A culture method of a highland adaptive denitrification composite microbial agent

Abstract

The application provides a culture method of a highland adaptive denitrification composite microbial agent and belongs to the technical field of water treatment and resource utilization. In view of the existing problem that the metabolism of sewage denitrification functional bacteria is limited under the combined stress of low temperature and low oxygen in the highlands, a specific composite microbial system with low-temperature and low-oxygen adaptability is constructed, the strain source is obtained from the in-situ environment of the highlands, the high-throughput sorting technology is used to efficiently and accurately obtain the functional single bacteria in the simulated highland environment, the best strain matching and inoculation ratio are determined through the control experiment, the synergistic denitrification of the composite strains is realized, the method can be used as a biological strengthening agent to enhance the denitrification effect of the highland sewage treatment unit, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a method for cultivating a high-altitude-adaptive denitrifying compound bacterial agent, belonging to the field of microbial technology. Background Technology

[0002] The low temperature, low pressure, and low oxygen environment of plateau regions inhibits the activity of wastewater biodegradation bacteria. Simultaneously, the low concentration and large fluctuations in influent concentration at wastewater treatment plants easily lead to microbial community imbalance, limiting the retention of core functional bacteria and reducing system stability. Microbial agents have important and widespread applications in environmental engineering. By adding dominant bacterial species with specific functions to wastewater biological treatment systems, the treatment effect on specific pollutants can be enhanced. Faced with the complex environmental stresses of plateau regions, screening, enrichment, and acclimatization of cold-adapted single or mixed microbial communities with highly efficient nitrogen removal functions, and then preparing them into agents and adding them to biological treatment systems, can shorten reactor start-up time and improve the system's operational efficiency and stability under stress.

[0003] Aerobic denitrifying bacteria typically exhibit "micro-aerobic denitrification," meaning they do not require the strictly anaerobic environment necessary for traditional denitrification, nor do they demand extremely high dissolved oxygen levels. This characteristic allows them to operate efficiently in the unique "low dissolved oxygen / micro-aerobic" environment of plateau regions, correspondingly reducing the demand for aeration intensity and energy consumption. Furthermore, compared to traditional anaerobic denitrifying bacteria, aerobic denitrifying bacteria generally have stronger environmental tolerance, faster growth rates, and more vigorous metabolism, making it easier to maintain high biomass and activity at low temperatures through bioaugmentation. In addition, aerobic denitrifying bacteria also have advantages such as promoting simultaneous nitrification and denitrification, lower greenhouse gas production, and lower sludge production. Therefore, aerobic denitrifying bacteria have significant research and application value in the biological treatment of wastewater in plateau regions.

[0004] However, in wastewater treatment systems, biodegradation efficiency and system stability often depend on complex, multi-species microbial systems, rather than single-species microorganisms. Microorganisms can achieve functions that cannot be accomplished by simply adding the properties of a single species through interspecies interactions such as metabolic specialization and cross-feeding. A well-structured functional microbial community typically has higher biomass yield and system stability. Therefore, expanding the research and application of functional microorganisms to synergistic multi-species agents centered on aerobic denitrifying bacteria, and through reasonable species combinations and inoculation ratios, is more likely to achieve enhanced pollutant degradation and long-term stable biofortification. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a cultivation method for a plateau-adaptive denitrification compound microbial agent, which can achieve efficient denitrification of wastewater in a plateau-simulated environment. This method and the microbial compound microbial agent are applicable to the construction of a bio-enhanced system for wastewater treatment in Tibet, thereby improving the biological denitrification effect of wastewater treatment in plateau areas.

[0006] Technical solution: This invention provides a method for cultivating a high-altitude adaptive denitrifying compound bacterial agent, comprising the following steps: (1) Strain isolation: Sludge was collected from the aerobic section of the wastewater treatment plant in the plateau region, and plateau-adapted aerobic denitrifying bacteria were selected as the screening target. Under simulated plateau low temperature conditions, the bacteria were pre-cultured at the first proportion of inoculum to enrich them and reach a stable growth period. Then, functional pure bacteria were isolated. (2) Cultivation and acclimatization of pure bacteria: The functional pure bacteria obtained in step (1) were inoculated into the denitrification basal medium BM to acclimate the aerobic denitrification pure bacteria to low pressure and low oxygen tolerance; under controlled low temperature, nitrate load and C / N ratio, a low pressure gradient was set up using a plateau environment simulation experimental chamber, and the culture was carried out step by step under the first constant low pressure / second constant low pressure / third constant low pressure. (3) Re-screening of denitrification performance of pure bacteria: using a culture containing 100 mg / L NO3 - -N or 16 mg / L NO2 - The nitrate or nitrite removal capacity of the domesticated pure bacteria was tested under low temperature and low pressure combined stress using BM medium with different single nitrogen sources of -N and a second inoculation ratio. (4) Co-culture test: The nitrate-reducing bacteria (type A) and nitrite-reducing bacteria (type B) that performed well in step (3) were combined into a dual-bacterial composite system and co-cultured under low temperature and low pressure composite stress to test the aerobic denitrification performance; the composite bacterial agent with excellent denitrification performance and significantly improved effect compared with pure culture was retained.

