Microbial agent for removing nitrate nitrogen at low temperature as well as preparation method and application of microbial agent

Abstract

The invention discloses a microbial agent for removing nitrate nitrogen at low temperature as well as a preparation method and application of the microbial agent, and belongs to the technical field of microorganisms. According to the invention, experiments prove that the pseudomontella GBW-HB2501 has the functions of low temperature resistance, salt resistance and nitrate nitrogen removal; the bacterial content of the prepared microbial agent is not less than 2.0 * 10 < 9 > CFU / g; the invention also provides a preparation method of the microbial agent. In the preparation method, the bacterial sludge yield is 30-50g / L; by utilizing the functional characteristics of low temperature resistance and nitrate nitrogen removal of the strain, the prepared microbial agent is used for removing nitrate nitrogen in wastewater, the removal rate of nitrate nitrogen is higher than 99.5%, and the method is efficient and convenient. The microbial agent provided by the invention is more suitable for wastewater treatment of photovoltaic enterprises in cold regions, solves the problem that the existing microbial denitrification technology is low in efficiency in low-temperature and high-salt environments, and has good application prospects and values.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a microbial agent for removing nitrate nitrogen at low temperature and a preparation method and application thereof. BACKGROUND

[0002] With the rapid development of the photovoltaic industry, high nitrate nitrogen wastewater (such as pickling and etching wastewater) generated in the production process has become a prominent environmental problem. Such wastewater usually contains high concentrations of nitrate (NO3⁻-N>100 mg / L), fluoride and salt, and is affected by seasons, with water temperature decreasing to below 15℃ in winter. At present, traditional biological denitrification processes generally have problems such as low microbial activity and significantly reduced denitrification efficiency (<50%) under low temperature conditions, while chemical treatment is high in cost and easy to cause secondary pollution.

[0003] Although the existing low-temperature resistant denitrifying bacteria (such as Pseudomonas Pseudomonas putida ) have certain low-temperature adaptability, their activity is obviously inhibited (degradation rate <40%) in high-salt and fluoride-containing photovoltaic wastewater. In addition, the survival rate of conventional microbial agents is low (<60%) in the freeze-drying process, which further limits their engineering application. Therefore, it is of great significance to develop a strain that has low-temperature resistance, high-salt resistance and can efficiently degrade nitrate nitrogen in photovoltaic wastewater, to solve the industry pain points. SUMMARY

[0004] To solve the above problems, the application aims to provide a microbial agent for removing nitrate nitrogen at low temperature and a preparation method and application thereof.

[0005] To achieve the above application purposes, the application is implemented by the following technical solutions: The application provides a microbial agent for removing nitrate nitrogen at low temperature, which is prepared by fermentation of Pseudomonas mendei GBW-HB2501, and the bacterial content of the microbial agent is not less than 2.0x10 9 CFU / g.

[0006] Further, the classification name of the Pseudomonas mendei GBW-HB2501 is Pseudomonas mendei Pseudomonas mandelii , and the accession number is CGMCC No. 34229.

[0007] Further, the 16S rDNA sequence of the Pseudomonas mendei GBW-HB2501 is shown in SEQ ID No. 1.

[0008] Further, the Pseudomonas mendei GBW-HB2501 can remove nitrate nitrogen in wastewater at 3-15℃.

[0009] Further, the salt tolerance concentration of the Pseudomonas monteilii GBW-HB2501 is 0.5%-5%; Further, based on the salt tolerance concentration, the viable bacterial count of the Pseudomonas monteilii GBW-HB2501 is 4×10 9 -5.5×10 9 CFU / mL.

[0010] The application also provides a preparation method of the microbial agent, and specifically as follows: (1) Seed liquid preparation: inoculate the Pseudomonas monteilii GBW-HB2501 into a nutrient broth to culture and prepare a seed liquid; (2) Fermentation culture: inoculate the seed liquid into a fermentation tank to ferment and prepare a fermentation bacterial liquid; (3) Centrifugal treatment: centrifuge the fermentation bacterial liquid to obtain a bacterial slurry; (4) Emulsion preparation: stir the bacterial slurry and a protective agent to prepare an emulsion; (5) Freeze-drying: vacuum freeze-dry the emulsion to obtain Pseudomonas monteilii GBW-HB2501 bacterial blocks, and then crush and sieve the bacterial blocks to obtain a bacterial powder; (6) Microbial agent preparation: compound the bacterial powder and a carrier to obtain a microbial agent.

