a mixture of several generations.

The invention also provides a microbial inoculum which contains the ocean Huang Lishi bacteria or/and the ocean Huang Lishi bacteria metabolite or/and the culture.

Herein, the metabolite may be obtained from a fermentation broth of Huang Lishi strain (Lihuangia oceani) D14. The metabolite of ocean Huang Lishi bacteria (Lihuangia oceanii) D14 may be a sterile metabolite of ocean Huang Lishi bacteria (Lihuangia oceanii) D14 or a bacterial metabolite of ocean Huang Lishi bacteria (Lihuangia oceanii) D14. The sterilized metabolite (sterile fermentation filtrate) of the ocean Huang Lishi strain (Lihuangia oceanii) D14 can be prepared by culturing the ocean Huang Lishi strain (Lihuangia oceanii) D14 in a liquid culture medium, and filtering to remove the ocean Huang Lishi strain (Lihuangia oceani) D14 in the liquid culture (fermentation broth) to obtain the sterilized metabolite of the ocean Huang Lishi strain (Lihuangia oceanii) D14. The bacterial metabolite of the ocean Huang Lishi bacterium (Lihuangia oceanii) D14 can be specifically prepared by culturing the ocean Huang Lishi bacterium (Lihuangia oceanii) D14 in a liquid fermentation medium, and collecting a fermentation broth (containing the ocean Huang Lishi bacterium (Lihuangia oceanii) D14 and a substance secreted into the liquid culture medium), which is the bacterial metabolite of the ocean Huang Lishi bacterium (Lihuangia oceanii) D14.

The active ingredient of the microbial inoculum may be a metabolite of ocean Huang Lishi bacteria (Lihuangia oceani) D14 or/and ocean Huang Lishi bacteria (Lihuangia oceani) D14 or/and the culture, and may further contain other biological components or non-biological components, and the other active ingredients of the microbial inoculum may be determined by one skilled in the art according to the effect of the microbial inoculum.

In the microbial inoculum, the microbial inoculum can further comprise a carrier. The carrier may be a solid carrier or a liquid carrier.

The solid carrier can be mineral material or biological material; the mineral material may be at least one of turf, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material can be at least one of straws, pine shells, straws, peanut shells, corn flour, bean flour, starch, turf and animal excrement of various crops; the liquid carrier may be water; in the microbial inoculum, the metabolite of the ocean Huang Lishi bacteria (Lihuangia oceanii) D14 or/and the ocean Huang Lishi bacteria (Lihuangia oceanii) D14 may exist in the form of living cells to be cultured, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and filtrate. The dosage form of the microbial inoculum can be various dosage forms, such as liquid, emulsion, suspending agent, powder, granule, wettable powder or water dispersible granule.

Surfactants (such as Tween 20, tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added into the microbial inoculum according to the need.

Further, the microbial inoculum can perform sulfur oxidization and oxidize sulfur compounds in low valence state into sulfate.

Further, the microbial inoculum can perform denitrification to reduce nitrogen in nitrate into nitrogen (N) ₂ ) Or nitrous oxide (N) ₂ O) and removed.

Specifically, it means that the ocean Huang Lishi bacteria (Lihuangia oceani) undergoes sulfur oxidation under anaerobic conditions using nitrate as an electron acceptor or under aerobic conditions using oxygen as an electron acceptor.

The invention also provides a nitrogen removal agent, which contains the metabolite of the ocean Huang Lishi bacteria or/and the ocean Huang Lishi bacteria or/and the culture or/and the bacterial agent. The nitrogen removing agent can simultaneously utilize organic matters and sodium thiosulfate to perform denitrification.

Further, the nitrogen removal agent may be a microbial agent that removes nitrogen in an anaerobic environment. The nitrogen scavenger may reduce nitrate to nitrogen by denitrification.

The invention also provides an application of the ocean Huang Lishi bacteria, the ocean Huang Lishi bacteria metabolite or the culture in preparing a bacterial agent for removing nitrogen and/or sulfur in the environment.

The environment may be a body of water, soil, and/or water sediment. The body of water may be wastewater.

In addition, the ocean Huang Lishi bacterium (Lihuangia oceania) and the polysaccharide macromolecule Polyhydroxyalkanoates (PHA) and the analogues thereof which can be produced in the cells in the using process can be applied to the field of the polysaccharide macromolecule Polyhydroxyalkanoates.

The invention also provides an application, in particular to an application of the ocean Huang Lishi bacteria, the ocean Huang Lishi bacteria metabolite, the culture and any one of the following bacterial agents:

n1, oxidizing sulfur in the wastewater from low valence to high valence or removing nitrogen in the wastewater through denitrification;

n2, preparing a product for oxidizing sulfur in the wastewater from low valence to high valence or removing nitrogen in the wastewater through denitrification;

n3, oxidizing sulfur in the sediment from low valence to high valence or removing nitrogen in the sediment through denitrification;

n4, preparing a product for oxidizing sulfur in the sediment from low valence to high valence or removing nitrogen in the sediment through denitrification;

n5, oxidizing sulfur in the soil from low valence to high valence or removing nitrogen in the soil through denitrification;

n6, preparing a product for oxidizing sulfur in the soil from low valence to high valence or removing nitrogen in the soil through denitrification;

n7, remediating nitrogen and/or sulfur contaminated deposits;

n8, preparing a product for repairing nitrogen and/or sulfur contaminated sediment;

n9, remediating nitrogen and/or sulfur contaminated soil;

n10, preparing a product for repairing the nitrogen and/or sulfur polluted soil;

n11, preparing a polysaccharide product;

n12, preparing a polyhydroxyalkanoate product.

In the above application, the wastewater comprises at least one of seafood culture wastewater and seafood treatment wastewater, and the sediment comprises seafood culture water sediment.

In such applications, the soil includes, but is not limited to, coastal or intertidal soils and the like.

The ocean Huang Lishi strain (Lihuangia oceania) D14 is a bacteria which can grow heterotrophically and has autotrophic sulfur oxidation denitrification function, has facultative sulfur oxidation denitrification function, can simultaneously utilize organic matters and sodium thiosulfate to perform denitrification, can utilize nitrate as an electron acceptor to perform sulfur oxidation under anaerobic conditions, and can also utilize oxygen as the electron acceptor to perform sulfur oxidation under aerobic conditions. Based on the above, ocean Huang Lishi bacteria (Lihuangia oceani) D14 can be used for simultaneously removing nitrate and thiosulfate in polluted water or sediment, and can be applied to treatment of (sewage) water such as landscape water, urban rivers and aquaculture, and treatment of eutrophic rivers and lakes.

