A marine bacterium capable of degrading lignin and scavenging nitrite

Abstract

The present application provides a kind of marine bacteria capable of degrading lignin and removing nitrite, which is a deep-sea seabed sulfide source, and has the function of degrading lignin and removing nitrite.The strain of Halomonas sp.has a preservation number of CGMCC No.34690.The strain screened in the present application can grow and reproduce in seawater, can utilize guaiacol as a carbon source, and can remove nitrite.The strain of the present application has laccase activity, has laccase gene and nitrite reductase gene.The experimental results show that the cumulative removal rate of guaiacol after 2 days of culture is 56%, and the removal rate of nitrite after 4 days of culture is close to 70%, which has great application potential in the marine aquaculture industry.

Description

Technical Field

[0001] This invention belongs to the field of functional microbial screening and application technology, specifically relating to a marine bacterium capable of degrading lignin and removing nitrite. Background Technology

[0002] Increased human activity and the rapid development of aquaculture have led to the presence of various pollutants, such as lignin and nitrite, in aquaculture seawater, pond bottom sediment, and aquaculture wastewater. These pollutants mainly originate from feed additives and fecal residues, seriously endangering the safety and health of farmed animals and humans. How to quickly, efficiently, and safely reduce the levels of these pollutants has always been a hot topic of social concern.

[0003] Lignin is a high-molecular-weight aromatic compound with a stable structure, making it difficult to degrade. In nature, microorganisms participate in the efficient degradation of lignin; however, the reported strains mainly originate from terrestrial environments, and may struggle to grow, reproduce, and function effectively in saline environments, making the treatment of lignin in saline bodies difficult. Furthermore, marine bacteria can reduce nitrite to gaseous nitrogen (N2) or assimilate it to ammonia through denitrification, further synthesizing organic nitrogen, thereby purifying inorganic nitrogen from aquaculture water. However, since aquaculture environments often contain multiple environmental pollutants such as lignin and nitrite, the treatment effect of single-function strains is often limited. Summary of the Invention

[0004] The purpose of this invention is to provide a strain derived from deep-sea seabed sulfides that possesses the functions of degrading lignin and scavenging nitrite, and its applications. The strain screened in this invention can grow and reproduce in seawater, utilize guaiacol as a carbon source, and is capable of scavenging nitrite. The strain of this invention exhibits laccase activity and possesses both laccase and nitrite reductase genes. Experimental results show that after 2 days of cultivation, the guaiacol scavenging rate is 56%, and after 4 days of cultivation, the nitrate scavenging rate approaches 70%, demonstrating significant application potential in the marine aquaculture industry.

[0005] The lignin-degrading and nitrite-removing bacterial strain provided by this invention is a marine bacterium isolated from Atlantic seabed sulfides (W13.551247, S 22.981238), belonging to the genus *Halomonas*. Halomonas litopenaei The strain F50-3 (Halomonas sp. F50-3) has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34690. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is May 27, 2025.

[0006] The aforementioned genus *Haloxylon* Halomonas sp. F50-3, its 16S rRNA gene is as follows:

[0007]

[0008] The genus Halomonas Halomonas sp. F50-3, its laccase gene YfiH The amino acid sequence is as follows:

[0009] MSKELGDERPLLWLDQVHGASVQHGFSDTIPKADASVATTRDYACVILTADCLPVFFCDRGGERVALAHAGWRSLAGGVLEATVASLGTAPGELMAWLGPAISNAQFEVGPEVREAFCGVHPEAASAFEPSPYRLGHYMADLYKLARLRLERLGVSHVSGGHFCTACESRFYSHRRRDDGVTGRMASVIWLR (SEQ ID NO: 2).

[0010] Among its nitrogen cycle-related genes, nirD The amino acid sequence of the gene is as follows:

[0011] MTASSISRSLDWIPLCQRSDLVPFSGVAAWIDSAEGPAQVALFYLPGHSPELFAIDHHDPIAKANVLARGIVGDIQGEPVVASPLYKQHFRLKDGSCVEDDSVSLRTWAVAFDQDRVMIAP (SEQ ID NO: 3);

[0012] nirB The amino acid sequence of the gene is as follows:

