CN113736700B

CN113736700B - Heterotrophic nitrification-aerobic denitrification bacteria and application thereof

Info

Publication number: CN113736700B
Application number: CN202111032868.6A
Authority: CN
Inventors: 薛亚平; 刘聪; 张剑峰; 柯霞; 郑裕国; 杨海本; 徐献忠
Original assignee: HANGZHOU ENVIRONMENTAL GROUP CO Ltd; Zhejiang University of Technology ZJUT
Current assignee: HANGZHOU ENVIRONMENTAL GROUP CO Ltd; Zhejiang University of Technology ZJUT
Priority date: 2021-09-03
Filing date: 2021-09-03
Publication date: 2023-09-05
Anticipated expiration: 2041-09-03
Also published as: CN113736700A

Abstract

The invention discloses heterotrophic nitrification-aerobic denitrification bacteria and application thereof. The heterotrophic nitrification-aerobic denitrification bacteria are preserved in China Center for Type Culture Collection (CCTCC), are classified and named as Acinetobacter indicum ZJB20129, and have a preservation number of CCTCC NO: m2020965, 12 months and 24 days of the preservation date 2020. The heterotrophic nitrification-aerobic denitrification Acinetobacter indicum B2-1%Acinetobacter indicusThe strain B2-1) ZJB20129 can use sodium succinate and sodium acetate as carbon sources, and has the optimal ammonia nitrogen removal effect of 99.27% when the sodium succinate is used as an optimal carbon source, and the sodium succinate is subjected to aerobic culture for 36 hours under the conditions of 15 mass ratio of C/N, 8 pH, 35 ℃ and 160rpm of rotating speed. Meanwhile, the strain has good effect of removing both nitrate nitrogen and nitrite nitrogen.

Description

Heterotrophic nitrification-aerobic denitrification bacteria and application thereof

Technical Field

The invention relates to the field of nitrogen-containing sewage treatment, in particular to heterotrophic nitrification-aerobic denitrification bacteria and application thereof.

Background

The rapid development of industry and agriculture and the production activities of human beings bring serious pollution to the water environment. Nitrogen out-of-standard is the most common pollution problem in water pollution, and can cause eutrophication of the water, so that algae are greatly propagated, dissolved oxygen of the water is reduced, aquatic organisms are greatly dead, the water safety of human beings is endangered, and the ecological balance of the water is seriously influenced; in addition, excessive nitrogen emission can also cause the nitrogen balance to be destroyed, thereby causing the water body to lose self-cleaning function. Therefore, the sewage rich in nitrogen can be discharged into the natural environment after the denitrification treatment reaches the standard.

Biological denitrification is a nitrogen-containing sewage treatment method widely applied at present, and has the advantages of good treatment effect, stable and reliable treatment process, convenient operation and management and the like. The biological denitrification sewage treatment technology mainly reduces ammoniacal nitrogen in sewage into nitrogen gas by the action of functional bacterial groups such as ammonia oxidizing bacteria, nitrifying bacteria, denitrifying bacteria and the like so as to achieve the aim of denitrification. In the treatment process, nitrifying bacteria and nitrosation bacteria convert ammonia nitrogen into nitrate nitrogen, and nitrate nitrogen has low chemical potential energy, high stability and easy accumulation of nitrate nitrogen. Denitrifying bacteria act on nitrate nitrogen in a water body to gradually reduce the nitrate nitrogen into nitrite nitrogen, nitric oxide and nitrous oxide, and finally the nitrite nitrogen is reduced into nitrogen to be released into the atmosphere, so that the circulation of nitrogen elements is completed, and the denitrifying bacteria are an important link of sewage denitrification. The traditional biological denitrification is completed by utilizing the aerobic autotrophic nitrification of nitrifying bacteria and the anaerobic heterotrophic denitrification of denitrifying bacteria, and the process needs to provide two independent reaction spaces with oxygen and without oxygen respectively for nitrification and denitrification, thereby achieving the aim of denitrification. Therefore, the treatment method occupies larger space, has higher running cost and has more complicated process.

