CN114958685B

CN114958685B - Hg-removing device 2+ Performance aerobic denitrification strain and application thereof

Info

Publication number: CN114958685B
Application number: CN202210722523.1A
Authority: CN
Inventors: 王莹; 陈虎; 丁欣; 吕永康; 陈宏平
Original assignee: Taiyuan University of Technology
Current assignee: Taiyuan University of Technology
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2023-09-15
Anticipated expiration: 2042-06-24
Also published as: CN114958685A

Abstract

The invention relates to the technical field of microorganisms, in particular to a method for removing Hg ²⁺ Aerobic denitrification strain with performance and application thereof; the strain is Acinetobacter genusAcinetobacter sp.) bacteria LV3 with a preservation number of CGMCC NO.25044, the strain can grow in mercury-free and mercury-containing environments by taking nitrate nitrogen or nitrite nitrogen as a sole nitrogen source, and can utilize an organic carbon source to perform aerobic denitrification and denitrification, which indicates that the strain LV3 has the capability of synchronous denitrification and carbon removal. Meanwhile, the strain can endure and completely remove Hg with the concentration not higher than 6mg/L in the denitrification process ²⁺ The method has important significance for biological denitration of the flue gas and treatment of mercury-containing and nitrogen-containing wastewater.

Description

Hg-removing device 2+ Performance aerobic denitrification strain and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a method for removing Hg ²⁺ Aerobic denitrifying bacterial strain with performance and application thereof.

Background

Nitrogen oxides (NOx) are one of the major atmospheric pollutantsMainly comprises NO and NO ₂ 、N ₂ O ₃ 、N ₂ O ₄ And the like. Wherein NO in flue gasxIs composed of NO and NO ₂ And is also a major component that causes atmospheric pollution. At present, for NO in flue gasxThe treatment method mainly comprises a Selective Catalytic Reduction (SCR), a selective non-catalytic reduction (SNCR), an absorption method, a biological method and the like, and the biological method has the advantages of low investment and operation cost, NO secondary pollution and the like, and becomes NO at home and abroadxThe hot spot and the key point of the research are removed.

Biological method for removing NOxThe history of exhaust gas research can be traced to the 90 s of the last century. In 1993, researchers Apel and the like in American Edahh national engineering laboratory carried out anaerobic denitrification for reducing NO in flue gasxIs the first to propose the reduction of denitrifying bacteria to eliminate NO from fumexThe reduction method is based on the traditional biological filter (biofilter) technology of the traditional anaerobic denitrification, and can finally reduce NO to form pollution-free N ₂ However, anaerobic conditions are difficult to realize, which severely restricts the practical application of the method.

The appearance of aerobic denitrification provides a brand new way for biological denitrification and also provides a new method for solving the problem of oxygen inhibition in the traditional biological denitrification process. In recent years, researchers apply aerobic denitrifying bacteria to different denitration treatment processes, and certain effects are achieved. The application of aerobic denitrifying bacteria in an oxidation absorption-biotrickling filter shows excellent NOxTreatment effect (Journal of Hazardous Materials, 2021, 404: 124109.).

However, for heavy metals represented by mercury in flue gas, whether the heavy metals can affect the aerobic denitrification flora in a biological filter tower is a worth discussing problem, considering that most microorganisms have a certain poison resistant zone, even can carry out biological adsorption or absorption degradation on mercury, the aerobic denitrification flora can be subjected to mercury resistant domestication, and the flora which has high-efficiency denitrification performance and can resist mercury to a certain extent or even remove mercury is screened out, so that precious microorganism strains can be provided for the combined treatment process of denitrification and mercury removal of the biological filter tower of a coal-fired power plant.

Although the research thought comes from the research of the subject group on the coal-fired flue gas of the thermal power plant, the final experimental part examines Hg in the liquid phase ²⁺ Influence on denitrification performance of aerobic denitrification strains. Thus the research result is not only suitable for simultaneously containing NOxAnd the mercury-containing thermal power plant coal-fired flue gas, nitric acid plant and coking flue gas, and is also suitable for treating certain industrial wastewater, tanning wastewater, domestic wastewater and the like containing nitrate nitrogen and mercury at the same time. Meanwhile, the water pollution status quo in China shows that the research result is also suitable for eutrophication, rivers, lakes, reservoirs, and the like polluted by heavy metals. Therefore, if the microbial flora which can consume organic pollutants and nitrogen-containing pollutants and has mercury resistance and even mercury removal performance can be screened and domesticated, great transformation and confidence can be brought to flue gas treatment and water body restoration.

Disclosure of Invention

The object of the present invention is to provide a process for removing Hg ²⁺ Aerobic denitrifying strain with performance. The strain can perform aerobic denitrification by taking nitrate nitrogen or nitrite nitrogen as a sole nitrogen source in mercury-free and mercury-containing environments, and can endure and completely remove Hg with the concentration not higher than 6mg/L in the denitrification process ²⁺ The method has important significance for biological denitration of the flue gas and treatment of mercury-containing and nitrogen-containing wastewater.

