Salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria

Abstract

The application discloses a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium. Halomonas elongata 5300, and the preservation number is CGMCC No.25768. The strain has a wide salt concentration tolerance range, can grow under a salt concentration of 0% to 26%, and can efficiently remove NO2 ‑ -N and NH4 + -N in water under a salt concentration of 4% to 16%. The strain 5300 can effectively solve the problem of activity inhibition of functional microorganisms in nitrogen-containing polluted water under high salt in a previous biological denitrification process, and is suitable for denitrification treatment of nitrogen-containing polluted water under high salt and aerobic conditions. ‑ -N removal efficiency reaches 100.00%, and NH4 + -N is more than 78.51%. The strain 5300 can effectively solve the problem of activity inhibition of functional microorganisms in nitrogen-containing polluted water under high salt in a previous biological denitrification process, and is suitable for denitrification treatment of nitrogen-containing polluted water under high salt and aerobic conditions.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering and environmental engineering technology, and relates to a salt-tolerant heterotrophic nitrification-aerobic denitrification strain, while also providing the denitrification efficiency of this strain. Background Technology

[0002] With economic development, industrial sectors such as petrochemicals, coal chemicals, fertilizers, plastics, pharmaceuticals, and dyes generate extremely difficult-to-treat high-salinity wastewater with a salinity of 10%-25%. Furthermore, coastal cities often use seawater for production and daily life, leading to a significant increase in the discharge of high-salinity wastewater. This wastewater is not only rich in highly difficult-to-degrade organic matter, but also has a high nitrogen content. If it enters the ecological environment, it will cause eutrophication in lakes and rivers, stressing and toxicizing aquatic organisms, and ultimately harming human health. Therefore, strengthening nitrogen treatment of high-salinity wastewater is urgently needed.

[0003] Currently, biological methods are the most economical and efficient wastewater treatment method, being green and without secondary hazards. However, high salinity inhibits the biochemical reaction process of microorganisms, significantly reducing wastewater treatment efficiency. To date, most heterotrophic nitrifying-aerobic denitrifying microorganisms have been found unable to stably perform nitrogen removal functions when salinity exceeds 2%. Under high salinity conditions, microbial growth is inhibited to varying degrees, and denitrification efficiency drops sharply. Therefore, screening for heterotrophic nitrifying-aerobic denitrifying strains capable of efficient biological denitrification in high-salt environments has significant application value for denitrification treatment of high-salt nitrogen-containing water bodies. Furthermore, while there are reports on the salt-tolerant heterotrophic nitrifying-aerobic denitrification capabilities of *Haloxylon* spp., no studies have yet investigated their application in this area. Halomonas elongata Research on the function of heterotrophic nitrifying-aerobic denitrifying bacteria.

[0004] Dong Lingxi. A heterotrophic nitrifying-aerobic denitrifying bacterium Halomonas piezotolerans Isolation and denitrification characteristics of HN2 [D]. Zhejiang Ocean University, 2022. Salinity tolerance is only 3.0%; Su Zhaopeng, Li Yun, Pan Luqing, et al. Denitrification performance and quantitative detection of a new heterotrophic nitrifying-aerobic denitrifying bacterium GJWA3 [J]. Journal of Ocean University of China (Natural Science Edition), 2021, 51(10). Halomonas meridiana strain SCSIO 43005 exhibits excellent NH4+ control under conditions of pH 7.0–8.5, temperature 25–35℃, C / N ratio 10–20, and salinity 24–40. + -N, NO2 - -N removal capacity. Ma Hongjing, Liu Ying, Mu Xinting, et al. Salt-tolerant, highly efficient aerobic denitrifying bacteria. HalomonasIsolation, identification and denitrification performance of sp. HRL-11 [J]. Journal of Dalian Ocean University, 2022, 37(02) verified a strain Halomonas zhaodongensis Under high salinity (31) conditions, strain HRL-11 was subjected to nitrate nitrogen (NO) testing. - The optimized conditions for NO3- (NO3) reduction were: shaking speed 150 r / min (initial DO concentration 7.48 mg / L), C / N ratio 10, and temperature 30℃. Under these conditions, strain HRL-11 reduced NO3- to 100 mg / L. - -N was the sole nitrogen source, and after 48 h of reaction, its OD 600 nm The value is 1.4 for NO3. - The removal rates of -N, TOC, and TN were 91.5%, 69.4%, and 65.6%, respectively.

