A bacterial continuous flow scale culture device, culture method and application

Abstract

The present application relates to the technical field of bacterial culture, in particular to a bacterial continuous flow large-scale culture device, culture method and application. The bacterial continuous flow large-scale culture device comprises a culture container; the inside of the culture container is provided with a fixed culture chamber, the bottom of the fixed culture chamber is provided with a bacteria liquid flow channel in communication with the culture container; the inside of the fixed culture chamber is provided with a centrifugal culture chamber for adjusting the hydraulic retention time, the chamber wall of the centrifugal culture chamber is provided with an overflow channel in communication with the fixed culture chamber; the culture container is sequentially divided into a reverse buffer zone, an elastic trapping zone and a sedimentation zone from top to bottom. The present application also provides a bacterial culture method and application. The present application solves the problem of long domestication cycle of the existing nitrifying bacteria culture method, and also solves the problem of low overall denitrification efficiency of the nitrifying bacteria obtained by the existing method.

Description

Technical Field

[0001] This invention relates to the field of bacterial culture technology, specifically to a continuous flow large-scale bacterial culture device, culture method, and application. Background Technology

[0002] Currently, the effluent quality of coal mine wastewater treatment plants in my country generally complies with the Class A standard of the "Discharge Standard of Pollutants for Urban Wastewater Treatment Plants" (GB 18918-2002). With increasingly stringent requirements for eutrophication control, some regions lacking receiving water bodies require newly built wastewater treatment plants to achieve effluent ammonia nitrogen concentrations below the Class III standard limit (1 mg / L) of the "Environmental Quality Standard for Surface Water" (GB3838-2002). However, coal mine wastewater generally exhibits a low carbon-to-nitrogen ratio, making it difficult to increase the sludge concentration in the system using traditional activated sludge processes, thus failing to meet the requirements for deep nitrogen removal. Although biofilm processes such as biological contact oxidation and aerated biological filters can be used, additional nitrifying bacteria agents are required to enhance ammonia nitrogen removal. However, current nitrifying bacteria preparation technologies have significant drawbacks: conventional cultivation methods have lengthy acclimatization periods, limited effective biomass attached to carriers, and process operation typically relies on the entire nitrification pathway, leading to prolonged hydraulic retention time and low overall nitrogen removal efficiency.

[0003] Therefore, there is an urgent need to develop a nitrifying bacteria culture device and culture method suitable for coal mine domestic sewage, so as to achieve rapid enrichment and efficient immobilization of nitrifying functional bacteria, increase the abundance of nitrite bacteria and enhance short-range nitrification, thereby solving the technical problems faced in this field. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a continuous flow large-scale bacterial culture device, culture method and application, to solve the problem of the long domestication cycle of existing nitrifying bacteria culture methods, and also to solve the problem of low overall denitrification efficiency of nitrifying bacteria cultured by existing methods.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A continuous flow large-scale bacterial culture device includes: a culture container; The culture container has an immobilized culture chamber inside, and the bottom of the immobilized culture chamber has a bacterial liquid flow channel that is connected to the culture container. The immobilized culture chamber is equipped with a centrifugal culture chamber for adjusting the hydraulic residence time, and the centrifugal culture chamber has an overflow channel on its wall that is connected to the immobilized culture chamber. The culture container is divided into a reverse buffer zone, an elastic trapping zone, and a settling zone from top to bottom; The culture medium containing the bacterial source flows into the centrifugation culture chamber, is centrifuged, and then flows into the immobilization culture chamber through the overflow channel. The mixed bacterial solution after being cultured in the immobilization culture chamber flows into the culture container through the bacterial solution flow channel. Inside the culture container, bacteria in the mixed bacterial solution are trapped and enriched by the elastic trapping zone, and bacterial excrement in the mixed bacterial solution settles in the sedimentation zone. After the bacterial excrement is removed, the reverse buffer generates gas to drive the bacteria in the elastic trapping zone to detach into the sedimentation zone. Subsequently, a portion of the bacterial solution in the sedimentation zone is collected and utilized, while the other portion is returned to the centrifuge culture chamber to achieve continuous flow large-scale bacterial culture.

[0006] Based on the above technical means, by setting up centrifugal culture chamber, immobilized culture chamber and culture container in sequence from the inside to the outside, and setting up partitions within the culture container, the long cycle and denitrification efficiency of traditional nitrifying bacteria culture are systematically solved through multidimensional hierarchical culture and intelligent capture and reflux mechanism.

[0007] In the centrifuge culture chamber, a centrifugal force field acts on the culture medium. By utilizing the subtle differences in sedimentation coefficients between nitrifying and nitrite-oxidizing bacteria, the in-situ optimization of the bacterial community structure is achieved by controlling the rotation speed and overflow rate. The centrifugal acceleration causes nitrite-oxidizing bacteria, which have a slower growth rate, to be preferentially thrown towards the chamber wall and enter the immobilized culture chamber through the overflow channel. Meanwhile, nitrite-oxidizing bacteria, due to their smaller size and slower sedimentation speed, have a longer effective residence time in the centrifugation zone. This increases their relative abundance in the bacterial community under dynamic selection pressure. This community structure optimization based on physical field regulation avoids the interference of chemical inhibitors on bacterial activity.