In order to solve the technical problems, the invention adopts the following technical scheme: hg-removing device ²⁺ Aerobic denitrifying strain with performance, classified and named Acinetobacter genusAcinetobacter sp.) bacteria LV3, deposited at the China general microbiological culture Collection center, address: the institute of microbiology, national academy of sciences, north chen xi lu 1, 3, the region of the morning sun in beijing; the preservation number is CGMCC NO. 25044.

The said process has Hg eliminating function ²⁺ The screening method of the aerobic denitrification strain LV3 with the performance comprises the following steps:

(1) 1g of soil in a mercury-rich region is taken, firstly, in Hg ²⁺ Enrichment culture in enrichment medium with concentration of 5mg/L at 30deg.CShaking culture at 120rpm for 2d.

(2) Then the enriched bacterial liquid of 1mL is continuously connected into Hg ²⁺ The culture was performed at 30℃and 120rpm in an enrichment medium at a concentration of 5mg/L for 2d, and this procedure was repeated 3 times.

(3) Subjecting the mixed bacterial liquid in the step (2) to 10 ⁻¹ ~10 ⁻⁹ And (3) carrying out gradient dilution, respectively coating the diluted solutions on a BTB solid culture medium, culturing in a constant temperature incubator until visible colonies are formed, selecting strains with blue halos in different forms, streaking in a DM culture medium by using an inoculating loop in an ultra-clean bench, and separating and purifying three times.

(4) The separated and purified strains are respectively picked and inoculated to Hg ²⁺ Culturing in DM culture medium with concentration of 4mg/L, and comprehensively investigating strain growth, nitrate nitrogen degradation, mercury resistance and Hg resistance ²⁺ Removing the condition, screening to obtain a strain with Hg ²⁺ The aerobic denitrifying strain with the performance was removed and designated LV3.

Strain LV3 has the following phenotypic characteristics on BTB solid medium: the colony has milky opaque dots with transparent halos, the surface is smooth and moist and is easy to pick up, the edges are regular, and the BTB solid flat plate turns blue under the denitrification of the strain. Negative under microscope after passing through gram stain.

The 16S rDNA gene sequence of the strain LV3 is SEQ ID NO:1, the length of the base sequence is 1427bp.

According to the form and GenBank database analysis of NCBI website, blast homology analysis shows that the strain LV3 has close relationship with Acinetobacter genus and with the strainAcinetobacter sp, strain IAE167 andAcinetobactersp and strain S1 sequence homology is up to 100%, thus determining that the strain LV3 is Acinetobacter @Acinetobacter sp.）。

In addition, the invention also provides a method for removing Hg ²⁺ The preparation method of the working solution of the aerobic denitrification strain with the performance comprises the following steps: acinetobacter with preservation number of CGMCC No.25044Acinetobacter sp.) bacterial LV3 was inoculated into denitrification medium for activation at 30Culturing in a shaking table at 120rpm at a constant temperature until the OD of the strain is at logarithmic phase ₆₀₀ Approximately equal to 1.3, the denitrification culture medium formula is 5.0625g/L, naNO of sodium succinate ₃ 0.607g/L、KH ₂ PO ₄ 0.4g/L、MnSO ₄ •H ₂ O 0.01g/L、MgSO ₄ •7H ₂ O 0.05g/L、FeSO ₄ •7H ₂ O0.01 g/L, and the pH of the medium was adjusted to 7.0 with NaOH or HCl.

The invention also provides the Hg-removing device ²⁺ Aerobic denitrifying strain LV3 or containing a strain with Hg removal ²⁺ The application of the working solution of the aerobic denitrification strain with the performance in the treatment of mercury-containing and nitrogen-containing wastewater.

Further, the mercury concentration of the wastewater ranges from 1mg/L to 6mg/L.

Further, the mercury concentration of the wastewater is in the range of 4mg/L.

Further, acinetobacter genusAcinetobacter sp.) bacteria LV3 are grown in mercury-containing environments with nitrate nitrogen or nitrite nitrogen as the sole nitrogen source, and can utilize organic carbon sources for aerobic denitrification. Strain LV3 in Hg ²⁺ The strain LV3 has the capability of synchronous denitrification and carbon removal, and can grow by taking 100mg/L nitrate nitrogen or nitrite nitrogen as the only nitrogen source under the condition of 4mg/L concentration, and can perform aerobic denitrification and nitrogen removal by utilizing an organic carbon source.

The invention also provides the Hg-removing device ²⁺ The application of the aerobic denitrification strain with the performance in the field of flue gas denitrification.

Acinetobacter genusAcinetobacter sp.) bacteria LV3 in Hg ²⁺ The method can also endure the heavy metal ions such as Pb (II) and Cr (VI) with a certain concentration under the condition of 4mg/L, and has more advantages in the biological denitration of the flue gas or the actual wastewater treatment process.