[0011] Further, the formula of the fermentation medium in the fermentation tank in the step (2) is as follows: sodium acetate 44-46 g / L, proteose peptone 28-30 g / L, beef extract 14-16 g / L, sodium chloride 1.5-2 g / L, potassium phosphate dibasic 1.2-1.4 g / L, magnesium sulfate 1.2-1.4 g / L, calcium chloride 0.1-0.3 g / L, and defoaming agent 1-1.5 g / L, and the pH value is 6.5-7.5.

[0012] Further, the fermentation conditions in the fermentation tank in the step (2) are as follows: adjust the tank pressure to 0.05-0.15 MPa, keep the temperature at 20-25℃, the dissolved oxygen is ≥30%, the stirring speed is 180-200 rpm, and the fermentation time is 18-20 h.

[0013] Further, the yield of the bacterial slurry in the step (3) is 30-50 g / L.

[0014] Further, the mass ratio of the bacterial slurry and the protective agent in the step (4) is 1:1-3.

[0015] Further, the formula of the protective agent in the step (4) is as follows: in terms of weight percentage, skimmed milk powder 15%, trehalose 10%, lactose 5%, β-cyclodextrin 2.5%, glycerol 3%, L-cysteine hydrochloride 2.5%, and the rest is distilled water.

[0016] Further, the temperature of vacuum freeze drying in step (5) is -40 to -50 DEG C.

[0017] Further, the carrier is at least one of calcium carbonate, talcum powder and diatomite.

[0018] Further, the mass ratio of the bacterial powder to the carrier in step (6) is 1:90-120.

[0019] Further, the bacterial content of the fermentation bacterial solution is not less than 2.0*10 10 CFU / mL; and the bacterial content of the bacterial powder is not less than 2*10 11 CFU / g.

[0020] The application further provides application of the microbial agent in removal of nitrate nitrogen in wastewater.

[0021] Further, the environmental temperature for removing the nitrate nitrogen is 3-13 DEG C, and the removal rate of the nitrate nitrogen is >96%.

[0022] Further, the removal rate of the nitrate nitrogen is >98% when the microbial agent is at 8-12 DEG C.

[0023] Further, the removal rate of the nitrate nitrogen is >99.5% when the microbial agent is at 12 DEG C.

[0024] Further, the conditions of the nitrate nitrogen in the wastewater are that the temperature is 3-5 DEG C, the pH is 7.0-8.0, the dosage concentration of the microbial agent is 90-200 ppm / L based on the volume of the wastewater, and the use time is 1-8 days.

[0025] Further, the method for removing the nitrate nitrogen in the wastewater by the microbial agent is that the microbial agent is directly added to the water inlet of a biochemical system or an aerobic tank, and the dissolved oxygen is >2 mg / L.

[0026] Further, the removal rate of the nitrate nitrogen in the wastewater is >99.5%.

[0027] Further, the wastewater includes photovoltaic wastewater.

[0028] Compared with the prior art, the application has the following advantages and technical effects: 1. The Pseudomonas mendei GBW-HB2501 is separated from an aerobic tank of chemical wastewater, has a salt concentration tolerance of 0.5-5% NaCI, and can grow normally at a temperature of 4-25 DEG C, so that the Pseudomonas mendei GBW-HB2501 has the growth characteristics of low-temperature tolerance and salt tolerance.

[0029] 2、The microbial agent is prepared by fermentation of Pseudomonas monteilii GBW-HB2501, and the bacterial content is not less than 2.0*10 9 The microbial agent can quickly remove nitrate nitrogen in a low-temperature environment of 4-15 DEG C, and is applied to winter to improve the efficiency of nitrate nitrogen removal in a low-temperature water environment. Experimental verification shows that the nitrate nitrogen removal rate of the microbial agent can be as high as 99.62% at 12 DEG C, thereby effectively solving the problems of low denitrification efficiency and poor stability of a sewage biochemical system caused by low temperature.