[0013] MSSTKHNLAAHNAPDSDATDTRPTLVVVGNGMVGHHLVEQLVERDQHRRYHIVVFGEERHLAYDRVHLSEYFSGRDAQSLALSTADYYAEHGIELKLHQAVTAIDRDARRVHTDAGYLDFDRLVLATGSFPFVPPIPGNDREGCLVYRTLDDLDAIRAAAATANTGVVVGGGLLGLEAANALRGLDLDTAVVEFAPRLMPMQVDEQGGELLKEKIEGLGVQVLTERATREIVPGEASRLRMVFQDDKVLETDLIVFSAGIRPRDELARECGLEIGERGGVVIDDHCLTSDPAILAVGEVALYNNSIFGLVAPGYQMAKAAADTLCGGDSTFTGADMSTKLKLMGVDVGAIGDAHANRHPGAKQYRYFDQIQQVYRKLVVSGDGKRLLGAMLVGDNSAYDSLMQYYANGLDLPADPASLIVPSSEPAPALGPDALPDSATICSCHNVTKGAISVAIDAGCEDLGAVKGTTKASTGCGGCAALLKNVFDHELAARGMEVDTSLCEHFAHTRQQLFDIVRVAGIKTFSELMTRHGTILDDGAPSLGCDVCKPAVASILASCFNEPITDAAHIPLQDTNDTFMANMQKNGTFSVVPRIAGGEITPDKLIVLGQVGKKYGLYTKITGGQRIDLFGARLEDLPAIWEELIDAGFETGHAYGKSLRTVKSCVGSSWCRYGVQDSVGMALRLEHRYKGLRSPHKLKFAVSGCTRECAEAQSKDIGVIATEHGWNLYVCGNGGMRPRHAELFATDLDDQALIQAIDRLLMFYVRTADRLQRTSVWRENLEGGLDYLKAVVLDDSLGIGEELERQMQHVIDTYECEWAGALADPDKLKRFRSYVNDSRPDPDIIVTEERGQLRPA (SEQ ID NO:4).

[0014] Halomonas provided by the present invention Halomonas sp. F50-3 can be used for efficiently degrading lignin and nitrite in water bodies.

[0015] The present invention also provides an article for degrading lignin or nitrite, comprising the aforementioned *Halomonas* species. Halomonas Live bacteria of sp.F50-3.

[0016] The strains screened in this invention have the following advantages:

[0017] 1. The first strain obtained from a marine environment that degrades lignin and removes nitrite was named *Halomonas*. Halomonas sp.F50-3.

[0018] 2. The strain is easy to propagate and grows rapidly, reaching a stable growth period after approximately 84 hours of cultivation at 15 ℃.

[0019] 3. The strain has laccase (Lac) activity.

[0020] 4. The strain possesses a key enzyme gene for assimilating nitrite reduction, which can reduce nitrite to ammonia for organic nitrogen synthesis, thereby removing nitrite.

[0021] 5. The strain of the present invention is salt tolerant and can treat lignin or nitrite in saline bodies. Attached Figure Description

[0022] Figure 1 Image showing the clear zone produced by strain F50-3 on guaiacol medium;

[0023] Figure 2 Electron micrograph of strain F50-3 cells;

[0024] Figure 3 Phylogenetic tree of 16S rRNA from strain F50-3;

[0025] Figure 4 Growth curve of strain F50-3;

[0026] Figure 5 Standard curve of guaiacol concentration;

[0027] Figure 6 : Bar graph of guaiacol degradation by strain F50-3;

[0028] Figure 7 Sodium nitrite standard curve.

[0029] Figure 8 : Column chart of nitrite removal by strain F50-3. Detailed Implementation

[0030] The present invention is a Halomonas bacterium Halomonassp. F50-3 possesses salt tolerance and other properties, enabling it to efficiently degrade guaiacol and remove nitrite. The *Haloxymonas* strain described in this invention... Halomonas sp. F50-3 has the advantages of simple operation and low cost, and can be widely used in the treatment and purification of marine aquaculture environments, with broad application prospects.

[0031] The present invention will be further described below with reference to the embodiments.

[0032] Example 1. Halomonas Obtaining strain F50-3 and screening for guaiacol degradation activity

[0033] Marine bacteria, Halomonas strain F50-3 was isolated from Atlantic seabed sulfides (W13.551247, S 22.981238) collected during the 83rd Chinese Ocean Expedition in 2024. It was cultured on Zobel 2216E solid medium at 1 / 4 strength (approximately 3% salt concentration): 1.25 g peptone, 0.25 g yeast extract, 20 g agar powder, 1000 mL natural seawater, pH 7.0–7.2. The culture conditions were 15 °C for 7 days.

