CN116553742A - Method for treating high-salt low-temperature wastewater by nitrifying bacteria and denitrifying bacteria - Google Patents

Abstract

The invention discloses a method for treating high-salt low-temperature wastewater by nitrifying bacteria and denitrifying bacteria, which adopts paracoccus denitrificans TD-20229 screened from ammonia nitrogen polluted water bodies to perform fermentation treatment on the wastewater in a high-salt low-temperature environment simultaneously in cooperation with alcaligenes FC-01052. The production strain can grow in high-salt and low-temperature wastewater environment, has better denitrification effect, and has the problems of low cost, secondary pollution reduction and the like compared with the traditional physical, chemical and other denitrification methods. The invention uses heterotrophic nitrifying bacteria and aerobic denitrifying bacteria to obviously reduce wastewater pollution from the biological denitrification technology, solves the problem of treatment of pesticide wastewater in a low-temperature high-salt environment, and has wide market prospect.

Description

A method for treating high-salt and low-temperature wastewater with nitrifying bacteria and denitrifying bacteria

technical field

The invention relates to the field of biotechnology, in particular to a method for treating high-salt and low-temperature wastewater with nitrifying bacteria and denitrifying bacteria.

Background technique

In recent years, human living standards have been rapidly improved, the economy has developed rapidly, and human production activities are frequent. The water environment is affected by human activities. It is rich in excessive nitrogen content, causing serious pollution of water resources and water bodies in my country, and greatly endangering the safety of ecosystems. Therefore, improving water resources and finding efficient denitrification methods are the key to the safety of water quality. Controlling the nitrogen content in water is the key to controlling water pollution, and the key to advanced treatment and recycling of sewage. The traditional denitrification method has disadvantages such as lengthy process, high cost, high energy consumption, and the need to add alkaline substances for neutralization. In recent years, the research on denitrification microorganisms has become more and more in-depth. As a new type of biological denitrification technology, the simultaneous reaction of heterotrophic nitrification-aerobic denitrification has also been gradually applied to the treatment of wastewater pollution. It is also an economical, efficient, and Environmentally friendly denitrification process.

Heterotrophic nitrification-aerobic denitrification bacteria break through the problem of oxygen demand in the denitrification process of the traditional biological nitrification box, strictly control and limit oxygen, can complete the process of nitrification and denitrification in the same reactor, and can remove COD and Reduce the content of ammonia nitrogen, control the nitrogen content, no need to adjust the acid and alkali, and at the same time, it can repair the water environment through its own growth metabolism, adsorption and mineralization of pollutants. Under aerobic conditions, nitrifying bacteria will degrade NH ₄ ⁺ -N or organic nitrogen into nitrite nitrogen and nitrate nitrogen, and then complete the denitrification process through denitrifying bacteria. Heterotrophic nitrifying bacteria have the advantages of strong adaptability and can grow with a variety of organic matter as carbon sources, so they are widely used by researchers. Aerobic denitrifying bacteria are a type of denitrifying bacteria that use the action of aerobic denitrifying enzymes to perform denitrification under aerobic conditions. Adding the cultivated aerobic denitrifying bacteria to the sewage treatment facilities can improve the effect of sewage denitrification treatment, and the aerobic denitrifying bacteria can effectively and quickly remove the nitrate inside the polluted water body. However, microbial denitrification is a series of denitrification reactions catalyzed by enzymes, which are affected by many factors, including wastewater temperature, dissolved oxygen, pH value, external carbon source, trace elements, etc. Among them, dissolved oxygen, pH value, exposure The amount of bacteria, carbon source, etc. can be adjusted through the process to improve the denitrification effect. Appropriate denitrification conditions can promote the growth and metabolism of nitrifying bacteria and denitrifying bacteria and the key conditions for improving the efficiency of nitrogen removal. At present, the prior art discloses that related nitrifying bacteria and denitrifying bacteria have problems such as poor denitrification effect, inability to grow and reproduce rapidly in low temperature environment and alkaline environment, thus reducing the sensitivity to ammonia nitrogen, NO ₂ ^- -N, NO ^3- -N removal effect. The nitrifying bacteria used in the prior art include Pseudomonas, Acinetobacter, and Nitrite bacteria, etc., and the denitrifying bacteria include Bacillus, Paracoccus, and Pseudomonas. The existing strains have certain removal effects on ammonium nitrogen, nitrate nitrogen and nitrite nitrogen, but their growth and denitrification effects are poor in low-temperature and high-salt environments.

Contents of the invention

The invention discloses a method for treating high-salt and low-temperature wastewater by using nitrifying bacteria and denitrifying bacteria. Combined use of Alcaligenes and Paracoccus denitrificans, combined with the characteristics of Alcaligenes to efficiently degrade ammonia nitrogen in a low-temperature and high-salt environment, combined with Paracoccus denitrification to synergistically and efficiently degrade ammonia nitrogen and NO ₂ ^- -N in a low-temperature and high-salt wastewater environment , NO ^{3 -} -N, the denitrification effect is more obvious, and the purpose of sewage treatment is further achieved. Compared with the traditional biological denitrification technology, the method of the present invention has obvious denitrification effect, can reduce ammonia nitrogen, NO ₂ ^- -N, and NO ^{3 -} -N, and can grow better in a low-temperature and high-salt environment. The process of the invention is low in price to a certain extent, simplifies the process, and simultaneously can solve the problem of sewage treatment in a low-temperature and high-salt environment.

