A method of preparing and screening biochar to facilitate extracellular electron transfer by anaerobic ammonium oxidizing bacteria

Abstract

The application provides a method for preparing and screening biochar for promoting extracellular electron transfer of anaerobic ammonia oxidation bacteria, and belongs to the technical field of wastewater treatment.The method comprises the following steps: pyrolyzing wood waste at high temperature, grinding the wood waste to a size less than 150 mu m after cooling, repeatedly soaking, oscillating and suction filtering the wood waste with deionized water until the pH of the filtrate is less than 9, drying the wood waste, and screening biochar containing rich aromatic ring structures and graphite defect points; then, the biochar is tested by adopting a nitrite starvation operation method, and candidate biochar is screened out so that the denitrification rate of anaerobic ammonia oxidation wastewater can reach the denitrification rate equivalent to that when nitrite is normally supplied. The application provides a protective testing and popularization application method, saves economic investment and time cost of pilot test, and is beneficial to obtaining electrically active substances at a material-microorganism interface, so as to further explore and popularize and migrate.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sewage treatment, and particularly relates to a method for preparing and screening biochar for promoting extracellular electron transfer of anammox bacteria. BACKGROUND

[0002] As a new nitrogen removal process, anammox has many advantages over traditional nitrification-denitrification process, such as saving carbon source and energy required for aeration, reducing greenhouse gas emissions and reducing excess sludge production. Anammox bacteria are highly sensitive to changes in the content of soluble electron acceptor nitrite, which makes the anammox process slow to start and requires coupling with partial nitrification and denitrification processes to regulate and stabilize the supply of nitrite. In recent years, it has been found that anammox bacteria have the ability to use graphene and electrodes as insoluble extracellular electron acceptors to directly oxidize ammonium to nitrogen, which is expected to reduce the risk of N2O (its greenhouse gas effect is three hundred times that of CO2) produced by decoupling partial nitrification and denitrification processes. However, graphene is expensive, and the graphene-assisted direct ammonium oxidation process has not been practically applied.

[0003] Currently, actual wastewater treatment plants generally use low-cost biochar to assist Anammox for rapid start-up and improve treatment effect. Biochar with redox activity is a very potential sustainable material for replacing graphene to establish microbial extracellular electron transfer, but there is no research to determine which process can prepare biochar with certain quantitative characteristics as an effective candidate. Since the actual application mainly uses powdered biochar, if the test fails and the system collapses, it is difficult to separate the powdered carbon from the bacteria, which will cause irreversible time and economic losses.

[0004] Biochar with superior electron-accepting ability often has abundant electron-accepting sites, including quinone groups (oxidized or semi-oxidized), graphite defect sites and carbon vacancies, however, it may cause certain oxidative stress in the water treatment system, thus causing a certain degree of microbial shock and autolysis. Therefore, in the process of preparing excellent biochar materials, a method is needed to quickly predict and evaluate whether the biochar can promote Anammox extracellular electron transfer, to develop a flexible alternative to nitrite supply, and also to facilitate the timely separation of powdered carbon materials in the system when the test ends or fails. At present, the literature mostly focuses on the conductive properties of biochar, that is, the sum of electron-accepting and electron-donating ability, and does not focus on the development of electron-accepting ability and electron-accepting sites, and there is also no quantitative mechanism research on the electron-accepting ability of biochar and the operation effect of Anammox process, resulting in no definite basis for the development process. Correspondingly, there is no good strategy to screen biochar in pre-experiment or pilot test while maximizing the consequences of avoiding inappropriate biochar test failure. SUMMARY

[0005] The purpose of the present application is to overcome the defects of the prior art and provide a biochar preparation idea that can promote Anammox extracellular electron transfer, and also to provide a method for reducing the negative impact of test failure on bacterial systems, and according to the test result regularity of the examples, to propose an optimal method for preparing biochar.

