CN114573099A

CN114573099A - Method for promoting enrichment of anaerobic ammonium oxidation bacteria by nitrogen-doped graphene

Info

Publication number: CN114573099A
Application number: CN202210200149.9A
Authority: CN
Inventors: 薛嵘; 张宝永; 郭艳; 李振; 燕锡尧; 张冠军; 杨文娟
Original assignee: Shandong Jingbo Holding Group Co ltd; Qilu University of Technology
Current assignee: Shandong Jingbo Holding Group Co ltd; Qilu University of Technology
Priority date: 2022-03-02
Filing date: 2022-03-02
Publication date: 2022-06-03
Anticipated expiration: 2042-03-02
Also published as: CN114573099B

Abstract

The invention provides a method for promoting enrichment of anaerobic ammonium oxidation bacteria by nitrogen-doped graphene, and particularly relates to application of nitrogen-doped graphene in promoting enrichment of anaerobic ammonium oxidation bacteria and denitrification of wastewater, wherein sludge and nitrogen-doped graphene are inoculated in the wastewater, and the anaerobic ammonium oxidation sludge and the denitrification of the wastewater are cultured by controlling operation conditions; the nitrogen-doped graphene promotes the growth of specific microorganisms participating in anammox, improves the key functions and gene abundance of anammox flora, and promotes the realization of higher denitrification efficiency of the reactor by the aid of the functions.

Description

Method for promoting enrichment of anaerobic ammonium oxidation bacteria by nitrogen-doped graphene

Technical Field

The invention belongs to the technical field of wastewater treatment, and particularly relates to a method for realizing efficient denitrification from wastewater by promoting enrichment of anammox bacteria and up-regulating key functional genes of flora through nitrogen-doped graphene.

Technical Field

Compared with the traditional nitrification-denitrification process, the anaerobic ammonia oxidation can reduce 50 percent of aeration quantity, 100 percent of organic carbon source and 90 percent of operating cost, and is an autotrophic nitrogen removal technology with more energy conservation and high efficiency. However, the anaerobic ammonium oxidation bacteria have long doubling time (10-12 days) and are sensitive to external environment change, so that the sewage treatment process mainly based on anaerobic ammonia oxidation is difficult to realize large-scale application. For a sewage treatment plant, the cost of starting the reaction system is increased undoubtedly by taking too long time to cultivate the anaerobic ammonia oxidation sludge, and a large amount of manpower and material resources are wasted. Based on the situation, realizing and maintaining the enrichment of a large number of anaerobic ammonia oxidizing bacteria and improving the tolerance of the anaerobic ammonia oxidizing bacteria to the variable external environment are the problems which are urgently needed to be solved for promoting the continuous forward development of the anaerobic ammonia oxidation process.

The document "comparison of different additives to reinforce low-abundance anammox bacteria" (yao li, zhang ya, etc., environmental engineering report, 5 months in 2018, vol. 12, 5 th phase) researches that graphene oxide can improve anammox denitrification performance, enrich phytophthora and increase number of genes of hzo in the metabolic process of anammox bacteria, and the action effect of nitrogen-doped graphene in the invention is obviously superior to that of graphene oxide in the document.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for promoting enrichment of anammox bacteria by nitrogen-doped graphene.