Preservation description

Ocean Huang Lishi bacteria latin name: lihuangingia oceani

Strain number: d14

Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

The preservation organization is abbreviated as: CGMCC

Address: beijing city, chaoyang area, north Chenxi Lu No.1 and 3

Preservation date: 2022, 6 and 30 days

Accession numbers of the preservation center: CGMCC No.1.13774.

Drawings

FIG. 1 is a transmission electron microscope and scanning electron microscope photograph of the ocean Huang Lishi bacterium (Lihuangia oceani) D14;

FIG. 2 is a phylogenetic cluster tree of the genus Dahurian Huang Lishi (Lihuangia oceani) D14;

FIG. 3 is a graph showing heterotrophic aerobic growth of Brevibacterium Dahurian Huang Lishi (Lihuangia oceania) D14;

FIG. 4 shows stable isotope labeling experiments to demonstrate that cells of the ocean Huang Lishi (Lihuangia oceania) D14 strain can undergo denitrification under autotrophic anaerobic conditions;

FIG. 5 is N at the time of autotrophic anaerobic sulfur oxidation denitrification by the microorganism (Lihuangia oceania) D14 of ocean Huang Lishi ₂ The content of O-N increases and changes along with the autotrophic culture time;

FIG. 6 is a graph showing the change in nitrate-nitrogen, thiosulfate-sulfur and sulfate-sulfur concentration during autotrophic anaerobic sulfur oxidation denitrification by ocean Huang Lishi bacteria (Lihuangia oceania) D14;

FIG. 7 is a graph showing the change in concentration of nitrous oxide-nitrogen, nitrate-nitrogen, thiosulfate-sulfur, and sulfate-sulfur in the presence of sodium thiosulfate and anaerobic conditions of ocean Huang Lishi bacteria (Lihuangia oceanii) D14;

FIG. 8 is a graph showing the change in concentration of nitrous oxide-nitrogen, nitrate-nitrogen, sulfate-sulfur when denitrification is performed by ocean Huang Lishi bacteria (Lihuangia oceania) D14 in the absence of sodium thiosulfate and under anaerobic conditions;

FIG. 9 shows the concentration changes of thiosulfate-sulfur and sulfate-sulfur when the sulfur oxidation of ocean Huang Lishi bacteria (Lihuangia oceania) D14 was carried out under heterotrophic aerobic conditions.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

2216E liquid medium composition, concentration and preparation: 5g/L peptone, 1g/L yeast extract, 0.1g/L ferric citrate, 30g/L sea salt (sea salt) solution (sea salt (Sigma, cat. S9883-1 KG) as solvent, pH 7.6-7.8.2216E solid culture medium, adding 15g/L agar into 2216E liquid culture medium, sterilizing at 121deg.C for 20min, and naturally cooling.

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

Example 1 isolation and identification of Strain D14

1. Isolation of Strain D14

A water sample at the water-sediment interface at 4213 m deep sea was taken as a sample from the North-west Indian ocean (69 DEG 20 '31.84' E4 DEG 00 '10.06' N) and collected at 21 st 6 in 2013 by the institute of microbiology of China academy of sciences participating in the study of 28 voyage of the ocean. Microbial cells in the samples were placed in 96-well plates (100 ul of medium/well) containing 2216E liquid medium by a flow cell sorter, and cultured at 25℃for about 40 days, and the single cell cultures with growth were taken out and spread on 2216E solid medium for 20-30 days, and single colonies were picked up and further purified by liquid medium and streaking to obtain pure culture strain, which was designated as strain D14.

2. Morphological and physiological Biochemical identification of Strain D14

Strain D14 grew slowly on 2216E solid medium at 30℃and after 3-4 weeks the colony had a diameter of about 0.5mm and was round, protruding, indicating smooth, translucent, white, and clean-edged. As shown in FIG. 1A, the cells were seen under a transmission electron microscope to be in the form of rods 1.0-2.0 μm long and 0.5-0.8 μm wide, and were single flagellum, and Polysaccharide (Polysaccharide) was present outside the cells.

Strain D14 grew faster at 30 ℃ in liquid 2216E liquid medium. In addition, intense light was found to inhibit the growth of the strain, and the culture was required to be carried out in a dark environment.

Strain D14 was grown under both aerobic and anaerobic conditions. The growth temperature is 15-40 ℃, the most suitable growth temperature is 30-35 ℃, the growth pH is 5.5-9.5, the most suitable growth pH is 6.5-8.5, the growth salinity is 0.5-6.5%, and the most suitable salinity is 2.5%.

Culturing the strain D14 in 2216E liquid culture medium at 30 ℃ shaking table 160rpm for 3 days, collecting cells, washing with sterile artificial seawater for 3 times, transferring to culture solution (sucrose 40g/L, yeast extract 0.4g/L, solvent is artificial seawater), standing for 3 days, fixing and slicing cells, and observing that the cells contain Polyhydroxyalkanoates (PHA) under a transmission electron microscope (shown in FIG. 1B); the bacteria were seen to be in a unique starfish arrangement under scanning electron microscopy (C in FIG. 1 and D in FIG. 1).

Biochemical identification results show that the strain D14 is a gram-negative bacterium, and cell respiration quinones are CoQ-10 (95.7%) and CoQ-8 (4.3%); the polar lipid composition (an important chemical classification index) of the cells of strain D14 includes Phosphatidylglycerol (PG), lecithin (PC), phosphatidylethanolamine (PE), one unknown Phospholipid (PL), and two unknown lipids (L).

3. Molecular classification identification of Strain D14

Extracting total DNA of the strain D14 according to a conventional method, and carrying out PCR amplification by using the total DNA as a template and adopting bacterial 16S rRNA gene universal primers 27F and 1492R, wherein the primer information is as follows:

27F：5’-AGAGTTTGATCCTGGCTCAG-3’；

1492R：5’-GGTTACCTTGTTACGACTT-3’；

the PCR system and the procedure are: 10 XPCR buffer 2.5. Mu.L, 10.0 mmol/L dNTP 2.0. Mu.L, 10.0 pmol/mu.L 27F primer 1.0. Mu.L, 10.0 pmol/mu.L 1492R primer 1.0. Mu.L, 2.5U/mu.L Taq enzyme 0.3. Mu.L, deionized water make up 25.0. Mu.L. After 5min of pre-denaturation at 95 ℃, denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 1.5min, and after 30 reaction cycles, extension at 72 ℃ for 10 min. Sending the PCR amplified product to a sequencing company for sequencing, and submitting the obtained sequence to a sequencing companyhttp:// www.ezbiocloud.net/identifyPerforming sequence phaseAnd (5) similarity analysis. The analysis results are shown in Table 1, and D14 is similar to 4 different genera of the order Rhizobium (Hyphomicroholes, redefined in 2020, including and replacing the previous Rhizobium) in 93.53-93.85% (as shown in Table 1), and the microorganism 16S rRNA gene similarity is less than 95% according to the conventional rule, and can be divided into new genera (Tindall BJ., et al 2010), indicating that the microorganism represents a new genus species in the order Rhizobium.