Since Paracoccus denitrificans was first identified as an aerobic denitrifying bacterium, aerobic denitrification techniques have been accepted and further generalized. The aerobic denitrifying bacteria can simultaneously perform nitrification-denitrification reaction by taking oxygen and nitrate nitrogen as electron acceptors under the aerobic condition, so that the rapid removal of organic nitrogen is realized, and the aerobic denitrifying bacteria has important application value in the biological denitrification technology of organic matters. The research in the field has been carried out for over thirty years, and various aerobic denitrification microbial strains are separated and identified, and the growth characteristics and the treatment capacity in the process of denitrification of the iso-oxo-denitrification organisms are quite different. Therefore, the strain which is high-efficiency and can perform nitrification-denitrification simultaneously is screened to have important application value for denitrification of polluted water.

Disclosure of Invention

The invention aims at overcoming the defects of higher running cost and complicated process of sewage treatment in the prior art and provides heterotrophic nitrification-aerobic denitrification acinetobacter and application thereof.

The technical scheme adopted by the invention is as follows:

heterotrophic nitrification-aerobic denitrifying bacteria, which are preserved in China Center for Type Culture Collection (CCTCC), address: china, university of Wuhan, classified name of Acinetobacter indicum B2-1 (Acinetobacter indicus strain B2-1), preservation number of CCTCC NO: m2020965, 12 months and 24 days of the preservation date 2020.

The heterotrophic nitrification-aerobic denitrification Acinetobacter ZJB20129 has the biological characteristics that: the bacterial colony is in a milky yellow round shape, the surface is moist and easy to pick up, and the bacterial body is in a rod shape. The strain is suitable for growing with sodium succinate or sodium acetate as carbon source, 15-18C/N, 7.0-9.0 pH and 30-40 deg.c.

The invention also relates to application of the heterotrophic nitrification-aerobic denitrification bacteria in preparation of denitrification biological bacterial agents. The denitrification biological bacterial agent comprises the heterotrophic nitrification-aerobic denitrification bacteria.

The invention also relates to application of the heterotrophic nitrification-aerobic denitrification bacteria in nitrogen-containing sewage denitrification treatment.

The invention also relates to a method for denitrifying nitrogen-containing sewage by using the heterotrophic nitrification-aerobic denitrification bacteria, which comprises the following steps: inoculating the heterotrophic nitrification-aerobic denitrification bacteria into the nitrogen-containing sewage, adding a carbon source to a C/N ratio of 15-18, and culturing at a pH of 7-9, a temperature of 30-40 ℃ and a speed of 80-200 rpm to remove ammonia nitrogen in the sewage.

Preferably, the carbon source is one or two of sodium succinate and sodium acetate. More stably, preferably, the carbon source is sodium succinate.

Preferably, the cultivation is carried out at a C/N ratio of 15, pH 8, temperature of 35℃and rotation speed of 160 rpm.

The beneficial effects of the invention are that

The heterotrophic nitrification-aerobic denitrification Acinetobacter indicum (Acinetobacter indicus strain) ZJB20129 can utilize sodium succinate and sodium acetate as carbon sources, and has an optimal ammonia nitrogen removal effect of 99.27% when the sodium succinate is an optimal carbon source, the C/N is 15, the pH is 8, the temperature is 35 ℃ and the rotating speed is 160rpm for aerobic culture for 36 hours. Meanwhile, the strain has good removal effect on nitrate nitrogen and nitrite nitrogen, and has important practical significance on developing high-efficiency denitrification biological bacterial agents.

Drawings

FIG. 1 is an optical microscope image of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 of the present invention.

FIG. 2 is a phylogenetic tree of the heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 16S rDNA of the invention.

Fig. 3 is a schematic diagram of the capability of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 of the present invention to degrade ammonia nitrogen.

Fig. 4 is a schematic diagram of the heterotrophic nitrification-aerobic denitrification bacteria ZJB 20129's ability to degrade nitrate nitrogen according to the present invention.

Fig. 5 is a schematic diagram of the heterotrophic nitrification-aerobic denitrification bacteria ZJB 20129's ability to degrade nitrite nitrogen according to the present invention.

Fig. 6 is a schematic diagram of ammonia nitrogen degradation of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 of the invention under different carbon sources.

Fig. 7 is a schematic diagram of ammonia nitrogen degradation of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 according to the invention at different carbon-nitrogen ratios.

