Low-temperature-resistant nitrosomonas and application thereof

Abstract

The invention relates to the technical field of microorganisms, and provides low-temperature-resistant nitrosomonas and application thereof. The nitrosomonas sp. Is nitrosomonas sp. H1, and the preservation number of the nitrosomonas sp. Is CCTCC (China Center For Type Culture Collection) NO: M 2024693. The nitrosomonas H1 disclosed by the invention has the characteristic of low temperature resistance, grows well at 10 DEG C, has relatively good ammonia oxidation capacity, is suitable for treating low-temperature wastewater in winter in northern China, and does not need to heat up or adopt other measures to maintain the stability of a biochemical system.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to low-temperature-resistant Nitrosomonas and applications thereof. Background Art

[0002] Ammonia nitrogen in water is a common water pollutant. Its main sources include agricultural discharge, industrial wastewater and urban sewage. In the water environment, ammonia nitrogen mainly exists in the form of ammonium ions (NH4 + ) and non-ionized ammonia (NH3). Excessive ammonia nitrogen concentration will not only promote eutrophication of water bodies and trigger algal blooms, but will also destroy the balance of aquatic ecosystems, causing serious negative impacts on human health and the environment. Nitrification and denitrification are key ways to deal with ammonia nitrogen pollution in water bodies. This process is regarded as an efficient, sustainable and economical solution. Specifically, the nitrification process first converts ammonia nitrogen into nitrite (NO2 - ), which is then further converted into nitrate (NO3 - ); during denitrification, nitrates are reduced to nitrogen gas (N2) or nitrous oxide (N2O).

[0003] In this conversion process, ammonia oxidizing bacteria (AOB) plays a key role in converting ammonia nitrogen in water into nitrite nitrogen. It is the rate-limiting step of the entire nitrification process and plays an important role in the global nitrogen cycle. This type of bacteria is widely used in wastewater treatment. However, ammonia oxidizing bacteria are very sensitive to environmental changes and usually need to establish a close symbiotic relationship with other microorganisms, which makes their isolation and cultivation difficult and time-consuming.

[0004] Nitrosomonas is a major species in ammonia oxidizing bacteria. It maintains its own growth by oxidizing ammonia nitrogen in water, and uses carbonate as a carbon source and ammonia nitrogen as a nitrogen source to synthesize the required substances. It is a typical autotrophic bacteria. The invention patent with publication number CN112625940A reports a high-temperature resistant application of Nitrosomonas, which exhibits good ammonia oxidation activity under high temperature conditions, and has important practical significance for stable denitrification under high temperature environments. However, there are few research reports on Nitrosomonas under low temperature environments. Therefore, the development of a Nitrosomonas that can survive under low temperature conditions and maintain good ammonia oxidation activity is of great value for the treatment of low-temperature wastewater in winter in northern regions. Summary of the invention

[0005] In view of this, the present invention proposes a low-temperature-resistant Nitrosomonas H1, which has good ammonia oxidation performance, can be quickly started in a short time, and has low-temperature resistance. It is applied to winter wastewater in northern regions and has good treatment effect.

[0006] The technical solution of the present invention is achieved as follows: In the first aspect, the present invention provides a low-temperature-resistant Nitrosomonas, wherein the Nitrosomonas is Nitrosomonas europaea. H1, and the deposit number is CCTCC M 2024693.

[0007] On the basis of the above technical solution, preferably, the 16SrDNA nucleotide sequence of the Nitrosomonas H1 is as shown in SEQ ID NO:1.

[0008] On the basis of the above technical solution, preferably, the tolerance temperature of the Nitrosomonas H1 is 10-40°C, the salinity is 0.2%-2%, and the pH value is 4-12.

[0009] In a second aspect, the present invention provides a sewage treatment bacterial agent, comprising the above-mentioned Nitrosomonas H1.

[0010] In a third aspect, the present invention provides an application of low-temperature-resistant Nitrosomonas in the treatment of low-temperature wastewater in winter, wherein the wastewater temperature is ≥10°C.