To achieve the above object, the technical scheme adopted in the present invention is as follows:

A method for treating high-salt low-temperature wastewater with nitrifying bacteria and denitrifying bacteria, comprising the steps of:

S1. Activate the nitrifying bacteria, insert the seed solution, and cultivate for 16-36 hours. According to the inoculum size of 1-10%, transfer it to the fermentation medium, the fermentation temperature is 5-42° C., and the fermentation time is 48-72 hours;

S2. Activate the denitrifying bacteria, insert the seed solution, and cultivate for 16-36 hours. According to the inoculum size of 1-10%, transfer it into the fermentation medium, the fermentation temperature is 5-42 °C, and the fermentation time is 48-60 hours. ;

S3. Add the two fermented liquids to the wastewater to be treated. According to the volume ratio of nitrifying bacteria and denitrifying bacteria is 1-10:1-10, first add nitrifying bacteria to treat wastewater for 10-18 hours, and then add denitrifying bacteria for 30 hours ~48h.

The nitrifying bacteria are Alcaligenes FC-01052; the denitrifying bacteria are Paracoccus denitrificans TD-20229.

Alcaligenes FC-01052 was deposited in the General Microorganism Center of China Microbiological Culture Collection Management Committee, the preservation number is CGMCC No.24294; Paracoccus denitrificans TD-20229 was deposited in China Microorganisms on September 15, 2022 General Microbiology Center of the Culture Collection Management Committee, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.25718.

The composition of the seed liquid is: glucose 5g/L, peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, pH7.

The composition of the nitrifying bacteria culture medium is: carbon source 5-30g/L, nitrogen source 5-30g/L, inorganic salt 1-10g/L, metal ion 0-2g/L, trace element 0.1-1g/L, and the rest It is water, pH4～9.0;

The composition of denitrifying bacteria medium is: carbon source 5-30g/L, nitrogen source 5-30g/L, inorganic salt 1-10g/L, metal ion 0-2g/L, trace element 0.1-1g/L, and the rest is Water, pH4～9.0.

The carbon source is at least one of glucose, trisodium citrate, sodium pyruvate, sodium acetate, molasses and the like.

The nitrogen source is at least one of ammonium chloride, ammonium nitrate, sodium nitrite, sodium nitrate, potassium nitrate, ammonium sulfate, urea, beef extract, ammonia water, corn steep liquor, fish meal and the like.

The inorganic salt is any one or a combination of magnesium salts, nitrates, potassium salts, calcium salts, phosphates or hydrochlorides.

The trace elements are ferrous sulfate 0.01-3g/L, copper sulfate 0.001-1g/L, zinc sulfate 0.01-0.5g/L, calcium chloride 0.1-2g/L, manganese sulfate 0.01-1g/L, chloride Cobalt 0.001 ~ 0.1g/L mixed.

Further preferably, the nitrifying bacteria are activated, inserted into the seed solution, cultivated for 24 hours, and transferred to the fermentation medium according to the inoculum size of 3%, and fermented and cultivated at 37°C for 48 hours;

Activate the denitrifying bacteria, insert the seed solution, and cultivate for 24 hours. According to the inoculum size of 3%, transfer it into the fermentation medium, and ferment and cultivate for 50 hours at 37°C;

Add the two fermentation liquids to the wastewater to be treated. According to the volume ratio of nitrifying bacteria and denitrifying bacteria is 1:1, first add nitrifying bacteria to treat wastewater for 10-18 hours, and then add denitrifying bacteria to treat wastewater for 30-48 hours.

More specifically, Bacillus alcaligenes FC-01052 and Paracoccus denitrificans TD-20229 were respectively activated, and inserted into the seed solution (glucose 5g/L, peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, pH 7), cultivated for 24 hours, according to the inoculum size of 3%, respectively inserted into the same fermentation medium, fermented at 15-45°C for 48-84 hours, in the fermentation stage, the aeration ratio was 1.0-2.5v/vm, and the stirring was 150-200rpm , wherein, the fermentation medium is: trisodium citrate 20g/L, ammonium nitrate 4.5g/L, potassium nitrate 3g/L, NaNO ₂ 0.3g/L, urea 1g/L, Na ₂ HPO ₄ ·3H ₂ O 2g/L, NaH ₂ PO ₄ 2g/L, MgSO ₄ 7H ₂ O 0.1g/L, ferrous sulfate 0.01g/L, copper sulfate 0.002g/L, zinc sulfate 0.01g/L, calcium chloride 0.5g/L, manganese sulfate 0.03g/L, cobalt chloride 0.002g/L, the rest is water, pH=7.5. At the end of the fermentation, the fermentation liquid is added to the wastewater to be treated according to (volume ratio 1:1) for wastewater treatment. The treatment method is to first add Alcaligenes FC-01052 to treat the wastewater for 15 hours, and then add Paracoccus denitrification TD-20229 treatment 40h.

The dosage of the fermentation broth added to the waste water to be treated is 2%-10%.

The Alcaligenes FC-01052 and Paracoccus denitrificans TD-20229 can grow well under low-temperature and high-salt environment and have obvious denitrification ability.

