CN113736700A

CN113736700A - Heterotrophic nitrification-aerobic denitrification bacteria and application thereof

Info

Publication number: CN113736700A
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: 2021-12-03
Anticipated expiration: 2041-09-03
Also published as: CN113736700B

Abstract

The invention discloses heterotrophic nitrification-aerobic denitrification bacteria and application thereof. The heterotrophic nitrification-aerobic denitrification bacteria are collected in China Center for Type Culture Collection (CCTCC), classified and named as Acinetobacter India ZJB20129, and the collection number is CCTCC NO: m2020965, preservation date 2020, 12.24.d. The heterotrophic nitrification-aerobic denitrification Acinetobacter indianii B2-1 (provided by the invention)Acinetobacter indicusstrain B2-1) ZJB20129 can use sodium succinate and sodium acetate as carbon sources, and the sodium succinate is used as the optimal carbon source, the mass ratio of C to N is 15, the pH is 8, the temperature is 35 ℃The optimal deamination nitrogen effect is achieved when the culture medium is cultured aerobically for 36 hours at the rotating speed of 160rpm, and the deamination nitrogen effect is 99.27 percent. Meanwhile, the strain has good effect of removing 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. The exceeding of nitrogen is the most common pollution problem in water body pollution, can cause water body eutrophication, cause mass propagation of algae, reduce dissolved oxygen in the water body and death of aquatic organisms, not only endangers the water safety of human beings, but also seriously influences the ecological balance of the water body; in addition, excessive nitrogen discharge can cause the nitrogen balance to be destroyed, thereby causing the water body to lose the self-cleaning function. Therefore, the sewage rich in nitrogen can be discharged into the natural environment after the sewage is subjected to denitrification treatment and 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 ammonia nitrogen in sewage into nitrogen gas under the action of functional floras such as ammonia oxidizing bacteria, nitrifying bacteria, denitrifying bacteria and the like so as to achieve the aim of denitrification. In the treatment process, nitrobacteria and nitrosobacteria convert ammonia nitrogen into nitrate nitrogen, and the nitrate nitrogen has low chemical potential energy and high stability and is easy to accumulate. Denitrifying bacteria act on nitrate nitrogen in a water body, gradually reduce the nitrate nitrogen into nitrite nitrogen, nitric oxide and nitrous oxide, finally reduce the nitrate nitrogen into nitrogen gas and release the nitrogen gas into the atmosphere to complete the circulation of nitrogen elements, and the method is an important link for 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 oxygen respectively for nitrification and denitrification respectively, so as to achieve the aim of denitrification. Therefore, the processing method occupies a large space, has high operation cost and is complicated in process.

FromParacoccus denitrificansSince the first identification of the aerobic denitrifying bacteria, the aerobic denitrifying technology is approved and further popularized. The aerobic denitrifying bacteria can simultaneously carry out nitration-denitrification reaction by taking oxygen and nitrate nitrogen as electron acceptors under aerobic condition, thereby realizing the rapid removal of organic nitrogen and having important application value in the biological denitrification technology of organic matters. Research in this field has been carried out for over thirty years, and various aerobic denitrification microbial strains have been identified separately, and their growth characteristics and treatment capacities in the process of heterotrophic nitrification-aerobic denitrification biological denitrification have been greatly different. Therefore, the screened bacterial strains which are efficient and can simultaneously carry out nitrification and denitrification have important application value for the denitrification of the polluted water body.

Disclosure of Invention

The invention aims to provide heterotrophic nitrification-aerobic denitrification acinetobacter and application thereof, aiming at the defects of higher operation cost and more complicated process of sewage treatment in the prior art.

The technical scheme adopted by the invention is as follows:

heterotrophic nitrification-aerobic denitrification bacteria are preserved in China Center for Type Culture Collection (CCTCC) with the address: chinese, Wuhan university, classified and named Acinetobacter India B2-1(Acinetobacter indicusstrain B2-1), and the preservation number is CCTCC NO: m2020965, preservation date 2020, 12.24.d.

The heterotrophic nitrification-aerobic denitrification acinetobacter ZJB20129 is characterized in that: the bacterial colony is identified as gram-negative bacteria, the bacterial colony is creamy yellow and round, the surface is wet and easy to pick up, and the bacteria are rod-shaped. The carbon source suitable for the growth of the strain is sodium succinate or sodium acetate, the suitable C/N is 15-18, the suitable pH is 7.0-9.0, and the suitable growth temperature is 30-40 ℃.