[0006] The purpose of the present application can be achieved by the following technical solutions:

[0007] To solve the above technical problems, the present application provides a method for preparing and screening activated carbon or biochar that promotes Anammox bacterial extracellular electron transfer, which comprises the following steps:

[0008] (1) Preparation of the biochar to be tested: after pulverizing the wood waste (such as sawdust, etc.), high-temperature pyrolysis is carried out, and after cooling, grinding to a size of less than 150 μm, repeated soaking, shaking and filtration with deionized water until the filtrate pH is less than 9, and finally drying at 60°C for 48h, screening the candidate biochar with abundant aromatic ring structure and graphite defect sites, which has theoretical extracellular electron transfer potential; preparing biochar containing abundant graphite defect structure (when characterized by the ratio of D peak and G peak area of Raman spectrum, the ratio is preferably greater than 1.35)

[0009] The method for preparing candidate biochar in step (1) includes at least two methods. One is directly pyrolyzing lignin waste at high temperature, reaching 600-900°C at a heating rate of 10°C / min, maintaining the highest temperature for at least 1 h, and then cooling down. The other is pyrolyzing lignin waste mixed with ferric salt at high temperature. The pyrolysis time depends on the pyrolysis method. If microwave pyrolysis is used, the temperature is maintained for 3-15 min after reaching the set highest temperature of 500-800°C. If tube furnace pyrolysis is used, the temperature is maintained for 1-4 h after reaching the set highest temperature.

[0010] (2) Biochar testing device assembly: The biochar to be tested is loaded into a pre-wetted dialysis bag with certain mechanical strength at an appropriate dose. The dialysis bag has a molecular weight cut-off of 3.5-10 k Dalton. The bag is tightly sealed, soaked in deionized water, and ultrasonicated for 30-40 min to remove air bubbles.

[0011] (3) Biochar testing reactor operation: The dialysis bag containing biochar obtained from (2) is taken out of the deionized water, washed three times with deionized water, and then placed in a sealed anaerobic digestion device. The device is set up with a blank group, a control group, and an experimental group (wastewater and anaerobic ammonia oxidation bacteria). The device is blown with high-purity nitrogen for 10-15 min, and then subjected to a continuous batch anaerobic ammonia oxidation test under constant temperature shaking at 35-37°C in the dark. The residence time is 12-24 h. The denitrification rate is tested after each batch. The test includes two stages: normal supply of nitrite and nitrite starvation. When the denitrification rate reaches a stable value for 3-5 consecutive days in the normal supply stage, the nitrite starvation test is started. When the denitrification rate reaches a stable value for 3-5 consecutive days in the nitrite starvation stage, the operation is completed.

[0012] (4) Result evaluation and subsequent processing: ① The results of the experimental group are adjusted by subtracting the effect of biochar adsorption of inorganic nitrogen in the influent from the blank group. ② After adjusting for the blank treatment, the biochar corresponding to the experimental group with a denitrification rate better than the control group is the candidate biochar that can promote extracellular electron transfer of anaerobic ammonia oxidation bacteria. ③ Among the candidate biochar, the change in electron gain and loss ability and the change in electron gain and loss site before and after the test are tested. The biochar with significant changes is the preferred candidate. ④ The preferred biochar can be subjected to a large-scale test. The non-preferred biochar can be removed from the sealed dialysis bag without affecting the morphology and reacclimation of anaerobic ammonia oxidation bacteria.

[0013] Further limitation, the specific method of step (2) is: the preferred mass ratio of biochar to anaerobic ammonia oxidation bacteria (wet weight) is 1:20-40. The biochar in the dialysis bag should be placed close to the anaerobic bacteria, and certain measures should be taken to fix it in place without floating and violent shaking.

[0014] Further limitation, the lignin-rich biomass waste for pyrolysis refers to one or both of wood chips and bamboo chips.

[0015] Further limitation, the high-temperature sintering is carried out in an oxygen-deficient atmosphere, and the sintering atmosphere is an argon atmosphere or a carbon dioxide atmosphere.

[0016] Further limitation, the mass-to-volume ratio of the biochar to the wastewater is 1g:(60-80)mL.

[0017] Further limitation, the specific method of step (2) is that the ratio of the diameter of the dialysis bag to the length of the dialysis bag (the part filled with biochar) is 1:3.5-5, and the length of the dialysis bag is preferably consistent with the inner diameter of the reactor.