[0030] 3、The microbial agent can effectively remove nitrate nitrogen in photovoltaic wastewater, and the removal rate of the nitrate nitrogen in the photovoltaic wastewater is more than 99.5% under the conditions of a temperature of 3-5 DEG C and a pH of 7.0-8.0. Further, the microbial agent can ensure the operation stability of a sewage treatment system in a low-temperature environment, has strong adaptability and fast start-up, and therefore has wide application prospect, market application potential and value. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Figure 1 is a colony morphology of Pseudomonas monteilii GBW-HB2501; Figure 2 Figure 3 is a gene sequence phylogenetic tree of Pseudomonas monteilii GBW-HB2501; Figure 3 Figure 5 is a photovoltaic wastewater nitrate nitrogen degradation application test of Pseudomonas monteilii GBW-HB2501 under a water temperature condition of 4 DEG C; Figure 4 Figure 6 is temperature measurement of the photovoltaic wastewater nitrate nitrogen degradation application test of Pseudomonas monteilii GBW-HB2501 under a water temperature condition of 4 DEG C; Figure 5 Figure 7 is the influence of Pseudomonas monteilii GBW-HB2501 on the nitrate nitrogen content of photovoltaic wastewater under a water temperature condition of 4 DEG C. DETAILED DESCRIPTION

[0032] The technical solutions of the present application are further described in combination with the following specific examples.

[0033] In the following examples, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies, unless otherwise specified.

[0034] Example 1: Screening and identification of Pseudomonas monteilii GBW-HB2501 1. Strain screening and separation The activated sludge of the aerobic section of a chemical plant in Binzhou was selected for strain enrichment and screening. 10 ml of activated sludge was added to a 250 ml conical flask containing 100 ml of enrichment medium, and the flask was incubated in a shaking incubator at 30°C and 200 rpm for 12 hours. The composition of the enrichment medium was as follows: ammonium chloride 1 g / L, sodium acetate 3.5 g / L, potassium hydrogen phosphate 0.2 g / L, potassium dihydrogen phosphate 0.2 g / L, sodium chloride 0.12 g / L, manganese sulfate monohydrate 0.01 g / L, ferrous sulfate heptahydrate 0.01 g / L, and the pH was 7.0-7.5.

[0035] The enrichment culture was diluted using gradient dilution plate coating method. 100 ul of bacterial enrichment liquid with 5 concentration gradients was coated on BTB agar plates, and the plates were incubated at 30°C for 12 hours. BTB medium was used for screening because BTB medium turns blue when it comes into contact with alkali, and aerobic denitrifying bacteria produce alkali. Therefore, BTB medium was used for screening aerobic denitrifying bacteria based on the color change principle. The composition of the BTB agar medium was as follows: sodium citrate 8.5 g / L, potassium nitrate 1 g / L, potassium dihydrogen phosphate 1 g / L, calcium chloride 0.2 g / L, magnesium sulfate 1 g / L, ferrous sulfate 0.5 g / L, 1% bromothymol blue 1 mL, and agar 20 g / L, and the pH was 7.0-7.3. Finally, 6 strains of bacteria with different morphologies were obtained on the color-changing BTB plates, and single colonies were picked and purified on BTB agar plates. The single colonies were stored in a 4°C refrigerator for future use.

[0036] 2. Strain preliminary screening The 6 strains of bacteria were named GBW-HB2501, GBW-HB2502, GBW-HB2503, GBW-HB2504, GBW-HB2505, and GBW-HB2506, respectively.

[0037] (1) Preparation of activation solution: In a sterile environment, the above 6 strains of bacteria were inoculated into 5 mL of nutrient broth test tubes (sterilized in a 116°C autoclave for 30 minutes) and incubated in a shaking incubator at 30°C and 200 rpm for 12 hours.

