New strain A39 of sodium nematode and application of new strain A39 in removal of heavy metals in high-salinity wastewater

Abstract

The invention provides a new strain A39 of nematode and application of extracellular crude polysaccharide produced by the new strain A39 in removal of heavy metals in high-salinity wastewater, and belongs to the technical field of bioengineering, the strain A39 is classified and named as Natinema sp. And is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC 35089, and the preservation date is July 3, 2025. The strain belongs to a potential new species of sodium nematodosis and has a remarkable removal effect on heavy metals such as Pb < 2 + >, Cu < 2 + >, Cr < 6 + >, Cd < 2 + > and Zn < 2 + > under the condition of 15% NaCl, and the removal rates of the produced extracellular crude polysaccharide on Pb < 2 + > and Cr < 6 + > are 71.07% and 69.43% respectively when the extracellular crude polysaccharide is treated for 72 hours. The strain A39 has a wide growth salinity range which is 12-35% NaCl, and the produced exopolysaccharide has an acidic characteristic and an amorphous structure and keeps stable activity in a pH range of 3-8. The invention provides a new biological material for high-salt industrial wastewater treatment and environmental restoration, and the thalli and exopolysaccharides have wide industrial application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to a new strain of genus Natrialba A39 and application of the strain in heavy metal removal in high-salt wastewater. BACKGROUND

[0002] Among the many proven water toxins / pollutants, heavy metal pollution is difficult to repair due to its biological accumulation and stubbornness, causing great harm to human health and the environment [Bolisetty S, Peydayesh M, Mezzenga R. Sustainable technologies for water purifcation from heavy metals: review and analysis. Chem Soc Rev, 2019, 48:463–487. ]. In the treatment of industrial wastewater containing heavy metals, traditional physical and chemical treatment technologies have many limitations due to low efficiency, high economic cost, and secondary pollution, etc. [Giovanella P, Vieira GAL, Ramos Otero IV, et al. Metal and organic pollutants bioremediation by extremophile microorganisms. J Hazard Mater, 2020, 382: 121024.]. Bioremediation has been proven to be an effective and low-cost method for removing heavy metals due to its green and sustainability [Pham VHT, Kim J, Chang S, et al. Bacterial biosorbents, an efcient heavy metals green clean-up strategy: prospects, challenges, and opportunities. Microorganisms, 2022, 10:1-16.].The industrial wastewater from textile, tanning and brewing industries usually has high salt concentration, and also contains heavy metals such as cadmium, nickel, zinc, hexavalent chromium, copper, lead and cobalt [Mubashar M, Naveed M, Mustafa A, et al. Experimental investigation of Chlorella vulgaris and Enterobacter sp. MN17 for decolorization and removal of heavy metals from textile wastewater. Water, 2020, 12(11): 3034.][Ali Z, Malik R N, Shinwari Z K, et al. Enrichment, risk assessment, and statistical apportionment of heavy metals in tannery-affected areas. International Journal of Environmental Science and Technology, 2015, 12(2): 537-550.]. In high-salt wastewater, ordinary heavy metal remediation microorganisms are difficult to effectively play a role and / or grow, which makes the treatment of such wastewater extremely thorny. In recent years, halophilic and alkaliphilic microorganisms have been used as an effective tool for heavy metal pollution control and resource recovery, but most of the related researches focus on halophilic bacteria [Varshney S, Bhattacharya A, Gupta A. Halo-alkaliphilic microbes as an effective tool for heavy metal pollution abatement and resource recovery: challenges and future prospects. 3 Biotech, 2023, 13(12): 400.].

[0003] Salt lakes are natural habitats of heavy metal enrichment. As an important biological group in salt environments, halophilic archaea have important applications in heavy metal removal and bioremediation of harmful substances [Martínez-Espinosa R M. Halophilic archaea as tools for bioremediation technologies. Applied Microbiology and Biotechnology, 2024, 108(1): 401.]. Based on the characteristics of screening and genomic analysis, halophilic archaea have good performance in screening and tolerating heavy metals [Shi Y, Huang J, Zeng G, et al. Exploiting extracellular polymeric substances (EPS) controlling strategies for performance enhancement of biological wastewater treatments: an overview [J]. Chemosphere, 2017, 180: 396-411.], and can be used to remove Cd 2+ , Cu 2+ , Ag +and have good removal effect [Saez-Zamacona I, Grindlay G, Martínez-Espinosa R M. Evaluation of Haloferax mediterranei strain R4 capabilities for cadmium removal from brines. Marine Drugs, 2023, 21(2): 72.][Llorca M G, Martínez-Espinosa R M. Assessment of Haloferax mediterranei genome in search of copper-molecular machinery with potential applications for bioremediation. Frontiers in Microbiology, 2022, 13: 895296.][Buda D M, Szekeres E, Tudoran L B, et al. Genome-wide transcriptional response to silver stress in extremely halophilic archaeon Haloferax alexandrinus DSM 27206 T. BMC microbiology, 2023, 23(1):381.] The halophilic archaea evolved unique detoxification mechanisms to adapt to high concentrations of heavy metals environment [Straková D, Sánchez-Porro C, de la Haba R R, et al. Strategies of Environmental Adaptation in the Haloarchaeal Genera Haloarcula and Natrinema. Microorganisms, 2025, 13(4):761.][Völkel S, Hein S, Benker N, et al. How to cope with heavy metal ions: cellular and proteome-level stress response to divalent copper and nickel in Halobacterium salinarum R1 planktonic and biofilm cells. Frontiers in Microbiology, 2020, 10: 3056.] Studies have shown that the extracellular polymeric substances secreted by microorganisms play an important role in heavy metal removal [Pagliaccia B, Carretti E, Severi M, et al. Heavy metal biosorption by Extracellular Polymeric Substances (EPS) recovered from anammox granular sludge. Journal of Hazardous Materials, 2022, 424: 126661.][Shi Y, Huang J, Zeng G, et al. Exploiting extracellular polymeric substances (EPS) controlling strategies for performance enhancement of biological wastewater treatments: an overview[J]. Chemosphere, 2017, 180: 396-411.]Exopolymers produced by halophilic archaea also have important functions in heavy metal removal [Voica D M, Bartha L, Banciu H L, et al. Heavy metal resistance in halophilic Bacteria and Archaea. FEMS Microbiology Letters, 2016, 363(14): fnw146.][Chouchane H, Najjari A, Neifar M, et al. Unravelling the characteristics of a heteropolysaccharide-protein from an Haloarchaeal strain with flocculation effectiveness in heavy metals and dyes removal. Environmental Technology, 2020.]. The composition of high-salinity wastewater is complex, and further screening of functional strains and functional biological materials is urgently needed. SUMMARY

[0004] In order to solve the problems in the prior art, the purpose of the present application is to provide a new strain of the genus Natrinema and its application in high-salinity organic wastewater.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is: In a first aspect, a new strain of the genus Natrinema A39 is provided, which is named Natrinema sp. and has been preserved in the China General Microbiological Culture Collection Center on July 3, 2025, with the preservation number CGMCC 35089.