The Alcaligenes FC-01052 has an obvious degrading effect on ammonia nitrogen in the wastewater under the environment of low-temperature and high-salt wastewater, and the degradation rate reaches more than 90%. The present invention reports the ammonia nitrogen degradation function of the strain for the first time.

The paracoccus denitrificans (Paracoccus denitrificans) TD-20229 has obvious degrading effects on ammonia nitrogen, NO ₂ ^- -N, and NO ₃ ^- -N in the wastewater under the low-temperature and high-salt wastewater environment, and the degradation rates are over 90% respectively , More than 99%, More than 99%.

Paracoccus denitrificans (Paracoccus denitrificans) TD-20229 was screened by the following method: five strains of Paracoccus denitrificans were isolated from a sewage plant, and were cultured in liquid LB medium for 1 day at 35°C. The biomass was measured, and a strain with the most vigorous activity was obtained, which was named TD-1 as the dominant strain.

Inoculate TD-1 in liquid LB seed medium for culture, dilute the cultured Paracoccus denitrificans by 10-fold dilution method until the number of cells is 10 ⁶ CFU/mL, take 1 mL of the bacteria solution and apply it evenly on sterile N ⁺ ion beam implantation ^was carried ^out in the empty flat plate after being air ^{- dried .} is 20keV. After the irradiation, the cells were washed with 1 mL of sterile water, diluted according to the 10-fold dilution method, and then applied to the plate medium, and cultured upside down at 37°C for 1 day. After picking a single colony, they were inserted into the LB seed solution for 12 hours at 37°C and 150 rpm. According to the inoculum amount of 5%, it nitrifies the medium (NH ₄ ⁺ -N medium, NO ₃ ^- -N medium and NO ₂ ^- -N medium) respectively, and screens out strains with better colony growth and better degradation effect , named Paracoccus denitrificans TD-20229. The strain adopts the method of aerobic culture, grows rapidly under aerobic conditions, and grows slowly under anoxic conditions. The suitable growth temperature is 25-30°C, and the growth pH is 6.5-9; spherical cells (0.5-0.9 μm in diameter) ) or Brevibacteria (length 0.9-1.2 μm), single, paired or stacked, forming poly-β-hydroxybutyrate particles, Gram-negative, immobile, aerobic, respiratory metabolism.

In the present invention, after the fermentation of Alcaligenes FC-01052 and Paracoccus denitrificans TD-20229 is completed for the first time, the fermentation broth is added (volume ratio 1:1) into the wastewater to be treated for wastewater treatment, The treatment method is to first add Alcaligenes FC-01052 to treat the wastewater, and then add the denitrifying Paracoccus TD-20229 for treatment.

The high-salt (1-10%) and low-temperature (5°C-20°C) sewage described in the present invention is pesticide chemical waste water.

The present invention mainly relates to the cultivation of Alcaligenes and Paracoccus denitrificans, optimization of fermentation process and application in low-temperature and high-salt sewage treatment, so as to avoid secondary pollution, achieve denitrification effect, and further achieve the purpose of sewage treatment. Compared with the traditional biological denitrification technology, the method of the present invention is compared with the prior art. After the fermentation of Alcaligenes FC-01052 and Paracoccus denitrificans (Paracoccus denitrificans) TD-20229 is completed, the fermented liquid is prepared according to (volume Ratio 1:1) is added to the wastewater to be treated for wastewater treatment, wherein the treatment method is to first add Alcaligenes FC-01052 to treat the wastewater and then add the denitrifying Paracoccus TD-20229 for treatment. The denitrification effect is obvious and can Reduce ammonia nitrogen, NO ₂ ^- -N, and NO ₃ ^- -N, and at the same time grow better in a low-temperature and high-salt environment Through a unique production strain that removes ammonia nitrogen, solve the problem of wastewater treatment and further provide a theoretical basis for solving related problems and technical guidance.

Beneficial effects: Compared with the existing technology, the present invention has the following advantages: the present invention utilizes Alcaligenes FC-01052 and Paracoccus denitrificans TD-20229, and combines the characteristics of Alcaligenes to efficiently degrade ammonia nitrogen in a low-temperature and high-salt environment, At the same time, combined with the denitrification paracoccus in the low-temperature and high-salt pesticide wastewater environment, the synergistic and efficient degradation of ammonia nitrogen, NO ^2- -N, and NO ^3- -N, the denitrification effect is more obvious.

1. Compared with other strains, the Alcaligenes and Paracoccus denitrificans of the present invention have high metabolic activity, can grow better under low temperature and high salt conditions, have better denitrification effect, and have better wastewater treatment effect .

2. Compared with the traditional nitrification-denitrification denitrification process, this process has the advantages of mixing Alcaligenes faecalis and Paracoccus demonococcus to reduce nitrogen content in wastewater, no need to neutralize acid and alkali, simplify process operation and save energy consumption.