In the invention, the highest total nitrogen removal rate of the reactor in which 50mg/L of nitrogen-doped graphene is added can reach 94.92% (shown in fig. 4 (B)), which is far higher than the highest total nitrogen removal rate of 76.01% in the above document in which graphene oxide with a final concentration of 0.1g/L is added, which indicates that compared with graphene oxide, nitrogen-doped graphene has a better effect on total nitrogen removal. In the invention, the nitrogen-doped graphene increases the relative abundance of the pumice phylum by 47.47%, which is higher than that of graphene oxide in the above documents, by 44.51%, which indicates that the nitrogen-doped graphene has a better enrichment effect on anammox bacteria than graphene oxide. In addition, while the graphene oxide is shown in the above documents to increase the number of the anammox-related gene hzo, the present invention shows that the nitrogen-doped graphene not only increases the abundance of the anammox-related genes (Hzs and Hdh), but also up-regulates the abundance of other related genes (NirS, NirK, NorB, NosZ and NrfA) involved in nitrogen metabolism. More importantly, the experiment in the invention is to continuously culture the seed sludge in a long time (110d), continuously increase the water inlet concentration and explore the influence of the nitrogen-doped graphene on the seed sludge; however, the above documents only explore the influence of graphene oxide on the inoculated sludge in a short time (45d) and under a constant water inlet concentration. The continuous culture mode with the constantly changing water inlet nitrogen load and the relatively long time strongly indicates that the nitrogen-doped graphene is a long-term and stable result for improving the denitrification performance of the anaerobic ammonia oxidation reactor, and is not a short-term phenomenon caused by the nature of the nitrogen-doped graphene. The technical effects of the present invention cannot be expected by those skilled in the art from the disclosures of the above documents.

The prepared nitrogen-doped graphene is added into an anaerobic ammonia oxidation reactor to realize the mass enrichment of anaerobic ammonia oxidation bacteria and the up-regulation of the abundance of key functional genes of flora, thereby achieving the purpose of high-quality effluent.

Technical scheme of the invention

The nitrogen-doped graphene is applied to promoting enrichment of anammox bacteria and denitrification of wastewater.

According to a preferred embodiment of the present invention, the nitrogen-doped graphene is used for promoting enrichment of anammox bacteria Candidatus Kuenenia, Candidatus Brocadia, Candidatus Jettenia and Candidatus Scalindua.

According to the invention, the nitrogen-doped graphene is preferably applied to the improvement of the abundance of the genes Hzs and Hdh encoding the key enzymes of anammox.

According to the invention, the nitrogen-doped graphene is preferably applied to improving the abundance of the related genes NirS, NirK, NorB, NosZ and NrfA of nitrogen metabolism in the denitrification of the anaerobic ammonia oxidation wastewater.

According to the invention, the nitrogen-doped graphene is preferably used for improving the abundance of synthesized cyclic diguanylate genes DgcB, PLeC, PLeD and quinolone signal molecule genes TrpE and TrpG in the anammox.

The method for promoting enrichment of anammox bacteria and denitrification of wastewater by nitrogen-doped graphene comprises the following steps:

inoculating sludge and nitrogen-doped graphene in the wastewater, and culturing anaerobic ammonia oxidation sludge and wastewater denitrification by controlling the operation conditions.

According to the invention, the main component of the waste water is preferably NH₄ ⁺-N and NO₂ ^--N。

Preferably, according to the invention, the method comprises the step of taking mature anaerobic ammonium oxidation sludge as the inoculation sludge.

According to the invention, in the method, the volume ratio of the inoculated sludge to the wastewater is 1 (4-4.5).

According to the preferable method, the final concentration of the nitrogen-doped graphene in the wastewater is 40-60 mg/L.

Preferably according to the invention, the reaction is carried out in an upflow anaerobic reactor.

Preferably, the upflow anaerobic reactor is operated continuously, and the operation period is 110-120 d.

Further preferably, the whole culture process is carried out in a dark place, the temperature of the reactor is controlled to be 30-35 ℃, and the hydraulic retention time is 24-25 h.

Further preferably, the feed water is NH₄ ⁺-N and NO₂ ^-N is used as a substrate, and is supplemented with trace elements required by the growth of microorganisms, and the pH of inlet water is controlled to be 7.0-7.5.

Further preferred, NH₄ ⁺-N and NO₂ ^-The concentration of N is 100-240 mg/L and 132-300 mg/L respectively; the microelements required for the growth of microorganisms mainly comprise Na⁺、Mg²⁺、Fe²⁺、Cu²⁺、Zn²⁺、K⁺。

The nitrogen-doped graphene contains C-N and C ═ N, contains nitrogen in two forms of pyridine and pyrrole, and has a shrunk and porous morphology structure.

