CN109402107B - Immobilization of high-efficiency denitrifying bacteria and preparation method and application of microbial inoculum package - Google Patents
Immobilization of high-efficiency denitrifying bacteria and preparation method and application of microbial inoculum package Download PDFInfo
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Abstract
The invention relates to immobilization of high-efficiency denitrifying bacteria and a preparation method and application of a microbial inoculum bag. The preparation of the microbial inoculum package comprises the steps of screening out high-efficiency heterotrophic nitrification-aerobic denitrifying bacteria, and then carrying out salt tolerance domestication on the high-efficiency heterotrophic nitrification-aerobic denitrifying bacteria to obtain salt tolerance heterotrophic nitrification-aerobic denitrifying bacteria; adsorbing immobilized salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria by using the modified zeolite to obtain immobilized salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria zeolite; and spreading in cloth bag. The zeolite granule has bacterial density of 1 × 109~5×109CFU/mL. The prepared immobilized microbial inoculum bag is widely used in shallow sea farms and culture ponds, and realizes high-efficiency removal in a high-salt environment; the microbial inoculum bag can be repeatedly used, the cost is greatly reduced, the problems that free state microbial strains which are directly used easily flow along with seawater tides and cannot be accurately positioned and the bacteria are fully contacted are effectively overcome, the threat of foreign microorganisms to the quality of aquaculture products is avoided, the ecological safety of water bodies in mariculture areas is protected, and the wide application is expected.
Description
Technical Field
The invention belongs to a water pollution treatment technology, and particularly relates to immobilization of high-efficiency denitrifying bacteria and a preparation method and application of a microbial inoculum bag.
Background
The marine aquaculture industry in China is rapidly developed and plays an important role in meeting the requirements of people on marine products. However, in the production process, a large amount of pollutants generated in mariculture areas and farms directly enter the marine environment, and the water quality is deteriorated and the marine ecosystem is unbalanced due to long-term accumulation. Nitrogen source pollution in the aquaculture process mainly comes from artificial feeding, animal wastes, residual feeds and dead animals and plants, and exists in the forms of nitrate nitrogen, ammonia nitrogen and the like. The existing fish gill-rot disease can destroy gill tissues of fishes, induce gill-rot disease, inhibit oxygen carrying capacity of blood, reduce respiratory function, and cause hypoxia or death of cultured animals due to poisoning. Therefore, controlling the nitrogen content in the aquaculture water is especially important for ensuring the safety of marine products and the ecological balance of the ocean.
The control method of ammonia nitrogen in the mariculture environment mainly adopts a physical method, a chemical method and a biological method at present. The physical method comprises water changing, aeration, zeolite powder or chitosan adsorption and other treatment methods; the chemical method comprises reducing ammonia nitrogen concentration by using appropriate amount of quicklime, ozone, hydrogen peroxide, chlorine dioxide and other chemical reagents; biological methods include the combined repair of microorganisms, plants and aquatic animals or a variety of organisms, among others. Compared with the traditional physical and chemical method, the biological method has the advantages of low investment, good environmental compatibility, no secondary pollution, no harm to the culture function and the like, and is the water body remediation technology with the greatest development prospect at present.
An integrated synchronous nitrification and denitrification sewage treatment device (CN 201510843362.1) is proposed in the prior patent, and the method simultaneously removes ammonia nitrogen and nitrate nitrogen in the culture water body, needs the nitrification and denitrification of microorganisms to be simultaneously matched, and needs the combined action of a plurality of microorganisms. However, this method is complicated in processing and increases the cost. Meanwhile, autotrophic ammonia-oxidizing bacteria capable of removing ammonia nitrogen are very fragile and are easy to cause the loss of normal ammonia nitrogen metabolism function directly under the change of environmental conditions (such as salinity increase).
Therefore, the prior art strains can not achieve better removal effect under high-salt conditions. Especially in mariculture areas. A method for acclimatizing and screening heterotrophic nitrification aerobic denitrifying bacteria (CN 201510077694.3) disclosed in the prior patent discloses an acclimatizing and screening method of heterotrophic nitrification aerobic denitrifying bacteria, but a strain obtained by adopting the method cannot achieve a good removal effect under a high-salt condition. Especially in mariculture areas, the following two parts mainly exist due to ammonia nitrogen and nitrate nitrogen pollution: (1) the residual bait, feces and other organic pollutants settle to the bottom of the culture environment through gravity, and then the decomposition products are high-concentration ammonia nitrogen, nitrate nitrogen ammonia nitrogen and nitrate nitrogen diffused from the bottom to the upper water area in a longer time; (2) free ammonia nitrogen and nitrate nitrogen present in the water.
Meanwhile, the free microbial inoculum directly used is easy to flow along with the seawater tide, cannot be accurately positioned, greatly reduces the utilization efficiency, cannot meet the removal effect, causes waste, and cannot be recycled and reused; the mariculture area is sensitive to water, and the introduction of foreign microorganisms can endanger the quality of aquaculture products and even prevent the aquaculture products from normally surviving.
Disclosure of Invention
The invention aims to provide a preparation method and application of an immobilized microbial inoculum bag of high-efficiency denitrifying bacteria, so as to overcome the problems in the prior art.
Another object of the present invention is to provide a microorganism belonging to genus Tolbecco (Zuobei) which is a microorganism having high denitrifying activityZobellellasp.) to obtain salt-tolerant heterotrophic nitrification aerobic denitrification bacteria and to enable the bacteria to exert high pollutant removal efficiency in both high-salt seawater and fresh water.
The invention also aims to overcome the defects that the existing free microbial agent is diffused along with water flow in the using process, the using amount of the microbial agent is large, the cost is high, the utilization efficiency is low, and the microbial agent cannot be recycled.