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

The invention also relates to the application of the heterotrophic nitrification-aerobic denitrification bacteria in the 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 nitrogen-containing sewage, adding a carbon source until the C/N ratio is 15-18, culturing at the pH value of 7-9, the temperature of 30-40 ℃ and the rpm of 80-200, and removing ammonia nitrogen in the wastewater.

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

Preferably, the culture is performed under the conditions of a C/N ratio of 15, pH of 8, temperature of 35 ℃ and rotation speed of 160 rpm.

The invention has the advantages of

The heterotrophic nitrification-aerobic denitrification Acinetobacter indiani (of the invention)Acinetobacter indicusStrain) ZJB20129 can use sodium succinate and sodium acetate as carbon source, and has optimal ammonia nitrogen removal effect of 99.27% when the sodium succinate is used as optimal carbon source, C/N is 15, pH is 8, temperature is 35 deg.C, and rotation speed is 160rpm, and aerobic culture is carried out for 36 h. Meanwhile, the strain has a good effect of removing nitrate nitrogen and nitrite nitrogen, and has important practical significance for developing high-efficiency denitrification biological agents.

Drawings

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

FIG. 2 is a phylogenetic tree of heterotrophic nitrification-aerobic denitrification bacteria ZJB 2012916S rDNA of the present invention.

FIG. 3 is a schematic diagram of the degradation capability of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 on ammonia nitrogen.

FIG. 4 is a schematic diagram of the capability of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 in degrading nitrate nitrogen.

FIG. 5 is a schematic diagram of the capability of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 of the invention in degrading nitrite nitrogen.

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

FIG. 7 is a schematic diagram of the degradation of ammonia nitrogen of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 in different carbon-nitrogen ratios.

FIG. 8 is a schematic diagram of the degradation of ammonia nitrogen of heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 under different pH values.

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

FIG. 10 is a schematic diagram of the degradation of ammonia nitrogen by heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 at different rotating speeds.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1: screening and identification of heterotrophic nitrification-aerobic denitrification bacteria

Adding 2-3 g of a biological rotating disc mud sample of a Hangzhou sewage treatment company into a conical flask filled with 100mL of sterile water, shaking on a shaking table for 3 hours, sucking 2mL of liquid by using a liquid transfer gun, inoculating the liquid into a sterilized LB culture medium, and culturing for 2-3 days at 30 ℃ and 180 rpm.

The LB medium described above: 10g of tryptone, 5g of yeast powder, 10g of sodium chloride and distilled water to a constant volume of 1000mL, wherein the pH value is natural; if a solid culture medium is needed, 1.5-2% agar is added.

Inoculating the strain culture solution into test tube containing sterile water, diluting at a certain proportion to obtain strain concentration of 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7And 10^-8A gradient of bacterial suspension; then, a plate coating method is adopted to respectively suck 100 mu L of the suspension liquid of each gradient bacterium to coat the suspension liquid on an LB solid culture medium, the suspension liquid is placed in a constant temperature incubator at 30 ℃ to be cultured, colonies with different forms are respectively picked to carry out streak separation, the colonies are separated and purified 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 by primary screening in an LB culture medium to logarithmic growth phase, respectively sucking 100 mu L of the strains, coating the strains on a bromothymol blue (BTB) solid culture medium, culturing for 4d at 30 ℃, and selecting a bacterial colony on the solid culture medium with the fastest blue change for preservation and protection to obtain the strain ZJB20129 which is used as a target strain for subsequent research.

The bromothymol blue (BTB) solid culture medium is prepared from the following components: potassium nitrate (KNO)₃) 1.01g, sodium succinate (C)₄H₄Na₂O₄) 8.5g, magnesium sulfate heptahydrate (MgSO)₄·7H₂O) 1.0g, potassium dihydrogen phosphate (KH)₂PO 4) 1.0g, ferrous sulfate heptahydrate (FeSO)₄·7H₂O) 0.59g, calcium chloride (CaCl)₂) 0.09g, 1mL of 1% bromothymol blue ethanol solution, distilled water to a constant volume of 1000mL, 20g of agar, and adjusting the pH value to 7.0-7.3 by using 1mol/L NaOH.

As shown in figure 1, the physiological and biochemical tests of the strain ZJB20129 are gram negative, and the strain is round in cream yellow, easy to pick up due to wet surface, and rod-shaped. Extracting bacterial genome of the target strain by using a genome kit; using bacterial universal primers 27F: 5 '-AGAGTTTGATCMTGGCTCAG-3', 1492R: 5 '-TACGGYTACCTTGTTACGACTT-3' and carrying out PCR amplification and agarose gel electrophoresis (1%) verification; electrophoresis detection, gel cutting purification sequencing, and sequencing by Scophthalmus sp.

