CN108587951B - Preparation method and application of salt-tolerant and cold-tolerant ammonia oxidizing bacteria biofilm-forming charcoal balls - Google Patents
Preparation method and application of salt-tolerant and cold-tolerant ammonia oxidizing bacteria biofilm-forming charcoal balls Download PDFInfo
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Abstract
The invention relates to a preparation method and application of a salt-tolerant and cold-tolerant ammonia oxidizing bacteria biofilm-forming charcoal ball. The method is characterized in that firstly, high-efficiency ammonia oxidizing bacteria are screened out, and then salt-tolerant and cold-tolerant domestication is carried out on the ammonia oxidizing bacteria to obtain salt-tolerant and cold-tolerant ammonia oxidizing bacteria; and secondly, preparing charcoal balls, and placing the prepared charcoal balls in the enriched liquid of the salt-tolerant and cold-tolerant ammonia oxidizing bacteria to obtain the salt-tolerant and cold-tolerant ammonia oxidizing bacteria biofilm-hanging charcoal balls. The salt-tolerant and cold-resistant biofilm-resistant biochar sphere is applied to purification of ammonia nitrogen polluted water under a low-temperature condition. The invention has the advantages that the salt-tolerant and cold-tolerant ammonia oxidizing bacteria biofilm-forming charcoal balls can have a good removing effect on ammonia nitrogen polluted water bodies in high-salt and low-temperature environments, and are particularly applied to flowing natural rivers, so that the microbial inoculum cannot be dispersed along with the flowing of the water bodies, and the sufficient retention time and the sufficient microbial concentration are ensured. Under the conditions that the salinity is 15 per mill and the temperature is 15 ℃, the ammonia nitrogen removal efficiency is still kept above 90 percent.
Description
The application is a divisional application of 2015100864643 'a salt-tolerant and cold-tolerant immobilization method and application', and the application date is 2015, 2 months and 17 days.
Technical Field
The invention belongs to a water pollution treatment technology, and particularly relates to a preparation method and application of a salt-tolerant and cold-tolerant ammonia oxidizing bacteria biofilm-hanging charcoal ball, which are a domestication and screening method and an immobilization method and application of salt-tolerant and cold-tolerant ammonia oxidizing bacteria.
Background
The excessive ammonia nitrogen is one of the main factors of water eutrophication and is the main index of water pollution control. At present, the method for removing ammonia nitrogen at home and abroad mainly comprises a physical method, a chemical method and a biological method. The physical method can only realize the displacement of the pollutants in the space and cannot realize the complete removal; the chemical method is quick and effective, but due to the introduction of chemical agents, secondary pollution can be caused to the water body when the treatment is improper; the biological method converts ammonia nitrogen into nitrogen by utilizing the metabolism of microorganisms, realizes the removal of pollutants, and simultaneously does not cause secondary pollution to the environment and is widely accepted. The prior patent of the method (ZL102001721A) for treating the ammonia nitrogen wastewater is to irradiate the mixed solution of the ammonia nitrogen wastewater and activated carbon in a microwave field to remove the ammonia nitrogen, although the removal efficiency is high, only the existing form of the ammonia nitrogen is changed, the ammonia nitrogen is not completely removed, and the method is not suitable for natural surface water bodies.
Research shows that the degradation efficiency is reduced due to the influence of environmental conditions such as salinity and temperature on the degradation of ammonia nitrogen in a water body by using microorganisms. The salinity of the estuary wetland water body is between that of fresh water and that of seawater, is higher than 10 per thousand, and is considered as a high salinity water body. Too high salinity in the water body can destroy the osmotic pressure of microbial cells and destroy the enzyme system of microbes. Meanwhile, the low-temperature environment can influence the activity and the reproduction rate of microbial enzymes, and inhibit the removal of ammonia nitrogen. At present, the best growth temperature of ammonia oxidizing bacteria is reported to be 30-35 ℃, and for northern cold regions, especially for natural wetlands with wide areas such as Liaohe wetlands, the low-temperature condition is not favorable for ammonia oxidizing bacteria to degrade ammonia nitrogen. The existing patent (ZL102268386A) screens out a strain of ammonia oxidizing bacteria with good degradation effect, the ammonia oxidizing efficiency of the strain can reach 90% under the laboratory culture condition of the temperature of 30 ℃, and obviously, the optimum temperature of the ammonia oxidizing bacteria screened out by the method is high, and the good removal effect cannot be generated in a low-temperature environment; even if there are low temperature resistant bacteria, they are often unable to adapt to high salinity environment.