Detailed ways

The present invention can be better understood by the following examples. Then, those skilled in the art will easily understand that the specific material ratios described in the examples, process conditions and results thereof are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

The influence of embodiment 1 temperature on bacterial strain growth and denitrification effect

Activate Bacillus Alcaligenes FC-01052 and Paracoccus denitrificans TD-20229 respectively, insert seed solution (peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, glucose 5g/L, pH7), and culture 20h, according to the inoculum size is 3%, respectively into the fermentation medium, fermentation at 5, 10, 15, 20, 25, 30, 37, 42 ℃ respectively for 48h, in the fermentation stage aeration ratio 1.5v/vm, Stirring at 180rpm, wherein, the fermentation medium of Alcaligenes faecalis and Paracoccus denitrificans is: trisodium citrate 20g/L, ammonium nitrate 4.5g/L, potassium nitrate 3g/L, NaNO ₂ 0.3g/L, Urea 1g/L, Na ₂ HPO ₄ 3H ₂ O 2g/L, NaH ₂ PO ₄ 2g/L, MgSO ₄ 7H ₂ O 0.1g/L, ferrous sulfate 0.01g/L, copper sulfate 0.002g/L , zinc sulfate 0.01g/L, calcium chloride 0.5g/L, manganese sulfate 0.03g/L, cobalt chloride 0.002g/L, the rest is water, pH=7.5. The effects of different temperatures on the strain growth and denitrification effect were investigated.

At the end of the fermentation, put the fermented broth into 2% nitrification medium (NH ₄ ⁺ -N medium, NO ₃ ^- -N medium and NO ₂ ^- -N medium) respectively, preferably at 35°C. After oxygen fermentation for 48 hours, denitrification tests and growth conditions were carried out in three different mediums.

Among them, NH ₄ ⁺ -N simulated wastewater medium: sodium acetate 5g/L, (NH ₄ ) ₂ SO ₄ 0.8g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L L, FeSO ₄ ·7H ₂ O 0.02g/L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

NaNO ₃ simulated wastewater medium: sodium acetate 5g/L, KNO ₃ 0.8g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L, FeSO ₄ 7H ₂ O 0.02g /L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

NaNO ₂ simulated wastewater medium: NaNO ₂ 0.8g/L, sodium acetate 5g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L, FeSO ₄ 7H ₂ O 0.02g /L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

Temperature has a great influence on microbial nitrification and denitrification denitrification. The results in Table 1 and 2 show that temperature has a great influence on the nitrification and denitrification activities of Alcaligenes FC-01052 and Paracoccus denitrification TD-20229. When When the temperature is 5-15°C, the removal rate of NO ₃ ^- -N and NO ₂ ^- -N is more than 40%. As the temperature increases, the removal rate of ammonia nitrogen also increases gradually. When the temperature is 35°C, the removal rate of ammonia nitrogen is above 92%; when the temperature is 42°C, the removal rate of total nitrogen decreases, and the ability of nitrification and denitrification decreases significantly, indicating that These two strains had better growth and denitrification effects in low temperature environment. Table 1 and Table 2 also show that under the same temperature conditions, the ability of Alcaligenes FC-01052 to degrade ammonia nitrogen is much higher than that of Paracoccus denitrificans TD-20229, while Paracoccus denitrificans TD-20229 removes NO The ability of ₃ ^- -N and NO ₂ ^- -N is significantly higher than that of Alcaligenes FC-01052, especially when the temperature is 5-25°C, the ability of Alcaligenes FC-01052 to degrade ammonia nitrogen is higher than 77%, and the degradation rate is 78%-88%, when the temperature is 30-35°C, the ability to degrade ammonia nitrogen is higher than 90%, and the degradation rate is 91%-99.3%.

Table 1 Effect of temperature on the growth and denitrification effect of Alcaligenes FC-01052

Table 2 Effect of temperature on the growth and denitrification effect of Paracoccus denitrificans TD-20229

Effect of embodiment 2 high salt on bacterial strain growth and denitrification effect

Activate Bacillus Alcaligenes FC-01052 and Paracoccus denitrificans TD-20229 respectively, insert seed solution (peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, glucose 5g/L, pH7), and culture 20h, according to the inoculum size of 3%, transfer to the fermentation medium, ferment for 48h at the concentration of sodium chloride respectively 1%, 2%, 4%, 6%, 8%, and 10%, and aerate during the fermentation stage Ratio 1.5v/vm, stirring 180rpm, wherein, the described fermentation medium of Alcaligenes and P. paracoccus is: trisodium citrate 20g/L, ammonium nitrate 4.5g/L, potassium nitrate 3g/L, NaNO ₂ 0.3g/L, urea 1g/L, Na ₂ HPO ₄ 3H ₂ O 2g/L, NaH ₂ PO ₄ 2g/L, MgSO ₄ 7H ₂ O 0.1g/L, ferrous sulfate 0.01g/L, sulfuric acid Copper 0.002g/L, zinc sulfate 0.01g/L, calcium chloride 0.5g/L, manganese sulfate 0.03g/L, cobalt chloride 0.002g/L, the rest is water, pH=7.5. The effects of different salinity on strain growth and denitrification effect were investigated.

At the end of the fermentation, put the fermented broth into 2% nitrification medium (NH ₄ ⁺ -N medium, NO ₃ ^- -N medium and NO ₂ ^- -N medium) respectively, preferably at 35°C. After oxygen fermentation for 48 hours, denitrification tests and growth conditions were carried out in three different mediums.