And (3) PCR reaction conditions: pre-denaturation at 94 deg.C for 3 min; denaturation for 30 s; annealing at 54 ℃ for 30 s; extension at 72 deg.C for 1min30 s; the cycle was repeated 30 times starting with the second step.

The length of the 16S rDNA sequence of the strain ZJB20129 is 1415bp, and the gene sequence is shown in SEQ ID number 1.

Uploading the result of the strain sequence to an NCBI database, comparing the result with the existing 16S rDNA gene sequence of the bacteria in the database, and indicating that the bacteria isAcinetobacter IndicusAnd (5) strain. Phylogenetic trees are constructed by the neighborhood joining method of Mega.7.0 software, the genetic characteristics of the strains are analyzed, the species condition and the evolutionary position of the strains in genetics are determined, and the result is shown in figure 2.

Strain ZJB20129 named Acinetobacter indifferens B2-1(Acinetobacter indicusstrain B2-1), strain number ZJB20129, preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2020965, preservation date is 2020, 12 months and 24 days, and addresses Wuhan university in Wuhan City, China.

Example 2: identification of nitrification/denitrification capability of strain ZJB20129 on different nitrogen source culture media

The formula of the nitrification culture medium is as follows: ammonium sulfate ((NH)₄)₂SO₄) 0.47g of sodium succinate (C)₄H₄Na₂O₄) 5.06g magnesium sulfate (MgSO)₄) 0.1g of potassium dihydrogen phosphate (KH)₂PO 4) 0.1g, trace element solution 2mL, distilled water with constant volume of 1000mL and pH value of 7.0-7.5; wherein the trace element solution comprises: EDTA 50g, Zinc sulfate heptahydrate (ZnSO)₄·7H₂O) 3.92g, calcium chloride (CaCl)₂) 5.5g of manganese chloride tetrahydrate (MnCl)₂·4H₂O) 5.1g, ferrous sulfate heptahydrate (FeSO)₄·7H₂O) 5.0g, ammonium molybdate tetrahydrate ((NH)₄)6Mo₇O₂₄·4H₂O) 1.1g, copper sulfate pentahydrate (CuSO)₄·5H₂O) 1.6g, cobalt chloride hexahydrate (CoCl)₂·6H₂O) 1.6g, and distilled water was added to make a volume of 1000 mL. Denitrifying Medium ammonium sulfate ((NH) in the nitrifying Medium₄)₂SO₄) 0.47g was replaced with potassium nitrate (KNO)₃) 0.72g or sodium nitrite (NaNO)₂) 0.49g, the remaining formulation was kept unchanged.

The treatment method comprises the following steps: inoculating 2% seed solution (the seed solution is LB culture medium 30 deg.C, bacterial suspension cultured at 160rpm for 12 h), and culturing at 30 deg.C and 160 rpm. Periodically takingSeparately determining NH in the supernatant₄ ⁺-N、NO₃⁻-N、NO₂⁻ -N and OD₆₀₀The results are shown in FIGS. 3 to 5.

As can be seen from FIG. 3, when the culture conditions are 30 ℃ and 160rpm, the ammonia nitrogen concentration of the strain ZJB20129 is reduced from 103.8mg/L to 1.32mg/L within 36h, the degradation rate is 98.73%, and the OD is OD₆₀₀The maximum is 2.08, which shows that ZJB20129 has good degradation capability on ammonia nitrogen. OD at 48h₆₀₀The ammonia nitrogen concentration is slightly increased, which is probably caused by the death of part of thalli and the release of nitrogenous organic matters in cells into a culture medium.

As can be seen from FIG. 4, when the culture conditions were 30 ℃ and 160rpm, the strain ZJB20129 reduced the nitrate nitrogen concentration from 109.1mg/L to 4.00mg/L within 48h, the degradation rate was 96.33%, and the OD was OD₆₀₀The maximum value is 2.03, which shows that ZJB20129 has good degradation capability on nitrate nitrogen.

As can be seen from FIG. 5, when the culture conditions were 30 ℃ and 160rpm, the strain ZJB20129 reduced the nitrite nitrogen concentration from 101.0mg/L to 4.03mg/L within 60h, the degradation rate was 96.01%, and the OD was OD₆₀₀The maximum value is 2.08, which shows that ZJB20129 has good capability of degrading nitrite nitrogen.