Acinetobacter genus as described aboveAcinetobacter sp.) bacteria LV3 in Hg ²⁺ When the concentration is not higher than 4mg/L, the proportion of the initial nitrogen converted into gaseous nitrogen is close to and is more than 60% when sodium pyruvate and nitrate nitrogen are respectively used as the only carbon source and the nitrogen source for growth, and the initial nitrogen is mainly converted into intracellular nitrogen, and almost no initial nitrogen is generated in the whole denitrification processAccumulation of nitrite nitrogen and ammonia nitrogen was detected, which suggests that strain LV3 is denitrified primarily by denitrification and assimilation.

Acinetobacter genus as described aboveAcinetobacter sp.) bacteria LV3 in Hg ²⁺ The mercury volatilizing efficiency under the conditions that the concentration is 4mg/L, the nitrate nitrogen concentration is 100mg/L and the sodium pyruvate content is 5.0625g/L shows that the strain LV3 can volatilize Hg ²⁺ Reducing into mercury simple substance with low toxicity and low solubility.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for removing Hg ²⁺ The aerobic denitrification strain with the performance is suitable for biological denitrification of flue gas and treatment of mercury-containing and nitrogen-containing wastewater, has wide application prospect and has good practical application value and social benefit.

Drawings

FIG. 1 shows colony morphology of strain LV3.

FIG. 2 shows a phylogenetic tree of strain LV3.

FIG. 3a shows different Hg ²⁺ Effect of concentration on strain LV3 growth.

FIG. 3b shows different Hg' s ²⁺ Effect of concentration on removal of nitrate nitrogen from strain LV3.

FIG. 4a shows strain LV3 against 2mg/L Hg in water ²⁺ Is not limited, and the removal performance of the catalyst is improved.

FIG. 4b shows strain LV3 against 4mg/L Hg in water ²⁺ Is not limited, and the removal performance of the catalyst is improved.

FIG. 4c shows strain LV3 against 6mg/L Hg in water ²⁺ Is not limited, and the removal performance of the catalyst is improved.

Figure 5 shows the aerobic denitrification performance of strain LV3 under mercury-containing conditions with nitrate nitrogen as the sole nitrogen source.

Figure 6 shows the aerobic denitrification performance of strain LV3 under mercury-containing conditions with nitrite nitrogen as the sole nitrogen source.

FIG. 7a shows the effect of heavy metal ions on the growth of strain LV3 in the presence of mercury.

FIG. 7b shows the effect of heavy metal ions in the presence of mercury on the removal of nitrate nitrogen from strain LV3.

FIG. 7c shows the effect of heavy metal ions in the presence of mercury on the removal rate (36 h) of nitrate nitrogen from strain LV3.

FIG. 8 shows different Hg' s ²⁺ Nitrogen balance analysis of strain LV3 at concentration with nitrate nitrogen as sole nitrogen source.

FIG. 9 shows strain LV3 versus Hg ²⁺ And volatilizing and developing the experimental result.

Detailed Description

The invention is further illustrated below with reference to specific examples.

In the following examples, the methods are conventional, unless otherwise specified. In the examples, nitrate nitrogen was measured by salicylic acid colorimetry, ammonia nitrogen was measured by Nashi reagent spectrophotometry, nitrite nitrogen was measured by N- (1-naphthyl) -ethylenediamine spectrophotometry, total nitrogen was measured by alkaline potassium persulfate ultraviolet spectrophotometry, mercury ion concentration was measured by ICP-OSE instrument, COD was measured by potassium dichromate method, dissolved oxygen was measured by portable dissolved oxygen meter (HQ 30D, HACH), pH was measured by pH meter (Seven 2Go pro, switzerland Meter Tolyduo), OD ₆₀₀ Measurement was performed at a wavelength of 600nm using a visible spectrophotometer, and pH adjustment was performed using 2mol/L hydrochloric acid and 2mol/L NaOH. The various units used in the examples all adopt the national standard in a unified way.

Example 1

Has Hg removal ²⁺ The screening of the aerobic denitrifying strain LV3 with the performance is as follows:

(1) Firstly, 1g of soil from a mercury-rich area is put into a 250mL conical flask containing 100mL of enrichment medium, and the enrichment medium comprises 10g/L peptone, 5g/L, naCl g/L, hg of yeast extract ²⁺ 5mg/L, ph=7.0. The mouth was then sealed with a sterile breath sealing membrane and placed in a shaker at 30℃and 120rpm for enrichment culture for 2d. ,

(2) Transferring 1mL of enriched bacterial suspension from the enriched culture medium in the step (1) into Hg ²⁺ The culture was acclimatized in enrichment medium at a concentration of 5mg/L at 30℃and 120rpm in a shaker for 2d. The domestication process is repeated for 3 times