Among them, NH ₄ ⁺ -N simulated wastewater medium: sodium acetate 5g/L, (NH ₄ ) ₂ SO ₄ 0.8g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L L, FeSO ₄ ·7H ₂ O 0.02g/L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

NaNO ₃ simulated wastewater medium: sodium acetate 5g/L, KNO ₃ 0.8g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L, FeSO ₄ 7H ₂ O 0.02g /L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

NaNO ₂ simulated wastewater medium: NaNO ₂ 0.8g/L, sodium acetate 5g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L, FeSO ₄ 7H ₂ O 0.02g /L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

Salinity has a great influence on microbial nitrification and denitrification denitrification. The following table shows that the salt concentration has a great influence on the nitrification and denitrification activities of Alcaligenes FC-01052 and Paracoccus denitrification TD-20229. Table 3 and Table 4 also shows that under the same salinity conditions, the ability of Alcaligenes FC-01052 to degrade ammonia nitrogen is much higher than that of Paracoccus denitrificans TD-20229. When the salinity is 1-10%, the removal rate of NO ₃ ^- -N and NO ₂ ^- -N is more than 40%, and the removal rate of ammonia nitrogen is gradually increasing with the increase of salinity. When the salinity is 1-6%, the removal rate of ammonia nitrogen is more than 80%; when the salinity is 1-4%, the removal rate of ammonia nitrogen is more than 90%; when the salinity is higher than 4%, NO ₃ ^- -N, NO The removal rate of ₂ ^- -N decreased, and the ability of nitrification and denitrification decreased obviously, which indicated that the growth and denitrification effect of these two strains were better in low temperature environment. Table 3 and Table 4 also show that under the same salinity conditions, the ability of Alcaligenes FC-01052 to degrade ammonia nitrogen is much higher than that of denitrification paracoccus TD-20229, while denitrification paracoccus TD-20229 can remove The ability of NO ₃ ^- -N and NO ₂ ^- -N is significantly higher than that of Alcaligenes FC-01052, especially when the salinity is 1-4%, the ability of Alcaligenes FC-01052 to degrade ammonia nitrogen is higher than 90%. The rate is 90.5%-99.3%. When the salinity is higher than 4%, the ability to degrade ammonia nitrogen is higher than 60%, and the degradation rate is 62.3%-89.8%.

Table 3 Effect of salinity on the growth and denitrification effect of Alcaligenes FC-01052

Table 4 The effect of salinity on the growth and denitrification effect of Paracoccus denitrificans TD-20229

The influence of embodiment 3 initial pH on strain growth and denitrification effect

Activate Bacillus Alcaligenes FC-01052 and Paracoccus denitrificans TD-20229 respectively, insert seed solution (peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, glucose 5g/L, pH7), and culture 20h, according to the inoculum size of 3%, transfer to the fermentation medium, ferment at 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9°C respectively for 48h, and the aeration ratio in the fermentation stage is 1.5v /vm, stirring 180rpm, wherein, the described fermentation medium of Alcaligenes and P. paracoccus is: trisodium citrate 20g/L, ammonium nitrate 4.5g/L, potassium nitrate 3g/L, NaNO ₂ 0.3g/ L, urea 1g/L, Na ₂ HPO ₄ 3H ₂ O 2g/L, NaH ₂ PO ₄ 2g/L, MgSO ₄ 7H ₂ O 0.1g/L, ferrous sulfate 0.01g/L, copper sulfate 0.002g /L, zinc sulfate 0.01g/L, calcium chloride 0.5g/L, manganese sulfate 0.03g/L, cobalt chloride 0.002g/L, the rest is water, pH=7.5. Investigate the effect of different initial pH on the strain growth and denitrification effect. Table 5 and Table 6 also show that under the same pH conditions, the ability of Alcaligenes FC-01052 to degrade ammonia nitrogen is much higher than that of Paracoccus denitrificans TD-20229, while Paracoccus denitrificans TD-20229 removes NO The ability of ^3- -N and NO ^2- -N was significantly higher than that of Alcaligenes FC-01052.

At the end of the fermentation, put the fermented broth into 2% nitrification medium (NH ₄ ⁺ -N medium, NO ₃ ^- -N medium and NO ₂ ^- -N medium) respectively, preferably at 35°C. After oxygen fermentation for 48 hours, denitrification tests and growth conditions were carried out in three different mediums.

Among them, NH ₄ ⁺ -N simulated wastewater medium: sodium acetate 5g/L, (NH ₄ ) ₂ SO ₄ 0.8g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L L, FeSO ₄ ·7H ₂ O 0.02g/L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

NaNO ₃ simulated wastewater medium: sodium acetate 5g/L, KNO ₃ 0.8g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L, FeSO ₄ 7H ₂ O 0.02g /L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

NaNO ₂ simulated wastewater medium: NaNO ₂ 0.8g/L, sodium acetate 5g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L, FeSO ₄ 7H ₂ O 0.02g /L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