In addition, the addition of the high-efficiency microbial inoculum in natural water is also a very important problem. The general high-efficiency microbial inoculum obtained by culture and acclimation is added into a reactor with a fixed volume, although a good degradation effect can be obtained by changing the conditions in the reactor, when the high-efficiency microbial inoculum is applied to a natural river with a field flow state, the microbial inoculum is dispersed in a water body along with the flow of the water body, and the sufficient retention time and the sufficient microbial concentration are difficult to ensure, so that the good removal effect cannot be realized. In addition, the Liaohe wetland in northeast is an important oil exploitation area, and oil leakage caused by oil exploitation, transportation and storage has toxic action on microorganisms, so that the degradation of ammonia nitrogen is greatly restricted. Obviously, the domestication and screening of the salt-tolerant and cold-tolerant ammonia oxidizing bacteria and the immobilization method thereof are needed for emergency preparation of the physical environment.
Disclosure of Invention
The invention aims to provide a preparation method and application of a salt-tolerant and cold-tolerant ammonia oxidizing bacteria biofilm-forming charcoal ball to overcome the problems in the prior art.
The invention firstly considers that the Liaozuo wetland in northern China is in a low-temperature state all the year round, and meanwhile, the estuary wetland ecosystem is a complex ecosystem with salt-fresh water intersection, high salinity and sensitive ecological environment. However, the existing traditional physical, chemical and biological methods can not achieve good repairing effect, and the treatment cost is high. Therefore, the salt-tolerant and cold-tolerant ammonia oxidizing bacteria screened and domesticated from the sediments of the wetland are specially used for treating ammonia nitrogen in the Liaohe water body, the treatment effect is good, the cost is low, the local conditions are met, the original ecological environment is not threatened, and the local resources can be fully utilized to reduce the cost. And considering that in a natural river with dynamic flow, in order to keep enough residence time and enough microorganism concentration of degradation bacteria in the water body to realize efficient removal effect, the important problem of adding in the natural water body on site is solved. The denitrifier immobilization technology taking the biochar pellets as carriers is provided for the first time, can ensure that microorganisms are not dispersed by water flow and microorganism biomass in unit volume, has a protection effect on salt-tolerant and cold-tolerant ammonia oxidizing bacteria, and realizes efficient removal of ammonia nitrogen.
Therefore, the invention firstly screens out high-efficiency ammonia oxidizing bacteria, then carries out salt-tolerant and cold-tolerant acclimation on the ammonia oxidizing bacteria to obtain salt-tolerant and cold-tolerant ammonia oxidizing bacteria, then places the prepared charcoal balls in the enrichment liquid of the salt-tolerant and cold-tolerant ammonia oxidizing bacteria to obtain biofilm-forming charcoal balls, then places the biofilm-forming charcoal balls in the embedding mixed liquid with the salt-tolerant and cold-tolerant ammonia oxidizing bacteria, takes out the biofilm-forming charcoal balls and places the biofilm-forming charcoal balls in the embedding mixed liquid into a saturated boric acid solution one by one, finally places the charcoal balls in the mixed liquid into the saturated boric acid solution for crosslinking for 12 hours at 4 ℃ in a refrigerator, and cleans the immobilized charcoal balls of the salt-tolerant and cold-tolerant ammonia oxidizing bacteria by using sterile water after the crosslinking is completed to obtain the immobilized charcoal balls of the salt-tolerant and cold-tolerant ammonia oxidizing bacteria.
The method for screening the high-efficiency ammonia oxidizing bacteria comprises the following steps:
(1) enrichment culture: weighing 10g-20g of sediment by dry weight, adding the sediment into a 250mL conical flask containing 100mL of enrichment medium, performing shake culture at the temperature of 28-30 ℃ for several days at the speed of 150-200 r/min, absorbing 10mL of the sediment into another enrichment medium, performing shake culture for several days under the conditions, repeating the steps for 3 times to improve the concentration of ammonia oxidizing bacteria, and finally obtaining ammonia oxidizing bacteria enrichment liquid; the enrichment medium comprises: (NH)4)2SO4 5.5g，CaCO3 5.5g，NaCl 2.0g，MgSO4 0.5g，K2HPO4 1.5g，MnSO4 0.01g，FeSO40.02g, 1000mL of distilled water, 5% Na2CO3Adjusting pH to 8.0, and sterilizing at 121 deg.C for 30 min.
(2) And (3) purification and separation: the ammonia oxidizing bacteria enrichment solution is added according to the proportion of 10-1-10-7Diluting in gradient, spreading on solid culture medium, culturing at 28-30 deg.c for several days, selecting different colony forms, streaking repeatedly on other solid culture medium until the colony forms different single bacteria, and separating to obtain pure ammonia oxidizing bacteria.