[0018] Further limitation, the specific method of step (3) is that a blank group is set as the dialysis bag filled with biochar + synthetic wastewater, a control group is anaerobic ammonia oxidation bacteria + synthetic wastewater, and an experimental group is the dialysis bag filled with biochar + anaerobic ammonia oxidation bacteria + synthetic wastewater, and the light-proof continuous batch anaerobic ammonia oxidation test is carried out by constant temperature shaking at 35-37℃.

[0019] The mass-to-volume ratio of the biochar to the wastewater is 1g:(60-80)mL.

[0020] Further limitation, the specific method of step (3) is that the preferred shaking speed is 100-120rpm, and the preferred two test conditions of nitrite normal supply and nitrite starvation are a nitrite nitrogen / ammonia nitrogen ratio of 1.32 and 0.

[0021] Further limitation, the wastewater composition is: KH2PO420 mg / L, CaCl2·2H2O 112mg / L, MgSO4·7H2O 45mg / L, KHCO3 1350 mg / L, trace element concentrate I and II each 1mL / L, NH4Cl and NaNO2 according to NO2 - -N / NH4 + -N=1.32, 0.90, 0.45, 0.30, 0.20, 0.10, 0 gradient supply.

[0022] Further limitation, trace element concentrate I is EDTA 5 g / L, and FeSO4.7H2O 5 g / L; trace element concentrate II is EDTA 15 g / L, HBO30.014 g / L, MnCl2.4H2O 0.90 g / L, CuSO4.5H2O 0.25 g / L, ZnSO4.7H2O 0.43 g / L, NiCl2.6H2O 0.19 g / L, Na2MoO4.2H2O 0.22 g / L, CoCl2.6H2O 0.24 g / L, and NaSeO4.10H2O 0.21 g / L.

[0023] Further limitation, the specific method of step (4) is: the effect of biochar can be evaluated by evaluating the activity of anaerobic ammonia oxidation bacteria and the assimilation rate of inorganic carbon (i.e. the amount of inorganic carbon consumption minus the part discharged in the form of carbon dioxide gas).

[0024] The carbon material corresponding to the experimental group with a denitrification rate superior to that of the control group is a candidate that can promote extracellular electron transfer of anaerobic ammonia oxidation bacteria; the preferred biochar can be subjected to a large-scale test, and the non-preferred biochar can be taken out in a sealed dialysis bag without affecting the morphology and re-acclimation of anaerobic ammonia oxidation bacteria.

[0025] Compared with the existing test scheme of directly mixing materials and microbial reaction systems, the present application provides a protective testing and popularization application method, saves the economic investment and time cost of pilot test, and is conducive to obtaining material-microbial interface electroactive substances, and is expected to be further explored and popularized. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The total nitrogen TN concentration and removal rate of the effluent of the continuous operation of Example 1 are shown in the schematic diagram;

[0027] Figure 2 The change in the degree of graphite defects of the biochar before and after the operation of Example 1 is shown in the schematic diagram;

[0028] Figure 3 The total nitrogen TN concentration and removal rate of the effluent of the continuous operation of Example 2 are shown in the schematic diagram;

[0029] Figure 4 The change in the degree of graphite defects of the biochar before and after the operation of Example 2 is shown in the schematic diagram. DETAILED DESCRIPTION

[0030] The present application will be described in detail below in conjunction with the drawings and specific examples. The present embodiment is implemented on the premise of the technical solution of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following examples.

[0031] Example 1

[0032] A method for preparing and screening biochar for promoting extracellular electron transfer of anaerobic ammonia oxidation bacteria, comprising the following steps:

[0033] (1) Four kinds of biochar were prepared: sawdust was heated to 800℃ at a heating rate of 10℃ / min under a nitrogen atmosphere, then maintained at the maximum temperature for 1h, 2h, 3h, and 4h, cooled to room temperature, and ground to a size of less than 150μm. The biochar was repeatedly soaked, shaken, and filtered with deionized water until the pH of the filtrate was less than 9, and finally dried at 60℃ for 48h;