[0011] On the basis of the above technical solution, preferably, the following steps are included:

[0012] S1, culturing Nitrosomonas H1 at 10°C for 3-5 rounds until the data is stable, then centrifuging Nitrosomonas H1 cultured to the logarithmic growth phase (10000g, 6min), removing the supernatant, and washing with sterilized saline for 3 times to obtain the mother liquor of Nitrosomonas H1;

[0013] The components of the autotrophic medium are: ammonium chloride (NH4Cl) 0.4-0.6 g / L, magnesium sulfate (MgSO4) 0.5-0.7 g / L, potassium dihydrogen phosphate (K2HPO4) 0.1-0.2 g / L, sodium bicarbonate (NaHCO3) 0.5-1.5 g / L, ferric chloride hexahydrate (FeCl3·6H2O) 0.34-0.70 g / 10 mL, EDTA 0.12-0.56 g / 10 mL (added separately to 1 L of culture medium after sterilization), sodium chloride (NaCl) 0.2-2 g / L, trace element stock solution 1 mL, distilled water 1000 mL, pH 6-9;

[0014] The components of the trace element stock solution are: 0.1 g / L of copper sulfate pentahydrate (CuSO4·5H2O), 0.5 g / L of zinc sulfate heptahydrate (ZnSO4·7H2O), 0.5 g / L of cobalt chloride hexahydrate (CoCl2·6H2O), 0.2 g / L of manganese sulfate tetrahydrate (MnSO4·4H2O), 0.1 g / L of sodium molybdate dihydrate Na2MoO4·2H2O, and 0.1 / L of boric acid (H3BO3);

[0015] Prepare autotrophic culture medium: sterilize the culture medium in an autoclave at 103.4 kPa and 121° C. for 20 min, and cool to room temperature;

[0016] S2, adding the mother liquor of Nitrosomonas H1 obtained into the autotrophic culture medium cooled to room temperature for fermentation culture to obtain the liquid bacterial liquid of Nitrosomonas H1; then adding the liquid bacterial agent of Nitrosomonas H1 obtained by fermentation into the low-temperature wastewater.

[0017] The low-temperature-resistant Nitrosomonas and its application of the present invention have the following beneficial effects compared with the prior art:

[0018] (1) The low-temperature-resistant Nitrosomonas europaea. H1 of the present invention has the characteristic of being resistant to low temperatures, grows well at 10°C, and has good ammonia oxidation ability, and is suitable for treating low-temperature wastewater in northern winter without the need to increase the temperature or take other measures to maintain the stability of the biochemical system. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is the morphological characteristics of Nitrosomonas H1 under a scanning electron microscope;

[0021] Figure 2 This is a graph of the denitrification performance of Nitrosomonas H1 at different salinities;

[0022] Figure 3 This is a graph showing the denitrification performance of Nitrosomonas H1 at different temperatures;

[0023] Figure 4 This is a graph of the denitrification performance of Nitrosomonas H1 at different pH values;

[0024] Figure 5 This is a graph of the denitrification performance of Nitrosomonas H1 at different rotation speeds;

[0025] Figure 6 This is a graph showing the denitrification performance of Nitrosomonas H1 at different salinities at low temperature.

[0026] Figure 7 This is a graph showing the denitrification performance of Nitrosomonas H1 at different pH values ​​at low temperature.

[0027] Figure 8 This is a graph of the denitrification performance of Nitrosomonas H1 at different rotation speeds under low temperature.

[0028] Fig. 9 This is the application effect of Nitrosomonas H1 in low-temperature wastewater.

[0029] Figure 2-9 In the figures, A is the ammonia nitrogen denitrification diagram, and B is the nitrite nitrogen denitrification diagram. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The present invention provides a low-temperature-resistant nitrosomonas, which is Nitrosomonas europaea. H1, deposited in China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, with a deposit date of April 15, 2024, a deposit number of CCTCCNO: M 2024693, and the strain was identified as being in an active state on April 22, 2024.