The initial pH has a great influence on microbial nitrification and denitrification denitrification. The results in Table 5 and 6 show that the temperature has a great influence on the nitrification and denitrification activities of Alcaligenes FC-01052 and Paracoccus denitrification TD-20229. When the initial pH is 4-9, the removal rate of NO ₃ ^- -N and NO ₂ ^- -N is more than 40%. As the pH increases, the removal rate of ammonia nitrogen also increases gradually. 8, the removal rate of ammonia nitrogen is more than 80%; when the initial pH is 4-5, the removal rate of ammonia nitrogen is more than 60%; when the pH is higher than 8, the removal rate of total nitrogen is reduced, and the ability of nitrification and denitrification significantly decreased, indicating that the two strains had better growth and denitrification effects in a low temperature environment. Table 5 and Table 6 also show that under the same pH conditions, the ability of Alcaligenes FC-01052 to degrade ammonia nitrogen is much higher than that of Paracoccus denitrificans TD-20229, while Paracoccus denitrificans TD-20229 removes NO The ability of ₃ ^- -N and NO ₂ ^- -N is significantly higher than that of Alcaligenes FC-01052, especially when the initial pH is 6.5-8, the ability of Alcaligenes FC-01052 to degrade ammonia nitrogen is higher than 90%, and the degradation rate is 90%-99.8%, when the initial pH is 4-6.5, the ability to degrade ammonia nitrogen is higher than 60%, and the degradation rate is 60.4%-89.9%.

Table 5 Effect of initial pH on the growth and denitrification effect of Alcaligenes FC-01052

Table 6 Effect of initial pH on the growth and denitrification effect of Paracoccus denitrificans TD-20229

The influence of embodiment 4 carbon source on bacterial strain growth and denitrification effect

Activate Bacillus Alcaligenes FC-01052 and Paracoccus denitrificans TD-20229 respectively, insert seed solution (peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, glucose 5g/L, pH7), and culture 20h, according to the inoculum size is 3%, transferred to the fermentation medium, fermented under carbon source conditions (sodium citrate, sodium acetate, sodium pyruvate) respectively for 48h, aeration ratio 1.5v/vm, stirring 180rpm in the fermentation stage , wherein, the described fermentation medium of Alcaligenes and Paracoccus denitrificans is: trisodium citrate 20g/L, ammonium nitrate 4.5g/L, potassium nitrate 3g/L, NaNO ₂ 0.3g/L, urea 1g/L L, Na ₂ HPO ₄ 3H ₂ O 2g/L, NaH ₂ PO ₄ 2g/L, MgSO ₄ 7H ₂ O 0.1g/L, ferrous sulfate 0.01g/L, copper sulfate 0.002g/L, zinc sulfate 0.01g/L, calcium chloride 0.5g/L, manganese sulfate 0.03g/L, cobalt chloride 0.002g/L, the rest is water, pH=7.5. Investigate the effect of different carbon sources on the growth of the strain and the denitrification effect.

Table 7 and Table 8 also show that under carbon source conditions, the ability of Alcaligenes FC-01052 to degrade ammonia nitrogen is much higher than that of Denitrification Paracoccus TD-20229, while denitrification Paracoccus TD-20229 removes NO The ability of ^3- -N and NO ^2- -N was significantly higher than that of Alcaligenes FC-01052.

At the end of the fermentation, put the fermented broth into 2% nitrification medium (NH ₄ ⁺ -N medium, NO ₃ ^- -N medium and NO ₂ ^- -N medium) respectively, preferably at 35°C. After oxygen fermentation for 48 hours, denitrification tests and growth conditions were carried out in three different mediums.

Among them, NH ₄ ⁺ -N simulated wastewater medium: sodium acetate 5g/L, (NH ₄ ) ₂ SO ₄ 0.8g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L L, FeSO ₄ ·7H ₂ O 0.02g/L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

NaNO ₃ simulated wastewater medium: sodium acetate 5g/L, KNO ₃ 0.8g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L, FeSO ₄ 7H ₂ O 0.02g /L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

NaNO ₂ simulated wastewater medium: NaNO ₂ 0.8g/L, sodium acetate 5g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L, FeSO ₄ 7H ₂ O 0.02g /L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

Carbon sources have a great influence on microbial nitrification and denitrification denitrification. The results in Table 7 and 8 show that temperature has a great influence on the nitrification and denitrification activities of Alcaligenes FC-01052 and Paracoccus denitrification TD-20229. Under different carbon sources, the removal rates of NO ₃ ^- -N and NO ₂ ^- -N are above 60%. When the nitrogen source is trisodium citrate, the ability of Alcaligenes FC-01052 to remove NO ₃ ^- -N and NO ₂ ^- -N is significantly higher than that when the carbon source is sodium acetate ^and _sodium pyruvate , NO ₂ ^- -N capacity, the removal rate of ammonia nitrogen is still 99.2% to 99.8%. It indicated that the two strains had better growth and denitrification effects in low temperature environment. Table 7 and Table 8 also show that the ability of Alcaligenes FC-01052 to degrade ammonia nitrogen is much higher than that of Paracoccus denitrificans TD-20229, while Paracoccus denitrificans TD-20229 removes NO ₃ ^- -N, NO The ability of ₂ ^- -N is higher than Alcaligenes FC-01052.