The solid culture medium: NH (NH)4SO4 2g，NaH2PO4 0.25g，MgSO4·7H2O 0.03g，MnSO4·4H2O 0.01g，K2HPO4 0.75g，NaCl 0.01g，CaCO35g, agar 20g, distilled water 1000mL, 5% Na2CO3Adjusting pH to 7.2, and sterilizing at 121 deg.C for 30 min.
The salt-tolerant and cold-tolerant domestication method of the ammonia oxidizing bacteria comprises the following steps:
(1) salt tolerance domestication: selecting a plurality of pure ammonia oxidizing bacteria obtained by separation into an enrichment medium, and performing shake culture for a plurality of days at the temperature of 28-30 ℃ at the speed of 150-200 r/min to respectively obtain single strain enrichment liquid. When the single strain enrichment solution enters a logarithmic growth period, sequentially inoculating 5% of the inoculation amount of each single strain enrichment solution and an enrichment culture medium into another 100mL of enrichment culture medium with the salinity of 5 per thousand, 10 per thousand, 15 per thousand and 20 per thousand, performing shake culture for several days under the conditions of 150r/min-200r/min and 28-30 ℃, and screening out ammonia oxidizing bacteria with good ammonia nitrogen degradation effect, namely salt-tolerant ammonia oxidizing bacteria;
(2) cold tolerance domestication: inoculating the screened salt-tolerant ammonia oxidizing bacteria into 100mL of enrichment medium with the salinity of 20 per thousand according to the inoculation amount of 5% of the enrichment medium and the enrichment medium, and carrying out shake culture for several days under the conditions of 25 ℃, 20 ℃, 15 ℃ and 10 ℃ at 150-180 r/min in sequence, thereby screening the ammonia oxidizing bacteria with good ammonia nitrogen degradation effect, namely the salt-tolerant cold-tolerant ammonia oxidizing bacteria.
The membrane hanging method of the charcoal spheres comprises the following steps: and (3) soaking the charcoal balls in the salt-tolerant and cold-tolerant ammonia oxidizing bacteria enriched liquid for 4-6 days to obtain the biofilm-forming charcoal balls.
The salt-tolerant and cold-tolerant ammonia oxidizing bacteria immobilized biochar ball comprises the following steps: mixing the biofilm-forming biochar balls with an embedding mixed solution, then putting the mixture into a saturated boric acid solution one by one, placing the mixture into a refrigerator for crosslinking for 12 hours at 4 ℃, and cleaning the mixture with sterile water after the crosslinking is finished to obtain the salt-resistant and cold-resistant ammonia oxidizing bacteria immobilized biochar balls, wherein the embedding mixed solution is a 10% polyvinyl alcohol aqueous solution and 20% wet bacteria by volume (10-15): 1 in a ratio of 1.
The wet thallus is obtained by centrifuging 50ml-100ml of microorganism enrichment liquid for 10-15min at 8000r/min of 5000-.
The salt-tolerant and cold-tolerant ammonia oxidizing bacteria are Ochrobactrum sp and Aquamibium sp which are domesticated.
The preparation method of the charcoal balls comprises the following steps:
(1) taking straws, cleaning, drying to constant weight, crushing and sieving the straws to obtain straw powder, and burning the straw powder into biochar through a muffle furnace;
(2) washing the fired biochar with deionized water for a plurality of times, using 0.1mol/L nitric acid as a modifier, fully and uniformly mixing the modifier and the biochar under the magnetic stirring condition of 200r/min, modifying for one day, and then washing with deionized water until the pH value is neutral to obtain chemically modified biochar; placing the chemically modified biochar in a microwave cleaning instrument with the frequency of 40Hz, performing microwave oscillation for 40-90 min, filtering, and drying at constant temperature to obtain the physicochemical modified biochar;
(3) drying the clay in a constant-temperature drying oven, and sieving with a 100-200 mesh sieve to obtain clay powder;
(4) the physicochemical modified charcoal and clay powder are mixed according to the mass ratio of (0.5-1): 10, adding the mixture into a mixed solution of sodium silicate and sodium bicarbonate dissolved by deionized water, preparing the mixture into spherical particles by a ball making machine, drying the spherical particles in a constant-temperature drying box, and finally firing the spherical particles in a muffle furnace to prepare the biochar balls, wherein the mass ratio of the sodium silicate to the clay is 3 percent, and the mass ratio of the sodium bicarbonate to the clay is 1.5 percent.
