A method for promoting anaerobic denitrification by electroactive bacteria

Abstract

The application discloses a method for promoting anaerobic denitrification by electroactive bacteria. The method is to promote denitrification by culturing electroactive bacteria and denitrifying bacteria in an anaerobic denitrification system. The method for promoting anaerobic denitrification by electroactive bacteria has the characteristics of improving denitrification rate, reducing accumulation of nitrite and nitrous oxide, reducing denitrification cost and no secondary pollution, and has important application prospects in the fields of wastewater denitrification and contaminated sediment remediation.

Description

Technical Field

[0001] This invention specifically relates to a method for promoting anaerobic denitrification with electroactive bacteria, belonging to the field of environmental protection treatment technology. Background Technology

[0002] The large-scale discharge of waste from aquaculture and livestock farming, excessive application of nitrogen fertilizers, and frequent industrial activities lead to excessive nitrates entering environmental water bodies. Biological denitrification, utilizing denitrifying bacteria, is currently the main method for removing nitrates from water. However, the biological denitrification process is slow and easily produces toxic and harmful intermediate products such as nitrite, which endangers aquatic life and human health, and nitrous oxide, an important greenhouse gas. Therefore, it is essential to develop a method to accelerate denitrification by denitrifying bacteria and reduce the accumulation of intermediate products during the denitrification process. Existing studies have shown that the addition of exogenous organic carbon sources can promote denitrification by denitrifying bacteria; however, adding carbon sources not only significantly increases the cost of denitrification but also poses a risk of secondary pollution.

[0003] Microbial respiration is essentially electron transfer; therefore, improving the denitrification efficiency of denitrifying bacteria is key to enhancing their electron transfer efficiency. Traditional research suggests that electron transfer in microorganisms occurs only within the cell, and the transfer distance is limited to the micrometer scale. Recent studies have discovered that electroactive bacteria possess a unique extracellular electron transfer function, capable of transferring electrons to electron acceptors tens of micrometers away, significantly increasing the distance and efficiency of electron transfer. Furthermore, it has been found that electroactive bacteria-mediated electron interoperation can significantly improve the electron transfer efficiency of microbial interoperation in processes such as anaerobic fermentation and methanogenesis. Clearly, electroactive bacteria have great potential for enhancing pollutant removal in water bodies. On the other hand, common denitrifying bacteria such as *Paracococcus denitrifyingus* and *Pseudomonas schwannii* have also been reported to possess electroactivity in wastewater denitrification. However, methods for enhancing denitrification using electroactive bacteria are still relatively few, and the number of highly efficient strains obtained is limited, typically confined to *Geobacterium* or *Shewanella*. Therefore, researching and developing methods for enhancing biological denitrification with electroactive bacteria can provide a reference for developing novel low-carbon, low-energy biological denitrification processes, and has significant application value. Summary of the Invention

[0004] To address the shortcomings of existing biological denitrification technologies, the present invention aims to provide a method for promoting anaerobic denitrification using electroactive bacteria. This method promotes denitrification by co-culturing electroactive and denitrifying bacteria in an anaerobic denitrification system. Compared with existing biological denitrification technologies, the method provided by the present invention features increased denitrification rate, reduced accumulation of nitrite and nitrous oxide, lower nitrogen removal costs, and no secondary pollution.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A method for promoting anaerobic denitrification with electroactive bacteria involves co-culturing electroactive bacteria and denitrifying bacteria in an anaerobic denitrification system to promote denitrification.

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

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

[0009] Preferably, the electroactive bacteria are inoculated by adding bacterial suspension or functional materials loaded with 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 when denitrifying bacteria and electroactive bacteria are co-cultured under anaerobic conditions is 2-6.

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

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

[0014] S1: Prepare electroactive bacterial suspension and denitrifying bacterial suspension.

[0015] Cultivate electroactive bacteria and denitrifying bacteria separately to the logarithmic growth phase, collect the bacterial cells by centrifugation, and resuspend the bacterial cells after rinsing them 2-3 times with phosphate buffer.

[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. After cooling to room temperature, the mixture is stirred evenly. Then, activated carbon powder, diatomaceous earth powder, carbon source and electroactive bacterial suspension obtained in step S1 are added. Add an appropriate amount of water to the required volume, stir and mix well to obtain a mixed solution and let it stand for later use.

[0018] Using anhydrous calcium chloride solution as the curing liquid, the mixture obtained in step S2 was slowly dripped into the curing liquid using a syringe. After curing, microspheres loaded with electroactive bacteria were obtained as functional materials. The microspheres were washed with sterile water and then moistened and stored in a refrigerator at 4°C.

