CN114752545A - High-salt-tolerance denitrifying bacterium flora screening and culturing method - Google Patents
High-salt-tolerance denitrifying bacterium flora screening and culturing method Download PDFInfo
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- 235000013619 trace mineral Nutrition 0.000 claims abstract description 9
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- 238000012423 maintenance Methods 0.000 claims abstract description 6
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- 229910052799 carbon Inorganic materials 0.000 claims abstract description 5
- 238000011081 inoculation Methods 0.000 claims abstract description 4
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- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 claims description 10
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 claims description 10
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- 238000009825 accumulation Methods 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
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- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 4
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 3
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 3
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 3
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 claims description 3
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 3
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 3
- 230000005764 inhibitory process Effects 0.000 claims description 3
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 3
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- 239000011684 sodium molybdate Substances 0.000 claims description 3
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- 239000011686 zinc sulphate Substances 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- YUWBVKYVJWNVLE-UHFFFAOYSA-N [N].[P] Chemical compound [N].[P] YUWBVKYVJWNVLE-UHFFFAOYSA-N 0.000 claims description 2
- 241001148470 aerobic bacillus Species 0.000 claims description 2
- 230000008030 elimination Effects 0.000 claims description 2
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
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Abstract
A method for screening and culturing denitrifying bacteria flora with high salt tolerance belongs to the field of sewage and wastewater treatment. The method comprises the steps of taking return sludge of a secondary sedimentation tank of a sewage treatment plant as inoculation sludge, manually preparing substances such as a carbon source, a nitrogen source, trace elements and the like required by microorganisms, realizing establishment and maintenance of a culture environment with a salinity value of 4% by utilizing high-concentration substrate feeding and intermittent operation, strictly controlling operation parameters such as pH, temperature and the like, and adjusting water inlet load by detecting various indexes of effluent water, so as to successfully screen and culture a denitrifying bacteria flora with high salt tolerance.
Description
Technical Field
The invention belongs to the field of sewage and wastewater treatment, and particularly relates to a denitrifying bacteria flora with high salt tolerance, which aims to screen and culture a denitrifying bacteria flora (Halomonas, the first dominant functional bacterium) with good tolerance to a high salinity environment.
Background
In the denitrification treatment process of various sewage and wastewater, the heterotrophic organism denitrification reaction plays a crucial role, and the reaction can oxidize organic matters under the anoxic environmental condition, and reduce nitrate nitrogen as an electron acceptor into gaseous nitrogen which is harmless to human bodies and the environment.
With the rapid development of the industrialization process in China, the discharge amount of the industrial comprehensive wastewater is increasing day by day and is influenced by the water quality of the industrial comprehensive wastewater, and the industrial comprehensive wastewater has the characteristics of complex pollutant components, high pollution intensity, high salt content and the like, which undoubtedly brings huge challenges to the corresponding denitrification treatment. The prior denitrification process by an activated sludge method is still adopted by most sewage treatment plants, wherein the activated sludge belongs to a single sludge system and has complex composition, and the functional floras have obvious physiological differences, so that the operation conditions cannot meet the growth requirements of each type of floras, and the denitrification capability of the sludge is severely limited. In addition, the high salinity environment of the industrial wastewater is very unfavorable for the growth of the conventional denitrifying bacteria, and has obvious adverse effect on the biological denitrification. Along with new requirements of national relevant departments on the total nitrogen discharge standard of a sewage plant, how to realize high-efficiency denitrification treatment of high-salinity industrial wastewater so as to reduce the treatment difficulty of a terminal sewage plant in an industrial area is a problem to be solved urgently.