The firing conditions of the muffle furnace are as follows: the temperature rising speed is 10 ℃/min, the temperature is 300-400 ℃, the heat preservation time is 180min, and the drying is carried out for 8-12 h under the conditions of constant temperature and drying conditions of 60-80 ℃.
The salt-tolerant ammonia oxidizing bacteria are applied to purification of ammonia nitrogen polluted water under high-salt conditions, and the salt-tolerant cold-tolerant ammonia oxidizing bacteria are applied to purification of ammonia nitrogen polluted water under low-temperature high-salt conditions.
The biofilm-forming biochar sphere is applied to purification of ammonia nitrogen polluted water under low-temperature and high-salt conditions, and the salt-tolerant and cold-tolerant ammonia oxidizing bacteria immobilized biochar sphere is applied to purification of ammonia nitrogen polluted water under low-temperature and high-salt conditions.
Detailed Description
The present invention will be described in further detail by way of examples.
Example 1
Sampling a region with serious pollution of Liaoriver in northeast, controlling the salinity to be 10 per mill by detecting the ammonia nitrogen concentration to be 3.4mg/L and adopting a salt-tolerant and cold-tolerant ammonia oxidizing bacteria immobilization method taking a biochar sphere as a carrier to carry out static treatment on the ammonia nitrogen in a water body under the condition that the temperature is 10 ℃, and the method comprises the following steps:
firstly, screening out efficient ammonia oxidizing bacteria: weighing 20g of sediment by dry weight, adding the sediment into a 250mL conical flask containing 100mL of enrichment medium, performing shake culture at 30 ℃ for 7 days at 200r/min, then sucking 10mL of the sediment into another enrichment medium, performing shake culture for 7 days under the conditions, repeating the steps for 3 times to improve the concentration of ammonia oxidizing bacteria, and finally obtaining an ammonia oxidizing bacteria enrichment solution; in order to ensure that the screened bacteria are ammonia oxidizing bacteria, a certain amount of the ammonia oxidizing bacteria enriched liquid is taken to be mixed with a certain amount of Griess reagent on a white porcelain plate, and the detection is finished if a mauve complex is generated; in order to obtain single bacterial colony, the ammonia oxidizing bacteria enriched liquid is added according to the proportion of 10-1-10-7Diluting in gradient, spreading on solid culture medium, culturing at 30 deg.C for 3 days, selecting different forms of colony, repeatedly streaking on another solid culture medium until the colony is single form bacterium, and obtaining pure ammonia oxidizing bacterium.
Then, domesticating salt tolerance and cold tolerance of ammonia oxidizing bacteria: and (3) selecting the pure ammonia oxidizing bacteria into an enrichment culture medium, and performing shake culture for 7 days at the temperature of 30 ℃ at 200r/min to obtain single strain enrichment liquid. When the strains enter a logarithmic growth period (the absorbance OD600 of the single-strain enrichment solution is approximately equal to 0.6), sequentially inoculating the strains into another 100mL enrichment medium with salinity of 5 per mill, 10 per mill, 15 per mill and 20 per mill, and carrying out shake culture for 7 days under the conditions to screen out salt-tolerant ammonia oxidizing bacteria; inoculating the screened salt-tolerant ammonia oxidizing bacteria into 100mL of enrichment medium with the salinity of 20 per mill in an inoculation amount of 5%, carrying out shake culture for 6 days under the conditions of 25 ℃, 20 ℃, 15 ℃ and 10 ℃ at 150r/min in sequence, screening the salt-tolerant cold-tolerant ammonia oxidizing bacteria, and identifying the salt-tolerant cold-tolerant ammonia oxidizing bacteria as Ochrobactrum.