[0034] (2) 1g of each of the four kinds of biochar prepared in step (1) was loaded into a pre-wetted dialysis bag with a molecular weight cut-off of 10k Dalton, mixed with 80mL of synthetic wastewater and 25mL of Anammox bacterial solution to form an experimental group. The blank group contained only the synthetic wastewater and Anammox bacteria. The blank group and the experimental group were moved into a sealed anaerobic digestion device, blown with high-purity nitrogen for 10min, and incubated at 36℃ under constant shaking (120rpm) for a sequencing batch SRT of 24h;

[0035] The synthetic wastewater composition was: KH2PO420 mg / L, CaCl2·2H2O 112mg / L, MgSO4·7H2O 45mg / L, KHCO31350 mg / L, trace element concentrate I (EDTA 5 g / L, FeSO4.7H2O 5g / L) and II (EDTA 15g / L, HBO30.014 g / L, MnCl2.4H2O 0.90g / L, CuSO4.5H2O 0.25g / L, ZnSO4.7H2O 0.43g / L, NiCl2.6H2O 0.19g / L, Na2MoO4.2H2O 0.22g / L, CoCl2.6H2O 0.24g / L, NaSeO4.10H2O 0.21g / L), 1mL / L each, NH4Cl and NaNO2 according to NO2 - -N / NH4 + -N=1.32 and 0 two-stage supply, i.e. normal supply of nitrite and zero supply. After the denitrification effect was stable in the normal nitrite supply stage, the zero supply stage was entered, and the denitrification effect was better than that of the experimental group of the blank group. Whether nitrite was generated was further verified, and the change of graphite defect site (electron accepting site) of the biochar was tested after the experiment was completed.

[0036] The removal rate of effluent TN in continuous operation was as follows: Figure 1 The results showed that the biochar prepared at 800℃ for 1h-4h could promote the extracellular electron transfer of Anammox bacteria and improve the denitrification effect. -- The stable denitrification rate of > 78% was obtained after 15-18 days of operation under normal NO2 - - The denitrification rate of the blank group did not exceed 50% after 8 days of operation under NO2 - - The denitrification rate of > 78% was obtained after 15-18 days of operation under normal NO2 - - The denitrification rate of > 78% was obtained after 15-18 days of operation under normal NO2 - - The denitrification rate of > 78% was obtained after 15-18 days of operation under normal NO2 Figure 2 The change in the degree of graphite structure defects (the ratio of the D peak and the G peak of the Raman spectrum) before and after the reaction of the biochar shown further indicates that the graphite defect structure before the reaction was the most, the anaerobic ammonia oxidation (NO2 - - The denitrification rate of > 78% was obtained after 15-18 days of operation under normal NO2

[0037] Example 2

[0038] A method for preparing and screening biochar that promotes extracellular electron transfer of anaerobic ammonia oxidation bacteria, the method comprising the following steps:

[0039] (1) Preparation of iron-modified biochar: 10 g of bamboo chips and FeCl3.6H2O were mixed in two proportions of 100:0.24 and 100:1.20 (since the carbon yield after pyrolysis of pure bamboo chips is about 24%, the proportion of FeCl3.6H2O to pure bamboo biochar is 1% and 5%), and then placed in a microwave-assisted device under a nitrogen atmosphere, with the temperature rising to 300°C at 0-3 min, 550°C at 3-10 min, 750°C at 10-12 min, holding for 3 min, and then cooling to room temperature. The biochar was ground to a size of less than 150 μm, and then repeatedly soaked, shaken, and filtered with deionized water until the pH of the filtrate was less than 8. The biochar was then dried at 60°C for 48 h;

[0040] (2) 1 g of each of the four biochars prepared in step (1) was placed in a pre-wetted dialysis bag with a molecular weight cutoff of 10 k Dalton, and then mixed with 80 mL of synthetic wastewater and 25 mL of Anammox bacterial solution to form an experimental group. The blank group contained only the synthetic wastewater and the Anammox bacteria. The blank group and the experimental group were moved into a sealed anaerobic digestion device and blown off with high-purity nitrogen for 10 min. The reaction was carried out at a constant temperature of 36°C by shaking (120 rpm) in a sequencing batch reactor with an SRT of 24 h.