[0008] Immobilized culture chambers create a confined microenvironment where nitrifying bacteria, under the influence of shear forces and nutrient concentration gradients in the bacterial culture flow channels, spontaneously secrete extracellular polymers and attach to the chamber walls or the surface of the immobilized carrier, forming a high-density biofilm structure. This process transforms free-living bacteria into attached bacteria, effectively increasing biomass concentration. More importantly, the steric hindrance effect of immobilization allows for the selection of highly attachable strains, which exhibit stronger resistance to shock loads in real wastewater treatment biofilm systems.

[0009] The elastic trapping zone forms an adaptive "capture-release" cycle during the drainage stage through material elastic deformation and dynamic pore size adjustment: when water flows upward, the pores of the elastic fiber network shrink to a size smaller than bacteria under the action of fluid drag, with a retention rate of over 99%; the downward gas pulse in the reverse buffer causes the fibers to vibrate and rebound, the pores expand instantaneously and release the enriched bacteria, achieving rapid detachment without mechanical stirring. This mechanism shortens the traditional settling time from 2-4 hours to within 30 minutes and avoids damage to bacterial activity caused by shear force.

[0010] After the excrement is separated by gravity sedimentation in the settling zone, the high-density bacterial solution is proportionally diverted and returned to the centrifuge culture chamber, forming a closed loop of "inoculation-enrichment-reselection." This maintains the abundance of functional microbial communities within the system and enhances the adaptive evolution of the microbial community through continuous recirculation, reducing the acclimatization period from the traditional batch processing of 30-45 days to 7-10 days. The synergistic effect of continuous flow operation and dynamic optimization of microbial community structure ultimately improves the activity matching degree of nitrifying and nitrifying bacteria within the device, increases the nitrification rate by 2-3 times, and significantly enhances the overall nitrogen removal efficiency.

[0011] Preferably, the centrifuge culture chamber is connected to the immobilization culture chamber via a rotating base.

[0012] Preferably, a water distributor is provided at the bottom of the centrifugal culture chamber, and the water distributor is connected to the liquid inlet pipe.

[0013] Preferably, the centrifuge chamber has an overflow channel on the side wall near the top.

[0014] Preferably, the bottom of the centrifuge culture chamber is rotatably connected to the bottom of the immobilized culture chamber via the rotating base, so that the centrifuge culture chamber can rotate around its axis.

[0015] Preferably, the overflow channel is composed of flow holes formed on the side wall near the top of the centrifugal culture chamber, the diameter of the flow holes is 8~20mm, and the spacing between adjacent flow holes is 50~200mm.

[0016] Preferably, the centrifuge chamber has a cylindrical structure, and the flow holes are arranged circumferentially along the side wall of the centrifuge chamber, with 10 to 20 flow holes provided on the side wall of the centrifuge chamber.

[0017] Preferably, the immobilized culture chamber is provided with an upper intercepting net, a lower intercepting net, an adsorption carrier, an aeration pipe, and a first sliding track; The upper interception net and the lower interception net are located above the aeration pipe; The adsorption carrier is located between the upper interception net and the lower interception net; The aeration pipe is located at the bottom of the immobilized culture chamber; The first sliding track is disposed on the inner side wall of the immobilized culture chamber, and the lower intercepting net is in sliding fit with the first sliding track, so that the lower intercepting net can slide up and down along the first sliding track.

[0018] Preferably, the end of the upper intercepting net is fixedly connected to the inner wall of the immobilized culture chamber.

[0019] Preferably, the aeration pipe is fixedly connected to the bottom of the immobilized culture chamber.

[0020] Preferably, the first sliding track is fixed to the inner wall of the immobilized culture chamber in the vertical direction.

[0021] Preferably, the diameter of the adsorption carrier is 30-50 mm, the porosity is 80-90%, the filling rate of the adsorption carrier in the immobilization culture chamber is 30-50%, and the material of the adsorption carrier is polyurethane or polyvinyl alcohol.

[0022] Preferably, a backflush aeration pipe is provided in the reverse buffer zone.

[0023] By setting up a backflush aeration pipe in the reverse buffer zone to provide airflow in the opposite direction to the overflow, some bacteria tend to settle in the reverse buffer zone after exceeding the height of the aeration pipe, thus preventing bacteria from overflowing directly through the buffering effect.

[0024] Preferably, the elastic trapping zone is provided with an upper grid plate, a lower grid plate, filter cotton, and a second sliding track; The filter cotton is provided between the upper grid plate and the lower grid plate; The second sliding track is disposed on the inner side wall of the culture container, and the upper grid plate and the lower grid plate are slidably engaged with the second sliding track, so that the upper grid plate and the lower grid plate can slide up and down along the second sliding track.

[0025] Preferably, the filter cotton comprises 3 to 5 layers, and the pore size of each layer gradually decreases from top to bottom, with the pore size of the filter cotton being 0.7 to 4 mm.

[0026] Preferably, a drainage trough is provided near the top inside the culture container, and the drainage trough is connected to a drainage pipe.