[0005] Chinese patent CN201210458579 discloses a strain of Haloxylon ammodendron BMEN3 with heterotrophic nitrification, aerobic denitrification, and hydrophilic properties. Halomonas campisalis .BMEN3) retains heterotrophic nitrification and aerobic denitrification capabilities even under high salinity and alkalinity conditions, but it is primarily designed for treating high-salt and high-nitrogen wastewater and flue gas. It can remove 100 mg / L of NH4+ within 12 hours at pH 9-10 and a salinity of only 4%. + -N and 140 mg / L NO3 - -N.

[0006] Chinese patent CN201410138167.4 discloses a marine halomonas strain HGMN521 with aerobic denitrification ability and its application, mainly used for treating seawater polluted by inorganic nitrogen.

[0007] CN201710421768 Alkalophilic monocytogenes ( Halomonas alkaliphila X3 was screened from the eutrophic sedimentary environment of the Xiangshan Port cage fish farming area, with an ammonia nitrogen content of 42 mg•L. -1 In the test solution, after 24 h, the removal rates of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen were 98.29%, 99.07% and 96.48% respectively, and it could grow under conditions of only 1%-8% NaCl.

[0008] Chinese patent CN201510315663 on Halomonas ( Halomonas lactosivorans Although CYQ1-6-1 has strong tolerance to high concentrations of nitrite and high denitrification efficiency, capable of removing 1000 mg / L NO2 within 72 hours... - -N was completely removed; however, this application discovered new bacterial strains. Halomonas elongata Previous studies have not reported this. Halomonas elongataIt has the function of heterotrophic nitrification-aerobic denitrification bacteria. Summary of the Invention

[0009] The purpose of this invention is to provide a salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacterium that can efficiently remove nitrogen under high-salt conditions, thereby enriching the current microbial resource library for the treatment of high-salt nitrogen-containing wastewater.

[0010] The technical solution of the present invention is as follows:

[0011] A salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacterium, this strain is classified and named Halomonas elongata It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on September 21, 2022, with accession number CGMCC No. 25768.

[0012] The heterotrophic nitrifying-aerobic denitrifying bacteria provided by this invention are salt-tolerant bacteria that can grow in a salt environment of 0% to 26% with good growth and a wider growth range compared to ordinary salt-tolerant bacteria.

[0013] This invention relates to salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria, characterized by pale yellow, round colonies with a moist surface that is easily picked up. These bacteria are inoculated into nitrogen-containing, high-salt artificial wastewater, with a salt concentration of 0%–24%, a nitrogen source concentration of 20–200 mg / L, an inoculum quantity of 1%–5%, different carbon sources (sucrose, glucose, sodium pyruvate, trehalose, inosine, sodium acetate, stachyose), a C / N ratio of 1–20, a pH of 5–9, a temperature of 28–40°C, and a speed of 0–220 r / min to induce heterotrophic nitrification-aerobic denitrification.

[0014] The optimal salt concentration for denitrification by the strain is 4%–16%, with NO2 as the primary nitrogen source. - -N and NH4 + The suitable nitrogen source concentrations, with -N as the indicator, are 20–40 mg / L and 20–80 mg / L, respectively, with NO2 as the primary indicator. - -N and NH4 + The suitable inoculum amounts for -N as an indicator are 2%~4% and 1%~2%, respectively. The suitable carbon source is sodium pyruvate, the C / N ratio is 15, the pH is 7.0, the culture temperature is 37℃~40℃, and the rotation speed is 165~220 r / min.

[0015] The beneficial effects of this invention are as follows:

[0016] The heterotrophic nitrifying-aerobic denitrifying bacteria of this invention are salt-tolerant bacteria, adapted to growth environments with salt concentrations of 0-26%, providing bacterial resources for nitrogen removal treatment of high-salt wastewater.