[0006] In a second aspect, a microbial inoculant for treating high-salinity organic wastewater is provided, which contains one of the cell bodies of the new strain of the genus Natrinema A39, the exopolysaccharide produced by the new strain of the genus Natrinema A39, or the fermentation broth, and the fermentation broth is a mixture containing the cell bodies of the new strain of the genus Natrinema A39 and the exopolysaccharide produced by the new strain of the genus Natrinema A39.

[0007] In a third aspect, the new strain of the genus Natrinema A39 or the microbial inoculant is applied in the removal of heavy metals in high-salinity organic wastewater.

[0008] Further, the high-salinity organic wastewater is treated with the microbial inoculant containing the exopolysaccharide produced by the new strain of the genus Natrinema A39, the treatment time is 48 h to 72 h, the pH is 3 to 8, and the concentration of the exopolysaccharide produced by the new strain A39 is 3 mg / mL.

[0009] Further, the high-salt organic wastewater contains at least one of copper ions, lead ions, chromium ions, zinc ions or cadmium ions.

[0010] According to the above technical solution, the application has the following beneficial effects: The application screens and separates a halophilic archaea new strain A39 of the genus Natinema, which is identified as a new species classification unit of the genus Natinema through multi-phase classification and identification, can grow well in a high-salt concentration of 12% to 35% NaCl, a wide range of pH 6.5 to 8.5 and a temperature range of 20 to 45 DEG C, has strong tolerance to heavy metals, and has a Pb 2+ tolerance concentration of 400 mg / L, a Cu 2+ tolerance concentration of 200 mg / L. The strain, the microbial agent composed of the extracellular crude polysaccharide and the fermentation liquor thereof have excellent heavy metal removal effects under high-salt conditions. The cell body, the fermentation liquor have Pb 6+ , Cu 2+ and Cr 2+ removal rates of 68.11%, 66.71% and 61.08% respectively, the extracellular crude polysaccharide has the best Pb 2+ and Cr 6+ removal effects at a concentration of 3 mg / mL and a treatment time of 72 h, has a Pb 2+ removal rate of 71.07% and a Cr 6+ removal rate of 69.43%, and also has good removal effects on Cu 2+ , Cd 2+ and Zn 2+ . The extracellular crude polysaccharide is an acidic polysaccharide, has irregular holes and a network structure and amorphous characteristics, is stable in heavy metal removal performance in high-salt / low-salt environments, and also has excellent flocculation characteristics; under high-salt conditions, the removal effect on heavy metals is significant, and the flocculation rate is high under conditions of 0% to 20% NaCl and pH 3 to 8, which is better than that of traditional chemical flocculants PAM and polysaccharide substances such as lentinan and xanthan gum. In summary, the strain A39 and the microbial agent thereof can adapt to the high-salt environment of high-salt wastewater, have high-efficiency heavy metal removal capacity, the extracellular crude polysaccharide is easy to store and transport and is convenient to use, and has a broad industrial application prospect in the treatment of high-salt organic wastewater, especially high-salt wastewater containing copper, lead, chromium, zinc and cadmium.

[0011] Strain preservation: The new strain A39 of the genus Natinema is named Natinema sp., has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC 35089 and the preservation date is July 3, 2025. DETAILED DESCRIPTION

[0012] Figure 1 Phylogenetic tree of Halococci A39 and close species based on 16S rRNA gene sequences; Figure 2 Phylogenetic N-J tree of Halococci A39 and close species based on rpoB' gene sequences; Figure 3 Circular map of Halococci A39 genome; Figure 4 COG functional classification map of Halococci A39 genome; Figure 5 Plate and microscopic morphology of Halococci A39; Figure 6 Polar lipids of Halococci A39; Figure 7 Heavy metal adsorption effect detection of different material samples of Halococci A39; Figure 8 Full wavelength ultraviolet spectrum of extracellular polymers produced by Halococci A39; Figure 9 Infrared spectrum of extracellular polymers produced by Halococci A39; Figure 10 Standard curve of glucose standard curve for determining the total sugar content in the extracellular crude polysaccharide of Halococci A39; Figure 11 Standard curve for determining the protein content in the extracellular crude polysaccharide of Halococci A39; Figure 12 Scanning electron microscope morphology map of extracellular crude polysaccharide produced by Halococci A39; Figure 13 X-ray diffraction spectrum of extracellular crude polysaccharide produced by Halococci A39; Figure 14 Heavy metal removal effect of different concentrations of extracellular crude polysaccharide produced by Halococci A39 under 15% NaCl conditions; Figure 15 Heavy metal removal effect of different concentrations of extracellular crude polysaccharide produced by Halococci A39 under 0% NaCl conditions; Figure 16 Heavy metal removal effect of extracellular crude polysaccharide produced by Halococci A39 under different treatment times; Figure 17 Flocculation effect of extracellular crude polysaccharide produced by Halococci A39; Figure 18 Flocculation effect of extracellular crude polysaccharide produced by Halococci A39 under different salinity; Figure 19 Flocculation effect of extracellular crude polysaccharide produced by Halococci A39 under different pH. DETAILED DESCRIPTION

[0013] The technical solutions of the present application will be clearly and completely described below in combination with the embodiments of the present application. If not specifically stated, the reagents or materials used in the following embodiments are all commercially available, and the operations used are all conventional technical means.