The preparation method of the nitrogen-doped graphene comprises the following steps:

(1) dissolving urea in a graphene oxide suspension, wherein the concentration of the graphene oxide suspension is 2-2.5 mg/mL, the mass ratio of urea to graphene oxide is (2-3) to (1-2), and stirring and ultrasonically mixing uniformly to obtain a mixture.

(2) Carrying out hydrothermal reaction on the mixture prepared in the step (1) at 160-200 ℃ for 14-18 h, cooling to room temperature, washing to be neutral by using deionized water, and carrying out freeze drying to obtain a dried product.

(3) And (3) grinding the dried substance prepared in the step (2), and calcining at 480-520 ℃ for 0.5-1.5 h under the protection of argon to prepare the nitrogen-doped graphene.

Preferably, according to the present invention, the graphene oxide in step (1) is prepared according to a modified Hummers method.

Further preferably, the preparation method of graphene oxide comprises the following steps:

firstly, dissolving graphite and sodium nitrate in 95-98 wt% of H₂SO₄Slowly adding potassium permanganate in ice-water bath to make graphite, sodium nitrate and H₂SO₄The molar ratio of the potassium permanganate is (38-42) to (2-4) to (220-230) to 1, and then the mixture is stirred and mixed for 2-2.5 hours to prepare a mixture I.

Secondly, stirring the mixture I obtained in the step I for 2-2.5 hours in an oil bath at the temperature of 30-35 ℃, and then adding deionized water to form H₂SO₄And (3) continuously stirring and reacting the mixed solution with deionized water in a volume ratio of (2-4) to 5 for 1.5-2 hours under the condition of oil bath at the temperature of 95-100 ℃ to obtain a mixture II.

Thirdly, adding deionized water into the mixture II obtained in the second step to enable the volume ratio of the deionized water to the mixture II to be (5:1) -2 to obtain a mixture III, and then adding the mixture III to enable the volume ratio of the mixture III to be 1.5-2.5%H of (A) to (B)₂O₂And sealing and standing for 12-16 h to obtain a mixture.

And fourthly, removing the step III to prepare a mixture supernatant, ultrasonically stripping the obtained mixed system for 3 to 3.5 hours, repeatedly washing the mixed system by using deionized water and 0.1 to 0.2mol/L HCl, and centrifuging the mixed system until the pH value of the mixed system is 7.0 to 7.5.

Fifthly, the mixture obtained in the step (iv) is frozen and dried to obtain the graphene oxide.

The invention has the advantages of

During the whole experiment, the effluent effect of the anaerobic reactor added with the nitrogen-doped graphene is better than that of the blank group, and the maximum nitrogen removal rate can reach 94.92% (shown in figure 4 (B)). In the experiment, the nitrogen-doped graphene promotes the growth of the biomass of the anaerobic ammonia oxidation bacteria, so that the relative abundance of the phytophthora phylum is increased by 47.47%, and the enrichment of the microorganisms (Candidatus Kuenenia, Candidatus Brocadia, Candidatus Jettenia and Candidatus Scalindua) participating in the key functions of the anaerobic ammonia oxidation reaction is realized on the microbial genus level. The nitrogen-doped graphene has a promoting effect on the metabolic function of the anaerobic ammonia oxidation flora, and the abundance of the amino acid and carbohydrate metabolic functions related to the production of extracellular proteins and polysaccharides is improved. In addition, nitrogen-doped graphene not only improves the abundance of the key enzyme genes (Hzs and Hdh) for coding anaerobic ammonia oxidation, but also improves the abundance of the key enzyme genes (NirS, NirK, NorB and NosZ) for coding nitrification, denitrification and the like. Meanwhile, the nitrogen-doped graphene up-regulates the gene abundance of synthetic cyclodiguanylic acid and quinolone signal molecules, further promotes the synthesis of Extracellular Polymeric Substances (EPS), enables the sludge to have better settling property, and is beneficial to the sludge to resist adverse environmental changes. These are important reasons for the better denitrification effect of the reactor with the nitrogen-doped graphene.