[0017] The salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria of this invention can efficiently carry out heterotrophic nitrification and aerobic denitrification reactions at salt concentrations of 4% to 16%, NO2 - -N removal rate is 100%, NH4 + The removal rate of -N is over 78.51%, and it can grow under salt concentration conditions of 4%~16%, using sodium pyruvate as the carbon source and NO2 as the nitrogen source. - -N and NH4 + The suitable nitrogen source concentrations for -N are 20–40 mg / L and 20–80 mg / L, respectively, for NO2. - -N and NH4 + The optimal inoculum concentrations (using -N as an indicator) were 2%–4% and 1%–2%, respectively. Under these conditions, with a C / N ratio of 15, pH 7.0, and a rotation speed of 165–220 r / min, after 7 days of aerobic culture, this strain showed resistance to NO2. - -N and NH4 + -N has a good denitrification effect, which is of great practical significance for realizing heterotrophic nitrification-aerobic denitrification in high-salt environments. Attached Figure Description

[0018] Figure 1 This is a phylogenetic tree of the 16S rRNA gene of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria 5300 of this invention.

[0019] Figure 2 This is a schematic diagram illustrating the effect of salt concentration on the denitrification of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria 5300 of this invention.

[0020] Figure 3 This is a schematic diagram illustrating the effect of nitrogen source concentration on the denitrification of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria 5300 of this invention.

[0021] Figure 4 This is a schematic diagram illustrating the effect of inoculum quantity on the denitrification of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria 5300 of this invention.

[0022] Figure 5 This is a schematic diagram showing the effect of different carbon sources on the denitrification of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria 5300 of the present invention, where 1: sucrose; 2: glucose; 3: sodium pyruvate; 4: trehalose; 5: inosine; 6: sodium acetate; 7: stachyose.

[0023] Figure 6 This is a schematic diagram illustrating the effect of the carbon-to-nitrogen ratio on the denitrification of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria 5300 of this invention.

[0024] Figure 7 This is a schematic diagram illustrating the effect of pH on the denitrification of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria 5300 of this invention.

[0025] Figure 8 This is a schematic diagram illustrating the effect of temperature on the denitrification of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria 5300 of this invention.

[0026] Figure 9 This is a schematic diagram illustrating the effect of rotational speed on the denitrification of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria 5300 of this invention. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: Example 1

[0028] Screening and identification of a salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacterium.

[0029] Samples were collected from Qijiaojing Salt Lake in Xinjiang. 5 g of mixed soil sample was added to a conical flask containing sterile water and shaken on a shaker for 0.5 h. 1 mL of the soil bacterial suspension was then transferred to a test tube containing sterile water and diluted sequentially until the bacterial concentration reached 10-1. -1 10 -2 Take 100 μL of each dilution and spread it on seawater 2216E medium. Incubate upside down at 37°C for 1 month. Select colonies with different morphological characteristics and streak them at least 3 times to isolate them. After maintaining consistent colony growth characteristics, identify them as single colonies. Transfer the single colonies to slant agar and incubate them at 4°C.

[0030] 2216E medium (g / L): peptone 5, yeast extract 1, ferric phosphate 0.1, agar 20, NaCl 80.0, trace element solution 2 mL, pH 7.2–7.5. The trace element solution (g / L) is: EDTA 50, ZnSO4 2.2, CaCl2 5.5, MnCl2•4H2O 2.06, FeSO4•7H2O 5.0, (NH4)6Mo7O 24 •4H2O 1.1, CuSO4•5H2O 1.6, CoCl2•6H2O 1.61.

[0031] The purified strain was inoculated into BTB medium for initial screening, inverted and cultured at 37℃ for 3 days. The strain that turned blue fastest and most obviously on the medium was named 5300, stored in 20% glycerol, and kept in a -80℃ refrigerator for long-term preservation.

[0032] BTB culture medium composition (g / L): C6H5Na3O7•2H2O 5.70, KNO3 2.0, K2HPO4 1.0, MgSO4•7H2O 0.2, NaCl 80.0, agar 20, 1% bromothymol blue ethanol solution 1 mL, water 1000 mL, pH 7.0.

