Method for promoting anaerobic denitrification by electroactive bacteria

Abstract

The invention discloses a method for promoting anaerobic denitrification by electroactive bacteria. According to the method, electroactive bacteria and denitrifying bacteria are co-cultured in an anaerobic denitrification system to promote denitrification. The method for promoting anaerobic denitrification by using the electroactive bacteria has the characteristics of increasing the denitrification rate, reducing the accumulation of nitrite and nitrous oxide, reducing the denitrification cost and avoiding secondary pollution, and has important application prospects in the fields of sewage denitrification and polluted sediment remediation.

Description

Technical Field

[0001] The invention specifically relates to a method for promoting anaerobic denitrification with electroactive bacteria, and belongs to the technical field of environmental protection treatment. Background Art

[0002] Large-scale discharge of aquaculture and animal husbandry waste, excessive application of nitrogen fertilizers and frequent industrial activities have led to excessive nitrate entering environmental water bodies. Biological denitrification using denitrifying bacteria to remove nitrogen is currently the main method for removing nitrate from water, but the biological denitrification process is slow and easily produces the accumulation of toxic and harmful intermediates such as nitrite, an important greenhouse gas, and nitrous oxide, which are harmful to the safety of aquatic organisms and human health. Therefore, it is extremely necessary to establish a method to accelerate denitrification by denitrifying bacteria and reduce the accumulation of intermediates in the denitrification process. Studies have shown that exogenous addition of organic carbon sources can promote denitrification by denitrifying bacteria, but the addition of carbon sources not only significantly increases the cost of denitrification, but also poses a risk of causing secondary pollution.

[0003] The essence of microbial respiration is electron transfer, so the key to improving the denitrification efficiency of denitrifying bacteria is to improve their electron transfer efficiency. Traditional studies believe that electron transfer of microorganisms only occurs within cells, and the distance of electron transfer is limited to the micrometer scale. Recent studies have found that electroactive bacteria have the unique function of transferring electrons outside the cell, which can transfer electrons to electron acceptors tens of micrometers away from themselves, significantly improving the distance and efficiency of microbial electron transfer. Furthermore, it has been found that electroactive bacteria-mediated electron interaction can significantly improve the electron transfer efficiency of microbial interaction in processes such as anaerobic fermentation and methanogenesis. It can be seen that electroactive bacteria have great potential in enhancing the removal of water pollutants. On the other hand, common denitrifying bacteria such as Paracoccus denitrificans and Pseudomonas stutzeri in sewage denitrification have also been reported to be electroactive, but there are still few methods for using electroactive bacteria to enhance denitrification of denitrifying bacteria, and the relevant high-efficiency strains obtained are relatively limited, usually limited to Geobacter or Shewanella. Therefore, the research and development of methods for enhancing biological denitrification with electroactive bacteria can provide a reference for the development of new biological denitrification processes with low carbon and low energy consumption, and has important application value. Summary of the invention

[0004] In view of the shortcomings of the existing biological denitrification technology, the purpose of the present invention is to provide a method for promoting anaerobic denitrification by electroactive bacteria. The method promotes denitrification by co-culturing electroactive bacteria and denitrifying bacteria in an anaerobic denitrification system. Compared with the existing biological denitrification technology, the method provided by the present invention has the characteristics of increasing the denitrification rate, reducing the accumulation of nitrite and nitrous oxide, reducing the denitrification cost, and no secondary pollution.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A method for promoting anaerobic denitrification by electroactive bacteria, which is to promote denitrification by co-culturing electroactive bacteria and denitrifying bacteria in an anaerobic denitrification system.

[0007] Preferably, the electroactive bacteria is Lysinibacillus sphaericus.

[0008] Preferably, the denitrifying bacteria are electroactive denitrifying bacteria, including but not limited to Alicycliphilus denitrificans, Paracoccus denitrificans and / or Pseudomonas stutzeri.

[0009] Preferably, the electroactive bacteria are inoculated by adding a bacterial suspension or adding a functional material that carries the electroactive bacteria.

[0010] Preferably, the inoculation biomass ratio of electroactive bacteria to denitrifying bacteria is 0.5-8.

[0011] Preferably, the initial C / N molar ratio of the denitrifying bacteria and the electroactive bacteria when co-cultured under anaerobic conditions is 2-6.

[0012] Preferably, the temperature for co-culturing the denitrifying bacteria and the electroactive bacteria is 20°C-35°C.

[0013] Preferably, the method is implemented by the following steps:

[0014] S1: Preparation of electroactive bacterial suspension and denitrifying bacterial suspension.