Drawings

Fig. 1 is an infrared (1), pore size (2) and X-ray photoelectron spectroscopy (3) analysis diagram of nitrogen-doped graphene.

Fig. 2 is a schematic view of a scanning electron microscope of nitrogen-doped graphene.

FIG. 3 is a diagram of an upflow anaerobic reactor apparatus.

FIG. 4 is a schematic of reactor nitrogen removal performance;

in the figure: (A) and (a) is a nitrogen removal scheme for R1; (B) and (b) is a nitrogen removal scheme for R2.

FIG. 5 is a plot of the structural composition of microbial communities (top 10 species);

in the figure: (A) schematic representation of microbial composition at the phylum level; (B) is a schematic diagram of the composition of microorganisms at the genus level.

FIG. 6 is a graph showing the comparison of the number of anammox bacteria and the total number of bacteria.

Fig. 7 is a graph of microbial community functional expression abundance, expressed in relative abundance (%).

FIG. 8 is a graph of microbial community functional gene abundance, expressed as absolute value CPM (copy per kilobase per million mapped reads).

Detailed Description

The invention is further illustrated by the following examples in connection with the accompanying drawings, without limiting the scope of the invention.

It is stated that since the application of graphene peroxide in anaerobic ammonia oxidation is reported in the document "comparison of different additives to enhance low-abundance anaerobic ammonia oxidation bacteria" (yao li, zhang juna, etc., report on environmental engineering, 5 months in 2018, vol. 12, 5 th), the experiment shows that the data and results obtained by the experiment are compared with the data and conclusions in the document: compared with graphene oxide, the nitrogen-doped graphene has more remarkable superiority to anaerobic ammonia oxidation denitrification, so that the experiment does not relate to experimental data of adding graphene oxide; in addition, the optimal addition amount of the graphene oxide in the literature is determined, and the experiment reduces (1/2) the addition amount of the nitrogen-doped graphene on the basis of the optimal addition amount of the graphene oxide and obtains a better denitrification effect than the nitrogen-doped graphene, so that the nitrogen-doped graphene is proved to be superior to the graphene oxide in improving the denitrification performance of the anaerobic ammonium oxidation reaction.

Example 1

preparing graphene oxide according to an improved Hummers method, which comprises the following steps:

(1) 5g of flake graphite and 2.5g of sodium nitrate are dissolved in 120ml of concentrated sulfuric acid (98 wt%), and 15g of potassium permanganate are slowly added in an ice-water bath and then stirred and mixed for 2 hours to prepare a mixture I.

(2) And (2) stirring the mixture I obtained in the step (1) for 2h under an oil bath at 35 ℃, adding 200ml of deionized water, and continuously stirring and reacting for 1.5h under an oil bath at 98 ℃ to obtain a mixture II.

(3) And (3) adding the mixture II obtained in the step (2) into deionized water until the total volume reaches 1000ml to obtain a mixture III, then adding 20ml of hydrogen peroxide, and sealing and standing for 12 hours to obtain a mixture.

(4) And (4) discarding the supernatant of the mixture obtained in the step (3), ultrasonically stripping the obtained mixed system for 3h, repeatedly washing with deionized water and 0.1mol/L HCl, and centrifuging until the pH value of the system is neutral.

(5) And (4) freeze-drying the mixture obtained in the step (4) to obtain the graphene oxide.

The preparation method of the (II) nitrogen-doped graphene comprises the following steps:

(1) and (2) dissolving 600mg of urea in 150mL of graphene oxide suspension prepared in the step (I), wherein the concentration of graphene oxide in the suspension is 2mg/mL, and stirring and then ultrasonically mixing the graphene oxide suspension uniformly.

(2) And (2) carrying out hydrothermal reaction on the mixture obtained in the step (1) at 180 ℃ for 16h, cooling to room temperature, washing to be neutral by using deionized water, and freeze-drying.

(3) And (3) grinding the dried substance prepared in the step (2), and calcining for 1h at 500 ℃ under the protection of argon to prepare the nitrogen-doped graphene.