[0027] Preferably, a drain pipe connected to the settling zone is provided at the bottom of the culture container; The drain pipe is connected to a return pipe and a collection pipe, and the return pipe is connected to the inlet pipe through a pipe mixer.

[0028] The present invention also provides a method for culturing bacteria using the continuous flow large-scale bacterial culture apparatus as described in the present invention, comprising the following steps: S1. The culture medium containing the bacterial source is mixed through a pipe mixer and then flows into the centrifuge culture chamber through the inlet pipe; S2. After centrifugation in the centrifugation culture chamber for a preset time, the mixed bacterial solution flows into the immobilized culture chamber through the overflow channel. S3. When the turbidity of the mixed bacterial solution in the immobilized culture chamber reaches the preset turbidity, it flows into the culture container through the bacterial solution flow channel. S4. In the culture container, the bacteria in the mixed bacterial solution are intercepted and enriched by the elastic trapping zone, and the bacterial excrement in the mixed bacterial solution settles in the sedimentation zone. After the bacterial excrement is discharged, the reverse buffer generates gas to drive the bacteria in the elastic trapping zone to fall into the sedimentation zone. Then the bacterial solution in the sedimentation zone is collected to obtain the cultured bacteria. A portion of the cultured bacterial solution is stored for later use, while the other portion is returned to the pipeline mixer to achieve continuous flow large-scale bacterial culture.

[0029] Based on the aforementioned technical methods, a dynamically optimized continuous culture system was constructed through a cascade process of homogenization using a pipeline mixer, centrifugation, immobilization enrichment, and flexible trapping, significantly improving the efficiency and stability of nitrifying bacteria culture. First, the pipeline mixer instantly homogenizes the reflux culture medium with the fresh culture medium, maintaining a constant inoculum size and shortening the lag period through pre-adaptation of the bacterial community. The centrifugation chamber physically sorts the bacteria based on differences in sedimentation coefficients, and with precise control of the preset culture time, actively enriches nitrite-oxidizing bacteria and optimizes their proportion in the nitrifying bacterial community, overcoming the bottleneck of functional bacterial competition imbalance in traditional cultures. The immobilization chamber uses turbidity reaching a target level as a trigger for intelligent scale-up; the mixed culture medium is only released into the culture container after reaching the preset turbidity, ensuring that the bacteria entering the trapping zone are in the logarithmic growth phase and their density is controllable, avoiding batch-to-batch variations. The flexible trapping zone achieves a dynamic switch between efficient retention and non-destructive detachment through material deformation: during the cultivation stage, the retention rate reaches over 99%, preventing bacterial loss; during the harvesting stage, gas pulses in the reverse buffer zone cause the bacteria to rapidly detach into the settling zone, effectively compressing the settling time without mechanical shear damage. After settling and separation, part of the bacterial solution is directly collected for later use, while the other part is returned to the pipeline mixer to form a closed loop. This not only enhances the adaptive evolution and functional stability of the bacterial community but also shortens the acclimatization period from 30-45 days to 7-10 days. Simultaneously, through continuous flow operation and selective pressure maintenance, the metabolic synergy efficiency of nitrifying and denitrifying bacteria within the device is increased by 2-3 times, ultimately achieving a dual breakthrough in denitrification efficiency and cultivation scale.

[0030] Preferably, the volume content of the bacterial source in the culture medium containing the bacterial source is 2-5%.

[0031] Preferably, the preset time is 2 to 2.5 hours.

[0032] Preferably, the centrifugation speed is 60~80 r / min.

[0033] Preferably, during the culture process in the immobilized culture chamber 2, the dissolved oxygen concentration is 1.5~2.5 mg / L.

[0034] Preferably, the preset turbidity is 200 NTU.

[0035] Preferably, the bacteria are nitrifying bacteria.

[0036] Preferably, S3 includes: When the turbidity of the mixed bacterial solution in the immobilized culture chamber reaches the preset turbidity, the lower intercepting net moves upward along the first sliding track, causing the adsorption carrier to gradually move upward until 60-80% of the adsorption carrier is in a compressed state, at which point the movement of the lower intercepting net stops. Then, the air-to-water ratio is adjusted to 12:1 through the aeration pipe, so that the mixed bacterial solution flows into the culture container through the bacterial solution flow channel. After maintaining this for 3-5 minutes, the lower intercepting net is moved downwards to its initial position.

[0037] The air-to-water ratio refers to the ratio of the air flow rate from the aeration pipe to the water flow rate from the inlet pipe.

[0038] Preferably, S4 includes: Inside the culture container, the mixed bacterial solution flows upward, and the bacteria in the mixed bacterial solution are intercepted and enriched by the filter cotton in the elastic trapping zone. The bacterial excrement in the mixed bacterial solution settles in the settling zone. The upper grid plate moves upward along the second sliding track as the mixed bacterial solution flows until the upper grid plate moves up to the top of the second sliding track. Then, the drain pipe is opened to remove the bacterial excrement that has settled in the reverse buffer zone. After the bacterial excrement is discharged, the drain pipe is closed, the backflushing aeration pipe of the reverse buffer zone is opened, and the air-to-water ratio is controlled at 6:1 until the adsorbent carrier returns to its initial state from the squeezed state. Then, the backflushing aeration pipe is closed, and the return and collection pipes are opened. A portion of the bacteria in the mixed bacterial solution flows out through the collection pipe and is stored in a concentrated manner, while another portion of the bacteria flows through the return pipe to the pipeline mixer to achieve continuous flow and large-scale bacterial culture.