[0034] The strain was inoculated onto guaiacol solid medium and incubated at 15 °C. The degradation effect of the strain on guaiacol was determined by the size of the clear zone. The guaiacol solid medium consisted of: 1.25 g peptone, 0.25 g yeast extract, 0.1 g ammonium tartrate, 1.0 g guaiacol, 20 g agar powder, 1000 mL natural seawater, and a pH of 7.0–7.2. Figure 1 As shown, after 15 days of cultivation, on guaiacol solid medium, Halomonas The presence of a clear zone around the sp. F50-3 colony indicates that it has significant degradation activity against guaiacol.

[0035] Example 2. Halomonas Preservation of sp. F50-3 strain

[0036] The strains selected:

[0037] 1) Short-term preservation: The preservation method is the test tube slant method, using Zobel 2216E solid medium at 1 / 4 strength: 1.25 g peptone, 0.25 g yeast extract, 20 g agar powder, 1000 ml natural seawater, pH 7.0–7.2. After sterilization of the medium, the strain is streaked onto the test tube slant and incubated at 15 ℃ for 36 h, then stored at 4 ℃.

[0038] 2) Long-term preservation: The bacterial cells were placed in seawater with a final glycerol concentration of 10% and a final trehalose concentration of 5% and preserved in glycerol tubes at a temperature of -80 ℃.

[0039] Example 3. Halomonas Classification, identification, nomenclature, and preservation of strain F50-3

[0040] 1) Morphological identification: Strawberry strain F50-3 was streaked onto 1 / 4 strength Zobel 2216E solid medium and incubated at 15℃ for 3 days. The colonies were approximately 2 mm in diameter, white, round, and with neat edges. Electron micrographs are shown below. Figure 2 As shown, the cells are short rod-shaped and do not produce spores.

[0041] 2) 16S rRNA molecular identification: Using the strain's genomic DNA as a template, the 16S rRNA gene sequence was amplified by PCR using universal primers 27F and 1492R. The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0042] The specific steps are as follows: Add 20–30 μL of ddH2O to a sterile 2 mL centrifuge tube. Pick a purified bacterial single colony, mix well, and heat in boiling water for 10 min to obtain a DNA template. Place on ice for later use. Using bacterial genomic DNA as a template, amplify the 16S rRNA gene fragment using the universal primers 27F (5'-AGAGTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). PCR amplification system: 1 μL TransStartTaq DNA polymerase (2.5 U, TransGold, Beijing, China), 2 μL DNA, 1 μL primer (0.2 μmol / L final concentration), 5 μL buffer, 4 μL dNTPs, and add ddH2O to a final volume of 50 μL. PCR reaction conditions: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 90 s, 30 cycles; final extension at 72 °C for 5 min. 2 μL of the PCR product was analyzed by 1% agarose gel electrophoresis and then sequenced.

[0043] The sequencing results were submitted to EzTaxon for comparison to determine the taxonomic position of the strain. The 16S rRNA gene sequence of strain F50-3 was compared with... Halomonas litopenaei The closest kinship is 99.85%, which is preliminarily determined to be... Halomonas sp., named Halomonassp. F50-3. The 16S rRNA gene sequence of strain F50-3 has been submitted to the NCBI database, sequence number (PV668911). A phylogenetic tree was constructed using MEGA 12.0 software with neighbor-joining to further confirm the strain's phylogenetic structure. Halomonas sp. F50-3 and Halomonas litopenaei They cluster on a single evolutionary branch and are most closely related in phylogeny. Figure 3 ).

[0044] The accession number for strain F50-3 is CGMCC No. 34690; the depository is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is June 9, 2025.

[0045] Example 4: Halomonas Growth curve of sp. F50-3

[0046] Bacterial cells were picked from the preserved slant and transferred to 50 mL of 1 / 4 strength Zobel 2216E seed culture medium and cultured with shaking at 15 °C for 48 h. At a 5% inoculum size, the cells were transferred to 200 mL of 1 / 4 strength Zobel 2216E liquid culture medium and cultured with shaking at 15 °C. Samples were taken at 12–24 h intervals, and OD values ​​were measured using a UV-Vis spectrophotometer. 600 The values ​​were calculated with uninoculated liquid culture medium as a blank control. OD values ​​were plotted on the x-axis as time. 600 The value is the ordinate, plot. Halomonas growth curve of sp. F50-3 ( Figure 4 The strain is easy to reproduce and grows rapidly, reaching a stable growth period in about 84 hours.