[0015] The electroactive bacteria and denitrifying bacteria were cultured to the logarithmic growth phase, the bacterial liquid was centrifuged to collect the bacterial cells, and the bacterial cells were rinsed 2-3 times with phosphate buffer and then resuspended;

[0016] S2: Preparation of functional materials loaded with electroactive bacteria.

[0017] In a constant temperature water bath, the embedding agent is stirred and dissolved in hot water, cooled to room temperature and mixed and stirred evenly, then activated carbon powder, diatomaceous earth powder, carbon source and the electroactive bacterial suspension obtained in step S1 are added, and an appropriate amount of water is added to the required volume, and the mixed solution is stirred and mixed to be allowed to stand for use;

[0018] Anhydrous calcium chloride solution is used as a solidifying liquid, and the mixed solution obtained in step S2 is slowly dripped into the solidifying liquid using a syringe. After solidification, a microsphere-shaped functional material loaded with electroactive bacteria is obtained. The microspheres are washed with sterile water and then stored in a 4°C refrigerator while moistened;

[0019] S3: Denitrifying bacteria and electroactive bacteria are inoculated into the reactor, and the denitrifying bacteria and electroactive bacteria are co-cultured under anaerobic conditions.

[0020] Preferably, the embedding agent is a mixed solution of sodium alginate and polyvinyl alcohol, and the dissolution temperatures of sodium alginate and polyvinyl alcohol are 60°C-75°C and 80°C-90°C, respectively.

[0021] Preferably, the mass concentration of sodium alginate is 0.1%-4%, the mass concentration of polyvinyl alcohol is 0.4%-16%, the mass concentration of diatomaceous earth powder is 5%-10%, the mass concentration of activated carbon powder is 1%-5%, the mass concentration of carbon source is 1%-3%, and the particle size of diatomaceous earth powder and activated carbon powder is 100-200 mesh.

[0022] Preferably, the concentration of the anhydrous calcium chloride solution is 10-30 g / L, and the curing time is 2-4 h.

[0023] The method for promoting anaerobic denitrification by electroactive bacteria provided by the present invention has the characteristics of increasing the denitrification rate, reducing the accumulation of nitrite and nitrous oxide, reducing the denitrification cost and no secondary pollution, and has important application prospects in the fields of sewage denitrification and contaminated sediment remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The nitric nitrogen concentration-time variation curve of the experimental group and the control group in Example 1 of the present invention;

[0025] Figure 2 The nitrite nitrogen concentration-time variation curve of the experimental group and the control group in Example 1 of the present invention;

[0026] Figure 3 The maximum accumulation concentration of nitrous oxide in the experimental group and the control group in Example 1 of the present invention;

[0027] Figure 4 The electron transfer system activity and ATP activity of the microorganisms in the experimental group and the control group in Example 1 of the present invention;

[0028] Figure 5 A physical photograph of the functional material loaded with electroactive bacteria in Example 4 of the present invention. DETAILED DESCRIPTION

[0029] The method for promoting anaerobic denitrification with electroactive bacteria provided by the present invention is further described below in conjunction with examples. The examples are intended to describe the present invention in detail, but are not intended to limit the claims of the present invention in any way.

[0030] Embodiment 1:

[0031] In a constant temperature shaker, sterile Luria-Bertani medium (LB medium) was used to aerobically culture spherical lysine Bacillus Lysinibacillus sphaericus (L.sphaericus) and denitrificant alicyclic bacteria Alicycliphilus denitrificans (A.denitrificans) for 13-16 hours at 180rpm and 30°C. The composition of the LB medium is as follows: each liter of culture medium contains 5g yeast powder, 10g peptone, 5g sodium chloride and 1L deionized water. The bacterial liquid of L.sphaericus and A.denitrificans was centrifuged at 6500rpm and 5min to collect the cells, and the cells were rinsed 3 times with phosphate buffer and then resuspended. The composition of the phosphate buffer is as follows: 4.54g / L Na 2 HPO 4 , 2.40g / L KH 2 PO 4 and deionized water.

[0032] The serum bottle was used as a reactor, and denitrification medium was added thereto. After exposure to high-purity nitrogen for 30 minutes to remove oxygen, the bottle was sealed with a butyl rubber stopper and an aluminum cap, and then sterilized with high-temperature steam at 121°C for 20 minutes. The composition of the denitrification medium is as follows: phosphate buffer, 0-5.06 g / L KNO 3 , appropriate amount of CH 3 COONa. The C / N molar ratio in the culture medium is 2.

[0033] The magnesium chloride solution, Wolfe trace element solution and Wolfe vitamin solution were filtered through a filter membrane with a pore size of 0.22 μm and added to the sterilized reactor. The final magnesium chloride concentration of the culture medium in the reactor was 0.05-0.2 g / L. 12.5 mL and 5 mL of Wolfe trace element solution and Wolfe vitamin solution were added per liter of culture medium in the reactor, respectively.