[0014] In order to effectively deal with the problem of heavy metal pollution under high salt conditions, the present study takes the sediment samples of Aiding Lake in Xinjiang as the research object, explores new groups of halophilic archaea, and studies the physiological and biochemical characteristics of potential new strains and the heavy metal removal characteristics of different biological samples, so as to explore the application in heavy metal removal of high-salt wastewater and lay a foundation for further mining and utilizing potential new halophilic archaea resources and exploring the development of heavy metal removal materials for high-salt organic wastewater.

[0015] I. Isolation of halophilic archaea A39 Take 5 g of salt lake sample and put it into a 100 mL 20% salt water flask and shake it gently for 4 h. Gradient dilution method is used to make the final dilution degree 10 -3 , take 100 μL and evenly coat on the isolation medium (modified Gao's medium: soluble starch 20 g / L, potassium phosphate dibasic 0.5 g / L, magnesium sulfate 0.5 g / L, potassium nitrate 1 g / L, sodium chloride 200 g / L, trace salt 1 mL / L, ultrapure water 1000 mL, agar 20 g / L, pH 7.2. Trace salt: MnCl2·4H2O 0.03 g, ZnSO4·7H2O 0.1 g, FeCl3 0.25 g, CuSO4·5H2O 0.01 g, ultrapure water 100 mL), and culture in a 37℃ incubator for 4 weeks. Remove the morphological duplicates on the same medium, describe and record the morphology and number of colonies. Inoculate the isolated strain on ISP4 medium (soluble starch 10 g / L, potassium phosphate dibasic 1 g / L, ammonium sulfate 2 g / L, calcium carbonate 2 g / L, magnesium sulfate heptahydrate 1 g / L, trace salt 1 mL / L, sodium chloride 200 g / L, ultrapure water 1000 mL, agar 20 g / L), and then transfer the slowly growing strain to Gao's No. 1 medium until a single colony is purified.

[0016] II. Determination method steps of halophilic archaea A39 1. Polyphase classification and identification of halophilic archaea A39 (1) Phylogenetic analysis of conserved genes of halophilic archaea A39 A small amount of bacteria was picked up by Chelex method, 50 μL Chelex-100 was added, and then lysis was performed in a PCR instrument (conditions 99℃ 15 min; 16℃ 10 min), and 12000 rpm centrifugation for 3 min was performed before storage for PCR amplification. Archaeal 16S rRNA gene universal primers: forward primer P1, 5'-ATTCCGGTTGATCCTGCCGGA-3'; reverse primer P2, 5'-AGGAGGTGATCCAGCCGCAG-3'. Halophilic archaeal rpoB´ gene sequencing primers: forward primer HrpoA 153R: 5'-GGGTCCATCAGCCCCATGTC-3'; reverse primer HrpoB2 1420F: 5'-TGTGGGCTNGTGAAGAACTT-3'. The amplification conditions were: 95℃ pre-denaturation for 4 min; 95℃ denaturation for 1 min; 55℃ annealing for 30 s; 72℃ extension for 5 min; 32 cycles. After observing the band size and sample purity by gel electrophoresis, it was sent to Shanghai Shengong Company for sequencing to determine whether it was a halophilic archaeon, and the measured sequence was spliced and treated to remove the carrier. CLUSTALX software was used for comparison and comparison on EzBiCloud (https: / / www.ezbiocloud.net / ). The classification standard for potential new species was that the 16S rRNA sequence similarity was less than 98.65%. MEGA7.0 software was used to construct phylogenetic NJ (Neighbor-Joining) trees based on 16S rRNA and rpoB´ gene sequences, respectively. The position characteristics of halophilic archaeon A39 in the phylogenetic tree were analyzed.

[0017] (2) Genome sequencing and analysis of halophilic archaeon A39 The whole genome sequencing of halophilic archaea used a double-end sequencing method. In Shengong Biological Engineering Co., Ltd. (Shanghai, China), Illumina NovaSeq PE150 was used to construct the library. The reads of each data set were filtered. High-quality double-end reads were assembled using SOAPdenovo v2.04 (http: / / soap.genomics.org.cn / ). The rRNA and tRNA contained in the genome were calculated using rRNAmmerv1.2 and tRNAscan-SE v1.3.1 software. The predicted coding sequences were translated into amino acid sequences and used as queries for BLAST comparison with GenBank, Swissprot, KEGG, COG and GO databases, respectively. The online intergenomic distance calculator (GGDC, http: / / ggdc.dsmz.de / ) was used for bioinformatics digital DNA-DNA hybridization (dDDH).

[0018] (3) Morphology and physiological and biochemical detection of halophilic archaeon A39 Inoculate the strain on modified Gao's medium, cultivate at 37°C for 2-3 weeks, and observe the strain plate morphology. Take fresh bacteria, use 15% NaCl solution to prepare a bacterial suspension with OD600=0.8-1.0, add glutaraldehyde solution to a final concentration of 0.5%, and place it in a 4°C refrigerator for 12 hours of fixation treatment. Take 30 microliters and evenly spread it on a cover glass. After natural air drying, dehydrate it in 40%, 60%, 90%, and 100% ethanol for 1 minute, and then naturally air dry. Observe the appropriate field under an optical microscope, cut the corresponding position glass, and paste it on a conductive tape. Spray gold for 120 seconds, and observe the strain microstructure under a scanning electron microscope. Culture the strain on modified Gao's medium and observe the physiological and biochemical experiments. Explore the physiological and biochemical indicators such as the required NaCl concentration range and temperature range, Mg 2+ concentration range and pH range, carbon and nitrogen source utilization, enzyme activity characteristics, antibiotic sensitivity characteristics, etc.

[0019] (4) Detection of polar ester class of halophilic archaea A39 Methanol method was used to extract the polar lipids of halophilic archaea. After spotting and layering, evenly spray the color developing agent (concentrated sulfuric acid: ethanol = 1:2) on the TLC Silica gel 60 plate (Merck), dry it, and place it in a 150°C oven for color development for 3 minutes until yellow or reddish-brown spots appear. Phospholipids are yellow, and glycolipids are reddish-brown. Scan the image with a scanner and record the experimental results.