[0038] (2) 1 mL of the activated bacterial solution was added to 100 mL of nitrate nitrogen degradation evaluation medium after washing, and 3 parallel samples were prepared for each group. The control group did not contain any bacterial agent. The nitrate nitrogen content of each group was measured after 24 hours, and the denitrification rate was calculated. The composition of the nitrate nitrogen degradation evaluation medium was as follows: glucose 2 g / L, potassium nitrate 0.722 g / L, disodium hydrogen phosphate dodecahydrate 10.55 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.1 g / L, and trace element solution 10 ppm, and the pH was 7.0. The experimental results are as follows: Table 1. Nitrate nitrogen degradation evaluation results - nitrate nitrogen content (mg / L)

[0039] From the experimental results, the strain GBW-HB2501 has obvious denitrification effect on nitrate nitrogen, and the denitrification rate can reach 86.66% after 24h of reaction, so the strain GBW-HB2501 is selected as the target strain for aerobic denitrification experimental study.

[0040] 3. Strain identification (1) Colony and cell characteristics As shown in Figure 1 , the colony of the strain GBW-HB2501 is round, white, small, regular edge, smooth surface, and easy to pick up.

[0041] (2) Molecular genetic characteristics The DNA of the strain Pseudomonas GBW-HB2501 was used as a template, and the 16S rDNA universal primer was used for amplification, and the amplified fragment was sequenced. The 16S rDNA sequencing result is shown as SEQ ID NO. 1. The obtained 16S rDNA sequence of the strain GBW-HB2501 was compared and analyzed with the sequence in NCBI GenBank by Blast, and a phylogenetic tree was constructed, and the result is shown in Figure 2 , the strain GBW-HB2501 has the highest homology with Pseudomonas mandelii, so the strain GBW-HB2501 is determined as Pseudomonas mandelii.

[0042] (3) Physiological and biochemical identification According to the physiological and biochemical detection methods in "Berger Bacterium Identification Manual" and "Common Bacterium System Identification Manual", the physiological and biochemical identification test of Pseudomonas mandelii GBW-HB2501 was carried out, and the results are as follows.

[0043] Table 2. Physiological and biochemical identification results of Pseudomonas mandelii GBW-HB2501

[0044] Note: + is positive, ± indicates that the result is not obvious, and - is negative.

[0045] (4) Preservation of Pseudomonas mandelii GBW-HB2501 strain The screened Pseudomonas monteilii GBW-HB2501 is preserved, and the preservation unit of the Pseudomonas monteilii GBW-HB2501 is the General Microbiological Center of China Microbiological Bacteria Preservation Management Committee (CGMCC), located at No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology of Chinese Academy of Sciences; the preservation date is April 16, 2025; and the preservation number of the Pseudomonas monteilii is CGMCC No. 34229. Pseudomonas mandelii

[0046] Example 2: Salinity tolerance experiment of Pseudomonas monteilii GBW-HB2501 In order to verify whether the Pseudomonas monteilii GBW-HB2501 can perform in high salinity conditions, the effective viable cell number of the culture solution under different salinity conditions is analyzed and detected. NB is used as a basic culture medium, and different amounts of sodium chloride in the NB culture medium are adjusted to prepare test culture media with different salt concentrations. Then the Pseudomonas monteilii GBW-HB2501 cultured on a slant is inoculated into the NB culture medium with different salt concentrations, and cultured in a constant temperature incubator at 25°C, 200r / min, pH 7.0-8.0 for 18h to obtain the culture solution of Pseudomonas monteilii GBW-HB2501. The effective viable cell number of Pseudomonas monteilii GBW-HB2501 after being cultured in different salt concentration culture media is analyzed and detected. The results are shown in Table 3, and the Pseudomonas monteilii GBW-HB2501 has good salt-tolerant growth characteristics and can grow well in the salinity range of 0.5-5%.

[0047] Table 3. Growth of Pseudomonas monteilii GBW-HB2501 under different salt concentrations

[0048] Example 3: Preparation of microbial inoculant The preparation method of the microbial inoculant of the low-temperature-resistant Pseudomonas monteilii GBW-HB2501 is as follows: (1) Seed liquid preparation: inoculate the Pseudomonas monteilii GBW-HB2501 into a nutrient broth for culture to obtain a Pseudomonas monteilii GBW-HB2501 seed liquid.