[0019] S3: Inoculate the reactor with denitrifying bacteria and electroactive bacteria, and co-culture the denitrifying bacteria and electroactive bacteria under anaerobic conditions.

[0020] Preferably, the encapsulating agent is a mixed solution of sodium alginate and polyvinyl alcohol, wherein the dissolution temperatures of sodium alginate and polyvinyl alcohol are 60℃-75℃ and 80℃-90℃, 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 electroactive bacteria-promoted anaerobic denitrification method provided by this invention has the characteristics of increasing the denitrification rate, reducing the accumulation of nitrite and nitrous oxide, reducing denitrification costs and eliminating secondary pollution, and has important application prospects in the fields of wastewater denitrification and polluted sediment remediation. Attached Figure Description

[0024] Figure 1 Nitrate concentration-time change curves of the experimental group and the control group in Example 1 of this invention;

[0025] Figure 2 Nitrite concentration-time change curves of the experimental group and the control group in Example 1 of this invention;

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

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

[0028] Figure 5 A photograph of the functional material loaded with electroactive bacteria in Embodiment 4 of the present invention. Detailed Implementation

[0029] The following examples further illustrate the method for promoting anaerobic denitrification by electroactive bacteria provided by the present invention. The examples are intended to provide a detailed description of the present invention, but are not intended to limit the scope of the claims of the present invention in any way.

[0030] Example 1:

[0031] *Lysinibacillus sphaericus* (L. sphaericus) and *Alicycliphilus denitrificans* (A. denitrificans) were aerobically cultured in a constant-temperature shaker at 180 rpm and 30 °C for 13-16 h using sterile Luria-Bertani medium (LB medium). The LB medium consisted of 5 g yeast extract, 10 g peptone, 5 g sodium chloride, and 1 L deionized water per liter. The bacterial suspensions of *L. sphaericus* and *A. denitrificans* were collected by centrifugation at 6500 rpm for 5 min. The bacterial cells were washed three times with phosphate-buffered saline (PBS) and resuspended. The PBS consisted of 4.54 g / L Na₂HPO₄, 2.40 g / L KH₂PO₄, and deionized water.

[0032] Using serum bottles as reactors, denitrification medium was added, and after aeration with high-purity nitrogen for 30 minutes to remove oxygen, the bottles were sealed with butyl rubber stoppers and aluminum caps, followed by high-temperature steam sterilization at 121°C for 20 minutes. The composition of the denitrification medium was as follows: phosphate buffer, 0-5.06 g / L KNO3, and appropriate amount of CH3COONa. The C / N molar ratio in the medium was 2.

[0033] Magnesium chloride solution, Wolfe trace element solution, and Wolfe vitamin solution were filtered through a 0.22 μm pore size filter membrane and then added to the sterilized reactor. The final magnesium chloride concentration in 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 to each liter of culture medium in the reactor, respectively.

[0034] The A. denitrificans suspension was first inoculated into the sterilized reactor to the initial OD of the culture medium. 600 Approximately 0.02, then inoculate L. sphaericus suspension to the OD of the culture medium. 600 Approximately 0.10. The inoculum biomass ratio of L. sphaericus and A. denitrificans was 4 (based on OD). 600 (Value measurement). The reactor was placed in a constant temperature shaker and co-cultured with *A. denitrificans* and *L. sphaericus* at 180 rpm and 30 °C. Only an isobaric biomass (OD) was inoculated. 600 Approximately 0.02) of A. denitrificans suspension and only inoculated with equal biomass (OD) 600 A suspension of L. sphaericus (approximately 0.08%) was used as a control.

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

[0036] The results showed that when the initial nitrate concentration in the culture medium was 349.99 ± 2.08 mg / L, the experimental group removed all nitrates within 45 hours, while the control group inoculated only with A. denitrificans suspension required 148 hours to remove all nitrates, and the control group inoculated only with L. sphaericus suspension could not remove nitrates at all. Figure 1 During biological denitrification, 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 only with 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 only with A. denitrificans suspension. Figure 3 Forty hours after inoculation, the electron transport 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 this invention, the co-culture of *Bacillus spheroidae* and denitrifying bacteria can enhance anaerobic denitrification because there is interspecific mutualistic growth between *Bacillus spheroidae* and denitrifying bacteria, which can promote interspecific electron transfer and thus improve the carbon and nitrogen metabolism capacity of denitrifying bacteria.

[0037] Example 2:

[0038] Compared to Example 1, the difference is that the denitrifying bacteria were 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. Only an isobiomass (OD) was inoculated. 600 Approximately 0.02) of P. denitrificans suspension and inoculation with only equal biomass (OD) 600 A suspension of L. sphaericus (approximately 0.12 g) was used as a control.