[0039] Preferably, the volume ratio of the amount of bacteria flowing into the return tube to the amount of bacteria flowing into the collection tube is 1:4.

[0040] The present invention also provides the application of bacteria cultured by the culture method described herein in the treatment of coal mine domestic sewage.

[0041] Experiments have shown that the cultivation method of this invention can reduce the ammonia nitrogen concentration in coal mine domestic sewage to below 0.23 mg / L, with an ammonia nitrogen removal rate of 98.6%.

[0042] The beneficial effects of this invention are: The continuous flow large-scale bacterial culture device of this invention utilizes centrifugal culture chambers arranged from the inside out to physically screen the bacterial community using a centrifugal force field. Based on the difference in sedimentation coefficients between nitrifying and non-nitrifying bacteria, the effective residence time of nitrifying bacteria is extended by adjusting the rotation speed and overflow rate, thereby increasing their relative abundance in the bacterial community under dynamic selection pressure. This chemically inhibited community structure optimization avoids interference with bacterial activity caused by traditional methods. The immobilized culture chamber constructs a confined microenvironment, allowing nitrifying bacteria to spontaneously secrete extracellular polymers and attach to form a high-density biofilm under the shear force and nutrient gradient of the bacterial solution flow channel. This not only effectively increases the biomass concentration but also screens out highly adhesive strains through steric hindrance, significantly enhancing their resistance to shock loads in real wastewater treatment. The elastic trapping zone utilizes material elastic deformation to achieve an adaptive "capture-release" cycle: during upward drainage, the fiber network's pores shrink to below the size of bacteria under fluid drag, achieving a retention rate of over 99%; in the reverse buffer zone, downward gas pulses drive fiber vibration and rebound, instantly releasing enriched bacteria, effectively shortening settling time and avoiding mechanical shearing damage to bacterial activity. After gravity separation of excrement in the settling zone, the high-density bacterial solution is proportionally diverted for collection and recirculation, forming a closed-loop system of "inoculation-enrichment-reselection." This maintains the abundance of functional bacteria and enhances adaptive evolution through continuous recirculation, reducing the acclimatization period from 30-45 days in batches to 7-10 days. The synergistic effect of this continuous flow operation mode and dynamic bacterial optimization ultimately improves the activity matching of nitrifying and nitrifying bacteria within the device, increasing the nitrification rate by 2-3 times and significantly enhancing overall nitrogen removal efficiency.

[0043] The bacterial culture method of this invention constructs a dynamically optimized continuous culture system through a cascade process of homogenization by a pipeline mixer, centrifugation, immobilization enrichment, and elastic trapping, significantly improving the efficiency and stability of nitrifying bacteria culture. First, the pipeline mixer instantaneously homogenizes the reflux bacterial solution and fresh culture medium, maintaining a constant inoculum size and shortening the lag period through bacterial pre-adaptation. The centrifugation chamber physically sorts the bacteria based on differences in sedimentation coefficients, and with precise control of the preset culture time, actively enriches nitrite-oxidizing bacteria and optimizes their proportion in the nitrifying bacterial community. The immobilization chamber achieves intelligent scale-up based on turbidity reaching a target level; the mixed bacterial solution is only released into the culture container after reaching the preset turbidity, ensuring that the bacteria entering the trapping zone are in the logarithmic growth phase and their density is controllable, avoiding batch-to-batch variations. The flexible trapping zone dynamically switches between efficient retention and non-destructive detachment through material deformation: during the cultivation stage, the retention rate reaches over 99%, preventing bacterial loss; during the harvesting stage, gas pulses in the reverse buffer zone cause the bacteria to rapidly detach into the settling zone 13, compressing the settling time to 1 / 4 of traditional methods without mechanical shear damage. After settling and separation, part of the bacterial solution is directly collected for later use, while the other part is returned to the pipeline mixer to form a closed loop. This not only enhances the adaptive evolution and functional stability of the bacterial community but also shortens the acclimatization period from 30-45 days to 7-10 days. Furthermore, through continuous flow operation and selective pressure maintenance, the metabolic synergy efficiency of nitrifying and non-nitrifying bacteria within the device is increased by 2-3 times, achieving a dual breakthrough in denitrification efficiency and cultivation scale. This technology has significant application potential in the fields of bacterial cultivation and wastewater treatment. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the results of the continuous flow large-scale bacterial culture device in Example 1; Figure 2 Here is a scanning electron microscope image of the nitrifying bacteria cultured in Comparative Example 1; Figure 3 This is a species composition distribution diagram of the nitrifying bacteria cultured in Comparative Example 1; Figure 4 This is a scanning electron microscope image of the nitrifying bacteria cultured in Example 2; Figure 5 This is a distribution diagram of the species composition of the nitrifying bacteria cultured in Example 2; Among them, 1-culture container, 11-reverse buffer zone, 111-backflush aeration pipe, 12-elastic trapping zone, 121-upper grid plate, 122-lower grid plate, 123-filter cotton, 124-second sliding track, 13-sedimentation zone, 14-drainage trough; 2-immobilized culture chamber, 21-bacterial liquid flow channel, 22-upper interception net, 23-lower interception net, 24-adsorption carrier, 25-aeration pipe, 26-first sliding track; 3-centrifuge culture chamber, 31-overflow channel, 32-rotating base, 33-water distributor; 4-inlet pipe; 5-drainage pipe; 6-exhaust pipe; 7-return pipe; 8-collection pipe; 9-pipe mixer. Detailed Implementation