Aiming at the problems, the invention provides a denitrifying bacteria flora with high salt tolerance, which is obtained by directionally screening and culturing, wherein the first dominant functional bacterium is Halomonas, which is a denitrifying bacterium with high salt tolerance and high-efficiency denitrification capability. The method is characterized in that the screening of Halomonas with high occupancy ratio is used as a target guide, the return sludge of a secondary sedimentation tank of a sewage treatment plant is used as seed sludge, the rapid establishment and the stable maintenance of a culture environment with the salinity of 4 percent are realized by utilizing high-concentration substrate feeding and intermittent operation according to the salinity range suitable for growth, and environmental parameters such as temperature, pH and the like suitable for growth are set at the same time. The Halomonas can become the first dominant functional bacteria in the cultured bacteria group by means of the methods, and other denitrifying bacteria exist as an auxiliary, so that the denitrification capability of the bacteria group in the high-salinity industrial wastewater treatment can be ensured. Based on the premise, the screened and cultured denitrifying bacteria flora with high salt tolerance can be used for preparing embedded bioactive fillers, and can also be directly added into a reaction tank to carry out denitrification treatment on industrial wastewater with high salt content. Different from the traditional denitrifying flora, the technology has the advantages that the culture flora has high-efficiency denitrification and high salt tolerance, and the denitrification performance of the industrial wastewater in the high-salinity environment is improved.
Disclosure of Invention
The invention aims to develop a screening culture method based on denitrifying bacteria floras with high salt tolerance, which improves the tolerance and adaptability of denitrifying bacteria to high salinity environment and further realizes high-efficiency and stable denitrification performance in water quality treatment.
A method for screening and culturing denitrifying bacteria flora with high salt tolerance is characterized by comprising the following steps:
(1) preparing materials: the return sludge of the secondary sedimentation tank of the sewage treatment plant is taken as inoculation sludge, and potassium nitrate (KNO) is respectively taken as inoculation sludge3) Sodium acetate (CH)3COONa) and potassium dihydrogen phosphate (KH)2PO4) The nitrogen source, the carbon source and the phosphorus element required by denitrifying bacteria are provided, the carbon-nitrogen ratio (C/N) is controlled to be between 3.0 and 3.5, and the phosphorus element is added according to the proportion that the nitrogen-phosphorus ratio (N/P) is 100: 1. In addition, a trace element solution is also added into the sludge culture system, and 1mL of the trace element solution is correspondingly added into every 1L of raw water liquid medicine, wherein the composition and the concentration of the trace element solution are as follows: ZnSO4·7H2O is 0.12mg/L, Na2MoO4·2H2O is 0.12mg/L, CoCl2·6H2O is 0.15mg/L, FeCl3·6H2O is 1.50mg/L, CuSO4·5H2O is 0.03mg/L, NiCl2·6H2O is 0.12mg/L, Mn2SO4·H2O is 0.12 mg/L. For the increase of pH during the sludge cultivation, 0.23mol/L diluted sulfuric acid solution was used for adjustment.
(2) The culture mode is as follows: the rapid establishment and stable maintenance of the culture environment with the salinity value of 4 percent are realized by utilizing the fed-batch and intermittent operation of the high-concentration substrate, and the substrate preparation concentration and the drainage proportion can be determined according to the salinity value. One period of the intermittent operation can be divided into a reaction stage, a precipitation stage, a drainage stage and a water inlet stage, and the reaction stage comprises the following specific steps: the quantitative feeding of the high-concentration nitrates, sodium acetate and other substrates is carried out by adjusting the flow of the dosing metering pump, meanwhile, the reaction temperature is controlled to be 26 ℃, the pH value is controlled within the range of 8.00 +/-0.50, and the stirring speed is controlled to be 16 r/s; a precipitation stage: monitoring the water level change, and when the water level reaches a preset water level, closing stirring and standing for 2 hours; a drainage stage: according to the determined water discharge proportion, carrying out quantitative water discharge through a water discharge port of the reaction device; a water inlet stage: and (3) supplementing water to the reaction device, starting to enter a reaction stage after the temperature is raised to 26 ℃, and circulating in sequence. The nitrate nitrogen, the sub-state nitrogen and the COD of the effluent are detected in time in the culture process: the carbon-nitrogen ratio (C/N) is adjusted according to the surplus of COD, so that the growth of other heterotrophic bacteria caused by higher COD in the reactor is avoided; according to the accumulation condition of nitrate nitrogen and nitrite nitrogen, a proper water inlet load is selected, the inhibition effect of high Free Nitrite (FNA) on denitrifying bacteria caused by the accumulation of high-concentration nitrite nitrogen is avoided, and the sum of the concentrations of the nitrate nitrogen and the nitrite nitrogen in the effluent is ensured to be within a certain safety range.