TABLE 1 analysis of similarity (%) of D1416S rRNA Gene/ANI/AAI Strain

Sequence number	Most similar sequence (16S rRNA GenBank accession number)	16S(％)	ANI(％)	AAI(％)	Root of Cork-Sitting
						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

* Phy: phyllobacterioceae; rho: rhodobiaceae; bre Breoghaniaceae; no genome data.

4. Genome assay for Strain D14

Extracting total DNA of the strain D14 according to a conventional CTAB method, purifying the DNA by using an MGIEasy DNA purification magnetic bead kit, adopting the PacBioSequenci II third generation sequencing of An Nuo company and combining with Illumina PE150 second generation sequencing, and splicing to obtain the complete genome sequence of the strain D14, wherein the genome size of the strain D14 is determined to be 3.53M, G+C% is determined to be 65.7%, and a set of rRNA (namely 16S-23S-5S rRNA) gene sequence is contained. The complete 16S rRNA gene sequence is extracted, as shown in SEQ ID NO.1, and is specifically as follows:

agagtttgatcctggctcagaacgaacgctggcggcaggcctaacacatgcaagtcgaacgtgaagctggagcttgctccagtggaaagtggcagacgggtgagtaacgcgtgggaacctacccaggagtacggaataactcagggaaacttgggctaataccgtatacgtcccccactagagatagtgggggagaaagatttatcgctcctggatgggcccgcgtcgcattagcttgttggtggggtaatggcctaccaaggcgacgatgcgtagctggtctgagaggatgatcagccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaatattggacaatgggcgcaagcctgatccagccatgccgcgtgggtgaagaaggccttagggttgtaaagccctttcagtcgtgaagataatgacggtagcgacagaagaagccccggctaactccgtgccagcagccgcggtaatacggagggggctagcgttgttcggaattactgggcgtaaagcgcgcgtaggcggatagttaagtcgggggtgaaatcccggggctcaaccccggaactgccctcgatactggctgtctagagtccgggagaggtgagtggaattcctagtgtagaggtgaaattcgtagatattaggaggaacaccagtggcgaaggcggctcactggcccggtactgacgctgaggtgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatggatgctagccgtcggacagcatgctgttcggtggcgcagctaacgcattaagcatcccgcctggggagtacggtcgcaagattaaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgcagaaccttaccagcccttgacatcccggtcgcggttaccagagatggtttccttcagttcggctggaccggtgacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaaccctcgcctttagttgccatcattcagttgggcactctagagggactgccggtgataagccggaggaaggtggggatgacgtcaagtcctcatggcccttacgggctgggctacacacgtgctacaatggcggtgacagtgggcagctaccccgcaaggggacgctaatctctaaaagccgtctcagttcggattgttctctgcaactcgagagcatgaagttggaatcgctagtaatcgtggatcagcatgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagttggttttacccgaaggcgctgcgctaacccgcaagggaggcaggcgaccacggtagggtcagcgactggggtgaagtcgtaacaaggtagccgtaggggaacctgcggctggatcacctcctttcta

according to the results of similarity analysis in Table 1, the 16SrRNA gene sequences of related genus species of the order Aplanophorales were downloaded from GenBank database, and systematic cluster analysis was performed using MEGA 7.0 software, and the resulting phylogenetic tree was shown in FIG. 2, in which "stress D14" was obtained ^T "means strain D14.

The results showed that strain D14 was clustered in one branch with 6 genera of the family Corynebacterium of the order Rhizoctonia (Parvibaculaceae), representing that D14 was a new genus species of this family, and that the 16S rRNA gene similarity (91.8-93.0%) between them was located between known similarity values (90.2-94.7%) (Table 2).

Average nucleotide similarity (Average Nucleotide Identity, ANI) of strain D14 to the genome of a similar genus species was calculated on the http:// jspecies. Ribohost. Com/jspecies ws/# analysis website, showing that strain D14 and the relevant strain each had an ANI value of less than 72% and lower than the ANI value (> 73%) of a different strain of the same genus (Table 1, table 3); since the relationship between species needs to be judged by average amino acid similarity (AverageAminoAcid Identity, AAI) after an ANI value of less than 75%, it is generally believed that AAI can be used for the division between genera with a threshold of about 65-72%, and thus average amino acid similarity (AAI) was calculated and further analyzed for strain D14 and related strain AAI values. The results show that the AAI values between strain D14 and the microorganisms of different genus of the order Rhizoles are all less than 57%, significantly less than the maximum value of 62.48% of the AAI of the different genus, and far less than the AAI values (> 73%) of the different species of the same genus (Table 1, table 4). It follows that the results of ANI and AAI both support a new genus of strain D14, micromycetoma (Hyphomicroholes).

TABLE 2 similarity matrix of the 16S rRNA genes of Strain D14 and species of genus modes of the family Corynebacteriaceae (Parvibaculaceae) (%)

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

TABLE 3 ANI matrix (%)

TABLE 4 AAI matrix between Strain D14 and species of genus modes of the family Corynebacteriaceae (Parvibaculaceae) (%)

Further analysis of the genome of strain D14 revealed that the gene contained 2 copies of PHA-synthesizing key enzyme, namely PHA synthase (Polyhydroxyalkanoic acid synthase) gene (SEQ ID NO.2, SEQ ID NO. 3), and that the genome of the strain also encoded three pathways of carbon dioxide fixation, denitrification and sulfur oxidation (sox) genes (clusters) including denitrification key enzyme nitric oxide reductase (nitric oxide reductase, norB) (SEQ ID NO. 4) and nitrous oxide reductase (nitrous oxide reductase, nosZ) genes (SEQ ID NO. 5), suggested that the strain has autotrophic sulfur oxidation and denitrification functions, and that nitrate (NO ₃ ^- ) Thoroughly reduce to nitrogen (N) ₂ )。

