CN114573099B - Method for promoting enrichment of anaerobic ammonium oxidation bacteria by nitrogen-doped graphene - Google Patents
Method for promoting enrichment of anaerobic ammonium oxidation bacteria by nitrogen-doped graphene Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/28—Anaerobic digestion processes
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
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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
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 ammonia oxidation bacteria have long multiplication time (10-12 days) and are sensitive to external environment changes, so that the sewage treatment process mainly using 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 for strengthening low-abundance anammox bacteria' (Yao Li, zhang Junya and the like, reported in environmental engineering, 5 months in 2018, 5 th volume of 12, 5 th phase) researches that graphene oxide can improve anammox denitrification performance, enrich phytophthora and increase the number of hzo genes 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 much higher than the highest total nitrogen removal rate of 76.01% in the above document, which is reached by adding graphene oxide with a final concentration of 0.1g/L, which indicates that compared with graphene oxide, the nitrogen-doped graphene has a better effect on removing total nitrogen. 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 (110 d), 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 (45 d) and under a constant water inlet concentration. The continuously changing nitrogen load of the inlet water and the relatively long-time continuous culture mode more strongly indicate that the nitrogen-doped graphene improves the denitrification performance of the anaerobic ammonia oxidation reactor as a long-term and stable result, rather than 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 gene Hzs and Hdh encoding the key enzyme for 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 a preferred embodiment of the invention, the main component of the waste water is NH 4 + -N and NO 2 - -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, the volume ratio of the inoculated sludge to the wastewater in the method is 1 (4-4.5).
According to the invention, in the preferable method, the final concentration of the nitrogen-doped graphene addition amount in the wastewater is 40-60 mg/L.
Preferably according to the invention, the reaction is carried out in an upflow anaerobic reactor.
More preferably, the upflow anaerobic reactor is operated continuously, and the operation period is 110 to 120 days.
Further preferably, the whole culture process is processed 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 NH 4 + -N and NO 2 - 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, NH 4 + -N and NO 2 - The concentration of N is 100-240 mg/L and 132-300 mg/L respectively; the trace elements required for the growth of microorganisms mainly include Na + 、Mg 2+ 、Fe 2+ 、Cu 2+ 、Zn 2+ 、K + 。
The nitrogen-doped graphene contains C-N and C = N, contains nitrogen in two forms of pyridine and pyrrole, and has a shrunken 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 neutrality by using deionized water, and carrying out freeze drying to obtain a dried substance.
(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:
(1) dissolving graphite and sodium nitrate in 95-98 wt% of H 2 SO 4 Slowly adding potassium permanganate in ice-water bath to make graphite, sodium nitrate and H 2 SO 4 The 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.
(2) Stirring the mixture I obtained in the step (1) in an oil bath at the temperature of 30-35 ℃ for 2-2.5H, and then adding deionized water to form H 2 SO 4 And the mixed solution is mixed with deionized water in the volume ratio of (2-4) to (5), and the mixture is continuously stirred and reacted for 1.5 to 2 hours under the condition of oil bath at the temperature of between 95 and 100 ℃ to prepare a mixture II.
(3) Adding the mixture II obtained in the step (2) into deionized water to ensure that the volume ratio of the deionized water to the mixture II is (5:1) -2 to prepare a mixture III, and then adding H accounting for 1.5-2.5% of the mixture III in volume ratio 2 O 2 And sealing and standing for 12-16 h to obtain a mixture.
(4) And (4) discarding the supernatant of the mixture prepared in the step (3), ultrasonically stripping the obtained mixed system for 3-3.5 h, repeatedly washing with deionized water and 0.1-0.2 mol/L HCl, and centrifuging until the pH value of the mixed system is 7.0-7.5.
(5) And (5) freezing and drying the mixture obtained in the step (4) to obtain the graphene oxide.
The invention has the advantages of
During the whole experiment period, the effluent effect of the anaerobic reactor added with the nitrogen-doped graphene is superior to that of a blank group, and the maximum nitrogen removal rate can reach 94.92% (shown in fig. 4 (B)). In the experiment, the nitrogen-doped graphene promotes the growth of the biomass of anammox bacteria, increases the relative abundance of the phytophthora, by 47.47%, and realizes the enrichment of microorganisms (Candidatus Kuenenia, candidatus Brocadia, candidatus Jettenia and Candidatus Scalindua) participating in the key functions of the anammox reaction at 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 analysis diagram of infrared (1), pore size (2) and X-ray photoelectron spectroscopy (3) 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) are schematic diagrams for R1 nitrogen removal; and (B) and (B) are R2 nitrogen removal schematic diagrams.