(3) Transferring 100 mu L of bacterial liquid, and carrying out gradient dilution to obtain 10 ⁻² ~10 ⁻⁷ Is diluted with a diluent of (2). Take 10 ⁻⁵ 、10 ⁻⁶ And 10 ⁻⁷ 100mL of gradient dilution is respectively coated on a bromothymol blue culture medium (BTB) solid culture medium, wherein the formula of the BTB solid culture medium comprises 0.683g/L sodium acetate, 0.675g/L sodium succinate and 0.5g/L, naNO glucose ₃ 0.607g/L、KH ₂ PO ₄ 0.4g/L、MnSO ₄ •H ₂ O 0.01g/L、MgSO ₄ •7H ₂ O 0.05g/L、FeSO ₄ •7H ₂ O0.01 g/L and 1% BTB 1mL/L, the pH of the culture medium was adjusted to 7.0 with NaOH or HCl, and 20 g/L of agar powder was added. Then placing the solid flat plate in a constant temperature incubator at 30 ℃ for culturing for 3-5 d until visible colonies are formed, selecting strains with blue halos and different forms, and streaking, separating and purifying the strains in a denitrification culture medium for three times by using an inoculating loop in an ultra-clean bench, wherein the denitrification culture medium is prepared from 5.0625g/L, naNO of sodium succinate ₃ 0.607g/L、KH ₂ PO ₄ 0.4g/L、MnSO ₄ •H ₂ O 0.01g/L、MgSO ₄ •7H ₂ O 0.05g/L、FeSO ₄ •7H ₂ O0.01 g/L, and the pH of the medium was adjusted to 7.0 with NaOH or HCl.

(4) The separated and purified strains were picked up and inoculated into 100mL of denitrification medium respectively to be activated (OD) at 30℃and 120rpm in a shaker ₆₀₀ About 1.3), 1mL of activated bacterial suspension is inoculated to Hg ²⁺ Culturing in 4mg/L denitrification culture medium at 30deg.C and 120rpm in shaking table, periodically measuring the content of nitrate nitrogen and nitrite nitrogen in the solution, and examining the growth, denitrification and Hg of the strain ²⁺ Tolerating and removing conditions. Finally screening to obtain a strain with Hg ²⁺ Removing the aerobic denitrification strain, namely LV3, and preserving the strain by adopting an ultralow temperature freezing method and a low temperature solid inclined plane preservation method.

Example 2

Has Hg removal ²⁺ The identification of the aerobic denitrifying strain LV3 in performance was as follows:

bacterial strain LV3 is inoculated on a BTB solid agar plate culture medium (same as in example 1), and biochemically cultured for 3-5 d at 30 ℃. The colony morphology of the strain LV3 is shown in FIG. 1, the colony has milky opaque dots with transparent halos, the surface is smooth and moist and easy to pick up, the edges are regular, and the color of the BTB solid flat plate changes blue under the denitrification of the strain.

Gram staining of strain LV3 revealed that the stained cells became red in color, indicating that strain LV3 was a gram negative bacterium.

The 16S rDNA sequencing base sequence of the strain LV3 is shown in the attached sequence table, the sequence is submitted to GenBank database analysis of NCBI website, and the Blast homology analysis shows that the strain LV3 has close relationship with Acinetobacter genus and with the strain (figure 2)Acinetobacter sp, strain IAE167 andAcinetobactersp and strain S1 sequence homology is up to 100%, thus determining that the strain LV3 is Acinetobacter @Acinetobacter sp.). The strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection) of microbiological culture Collection center, the academy of sciences of China, beijing, at a position of 2022, 6 and 10: the registration number of the collection center is CGMCC No.25044, and the registration number of the North Chen Xili No. 1 and 3 in the Chaoyang area of Beijing city is CGMCC No. 25044.

Example 3

Different Hg ²⁺ The growth and aerobic denitrification experiments of strain LV3 under concentration are as follows:

the working solution was purified from the preserved strain LV3 and activated in a 250mL Erlenmeyer flask containing 100mL of denitrification medium (same as in example 1) and cultured in a constant temperature shaker at 30℃and 120rpm until the logarithmic phase (OD) ₆₀₀ ≈1.3）。

Preparing Hg by taking sodium succinate and nitrite nitrogen as unique carbon sources and nitrogen sources respectively ²⁺ In 0, 1mg/L, 4mg/L, 6mg/L and 7mg/L of denitrification medium (same as in example 1), 5mL of working solution was inoculated into the 100mL of the above culture medium, sealed with a sealing film, and placed in a shaking table at 30℃and 120rpm to study the growth of strain LV3 under different mercury concentrations and the degradation of nitrate nitrogen. As shown in FIG. 3a, and no Hg was added ²⁺ In comparison with the blank control group of (C), the lag phase of strain LV3 was increased with Hg ²⁺ The increase in concentration prolonged with little degradation of nitrate nitrogen during the lag phase (fig. 3 b); once strain LV3 enters fast speedThe logarithmic phase of growth, the nitrate nitrogen content decreased rapidly (fig. 3 b), indicating that degradation of nitrate nitrogen by strain LV3 is closely related to its growth. But when Hg ²⁺ At a concentration of 7mg/L, no growth and no degradation of nitrate nitrogen was observed in strain LV3 throughout the experimental period, indicating that the strain had a maximum mercury tolerance concentration of 6mg/L in the denitrification medium. Duxbury's study showed that it was capable of measuring 4. 4mg/L Hg ²⁺ The strain grown in (a) is called "mercury resistant" strain, so strain LV3 is considered to be mercury resistant denitrification strain. To sum up, strain LV3 is available in the absence of mercury and Hg ²⁺ And (3) in an aerobic environment with the concentration not higher than 6mg/L, the nitrate nitrogen is used as the sole nitrogen source for growth and denitrification, and the nitrate nitrogen removal rate can finally exceed 95%.