Table 7 Effect of carbon source on the growth and denitrification effect of Alcaligenes FC-01052

Table 8 Effect of carbon source on the growth and denitrification effect of Paracoccus denitrificans TD-20229

Example 5 Effect of different treatment methods on denitrification effect

Activate Bacillus Alcaligenes FC-01052 and Paracoccus denitrificans TD-20229 respectively, insert seed solution (peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, glucose 5g/L, pH7), and culture 20h, according to the inoculum size is 3%, transferred to the fermentation medium, fermented under carbon source conditions (sodium citrate, sodium acetate, sodium pyruvate) respectively for 48h, aeration ratio 1.5v/vm, stirring 180rpm in the fermentation stage , wherein, the described Alcaligenes faecalis and P. paracoccus fermentation medium are: trisodium citrate 20g/L, ammonium nitrate 4.5g/L, potassium nitrate 3g/L, NaNO ₂ 0.3g/L, urea 1g /L, Na ₂ HPO ₄ 3H ₂ O 2g/L, NaH ₂ PO ₄ 2g/L, MgSO ₄ 7H ₂ O 0.1g/L, ferrous sulfate 0.01～3g/L, copper sulfate 0.002g/L, Zinc sulfate 0.01g/L, calcium chloride 0.5g/L, manganese sulfate 0.03g/L, cobalt chloride 0.002g/L, the rest is water, pH=7.5. At the end of the fermentation, it was added to the following simulated wastewater medium for denitrification according to different treatment methods.

At the end of the fermentation, put the fermented broth into 2% nitrification medium (NH ₄ ⁺ -N medium, NO ₃ ^- -N medium and NO ₂ ^- -N medium) respectively, preferably at 35°C. After oxygen fermentation for 48 hours, denitrification tests and growth conditions were carried out in three different mediums.

Among them, NH ₄ ⁺ -N simulated wastewater medium: sodium acetate 5g/L, (NH ₄ ) ₂ SO ₄ 0.8g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L L, FeSO ₄ ·7H ₂ O 0.02g/L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

NaNO ₃ simulated wastewater medium: sodium acetate 5g/L, KNO ₃ 0.8g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L, FeSO ₄ 7H ₂ O 0.02g /L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

NaNO ₂ simulated wastewater medium: NaNO ₂ 0.8g/L, sodium acetate 5g/L, NaCL 0.5g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1g/L, FeSO ₄ 7H ₂ O 0.02g /L, MgSO ₄ ·7H ₂ O 0.05g/L, zinc sulfate 0.01g/L, manganese chloride 0.01g/L, pH7.2.

Treatment method ①: Add the fermentation liquid to the simulated wastewater according to (volume ratio 1:1) for wastewater treatment. The treatment method is to first add Alcaligenes FC-01052 to treat the wastewater for 15 hours, and then add Paracoccus denitrification TD-20229 treatment 40h.

Treatment method ②: Add the fermentation broth to the wastewater to be simulated at the same time (volume ratio 1:1) for wastewater treatment.

Treatment method ③: Add the two fermentation broths to the wastewater to be treated respectively to simulate wastewater treatment.

Treatment method ④: Add the fermentation broth to the simulated wastewater according to (volume ratio 1:1) for wastewater treatment. The treatment method is to add Paracoccus denitrificans TD-20229 for 15 hours before adding Alcaligenes FC -01052 processing 40h.

Different treatment methods have significantly different denitrification effects on the two strains in the low-temperature and high-salt wastewater environment. When the treatment method is to add the fermentation broth (volume ratio 1:1) to the wastewater to be treated for wastewater treatment, first Add Alcaligenes FC-01052 to treat the wastewater for 15 hours, and then add Paracoccus denitrificans TD-20229 to treat it for 40 hours. The removal rates of NO ₃ ^- -N, NO ₂ ^- -N and NH ₄ ⁺ -N in treatment method ① were 99.7%, 99.2% and 99.3%, respectively, and the removal effect of treatment method ① was the best.

Table 9 Effect of different treatment methods on denitrification effect

The research of embodiment 6 bacterial strains in low-temperature high-salt industrial wastewater

Activate Bacillus Alcaligenes FC-01052 and Paracoccus denitrificans TD-20229 respectively, insert seed solution (peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, glucose 10g/L, pH7), and culture 20h, according to the inoculum size is 3%, transferred to the fermentation medium, fermented under carbon source conditions (sodium citrate, sodium acetate, sodium pyruvate) respectively for 48h, aeration ratio 1.5v/vm, stirring 180rpm in the fermentation stage , wherein, the described fermentation medium of Alcaligenes and Paracoccus denitrificans is: trisodium citrate 20g/L, ammonium nitrate 4.5g/L, potassium nitrate 3g/L, NaNO ₂ 0.3g/L, urea 1g/L L, Na ₂ HPO ₄ 3H ₂ O 2g/L, NaH ₂ PO ₄ 2g/L, MgSO ₄ 7H ₂ O 0.1g/L, ferrous sulfate 0.01g/L, copper sulfate 0.002g/L, zinc sulfate 0.01g/L, calcium chloride 0.5g/L, manganese sulfate 0.03g/L, cobalt chloride 0.002g/L, the rest is water, pH=7.5.

At the end of the fermentation, the fermented fermentation liquid was transferred to the fermentation medium according to the inoculation amount of 2%, aerobically fermented at 35°C for 48 hours, and the fermented liquid was added to the pesticide chemical industry according to (volume ratio 1:1) In the waste water, waste water treatment is carried out, and the treatment method is to add Alcaligenes FC-01052 to treat the waste water for 15 hours, and then add Paracoccus denitrificans TD-20229 to treat the waste water for 40 hours.