SEQ ID NO.2：

atgagcggagacgacaaacaggaccgaacgaaagcgcgcgaaaccgcttcccgaccgacgggcgagagcgaggccgcgagcgccgatccggaggagcttgcccgcaacatggtgcgtctgctggaggaaagcggccgcgccgtgtcggcctacctcaagccgcgcgagaagggcgaaacgccgctgctaccgttcgacgaattcgcccgtgtcgccaagacgatgggcgcggtggcggagaactggctgtccgacccgcagcgggcggtgaaggcgcaatcggagcttttcggcggttatatggacgtgtggtcgcgctccatgcggaggttttggggcgggccggaggctaacgagggcgagggccaaagcgaagctcctacgagcgcgcgccctgcaaccggcgacaagcgcttcgccgatccggagtggtccgacaatcagttcttcgacctgatcaaagagacctatctggtgacctcgcaatgggccgagcggatggcccgcgacgccgaagatctcgacgaccacacccggcacaaggcggagttctacgtcaagcagatcgccaacgcgctggcgcccagcaacttcgtgctgaccaacccggcactgctgcgggaaactctttcccagaacggcgagaacctggtgcgcggcatgcgcatgctggccgaggacatcgaacggggcgagggcgaactgcgcatccgccagtcggacccggaggcgttcaaggtcggcgagaacctggcgctgacgccgggcaaggtgatctatcagaacgatctgatgcagctcatccaatacaatccgaccaccgaaaaggtgctgaagcggccgctgctgatcgtgccgccgtggatcaacaagttctacattctcgacctgacgccccagaagagcttcatcaaatggtgcgtcgaccaggggctgactgtgttcgtcatctcctgggccaaccccgaccggcggctggcgcagaaaagcttcgagagctacatgctcgagggcatcgtcgaggcggtgaaggtggtcgagaaggtcgccaagtgcgaccacatcaacaccatcggctattgcgtcggcggcacgctgctggccaccgcgctggcctatctgacggctcgcggcgacaagcgcatcgcctcggccaccttgttcaccacccaggtcgatttcacccacgccggcgatctgaaggtgttcgtcgacgaggagcagatcgcggcgatcgagcggcagatgcaggagcgcggctttctggagggcaagaagatggctggcgccttcaaccttttgcgctccaacgatctgatctggccctatgtcgtcaacaactacatgaagggccaggcgcccttccccttcgacctgctgttctggaactccgacgcgacccgcatgccggccgccaaccactccttctacctgcgcaagtgctatttggagaacgcgctgaccaaaggcgaaatggaacttgccggcgagcggctcgacctcggcgcggtgaccgtcccgatctacaatctggcggcccgcgaggaccatatcgcgccggccaggtcggtgttcatcggctgccggttctttggcggtccggtgcgttacgtgatggcggggtccggccatatcgccggcgtcatcaacccgccggcgaagaacaagtatcaatattggaccggcgaggcaccggccggcgagttcgaggagtgggtcgaacgcgccgaggagcaccccggctcctggtggccggattggctgtcatggatcacctcgctcgacgaggagacggtgaaggcgcgcaagcccggcggcggcaagtacaagccgatcgaggacgcaccgggttcctacgtcaaggtgcgcgcctga

SEQ ID NO.3：

atgttggataaagccactccacgcccaagcggtcataccggcacggcgtcgccgccgcccaccgatccgctgataccgccgatgccgtcaaccgagcgccgcaagatgctcgaccagccgctgcacgcggcactcgggtggttcaccgccgggctgtcgccggtctccctggcgctggcctatgcggactgggtccaacatctcagcctgtcgcccgacaagcagatcgaactggtcgaaaaagcgctgcgcaaactgacccggcatcaaaggttttctcgccgggcgctggctggcggcgactgcccggcctgcatcgagccgctgccccaggacacccgcttttccagcgatgcctggcaggcctggccctacaacgcaatttatcagggtttcctgctgacccaacaatggtggcacaacgccaccaccgaggtgcccggcgtctctaagcaccacgaggacgtcgtcgccttcatcgcccggcagatgctcgacgtggtgtcgcccgccaatttccccctcaccaacccggaagtgctcaccgccacgctcgaaagcggcggcgagaacctgatgcgcggcgcggtgaatttcatcgaggactggcagcgcatgctgcagggcgaccggcccgccggcgccgagcagtttcgtgtcggccgcgacctcgccatcacccccggcaaggtcgtgttccgcaacgagctgatggagctcatccaatacacgccgaccacgcagacggtgcaccccgagccggtgctgatcgtgccggcctggattatgaagtactacatcctcgatctcagcccgcacaattcgctggtcaaatatttggtcgaccagggccacacggtgttcatgatgtcctggaagaaccctgtcgccgccgaccgcgatctgagcctggaggactaccgcgagaagggcgtcatggcggccctcgacgcagtaggcgcgatcgtcccggatcagccgatccacggcgccggctattgcctcggcggcacgctgctgtcgatcgccgccgcggcgatggcgcgcgacggcgaccggcggctcgcaaccatgaccatgttcgccgcccaaaccgacttcaccgaggccggcgaactgatgctgttcatcgacgaggcgcaggtctccttcctggagaacatcatggccgagcagggctatctcgacaccaagcagatggccggcgctttccaattgctgcgctccaacgatctgatctggtcggcgatggtgcacaactatcttctcggccagcgccgcccgatgttcgacctgatggcgtggaacgccgacgctacccgcatgccggcgaaaatgcattcgcagtatctgcggcagttgttcctcaacaacgatctcgccgacgagcgctaccgcgtcgacgggcggccggtgtctctgcgcgatatccgcctgccggtgttcgcggtctccaccctcaccgaccacgtcgcgccctggcgctcggtctacaagatccacgcccagaccaacgccgacgtgaccttcgtgctgtccaacggcggccacaacgccggcatcgtcaacccgccgggcaacggcaagcgcttccaccaaatcgccacccacgacgatctggaggcctatctggaccccgacgcctggcaggagcacgccgcccgccacgacggctcttggtggccgtgctggcgggactggctcgccgaacgttccagcgccaagcaggcgccgcccgcaatgggggcgcctgacaagggctatacgcccctcggcgaggcgcctggcgactatgtgctgcaaccgtag