[0041] wherein the synthetic wastewater components are: KH2PO420 mg / L, CaCl2.2H2O 112 mg / L, MgSO4.7H2O 45 mg / L, KHCO3 1350 mg / L, trace element concentrate I (EDTA 5 g / L, FeSO4.7H2O 5 g / L) and II (EDTA 15 g / L, HBO3 0.014 g / L, MnCl2.4H2O 0.90 g / L, CuSO4.5H2O 0.25 g / L, ZnSO4.7H2O 0.43 g / L, NiCl2.6H2O 0.19 g / L, Na2MoO4.2H2O 0.22 g / L, CoCl2.6H2O 0.24 g / L, NaSeO4.10H2O 0.21 g / L), each 1 mL / L, NH4Cl and NaNO2 according to NO2 - -N / NH4 + -N = 1.32 and 0 two-stage supply, namely normal supply and zero supply of nitrite. After the denitrification effect is stable in the normal supply stage of nitrite, the zero supply stage is entered, and the denitrification effect is better than that of the experimental group of the blank group. Whether nitrite is generated is further verified, and the electron accepting site change of the used biochar is tested after the test is completed.

[0042] Trace element concentrate I is EDTA 5 g / L and FeSO4.7H2O 5 g / L; trace element concentrate II is EDTA 15 g / L, HBO3 0.014 g / L, MnCl2.4H2O 0.90 g / L, CuSO4.5H2O 0.25 g / L, ZnSO4.7H2O 0.43 g / L, NiCl2.6H2O 0.19 g / L, Na2MoO4.2H2O 0.22 g / L, CoCl2.6H2O 0.24 g / L, and NaSeO4.10H2O 0.21 g / L.

[0043] The removal rate of effluent TN in continuous operation is as follows Figure 3 According to the proportion of ferric chloride mixed, the prepared biochar is named as BC750-1%Fe and BC750-5%Fe. The results show that BC750-1%Fe and BC750-5%Fe can obtain about 84% of denitrification effect after 18 days of operation under normal supply of NO2 - -N, while the denitrification rate of the blank group at this time is only about 72%; NO2 - -N starvation, the denitrification rate of the blank group after 8 days of operation is not more than 50%, which shows that it cannot recover NO2 -The nitrogen removal effect of BC750-1%Fe and BC750-5%Fe is better than that of BC750-0%Fe (normal supply of N) and BC750-0%Fe (starvation supply of N), and the nitrogen removal rates of BC750-1%Fe and BC750-5%Fe after 8 days of reaction are about 52% and 64% respectively. Figure 4 The change of the degree of graphite structure defect (the ratio of the area of D peak to the area of G peak in Raman spectrum) is shown in Table 1. The defect site of BC750-5%Fe before reaction is the most, and the defect site of BC750-0%Fe (normal supply of N) and BC750-0%Fe (starvation supply of N) after reaction is repaired most significantly, which indicates that BC750-5%Fe can be used as an insoluble electron acceptor to maintain the denitrification reaction of Anammox bacteria, especially in the absence of NO2 - -N as an electron acceptor. In addition, according to Examples 1 and 2, it can be found that when the ratio of the area of D peak to the area of G peak in Raman spectrum (characterizing the degree of graphite defect) of the biochar before reaction is greater than 1.35, the biochar can assist the Anammox process to obtain a better denitrification effect and exhibit a better extracellular electron transfer potential for promoting Anammox than other biochars. - -N as an electron acceptor. In addition, according to Examples 1 and 2, it can be found that when the ratio of the area of D peak to the area of G peak in Raman spectrum (characterizing the degree of graphite defect) of the biochar before reaction is greater than 1.35, the biochar can assist the Anammox process to obtain a better denitrification effect and exhibit a better extracellular electron transfer potential for promoting Anammox than other biochars.

[0044] The above merely describes the preferred embodiments of the present application, but does not represent other forms of the present application. Any person skilled in the art can make changes or modifications to the above disclosed technical contents to obtain equivalent embodiments. However, any simple modification, equivalent change and modification made to the above embodiments without departing from the technical solution of the present application and according to the technical essence of the present application still falls within the protection scope of the present application.