Example 4

Strain LV3 against different Hg ²⁺ The degradation experiments of the concentration are as follows:

experiments were performed in denitrification media (same as in example 1) with sodium succinate and nitrite nitrogen as the only carbon source and nitrogen source, respectively, and with Hg < 2+ > concentrations of 2mg/L, 4mg/L and 6mg/L. Inoculating 5mL of working solution (same as in example 3) into the 100mL of culture medium, sealing with sealing film, and inoculating Hg corresponding to the non-inoculated strain ²⁺ Concentration medium was used as a blank and placed on a shaker at 30℃and 120rpm to investigate the degradation of strain LV3 to different mercury concentrations.

As shown in fig. 4a, 4b, 4c, strain LV3 is mercury tolerant and is effective in removing mercury from solution to a final effluent mercury concentration of 0mg/L with a mercury removal of 100%. In addition, the fact that the mercury concentration of the control group also showed a decreasing trend in the experimental time is consistent with the research results of Hu Liang and the like, which shows that the medium component has a certain influence on the removal of mercury, and the chelating effect between the medium component and mercury ions is probably caused. At mercury concentrations of 2mg/L, 4mg/L and 6mg/L, the amount of mercury removed by strain LV3 at 48 hours was 44.6%,39.33% and 41.5% of the total amount, respectively, indicating that strain LV3 also had a mercury removal effect. In combination, 6mg/L Hg can be tolerated and completely removed in the aerobic denitrification process of the strain LV3 with nitrate nitrogen as the sole nitrogen source ²⁺ 。

Example 5

The aerobic denitrification experiment of the strain LV3 with nitrate nitrogen as the sole nitrogen source under the mercury-containing condition is as follows:

in the experiment, sodium pyruvate and nitrate nitrogen are respectively used as the sole carbon source and nitrogen source, the carbon-nitrogen ratio is 16, and the nitrate nitrogen concentration is 100mg/L, hg ²⁺ The denitrification is carried out in a denitrification culture medium with the concentration of 4. 4mg/L, and the formula of the denitrification culture medium is sodium pyruvate of 4.80g/L, naNO ₃ 0.607g/L、KH ₂ PO ₄ 0.4g/L、MnSO ₄ •H ₂ O 0.01g/L、MgSO ₄ •7H ₂ O 0.05g/L、FeSO ₄ •7H ₂ O0.01 g/L, pH 7.0. 5mL of working solution (the same as in example 3) is inoculated into 100mL of culture medium, the culture medium is sealed by a sealing film, and the culture medium is placed in a shaking table at 30 ℃ and 120rpm to study the aerobic denitrification performance of the strain LV3 under the condition of mercury and taking nitrate nitrogen as the sole nitrogen source.

As shown in FIG. 5, after optimization of carbon source, C/N, temperature, pH and other conditions, the growth delay of strain LV3 under mercury-containing conditions is shortened, and OD at 12h ₆₀₀ The value can reach 0.512, and the concentration of the nitrate nitrogen is reduced to 81.85mg/L from the initial 101.59 mg/L at 12 h. And then the strain LV3 enters the logarithmic growth phase at 12-36 h, and the values of nitrate nitrogen and COD show a rapid decrease trend, so that the removal of the nitrate nitrogen is closely related to the growth of the strain LV3. The removal rates of nitrate nitrogen and total nitrogen at 36h can reach 100% and 93.94%, respectively, and the residual total nitrogen concentration is 6.16mg/L. Subsequent growth of strain LV3 into the decay phase, OD ₆₀₀ The value showed a decreasing trend, and the total nitrogen content in the solution was slightly increased, probably due to ammonia nitrogen release caused by strain death. The accumulation of nitrite nitrogen and ammonia nitrogen is basically avoided in the whole experimental process, which shows that the strain LV3 can effectively remove the nitrite nitrogen and does not cause secondary pollution when being used for sewage treatment. The removal of COD and the removal of nitrate nitrogen are synchronously carried out, and the maximum removal rates of COD, nitrate nitrogen and total nitrogen are 85.38%, 100% and 93.94%, respectively, which indicates that the strain LV3 can simultaneously carry out denitrification and decarbonization.