[0020] 2. Heavy metal tolerance of halophilic archaea A39 and heavy metal removal characteristics of different biological materials (1) Heavy metal tolerance plate detection of halophilic archaea A39 Prepare modified Gao's liquid medium, add Cr 6+ , Zn 2+ , Cu 2+ , Pb 2+ , Cd 2+ to a final concentration of 0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, and 400 mg / L, respectively, inoculate the bacterial liquid of halophilic archaea A39 (OD600=0.8-1.0), and cultivate at 37°C for 2-3 weeks. Observe and measure the difference in OD value before and after cultivation to determine the growth tolerance of halophilic archaea A39 under the corresponding heavy metal concentration.

[0021] (2) Detection of heavy metal removal of different biological materials of halophilic archaea A39 A certain amount of heavy metal was weighed, dissolved in ultrapure water to 1 L, and prepared into a 1 g / L heavy metal ion solution. 5 mL of the 1 g / L heavy metal ion solution was taken and diluted to 100 mL to prepare a 50 mg / L heavy metal ion standard solution. First dilution: 60 μL of the 50 mg / L copper ion standard solution was precisely taken and diluted to 12 mL with ultrapure water. Second dilution: 20 μL, 40 μL, 200 μL, and 400 μL of the first dilution were taken and diluted to 10 mL with ultrapure water to obtain standard solutions with concentrations of 0.5, 1, 5, and 10 μg / L. The heavy metal pollutant sample of the high-salt organic wastewater was filtered through a 0.22 μm filter head to remove large particulate matter in the solution, and the concentration of heavy metal ions was detected by inductively coupled plasma (ICP-MS) to draw a heavy metal ion standard curve. 10 mL of the 50 mg / L heavy metal ion standard solution was taken with a pipette, and the cell body, supernatant, fermentation broth, and crude polysaccharide solution of the halophilic archaeon A39 were added to the solution to investigate the heavy metal removal effect of the strain A39 under high-salt (15%) conditions at concentrations of 1 mg / mL, 2 mg / mL, and 3 mg / mL. After 48 h of treatment, first and second dilutions were performed using the above method, and the concentration of heavy metal ions was detected by ICP-MS.

[0022] 3. Morphology and characteristics of the extracellular polymers produced by the halophilic archaeon A39 (1) Fermentation of the halophilic archaeon and extraction of extracellular polymers The screened strain was inoculated into modified Gao's liquid medium and fermented at 37°C and 140 rpm for 21 d. The obtained fermentation broth was subjected to the following extraction process: the fermentation broth was centrifuged (8000 rpm, 10 min), and the cell body was collected and dried for weighing; the upper liquid was collected, diluted by equal volume, and filtered through a 0.45 μm filter membrane; rotary evaporation was performed for concentration to the original volume; dialysis was performed for 48 h using a dialysis bag (MD44-8000) to remove salt and impurities in the fermentation broth; 10% trichloroacetic acid was added to remove protein, and the supernatant was obtained by centrifugation at 7000 rpm; the precipitate was collected after alcohol precipitation (3 times the volume of pre-cooled anhydrous ethanol, 4°C, 4 h) and re-dissolved in pure water to the original volume, and the trichloroacetic acid protein removal + alcohol precipitation step was repeated 2-3 times; the precipitate was dried in a 55°C oven after centrifugation at 7000 rpm for 10 min; the yield of extracellular polymers was calculated, and the yield of extracellular polymers per 1 g of cell body was converted.

[0023] (2) Detection of the extracellular polymers produced by the halophilic archaeon A39 by full-wavelength ultraviolet scanning The extracted extracellular polymer was scanned at full wavelength, and the full wavelength scanning range of the ultraviolet spectrum of the polymer was usually set between 190-400 nm. Whether the polymer was extracellular polysaccharide was determined according to the characteristic absorption peak shape of the polymer in the ultraviolet spectrum, and whether the polymer contained nucleic acid and protein was determined according to the special absorption peak wavelength of nucleic acid and protein.

[0024] (3) Determination of extracellular polymer produced by halophilic archaea A39 by Fourier infrared spectroscopy Take 1.0 mg of dry sample of extracellular polymer, add 40.0 mg of dry KBr powder, mix thoroughly and grind, and use a tablet press to make transparent thin slices. Use a Fourier infrared spectrometer to scan the infrared spectrum in the range of 400-4000 cm -1 , with a resolution of 4 cm -1 . Infrared spectroscopy is used for preliminary characterization of polymer structure, and infrared spectrum is drawn to analyze characteristic groups and determine the properties of extracellular polymer based on the results of full wavelength ultraviolet analysis.

[0025] (4) Detection of sugar content of extracellular polymer produced by halophilic archaea A39 The phenol-sulfuric acid method was used to draw the calibration curve and determine the sugar content. Accurately weigh 10 mg of dried glucose, and make a stock solution of 0.1 mg / mL. Take 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mL of the stock solution, and add deionized water to 1 mL. Take 500 μL of each into a glass test tube, and quickly add 500 μL of 6% phenol solution and 2.5 mL of 98% concentrated sulfuric acid in a dark environment, mix well and react at room temperature for 30 min. The blank control is distilled water, and the extracellular polymer is dissolved in 5 mL of deionized pure water, and the sugar content is determined by the phenol-sulfuric acid method. Measure the absorbance at 490 nm and calculate the sugar content of the polymer.

[0026] (5) Detection of protein content of extracellular polymer produced by halophilic archaea A39 The content of protein was determined by Coomassie brilliant blue method. The bovine serum albumin standard was prepared: 10 mg of dried bovine serum albumin was accurately weighed, and the volume was made to 0.1 mg / mL of mother liquor. 0, 20, 40, 60, 80, 100, and 120 μL of 0.1 mg / mL protein standard solution were respectively taken into an EP tube with a pipette, and deionized water was added to 200 μL, 300 μL of prepared Coomassie brilliant blue solution was added into the EP tube in turn, shaken uniformly, and placed at room temperature for 30 min, and the absorbance was measured at 595 nm, and the standard curve was drawn by parallel determination for three times, and the regression curve equation was obtained. The exopolysaccharide sample produced by strain A39 was accurately weighed and prepared into a 0.1 mg / mL solution, 60 μL of which was taken into an EP tube with a pipette, deionized water was added to 200 μL, and 300 μL of Coomassie brilliant blue solution was added, shaken uniformly, and placed at room temperature for 30 min, and the absorbance was measured at 595 nm, and the average value of the absorbance was calculated by parallel determination for three times, and the average value of the absorbance was substituted into the regression equation to calculate the protein content in the sample.