Example 2

2 anaerobic reactors (2L) with the same specification are constructed, R1 only contains sludge as a blank group, and R2 contains a mixture of the sludge and nitrogen-doped graphene as an experimental group. The sludge used in the experiment was mature sludge from a laboratory scale upflow anaerobic sludge bed reactor, the concentration of volatile suspended solids in the inoculated sludge was about 1774mg/L, and the volume ratio of inoculated sludge to wastewater was 1: 4. The final concentration of the nitrogen-doped graphene is 50mg/L, and the nitrogen-doped graphene is added at one time. In the experiment, the inoculated sludge is continuously cultured for a period of 110d, the reactor is operated in a dark place, the temperature is controlled to be 34 +/-1 ℃, and the wastewater mainly comprises NH₄ ⁺-N and NO₂ ^-N composition supplemented with growth elements required for microbial growth and wastewater pH adjusted to 7.5 + -0.2, reactor hydraulic retention time maintained at 24h throughout the culture process, nitrogen load increased by increasing feed water concentration step by step (see Table 1).

The wastewater is a main component of synthetic wastewater and is supplied by the following reagents:

NH₄Cl，NaNO₂，NaHCO₃，MgSO₄，KH₂PO₄trace elements I (5g/L EDTA and 9.14g/L FeSO)₄·7H₂O), trace element II (15g/L EDTA,0.014g/L H)₃BO₄,0.99g/L MnCl₂·4H₂O,0.25g/L CuSO₄·5H₂O,0.43g/L ZnSO₄·7H₂O,0.21g/L NiCl₂·6H₂O,0.22g/L NaMoO₄·2H₂O and 0.24g/L CoCl₂·6H₂O). The above reagents are all common commercial products.

TABLE 1

Note: InfNH₄ ⁺-N and InfNO₂ ^-N refers to the concentration of influent ammonia nitrogen and nitrite nitrogen, respectively.

Examples of effects

(1) Material characterization

As shown in FIG. 1(1), the nitrogen-doped graphene is 1165cm^-1And 1550cm^-1The characteristic absorption peaks C-N and C ═ N are shown, and the urea reduces the graphene oxide under the hydrothermal condition and dopes the graphene oxide with nitrogen.

As shown in figure 1(2), the aperture of the nitrogen-doped graphene is micropore (less than or equal to 2nm) and mesopore (2-50 nm).

As shown in fig. 1(3), nitrogen-doped graphene forms two forms of nitrogen, pyridine and pyrrole, at 398eV and 401 eV.

As shown in fig. 2(a) and (B), the nitrogen-doped graphene has a morphology of interconnected shrinkage porosity.

(2) Comparison of denitrification Effect of the reactor

FIG. 4 shows the denitrification performance of the two reactors in combination, and the comparison of the denitrification performance of the reactors at different stages is shown in Table 2.

As can be seen from Table 2, Δ NO of both reactors₂ ^-/ΔNH₄ ⁺And Δ NO₃ ^-/ΔNH₄ ⁺The ratio is close to the theoretical value (1.32 and 0.26) of the anaerobic ammonium oxidation reaction, but the denitrification efficiency of R2 is obviously higher than that of R1 in the 110-day experiment period, which indicates that the nitrogen-doped graphene promotes NH₄ ⁺-N and NO₂ ^--removal of N. NH accompanying the inflow of water, as shown in FIG. 4₄ ⁺-N and NO₂ ^-The N concentration is continuously increased, the reactor for adding the nitrogen-doped graphene is not inhibited, and the denitrification efficiency is continuously and stably increased; the nitrogen removal efficiency of the blank group without the nitrogen-doped graphene is sharply reduced under the influence of fluctuation of the concentration of the inlet water, but the nitrogen removal performance of the reactor can be restored to the original effect through long-time domestication along with regulation and control of the concentration of the inlet water, which also shows that the nitrogen-doped graphene has a positive promotion effect on the adaptation and resistance of a reaction system to a variable environment.

TABLE 2

Note: NLR, NRR and NRE refer to nitrogen loading rate, nitrogen removal rate and denitrification efficiency, respectively.