[0047] Example 5. Halomonas Application of sp. F50-3 in the degradation of guaiacol lignin

[0048] (1) Preparation of standard absorbance curve of guaiacol

[0049] Accurately weigh 1.00 g of guaiacol, dissolve it in 1 / 4 strength Zobel 2216E liquid medium, and bring the volume to 1000 mL. Mix well. Take 0, 2.0, 4.0, 6.0, 8.0, and 10 mL of the guaiacol solution (1 mg / mL), respectively, and bring the volume to 10 mL with 1 / 4 strength Zobel 2216E liquid medium, resulting in final concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 g / L. Measure the OD using a UV-Vis spectrophotometer. 290Values ​​were obtained by plotting the standard absorbance curve of guaiacol. y =0.9057 x +0.0148, R 2 The linear relationship is 0.9804. Figure 5 ).

[0050] (2) Strains Halomonas Application of sp. F50-3 in the degradation of guaiacol

[0051] Step 1: Preparation of guaiacol liquid culture medium at different concentrations

[0052] 1.25 g peptone, 0.25 g yeast extract, 0.1 g ammonium tartrate, and 1000 mL natural seawater (pH 7.0–7.2) were dispensed into five 100 mL Erlenmeyer flasks (250 mL each). 0, 0.02, 0.04, 0.06, 0.08, and 0.1 g guaiacol were added respectively to prepare culture solutions with guaiacol concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 g / L.

[0053] Step 2: Seed culture preparation and cultivation

[0054] from Halomonas Bacterial cells from sp. F50-3 slant were picked and placed in 100 mL of 1 / 4 strength Zobel 2216E liquid medium and cultured at 15 °C for 48 h to obtain the seed culture. At a 5% inoculum size, the culture was transferred to different concentrations of guaiacol liquid medium and cultured at 15 °C and 150 rpm / min. After 24 h intervals, the bacterial culture was collected, filtered through a 0.22 µm membrane, and the supernatant was used to determine the OD. 290 value.

[0055] Step 3: Determination of guaiacol content

[0056] The absorbance values ​​were input into the regression equation to calculate the guaiacol degradation rate. (Strain) Halomonas sp. F50-3 achieved a cumulative degradation rate of 56% for guaiacol on the second day of cultivation. Figure 6 ).

[0057] (3) Determination of laccase (Lac) activity

[0058] Lac activity was determined using the ABTS method with a laccase kit (Suzhou Grace Biotechnology Co., Ltd.). F50-3 strain culture was collected every 1 day and incubated at 4 ℃ and 10000× g Centrifuge for 20 min, collect the bacterial cells, add 1 mL of extraction solution, and sonicate the cells in an ice bath to disrupt them. Then, take the supernatant enzyme solution to determine the enzyme activity.

[0059] Mix 30 μL of 1 mol / L ABTS, 140 μL of 100 mol / L sodium acetate buffer (pH=5), and 30 μL of enzyme solution in an ELISA plate and carry out the reaction at 15 ℃. Record the OD of the reaction solution within 1 min using an ELISA reader. 410 Value. Uninoculated culture medium serves as a blank control. Enzyme activity definition: The amount of enzyme required to catalyze the formation of product from 1 μmol of substrate per minute is defined as one enzyme activity unit (U), expressed in U / L.

[0060] strains Halomonas The Lac enzyme activity produced on day 2 of culture of sp. F50-3 reached 28.0 U / L.

[0061] Example 6. Halomonas Application of sp. F50-3 in nitrite reduction

[0062] (1) Preparation of the standard absorbance curve of nitrite

[0063] Step 1: Preparation of standard solution

[0064] Accurately weigh 1.00 g of sodium nitrite, dissolve it in distilled water, and bring the volume to 1000 mL. Mix well. The nitrite concentration is 1.0 mg / mL. Take 0.25, 0.5, 1.0, 1.5, and 2.0 mL of the sodium nitrite solution (1 mg / mL), respectively, and bring the volume to 1000 mL using Zobel 2216E liquid medium with a strength of 1 / 4, so that the final concentrations are 0.25, 0.5, 1.0, 1.5, and 2.0 mg / L.

[0065] Step 2: Prepare Griess A and Griess B reagents for nitrite content detection.