[0032] The present invention also provides a sewage treatment bacterial agent comprising Nitrosomonas H1, and application of low-temperature-resistant Nitrosomonas H1 in winter low-temperature wastewater treatment.

[0033] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0034] Example 1: Isolation and identification of Nitrosomonas H1

[0035] The present invention provides low-temperature-resistant Nitrosomonas europaea. H1, which was isolated from a natural smelly water body in 2022.

[0036] 1.1 Separation method

[0037] The isolation method of Nitrosomonas H1 is as follows: take 2mL of natural malodorous wastewater and inoculate it into 100mL of sterilized autotrophic nitrification medium, place it in a constant temperature incubator (10°C, 150rpm) and culture it for 3-5 days, take 5mL of enriched bacterial solution, transfer it to an autotrophic nitrification medium containing 100mg / L ammonia nitrogen, and culture it in a constant temperature incubator. Alternately detect ammonia nitrogen and nitrite nitrogen, test ammonia oxidation performance, draw a growth curve, and perform the next round of culture at a 1% ratio during the logarithmic growth period. After repeating 3-5 rounds of enrichment culture, dilute the enriched culture and then spread it on a solid culture medium. When the colonies can be observed by the naked eye, select a single colony and inoculate it into a sterilized autotrophic culture medium for 3-5 days, and perform gradient dilution (10 -1 ~10 -8 ), select 10 -8 The diluted solution is expanded and cultured, and the plate is spread to confirm that there are no foreign bacteria, thus obtaining a pure culture of low-temperature-resistant Nitrosomonas H1.

[0038] The autotrophic nitrification medium for strain enrichment and separation includes the following components: ammonium chloride (NH4Cl) 0.4g / L, magnesium sulfate (MgSO4) 0.05g / L, dipotassium hydrogen phosphate (K2HPO4) 0.01g / L, sodium bicarbonate (NaHCO3) 0.5g / L, ferric chloride hexahydrate (FeCl3·6H2O) 0.052g / 10mL (added separately to 1L of culture medium after sterilization), sodium chloride (NaCl) 2g / L, trace elements 1mL, distilled water 1000mL, pH 7-8.

[0039] The formula of trace element stock solution is: copper sulfate pentahydrate (CuSO4·5H2O) 0.1g / L, zinc sulfate heptahydrate (ZnSO4·7H2O) 0.5g / L, cobalt chloride hexahydrate (CoCl2·6H2O) 0.5g / L, manganese sulfate tetrahydrate (MnSO4·4H2O) 0.2g / L, sodium molybdate dihydrate Na2MoO4·2H2O 0.1g / L, boric acid (H3BO3) 0.1 / L.

[0040] The solid culture medium is prepared by adding 1.5% by mass of agar powder to the enriched culture medium.

[0041] 1.2 Identification of bacterial species

[0042] 1.2.1 Bacterial morphology and physiological and biochemical characteristics

[0043] Bacterial morphological characteristics: The colony morphology of strain H1 is transparent and light yellow, the size of a needle tip, and the cells are rod-shaped, 1 to 1.5 μm long and about 0.5 μm wide. The cell suspension of the strain is light yellow during enrichment culture (see Figure 1 ).

[0044] Physiological and biochemical characteristics: Strain H1 is a Gram-negative bacterium with good ammonia oxidation ability.

[0045] 1.2.2 16S rDNA Identification of Strain H1

[0046] The bacterial genome was used as a DNA template, and 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used as upstream and downstream primers for 16S rRNA amplification. The PCR reaction conditions were: 94°C pre-denaturation for 4 min; 94°C denaturation for 25 s, 58°C annealing for 30 s, 72°C extension for 30 s, for a total of 25 cycles; 72°C final extension for 5 min. After amplification, the PCR product was subjected to 1% agarose gel electrophoresis to detect the integrity of the PCR product. Finally, the PCR product was sent to the sequencing department of Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0047] The effective length of the amplified strain 16S rDNA gene is about 1443bp nucleotide sequence, and the 16SrDNA sequence is shown in SEQ ID NO: 1. The sequence was entered into GenBank, and the database sequence was compared and analyzed using Blast software. The results showed that the 16S rDNA sequence of Nitrosomonas europaea. had a high similarity of 99.93%. Based on the phylogenetic analysis results of the 16S rDNA gene and the physiological and biochemical characteristics, it was identified as a new strain of Nitrosomonas europaea., named Nitrosomonas europaea. H1 (hereinafter referred to as "strain H1"); and the strain was deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2024693.