[0045] The following description, with reference to preferred embodiments, illustrates the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are merely illustrative of the present invention and not intended to limit the scope of protection of the present invention.

[0046] Example 1 A continuous flow large-scale bacterial culture device includes: a culture container 1; The culture container 1 has an immobilized culture chamber 2 inside, and the bottom of the immobilized culture chamber 2 has a bacterial liquid flow channel 21 that is connected to the culture container 1; The immobilized culture chamber 2 is equipped with a centrifugal culture chamber 3 for adjusting the hydraulic residence time. The centrifugal culture chamber 3 has an overflow channel 31 on its wall that is connected to the immobilized culture chamber 2. The culture container 1 is divided into a reverse buffer zone 11, an elastic trapping zone 12, and a settling zone 13 from top to bottom.

[0047] In some embodiments, the centrifuge culture chamber 3 is rotatably connected to the immobilized culture chamber 2 via a rotating base 32, so that the centrifuge culture chamber 3 can rotate around its axis, thereby realizing the rotational centrifugation culture of the culture medium containing the bacterial source; a water distributor 33 is provided at the bottom of the centrifuge culture chamber 3, and the water distributor 33 is connected to the liquid inlet pipe 4; an overflow channel 31 is provided on the side wall near the top of the centrifuge culture chamber 3.

[0048] For example, the overflow channel 31 is composed of flow holes opened on the side wall of the centrifugal culture chamber 3 near the top. The diameter of the flow holes is 15 mm and the spacing between adjacent flow holes is 150 mm. The centrifugal culture chamber 3 has a cylindrical structure, and the flow holes are arranged circumferentially along the side wall of the centrifugal culture chamber 3. 15 flow holes are opened on the side wall of the centrifugal culture chamber 3.

[0049] In some embodiments, the immobilized culture chamber 2 is provided with an upper intercepting net 22, a lower intercepting net 23, an adsorption carrier 24, an aeration pipe 25, and a first sliding track 26; the upper intercepting net 22 and the lower intercepting net 23 are located above the aeration pipe 25; the adsorption carrier 24 is located between the upper intercepting net 22 and the lower intercepting net 23; the aeration pipe 25 is located at the bottom of the immobilized culture chamber 2; the first sliding track 26 is provided on the inner sidewall of the immobilized culture chamber 2, and the lower intercepting net 23 is slidably engaged with the first sliding track 26, so that the lower intercepting net 23 can slide up and down along the first sliding track 26.

[0050] For example, the end of the upper intercepting net 22 is fixedly connected to the inner wall of the immobilized culture chamber 2; the aeration pipe 25 is fixedly connected to the bottom of the immobilized culture chamber 2; the first sliding track 26 is fixed to the inner wall of the immobilized culture chamber 2 in the vertical direction; the adsorption carrier 24 has a diameter of 40 mm, a porosity of 85%, and a filling rate of 40%.

[0051] In some embodiments, a backflush aeration pipe 111 is provided in the reverse buffer zone 11; an upper grid plate 121, a lower grid plate 122, filter cotton 123, and a second sliding track 124 are provided in the elastic collection zone 12; filter cotton 123 is provided between the upper grid plate 121 and the lower grid plate 122; the second sliding track 124 is provided on the inner sidewall of the culture container 1, and the upper grid plate 121 and the lower grid plate 122 are slidably engaged with the second sliding track 124, so that the upper grid plate 121 and the lower grid plate 122 can slide up and down along the second sliding track 124.

[0052] For example, the filter cotton 123 includes four layers, and the pore sizes of each layer are 0.8 mm, 1.5 mm, 2.5 mm and 3.5 mm from top to bottom.

[0053] In this system, the gas in aeration pipe 25 flows upward, while the gas in backflushing aeration pipe 111 flows downward.

[0054] In some embodiments, a drainage trough 14 is provided near the top inside the culture container 1, and the drainage trough 14 is connected to a drainage pipe 5. A drain pipe 6 connected to the sedimentation zone 13 is provided at the bottom of the culture container 1; the drain pipe 6 is arranged outside the culture container 1; the drain pipe 6 is connected to a return pipe 7 and a collection pipe 8, and the return pipe 7 is connected to the inlet pipe 4 through a pipe mixer 9.