SEQ ID NO.4：

atgaaatatgcaactcagcaaatcggattttattacttcctggccgcgatgatcctattcgccatccaggtctccggcggcctgctggcgggttatatctatgtcgacccgaacttcctcagcgagctgctgcccttcaacatcgcccgcatgctgcacaccaacgcgttgatcgtgtggctgctgctcggcttcttcggtgccgcctatttcatcatccccgaagagtccgaacgcgagatccattcgccgctgctcgcctatgtgcaactggcaatcctgctgctcggcaccctcggcgtggtggtgacctatctgttcaacctgttcgagggcaactggctgctcggcaaggaggggcgcgagttcctcgagcagcccttgtgggtcaagctcggcataatcgtcgccgcgctgatctttctctacaacgtcaccttgacggtcctaaagggcaagaagaccgccatcaccaacgtcctgctgatcggtctgtggggcttggcgctgctgttcttgttctccctctacaatccggagaacctcggcctcgacaagatgtactggtggtacgtcgtgcacctgtgggtggaaggcgtgtgggagctgatcatggcctccattctcgcctatttgatgctgaagctgaccggcgtcgaccgcgaggtggtggagaaatggctctatgtcatcgtcgccgcggcgctgttcaccggcatcctcggcaccggccaccactattactggatctccacccccgcttactggcaatggctcggctcgatcttctcctcgctggaggtgatccccttcttcctgatggtggtgttcgccttcgccatggtgtggaagggccgtcgcgaccatcccaacaaggccgccatgctgtggtcgctgggctgcgtcgttctcgccttcttcggcgccggcgtgtggggcttcctgcacaccctgcacggcatcaactactacacccacggcacgcagatcacctccgcccacgggcatctcgccttcttcggcgcctatgtctcgctgaacctggcgatcttcacctatgccttcccgatcctgcgcaaccgcgatccctacaaccaggtgctcaacatggcgagcttctggctgatggccggcggcatgacgttcatgaccttcgtgctgaccttcgccggcgtcatccagacccacctgcagcgggtgctcggcgacggttatatggacgtgcaggcgcagctcgagctgttctacctgatgcgtctgggtgccggagcgctggtcgtggtcggcgcgctgctgttcatctacgcggtgctgtcgacgcccaaggccgagctgatctcgcgtggccggccggcgccggccgagtga

SEQ ID NO.5：

atggcacggaaaaccgatgacaatggcttcacacgccgcgacctgatgtcaggctcggccaagacggcgctgctggtcggcgccggcggggccgccggcttgagcggtgcaacgctgttgcagaccggggcggtgagcccggcggcggcggccgagaatggttcggccaacctcaagcccggcgagctcgacgattactacggcttctggtcgagcgggcagaccggcgaactgcggatcctgggcgtgccctcgatgcgcgaattcatgcgcgtgccggtgttcaaccgcgacagcgccagcggctggggacagaccaacgaatcgctcaagatcctgaccgagggcctgctgccggagaccaaggaatttctcgccaagcgcggcaaggtcacctacgacaacggcgatctgcaccatccgcacatgtcgttcaccgacggcacctacgatggccgctacttgttcatgaacgacaaggccaacacgcgggtggcgcgggtgcgctgcgacgtgatgaagtgcgacaagatcatccagatccccaacgcgcaggacatccacggcatgcggccgcagaaattcccgcgcaccggctatgtgttcgccaacggcgagcacgagacgccgctggtcaacgacggcaagatcctcgacgagccggacaagtacgtgaacatcttcaccgccatcgacggcgacaagatggaggtcgcctggcaggtgatcgtctccggcaacctcgacaacgtcgattgcgactaccagggcaaatatgccttctcctcctcttacaactcggagatgggcatgaacctggcggagatgactgccagcgagttcgaccatgtggtgatcttcgacatcgccgagatcgagcggggggttgccgccggcgactatcaaacgctgaacggggtgccggtgctcgacgggcgcaagggccgcaacaagaagtacacccgctacgtgccgatccccaacagcccgcacggcgtcaacgtcgcccccgacaagcgtcacgtgatgatcaacggcaagctgtcgccgaccgtctcggtgatcgacgtgaccaaggtcgacgcgctgtttgccgaggacgccgaaccgcgttcgtgcatcgtcgcagagccgcagctcggccttgggccccttcacaccgccttcgacggcaagggtaatgccttcaccaccttgttcctcgacagccaggtggtgaagtgggacatcgacaaggccgttcgcgcctatgccggcgaggacgtcgacccgatcctcgacaaagtggatgtcgcctatcagcccggccacaactcgacctcgatgggcgagaccctggaggccgacggccgctggctggtgtcgatgaacaagttctccaaggaccgcttcctcaacgtcggtccgctgaagccggagaacgagcagctcatcgacctcaccagtgagaagatggaggtgatccacgacgggccgaccttcgccgagccgcacgacagcatcatcgtgcgcgccgatatcgtcaatccggtcaacgtgtggaagcgcgacgacccgacgtgggaagacgcgcgcaagcaggccgaggcggacggcgtggtgctcgaggacggcgccgaggagccgatccgcgacggcaacaaggtgcgggtctatatgaccagcctggcaccgcagttctctttggagaagttcacggtgaaacagggcgacgaggtgacggtgtacatcaccaacctcgacgatgtcgacgacgtcacccacggcttcacgctctccaaccatggcatcgccttcgaggtcgcgccgcaggcaaccgcctcgatcacgtttaccgccgacatgccgggcgtgcactggttctactgtcagtggttctgccatgcgctgcacatggaaatgcgcggccgcatgctggtcgagccggccggagcctga

In combination with morphological, physiological and biochemical and molecular taxonomic identification results, the strain D14 was identified as a novel species of genus Huang Lishi (Lihuangia) belonging to the family Corynebacterium (Parvibaculaceae) of the order Rhizoctonia (Hyphomicrobiological), designated as Dayang Huang Lishi (Lihuangia oceani). The strain is preserved in China general microbiological culture Collection center (CGMCC; address: north Chen West Lu No.1, 3 of the Korean area of Beijing, national academy of sciences of China, and the microorganism research institute; postal code: 100101) at 30 days of 2022, with a preservation number of CGMCC No.1.13774, and is subsequently called as Dayang Huang Lishi bacterium (Lihuanginia oceania) D14 or strain D14.