[0048] Example 2 Preparation of strain H1

[0049] The preparation method of strain H1 comprises the following steps:

[0050] S1, culturing Nitrosomonas H1 at 0.2% salinity and 10°C for 3-5 rounds until the data is stable, then centrifuging Nitrosomonas H1 cultured to the logarithmic growth phase, removing the supernatant, and washing three times with sterilized physiological saline to obtain the mother solution of Nitrosomonas H1;

[0051] S2, preparing an autotrophic nitrification medium, sterilizing the medium in an autoclave at 103.4 kPa and 121° C. for 20 min, and cooling to room temperature;

[0052] S3, adding the mother liquor of Nitrosomonas H1 obtained in step S1 to the culture medium cooled to room temperature in step S2 for fermentation culture to obtain a liquid bacterial liquid of Nitrosomonas H1, i.e., a bacterial suspension.

[0053] Example 3 Bacterial strain H1 test

[0054] 3.1 Denitrification effect of strain H1 at different salinities

[0055] Under the conditions of ammonia nitrogen concentration of 100 mg / L, temperature of 30°C, and pH of 8, a bacterial suspension of strain H1 was inoculated into a 250 mL triangular flask containing 100 mL of autotrophic nitrification medium at a 1% inoculation ratio, and cultured at different salinities (0.2%, 0.5%, 1%, 1.5%, and 2%). The shaking speed during the culture process was set to 150 rpm. During the culture process, ammonia nitrogen and nitrite nitrogen were detected alternately, the denitrification performance was tested, and the growth curve was drawn. The results are shown in Figure 2 .

[0056] like Figure 2 As shown, the strain can grow and show autotrophic nitrification activity in the salinity range of 0.2%-2%, and the ammonia nitrogen removal rate can reach 57.78%-100% in 72 hours. The data show that strain H1 can remove ammonia nitrogen in 72 hours at salinity below 1%.

[0057] 3.2 Denitrification effect of H1 strain at different temperatures

[0058] Under the conditions of ammonia nitrogen concentration of 100 mg / L, salt concentration of 0.2%, and pH of 8, a bacterial suspension of strain H1 was inoculated into a 250 ml triangular flask containing 100 mL of autotrophic nitrification medium at a 1% inoculation ratio, and cultured at different temperatures (10°C, 20°C, 30°C, 40°C, and 50°C). During the culture process, the shaking speed was set to 150 rpm. During the culture process, ammonia nitrogen and nitrite nitrogen were detected alternately, the denitrification performance was tested, and the growth curve was drawn. The results are shown in Figure 3 .

[0059] like Figure 3 As shown, the strain can grow in the range of 10-40°C, and as the temperature increases, the strain growth and ammonia nitrogen conversion efficiency are higher, the ammonia nitrogen degradation rate reaches more than 99% in 72 hours, and the strain stops growing at 50°C.

[0060] 3.3 Denitrification effect of strain H1 at different pH

[0061] Under the conditions of ammonia nitrogen concentration of 100 mg / L, culture temperature of 30°C, and salt concentration of 0.2%, a bacterial suspension of strain H1 was inoculated into a 250 ml triangular flask containing 100 mL of autotrophic nitrification medium at a 1% inoculation ratio, and cultured at different pH values ​​(4, 6, 8, 10, 12). The speed was set to 150 rpm during the culture process. During the culture process, ammonia nitrogen and nitrite nitrogen were detected alternately, the denitrification performance was tested, and the growth curve was drawn. The results are shown in Figure 4 .