(3) And (3) working condition adjustment: an adaptation stage of 7-10 days occurs in the initial culture period, in which the dissolved oxygen and the oxidation-reduction potential of the sludge environment are strictly controlled, wherein the dissolved oxygen is controlled below 0.5mg/L, the oxidation-reduction potential is controlled between-200 mv and-400 mv so as to ensure the oxygen-poor condition required by denitrifying bacteria and realize effective elimination of aerobic bacteria in inoculated sludge, and in addition, the carbon-nitrogen ratio (C/N) is adjusted to 3.5 so as to ensure that sufficient carbon sources are provided, and the water inlet load is kept at a relatively stable level; after the adaptation stage, denitrifying bacteria enter a rapid growth stage, and the denitrification rate is rapidly increased in the adaptation stage, so that effluent water is detected in time, the influent water load can be adjusted in time to avoid that sludge is in a low-load culture state for a long time, and the carbon-nitrogen ratio (C/N) of the adaptation stage is controlled to be 3.0-3.2 to avoid more residual COD. Along with the culture, the sludge concentration is continuously increased, denitrifying bacteria flora is successfully screened and cultured in a high-salt environment, and when the sludge concentration is increased to more than 13000mg/L, sludge is timely taken to keep a good growth effect of the sludge, so that the cost performance of the bacteria screening and culturing is improved.
The beneficial effects of the invention are mainly reflected in that:
1. the method for acclimating the activated sludge is used, the return sludge of the secondary sedimentation tank of the sewage plant is used as the inoculated sludge for screening and culturing, the characteristics of flora diversity are fully exerted, the screening and culturing of the denitrifying functional flora are realized through constantly enhanced acclimation conditions, namely the screened denitrifying bacteria maintain rich flora structures while the denitrification capability is enhanced, and the method has good adaptability to different water qualities.
2. The establishment and maintenance of a culture environment with a salinity value of 4% are realized by utilizing high-concentration substrate feeding and intermittent operation, along with the culture, the denitrifying bacteria flora also gradually completes the screening process under high salinity, and the screened bacteria flora simultaneously has high-efficiency denitrification capability and good salt tolerance and can be well applied to the denitrification treatment of high-salinity industrial wastewater.
3. According to the high-throughput sequencing technology, under the dual effects of high salinity and high-concentration substrates, the first dominant bacterium in the screened denitrifying bacteria flora is high-salt-tolerance Halomonas, the proportion of the first dominant bacterium can be rapidly improved in a short time, and the structure of the dominant bacterium flora still keeps good stability after mud is taken for many times.
4. The method realizes the stable increase of the sludge amount while screening the denitrifying bacteria, namely, the biomass of the functional bacteria is continuously improved while ensuring the high proportion of the denitrifying bacteria with high salt tolerance, so that the large-scale application of the denitrifying bacteria flora with high salt tolerance is feasible.
5. The Halomonas belonging to the first dominant genus has good tolerance to a high salinity environment and high-efficiency nitrite nitrogen reduction capability, namely accumulation of high-concentration nitrite nitrogen is not easy to occur in denitrification treatment of high-pollution-intensity industrial wastewater, inhibition of high Free Nitrite (FNA) on bacterial activity can be effectively avoided, and efficient and stable denitrification performance can be better realized.
Drawings
FIG. 1 is a diagram of a screening and culturing device for denitrifying bacteria flora with high salt tolerance.
1. Industrial grade bioreactors (fermenters); 2. a stirrer; 3. a dosing pipeline; 4. an acid adding pipeline; 5. a pH probe; 6. a temperature probe; 7. a PLC automatic control cabinet; 8. a dilute acid solution cylinder; 9. a dispensing water tank; 10. adding an acid metering pump; 11. a dosing metering pump.
Detailed Description
The present invention will be further described with reference to the following examples, but the embodiments of the present invention are not limited thereto.