[0007] The beneficial effects of this method include: the introduction of highly efficient type B bacteria capable of rapidly reducing nitrite, effectively promoting NO2 reduction. - The timely removal of nitrate metabolic intermediates such as N2O reduces the accumulation of inhibitory products, establishes a positive interaction of metabolic specialization and cross-feeding between the bacteria and the highly efficient nitrate-reducing type A bacteria in pure culture, and offsets the potential resource competition between the two bacteria. This application utilizes its constructed denitrifying compound bacterial agent, which enhances the synergistic nitrogen metabolism between the two bacterial species, simultaneously strengthening the compound bacterial agent's ability to withstand the dual stresses of low temperature and low pressure. Attached Figure Description

[0008] Figure 1 The results of the control experiment for the optimal co-culture strain combination under different inoculation ratios are shown, where (a) OD 600 ; (b) Changes in nitrate nitrogen content. Detailed Implementation

[0009] The present invention will be described below with reference to specific embodiments to enable those skilled in the art to further understand the invention. It should be noted that the present invention is not limited to the following embodiments.

[0010] Example 1: A method for cultivating a high-altitude adaptive denitrifying compound bacterial agent, comprising the following steps: (1) Strain Isolation Stage: In this embodiment, aerobic denitrifying bacteria adapted to high altitudes were selected as the screening target. The strain was collected from sludge in a typical wastewater treatment plant in Linzhi City, Tibet Autonomous Region. The collection point was located in the last aerobic stage of the multi-stage AO process in the biochemical treatment unit of the wastewater treatment plant. The strain was inoculated from the initial sample into the enrichment medium at an inoculation ratio of 10%. The low temperature conditions (13-15℃) of the high altitude were simulated using a constant temperature and humidity incubator. After pre-culturing, the strain was enriched and reached a stable growth period. Using high-throughput droplet microfluidic cell sorting technology, empty droplets that failed to capture single cells or failed the initial screening of denitrification function were removed. Single colonies with aerobic denitrification potential were isolated and amplified.

[0011] (2) Cultivation and acclimatization stage of pure bacteria: The pure bacteria obtained in step (1) were inoculated into the denitrification basic medium BM. Under the control of low temperature of 15℃, nitrate load and C / N ratio, the plateau environment simulation experimental chamber was used to set a low pressure gradient and gradually carry out the subculture under constant low pressure of 82-85Kpa, 72-75Kpa and 62-65Kpa: The simulation chamber parameters were controlled and the constant air pressure value was gradually reduced. The air pressure was reduced by 10Kpa in each stage. After the growth of aerobic denitrifying bacteria in the previous stage was stable, the air pressure was further reduced to gradually increase the low pressure stress and enhance the adaptability of the initial screening strains to the combined stress.

[0012] (3) Re-screening stage of denitrification performance of pure bacteria: using a culture containing 100 mg / L NO3 - -N or 16 mg / L NO2 - BM medium with different single nitrogen sources and an inoculum ratio of 2% was used, with a C / N ratio controlled at 5, to simulate the characteristics of typical domestic sewage with a low C / N ratio in the Tibetan region. The nitrate or nitrite removal capacity of the acclimated pure bacteria was tested under low-temperature and low-pressure combined stress (13-15℃, 62-65KPa). This involved a secondary screening of denitrification capabilities to identify target type A bacteria with high nitrate reduction capacity and target type B bacteria with high nitrite reduction capacity, providing a selection of bacterial strains for the subsequent establishment of compound bacterial agents. The aerobic denitrification performance of some pure bacteria in the functional secondary screening is shown in Tables 1 and 2.

[0013] Table 1. Detection results at different time points during the nitrate reducing capacity test.

[0014] Table 2. Detection results at different time points during the nitrite reducing capacity test.

[0015] (4) Co-culture test stage: The type A and type B bacteria that performed well in step (3) were combined into a two-strain compound system. Control experiments with different bacterial species combinations were set up. Co-culture was carried out under the same low temperature and low pressure compound stress as in step (3). The growth of the bacterial agent and the aerobic denitrification performance were tested. The denitrification performance test results with nitrate as the only nitrogen source are shown in Table 3. Verify whether the compound bacterial agent with a specific composition has a significant advantage over other co-culture groups and pure culture control groups. For the co-culture combination that showed a significant advantage, a control experiment was further set up with different bacterial species inoculation ratios. The specific results are shown in Table 3. Figure 1 As shown. The compound bacterial agent with outstanding denitrification performance is retained.