Secondly, preparing a charcoal ball and hanging a membrane on the charcoal ball: weighing 200g of reed straw, washing with deionized water, drying to constant weight, crushing the reed straw by using a plant straw crusher, sieving with a 20-mesh sieve to obtain reed straw powder, and burning the reed straw powder into biochar by using a muffle furnace. The firing conditions of the muffle furnace are as follows: the temperature rising speed is 10 ℃/min, the temperature is 300 ℃ and the heat preservation time is 180 min; in order to increase the specific surface area of the biochar, namely to increase the porosity of the biochar and remove ash in the biochar, the biochar is subjected to surface modification by adopting a physicochemical combined means: adding 0.1mol/L nitric acid solution into the baked 5g of biochar, fully and uniformly mixing, stirring for 24 hours under the condition of room temperature of 200r/min in a magnetic stirrer, and washing the biochar to be neutral by deionized water, so that the biochar is chemically modified. Placing the chemically modified biochar in a microwave cleaning machine, and vibrating for 40min by microwaves with the frequency of 40Hz to further remove ash and increase the porosity of the biochar. Filtering the biochar subjected to physicochemical modification, drying in a drying box at the constant temperature of 60 ℃ for 8h, drying to prepare physicochemical biochar, and storing in a dark place; drying 100g of Liaohe mouth wetland clay in a constant-temperature drying oven at 60 ℃ for 12h, sieving with a 100-mesh sieve to obtain clay powder, and placing the clay powder in a brown bottle for later use; dissolving sodium silicate and sodium bicarbonate respectively by using deionized water according to the mass ratio of clay of 3% and 1.5% and uniformly mixing, drying the materialized modified biochar, mixing the biochar with clay powder according to the proportion of 1:10, adding the mixture into the mixed solution of the sodium silicate and the sodium bicarbonate, preparing a spherical particle with the diameter of 0.8cm by using a ball making machine, and drying the spherical particle in a constant-temperature drying box at 80 ℃ for 8 hours to obtain dried carbon spherical particles; and (3) placing the prepared charcoal balls in a muffle furnace to be fired under the conditions of a heating rate of 10 ℃/min, a temperature of 300 ℃ and a heat preservation time of 180 min. And (3) soaking the charcoal balls in the salt-tolerant and cold-tolerant ammonia oxidizing bacteria enriched liquid for 4 days to obtain the biofilm-forming charcoal balls.
Finally, 200g of charcoal balls are placed in 400mL of salt-resistant and cold-resistant ammonia oxidizing bacteria to be soaked for 6 days, and membrane-hanging charcoal balls are obtained; centrifuging 100ml of the salt-tolerant and cold-tolerant ammonia oxidizing bacteria at 8000r/min for 15min, removing supernatant, and washing with sterile water for 4 times to obtain wet bacteria; by volume 15: 1, weighing 10 percent of polyvinyl alcohol aqueous solution and 20 percent of the wet thalli according to the proportion, and mixing to obtain an embedding mixed solution; and mixing the biofilm-forming biochar balls with the embedding mixed solution, then putting the mixture into a saturated boric acid solution one by one, placing the mixture in a refrigerator for crosslinking for 12 hours at 4 ℃, and cleaning the mixture with sterile water after the crosslinking is finished to obtain the salt-resistant and cold-resistant ammonia oxidizing bacteria immobilized biochar balls.
Static test: polluted river water is taken to be placed in a reactor, the cross-linked immobilized biochar spheres are spread in the reactor, a static removal experiment is carried out in the environment with the temperature of 10 ℃, and the ammonia nitrogen concentration is measured once every 5 days.
The results of the measurements are given in the following table:
example 2
Sampling the surface water body of the wetland, controlling the salinity to be 15 per mill by detecting the ammonia nitrogen concentration to be 1.6mg/L and adopting a salt-tolerant and cold-tolerant ammonia oxidizing bacteria immobilization method taking a biochar sphere as a carrier to dynamically treat the ammonia nitrogen in the water body at the temperature of 15 ℃, and the method comprises the following steps:
firstly, screening out efficient ammonia oxidizing bacteria: : weighing 10g of sediment by dry weight, adding the sediment into a 250mL conical flask containing 100mL of enrichment medium, performing shake culture at 28 ℃ for 7 days at 150r/min, then sucking 10mL of the sediment into another enrichment medium, performing shake culture for 7 days under the conditions, repeating the steps for 3 times to improve the concentration of ammonia oxidizing bacteria, and finally obtaining an ammonia oxidizing bacteria enrichment solution; in order to ensure that the screened bacteria are ammonia oxidizing bacteria, a certain amount of the ammonia oxidizing bacteria enriched liquid is taken to be mixed with a certain amount of Griess reagent on a white porcelain plate, and the detection is finished if a mauve complex is generated; in order to obtain single bacterial colony, the ammonia oxidizing bacteria enriched liquid is added according to the proportion of 10-1-10-7Diluting in gradient, spreading on solid culture medium,culturing at 30 deg.C for 3 days, after the colony grows, selecting colony of different forms, repeatedly streaking on another solid culture medium until the generated colony is single bacterium of single form, and obtaining pure ammonia oxidizing bacteria.