[0033] Genomic DNA was extracted from the bacterial strain according to the TaKaRa kit instructions (model 9769S). Using the extracted genomic DNA as a template, the bacterial 16S rRNA gene was amplified using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′). After passing PCR detection by 1% agarose gel electrophoresis, the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0034] The sequence length of strain 5300 is 1372 bp. The strain sequence was uploaded to the NCBI database and compared with known bacterial 16S rRNA gene sequences. The results showed that strain 5300 is... Halomonas elongata A phylogenetic tree of the strains was constructed using MEGA 7.0, and the results are as follows: Figure 1 . Example 2

[0035] The optimal salt concentration range for the growth of strain 5300.

[0036] Strain 5300 was pre-activated, and 2216E medium was prepared with salt concentration gradients of 0%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 25%, and 26%. The strain was inoculated onto medium plates with different salt concentrations and incubated upside down at 37°C for 7 days. The growth of the strain under different salt concentrations was observed.

[0037] The results showed that strain 5300 could grow normally in salt concentrations of 0% to 26%, with the optimal growth range also being 0% to 26%. This bacterium has a wide salt tolerance range and strong adaptability.

[0038] Table 1: Growth of strain 5300 at different salt concentrations

[0039]

[0040] Note: "++" indicates good growth. Example 3

[0041] The appropriate salt concentration for denitrification treatment of strain 5300.

[0042] Nitrification medium (g / L): (NH4)2SO4 0.0896, CH3COONa 2.0, K2HPO4 0.5, MgSO4•7H2O 0.41, pH=7.0-7.2, the remainder is water.

[0043] Denitrification media 1 and 2: Replace (NH4)2SO4 0.0896 in the nitrification medium with NaNO2 0.1, and keep the rest of the formula the same.

[0044] Treatment method: The bacterial strain was inoculated at 1% into liquid nitrification medium, denitrification medium 1 and 2, and then cultured at 37℃ and 165 r / min for 7 days. Samples were taken periodically to determine the NO3 content in the liquid. - -N, NO2 - -N and NH4 + -N denitrification efficiency.

[0045] The salt concentrations of nitrification medium, denitrification medium 1, and denitrification medium 2 were set to 0, 4, 8, 12, 16, 20, and 24%, respectively. 1% bacterial suspension was inoculated, and the medium was cultured for 7 days under the same other culture conditions. The nitrogen removal capacity was then determined.

[0046] The results are as follows Figure 2 As shown, this strain can efficiently carry out heterotrophic nitrification and aerobic denitrification reactions at salt concentrations of 4%–16%, with NO2... - -N removal rate is 100%, NH4 + The removal rate of -N was over 78.51%. Example 4

[0047] The effects of different growth conditions on the high-efficiency heterotrophic nitrification-aerobic denitrification characteristics of strain 5300.

[0048] Strain 5300 was pre-activated, and different gradients of nitrogen source concentration (20, 40, 80, 120, 160, and 200 mg / L), inoculum quantity (1, 2, 3, 4, and 5%), carbon source (sucrose, glucose, sodium pyruvate, trehalose, inosine, sodium acetate, and stachyose), carbon-to-nitrogen ratio (1, 5, 10, 15, and 20), pH (5, 6, 7, 8, and 9), culture temperature (28, 37, and 40 °C), and rotation speed (0, 85, 165, and 220 r / min) were sequentially set. The experimental procedure was the same as in Example 3, and samples were taken periodically to determine the denitrification efficiency.

[0049] The results showed that ( Figures 3 to 9 This strain is resistant to NO2. - -N and NH4 + The optimal removal rates of -N reached 100.00% and 86.63%. When this strain performs denitrification, it uses NO2... - -N and NH4 + The suitable nitrogen source concentrations, with -N as the indicator, are 20–40 mg / L and 20–80 mg / L, respectively, with NO2 as the primary indicator. - -N and NH4 +The suitable inoculum amounts for -N indicators are 2%~4% and 1%~2%, respectively. The suitable carbon source is sodium pyruvate, the suitable C / N ratio is 15, the suitable pH is 7, the suitable culture temperature is 37℃~40℃, and the suitable rotation speed is 165~220 r / min.

Claims

1. A salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacterium, characterized by: The salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria are Halomonas elongata 5300, Collection No.: CGMCC No.25768.

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