Example 3: determination of ZJB20129 efficient denitrification conditions

(1) Influence of different carbon sources on ammonia nitrogen degradation rate of ZJB 20129: changing the carbon sources of the nitrification medium in example 2 to be sodium acetate, sodium succinate, sodium citrate, sucrose and glucose respectively, the fixed carbon source content is 1.5g/L (calculated by C), inoculating 2% ZJB20129 seed solution, culturing for 36h at 30 ℃ and 160rpm, and measuring the ammonia nitrogen degradation capability, wherein the results are shown in FIG. 6. The results show that 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 rates of glucose, sucrose and sodium citrate with the same content (calculated by C) as the carbon source.

(2) Influence of different carbon-nitrogen ratios on ammonia nitrogen degradation rate of ZJB 20129: the content of sodium succinate in the nitrification culture medium in example 2 was changed to make the mass ratios of C/N in the

medium

6, 9, 12, 15 and 18, respectively, 2% ZJB20129 seed solution was inoculated, cultured at 30 ℃ and 160rpm for 36 hours, and the ammonia nitrogen degradation capacity was measured, with the results shown in fig. 7. The method shows that when the mass ratio of C to N is 15, the ammonia nitrogen clearance is highest and reaches 98.31 percent.

(3) Influence of different pH values on the ammonia nitrogen degradation rate of ZJB 20129: the pH values of the nitrification media in example 2 were changed to 5, 6, 7, 8 and 9, respectively, 2% ZJB20129 seed solution was inoculated, the seeds were cultured at 30 ℃ and 160rpm for 36 hours, and the ammonia nitrogen degradation capability was measured, and the results are shown in FIG. 8. When the pH value of the culture medium is 8, the ammonia nitrogen clearance rate reaches 99.27 percent, and the bacterial strain has the strongest denitrification capability when the pH value is about 8.

(4) Influence of different temperatures on the ammonia nitrogen degradation rate of ZJB 20129: the nitrifying medium in example 2 was used, the culture temperatures were changed to 20, 25, 30, 35 and 40 ℃, and 2% ZJB20129 seed solutions were inoculated and cultured at 160rpm for 36 hours, and the ammonia nitrogen degradation ability was measured, with the results shown in fig. 9. When the temperature is 35 ℃, the ammonia nitrogen clearance is the highest and reaches 98.94 percent.

(5) Influence of different rotating speeds on the ammonia nitrogen degradation rate of ZJB 20129: the nitrifying medium in example 2 was used, and 2% of ZJB20129 seed solutions were inoculated by changing the culture rotation speed to 40, 80, 120, 160 and 200rpm, respectively, and cultured at 30 ℃ for 36 hours, and the ammonia nitrogen degradation ability was measured, and the results are shown in fig. 10. When the rotating speed is 160rpm, the ammonia nitrogen clearance is the highest and reaches 99.05 percent.

Example 4: application of heterotrophic nitrification-aerobic denitrification bacteria in sewage treatment

The sewage sample is taken from a certain Hangzhou sewage treatment plant. 1L of the above wastewater sample was inoculated with 2% seed solution (ZJB 20129 seed solution is a bacterial suspension obtained by culturing LB medium in example 2 at 30 ℃ and 160rpm for 12 h), and shake-cultured at 30 ℃ and 160rpm for 36 h. The total nitrogen, ammonia nitrogen and nitrite nitrogen concentrations before and after sewage treatment were measured by sampling, and the measurement results are shown in table 1.

TABLE 1 Change in Nitrogen concentration before and after treatment of Sewage samples

As can be seen from Table 1, the strain has good effect of removing ammonia nitrogen and nitrate nitrogen in sewage, and does not generate accumulation of nitrite nitrogen. Therefore, compared with the existing strains, the strain has wide application range, can enrich the strain stock of the environmental microorganisms, provides the environmental microorganisms 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 bacteria and application thereof

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Claims

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

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

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

4. The method for denitrifying nitrogen-containing sewage by using the heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 in claim 1, wherein: inoculating the heterotrophic nitrification-aerobic denitrification bacteria ZJB20129 into nitrogen-containing sewage, adding a carbon source until the C/N ratio is 15-18, culturing at the pH of 7-9, the temperature of 30-40 ℃ and the rpm of 80-200, and removing ammonia nitrogen in the sewage.

5. The method of claim 4, wherein: the carbon source is one or two of sodium succinate and sodium acetate.

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