(3) Comparison of microbial composition, abundance and quantity

The microbial community composition changes for both reactors are shown in figure 5. At the phylum and genus level, the species compositions of the two reactors were identical, but the species abundances varied significantly. At the gate level, nitrogen-doped graphene significantly increased the relative abundance of planctomycete gates (Planctomycetes), with the occupancy at R1 and R2 being 16.77% and 24.73%, respectively. Currently known anammox bacteria belong to the phylum Aphyllophorales. At the genus level, the genera Candidatus Kuenenia, Candidatus Brocadia, Candidatus Jettenia and Candidatus Scalindua, which are involved in anammox, were detected in both reactors. Wherein the ratio of Candidatus Kueneni at R1 and R2 is 18.10% and 28.30%, respectively; the percentage of Candidatus Brocadia in R1 and R2 was 11.18% and 11.79%, respectively; the percentages of Candidatus Jettenia at R1 and R2 were 8.05% and 11.06%, respectively; the percentage of Candidatus Scalindua in R1 and R2 is 0.26% and 1.07%, respectively, which shows that the nitrogen-doped graphene effectively increases the relative abundance of anammox bacteria. In addition, fig. 6 further shows that the biomass of anammox bacteria in R2 is significantly higher than that of R1, which further illustrates that the nitrogen-doped graphene has the potential of promoting the growth of anammox bacteria.

(4) Comparison of changes in abundance and EPS content of community function and key function genes

Figure 7 shows the difference in colony function within the two reactors. Nitrogen-doped graphene increases the relative abundance of Amino acid Metabolism (Amino acid Metabolism), "Carbohydrate Metabolism (Carbohydrate Metabolism)" and "Lipid Metabolism (Lipid Metabolism)" under the "metabolic pathway (Metabolism)," which are closely related to the formation of extracellular polymers. Fig. 8 shows that nitrogen-doped graphene increases abundance of genes encoding key enzymes for anammox (Hzs and Hdh), which allows R2 to have better anammox performance; in addition, the abundance of other genes involved in nitrogen metabolism (NirS, NirK, NorB, NosZ and NrfA) was also increased. Meanwhile, nitrogen-doped graphene up-regulates the abundance of synthetic cyclic diguanylate genes (DgcB, PLeC and PLeD) and quinolone signal molecule genes (TrpE and TrpG), further making the EPS content from 96.1mg g in R1^-1VSS increase to 122.7mg g in R2^-1VSS and better settling properties of the sludge in R2, which are beneficial for the sludge to resist adverse external environments.

In conclusion, compared with the graphene oxide in the reported literature, the application effect of the nitrogen-doped graphene disclosed by the invention is significantly better than that of the graphene oxide. According to the invention, the nitrogen-doped graphene with less dosage can obtain better denitrification performance of the anaerobic ammonia oxidation reactor. Specifically, the nitrogen-doped graphene promotes the growth of specific microorganisms participating in anammox, improves the key functions and gene abundance of anammox flora, and promotes the realization of higher denitrification efficiency of the reactor under the action of the functions, and is also a root cause of long-term stable operation of the reactor.

Claims

1. The nitrogen-doped graphene is applied to promoting enrichment of anammox bacteria and denitrification of wastewater.

2. Use according to claim 1, wherein the nitrogen-doped graphene is used in promoting enrichment of anammox bacteria Candidatus Kuenenia, Candidatus Brocadia, Candidatus Jettenia and Candidatus Scalindua.

3. The use of claim 1, wherein the nitrogen-doped graphene is used for increasing the abundance of genes Hzs and Hdh encoding key enzymes for anammox.

4. The use of nitrogen-doped graphene according to claim 1, wherein the use of nitrogen-doped graphene for improving the abundance of the genes NirS, NirK, NorB, NosZ and NrfA associated with nitrogen metabolism in the denitrification of anammox wastewater.

5. Use of nitrogen-doped graphene for increasing the abundance of the synthetic cyclic diguanylate genes DgcB, PLeC, PLeD and quinolone signaling molecule genes TrpE, TrpG in anammox bacteria according to claim 1.