Then, domesticating salt tolerance and cold tolerance of ammonia oxidizing bacteria: and (3) selecting the pure ammonia oxidizing bacteria into an enrichment culture medium, and performing shake culture for 7 days at the temperature of 28 ℃ at the speed of 150r/min to obtain single strain enrichment liquid. When the strains enter a logarithmic growth period (the absorbance OD600 of the single-strain enrichment solution is approximately equal to 0.6), sequentially inoculating the strains in another 100mL of enrichment medium with salinity of 5 per mill, 10 per mill, 15 per mill and 20 per mill, performing shake culture for 7 days under the conditions, and screening out salt-tolerant ammonia oxidizing bacteria; inoculating the screened salt-tolerant ammonia-oxidizing bacteria into 100mL of enrichment medium with 20 per mill of salinity by 5 percent of inoculation amount, sequentially carrying out shake culture for 5 days at 25 ℃, 20 ℃, 15 ℃ and 10 ℃ at 180r/min, screening the salt-tolerant cold-tolerant ammonia-oxidizing bacteria, and identifying the salt-tolerant cold-tolerant ammonia-oxidizing bacteria as Aquamibium.
Secondly, preparing a charcoal ball and hanging a membrane on the charcoal ball: weighing 100g of reed straw, washing with deionized water, drying to constant weight, crushing the reed straw by using a plant straw crusher, sieving with a 20-mesh sieve to obtain reed straw powder, and burning by using a muffle furnace to obtain biochar. The firing conditions of the muffle furnace are as follows: the temperature rising speed is 10 ℃/min, the temperature is 400 ℃ and the heat preservation time is 180 min; in order to increase the specific surface area of the biochar, namely to increase the porosity of the biochar and remove ash in the biochar, the biochar is subjected to surface modification by adopting a physicochemical combined means: adding 0.1mol/L nitric acid solution into the baked 10g of biochar, fully and uniformly mixing, stirring for 24 hours under the condition of room temperature of 200r/min in a magnetic stirrer, and washing the biochar to be neutral by deionized water, so that the biochar is chemically modified. Placing the chemically modified biochar in a microwave cleaning machine, and oscillating for 60min by microwaves with the frequency of 40Hz to further remove ash and increase the porosity of the biochar. Filtering the biochar subjected to physicochemical modification, drying the biochar in a drying box at the temperature of 80 ℃ for 8 hours at constant temperature, drying the biochar to prepare physicochemical biochar, and storing the physicochemical biochar in a dark place; based on the consideration of protection and economy of the original ecological environment of the Liaohe estuary wetland, local clay of the Liaohe river is adopted as a raw material. Drying 110g of Liaohe mouth wetland clay in a constant-temperature drying oven at 80 ℃ for 8h, sieving with a 100-mesh sieve to obtain clay powder, and placing the clay powder in a brown bottle for later use; dissolving sodium silicate and sodium bicarbonate by using deionized water according to the mass ratio of clay of 3% and 1.5% respectively, uniformly mixing, drying the materialized modified biochar, mixing with clay powder according to the proportion of 0.5:10, adding the mixture into the mixed solution of the sodium silicate and the sodium bicarbonate, preparing into spherulites with the diameter of 1.0cm by using a ball making machine, and drying in a constant-temperature drying box at 80 ℃ for 8 hours to obtain dried carbon sphere particles; and (3) placing the prepared charcoal balls in a muffle furnace to be fired under the conditions of a heating rate of 10 ℃/min, a temperature of 400 ℃ and a heat preservation time of 180 min. And (3) soaking the charcoal balls in the salt-tolerant and cold-tolerant ammonia oxidizing bacteria enriched liquid for 4 days to obtain the biofilm-forming charcoal balls.
Finally, 50g of charcoal balls are placed in 100mL of salt-resistant and cold-resistant ammonia oxidizing bacteria to be soaked for 4 days, and membrane-hanging charcoal balls are obtained; centrifuging 100ml of the salt-tolerant and cold-tolerant ammonia oxidizing bacteria at 8000r/min for 15min, removing supernatant, and cleaning with sterile water for 3 times to obtain wet bacteria; by volume 10: 1, weighing 10 percent of polyvinyl alcohol aqueous solution and 20 percent of the wet thalli according to the proportion, and mixing to obtain an embedding mixed solution; and mixing the biofilm-forming biochar balls with the embedding mixed solution, then putting the mixture into a saturated boric acid solution one by one, placing the mixture in a refrigerator for crosslinking for 12 hours at 4 ℃, and cleaning the mixture with sterile water after the crosslinking is finished to obtain the salt-resistant and cold-resistant ammonia oxidizing bacteria immobilized biochar balls.