Example 1
The embodiment performs industrial-grade screening culture, the device is shown in figure 1, the effect is obvious, and the specific contents are as follows:
(1) preparing materials: the experimental facility is an industrial-grade biological fermentation tank with the volume of 3000L, and the effective volume is controlled to be 2500L in the actual operation process. The returned sludge of a secondary sedimentation tank of a sewage treatment plant is used as inoculated sludge, and the sludge concentration MLSS is 3339 mg/L. The concentrations of nitrate nitrogen, COD and phosphorus were 13000mg/L, 39000-45500mg/L and 130mg/L, respectively. In addition, the supplemented trace element solution specifically comprises the following components: ZnSO4·7H2O is 0.12mg/L, Na2MoO4·2H2O is 0.12mg/L, CoCl2·6H2O is 0.15mg/L, FeCl3·6H2O is 1.50mg/L, CuSO4·5H2O is 0.03mg/L, NiCl2·6H2O is 0.12mg/L, Mn2SO4·H2O is 0.12mg/L, and 1mL of trace element solution is correspondingly added into every 1L of original water liquid medicine. The pH was adjusted using a 0.23mol/L dilute sulfuric acid solution.
(2) The culture mode comprises the following steps: the establishment and maintenance of a culture environment with the salinity value of 4% are realized by utilizing the fed-batch and intermittent operation of high-concentration substrates, and the drainage proportion is determined to be 80% by combining the preparation concentration of the substrates. One cycle of the batch operation can be divided into a reaction stage, a precipitation stage, a drainage stage and a water inlet stage, and specifically, the reaction stage comprises the following steps: the quantitative flow addition is carried out on the high-concentration nitrates, sodium acetate and other substrates by adjusting the flow of a dosing metering pump, the temperature and the pH are respectively kept at 26 ℃ and 8.00 +/-0.50 by automatically controlling a heating switch and an acid pump switch through a PLC (programmable logic controller) automatic control cabinet, and the stirring speed is controlled to be 16 r/s; a precipitation stage: adding medicine and acid to ensure that the volume of the internal mixed solution reaches 2500L, closing stirring, and standing and precipitating for 2 h; a drainage stage: according to the water drainage proportion, after fully standing, discharging supernatant liquid by using a water outlet of a fermentation tank until the liquid volume is 500L; a water inlet stage: adding water to the reaction kettle until the volume of the liquid is 1500L, so that the temperature probe and the pH probe can be immersed in the water, starting to enter a reaction stage after the temperature is raised to 26 ℃, and circulating in sequence. In the culture process, various indexes (nitrate nitrogen, nitrite nitrogen, COD and sludge concentration) are detected in time: controlling the concentration of COD in the effluent to be less than 100 mg/L; ensuring that the sum of the concentrations of nitrate nitrogen and nitrite nitrogen in effluent is less than 800 mg/L; when the total nitrogen of the effluent is less than 10mg/L, the operation load can be increased to the next gradient; when the sludge concentration is increased to more than 13000mg/L, sludge should be taken out in time.
(3) Through adjusting the flow that adds the medicine metering pump, promote the operating load, the operating load of setting is specifically as follows: 50,100,150,200,250,300,350,400,450,500,550 mg/L. The running time was set to 8h per day. The denitrification rate of denitrifying bacteria can be steadily increased, the maximum denitrification rate is 534.7 mg/(L.h), the specific denitrification rate is 41.7mg N/(g MLSS.h) at most, and the fastest sludge increasing effect, namely 696.5 mg/(L.d), can be achieved under the sludge concentration of 7515.3 mg/L. The result of high-throughput analysis shows that the denitrifying bacteria can realize effective screening and production after being cultured for 30d, the total proportion of functional bacteria is increased from the original 17.97% to 83.38%, wherein the proportion of Halomonas is increased from the first undetected state to 57.79%, and the denitrifying bacteria are always the first dominant functional bacteria. The flora structure in the sludge still keeps higher stability after the sludge is taken for many times. In an industrial bioreactor, a denitrifying bacteria flora (Halomonas, the first dominant genus) with high salt tolerance realizes successful screening culture, and can be well applied to denitrification treatment of high-salinity industrial wastewater.