Claims

1. A method for preparing and screening biochar that promotes extracellular electron transfer in anaerobic ammonia-oxidizing bacteria, characterized in that, The method is implemented through the following steps: S1: The wood waste was crushed and pyrolyzed at high temperature. After cooling, it was ground to a size of less than 150μm. It was repeatedly soaked, shaken and filtered with deionized water until the pH of the filtrate was less than 9. Finally, it was dried at 60℃ for 48h. Biochar with rich aromatic ring structure and graphite defect sites was selected as candidate biochar. S2: The test was conducted using a nitrite starvation method: The biochar prepared in step S1 was loaded into a pre-wetted dialysis bag with a molecular weight cutoff of 3.5k-10k Dalton, then mixed with wastewater and anaerobic ammonia-oxidizing bacteria, and then transferred into a closed anaerobic digestion device. High-purity nitrogen was used to strip the biochar for 10 min to 15 min. The reaction was carried out in a continuous batch anaerobic ammonia oxidation reaction in the dark by shaking under constant temperature conditions of 35℃ to 37℃, with a residence time of 12 h to 24 h. S3: Screening showed that the denitrification rate of anaerobic ammonia oxidation wastewater with zero nitrite supply could reach the same level as the denitrification rate with normal nitrite supply. In step S2, the wastewater includes nitrite nitrogen and ammonia nitrogen; In S1, when screening biochar with graphite defect sites and characterizing it by the ratio of the D and G peak areas in the Raman spectrum, those with a ratio greater than 1.35 were selected. In S2, the wastewater composition is as follows: KH2PO4 20 mg / L, CaCl2•2H2O 112 mg / L, MgSO4•7H2O 45 mg / L, KHCO3 1350 mg / L, trace element concentrate I 1 mL / L and trace element concentrate II 1 mL / L, NH4Cl and NaNO2 are supplied according to the normal nitrite supply stage. - -N / NH4 + -N=1.32 and nitrite starvation stage NO2 - -N / NH4 + Supply is carried out sequentially with N=0; The trace element concentrate I consisted of 5 g / L EDTA and 5 g / L FeSO4·7H2O; the trace element concentrate II consisted of 15 g / L EDTA, 0.014 g / L HBO3, 0.90 g / L MnCl2·4H2O, 0.25 g / L CuSO4·5H2O, 0.43 g / L ZnSO4·7H2O, 0.19 g / L NiCl2·6H2O, 0.22 g / L Na2MoO4·2H2O, 0.24 g / L CoCl2·6H2O, and 0.21 g / L NaSeO4·10H2O.

2. The method for preparing and screening biochar that promotes extracellular electron transfer in anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: In step S1, the method for preparing candidate biochar by high-temperature pyrolysis includes at least two methods: one is to directly pyrolyze lignin waste at high temperature, reaching 600℃~900℃ at a heating rate of 10℃ / min, maintaining the highest temperature for at least 1 hour, and then cooling down after the reaction is complete; the other is to pyrolyze lignin waste mixed with trivalent iron salts at high temperature. The pyrolysis time depends on the pyrolysis method. If microwave pyrolysis is used, it is sufficient to maintain the set highest temperature of 500-800℃ for 3-15 minutes. If tube furnace pyrolysis is used, it is sufficient to maintain the set highest temperature for 1-4 hours.

3. The method for preparing and screening biochar that promotes extracellular electron transfer in anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: In S1, the wood waste used for pyrolysis refers to biomass waste containing abundant lignin structure.

4. The method for preparing and screening biochar that promotes extracellular electron transfer in anaerobic ammonia-oxidizing bacteria according to claim 3, characterized in that: Wood waste includes one or both of wood chips and bamboo chips.

5. The method for preparing and screening biochar that promotes extracellular electron transfer in anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: In S1, high-temperature pyrolysis is carried out under an oxygen-deficient atmosphere, which is either a nitrogen atmosphere or an argon atmosphere.

6. The method for preparing and screening biochar that promotes extracellular electron transfer in anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: In S2, the wet weight ratio of biochar to anaerobic ammonia-oxidizing bacteria is 1:(20-40).

7. The method for preparing and screening biochar that promotes extracellular electron transfer in anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: In S2, the mass ratio of biochar to wastewater is 1 g : (60~80) mL.

8. The method for preparing and screening biochar that promotes extracellular electron transfer in anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: In S2, the oscillation speed is 100 rpm-120 rpm.

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