[0062] Figure 4 As shown in the results, the strain showed good growth adaptability in the pH range of 4-12. In the pH range of 6-10, the growth and ammonia nitrogen conversion efficiency of the strain increased with the increase of pH value. Within 72 hours, the ammonia nitrogen degradation rate of strain H1 was as high as more than 99%, and then the activity of the strain decreased with the increase of pH. It is worth noting that under pH = 4 and pH = 12 conditions, the growth metabolism of the strain was inhibited to a certain extent, but it still maintained a certain ammonia nitrogen conversion ability. Compared with alkaline conditions, the strain showed good acid resistance.

[0063] 3.4 Denitrification effect of strain H1 at different rotation speeds

[0064] Under the conditions of 100 mg / L ammonia nitrogen concentration, sodium acetate as carbon source, pH 8.0, culture temperature 30°C, and salt concentration 0.2%, a 250 ml triangular flask containing 100 ml autotrophic nitrification medium was inoculated with a bacterial suspension of strain H1 at a 1% inoculation ratio and cultured at different speeds (0 rpm, 50 rpm, 100 rpm, 150 rpm, 200 rpm). During the culture process, ammonia nitrogen and nitrite nitrogen were detected alternately, the denitrification performance was tested, and the growth curve was drawn. The results are shown in Figure 5 .

[0065] like Figure 5 As shown in the figure, within the shaking table speed range of 0-200rpm, the experiment observed that the removal efficiency of ammonia nitrogen by strain H1 increased significantly with the increase of the speed. When the shaking table speed exceeded 50rpm, the ammonia nitrogen degradation rate of strain H1 within 72 hours was maintained at about 100%. Further increasing the speed to above 100rpm, strain H1 only took 48 hours to completely degrade ammonia nitrogen with an initial concentration of 100mg / L. These results show that the optimization of the shaking table speed has a significant effect on improving the efficiency of strain H1 in removing ammonia nitrogen.

[0066] Example 4 Tolerance of strains to salinity, rotation speed, and pH at 10°C

[0067] 4.1 Denitrification effect of strain H1 at low temperature and different salinities

[0068] Under the conditions of ammonia nitrogen concentration of 100 mg / L, temperature of 10°C, and pH of 8, a bacterial suspension of strain H1 was inoculated into a 250 mL triangular flask containing 100 mL of autotrophic nitrification medium at a 1% inoculation ratio, and cultured at different mass percentage salinities (0.2%, 0.5%, 1%, 1.5%, and 2%). The shaking speed during the culture process was set to 150 rpm. Ammonia nitrogen and nitrite nitrogen were detected every 24 hours, the denitrification performance was tested, and the growth curve was drawn. The results are shown in Figure 6 .

[0069] Figure 6 As shown, under the synergistic effect of low temperature and high salt, the denitrification performance of strain H1 decreased. At 10°C and 0.2% salinity, strain H1 could completely metabolize 100 mg / L of ammonia nitrogen within 72 hours. With the increase of salinity, the performance decreased significantly. It could still metabolize ammonia nitrogen at 2% salinity, but the conversion rate was less than 10% in 120 hours.

[0070] 4.2 Denitrification effect of strain H1 at low temperature and different pH

[0071] Under the conditions of ammonia nitrogen concentration of 100 mg / L, culture temperature of 10°C, and salt concentration of 0.2%, a 250 ml triangular flask containing 100 ml of autotrophic nitrification medium was inoculated with a bacterial suspension of strain H1 at a 1% inoculation ratio, and cultured at different pH values ​​(4, 6, 8, 10, 12). The speed was set to 150 rpm during the culture process. Ammonia nitrogen and nitrite nitrogen were detected every 24 hours, the denitrification performance was tested, and the growth curve was drawn. The results are shown in Figure 7 .