[0027] (6) Morphology detection of the exopolysaccharide produced by halophilic archaea A39 The morphology of the exopolysaccharide was observed by scanning electron microscope. A small amount of exopolysaccharide powder was adhered to a double-sided adhesive conductive carbon film. Gold spraying treatment was performed for 120 s. The microstructure characteristics of the exopolysaccharide produced by strain A39 were observed by scanning electron microscope at an acceleration voltage of 2 kV.

[0028] (7) X-ray diffraction analysis of the exopolysaccharide produced by halophilic archaea A39 The dried crude exopolysaccharide powder was determined on an X-ray diffractometer at room temperature. The sample scanning range was set to 10°-90°, the scanning step was 0.013, and the speed was 15 s / step.

[0029] 4. Heavy metal removal characteristics of the exopolysaccharide produced by halophilic archaea A39 (1) Detection of heavy metal removal performance of different concentrations of exopolysaccharide produced by halophilic archaea A39 under 0%, 15% NaCl conditions 10 mL of 50 mg / L different heavy metal ion standard solution was taken with a pipette, and different concentrations of exopolysaccharide produced by strain A39 were added to the solution to a final concentration of 1 mg / mL, 2 mg / mL, and 3 mg / mL, respectively, and oscillated at 100 r / min for 72 h. The first and second dilutions were performed and filtered by the above-mentioned method 2(2), and ICP-MS was selected to detect the concentration of heavy metal ions under 0%, 15% NaCl conditions.

[0030] (2) Detection of heavy metal removal performance of the exopolysaccharide produced by halophilic archaea A39 under different treatment times The extracellular crude polysaccharide produced by strain A39 was selected, and the concentration was 3 mg / mL. The heavy metal removal performance of the sample at different time periods of 24, 48, 72 and 96 h was explored. The heavy metal ion concentration under the condition of 15% NaCl was detected by the above-mentioned method 2(2), and the removal effect was analyzed. The heavy metal removal capacity of the crude polysaccharide was quantitatively compared by the heavy metal removal rate Q. The calculation formula is: Q = (C0-C) / C0 (%). Wherein, C0 is the initial concentration; C is the final concentration. Note: all detection experiments are set in triplicate.

[0031] 5. Flocculation detection of extracellular polysaccharide produced by halophilic archaea A39 (1) Flocculation detection of extracellular polysaccharide produced by halophilic archaea A39 at different concentrations Prepare 5 g / L kaolin suspension containing 15% NaCl: 25°C, 100 r / min magnetic stirrer, stir uniformly, solution present use; experimental sample preparation: the concentration of extracellular polysaccharide solution produced by strain A39 is 1 mg / mL, 2 mg / mL, 3 mg / mL; add 93 mL suspension, 5 mL CaCl2(1%) solution to the beaker, respectively add 2 mL extracellular polysaccharide solution with different concentrations; magnetic stirrer 250 rpm stirring for 5 min, standing for 20 min; take 5 mL liquid in the middle layer of the beaker, measure the absorbance at 550 nm.

[0032] (2) Flocculation detection of extracellular polysaccharide produced by halophilic archaea A39 at different salinity and pH range The concentration of extracellular polysaccharide produced by strain A39 is set to 3 mg / L, and the flocculation effect under different salt concentrations and pH is explored. The salinity (0%, 3%, 5%, 8%, 10%, 13%, 15%, 18%, 20%) and pH (3.0, 5.0, 6.0, 7.0, 8.0, 9.0, 11.0, 15% NaCl) gradient are set for related flocculation rate detection to determine the stability of the extracellular polysaccharide. The calculation formula is: E (%) = (A-B) / A, wherein E is the flocculation rate, A is the blank control, and B is the experimental group.

[0033] III. Experimental results (A) Polyphase classification and identification of halophilic archaea A39 1. Molecular biology identification of halophilic archaea A39 (1) Evolution analysis of conserved genes of halophilic archaea A39 The 16S rRNA gene sequence of halophilic archaea A39 and Natrinema versiforme XF-10 TThe similarity of 16S rRNA gene is 97.77%, and it is contained in Natrinema genus, and is clustered in a branch alone. The N-J (Neighbor-Joining Algorithm) tree of 16S rRNA gene phylogeny is shown in Figure 1 The N-J tree of rpoB' gene phylogeny is shown in Figure 2 Based on the similarity of 16S rRNA gene, strain A39 is clustered in a branch alone, and is distributed in the branch range of Natrinema genus, and is preliminarily judged as a potential new species classification unit of Natrinema genus.

[0034] (2) Genome sequencing analysis of halophilic archaea A39 The genome data of halophilic archaea A39 has two spliced sequences, wherein Chrom1 is the whole genome map as shown in Figure 3 The size is 3.8 M, the GC content is 64.23%, the coverage reaches 100%, contains 52 tRNA genes, 14 rRNA genes, and 4438 CDS (coding sequence). From the COG functional classification results of strain A39.Chrom1 genome Figure 4 It can be seen that the R (General function prediction only) general metabolic function related genes are the most, followed by the E (Amino acid transport and metabolism) amino acid metabolism and transport related genes, and the D (Cell cycle control, cell division, chromosome partitioning) cell cycle control, cell division, and chromosome separation related genes are the least. In the COG functional classification result diagram of strain A39.Chrom1 genome, the number of S (Function unknown) is relatively large, which indicates that there are still a large number of unknown function genes in the genome, which need to be further analyzed and verified. The ANI value of strain A39 and the closest strain Natrinema versiforme XF-10 T is 85.19%, and the dDDH value is 47.13%, so strain A39 is judged as a potential new species classification unit of Natrinema genus.

[0035] 2. Morphology and physiological and biochemical detection of halophilic archaea A39 (1) Morphology detection of halophilic archaea A39 The halophilic archaea A39 is a red colony on the modified Gause culture medium, as shown in Figure 5 The cell morphology is spherical by scanning and transmission electron microscope observation, and the A39 strain cell periphery has nano-particle extracellular polymer.