Fig. 8 is a schematic diagram of ammonia nitrogen degradation of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 of the invention at different pH.

Fig. 9 is a schematic diagram of ammonia nitrogen degradation of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 of the invention at different temperatures.

Fig. 10 is a schematic diagram of ammonia nitrogen degradation of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 of the present invention at different rotational speeds.

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto: example 1: screening and identifying heterotrophic nitrification-aerobic denitrification bacteria, adding 2-3 g of biological turntable mud sample of a certain sewage treatment company in Hangzhou into a conical flask filled with 100mL of sterile water, shaking for 3 hours on a shaking table, sucking 2mL of liquid by a pipetting gun, inoculating the liquid into a sterilized LB culture medium, and culturing for 2-3 d at the temperature of 30 ℃ and the speed of 180 rpm.

LB medium as described above: 10g of tryptone, 5g of yeast powder, 10g of sodium chloride, and distilled water to 1000mL, and naturally pH; if a solid medium is required, 1.5-2% agar is added.

Inoculating strain culture solution into test tube containing sterile water, and diluting at a certain proportion to obtain strain concentration of 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 And 10 ^-8 A gradient of bacterial suspension; then, 100 mu L of each gradient bacterial suspension is respectively absorbed from each gradient bacterial suspension by adopting a flat plate coating method and coated on an LB solid culture medium, the LB solid culture medium is placed in a constant temperature incubator at 30 ℃ for culture, colonies with different forms are respectively picked up for streak separation, separation and purification are carried out for more than 3 times until the characteristics of the colonies are basically consistent, and then the colonies are inoculated on a slant culture medium and stored in a refrigerator at 4 ℃.

Culturing 15 strains obtained through preliminary screening in LB culture medium to logarithmic phase, respectively sucking 100 mu L of the strain, coating the strain on bromothymol blue (BTB) solid culture medium, culturing for 4d at 30 ℃, and selecting the colony on the solid culture medium with the fastest bluing for preservation and protection, thus obtaining the strain ZJB20129 as a target strain for subsequent research.

The bromothymol blue (BTB) solid medium is prepared according to the following components: potassium nitrate (KNO) ₃ ) 1.01g of sodium succinate (C ₄ H ₄ Na ₂ O ₄ ) 8.5g of magnesium sulfate heptahydrate (MgSO ₄ ·7H ₂ O)1.0g, monopotassium phosphate (KH) ₂ PO 4) 1.0g, ferrous sulfate heptahydrate (FeSO) ₄ ·7H ₂ O) 0.59g, calcium chloride (CaCl) ₂ ) 0.09g,1% bromothymol blue ethanol solution 1mL, distilled water to 1000mL, agar 20g, and pH 7.0-7.3 with 1mol/L NaOH.

As shown in fig. 1, the physiological and biochemical test of the strain ZJB20129 is gram negative, milky yellow circular, and the surface is wet and easy to pick up, and the thallus is rod-shaped. Extracting bacterial genome of the target strain by using a genome kit; using bacterial universal primer 27F:5'-AGAGTTTGATCMTGGCTCAG-3',1492R:5'-TACGGYTACCTTGTTACGACTT-3' to carry out PCR amplification and agarose gel electrophoresis (1%) verification; electrophoresis detection, gel cutting, purification and sequencing, and sequencing by the Optimago.

PCR reaction conditions: pre-denaturing for 3min at 94 ℃; denaturation for 30s; annealing at 54 ℃ for 30s; extending at 72 ℃ for 1min and 30s; the 30 cycles are repeated from the second step.

The 16S rDNA sequence length of the strain ZJB20129 is 1415bp, and the gene sequence is shown in SEQ ID NO.1.

The strain sequence results were uploaded to the NCBI database and compared with the existing bacterial 16S rDNA gene sequences in the database, and the results showed that the bacteria were Acinetobacter Indicus strain. Phylogenetic tree was constructed by the orthotopic ligation method of mega.7.0 software, and the genetic characteristics of the strains were analyzed, their species and their evolutionary positions were determined, and the results are shown in FIG. 2.

The strain ZJB20129 is named as Acinetobacter indicum B2-1 (Acinetobacter indicus strain B2-1), the strain number ZJB20129 is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m2020965, the preservation date is 12 months and 24 days in 2020, and the address is university of Wuhan in Wuhan, china.