6. The method for promoting enrichment of anammox bacteria and denitrification of wastewater by nitrogen-doped graphene comprises the following steps:

sludge and nitrogen-doped graphene are inoculated in the wastewater, and anaerobic ammonium oxidation sludge and wastewater denitrification are cultured by controlling the operation conditions.

7. The method of claim 6, wherein the main component of the wastewater is NH₄ ⁺-N and NO₂ ^--N；

Preferably, in the method, mature anaerobic ammonium oxidation sludge is taken as the inoculation sludge;

preferably, in the method, the volume ratio of the inoculated sludge to the wastewater is 1 (4-4.5);

preferably, in the method, the final concentration of the nitrogen-doped graphene added in the wastewater is 40-60 mg/L;

preferably, the reaction is carried out in an upflow anaerobic reactor;

preferably, the upflow anaerobic reactor is continuously operated, and the operation period is 110-120 d;

preferably, the whole culture process is carried out in a dark place, the temperature of the reactor is controlled to be 30-35 ℃, and the hydraulic retention time is 24-25 h;

preferably, the feed water is NH₄ ⁺-N and NO₂ ^-N is used as a substrate, and is supplemented with trace elements required by the growth of microorganisms, and the pH of inlet water is controlled to be 7.0-7.5;

preferably, NH₄ ⁺-N and NO₂ ^-The concentration of N is 100-240 mg/L and 132-300 mg/L respectively; the microelements required for the growth of microorganisms mainly comprise Na⁺、Mg²⁺、Fe²⁺、Cu²⁺、Zn²⁺、K⁺。

8. The nitrogen-doped graphene is characterized by containing C-N and C ═ N, containing nitrogen in two forms of pyridine and pyrrole, and having a morphology structure of shrinkage porosity.

9. The preparation method of the nitrogen-doped graphene is characterized by comprising the following steps:

(1) dissolving urea in a graphene oxide suspension, wherein the concentration of the graphene oxide suspension is 2-2.5 mg/mL, the mass ratio of urea to graphene oxide is (2-3) to (1-2), and stirring and then ultrasonically mixing uniformly to prepare a mixture;

(2) carrying out hydrothermal reaction on the mixture prepared in the step (1) at 160-200 ℃ for 14-18 h, cooling to room temperature, washing to be neutral by using deionized water, and carrying out freeze drying to obtain a dried substance;

10. The method according to claim 9, wherein the graphene oxide in the step (1) is prepared according to a modified Hummers method;

preferably, the preparation method of graphene oxide includes the following steps:

firstly, dissolving graphite and sodium nitrate in 95-98 wt% of H₂SO₄In the method, potassium permanganate is slowly added in an ice water bath to ensure that graphite, sodium nitrate and H₂SO₄The molar ratio of potassium permanganate is (38-42) to (2-4) to (220-230) to 1, and then stirring and mixing are carried out for 2-2.5 hours to prepare a mixture I;

secondly, stirring the mixture I obtained in the step I for 2-2.5 hours in an oil bath at the temperature of 30-35 ℃, and then adding deionized water to form H₂SO₄And the mixed solution with deionized water in a volume ratio of (2-4) to (5) is continuously stirred and reacted for 1.5-2 hours under the condition of oil bath at the temperature of 95-100 ℃ to prepare a mixture II;

thirdly, adding deionized water into the mixture II obtained in the step II to enable the volume ratio of the deionized water to the mixture II to be (5:1) -2 to obtain a mixture III, and then adding H accounting for 1.5-2.5% of the mixture III in volume ratio₂O₂Sealing and placing for 12-16 h to prepare a mixture;

fourthly, after the supernatant of the mixture is prepared, ultrasonically stripping the obtained mixed system for 3 to 3.5 hours, repeatedly washing the mixed system by using deionized water and 0.1 to 0.2mol/L HCl, and centrifuging the mixed system until the pH value of the mixed system is 7.0 to 7.5;