[0036] (2) Physiological and biochemical analysis of halophilic archaea A39 The growth temperature range of halophilic archaea A39 is 20-45℃ (optimum 37℃); the growth salt concentration range is 12%-35% (w / v) (optimum 22% (w / v)); the MgCl2 concentration range is 0-0.8 M (optimum 0.2-0.3 M); and the pH range is 6.5-8.5 (optimum 7.5).

[0037] The carbon sources that can be used as the sole carbon source for growth are trehalose, starch, sorbitol, sucrose, glycerol, glucose, mannitol, raffinose; and the carbon sources that cannot be used are maltose, mannose, galactose, cellobiose, ribose, fructose, xylose, lactose, sodium lactate, and sodium malate.

[0038] The nitrogen sources that can be used as the sole nitrogen source for growth are lysine, arginine, glycine, serine, and alanine; and the nitrogen sources that cannot be used are phenylalanine, tyrosine, methionine, and glutamic acid.

[0039] The cellulase activity and esterase activity reactions are both negative, and the amylase, protease, oxidase, and catalase activities are all positive; nitrate is reduced to produce gas.

[0040] Sensitivity to rifampicin and novobiocin, and insensitivity to trimethoprim, streptomycin, kanamycin, tetracycline, vancomycin, gentamicin, ampicillin, chloramphenicol, neomycin, norfloxacin, bacitracin, nystatin, and ciprofloxacin.

[0041] Legend: + is positive, - is negative, w is weakly positive, and ND is not detected.

[0042] (3) Analysis of polar lipids of halophilic archaea A39 The polar lipid thin layer chromatography results of halophilic archaea A39 show that Figure 6 Compared with the standard strain Natrinema versiforme CGMCC 1.2365 T The polar lipids contained by strain A39 include PG (phosphatidylglycerol), PGP-Me (phosphatidylglycerol phosphatidylmethanol), PGS (phosphatidylglycerol sulfate), UGL-1 (unknown glycolipid 1), and UGL-2 (unknown glycolipid 2). The yellow spots in the TLC plate are phospholipid types, and the red-brown spots are glycolipid types. The polar lipid components of strain A39 are basically consistent with those of the standard strain, which are consistent with the polar lipid types of the genus Natrinema, and are preliminarily judged to be the group of the genus Natrinema.

[0043] Based on the comprehensive analysis of molecular indicators, morphological indicators, physiological and biochemical indicators, and chemical indicators, strain A39 is determined to be a new species classification unit of the genus Natrinema.

[0044] (II) Application of different biological materials of halophilic archaea A39 in high-salinity wastewater 1. Heavy metal removal characteristics of halophilic archaea A39 under high-salinity conditions (1) Plate tolerance of halophilic archaea A39 to different heavy metals Halophilic archaea A39 can tolerate 0 mg / L to 400 mg / L of Pb 2+ , and grows well; it can tolerate 0 mg / L to 200 mg / L of Cu 2+ , and the growth gradually weakens with increasing metal concentration; it can tolerate 0 mg / L to 50 mg / L of Cd 2+ , Zn 2+ , and Cr 6+ . The liquid culture tolerance results show that the tolerance of strain A39 to Pb 2+ is the strongest, followed by Cu 2+ .

[0045] (2) Heavy metal removal of different biological materials of halophilic archaea A39 Under the condition of 15% NaCl, the standard curve formulas of Pb 2+ , Cr 6+ , Cu 2+ , Cd 2+ , and Zn 2+ are y = 8677.2x - 5362.7, y = 815.26x - 777.14, y = 4091.1x - 3187.4, y = 3056.4x - 3249.1, and y = 2539.8x - 3203.9, respectively, and the linear correlation coefficients R 2 are 0.9991, 0.9965, 0.9976, 0.9977, and 0.9907, respectively. R 2 all reach above 0.99, indicating a good linear relationship, which can be used for subsequent calculation of the heavy metal content in the solution.

[0046] The heavy metal removal effects of the cell body, fermentation broth, supernatant, and produced extracellular polymer of halophilic archaea A39 under high-salinity conditions (15% NaCl) are shown in Table 2. Figure 7 For copper ions (Cu 2+ ), the cell body of strain A39 has the highest removal rate, reaching 61.08%; followed by the fermentation broth, extracellular polymer, and supernatant, with removal rates of 59.87%, 57.53%, and 53.18%, respectively. For lead ions (Pb 2+ ), the cell body of strain A39 has the highest removal rate, reaching 66.71%; followed by the fermentation broth, supernatant, and extracellular polymer, with removal rates of 62.03%, 58.12%, and 52.38%, respectively. For chromium ions (Cr6+ The removal rate of the fermentation broth of strain A39 was the highest, reaching 68.11%; followed by the extracellular polymer, the cell body, and the supernatant, and the removal rates were 68.03%, 57.83%, and 52.17%, respectively. For cadmium ions (Cd 2+ The removal rate of the cell body of strain A39 was the highest, reaching 60.71%; followed by the extracellular polymer, the fermentation broth, and the supernatant, and the removal rates were 54.31%, 37.65%, and 30.18%, respectively. For zinc ions (Zn 2+ The removal rate of the extracellular polymer of strain A39 was the highest, reaching 41.32%; followed by the fermentation broth, the supernatant, and the cell body, and the removal rates were 40.83%, 38.74%, and 27.99%, respectively.

[0047] It can be seen from the experimental results that under high-salt conditions, the removal rates of the cell body and the fermentation broth for Cu 2+ , Pb 2+ , and Cr 6+ are relatively high as a whole compared with the extracellular polymer. The possible reason is that the fermentation broth contains both the extracellular polymer and the cell body. The cell body surface contains a large number of negatively charged functional groups (such as carboxyl and amino groups), which can directly capture heavy metals through electrostatic adsorption or ion exchange, or metabolites (polysaccharides) produced in the cell metabolism can adsorb heavy metals. However, considering the convenience of the material, the extracellular polymer is easy to store, transport, and use, and the heavy metal removal performance does not decrease significantly in a high-salt environment, and it is suitable for the treatment of high-salt wastewater of different concentrations. Therefore, the extracellular polymer produced by halophilic archaea has a good advantage in the treatment of complex high-salt wastewater.