Example 6

The aerobic denitrification experiment of the strain LV3 with nitrite nitrogen as the sole nitrogen source under the condition of mercury is as follows:

in the experiment, sodium pyruvate and nitrate nitrogen are respectively used as the sole carbon source and nitrogen source, the carbon-nitrogen ratio is 16, and the nitrite nitrogen concentration is 100mg/L, hg ²⁺ The denitrification is carried out in a denitrification culture medium with the concentration of 4mg/L, and the formula of the culture medium is sodium pyruvate of 4.80g/L, naNO ₂ 0.493g/L、KH ₂ PO ₄ 0.4g/L、MnSO ₄ •H ₂ O 0.01g/L、MgSO ₄ •7H ₂ O 0.05g/L、FeSO ₄ •7H ₂ O0.01 g/L, pH 7.0. 5mL of working solution (the same as in example 3) is inoculated into 100mL of culture medium, the culture medium is sealed by a sealing film, and the culture medium is placed in a shaking table at 30 ℃ and 120rpm to study the aerobic denitrification performance of the strain LV3 under the condition of mercury and taking nitrite nitrogen as the sole nitrogen source.

Hg-containing ²⁺ Under the conditions, strain LV3 grew similarly to the growth with nitrite nitrogen as sole nitrogen source (FIG. 6), and at 12h the OD of strain LV3 was similar to that of the strain with nitrate nitrogen as sole nitrogen source ₆₀₀ The value can reach 0.529. At the same time, the concentration of nitrite nitrogen was decreased from the initial 99.46mg/L to 68.93mg/L. The growth phase is started at 12-36 h, the highest growth value is reached at 36h, the removal rates of nitrite nitrogen and total nitrogen at the time respectively reach 99.96% and 94.69%, and the average removal rate of the corresponding nitrite nitrogen is 2.76mg/L/h. Pseudomonas putida in Zhang Ni et alPseudomonas putida The average nitrite nitrogen removal rate of ZN1 in the 100mg/L nitrite nitrogen culture solution is 1.97mg/L/h. In the research of Yan et al, bacillus renmizuBacillus hwajinpoensis SLWX ₂ The average removal efficiency at an initial nitrite nitrogen concentration of 100mg/L was 0.96mg/L/h. Compared with the average removal efficiency of nitrite nitrogen by the two, the strain LV3 has better denitrification capability. No ammonia nitrogen and nitrate nitrogen are accumulated in the experimental process, which shows that the path of removing nitrite nitrogen by the strain LV3 is reduced to gas nitrogen instead of oxidized to nitrate nitrogen or converted to ammonium.

Example 7

The heavy metal ion pair strain LV3 has the following nitrate nitrogen removal experiment under the mercury-containing condition:

sodium pyruvate and nitrate nitrogen are respectively used as the only carbon source and nitrogen source,in the presence of Hg ²⁺ 2mg/L or 4mg/L of heavy metal ion Cr is added respectively on the basis of 4mg/L of denitrification culture medium (same as in example 5) ⁶⁺ And Pb ²⁺ . Inoculating 5mL of working solution (same as in example 3) into the 100mL of culture medium, sealing with sealing film, and researching other heavy metals (Pb) in flue gas in the presence of mercury in a constant temperature shaker at 30 ℃ and 120rpm ²⁺ 、Cr ⁶⁺ ) Influence on the growth and denitrification performance of the strain LV3.

The coexisting heavy metal ions inhibited the growth of the strain and denitrification to varying degrees at different concentrations (FIGS. 7a and 7 b). When Pb ²⁺ When the addition amount of (C) was 2mg/L, the maximum growth amount and the final denitrification efficiency were not significantly different from those of the blank group (p>0.05). However when Cr ⁶⁺ When the amount of the catalyst to be added was 2mg/L, the denitrification efficiency at 36 hours was only 84.09% (FIG. 7 c). At the same time, when Pb ²⁺ And Cr (V) ⁶⁺ When the addition amounts of the bacterial strain are all 4mg/L, the removal rate of the nitrate nitrogen of the bacterial strain at 36h is 94.87 percent and 7.58 percent respectively, and Cr is calculated ⁶⁺ Has a far greater inhibition than Pb ²⁺ (FIG. 7 c). Hu et al research has shown that Cr ⁶⁺ The presence of (a) not only strongly inhibits the expression of the strain denitrification gene, but also up-regulates the metabolic activity associated with apoptosis.Shewanella oneidensis MR-1 in Cr ⁶⁺ The denitrification performance is strongly inhibited at 45 mu M concentration, while strain LV3 is at 4mg/L Hg ²⁺ And 2mg/LCr ⁶⁺ Still has higher nitrate nitrogen removal rate in the presence. And when Cr ⁶⁺ When the concentration is 4mg/L, the inhibition effect is realized, but the inhibition effect is mainly reflected in the increase of the delay period, and the removal rate of the bacterial strain LV3 to nitrate nitrogen can reach 99.45% at 60 h. The strain LV3 has strong heavy metal tolerance in the experimental range.