FIG. 5 is a plot of the structural composition of microbial communities (top 10 species);
in the figure: (A) is a schematic representation of the microbial composition at the phylum level; and (B) is a schematic diagram of the composition of microorganisms on 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 demonstrated that since the application of graphene peroxide in anammox is reported in the document "comparison of different additives reinforced low-abundance anammox bacteria" (Yao Li, zhang Junya, etc., reported in environmental engineering, 5 months in 2018, volume 12, phase 5), the data and results obtained in this experiment are compared with the data and conclusions in the document to obtain: 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
The preparation method of the nitrogen-doped graphene comprises the following steps:
preparing graphene oxide according to an improved Hummers method, which specifically 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 2 hours under 35 ℃ oil bath, adding 200ml of deionized water, and continuously stirring and reacting for 1.5 hours under 98 ℃ oil bath 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 (3) dissolving 600mg of urea in 150mL of the 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, wherein R1 only contains sludge as a blank group, and R2 contains a mixture of the sludge and the nitrogen-doped graphene as an experimental group. The sludge used in the experiment is mature sludge from a laboratory-scale upflow anaerobic sludge bed reactor, the concentration of volatile suspended solids of the inoculated sludge is about 1774mg/L, and the volume ratio of the inoculated sludge to the wastewater is 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 NH 4 + -N and NO 2 - 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:
NH 4 Cl，NaNO 2 ，NaHCO 3 ，MgSO 4 ，KH 2 PO 4 trace elements I (5 g/L EDTA and 9.14g/L FeSO) 4 ·7H 2 O), trace element II (15 g/L EDTA,0.014g/L H) 3 BO 4 ,0.99g/L MnCl 2 ·4H 2 O,0.25g/L CuSO 4 ·5H 2 O,0.43g/L ZnSO 4 ·7H 2 O,0.21g/L NiCl 2 ·6H 2 O,0.22g/L NaMoO 4 ·2H 2 O and 0.24g/L CoCl 2 ·6H 2 O). The above reagents are all common commercial products.
TABLE 1
Note: infNH 4 + -N and InfNO 2 - 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 -1 And 1550cm -1 The characteristic absorption peaks C-N and C = N show that under the hydrothermal condition, the urea reduces the graphene oxide and simultaneously performs nitrogen doping on the graphene oxide.
As shown in FIG. 1 (2), the aperture of the nitrogen-doped graphene is micropore (less than or equal to 2 nm) 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 a comparison of the denitrification performance of the reactors in different stages is shown in Table 2.
As can be seen from Table 2, Δ NO of both reactors 2 - /ΔNH 4 + And Δ NO 3 - /ΔNH 4 + The ratio is close to the theoretical value (1.32 and 0.26) of the anammox 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 NH 4 + -N and NO 2 - -removal of N. NH accompanying the inflow of water, as shown in FIG. 4 4 + -N and NO 2 - 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 influenced by the fluctuation of the concentration of the inlet water, but the nitrogen removal efficiency is rapidly reduced through long-time domestication along with the regulation and control of the concentration of the inlet waterThe denitrification performance of the reactor can be restored to the original effect, which also shows that the nitrogen-doped graphene has a positive promotion effect on adapting and resisting the variable environment of the reaction system.
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 plancomycota gates (Planctomycetes), at ratios of R1 and R2 of 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 in R1 and R2 is 18.10% and 28.30%, respectively; candidatus Brocadia has a ratio of R1 to R2 of 11.18% and 11.79%, respectively; the percentage of Candidatus Jettenia in R1 and R2 is 8.05% and 11.06%, respectively; the percentage of the 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 the 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 Amino acid Metabolism (Amino acid Metabolism) "under the metabolic pathway (Metabolism)," "Carbohydrate Metabolism (Carbohydrate Metabolism)" and "lipid MetabolismRelative abundance of Lipid metabolism (metabolism), several metabolic functions that are closely related to extracellular polymer formation. Figure 8 shows that nitrogen-doped graphene increases the 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 -1 VSS increases to 122.7mg g in R2 -1 VSS and better settling properties of the sludge in R2, which all contribute to the sludge's resistance to adverse external conditions.