[0048] 2. Yield and structural characteristics of the extracellular polymer produced by halophilic archaea A39 (1) Yield of the extracellular polymer produced by halophilic archaea A39 The yield of the extracellular polymer produced by halophilic archaea A39, the dry weight of the cell body, and the sugar production of 1 g of the cell body were 1.20±0.10 g / L, 0.65±0.02 g / L, and 1.86±0.18 g / g, respectively.

[0049] (2) Full-wavelength ultraviolet scanning spectrum characteristics of the extracellular polymer produced by halophilic archaea A39 The full-wavelength results of each extracellular crude polysaccharide are shown in Table 1. Figure 8 The extracellular polymer has absorption values at 260 nm and 280 nm, indicating that it may also contain some proteins and nucleic acid substances, but no obvious absorption peak is observed, indicating that the content is low. There is no obvious absorption at 400 nm, that is, the polysaccharide sample almost does not contain pigments. The maximum absorption peak of the extracellular polymer produced by strain A39 is 202 nm. This is consistent with the spectral characteristics of the extracellular polysaccharide produced by halophilic archaea.

[0050] (3) Fourier transform infrared spectroscopy characteristics of extracellular polymers produced by halophilic archaea A39 The Fourier transform infrared spectroscopy characteristics of extracellular polymers produced by halophilic archaea A39 Figure 9 The position information of the characteristic peaks related to polysaccharides in the Fourier transform infrared spectroscopy characteristics of extracellular polymers produced by halophilic archaea A39 is basically similar, and has the core characteristics of polysaccharides: glycosidic bond (1079 cm -1 , 1145 cm -1 ), hydroxyl group (3295 cm -1 ), carboxylic acid group (1648 cm -1 ). The vibration of methylene (2917 cm -1 ) and C-O (1105 cm -1 ) indicates the presence of a typical polysaccharide skeleton. The extracellular polymers produced by strain A39 have a wide band in the 3000-3750 cm -1 region, and a strong absorption peak around 3295 cm -1 , which is caused by the stretching vibration of polysaccharide hydroxyl group (O-H) and is consistent with the characteristics of alcohol hydroxyl group of polysaccharides. There may be C=O at 1648 cm -1 , which corresponds to the absorption peak of the carbonyl group of carboxylic acid group (-COOH), indicating that the polysaccharide contains carboxylic acid modification and is an acidic polysaccharide. There is a C-H absorption peak at 1419 cm -1 , indicating that there may be a bending vibration of methyl or methylene. There is a C-O-C absorption peak around 1145 cm -1 . The characteristic absorption peak of glycosidic bond is at 1079 cm -1 , which can confirm that there is a glycosidic bond between the molecular units of the extracellular polymers produced by strain A39. Based on the comprehensive analysis of full-wave ultraviolet and infrared, it is determined that the extracellular polymers produced by halophilic archaea A39 are extracellular polysaccharides, which contain carboxyl groups and are acidic polysaccharides.

[0051] (4) Sugar content of extracellular crude polysaccharides produced by halophilic archaea A39 Figure 10 To determine the standard curve of the total sugar content of extracellular crude polysaccharides, by comparing with the standard curve, the total sugar content of different crude polysaccharides was calculated as 56.96±3.97%.

[0052] (5) Protein content of extracellular crude polysaccharides produced by halophilic archaea A39 Figure 11 To determine the standard curve of the protein content of extracellular crude polysaccharides, by comparing with the standard curve, the protein content of different crude polysaccharides was calculated as 2.37±0.26%.

[0053] (6) Morphological characteristics of extracellular polysaccharides produced by halophilic archaea A39 The morphological characteristics of extracellular crude polysaccharides produced by halophilic archaea A39 were observed under a scanning electron microscope (SEM) asFigure 12 As shown, the crude polysaccharide produced by strain A39 exhibits irregular pores and a network structure. This network structure helps improve the product's viscosity and adsorption properties.

[0054] (7) X-ray diffraction analysis of crude extracellular polysaccharides produced by halophilic archaea A39 X-ray diffraction results of the crude extracellular polysaccharide of the halophilic archaea A39 are as follows: Figure 13 As shown, the crude extracellular polysaccharide produced by strain A39 has obvious diffuse, weak broad peaks, indicating that the crude extracellular polysaccharide has very weak crystallinity and belongs to an amorphous structure.

[0055] 3. Heavy metal scavenging properties of extracellular crude polysaccharides produced by halophilic archaea A39 (1) Heavy metal scavenging characteristics of different concentrations of extracellular crude polysaccharide produced by halophilic archaea A39 under 0% and 15% NaCl conditions. Pb under 15% NaCl conditions 2+ Cr 6+ Cu 2+ Cd 2+ and Zn 2+ The standard curve formulas are y=8677.2x-5362.7, y=815.26x-777.14, y=4091.1x-3187.4, y=3056.4x-3249.1 and y=2539.8x-3203.9, respectively, and the linear correlation coefficient R0 is given. 2 The values ​​are 0.9991, 0.9965, 0.9976, 0.9977, and 0.9907, respectively. R 2 A linearity of 0.99 or higher indicates good linearity and can be used for subsequent calculations of heavy metal content in the solution. The heavy metal removal effects of different concentrations of crude polysaccharide samples under 15% NaCl conditions are shown below. Figure 14 As shown, under high-salt conditions, the extracellular crude polysaccharides produced by different concentrations of strains exhibited varying scavenging abilities for different heavy metal ions. The removal efficiency of the extracellular crude polysaccharides for heavy metals increased with increasing concentration, reaching a peak at a polysaccharide concentration of 3 mg / mL. The extracellular crude polysaccharide produced by the halophilic archaea A39 showed resistance to Pb. 2+ and Cr 6+ It has good removal performance for Pb. 2+ The removal rate reached 71.07%, and the removal rate of Cr was [missing information]. 6+ The removal rate reached 69.43%, for Cu 2+ The removal rate reached 57.53%, and the removal rate of Cd was [missing information]. 2+ The clearance rate reached 54.31%, for Zn 2+ The clearance rate reached 41.32%. Under high-salt conditions, compared with the positive control PAM, lentinan, and xanthan gum, the crude extracellular polysaccharide produced by strain A39 showed a significant effect on lead (Pb) removal at a concentration of 3 mg / L.2+ Cr 6+ Cu 2+ Cd 2+ and Zn 2+ All showed good removal effects.