Example 8

Strains LV3 at different Hg ²⁺ The nitrogen balance analysis experiment using nitrate nitrogen as the only nitrogen source at the concentration is as follows:

in the experiment, sodium pyruvate and nitrate nitrogen are respectively used as the only carbon source and nitrogen source, hg is respectively configured ²⁺ Denitrification media (same as in example 5) at concentrations of 0, 2mg/L, 4mg/L and 6mg/L were run in 250mL Erlenmeyer flasks and 5mL of the process was takenInoculating the culture solution (same as in example 3) into the 100mL culture medium, fully aerating the conical flask with high purity oxygen, sealing with sealing film, culturing at 30deg.C and 120rpm for 36 hr, collecting gas samples (400 μl) in the flask at 0 hr and 36 hr respectively by syringe to measure nitrogen, and collecting bacterial liquid in the flask at 0 hr and 36 hr to measure TN before centrifugation ₁ NO after centrifugation ₃ ⁻ –N、NO ₂ ⁻ –N、NH ₄ ⁺ –N、TN ₂ . Gaseous nitrogen (Ngas), intracellular nitrogen (TN) _in ) And the concentration of extracellular organic nitrogen (Ne-org) were calculated with reference to the relevant literature (Bioresource Technology, 2020, 314: 123733.).

As shown in FIG. 8, most of the nitrate nitrogen can be removed within 36 hours under the condition that the nitrate nitrogen is the only nitrogen source and the mercury concentration is 0mg/L, 2mg/L and 4mg/L. The main product is gaseous nitrogen with conversion rates of 67.2%, 67.83%, 64.27%, respectively, to ammonia nitrogen in an amount of about 2%. There is little accumulation of nitrate nitrogen and nitrite nitrogen. The proportion of TNin in TN increases with increasing mercury concentration, probably due to the effect of mercury on enzyme activity, but this proportion is below 30%, which is significantly lower than the intracellular nitrogen accumulation of most of the denitrifying strains that have been reported. The degradation of the strain LV3 on the nitrate nitrogen mainly comprises denitrification to generate gaseous nitrogen and assimilation into self intracellular nitrogen, and the denitrification is mainly adopted.

Example 9

Strain LV3 against Hg ²⁺ The volatilizing development experiments were as follows:

simple judgment of Hg in strain LV3 by X-ray film method ²⁺ Volatilizing into Hg ⁰ Is based on Hg ⁰ Will be in contact with Ag on film ⁺ The reaction takes place to form a vaporous region, and then whether the strain has volatility to mercury is judged. First, 5mL of working solution (same as in example 3) was inoculated into Hg-containing solution ²⁺ And (3) placing the strain in 100mL of denitrification medium with the concentration of 4mg/L, culturing the strain for 36h at 30 ℃ and 120rpm by a shaking table, and collecting bacterial liquid cultured in the logarithmic phase for mercury volatilization development experimental study of the strain. Experiment Co-set experiment group (slow)Three groups of flushing liquid, mercury solution and strain LV 3), control group 1 (buffer solution and mercury solution) and control group 2 (buffer solution and strain LV 3) are respectively placed in a porous plate, an X-ray film is placed above the porous plate, and the reaction is carried out at room temperature under the conditions of sealing and darkness for 1 h.

FIG. 9 shows the detection of the volatility of bacteria to mercury by X-ray film method, i.e., the verification of whether the strain has reduced Hg ²⁺ Is provided). The developed part of the experimental group was more evident than that of the control group 1, indicating that although there was a small volatilization of mercury in the solution when no strain was added, the addition of strain enhanced this effect. The control group 2 was not developed substantially, so that it was confirmed that the strain had a volatilization effect on mercury, and that the strain LV3 could gash Hg ²⁺ Reduced to less toxic and soluble Hg ⁰ 。

The foregoing description is only illustrative of the invention and is not intended to limit the scope of the invention, and all changes and substitutions made herein without departing from the spirit and scope of the invention as defined by the appended claims.

<110> university of Tai principle engineering

<120> an aerobic denitrification strain having Hg < 2+ > removal performance and use thereof