[0049] (2) Fermentation culture: inoculate the Pseudomonas monteilii GBW-HB2501 seed liquid into a fermenter at a volume ratio of 10%, adjust the tank pressure to 0.05MPa, keep the temperature at 20-25°C, the dissolved oxygen ≥30%, the stirring speed is 180-200rpm, and the fermentation time is 18-20h. The Pseudomonas monteilii GBW-HB2501 fermentation broth is obtained.

[0050] ​(3) Centrifugal treatment: the Pseudomonas mesoatlantica GBW-HB2501 fermentation broth obtained in step (2) is subjected to centrifugal treatment by a tubular centrifuge at a flow rate of 150 L-200 L / h to obtain Pseudomonas mesoatlantica GBW-HB2501 slurry, and the yield of the slurry is measured to be 30-50 g / L.

[0051] (4) Preparation of protective agent: the components in the protective agent are mixed in a weight percentage ratio of 15% skimmed milk powder, 10% trehalose, 5% lactose, 2.5% β-cyclodextrin, 3% glycerol, 2.5% L-cysteine hydrochloride, and the rest is distilled water.

[0052] (5) Addition of protective agent: the slurry obtained in step (3) is mixed with the protective agent obtained in step (4) at a weight ratio of 1:2.5, and stirred uniformly to prepare an emulsion.

[0053] (6) Freeze-drying: the emulsion obtained in step (5) is subjected to vacuum freeze-drying at a temperature of -40 to -50°C to obtain Pseudomonas mesoatlantica GBW-HB2501 blocks, and the Pseudomonas mesoatlantica GBW-HB2501 blocks are crushed by a crusher and sieved through a 60-mesh screen to obtain Pseudomonas mesoatlantica GBW-HB2501 powder.

[0054] (7) Preparation of microbial agent: the Pseudomonas mesoatlantica GBW-HB2501 powder is compounded with diatomite at a weight ratio of 1:100 to obtain a microbial agent of Pseudomonas mesoatlantica GBW-HB2501.

[0055] The bacterial content of the fermentation broth prepared by the above method is not less than 2.0 x 10 10 CFU / mL; the bacterial content of the obtained powder is not less than 2 x 10 11 CFU / g; and the bacterial content of the obtained microbial agent is not less than 2.0 x 10 9 CFU / g.

[0056] Example 4: Test of nitrate nitrogen removal efficiency under different low-temperature conditions Different low-temperature conditions are set to simulate winter low-temperature environment. The low temperatures are set to be 4°C, 8°C, and 12°C, respectively, the pH range is 7.0-8.0, the dissolved oxygen is ≥2 mg / L, and the Pseudomonas mesoatlantica GBW-HB2501 is used to treat the nitrate nitrogen in the water. The initial nitrate nitrogen concentration of each group is 100 mg / L. The concentration change of nitrate nitrogen in each group is detected during the reaction process, and the removal rate of nitrate nitrogen is calculated. Three repeated experiments are set for each concentration gradient.

[0057] The microbial inoculum prepared from Pseudomonas monteilii GBW-HB2501 was added to each test solution in the system at a dosage of 100 ppm according to the method of Example 3. The control group was not added with the microbial inoculum. Then, the control group and the experimental group were placed in different temperature conditions for 72 h of nitrate removal experiment.

[0058] The results are shown in Table 4. After 72 h of treatment, Pseudomonas monteilii GBW-HB2501 can effectively remove nitrate under different low temperature conditions, and the nitrate removal rate is higher than 95%. The experiment shows that Pseudomonas monteilii GBW-HB2501 can effectively degrade nitrate under different low temperature conditions. The ability of the bacteria to quickly remove nitrate in wastewater at low temperature has important practical application value.

[0059] Table 4 Nitrate removal rate test results of Pseudomonas monteilii GBW-HB2501 under low temperature conditions