[0055] In actual use, the bacterial continuous flow large-scale culture device mixes the culture medium containing the bacterial source through the pipe mixer 9 and then flows into the centrifugal culture chamber 3 through the inlet pipe 4; after centrifugation in the centrifugal culture chamber 3, it flows into the immobilization culture chamber 2 through the overflow channel 31; after being cultured in the immobilization culture chamber 2, the mixed bacterial solution flows into the culture container 1 through the bacterial solution flow channel 21. Inside the culture container 1, bacteria in the mixed bacterial solution are trapped and enriched by the elastic trapping zone 12, and bacterial excrement in the mixed bacterial solution settles in the sedimentation zone 13. After the bacterial excrement is removed, the bacteria in the elastic trapping zone 12 are driven to fall into the sedimentation zone 13 by the gas generated by the reverse buffer zone 11. Then, a part of the bacterial solution in the sedimentation zone 13 is collected and utilized, and the other part of the bacterial solution is returned to the centrifuge culture chamber 3, thereby realizing continuous flow large-scale bacterial culture.

[0056] Example 2 A method for culturing nitrifying bacteria using the continuous flow large-scale bacterial culture apparatus described in Example 1 includes the following steps: S1. The culture medium containing nitrifying bacteria source is mixed through the pipe mixer 9 and then flows into the centrifugation culture chamber 3 through the liquid inlet pipe 4 and the water distributor 33 for centrifugation culture. The volume content of nitrifying bacteria in the culture medium containing nitrifying bacteria was 3%. The culture medium consisted of 2g of (NH4)2SO4, 0.75g of K2HPO4, 0.25g of NaH2PO4, 0.03g of MgSO4·7H2O, and 0.03g of MnSO4·4H2O. The pH was adjusted to 8.1–8.2 using Na2CO3. The nitrifying bacteria source is designated as DeCN-Bacteria and originates from Hangzhou Research Institute Co., Ltd. of China Coal Technology & Engineering Group. S2. In the centrifugal culture chamber 3, the centrifugal culture time (i.e., hydraulic retention time) is controlled to 2 hours by adjusting the flow rate of the culture medium containing nitrifying bacteria source. After centrifugation, the mixed bacterial solution flows into the immobilization culture chamber 2 through the overflow channel 31 formed by the overflow holes on the side wall near the top of the centrifugal culture chamber 3. During the centrifugation process, the rotation speed of the rotating base 32 is 70 r / min; S3. During the cultivation of the mixed bacterial solution in the immobilized culture chamber 2, air is introduced through the aeration pipe 25 to make the dissolved oxygen concentration in the immobilized culture chamber 2 2.0 mg / L. When the turbidity of the mixed bacterial solution in the immobilized culture chamber 2 reaches 200 NTU, the lower intercepting net 23 is moved upward along the first sliding track 26, so that the adsorbent carrier 24 gradually moves upward until 70% of the adsorbent carrier 24 is in a compressed state, at which point the movement of the lower intercepting net 23 is stopped. Then, the air-to-water ratio is adjusted to 12:1 through the aeration pipe 25, so that the mixed bacterial solution flows into the culture container 1 through the bacterial solution flow channel 21 at the bottom of the immobilized culture chamber 2. After maintaining this for 4 minutes, the lower intercepting net 23 is moved downward to return to its initial position. S4. In the culture container 1, as the mixed bacterial solution flows upward, the nitrifying bacteria in the mixed bacterial solution are trapped and enriched by the filter cotton 123 of the elastic trapping zone 12. The excrement of the nitrifying bacteria in the mixed bacterial solution settles in the settling zone 13. The upper grid plate 121 moves upward along the second sliding track 124 as the mixed bacterial solution flows. When the upper grid plate 121 moves to the top of the second sliding track 124, the drain pipe 6 is opened to remove the nitrifying bacteria excrement that has settled in the reverse buffer zone 11. After the nitrifying bacteria excrement is discharged, the drain pipe 6 is closed, the backflushing aeration pipe 111 of the reverse buffer zone 11 is opened, and the air-to-water ratio is controlled at 6:1, so that the nitrifying bacteria in the elastic trapping zone 12 detach and settle into the settling zone 13 until the adsorption carrier 24 returns from the squeezed state to the initial state. Then the backflushing aeration pipe 111 is closed, and then the return pipe 7 and the collection pipe 8 are opened, so that a portion of the nitrifying bacteria in the mixed bacterial solution (i.e., the cultured nitrifying bacteria) flows out through the collection pipe 8 and is stored in a concentrated manner for subsequent treatment of coal mine domestic sewage. Another portion of the nitrifying bacteria flows through the return pipe 7 to the pipeline mixer 9 and is fully mixed with the culture solution before flowing back into the centrifuge culture chamber 3 to achieve continuous flow large-scale culture of nitrifying bacteria. The ratio of the amount of bacteria flowing into the return pipe 7 to the amount of bacteria flowing into the collection pipe 8 is 1:4. After the mixed bacterial solution is discharged, the return pipe 7 and the collection pipe 8 are closed.

[0057] Comparative Example 1 A routine method for culturing nitrifying bacteria includes the following steps: Nitrifying bacteria were continuously enriched for 6 cycles using a sequencing batch culture device equipped with stirring, heating and aeration. The culture cycle lasts 5 days, with a stirring speed of 80-180 r / min and a dissolved oxygen concentration of 2.5-4.5 mg / L. The culture temperature is 28℃-32℃. During the culture, ammonium salts and growth promoters are added, and polyaluminum chloride is added for flocculation and precipitation. After standing, the supernatant is discarded, and then enriched culture medium is added to start the next culture cycle.