Example 2 characterization of growth of the microorganism Dayang Huang Lishi (Lihuangia oceania) D14 under heterotrophic aerobic conditions

Strain D14 was activated on 2216E solid medium, inoculated in 2216E liquid medium, placed in intelligent honest cell culture shaker (OD value on-line detection) ZWYC-290C for 5 days at 30 ℃ at 160rpm, with the time of cultivation as horizontal axis and the OD600nm corresponding to each detection time point as vertical axis, and strain growth curve was drawn, as shown in fig. 3, it was seen that strain D14 had logarithmic growth phase for 24-48 hours (1-2 days) under heterotrophic aerobic condition and OD600nm reached maximum value of 1.73 at 72 hours (3 days).

Example 3 Brevibacterium Davidii (Lihuangia oceania) D14 has Sulfur oxidation denitrification function under anaerobic and autotrophic conditions

1. The cells of the ocean Huang Lishi (Lihuangia oceania) D14 strain have denitrification function under anaerobic and autotrophic conditions

The media used in this experiment were as follows:

containing 1g/L ¹⁵ N stabilizationIsotopically labeled NaNO ₃ (wherein N is ¹⁵ N) and 1g/L unlabeled NaNO ₃ (wherein N is ¹⁴ Preparation of sodium thiosulfate-containing autotrophic medium of N): naNO ₃ (wherein N is ¹⁵ N)1g/L，NaNO ₃ (wherein N is ¹⁴ N) 1g/L, sodium bicarbonate 2g/L, potassium nitrate 2g/L, sodium thiosulfate 2.5g/L, the solvent is the above 30g/L sea salt solution, pH7.6-7.8. Wherein sodium bicarbonate and sodium thiosulfate are filtered and sterilized by a 0.22 μm filter membrane, and the other components are sterilized at 121 ℃ for 20min and naturally cooled for use.

Experiments were repeated three times, two treatments were set up each time: experimental and control groups.

1. Experimental group: strain D14 was cultured on 2216E liquid medium to an OD600nm of 0.6 (2216E liquid medium was used as a blank), 10ml of cells were collected and washed three times with sterilized artificial seawater, and 1g/L of the cells were added ¹⁵ N-stable isotope labeled NaNO ₃ (wherein N is ¹⁵ N) and 1g/L unlabeled NaNO ₃ (wherein N is ¹⁴ N) sodium thiosulfate-containing autotrophic culture medium, incubating for 1 day at a dark place at 30 ℃ and then (namely, denitrification reaction of the somatic cells), and detecting the headspace gas by using an Agilent 7890A/5975C GC/MS gas chromatography-mass spectrometer, namely, carrying out gas chromatography-mass spectrometry analysis on the headspace gas.

2. Control group: will contain 1g/L ¹⁵ N stable isotope labeled ¹ NaNO ₃ (wherein N is ¹⁵ N) and 1g/L unlabeled NaNO ₃ (wherein N is ¹⁴ N) sodium thiosulfate-containing autotrophic medium (without access to strain D14), incubating at 30℃for 1 day in the absence of light, and analyzing the presence of N with an Agilent 7890A/5975C GC/MS gas chromatograph-mass spectrometer ₂ O。

Detection of N in headspace of experimental and control groups by GC/MS ₂ The abundance of O and the GC/MS detection conditions are as follows: the sample was applied to a GS-Carbon Plot (30 mX0.32 mm X3.0. Mu.m, agilent, USA) analytical column with a loading of 20. Mu.L headspace gas (5190-1503 Agilent 50. Mu.L gas-tight needle sampling); the sample inlet is 160 ℃, the split ratio is 5:1, the initial temperature is 35 ℃, the temperature is kept for 5min, then the temperature rises from 140 ℃ at 20 ℃/min, the temperature is kept for 2min, the carrier gas is helium, and the flow rate is 1.3ml/min.

The results are shown in FIG. 4, the upper graph in FIG. 4 shows that the gas chromatograph mass spectrum detects N with a molecular weight of 44 ₂ O, the lower panel shows the N with molecular weight of 45 detected by gas chromatography mass spectrometry ₂ The peak diagram of O is the retention time on the abscissa and the response signal of the gas chromatograph mass spectrum on the ordinate, so that the experiment group can clearly detect the N containing the stable isotope label ₂ O, control group failed to detect N ₂ O。

To determine N generated by denitrification ₂ Whether O can be further reduced to N ₂ The headspace gas of the above experimental group was detected by means of isotope ratio mass spectrometry ThermoFisher Trace GC/Delta VAdvantage IRMS (company Thermo Fisher Scientific, U.S.) by the method reference (Ai GM., et al, 2011), specifically: mu.L of gas-tight needle was used to take 5. Mu.L of headspace gas and analyzed for N by isotope ratio mass spectrometry (GC-IRMS) ₂ Delta of (2) ¹⁵ The analysis conditions for N were as follows: GS-Carbon Plot column (30 m. Times.0.32 mm. Times.3.0 μm); sample inlet 160 ℃, split ratio 30:1, the initial temperature of the column is 35 ℃ and kept for 6min, and then the temperature is increased to 140 ℃ at 20 ℃/min and kept for 2min; the carrier gas is He gas with the flow rate of 1mL/min. IRMS is nitrogen analysis mode, detecting ions m/z 28 and 29. The detection result confirms that the water-soluble polymer contains ¹⁵ N-labelled nitrogen, indicating that strain D14 can reduce nitrate to N ₂ O and further reduced to N ₂ Thereby realizing the complete conversion of nitrate into nitrogen for denitrification.

2. Ocean Huang Lishi bacteria (Lihuangia oceania) D14 has sulfur oxidation denitrification function under anaerobic and autotrophic conditions

The preparation method of the culture medium used in the experiment is as follows:

autotrophic sulfur oxidation denitrification culture medium components and preparation: sodium bicarbonate 2g/L, potassium nitrate 2g/L, sodium thiosulfate 2.5g/L, 30g/L sea salt solution (water as solvent, sea salt (Sigma, cat. No. S9883-1 KG) as solute), pH7.6-7.8, wherein sodium bicarbonate and sodium thiosulfate are sterilized by filtration with 0.22 μm filter membrane, and the other components are autoclaved at 121deg.C for 20min, and naturally cooled.

Sodium thiosulfate-free medium composition and preparation: sodium bicarbonate 2g/L, potassium nitrate 2g/L, and sea salt solution 30g/L, pH7.6-7.8. Wherein sodium bicarbonate is filtered and sterilized by a 0.22 μm filter membrane, and other components are sterilized at 121deg.C for 20min, and naturally cooled for use.

Experiments were repeated three times, 3 treatments were set up each time: experimental group, sodium thiosulfate-free control group and blank control group without bacteria.