Example 2: the nitrifying medium formula for identifying the nitrifying/denitrifying capacity of the strain ZJB20129 on different nitrogen source media is as follows: ammonium sulfate ((NH) ₄ ) ₂ SO ₄ ) 0.47g, sodium succinate (C) ₄ H ₄ Na ₂ O ₄ ) 5.06g, magnesium sulfate (MgSO ₄ ) 0.1g, monopotassium phosphate (KH) ₂ PO 4) 0.1g, trace elements2mL of plain solution, distilled water to 1000mL, and pH value of 7.0-7.5; wherein the trace element solution comprises: EDTA 50g, zinc sulfate heptahydrate (ZnSO) ₄ ·7H ₂ O) 3.92g, calcium chloride (CaCl) ₂ ) 5.5g, manganese chloride tetrahydrate (MnCl) ₂ ·4H ₂ O) 5.1g, ferrous sulfate heptahydrate (FeSO) ₄ ·7H ₂ O) 5.0g of ammonium molybdate tetrahydrate ((NH) ₄ )6Mo ₇ O ₂₄ ·4H ₂ O) 1.1g, copper sulfate pentahydrate (CuSO) ₄ ·5H ₂ O) 1.6g, cobalt chloride hexahydrate (CoCl) ₂ ·6H ₂ O) 1.6g, distilled water was used to volume to 1000mL. Denitrification Medium ammonium sulfate ((NH) in the nitrification medium ₄ ) ₂ SO ₄ ) 0.47g of potassium nitrate (KNO) ₃ ) 0.72g or sodium nitrite (NaNO) ₂ ) 0.49g, the remaining formulation remained unchanged.

The processing mode is as follows: inoculating 2% seed solution (the seed solution is bacterial suspension cultured for 12h at 160rpm at 30 ℃ in LB culture medium), and culturing at 160rpm at 30 ℃. Periodic sampling to determine the NH in the supernatant, respectively ₄ ⁺ -N、NO ₃ --N、NO ₂ - -N and OD ₆₀₀ Values, results are shown in figures 3 to 5.

As can be seen from FIG. 3, when the culture condition is 30 ℃ and 160rpm, the strain ZJB20129 reduces the ammonia nitrogen concentration from 103.8mg/L to 1.32mg/L in 36h, the degradation rate is 98.73%, and the OD ₆₀₀ The maximum is 2.08, which shows that the ZJB20129 has good degradation capability on ammonia nitrogen. OD at 48h ₆₀₀ The decrease in ammonia nitrogen concentration and the slight increase in ammonia nitrogen concentration may be due to death of some of the cells and release of intracellular nitrogen-containing organic matter into the medium.

As can be seen from FIG. 4, when the culture condition is 30℃and 160rpm, the strain ZJB20129 reduces the nitrate nitrogen concentration from 109.1mg/L to 4.00mg/L within 48 hours, the degradation rate is 96.33%, and the OD ₆₀₀ The maximum is 2.03, which shows that the ZJB20129 has good degradation capability on the nitrate nitrogen.

As can be seen from FIG. 5, strain ZJB20129 reduced the concentration of nitrous nitrogen from 101.0mg/L to 4.03mg/L in 60 hours at 30℃and 160rpm, the degradation rate was 96.01%, OD ₆₀₀ Maximum 2.08, indicating that ZJB20129 has good degradation capability on nitrous nitrogen.

Example 3: determination of ZJB20129 efficient denitrification condition

(1) Influence of different carbon sources on the degradation rate of ZJB20129 ammonia nitrogen: the carbon sources of the nitrifying culture medium in example 2 were changed to sodium acetate, sodium succinate, sodium citrate, sucrose and glucose respectively, the fixed carbon source content was 1.5g/L (calculated as C), 2% ZJB20129 seed solution was inoculated, and the culture was carried out at 30℃and 160rpm for 36 hours to determine the ammonia nitrogen degradation capacity, and the results are shown in FIG. 6. The ammonia nitrogen clearance rate is 98.26% when sodium acetate is used as a carbon source, and 99.01% when sodium succinate is used as a carbon source, which is far higher than the clearance rate when glucose, sucrose and sodium citrate with the same content (calculated by C) are used as the carbon source.