Investigate the pesticide and chemical wastewater environment at a temperature of 15°C, a salinity of 4%, an ammonia nitrogen concentration of 400mg/L, a NO ₃ ^- -N concentration of 300mg/L, and a NO ₂ ^- -N concentration of 300mg/L. It can be seen from Table 10 that Alcaligenes FC-01052 and Paracoccus denitrificans TD-20229 have excellent denitrification effects in low-temperature high-salt wastewater, NO ₃ ^- -N removal rate, NO ₂ ^- -N removal rate and NH ₄ ⁺ -N removal rates were 89.7%, 88.4% and 90.6%, respectively.

Table 10 The growth of bacterial strains in low-temperature high-salt industrial wastewater and the effect of nitrogen removal

The detection method of the process in the present invention: NO ₃ ^- -N concentration is measured by ultraviolet spectrophotometry, NO ₂ ^- -N is measured by N-(1-naphthyl)-ethylenediamine spectrophotometry, NH ₄ ⁺ -N The concentration was determined by Nessler's reagent spectrophotometry.

The technical solutions provided by the embodiments of the present invention have been introduced in detail above, and the principles and implementation modes of the embodiments of the present invention have been explained by using specific examples in this paper. The descriptions of the above embodiments are only applicable to help understand the embodiments of the present invention Principle At the same time, for those skilled in the art, according to the embodiment of the present invention, there will be changes in the specific implementation and application scope. In summary, the contents of this specification should not be construed as limiting the present invention.

Claims (10)

1. A method for treating high-salt low-temperature wastewater by nitrifying bacteria and denitrifying bacteria is characterized by comprising the following steps:

s1, activating nitrifying bacteria, inoculating seed liquid, culturing for 16-36 h, inoculating the nitrifying bacteria into a fermentation medium according to the inoculum size of 1-10%, and fermenting at the temperature of 5-42 ℃ for 48-72 h;

s2, activating denitrifying bacteria, inoculating seed liquid, culturing for 16-36 h, inoculating into a fermentation medium according to the inoculum size of 1-10%, and fermenting at 5-42 ℃ for 48-60 h;

s3, adding the fermentation liquor into the wastewater to be treated, wherein the volume ratio of nitrifying bacteria to denitrifying bacteria is 1-10: 1-10, adding nitrifying bacteria to treat wastewater for 10-18 hours, and then adding denitrifying bacteria to treat wastewater for 30-48 hours.

2. The method for treating high-salt low-temperature wastewater by nitrifying bacteria and denitrifying bacteria according to claim 1, wherein the nitrifying bacteria is alcaligenes FC-01052; the denitrifying bacteria are Paracoccus denitrificans TD-20229.

3. The method for treating high-salt low-temperature wastewater by nitrifying bacteria and denitrifying bacteria according to claim 1, wherein the fermentation media in S1 and S2 are the same, the components are 5-30 g/L carbon source, 5-30 g/L nitrogen source, 1-10 g/L inorganic salt, 0-2 g/L metal ion, 0.1-1 g/L trace element, and the balance water, wherein the pH is 4-9.0.

4. The method for treating high-salt low-temperature wastewater by nitrifying bacteria and denitrifying bacteria according to claim 3, wherein the carbon source is at least one of glucose, trisodium citrate, sodium pyruvate, sodium acetate, molasses and the like.

5. A method for treating high-salt low-temperature wastewater by nitrifying bacteria and denitrifying bacteria according to claim 3, wherein the fermentation temperature of the nitrifying bacteria is 37 ℃ and the fermentation time is 48h; the fermentation temperature of denitrifying bacteria is 37 ℃ and the fermentation time is 50 h.

6. The method for treating high-salt low-temperature wastewater by nitrifying bacteria and denitrifying bacteria according to claim 3, wherein the nitrogen source is at least one of ammonium chloride, ammonium nitrate, sodium nitrate, ammonium sulfate, urea, beef extract, ammonia water, corn steep liquor and fish meal.

7. The method for treating high-salt low-temperature wastewater by nitrifying bacteria and denitrifying bacteria according to claim 3, wherein the inorganic salt is any one or a combination of a plurality of magnesium salt, nitrate, potassium salt, calcium salt, phosphate or hydrochloride.

8. The method for treating high-salt low-temperature wastewater by nitrifying bacteria and denitrifying bacteria according to claim 3, wherein the trace elements are prepared by mixing 0.01-3 g/L of ferrous sulfate, 0.001-1 g/L of copper sulfate, 0.01-0.5 g/L of zinc sulfate, 0.1-2 g/L of calcium chloride, 0.01-1 g/L of manganese sulfate and 0.001-0.1 g/L of cobalt chloride.

9. The method for treating high-salt low-temperature wastewater by nitrifying bacteria and denitrifying bacteria according to claim 4, wherein the method comprises the steps of adding alcaligenes FC-01052 to treat wastewater 15h and adding paracoccus denitrificans TD-20229 to treat wastewater 40h.

10. The method for treating high-salt low-temperature wastewater by nitrifying bacteria and denitrifying bacteria according to claim 9, wherein the adding amount of the fermentation liquor to the wastewater to be treated is 2% -10%.