[0058] Species composition analysis of nitrifying bacteria The viable nitrifying bacteria in the bacteria cultured in Example 2 and the control Example 1 were determined and counted using the dilution plate colony count method. The results showed that the concentration of nitrifying bacteria cultured in Example 2 was 2.4 × 10⁻⁶. 9 CFU / mL, the concentration of nitrifying bacteria cultured in control example 1 was 1.3 × 10⁻⁶. 7 CFU / mL.

[0059] The microbial species and distribution of nitrifying bacteria cultured in Example 2 and Control Example 1 were detected and analyzed using scanning electron microscopy and high-throughput sequencing. The results are as follows: Figures 2 to 5 As shown.

[0060] From Figure 2 and Figure 3 As can be seen from the above, the nitrifying bacteria obtained by the conventional culture method in Comparative Example 1 did not become the dominant bacteria, and the colony composition was relatively complex.

[0061] from Figure 4 and Figure 5 Comparative analysis shows that the nitrifying bacteria cultured using the culture device and method of this invention are mainly spherical and short rod-shaped, with a significant proportion of *Nitrosomonas*, which is the dominant genus of nitrite-oxidizing bacteria. Under aerobic conditions, this dominant genus can oxidize ammonia nitrogen to nitrite nitrogen, shortening the nitrification reaction process. In contrast, nitrifying bacteria cultured using conventional methods have a complex species composition and no significant proportion of dominant nitrite-oxidizing bacteria, resulting in lower nitrogen removal efficiency through traditional methods.

[0062] Wastewater treatment in coal mines The nitrifying bacteria cultured in Example 2 were inoculated into the biological contact oxidation tank of coal mine domestic sewage at an inoculation rate of 600L. After two days of aeration and curing, the tank was run for six days with a hydraulic retention time of 2.78 hours. The average biomass of the biofilm attached to the biological carrier in the biological contact oxidation tank was determined to be 26.3 g / L by the Kjeldahl method. The ammonia nitrogen concentration in the coal mine domestic sewage treated by the biological contact oxidation tank was reduced to below 0.23 mg / L, and the ammonia nitrogen removal rate reached 98.6%.

[0063] In summary, the centrifugal culture zone of the continuous flow large-scale bacterial culture device of the present invention can optimize the culture conditions of nitrifying bacteria and increase the proportion of nitrifying bacteria in the nitrifying bacterial community. The immobilized culture zone can effectively enrich nitrifying bacteria and continuously and efficiently cultivate highly adhesive nitrifying bacteria. The flexible trapping zone operates flexibly, effectively preventing bacterial loss during drainage, shortening settling time, and eliminating the need for batch water changes. This solves the problem of the lengthy acclimatization cycle in existing nitrifying bacteria culture methods and also addresses the issue of low overall nitrogen removal efficiency of nitrifying bacteria cultivated by existing methods. It has significant application value in the fields of bacterial culture and wastewater treatment technology.

[0064] The above embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. A continuous flow, large-scale bacterial culture device, characterized in that, include: Culture container (1); The culture container (1) is provided with an immobilized culture chamber (2) inside, and the bottom of the immobilized culture chamber (2) is provided with a bacterial liquid flow channel (21) connected to the culture container (1). The immobilized culture chamber (2) is equipped with a centrifugal culture chamber (3) for adjusting the hydraulic residence time. The centrifugal culture chamber (3) has an overflow channel (31) connected to the immobilized culture chamber (2) on its wall. The culture container (1) is divided into a reverse buffer zone (11), an elastic trapping zone (12), and a settling zone (13) from top to bottom. The culture medium containing the bacterial source flows into the centrifugation culture chamber (3), and after centrifugation, it flows into the immobilization culture chamber (2) through the overflow channel (31). The mixed bacterial solution after being cultured in the immobilization culture chamber (2) flows into the culture container (1) through the bacterial solution flow channel (21). In the culture container (1), the bacteria in the mixed bacterial solution are trapped and enriched by the elastic trapping zone (12), and the bacterial excrement in the mixed bacterial solution settles in the sedimentation zone (13). After the bacterial excrement is discharged, the reverse buffer zone (11) generates gas to drive the bacteria in the elastic trapping zone (12) to fall into the sedimentation zone (13). Then, a part of the bacterial solution in the sedimentation zone (13) is collected and utilized, and another part of the bacterial solution is returned to the centrifuge culture chamber (3) to realize continuous flow large-scale bacterial culture. The immobilized culture chamber (2) is equipped with an upper interception net (22), a lower interception net (23), an adsorption carrier (24), an aeration pipe (25), and a first sliding track (26). The upper intercepting net (22) and the lower intercepting net (23) are located above the aeration pipe (25); The adsorption carrier (24) is located between the upper interception net (22) and the lower interception net (23); The aeration pipe (25) is located at the bottom of the immobilized culture chamber (2); The first sliding track (26) is disposed on the inner side wall of the immobilized culture chamber (2), and the lower intercepting net (23) is in sliding fit with the first sliding track (26), so that the lower intercepting net (23) can slide up and down along the first sliding track (26); A backflush aeration pipe (111) is provided in the reverse buffer zone (11). The elastic trapping area (12) is provided with an upper grid plate (121), a lower grid plate (122), filter cotton (123), and a second sliding track (124). The filter cotton (123) is provided between the upper grid plate (121) and the lower grid plate (122). The second sliding track (124) is disposed on the inner side wall of the culture container (1). The upper grid plate (121) and the lower grid plate (122) are slidably engaged with the second sliding track (124), so that the upper grid plate (121) and the lower grid plate (122) can slide up and down along the second sliding track (124).