[0072] Figure 7 As shown, under low temperature and alkaline environment, strain H1 was significantly inhibited and stopped growing at pH = 12, but it still had a certain denitrification efficiency under acidic conditions. Strain H1 could tolerate a minimum pH of 4 under low temperature conditions, and at pH = 6, the strain could metabolize 100 mg / L ammonia nitrogen within 120 hours.

[0073] 4.3 Denitrification effect of strain H1 at low temperature and different rotation speeds

[0074] Under the conditions of 100 mg / L ammonia nitrogen concentration, sodium acetate as carbon source, pH 8.0, culture temperature 10°C, and salt concentration 0.2%, a 250 ml triangular flask containing 100 ml of the above culture medium was inoculated with a bacterial suspension of strain H1 at a 1% inoculation ratio and cultured at different speeds (0 rpm, 50 rpm, 100 rpm, 150 rpm, 200 rpm). Ammonia nitrogen and nitrite nitrogen were detected every 24 hours, denitrification performance was tested, and growth curves were drawn. The results are shown in Figure 8 .

[0075] like Figure 8As shown in the figure, at 0 speed, the growth of the strain was significantly inhibited and was in a stagnant period within 120 hours. After that, the denitrification ability increased with the increase of speed. The optimal speed was still 150 rpm. Under the conditions of 100 rpm and 200 rpm, strain H1 had almost similar denitrification rates.

[0076] Example 5 Effect of application of strain H1 in low-temperature wastewater

[0077] 30L of sewage was collected from a garbage transfer station in Xiaoting District, Yichang City, Hubei Province. The initial water quality parameters of the sewage were: ammonia nitrogen 2236mg / L, nitrite nitrogen 30mg / L, and salinity 2%. The sewage was divided into 6 plastic cups with a volume of 8L, 3 of which were added with 1% bacterial suspension of strain H1 (OD600≈0.01), and the other 3 were used as control groups (no bacterial agent was added); all barrels were placed in a 10℃ incubator for cultivation, and aerated with a microporous sand core air head at a ventilation volume of 180mL / min; during the test, the changes of ammonia nitrogen and nitrite nitrogen in the system were regularly detected, and the denitrification ability of the strain was analyzed. The results are shown in Fig. 9 .

[0078] Fig. 9 As shown, after 9 days, the ammonia nitrogen removal rate of the bacteria-added group reached 100%, while almost no ammonia nitrogen was removed in the control group. The denitrification efficiency of the strain in the sewage was very significant; this shows that the strain H1 of the present invention exhibits good resistance to low-temperature ammonia oxidation.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0080]

[0081]

Claims

1. A low-temperature resistant Nitrosomonas, characterized in that: The nitrosomonas is Nitrosomonas europaea. H1, and its preservation number is CCTCC NO: M 2024693.

2. A low-temperature resistant Nitrosomonas according to claim 1, characterized in that: The 16SrDNA nucleotide sequence of the Nitrosomonas H1 is shown in SEQ ID NO:

1.

3. The low-temperature resistant Nitrosomonas according to claim 1, characterized in that: The nitrosomonas H1 has a tolerance temperature of 10-40°C, a salinity of 0.2%-2%, and a pH value of 4-12.

4. A sewage treatment bacterial agent, characterized in that: The method comprises the Nitrosomonas H1 according to any one of claims 1 to 3.

5. The use of a low-temperature resistant Nitrosomonas according to any one of claims 1 to 3 in the treatment of low-temperature wastewater in winter, characterized in that: Wastewater temperature ≥10℃.

6. The use of a low-temperature resistant Nitrosomonas according to claim 5 in the treatment of low-temperature wastewater in winter, characterized in that: The following steps are involved: S1, culturing Nitrosomonas H1 at 10°C to the logarithmic growth phase, centrifuging, removing the supernatant, and washing to obtain the mother liquor of Nitrosomonas H1; S2, fermenting and culturing the mother liquor of Nitrosomonas H1 obtained in step S1, and then adding it to low-temperature wastewater to degrade ammonia nitrogen.

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