[0066] Griess A reagent (10 g / L p-aminobenzenesulfonic acid solution): Accurately weigh 0.1 g of sulfanilamide, dissolve in 20% hydrochloric acid and dilute to 100 mL, mix well, and store in a brown bottle protected from light. Griess B reagent (1 g / L naphthylethylenediamine hydrochloride solution): Accurately weigh 0.1 g of naphthylethylenediamine hydrochloride, dissolve in distilled water and dilute to 100 mL, mix well, and store in a brown bottle protected from light.

[0067] Step 3: Nitrite content determination

[0068] In the test tube, add 525 μL of standard working solution of different concentrations, 175 μL each of Griess A reagent and Griess B reagent sequentially; after mixing, react accurately at room temperature (25 ℃) for 15 min, and immediately measure the OD. 540Read the absorbance value A, using the standard solution with a final sodium nitrite concentration of 0 as the blank. △A = Adetermined - Ablank. Plot the nitrite standard curve as follows: Figure 7 As shown. Obtain y =0.0379 x + 0.0013, R 2 A linear relationship of 0.997.

[0069] (2) Halomonas Degradation of nitrite at different concentrations by sp. F50-3

[0070] Step 1: Preparation of liquid culture medium with different concentrations of nitrite

[0071] Prepare 1 / 4 strength Zobel 2216E liquid culture medium and dispense it into 5 Erlenmeyer flasks (250 mL each) with volumes of 80 mL, 85 mL, 90 mL, 95 mL and 100 mL.

[0072] Weigh 1.00 g of sodium nitrite and dissolve it completely in 100 mL of distilled water (nitrite concentration of 10 g / L) to prepare sodium nitrite storage solution.

[0073] Pipette 20 mL of the stock solution into 80 mL of Zobel 2216E liquid medium at 1 / 4 strength, with a nitrite concentration of 2.0 g / L;

[0074] Pipette 15 mL of the stock solution into 85 mL of Zobel 2216E liquid medium at 1 / 4 strength, with a nitrite concentration of 1.5 g / L;

[0075] Pipette 10 mL of the stock solution into 90 mL of Zobel 2216E liquid medium at 1 / 4 strength, with a nitrite concentration of 1.0 g / L;

[0076] Pipette 5 mL of the stock solution into 95 mL of Zobel 2216E liquid medium at 1 / 4 strength, with a nitrite concentration of 0.5 g / L.

[0077] Pipette 2.5 mL of the stock solution into 97.5 mL of Zobel 2216E liquid medium at 1 / 4 strength, with a nitrite concentration of 0.25 g / L.

[0078] Step 2: Seed culture preparation and cultivation

[0079] from HalomonasBacterial cells from sp. F50-3 slant were picked and placed in 100 mL of 1 / 4 strength Zobel 2216E liquid medium and cultured at 15 ℃ for 48 h to obtain the seed culture. At a 5% inoculum, the culture was transferred to liquid medium of different concentrations of nitrite and cultured at 15 ℃ and 60 rpm / min. After 4 days, the bacterial culture was collected, filtered through a 0.22 µm membrane, and the supernatant was used for analysis.

[0080] Step 3: Nitrite content determination

[0081] In the test tube, add 525 μL of supernatant, 175 μL each of Griess A and Griess B reagents sequentially; mix well, and react accurately at room temperature (25 °C) for 15 min. Immediately measure the OD value. 540 Read the absorbance value A, using the standard solution with a final sodium nitrite concentration of 0 as the blank. △A = Adetermined - Ablank. Calculate the corresponding nitrite amount based on the nitrite standard curve, plotting nitrite concentration on the x-axis and nitrite clearance rate on the y-axis, and calculate... Halomonas The nitrite scavenging effect of sp. F50-3.

[0082] Experimental results show that ( Figure 8 Under the above experimental conditions, when the nitrite content is 0.25 g / L, Halomonas strain F50-3 was able to remove nearly 70% of nitrite within 4 days.

Claims

1. A strain of the genus *Halomonas*, characterized in that, The strain is Halomonaslitopenaei. The strain has the CGMCC No. 34690.

2. The use of the *Haloxylon* strain according to claim 1 in the degradation of lignin or nitrite.

3. A product for degrading lignin or nitrite, characterized in that, The product contains live bacteria of the *Haloxylon* strain as described in claim 1.

4. A method for degrading lignin or nitrite, characterized in that, The method involves using the *Haloxylon* strain described in claim 1 for degradation.

5. The method as described in claim 4, characterized in that, The method described is to degrade lignin or nitrite in brine.

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