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

[0040] Example 3:

[0041] Compared to Example 1, the differences are: the denitrifying bacteria were changed from *A. denitrificans* to *P. denitrificans*, the carbon source in the denitrification medium was changed from CH3COONa to HCOONa, and the C / N molar ratio in the medium was changed from 2 to 3. The inoculation biomass ratio of electroactive bacteria to denitrifying bacteria was changed from 4 to 3. Only an equal biomass (OD) was inoculated. 600 Approximately 0.02) of P. denitrificans suspension and inoculation with only equal biomass (OD) 600 A suspension of L. sphaericus (approximately 0.06%) was used as a control.

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

[0043] Example 4:

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

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

[0046] In a constant temperature water bath, 0.9g of sodium alginate and 3.6g of polyvinyl alcohol were dissolved separately in appropriate amounts of deionized water at 70℃ and 85℃, respectively. After cooling to room temperature, they were mixed and stirred thoroughly. Then, 1.8g of 200-mesh diatomaceous earth powder, 0.6g of 100-mesh activated carbon powder, 0.6g of anhydrous sodium acetate, and 30mL of OD were added to the sodium alginate and polyvinyl alcohol mixture. 600A suspension of approximately 1.8 g of *L. sphaericus* was added, and deionized water was added to bring the total volume to approximately 60 mL. The mixture was stirred and allowed to stand. Then, the mixture was slowly added dropwise to a 20 g / L anhydrous calcium chloride solution using a 20 mL syringe. After solidification for 3 hours, microspheres loaded with electroactive bacteria were obtained as functional materials. Figure 5 The microspheres were washed with sterile water and then kept moist in a refrigerator at 4°C.

[0047] Experimental groups were set up with 5, 10, and 20 microspheres, corresponding to inoculation biomass ratios of approximately 2, 4, and 8 for *L. sphaericus* and *P. denitrificans*, respectively. Inoculation with only equal biomass (OD) was also performed. 600 A suspension of P. denitrificans at approximately 0.02 g was used as a control.

[0048] The results showed that 22 h after inoculation in the reactor, the nitrate nitrogen concentrations in the culture media of the experimental groups with biomass of L. sphaericus and P. denitrificans were 145.14±7.05 mg / L, 70.95±13.93 mg / L, and 18.46±2.68 mg / L, respectively, which were significantly lower than those in 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 with electroactive bacteria, characterized in that, The application discloses a method for promoting denitrification by culturing electroactive bacteria and denitrifying bacteria in an anaerobic denitrification system, wherein the electroactive bacteria is a spherical lysinibacillus, the denitrifying bacteria is electroactive denitrifying bacteria, specifically, denitrosomonas guttae or denitrobacterium paraffinicum. Lysinibacillus sphaericu Alicycliphilus denitrificans Paracoccus denitrifican ​​​ 2. The method of claim 1, wherein, Electroactive bacteria are inoculated by adding bacterial suspension or functional materials loaded with electroactive bacteria.

3. The method of claim 1, wherein, The inoculation ratio of electroactive bacteria to denitrifying bacteria is 0.5-8.

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

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

6. The method according to claim 1, characterized in that, This method is implemented through the following steps: S1: Preparation of electroactive bacterial suspension and denitrifying bacterial suspension Cultivate electroactive bacteria and denitrifying bacteria separately to the logarithmic growth phase, collect the bacterial cells by centrifugation, and resuspend the bacterial cells after rinsing them 2-3 times with phosphate buffer. 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. After cooling to room temperature, the mixture is stirred evenly. Then, activated carbon powder, diatomaceous earth powder, carbon source and electroactive bacterial suspension obtained in step S1 are added. Add an appropriate amount of water to the required volume, stir and mix well to obtain a mixed solution and let it stand for later use. Anhydrous calcium chloride solution was used as the curing liquid. The obtained mixture was slowly dripped into the curing liquid using a syringe. After curing, microspheres loaded with electroactive bacteria were obtained. The microspheres were washed with sterile water and then moistened and stored in a 4°C refrigerator. S3: Inoculate the reactor with denitrifying bacteria and electroactive bacteria, and co-culture the denitrifying bacteria and electroactive bacteria under anaerobic conditions.

7. The method according to claim 6, characterized in that, The embedding agent is a mixed solution of sodium alginate and polyvinyl alcohol, with the dissolution temperatures of sodium alginate and polyvinyl alcohol being 60°C-75°C and 80°C-90°C, respectively.

8. The method according to claim 6, 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%, the particle size of diatomaceous earth powder and activated carbon powder is 100-200 mesh; the concentration of anhydrous calcium chloride solution is 10-30 g / L, and the curing time is 2-4 h.

Images