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 (13)
1. The application of the nitrogen-doped graphene oxide in promoting the enrichment of anammox bacteria and the denitrification of wastewater comprises the following steps:
inoculating sludge and nitrogen-doped graphene oxide in the wastewater, and culturing anaerobic ammonium oxidation sludge and wastewater denitrification by controlling the operation conditions;
the anaerobic ammonium oxidation bacteria belong to the genus ofCandidatus Kuenenia, Candidatus Brocadia, Candidatus Jettenia AndCandidatus Scalindua；
the preparation method of the nitrogen-doped graphene oxide 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 the urea to the graphene oxide is (2~3) - (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 14-18h, cooling to room temperature, washing to neutrality by using deionized water, and carrying out freeze drying to obtain a dried product;
(3) Grinding the dried substance prepared in the step (2), and calcining at 480 to 520 ℃ for 0.5 to 1.5 hours under the protection of argon to prepare nitrogen-doped graphene oxide;
in the step (1), the preparation method of the graphene oxide comprises the following steps:
(1) dissolving graphite and sodium nitrate in H of 95 to 98wt percent 2 SO 4 Slowly adding potassium permanganate in ice-water bath to make graphite, sodium nitrate and H 2 SO 4 The molar ratio of potassium permanganate is (38-42): 2~4): 220-230): 1, and then the mixture is stirred and mixed for 2-2.5 h to prepare a mixture I;
(2) stirring the mixture I obtained in the step (1) in an oil bath at the temperature of 30-35 ℃ for 2-2.5H, and adding deionized water to form H 2 SO 4 And deionized water in the volume ratio of 2~4 to 5, and continuously stirring for reaction for 1.5 to 2h under the oil bath at the temperature of 95 to 100 ℃ to prepare a mixture II;
(3) adding deionized water into the mixture II obtained in the step (2) to enable the volume ratio of the deionized water to the mixture II to be (5:1) - (2) to prepare a mixture III, and then adding H accounting for 1.5-2.5% of the mixture III in volume ratio 2 O 2 Sealing and placing for 12 to 169h to prepare a mixture;
(4) discarding the supernatant of the mixture prepared in the step (3), ultrasonically stripping the obtained mixed system for 3 to 3.5 hours, repeatedly washing the mixed system with deionized water and 0.1 to 0.2mol/L HCl, and centrifuging the mixed system until the pH of the mixed system is 7.0 to 7.5;
(5) and (4) freeze-drying the mixture obtained in the step (4) to obtain the graphene oxide.
2. The use of nitrogen-doped graphene oxide according to claim 1 for increasing the gene encoding key enzyme for anammoxHzsAndHdhthe use in abundance.
3. The application of the nitrogen-doped graphene oxide of claim 1, wherein the nitrogen-doped graphene oxide is related to improving nitrogen metabolism in denitrification of anaerobic ammonia oxidation wastewaterNirS、NirK、NorB、NosZAndNrfAand (4) application of abundance.
4. The use of nitrogen-doped graphene oxide according to claim 1, for increasing the synthesis of cyclic diguanylate gene in anammox bacteriaDgcB、PLeC、PLeDAnd quinolone signaling molecule genesTrpE、TrpGAnd (4) application of abundance.
5. Use according to claim 1, wherein the main component of the waste water is NH 4 + -N and NO 2 - -N。
6. The use according to claim 1, wherein the seeded sludge is mature anammox sludge.
7. The application of claim 1, wherein the volume ratio of the inoculated sludge to the wastewater is 1 (4 to 4.5).
8. The application of claim 1, wherein the final concentration of the nitrogen-doped graphene oxide in the wastewater is 40-60mg/L.
9. The use according to claim 1, wherein the reaction is carried out in an upflow anaerobic reactor.
10. The application of claim 9, wherein the upflow anaerobic reactor is operated continuously for a period of 110 to 120d.
11. The application as claimed in claim 10, characterized in that the whole cultivation process is protected from light, the temperature of the reactor is controlled to be 30-35 ℃, and the hydraulic retention time is 24-25h.
12. The use according to claim 11, wherein the feed water is NH 4 + -N and NO 2 - the-N is taken as a substrate, and is supplemented with trace elements required by the growth of microorganisms, and the pH value of water is controlled to be 7.0 to 7.5.
13. Use according to claim 12, characterised in that NH is present 4 + -N and NO 2 - The concentration of the-N is respectively 100 to 240mg/L and 132 to 300mg/L.
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