[0016] Table 3. Nitrate removal during the test process for different co-culture combinations.

Claims

1. A method for cultivating a high-altitude-adaptive denitrifying compound bacterial agent, characterized in that, This can be achieved through the following steps: (1) Strain isolation: Sludge was collected from the aerobic section of the wastewater treatment plant in the plateau region, and plateau-adapted aerobic denitrifying bacteria were selected as the screening target. Under simulated plateau low temperature conditions, the bacteria were pre-cultured at the first proportion of inoculum to enrich them and reach a stable growth period. Then, functional pure bacteria were isolated. (2) Cultivation and acclimatization of pure bacteria: The functional pure bacteria obtained in step (1) were inoculated into the denitrification basal medium BM to acclimate the aerobic denitrification pure bacteria to low pressure and low oxygen tolerance; under controlled low temperature, nitrate load and C / N ratio, a low pressure gradient was set up using a plateau environment simulation experimental chamber, and the culture was carried out step by step under the first constant low pressure / second constant low pressure / third constant low pressure. (3) Re-screening of denitrification performance of pure bacteria: using a culture containing 100 mg / L NO3 - -N or 16 mg / L NO2 - The nitrate or nitrite removal capacity of the domesticated pure bacteria was tested under low temperature and low pressure combined stress using BM medium with different single nitrogen sources of -N and a second inoculation ratio. (4) Co-culture test: The nitrate-reducing bacteria (type A) and nitrite-reducing bacteria (type B) that performed well in step (3) were combined into a dual-bacterial composite system and co-cultured under low temperature and low pressure composite stress to test the aerobic denitrification performance; the composite bacterial agent with excellent denitrification performance and significantly improved effect compared with pure culture was retained.

2. The cultivation method of the plateau-adaptive denitrifying compound bacterial agent according to claim 1, characterized in that, In step (1), the enrichment medium used in the enrichment stage has the following composition: KNO3 1.5-2.0 g / L, CH3COONa 8.0-10.0 g / L, KH2PO4 1.0-1.5 g / L, K2HPO4 1.0-1.5 g / L, MgSO4·7H2O 0.1-0.2 g / L, FeSO4 0.01-0.02 g / L.

3. The cultivation method of the plateau-adaptive denitrifying compound bacterial agent according to claim 1, characterized in that, In step (2), the nutrient composition of the culture medium BM is as follows: KNO3 0.361 g / L, CH3COONa 3.416 g / L, Na2HPO4 4.20-4.50 g / L, KH2PO4 1.20-1.50 g / L, MgSO4·7H2O 0.10-0.15 g / L, and trace element solution 1 mL.

4. The cultivation method of the plateau-adaptive denitrifying compound bacterial agent according to claim 3, characterized in that, The composition of the trace element solution is as follows: EDTA-2Na 45.0-50.0 g / L, ZnSO4 2.00-2.20 g / L, CaCl2 5.00-5.50 g / L, MnCl2·4H2O 5.00-5.20 g / L, FeSO4 5.00-5.20 g / L, CuSO4·5H2O 1.50-1.60 g / L, (NH4)6Mo7O2·4H2O 1.00-1.20 g / L and CoCl2·6H2O 1.50-1.60 g / L.

5. The cultivation method of the plateau-adaptive denitrifying compound bacterial agent according to claim 1, characterized in that, In step (1), the low temperature condition is 13-15℃, and the first proportion is 10%.

6. The cultivation method of the plateau-adaptive denitrifying compound bacterial agent according to claim 1, characterized in that, In step (2), the low temperature is 13-15℃.

7. The cultivation method of the plateau-adaptive denitrifying compound bacterial agent according to claim 1, characterized in that, In step (3), the low temperature and low pressure are 13-15℃ and 62-65KPa; the second ratio is 2%.

8. The cultivation method of the plateau-adaptive denitrifying compound bacterial agent according to claim 1, characterized in that, In step (3), the low temperature and low pressure are 13-15℃ and 60-65KPa.

9. The cultivation method of the plateau-adaptive denitrifying compound bacterial agent according to claim 1, characterized in that, In step (2), the first constant low pressure is 82-85 kPa, the second constant low pressure is 72-75 kPa, and the third constant low pressure is 62-65 kPa.

10. The cultivation method of the plateau-adaptive denitrifying compound bacterial agent according to claim 1, characterized in that, In the denitrification performance test in steps (3) and (4), the C / N ratio was controlled to 5 based on the low temperature and low pressure simulation conditions, to further simulate the typical characteristics of domestic sewage with low C / N ratio in the Tibetan Plateau region.

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