Static test: and (3) putting the wetland water body into a reactor, paving 50g of the cross-linked immobilized pellets into the reactor, performing a dynamic removal experiment at the temperature of 15 ℃, and measuring the ammonia nitrogen concentration once every 5 days, wherein the ammonia nitrogen removal rate is about 78%.
Claims (1)
1. The application of the salt-tolerant and cold-tolerant ammonia oxidizing bacteria biofilm formation charcoal balls is characterized in that the biofilm formation charcoal balls are applied to purification of ammonia nitrogen polluted water under low-temperature and high-salt conditions;
the salt-tolerant and cold-tolerant ammonia oxidizing bacteria biofilm-forming charcoal ball is prepared by the following method:
firstly, screening out efficient ammonia oxidizing bacteria, then performing salt-tolerant and cold-tolerant domestication on the ammonia oxidizing bacteria to obtain salt-tolerant and cold-tolerant ammonia oxidizing bacteria, then preparing charcoal balls, and placing the prepared charcoal balls in a concentrated solution of the salt-tolerant and cold-tolerant ammonia oxidizing bacteria to obtain salt-tolerant and cold-tolerant ammonia oxidizing bacteria biofilm-hanging charcoal balls; placing the biofilm-forming biochar balls in a cold-resistant ammonia oxidizing bacteria embedding mixed solution, taking out the biofilm-forming biochar balls, placing the biofilm-forming biochar balls into a saturated boric acid solution one by one, crosslinking for 12 hours at 4 ℃, and cleaning with sterile water after the crosslinking is finished; the embedding mixture is 10% polyvinyl alcohol aqueous solution and 20% wet bacteria by volume (10-15): 1 in a certain proportion;
the wet thallus is obtained by centrifuging 50ml-100ml of microorganism enrichment liquid for 10-15min at 8000r/min of 5000-
Washing the solution with sterile water for multiple times to obtain the final product;
the method for screening the high-efficiency ammonia oxidizing bacteria comprises the following steps:
(1) enrichment culture: weighing 10g-20g of sediment by dry weight, adding the sediment into a 250mL conical flask containing 100mL of enrichment medium, performing shake culture at the temperature of 28-30 ℃ for several days at the speed of 150-200 r/min, absorbing 10mL of the sediment into another enrichment medium, performing shake culture for several days under the conditions, repeating the steps for 3 times to improve the concentration of ammonia oxidizing bacteria, and finally obtaining ammonia oxidizing bacteria enrichment liquid; (ii) a In order to ensure that the screened bacteria are ammonia oxidizing bacteria, a certain amount of the ammonia oxidizing bacteria enriched liquid is taken to be mixed with a certain amount of Griess reagent on a white porcelain plate, and the detection is finished if a mauve complex is generated; the enrichment medium comprises: (NH4)2 so45.5g, caco 35.5g, NaCl 2.0g, mgso40.5g, k2hpo41.5g, mnso40.01g, feso40.02g, 1000mL of distilled water, 5% Na2CO3 adjusted pH =8.0, sterilized at 121 ℃ for 30 min;
(2) and (3) purification and separation: the ammonia oxidizing bacteria enrichment solution is added according to the proportion of 10-1-10-7Diluting in gradient, spreading on solid culture medium, culturing at 28-30 deg.C for several days, selecting colonies of different forms, repeatedly streaking on another solid culture medium until the colonies are single bacteria of different forms, and separating to obtain multiple pure ammonia oxidation strainsBacteria, the culture medium is: NH4SO 42 g, NaH2 PO40.25g, MgSO4 & 7H2O 0.03.03 g, MnSO4 & 4H2O 0.01.01 g, K2HPO40.75g, NaCl 0.01g, CaCO 35 g, agar 20g, distilled water 1000mL, 5% Na2CO3 with pH =7.2 adjusted, and sterilizing at 121 ℃ for 30 min;
the salt tolerance and cold tolerance domestication method of the ammonia oxidizing bacteria comprises the following steps:
(1) salt tolerance domestication: selecting a plurality of pure ammonia oxidizing bacteria obtained by separation into an enrichment medium, and performing shake culture at the temperature of 28-30 ℃ for several days at the speed of 150-200 r/min to respectively obtain single strain enrichment liquid; when the single strain enrichment solution enters a logarithmic growth period, sequentially inoculating 5% of the inoculation amount of each single strain enrichment solution and an enrichment culture medium into another 100mL of enrichment culture medium with the salinity of 5 per mill, 10 per mill, 15 per mill and 20 per mill, performing shake culture for several days under the conditions of 150r/min-200r/min and 28-30 ℃, and screening out ammonia oxidizing bacteria with good ammonia nitrogen degradation effect, namely salt-tolerant ammonia oxidizing bacteria;