[0060] Example 5: Photovoltaic wastewater nitrate removal efficiency test under 4°C conditions The Pseudomonas monteilii GBW-HB2501 was combined with actual photovoltaic wastewater for application verification. The water sample was provided by a silicon wafer processing plant in Xingtai, Hebei Province for winter low temperature nitrate degradation application test. The nitrate in the influent of the photovoltaic wastewater biochemical system of the silicon wafer processing plant was 462.8 mg / L, and the nitrate in the effluent was 30.25 mg / L, which was far more than the discharge requirement of <15 mg / L. 1 L of influent water sample was taken from the biochemical system and added to a 2 L plastic container, 100 ppm of microbial inoculum of Pseudomonas monteilii GBW-HB2501 (prepared in Example 3) was added and stirred uniformly. An oxygenation pump was used to introduce air, and the device was placed in a low temperature environment for aeration culture. Three groups of parallel experiments were set up, and the experimental device is as shown in Figure 3 The water temperature of this experiment was about 4°C as shown in Figure 4 The experimental results are shown in Figure 5 After 7 days of aeration culture, the nitrate content decreased from the initial 462.8 mg / L to 1.06 mg / L, and the degradation rate of nitrate was 99.77%. The results show that the microbial inoculum prepared from Pseudomonas monteilii GBW-HB2501 can efficiently degrade nitrate in photovoltaic wastewater under low temperature conditions.

[0061] In summary, the indoor test and the application evaluation results of the water sample taken in the field both show that the Pseudomonas monteilii GBW-HB2501 can significantly enhance and improve the removal ability of nitrate nitrogen in photovoltaic wastewater under low temperature conditions, and the preparation operation is simple and convenient to apply, and meanwhile has the characteristics of strong system adaptability and fast start, so it has broad application prospect and market application potential and value.

[0062] The above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified or some technical features can be replaced by equivalents for ordinary skilled in the art; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. A microbial agent for removing nitrate nitrogen at low temperature, characterized in that, The microbial inoculant is prepared by fermentation of *Pseudomonas meningitidis* GBW-HB2501, and the microbial content of the inoculant is not less than 2.0 × 10⁻⁶. 9 CFU / g.

2. The microbial agent for removing nitrate nitrogen at low temperature according to claim 1, characterized in that, The preservation number of the *Pseudomonas moniliforme* GBW-HB2501 is CGMCC No. 34229.

3. The microbial agent for low-temperature nitrate removal according to claim 1, characterized in that, The *Pseudomonas meningitidis* GBW-HB2501 strain can remove nitrate nitrogen from wastewater at 3-15°C.

4. The method for preparing the microbial inoculant according to claim 1, characterized in that, The preparation method specifically includes the following steps: (1) Seed culture preparation: Pseudomonas meningitidis GBW-HB2501 was inoculated into nutrient broth to prepare seed culture; (2) Fermentation culture: The seed liquid is inoculated into a fermenter for fermentation to prepare a fermentation liquid; (3) Centrifugation: The fermentation liquid is centrifuged to obtain bacterial sludge; (4) Emulsion preparation: The bacterial sludge and the protective agent are stirred to prepare an emulsion; (5) Freeze-drying: The emulsion was freeze-dried under vacuum to obtain Pseudomonas meningitidis GBW-HB2501. The mushroom blocks are then crushed and sieved to obtain mushroom powder. (6) Preparation of microbial inoculants: The microbial powder and the carrier are compounded to obtain microbial inoculants.

5. The method for preparing the microbial inoculant according to claim 4, characterized in that, In step (4), the mass ratio of the fungal mud and the protective agent is 1:1~3.

6. The method for preparing the microbial inoculant according to claim 4, characterized in that, In step (6), the carrier is at least one of calcium carbonate, talc and diatomaceous earth, and the mass ratio of the bacterial powder to the carrier is 1:90-120.

7. The method for preparing the microbial inoculant according to claim 4, characterized in that, The bacterial content of the fermentation broth is not less than 2×10⁻⁶. 10 CFU / mL; the bacterial content of the bacterial powder is not less than 2×10⁻⁶. 11 CFU / g.

8. The application of the microbial agent according to claim 1 or 2 in the removal of nitrate nitrogen from wastewater.

9. The application according to claim 8, characterized in that, The wastewater is treated under the following conditions: temperature 3-25℃, pH 7.0-8.0; the concentration of the microbial agent is 90-200 ppm / L based on the volume of wastewater, and the treatment period is 1-8 days.

10. The application according to claim 8, characterized in that, The method for removing nitrate nitrogen from wastewater using the microbial agent is as follows: the microbial agent is directly added to the inlet of the biochemical system or aerobic tank.

Images