[0034] Inoculate the sterilized reactor with A. denitrificans suspension until the initial OD value of the culture medium is 600 About 0.02, and then inoculate the L. sphaericus suspension to the OD of the culture medium 600 The inoculum biomass ratio of L. sphaericus and A. denitrificans was 4 (measured in OD 600 The reactor was placed in a constant temperature shaker at 180 rpm and 30°C for co-cultivation of A. denitrificans and L. sphaericus. 600about 0.02) of A. denitrificans suspension and inoculated with the same biomass (OD 600 About 0.08) of L. sphaericus suspension was used as a control.

[0035] The nitrate and nitrite concentrations in the culture medium were determined by spectrophotometry, the nitrous oxide concentration in the reactor headspace was determined by gas chromatography, and the electron transport system activity and ATP activity of the experimental group and control group were determined by iodonitrotetrazolium violet method and ATP detection kit, respectively.

[0036] The results showed that when the initial nitrate nitrogen concentration of the culture medium was 349.99±2.08 mg / L, the experimental group removed all nitrates within 45 h, while the control group inoculated only with A. denitrificans suspension required 148 h to remove all nitrates, and the control group inoculated only with L. sphaericus suspension could not remove nitrates ( Figure 1 During the biological denitrification process, the maximum accumulation concentration of nitrite in the experimental group was 8.79±0.87 mg / L, which was 73.8% lower than that in the control group inoculated with only A. denitrificans suspension ( Figure 2 The maximum accumulation concentration of nitrous oxide in the experimental group was 79.3% lower than that in the control group inoculated with only A. denitrificans suspension ( Figure 3 ). 40 hours after inoculation, the electron transfer system activity and ATP activity of the microorganisms in the experimental group increased by 119.3% and 163.4% respectively compared with the control group ( Figure 4 ). In the present invention, the reason why co-cultivation of spherical lysine Bacillus and denitrifying bacteria can enhance anaerobic denitrification is that there is an interspecific symbiotic growth effect between spherical lysine Bacillus and denitrifying bacteria, which can promote interspecific electron transfer, thereby improving the carbon and nitrogen metabolism capacity of denitrifying bacteria.

[0037] Embodiment 2:

[0038] Compared with Example 1, the difference is that the denitrifying bacteria was changed from A. denitrificans to Paracoccus denitrificans (P. denitrificans), and the inoculation biomass ratio of electroactive bacteria to denitrifying bacteria was changed from 4 to 6. 600 about 0.02) of P. denitrificans suspension and inoculated with the same biomass (OD 600 About 0.12) of L. sphaericus suspension was used as a control.

[0039] The results showed that the experimental group removed all nitrates within 24 hours, while the control group inoculated with only P.denitrificans suspension needed 30 hours to remove all nitrates, and the control group inoculated with only L.sphaericus suspension could not remove nitrates. During the biological denitrification process, no nitrite accumulation occurred in the experimental group, while the maximum accumulation concentration of nitrite nitrogen in the control group inoculated with only P.denitrificans suspension could reach 35.18±9.44mg / L.

[0040] Embodiment 3:

[0041] Compared with Example 1, the difference is that the denitrifying bacteria is changed from A. denitrificans to P. denitrificans, and the carbon source in the denitrification medium is changed from CH 3 COONa was changed to HCOONa, and the C / N molar ratio in the culture medium was changed from 2 to 3. The inoculation biomass ratio of electroactive bacteria and denitrifying bacteria was changed from 4 to 3. 600 about 0.02) of P. denitrificans suspension and inoculated with the same biomass (OD 600 About 0.06) of L. sphaericus suspension was used as a control.

[0042] The results showed that the experimental group removed all nitrates within 84 hours, while the control group inoculated with only P.denitrificans suspension needed 123 hours to remove all nitrates, and the control group inoculated with only L.sphaericus suspension could not remove nitrates. During the biological denitrification process, the maximum accumulation concentration of nitrite in the experimental group was 1.27±0.31mg / L, which was 93.3% lower than that of the control group inoculated with only P.denitrificans suspension.

[0043] Embodiment 4:

[0044] Compared with Example 1, the difference is that the denitrifying bacteria is changed from A. denitrificans to P. denitrificans, and the inoculation method of the electroactive bacteria is changed from adding a bacterial suspension to adding a functional material that loads the electroactive bacteria.