(2) cold tolerance domestication: inoculating the screened salt-tolerant ammonia oxidizing bacteria into 100mL of enrichment medium with the salinity of 20 per thousand according to the inoculation amount of 5% of the enrichment medium and the enrichment medium, and carrying out shake culture for several days under the conditions of 25 ℃, 20 ℃, 15 ℃, 10 ℃ and 150-180 r/min in sequence to screen out ammonia oxidizing bacteria with good ammonia nitrogen degradation effect, namely salt-tolerant cold-tolerant ammonia oxidizing bacteria;
the preparation method of the charcoal balls comprises the following steps:
(1) cleaning straws, drying to constant weight, crushing the straws, sieving to obtain straw powder, and burning the straw powder into biochar through a muffle furnace;
(2) washing the fired biochar with deionized water for a plurality of times, using 0.1mol/L nitric acid as a modifier, fully and uniformly mixing the modifier and the biochar under the magnetic stirring condition of 200r/min, modifying for one day, and then washing with deionized water until the pH value is neutral to obtain chemically modified biochar; placing the chemically modified biochar in a microwave cleaning instrument with the frequency of 40KHz, performing microwave oscillation for 40-90 min, filtering, and drying at constant temperature to obtain the physicochemical modified biochar;
(3) drying the clay in a constant-temperature drying oven, and sieving with a 100-200 mesh sieve to obtain clay powder;
(4) the physicochemical modified charcoal and clay powder are mixed according to the mass ratio of (0.5-1): 10, adding the mixture into a mixed solution of sodium silicate and sodium bicarbonate dissolved by deionized water, preparing the mixture into spherical particles by a ball making machine, drying the spherical particles in a constant-temperature drying box, and finally firing the spherical particles in a muffle furnace to prepare the biochar balls, wherein the mass ratio of the sodium silicate to the clay is 3 percent, and the mass ratio of the sodium bicarbonate to the clay is 1.5 percent;
the membrane hanging method of the charcoal spheres comprises the following steps:
soaking the charcoal balls in the salt-tolerant and cold-tolerant ammonia oxidizing bacteria enriched liquid for 4-6 days to obtain biofilm-forming charcoal balls;
in the preparation method of the charcoal balls, the firing conditions of the muffle furnace are as follows: the temperature rising speed is 10 ℃/min, the temperature is 300-400 ℃, the heat preservation time is 180min, and the drying is carried out for 8-12 h under the conditions of constant temperature and drying conditions of 60-80 ℃.
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| CN114890555B (en) * | 2022-05-17 | 2023-09-22 | 沈阳建筑大学 | Solid microbial preparation for treating rural black and odorous water body and preparation method and application thereof |
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| CN102286390A (en) * | 2010-06-18 | 2011-12-21 | 辽宁北方环境保护有限公司 | Method for screening low temperature resisting nitrifying bacteria |
| CN102382767A (en) * | 2010-08-31 | 2012-03-21 | 中国石油化工股份有限公司 | Enrichment culture method of salt-tolerant nitrifying bacterium communities |
| CN102070251A (en) * | 2010-11-29 | 2011-05-25 | 哈尔滨工业大学 | Preparation method of immobilized cold resistant nitrobacteria |
| CN102260017B (en) * | 2011-05-24 | 2013-03-06 | 哈尔滨工程大学 | Method for effectively removing ammonia nitrogen from polluted water body |
| CN102329753A (en) * | 2011-09-27 | 2012-01-25 | 哈尔滨工业大学（威海） | Polyculture enrichment screening method for ocean heterotrophic denitrifying bacteria |
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| CN103013973A (en) * | 2012-12-17 | 2013-04-03 | 常州大学 | Method for preparing aerobiotic nitrifier immobilizing vector |
| CN103193315B (en) * | 2013-04-12 | 2014-05-07 | 北京工业大学 | Method for preparing bioactive filler through immobilization of anaerobic ammonia oxidizing bacterium |
| CN103773722B (en) * | 2014-01-16 | 2015-08-19 | 中国药科大学 | Salt tolerant also has subtilis and the application thereof of low-temperature biological deamination function |
| CN104164386A (en) * | 2014-07-02 | 2014-11-26 | 中国科学院成都生物研究所 | Method of medium-temperature enrichment culturing and low-temperature application of low-temperature resistant nitrobacterium flora |
| KR20160116647A (en) * | 2015-03-31 | 2016-10-10 | 충북대학교 산학협력단 | Hybid nutrient salt removal system |
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