<160>1

<210>1

<211>1427

<212>DNA

<213> Acinetobacter (Acinetobacter sp.) bacterium LV3

<220>

<223> 16S rDNA of Acinetobacter genus bacterium LV3

<400>1

GCTCAGATTG AACGCTGGCG GCAGGCTTAA CACATGCAAG TCGAGCGGGG AAATGTAGCT 60

TGCTACATTA CCTAGCGGCG GACGGGTGAG TAATGCTTAG GAATCTGCCT ATTAGTGGGG 120

GACAACATTC CGAAAGGAAT GCTAATACCG CATACGTCCT ACGGGAGAAA GCAGGGGACC 180

TTCGGGCCTT GCGCTAATAG ATGAGCCTAA GTCGGATTAG CTAGTTGGTG GGGTAAAGGC 240

CTACCAAGGC GACGATCTGT AGCGGGTCTG AGAGGATGAT CCGCCACACT GGGACTGAGA 300

CACGGCCCAG ACTCCTACGG GAGGCAGCAG TGGGGAATAT TGGACAATGG GGGGAACCCT 360

GATCCAGCCA TGCCGCGTGT GTGAAGAAGG CCTTTTGGTT GTAAAGCACT TTAAGCGAGG 420

AGGAGGCTAC TAGTATTAAT ACTACTGGAT AGTGGACGTT ACTCGCAGAA TAAGCACCGG 480

CTAACTCTGT GCCAGCAGCC GCGGTAATAC AGAGGGTGCG AGCGTTAATC GGATTTACTG 540

GGCGTAAAGC GTGCGTAGGC GGCTGATTAA GTCGGATGTG AAATCCCTGA GCTTAACTTA 600

GGAATTGCAT TCGATACTGG TCAGCTAGAG TATGGGAGAG GATGGTAGAA TTCCAGGTGT 660

AGCGGTGAAA TGCGTAGAGA TCTGGAGGAA TACCGATGGC GAAGGCAGCC ATCTGGCCTA 720

ATACTGACGC TGAGGTACGA AAGCATGGGG AGCAAACAGG ATTAGATACC CTGGTAGTCC 780

ATGCCGTAAA CGATGTCTAC TAGCCGTTGG GGCCTTTGAG GCTTTAGTGG CGCAGCTAAC 840

GCGATAAGTA GACCGCCTGG GGAGTACGGT CGCAAGACTA AAACTCAAAT GAATTGACGG 900

GGGCCCGCAC AAGCGGTGGA GCATGTGGTT TAATTCGATG CAACGCGAAG AACCTTACCT 960

GGTCTTGACA TAGTAAGAAC TTTCCAGAGA TGGATTGGTG CCTTCGGGAA CTTACATACA 1020

GGTGCTGCAT GGCTGTCGTC AGCTCGTGTC GTGAGATGTT GGGTTAAGTC CCGCAACGAG 1080

CGCAACCCTT TTCCTTATTT GCCAGCGGGT TAAGCCGGGA ACTTTAAGGA TACTGCCAGT 1140

GACAAACTGG AGGAAGGCGG GGACGACGTC AAGTCATCAT GGCCCTTACG ACCAGGGCTA 1200

CACACGTGCT ACAATGGTCG GTACAAAGGG TTGCTACCTA GCGATAGGAT GCTAATCTCA 1260

AAAAGCCGAT CGTAGTCCGG ATTGGAGTCT GCAACTCGAC TCCATGAAGT CGGAATCGCT 1320

AGTAATCGCG GATCAGAATG CCGCGGTGAA TACGTTCCCG GGCCTTGTAC ACACCGCCCG 1380

TCACACCATG GGAGTTTGTT GCACCAGAAG TAGGTAGTCT AACCGCA 1427

Claims

1. Hg-removing device ²⁺ The aerobic denitrifying strain with the performance is characterized in that the strain is classified as Acinetobacter genus @Acinetobacter sp.) bacteria LV3, which are preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 25044.

2. A process according to claim 1 having Hg removal ²⁺ The aerobic denitrification strain with the performance is characterized in that the acinetobacter is [ ]Acinetobacter sp.) bacterial LV3 16S rDNA gene sequence SEQ ID NO:1.

3. a process according to claim 1 having Hg removal ²⁺ The aerobic denitrification strain with the performance is characterized in that the acinetobacter is [ ]Acinetobacter sp.) bacterial LV3 colony morphology: the bacterial colony is provided with milky opaque dots with transparent halos, the surface is smooth and moist and is easy to pick up, and the edges are regular; under the denitrification of the strain, the color of the BTB solid flat plate changes blue; negative under microscope after passing through gram stain.

4. A composition with Hg removing effect ²⁺ The preparation method of the working solution of the aerobic denitrification strain with the performance is characterized by comprising the following steps: acinetobacter with preservation number of CGMCC No.25044Acinetobacter sp.) bacterial LV3 was inoculated into denitrification medium for activation and cultured in a thermostatic shaker at 30℃and 120rpm to logarithmic phase OD of strain growth ₆₀₀ Approximately equal to 1.3, the denitrification culture medium formula is 5.0625g/L, naNO of sodium succinate ₃ 0.607g/L、KH ₂ PO ₄ 0.4g/L、MnSO ₄ •H ₂ O 0.01g/L、MgSO ₄ •7H ₂ O 0.05g/L、FeSO ₄ •7H ₂ O0.01 g/L, and the pH of the medium was adjusted to 7.0 with NaOH or HCl.

5. A process according to claim 1 having Hg removal ²⁺ Aerobic denitrifying strains with properties or as claimed in claim 4The working solution prepared by the preparation method is applied to the treatment of mercury-containing and nitrogen-containing wastewater.

6. The use according to claim 5, characterized in that: the mercury concentration of the wastewater is 1 mg/L-6 mg/L.

7. The use according to claim 5, characterized in that: the mercury concentration range of the wastewater is 4mg/L.

8. The use according to claim 5, characterized in that: acinetobacter genusAcinetobacter sp.) bacteria LV3 are grown in mercury-containing environments with nitrate nitrogen or nitrite nitrogen as the sole nitrogen source, and can utilize organic carbon sources for aerobic denitrification.

9. A process according to claim 1 having Hg removal ²⁺ The application of the aerobic denitrification strain with the performance in flue gas denitrification.