[0045] The preparation process of the functional material loaded with electroactive bacteria is as follows:

[0046] In a constant temperature water bath, 0.9 g of sodium alginate and 3.6 g of polyvinyl alcohol were stirred and dissolved in appropriate amounts of deionized water at 70°C and 85°C, respectively. After cooling to room temperature, the two were mixed and stirred evenly. Then, 1.8 g of 200 mesh diatomaceous earth powder, 0.6 g of 100 mesh activated carbon powder, 0.6 g of anhydrous sodium acetate and 30 mL of OD 600About 1.8 L. sphaericus suspension, add appropriate amount of deionized water to a total volume of about 60mL, stir and mix well and let stand. Then, the mixture was slowly dripped into 20g / L anhydrous calcium chloride solution using a 20mL syringe, and after curing for 3h, a microsphere-loaded electroactive bacteria functional material ( Figure 5 ), the microspheres were washed with sterile water and then stored moist in a 4°C refrigerator.

[0047] The experimental groups were set up with 5, 10 and 20 microspheres, and the corresponding inoculation biomass ratios of L. sphaericus and P. denitrificans were about 2, 4 and 8, respectively. 600 About 0.02) of P. denitrificans suspension was used as a control.

[0048] The results showed that 22 hours after the reactor was inoculated, the nitrate-nitrogen concentrations of the culture medium in the experimental groups with inoculation biomass ratios of L. sphaericus and P. denitrificans of about 2, 4 and 8, respectively, were 145.14±7.05 mg / L, 70.95±13.93 mg / L and 18.46±2.68 mg / L, which were significantly lower than those of the control group (176.50±6.98 mg / L) by 17.8%, 59.8% and 89.5%, respectively.

Claims

1. A method for promoting anaerobic denitrification by electroactive bacteria, characterized in that: The method promotes denitrification by co-culturing electroactive bacteria and denitrifying bacteria in an anaerobic denitrification system.

2. The method according to claim 1, characterized in that The electroactive bacteria is spherical lysine bacillus Lysinibacillus sphaericus.

3. The method according to claim 1, characterized in that The denitrifying bacteria are electrically active denitrifying bacteria, including but not limited to Alicycliphilus denitrificans, Paracoccus denitrificans and / or Pseudomonas stutzeri.

4. The method according to claim 1, characterized in that The electroactive bacteria are inoculated by adding a bacterial suspension or adding a functional material loaded with the electroactive bacteria.

5. The method according to claim 1, characterized in that The inoculation ratio of electroactive bacteria to denitrifying bacteria is 0.5-8.

6. The method according to claim 1, characterized in that The initial C / N molar ratio of denitrifying bacteria and electroactive bacteria when co-cultured under anaerobic conditions was 2-6.

7. The method according to claim 1, characterized in that The temperature for co-culturing denitrifying bacteria and electroactive bacteria is 20°C-35°C.

8. The method according to claim 1, characterized in that The method is implemented by the following steps: S1: Preparation of electroactive bacterial suspension and denitrifying bacterial suspension. The electroactive bacteria and denitrifying bacteria were cultured to the logarithmic growth phase, the bacterial liquid was centrifuged to collect the bacterial cells, and the bacterial cells were rinsed 2-3 times with phosphate buffer and then resuspended; S2: Preparation of functional materials loaded with electroactive bacteria. In a constant temperature water bath, the embedding agent is stirred and dissolved in hot water, cooled to room temperature and mixed and stirred evenly, then activated carbon powder, diatomaceous earth powder, carbon source and the electroactive bacterial suspension obtained in step S1 are added, and an appropriate amount of water is added to the required volume, and the mixed solution is stirred and mixed to be allowed to stand for use; Anhydrous calcium chloride solution is used as a solidifying liquid, and the mixed solution obtained in step S2 is slowly dripped into the solidifying liquid using a syringe. After solidification, a microsphere-shaped functional material loaded with electroactive bacteria is obtained. The microspheres are washed with sterile water and then stored in a 4°C refrigerator while moistened; S3: Denitrifying bacteria and electroactive bacteria are inoculated into the reactor, and the denitrifying bacteria and electroactive bacteria are co-cultured under anaerobic conditions.

9. The method according to claim 8, characterized in that The embedding agent is a mixed solution of sodium alginate and polyvinyl alcohol, and the dissolution temperatures of sodium alginate and polyvinyl alcohol are 60°C-75°C and 80°C-90°C respectively.

10. The method according to claim 8, characterized in that The mass concentration of sodium alginate is 0.1%-4%, the mass concentration of polyvinyl alcohol is 0.4%-16%, the mass concentration of diatomaceous earth powder is 5%-10%, the mass concentration of activated carbon powder is 1%-5%, the mass concentration of carbon source is 1%-3%, and the particle size of diatomaceous earth powder and activated carbon powder is 100-200 mesh; The concentration of anhydrous calcium chloride solution is 10-30g / L, and the curing time is 2-4h.

Images