(2) Influence of different carbon-nitrogen ratios on the degradation rate of the ZJB20129 ammonia nitrogen: the sodium succinate content of the nitrifying medium in example 2 was changed to have the mass ratios of C/N of 6, 9, 12, 15 and 18, respectively, and 2% ZJB20129 seed solution was inoculated, and the culture was performed at 30℃and 160rpm for 36 hours to determine the ammonia nitrogen degradation capacity, and the results are shown in FIG. 7. The ammonia nitrogen clearance rate is highest and reaches 98.31% when the C/N mass ratio is 15.

(3) Influence of different pH on the degradation rate of ZJB20129 ammonia nitrogen: the pH of the nitrifying medium in example 2 was changed to 5, 6, 7, 8 and 9, respectively, and 2% ZJB20129 seed solution was inoculated, and the culture was performed at 30℃and 160rpm for 36 hours to determine the ammonia nitrogen degradation capacity, and the results are shown in FIG. 8. When the pH of the culture medium is 8, the ammonia nitrogen clearance rate reaches 99.27%, and the strain has the strongest denitrification capability when the pH is near 8.

(4) Influence of different temperatures on the degradation rate of the ZJB20129 ammonia nitrogen: the nitrifying medium in example 2 was used, the culture temperatures were changed to 20, 25, 30, 35 and 40℃respectively, 2% ZJB20129 seed solution was inoculated, and the culture was carried out at 160rpm for 36 hours to determine the ammonia nitrogen degradation capacity, and the results are shown in FIG. 9. The ammonia nitrogen clearance rate is highest when the temperature is 35 ℃, and reaches 98.94%.

(5) Influence of different rotational speeds on the degradation rate of ZJB20129 ammonia nitrogen: the nitrifying medium in example 2 was used, the culture speeds were changed to 40, 80, 120, 160 and 200rpm, 2% ZJB20129 seed solution was inoculated, and the culture was performed at 30℃for 36 hours to determine the ammonia nitrogen degradation capacity, and the results are shown in FIG. 10. The ammonia nitrogen clearance rate is highest when the rotating speed is 160rpm, and reaches 99.05 percent.

Example 4: application of heterotrophic nitrification-aerobic denitrification bacteria in sewage treatment a sewage sample is taken from a sewage treatment plant in Hangzhou. 1L of the sewage water sample is taken, 2% seed solution (ZJB 20129 seed solution is bacterial suspension obtained by culturing the LB culture medium in example 2 for 12 hours at 30 ℃ and 160 rpm) is inoculated, and the culture is carried out by shaking at 30 ℃ and 160rpm for 36 hours. The total nitrogen, ammonia nitrogen and nitrite nitrogen concentration before and after sewage treatment were measured by sampling, and the measurement results are shown in Table 1.

TABLE 1 Nitrogen concentration variation before and after wastewater sample treatment

As shown in Table 1, the strain has good effect of removing ammonia nitrogen and nitrate nitrogen in sewage, and no accumulation of nitrite nitrogen is generated. Therefore, compared with the existing strain, the strain has wide application range, can enrich the strain library of the environmental microorganism, provides the environmental microorganism with high-efficiency denitrification, and provides a solution for polluted water bodies with different degrees.

Sequence listing

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<120> heterotrophic nitrification-aerobic denitrification bacterium and application thereof

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Claims

1. Heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 with a preservation number of CCTCC NO: m2020965.

2. The use of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 as claimed in claim 1 in the preparation of denitrification biological agents.

3. The use of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 in nitrogen-containing wastewater denitrification treatment as claimed in claim 1.

4. A method for denitrification treatment of nitrogen-containing sewage by using heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 according to claim 1, which is characterized in that: inoculating heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 into the nitrogen-containing sewage, adding a carbon source until the C/N ratio is 15-18, culturing at the pH value of 7-9, 30-40 ℃ and 80-200 rpm, and removing ammonia nitrogen in the sewage; the carbon source is one or two of sodium succinate and sodium acetate.

5. The method of claim 4, wherein: the culture was carried out at a C/N ratio of 15, pH 8, temperature of 35℃and rotation speed of 160 rpm.