1. Experimental group: strain D14 was activated on 2216E liquid medium, bacterial cells with an OD600nm of 0.6 (with 2216E liquid medium as a blank) were collected, washed three times with sterilized artificial seawater, inoculated into sterilized headspace bottles (100 mL in volume) containing 50mL of autotrophic sulfur oxidation denitrification medium in an inoculum size of 2%, and repeated three times. The headspace bottle is blown off by nitrogen, then is capped and is placed in an anaerobic box for anaerobic culture at 30 ℃. Shaking the culture once every 24h, taking headspace gas at 0 day, 14 day, 21 day, and 28 day, and measuring N according to the method of step one ₂ O。

2. Sodium thiosulfate-free control group: strain D14 was activated on 2216E liquid medium, bacterial cells with an OD600nm of 0.6 (with 2216E liquid medium as a blank) were collected, washed three times with sterilized artificial seawater, and inoculated into sterilized headspace bottles (100 mL in volume) containing 50mL of sodium thiosulfate-free medium in an inoculum size of 2%, and three replicates were set. The headspace bottle is blown off by nitrogen, then is capped and is placed in an anaerobic box for anaerobic culture at 30 ℃. Shaking the culture once every 24h, taking headspace gas at 0 day, 14 day, 21 day, and 28 day, and measuring N according to the method of step one ₂ O。

3. Blank group without bacteria: the sterilized headspace bottle (volume 100 mL) contained 50mL of autotrophic sulfur oxidation denitrification medium, and was repeated three times. The headspace bottle is blown off by nitrogen, then is capped and is placed in an anaerobic box for anaerobic culture at 30 ℃. Shaking the culture once every 24h, taking headspace gas at 0 day, 14 day, 21 day, and 28 day, and measuring N according to the method of step one ₂ O。

The results are shown in FIG. 5, N of the experimental group ₂ The O content increases with autotrophic culture time; when the culture medium does not contain sodium thiosulfate, the sodium thiosulfate-free control group cannot be detectedN is measured ₂ O, at the same time, since the strain D14 was not inoculated, N could not be detected in the blank group without inoculation ₂ O, indicating that the denitrification of strain D14 in the absence of organics requires sodium thiosulfate as an electron donor.

In addition, NO at 14 days of culture of the experimental group was measured by ion chromatography ₃ ^- -N、SO ₄ ^2- -S、S ₂ O ₃ ^2- -S, ion chromatography assay method: sample pretreatment: after the sample is centrifuged for 5min at 13000r/min, the supernatant is diluted 100-200 times by water and filtered by a microporous membrane of 0.22 μm. Chromatographic conditions: automatic online leaching liquid: EGCIII KOHRFIC ^TM The method comprises the steps of carrying out a first treatment on the surface of the Chromatographic column: dionex IonPac ^TM AS19-4um IC analytical column; column temperature: 30 ℃; the flow rate is 1mL/min; anion suppressor ADRS600: an automatic regeneration suppression mode, suppressing a current of 50mA; detecting by a conductivity detector; the sample injection amount was 25. Mu.L.

The results are shown in FIG. 6, NO after 14 days of culture ₃ ^- -N and S ₂ O ₃ ^2- The amount of S is slightly reduced, while SO ₄ ^2- The amount of S rises slightly. It is generally seen that strain D14 oxidizes thiosulfate to sulfate under anaerobic conditions without organics and reduces nitrate to N ₂ O and N ₂ 。

EXAMPLE 4 denitrification function of the microorganism Dayang Huang Lishi (Lihuangia oceani) D14 under organic anaerobic conditions

The media used in this experiment were as follows:

heterotrophic denitrification medium components and preparation: 2216E liquid culture medium is added with 2g/L potassium nitrate and 2.5g/L sodium thiosulfate, wherein the sodium thiosulfate is filtered and sterilized by a 0.22 mu m filter membrane, and other components are sterilized at 121 ℃ for 20min under high pressure and naturally cooled for use.

Heterotrophic denitrification culture medium without sodium thiosulfate comprises the following components: adding 2g/L potassium nitrate into 2216E liquid culture medium, autoclaving at 121deg.C for 20min, and naturally cooling.

Experiments were repeated three times, three treatments were set up each time: experimental group, sodium thiosulfate-free control group and blank control group without bacteria.

1. Experimental group: strain D14 was activated on 2216E liquid medium, somatic cells with an OD600nm of 0.6 were collected, washed three times with sterilized artificial seawater, and inoculated into a sterilized headspace bottle (volume 100 mL) containing 50mL of heterotrophic denitrification medium at an inoculum size of 2%. And (5) after nitrogen stripping, capping, and placing the mixture in an anaerobic box for anaerobic culture at 30 ℃. The culture was shaken once every 24 hours, and after 7 days of culture, samples were taken and the cell concentration of the cells was measured by a spectrophotometer.

2. Control group without sodium thiosulfate: strain D14 was activated on 2216E liquid medium, cells with OD600nm of 0.6 were collected, washed three times with sterilized artificial seawater, and inoculated into a headspace bottle (volume 100 mL) of sterilized heterotrophic denitrification medium containing 50mL of sodium thiosulfate-free medium at an inoculum size of 2%. And (5) after nitrogen stripping, capping, and placing the mixture in an anaerobic box for anaerobic culture at 30 ℃. The culture was shaken once every 24 hours, and after 7 days of culture, samples were taken and the cell concentration of the cells was measured by a spectrophotometer.

3. Blank group without bacteria: the sterilized headspace bottle (volume 100 mL) contained 50mL heterotrophic denitrification medium, and was repeated three times. And (5) after nitrogen stripping, capping, and placing the mixture in an anaerobic box for anaerobic culture at 30 ℃. The culture was shaken once every 24 hours, and after 7 days of culture, samples were taken and the cell concentration of the cells was measured by a spectrophotometer.

The results showed that the average value of OD600nm of the experimental group was 0.24.+ -. 0.016, the average value of OD600nm of the sodium thiosulfate-free control group was 0.12.+ -. 0.023, and the OD600nm of the non-inoculated blank control group was unchanged. It was shown that ocean Huang Lishi bacteria (Lihuanginia oceania) D14 can proliferate under organic anaerobic conditions.