Claims (2)
1. A method for screening and culturing denitrifying bacteria flora with high salt tolerance is characterized by comprising the following steps:
(1) preparing materials: taking return sludge of a secondary sedimentation tank of a sewage treatment plant as inoculation sludge, and respectively taking potassium nitrate (KNO)3) Sodium acetate (CH)3COONa) and potassium dihydrogen phosphate (KH)2PO4) The nitrogen source, the carbon source and the phosphorus element required by denitrifying bacteria are provided, the carbon-nitrogen ratio (C/N) is controlled to be between 3.0 and 3.5, and the phosphorus element is added according to the proportion that the nitrogen-phosphorus ratio (N/P) is 100: 1. In addition, a trace element solution is also added into the sludge culture system, and 1mL of the trace element solution is correspondingly added into every 1L of raw water liquid medicine, wherein the composition and the concentration of the trace element solution are as follows: ZnSO4·7H2O is 0.12mg/L, Na2MoO4·2H2O is 0.12mg/L, CoCl2·6H2O is 0.15mg/L, FeCl3·6H2O is 1.50mg/L, CuSO4·5H2O is 0.03mg/L, NiCl2·6H2O is 0.12mg/L, Mn2SO4·H2O is 0.12 mg/L. For the increase of pH in the sludge culture process, 0.23mol/L diluted sulfuric acid solution is used for adjustment;
(2) the culture mode is as follows: the rapid establishment and stable maintenance of the culture environment with the salinity value of 4 percent are realized by utilizing the fed-batch and intermittent operation of the high-concentration substrate, and the substrate preparation concentration and the drainage proportion can be determined according to the salinity value. One cycle of the batch operation can be divided into a reaction stage, a precipitation stage, a drainage stage and a water inlet stage, and specifically, the reaction stage comprises the following steps: the method comprises the steps of carrying out quantitative flow addition on high-concentration nitrates, sodium acetate and other substrates by adjusting the flow of a dosing metering pump, controlling the reaction temperature to be 26 ℃, controlling the pH value to be within the range of 8.00 +/-0.50, and controlling the stirring speed to be 16 r/s; a precipitation stage: monitoring the water level change, and when the water level reaches a preset water level, closing stirring and standing for 2 hours; a drainage stage: according to the determined water discharge proportion, carrying out quantitative water discharge through a water discharge port of the reaction device; a water inlet stage: and (3) supplementing water to the reaction device, starting to enter a reaction stage after the temperature is raised to 26 ℃, and circulating in sequence. The nitrate nitrogen, the metastable nitrogen and the COD of the effluent are detected in time in the culture process: the carbon-nitrogen ratio (C/N) is adjusted according to the surplus of COD, so that the growth of other heterotrophic bacteria caused by higher COD in the reactor is avoided; according to the accumulation condition of nitrate nitrogen and nitrite nitrogen, selecting proper water inlet load, avoiding the inhibition effect of high Free Nitrite (FNA) on denitrifying bacteria caused by the accumulation of high-concentration nitrite nitrogen, and ensuring the sum of the concentrations of nitrate nitrogen and nitrite nitrogen in the effluent to be within a certain safety range;
(3) and (3) working condition adjustment: an adaptation stage of 7-10 days occurs at the initial stage of culture, in which the dissolved oxygen and the oxidation-reduction potential of the environment where the sludge is located need to be strictly controlled, wherein the dissolved oxygen is controlled below 0.5mg/L, the oxidation-reduction potential is controlled between-200 mv and-400 mv, so as to ensure the oxygen-poor condition required by denitrifying bacteria and realize effective elimination of aerobic bacteria in inoculated sludge, and in addition, the carbon-nitrogen ratio (C/N) is adjusted to 3.5 so as to ensure that sufficient carbon sources are provided, and the water inlet load is kept at a relatively stable level; after the adaptation stage, denitrifying bacteria enter a rapid growth stage, and the denitrification rate is rapidly improved in the stage, so that effluent water is detected in time, the water inflow load can be adjusted in time to avoid that the sludge is in a low-load culture state for a long time, and in addition, the carbon-nitrogen ratio (C/N) of the stage is controlled to be between 3.0 and 3.2 to avoid more residual COD; along with the culture, the sludge concentration is continuously increased, denitrifying bacteria flora is successfully screened and cultured in a high-salt environment, and when the sludge concentration is increased to more than 13000mg/L, sludge is timely taken to keep a good growth effect of the sludge, so that the cost performance of the bacteria screening and culturing is improved.
2. The method according to claim 1, characterized in that the first dominant functional genus of the denitrifying bacteria flora is Halomonas Halomonas.
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