[0056] Under 0% NaCl conditions, Pb 2+ Cr 6+ Cu 2+ Cd 2+ and Zn 2+ The standard curve formulas are y=8702.1x-5387.8, y=807.86x-769.74, y=4123.6x-3219.9, y=3081.5x-3274.2 and y=2389.34x-3035.44, respectively, and the linear correlation coefficient R0 is... 2 The values ​​were 0.9969, 0.9983, 0.9986, 0.9928, and 0.9957, respectively. Under 0% NaCl conditions, the removal efficiency of different concentrations of extracellular crude polysaccharide samples for heavy metals increased with increasing concentration, reaching a peak at a crude polysaccharide concentration of 3 mg / mL. The extracellular crude polysaccharide produced by strain A39 showed improved removal efficiency for Pb. 2+ Cr 6+ Cu 2+ Cd 2+ Zn 2+ The removal rates reached 68.18%, 61.36%, 53.27%, 56.12%, and 44.51%. The heavy metal removal effects of crude polysaccharide samples with different concentrations under 0% NaCl conditions are shown below. Figure 15 As shown, the positive controls PAM, lentinan, and xanthan gum showed good scavenging effects on metal ions under salt-free conditions, but their scavenging effects were greatly reduced under high-salt conditions. Compared with PAM and representative polysaccharides produced by fungi and bacteria (lentinan and xanthan gum), the extracellular crude polysaccharide produced by strain A39 showed good adsorption performance under high-salt conditions and maintained a stable and good heavy metal scavenging effect even at low concentrations, demonstrating potential for the development of novel biosorbents.

[0057] (2) Heavy metal removal effect of extracellular crude polysaccharide produced by halophilic archaea A39 under different treatment times The extracellular crude polysaccharide produced by the halophilic archaea A39 at a concentration of 3 mg / mL, under 15% NaCl conditions, showed different effects on Pb at different treatment times. 2+ Cr 6+ Cu 2+ Cd 2+ Zn 2+ The cleaning effect is as follows Figure 16As shown in the figure, the removal of heavy metals by the exopolysaccharide produced by strain A39 increased with the increase of treatment time. When the treatment time reached 48 h, the removal effect of Cd 2+ , Zn 2+ was optimal, with removal rates of 53.10% and 44.50%, respectively. Subsequently, the removal efficiency decreased slightly with the increase of treatment time. When the treatment time reached 72 h, the removal effect of Pb 2+ , Cr 6+ , Cu 2+ was optimal, with removal rates of 71.07%, 69.43%, and 47.63%, respectively.

[0058] 4. Flocculation properties of the exopolysaccharide produced by halophilic archaea A39 (1) Flocculation properties of the exopolysaccharide produced by halophilic archaea A39 at different concentrations The flocculation effect of the exopolysaccharide produced by halophilic archaea A39 at different concentrations is shown in Figure 17 . With the increase of the concentration of the exopolysaccharide, the flocculation effect of each group of samples increased. When the sample concentration was 3 mg / mL, the flocculation effect was best, and the flocculation rate of the exopolysaccharide produced by strain A39 was 61.40%. The flocculation effect of the exopolysaccharide produced by strain A39 was higher than that of PAM, xanthan gum, and lentinan. This indicates that under high salt concentration (15% NaCl), the flocculation effect of traditional chemical flocculants PAM and fungal polysaccharides (lentinan) and bacterial polysaccharides (xanthan gum) is poor, while the exopolysaccharide produced by halophilic archaea shows better flocculation effect. The exopolysaccharide produced by strain A39 can become a good research material for the treatment of high-salt industrial wastewater.

[0059] (2) Stability of the exopolysaccharide produced by halophilic archaea A39 to salinity and pH The flocculation effect of the exopolysaccharide produced by halophilic archaea A39 under different salt concentrations and different pH is shown in Figure 18 and 19 . Under NaCl salinity of 0% to 20%, the exopolysaccharide showed good flocculation effect, and with the increase of the concentration of the exopolysaccharide, the flocculation effect of each group of samples increased, showing a dose-effect relationship. The exopolysaccharide produced was better than PAM in flocculation effect, especially under NaCl greater than 10%. When the sample concentration was 3 mg / mL, the flocculation effect was best, and the flocculation rate of the exopolysaccharide produced by strain A39 was 62.07%. Under pH 3 to 8, the flocculation effect was better than PAM, and under pH 9 to 11, the flocculation effect was worse than PAM. Therefore, the exopolysaccharide produced by strain A39 is not suitable for treating alkaline wastewater (pH 10 to 11) as a flocculant.

[0060] It should be noted that the above-mentioned embodiments are only illustrative, but not intended to limit the scope of protection of the present application, and the scope of protection of the present application is subject to the claims. For those skilled in the art, some non-essential improvements and adjustments of the present application without departing from the spirit and scope of the present application still belong to the protection scope of the present application.

Claims

1. A new strain of the genus Naiinobacterium, A39, characterized in that, The classification name is Natrinema sp., which has been preserved in China General Microbiological Culture Collection Center, with the preservation number of CGMCC 35089 and the preservation date of July 3, 2025.

2. A microbial inoculant for treating high-salinity organic wastewater, characterized by, One of the cell bodies of the new strain of sodium line bacteria A39, the produced extracellular crude polysaccharide, or the fermentation liquor, wherein the fermentation liquor is a mixture including the cell bodies of the new strain of sodium line bacteria A39 and the produced extracellular crude polysaccharide.

3. The application of the new strain of sodium line bacteria A39 in claim 1 or the microbial inoculant in claim 2 in the heavy metal removal of high-salt organic wastewater.

4. Use according to claim 3, characterized in that, The high-salt organic wastewater is treated by the microbial inoculant containing the extracellular crude polysaccharide produced by the new strain of sodium line bacteria A39, the treatment time is 48 h-72 h, the pH is 3-8, and the concentration of the extracellular crude polysaccharide produced by the strain A39 is 3 mg / mL.

5. Use according to claim 3, characterized in that: The high-salt organic wastewater contains at least one of copper ions, lead ions, chromium ions, zinc ions, or cadmium ions.

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