At the same time, headspace gas NO was detected by Agilent 7890A/5975C GC/MS as described in example 3 ₂ -N and measuring NO by ion chromatography according to the method of example 3 II ₃ ^- -N、SO ₄ ^2- -S、S ₂ O ₃ ^2- -S. As shown in FIG. 7, thiosulfate was detected after 7 days of culture under organic anaerobic conditions (experimental group) (S ₂ O ₃ ^2- -S) concentration decrease, sulfate (SO) ₄ ^2- -S) an increase in concentration, indicating that thiosulfate is oxidized to sulfate; at the same time nitrate Nitrogen (NO) ₃ ^- -N) concentration decrease, and there is N ₂ O is generated, which indicates that denitrification process exists in the experimental group containing sodium thiosulfate; the results of the sodium thiosulfate-free control group are shown in FIG. 8, in which sulfate (SO ₄ ^2- -S) concentration unchanged, nitrate Nitrogen (NO) ₃ ^- -N) concentration is slightly reduced with a small amount of N ₂ O is produced but the amount of change is significantly less than in the presence of sodium thiosulfate; the blank group without bacteria was unchanged. Thus, it can be seen that the strain D14 can perform both the sulfur oxidation denitrification by using thiosulfate and the anaerobic denitrification in the presence of organic matters.

EXAMPLE 5 Sulfur Oxidation function of Brevibacterium Dacron (Lihuangia oceanii) D14 under heterotrophic aerobic conditions

Experiments were repeated three times, 3 treatments were set up each time: experimental and non-inoculated blank control groups.

1. Experimental group: strain D14 was activated in 2216E liquid medium, bacterial cells with an OD600nm of 0.6 were collected ((with 2216E liquid medium as a blank)), washed three times with sterilized artificial seawater, and inoculated into sterilized conical flasks (volume 250 mL) containing Na in advance at an inoculum size of 2% ₂ S ₂ O ₃ ·5H ₂ 100mL of 2216E liquid medium with O5.0 g/L (i.e. Na with a final concentration of 5.0g/L is added into the 2216E liquid medium) ₂ S ₂ O ₃ ·5H ₂ O), three repetitions are set. Culturing in shaking table at 180rpm at 30deg.C for 3 days, sampling to measure OD600nm value and SO ₄ ^2- -S、S ₂ O ₃ ^2- S concentration, the assay method was as in example 4.

2. Blank group without bacteria: the sterilized Erlenmeyer flask had a volume of 250mL and was filled with a solution containing Na ₂ S ₂ O ₃ ·5H ₂ 100mL of 2216E liquid medium with O5.0 g/L (i.e. Na with a final concentration of 5.0g/L is added into the 2216E liquid medium) ₂ S ₂ O ₃ ·5H ₂ O), placing the mixture on a shaking table for 180rpm and culturing at 30 ℃ for 3 days, and sampling and measuring the OD600nm value of the liquid in the conical flask.

As a result, the average value of OD600nm after 7 days of culture was 0.75.+ -. 0.015, indicating that ocean Huang Lishi bacteria (Lihuanginiaoceania) D14 were proliferated under heterotrophic aerobic conditions. SO (SO) ₄ ^2- -S、S ₂ O ₃ ^2- The S change is shown in FIG. 9, sodium thiosulfate in the culture broth of the experimental group strain D14 (S ₂ O ₃ ^2- -S) is significantly reduced, SO ₄ ^2- The S concentration increases, i.e. sulphate (SO ₄ ^2- S) whereby it is seen that strain D14 can undergo sulphur oxidation under heterotrophic aerobic conditions.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims (10)

1. Ocean Huang Lishi fungus, its characterized in that: the ocean Huang Lishi bacteria is Lihuanginia oceania, the strain number of the ocean Huang Lishi bacteria is D14, and the registration number of the ocean Huang Lishi bacteria in the common microorganism center of the China Committee for culture Collection of microorganisms is CGMCC No.1.13774.

2. A culture of the microorganism Huang Lishi strain according to claim 1, wherein the microorganism is the microorganism Huang Lishi strain according to claim 1.

3. The microbial inoculum is characterized in that: the microbial inoculum comprises the ocean Huang Lishi bacterium according to claim 1 or/and the ocean Huang Lishi bacterium metabolite according to claim 1 or/and the culture according to claim 2.

4. A microbial agent according to claim 3, wherein: the microbial inoculum can perform sulfur oxidization and oxidize low-valence sulfur compounds into sulfate.

5. A microbial agent according to claim 3, wherein: the microbial inoculum can perform denitrification, and nitrogen in the nitrate is reduced into nitrogen gas or nitrous oxide to be removed from the environment.

6. The nitrogen removing agent is characterized in that: the nitrogen removal agent contains the ocean Huang Lishi bacterium of claim 1 or/and the ocean Huang Lishi bacterium metabolite of claim 1 or/and the culture of claim 2 or/and the bacterial agent of claim 3.

7. Use of the ocean Huang Lishi bacterium of claim 1, the metabolite of the ocean Huang Lishi bacterium of claim 1 or the culture of claim 2 for the preparation of a nitrogen-removing bacterial agent in an environment.

8. Use of the ocean Huang Lishi bacterium of claim 1, the metabolite of the ocean Huang Lishi bacterium of claim 1 or the culture of claim 2 for the preparation of a sulfur oxide microbial agent.

9. The use of any of the ocean Huang Lishi bacteria of claim 1, the metabolite of the ocean Huang Lishi bacteria of claim 1, the culture of claim 2, the microbial inoculum of claim 3, the nitrogen scavenger of claim 6, as follows:

n1, oxidizing sulfur in the wastewater from low valence to high valence or removing nitrogen in the wastewater through denitrification;

n2, preparing a product for oxidizing sulfur in the wastewater from low valence to high valence or removing nitrogen in the wastewater through denitrification;

n3, oxidizing sulfur in the sediment from low valence to high valence or removing nitrogen in the sediment through denitrification;

n4, preparing a product for oxidizing sulfur in the sediment from low valence to high valence or removing nitrogen in the sediment through denitrification;

n5, oxidizing sulfur in the soil from low valence to high valence or removing nitrogen in the soil through denitrification;

n6, preparing a product for oxidizing sulfur in the soil from low valence to high valence or removing nitrogen in the soil through denitrification;

n7, remediating nitrogen and/or sulfur contaminated deposits;

n8, preparing a product for repairing nitrogen and/or sulfur contaminated sediment;

n9, remediating nitrogen and/or sulfur contaminated soil;

n10, preparing a product for repairing the nitrogen and/or sulfur polluted soil;

n11, preparing a polysaccharide product;

n12, preparing a polyhydroxyalkanoate product.

10. The use according to claim 9, characterized in that: the wastewater includes at least one of seafood culture wastewater and seafood treatment wastewater, and the sediment includes seafood culture water sediment.

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