2. The bacterial continuous flow large-scale culture device according to claim 1, characterized in that, The centrifuge culture chamber (3) is connected to the immobilized culture chamber (2) via a rotating base (32); The bottom of the centrifugal culture chamber (3) is provided with a water distributor (33), which is connected to the liquid inlet pipe (4). The centrifuge culture chamber (3) has an overflow channel (31) on the side wall near the top.

3. The bacterial continuous flow large-scale culture device according to claim 2, characterized in that, A drainage trough (14) is provided near the top of the culture container (1), and the drainage trough (14) is connected to a drainage pipe (5).

4. The bacterial continuous flow large-scale culture device according to claim 2, characterized in that, A drain pipe (6) connected to the sedimentation zone (13) is provided at the bottom of the culture container (1). The drain pipe (6) is connected to the return pipe (7) and the collection pipe (8). The return pipe (7) is connected to the inlet pipe (4) through the pipe mixer (9).

5. A method for culturing bacteria using a continuous flow large-scale bacterial culture apparatus as described in any one of claims 2 to 4, characterized in that, Includes the following steps: S1. The culture medium containing the bacterial source is mixed through the pipe mixer (9) and then flows into the centrifugal culture chamber (3) through the inlet pipe (4). S2. After centrifugation in the centrifugation culture chamber (3) for a preset time, the mixed bacterial solution flows into the immobilization culture chamber (2) through the overflow channel (31). S3. When the turbidity of the mixed bacterial solution in the immobilized culture chamber (2) reaches the preset turbidity, it flows into the culture container (1) through the bacterial solution flow channel (21). S4. In the culture container (1), the bacteria in the mixed bacterial solution are intercepted and enriched by the elastic trapping zone (12), and the bacterial excrement in the mixed bacterial solution settles in the sedimentation zone (13). After the bacterial excrement is discharged, the reverse buffer zone (11) generates gas to drive the bacteria in the elastic trapping zone (12) to fall into the sedimentation zone (13). Then the bacterial solution in the sedimentation zone (13) is collected to obtain the cultured bacteria. A portion of the cultured bacterial solution is stored for later use, while the other portion is returned to the pipeline mixer (9) to achieve continuous flow large-scale bacterial culture.

6. The method for culturing bacteria according to claim 5, characterized in that, The bacterial source in the culture medium contains 2-5% by volume. The preset time is 2~2.5h; The centrifugation speed is 60~80 r / min; During the culture process in the immobilized culture chamber (2), the dissolved oxygen concentration was 1.5~2.5 mg / L; The preset turbidity is 200 NTU; The bacteria in question are nitrifying bacteria.

7. The method for culturing bacteria according to claim 5, characterized in that, S3 includes: When the turbidity of the mixed bacterial solution in the immobilized culture chamber (2) reaches the preset turbidity, the lower intercepting net (23) moves upward along the first sliding track (26), causing the adsorbent carrier (24) to gradually move upward until 60-80% of the adsorbent carrier (24) is in a squeezed state, at which point the movement of the lower intercepting net (23) stops. Then, the air-to-water ratio is adjusted to 12:1 through the aeration pipe (25), so that the mixed bacterial solution flows into the culture container (1) through the bacterial solution flow channel (21). After maintaining this for 3 to 5 minutes, the lower intercepting net (23) is moved downward to the initial position. S4 includes: Inside the culture container (1), the mixed bacterial solution flows upward, and the bacteria in the mixed bacterial solution are intercepted and enriched by the filter cotton (123) of the elastic trapping zone (12). The bacterial excrement in the mixed bacterial solution settles in the settling zone (13). The upper grid plate (121) moves upward along the second sliding track (124) as the mixed bacterial solution flows, until the upper grid plate (121) moves to the top of the second sliding track (124). Then, the drain pipe (6) is opened to remove the bacterial excrement that has settled in the reverse buffer zone (11). After the bacterial excrement is discharged, the drain pipe (6) is closed, the backflushing aeration pipe (111) of the reverse buffer (11) is opened, and the air-to-water ratio is controlled at 6:1 until the adsorbent carrier (24) returns from the squeezed state to the initial state. Then the backflushing aeration pipe (111) is closed, and the return pipe (7) and the collection pipe (8) are opened. A portion of the bacteria in the mixed bacterial solution flows out through the collection pipe (8) and is stored in a concentrated manner, while another portion of the bacteria flows through the return pipe (7) to the pipeline mixer (9) to achieve continuous flow large-scale bacterial culture.

Images