The invention firstly considers that the seawater culture wastewater has higher salinity than surface water body, common functional microorganisms can not play a role, and the seawater culture area has sensitive water body, and the introduction of external microorganisms can endanger the quality of aquaculture products and even cause the aquaculture products to be incapable of normally surviving; but the existing traditional physical, chemical and biological methods can not achieve better repairing effect and the treatment cost is high. Therefore, the invention screens and domesticates the salt-tolerant heterotrophic nitrification aerobic denitrification bacteria from the sediments in the mariculture area to treat ammonia nitrogen and nitrate nitrogen in the culture water body and the sediments, thereby realizing good treatment effect and low cost, and not threatening the original ecological environment due to local conditions.
In addition, in the case of seawater having a dynamic flow, since the free microbial inoculum used as it is tends to flow with the tide of seawater due to the flow of the water, the localization cannot be performed accurately, and therefore, the use efficiency is low, and the removal effect cannot be satisfied. Therefore, the invention provides the immobilized microbial inoculum bag for removing the ammonia nitrogen and the nitrate nitrogen in the aquaculture water for the first time, so that the microorganisms in the immobilized microbial inoculum bag can not be dispersed by water flow and can not only ensure enough microbial biomass in unit volume, but also have a protection effect on the salt-tolerant heterotrophic nitrification aerobic denitrification bacteria, thereby realizing the important problem of effectively adding the high-efficiency microbial inoculum in the seawater and achieving the practical and high-efficiency removal of the ammonia nitrogen and the nitrate nitrogen.
The immobilized bacteria agent bag is characterized in that a plurality of immobilized salt-tolerant heterotrophic nitrification aerobic denitrification bacteria zeolite particles are evenly spread and distributed in a corrosion-resistant and water-permeable geotextile bag, wherein the particle size of the modified zeolite is between 3 and 8mm, and the density of bacteria in the modified zeolite is 1 multiplied by 109~5×109CFU/mL, the bacteria are acclimatized heterotrophic nitrification aerobic denitrification bacteria.
The preparation method of the immobilized bacteria package comprises the steps of screening out high-efficiency heterotrophic nitrifying and aerobic denitrifying bacteria, and then carrying out salt tolerance acclimation on the heterotrophic nitrifying and aerobic denitrifying bacteria to obtain salt-tolerant heterotrophic nitrifying and aerobic denitrifying bacteria; further taking the modified zeolite as a carrier, adsorbing the immobilized salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria to obtain immobilized salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria zeolite; and finally, flatly paving the immobilized salt-tolerant heterotrophic nitrification and aerobic denitrification bacterial zeolite in a square geotextile bag to obtain the immobilized microbial inoculum bag of the heterotrophic nitrification and aerobic denitrification bacteria.
The screening method of the high-efficiency heterotrophic nitrification aerobic denitrification bacteria comprises the following steps:
(1) enrichment culture: weighing 10g-20g of seabed sediment by dry weight, adding the seabed sediment into a 250mL conical flask containing 100mL of enrichment culture medium, performing shake culture for several days at the temperature of 28-30 ℃ at the speed of 150-200r/min, taking supernatant into a separation set culture medium, and performing shake culture for several days under the conditions. Then, taking 10mL of supernatant to a fresh separation culture medium, performing shake culture for several days at the temperature of 28-30 ℃ at the speed of 150-200r/min, repeating the steps for 3 times to improve the concentration of the heterotrophic nitrification aerobic denitrifying bacteria, and finally obtaining the heterotrophic nitrification aerobic denitrifying bacteria enrichment solution; the enrichment medium comprises: 5g of NaCl; 5g of yeast extract; 10g of tryptone and 1000mL of distilled water, adjusting the pH to be 8.0, and sterilizing at 121 ℃ for 30 min; the separation culture medium comprises: 5g of NaCl, KH2PO4 1.5g，MgSO4﹒7H2O 0.01g，Na2HPO47.9g, sodium citrate 5.96g, NaNO30.4268g，NH40.2686g of Cl, 2mL of trace element solution, 1000mL of deionized water, pH =8.0 adjustment and sterilization at 121 ℃ for 30 min; the trace element solution comprises: ZnSO4 2.2g，CaCl2 5.5g，Na2EDTA 63.70g，MnCl2·4H2O 5.06g，FeSO4·7H2O 5.0g，CuSO4·5H2O 1.57g，Na2MoO4·4H2O 1.1g，CoCl2·6H2O1.61 g, deionized water 1000 mL.
(2) And (3) purification and separation: the heterotrophic nitrification aerobic denitrifying bacteria enrichment solution is added according to the proportion of 10-1-10-7The dilution is carried out in a gradient manner,coating the mixture on a solid culture medium, culturing for several days at 28-30 ℃, selecting colonies with different forms on another solid culture medium after the colonies grow, repeatedly streaking until the generated colonies are single bacteria with different forms, and separating to obtain a plurality of pure heterotrophic nitrification aerobic denitrifying bacteria, wherein the culture medium is as follows: 5g of NaCl, KH2PO4 1.5g，MgSO4﹒7H2O 0.01g，Na2HPO47.9g, sodium citrate 5.96g, NaNO30.4268g，NH4Cl 0.2686g, trace element solution 2mL, agar 20g, distilled water 1000mL, pH =7.2 adjusted, sterilized at 121 ℃ for 30 min.
The method for domesticating the salt tolerance of the heterotrophic nitrification aerobic denitrifying bacteria is characterized in that a plurality of pure-breed nitrification aerobic denitrifying bacteria obtained by separation are picked into a new enrichment medium and are subjected to shake culture for several days under the conditions of 150-200r/min and 28-30 ℃, so that single-strain enrichment liquid is respectively obtained. 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 a new enrichment culture medium into another enrichment culture medium with 100mL of salinity (in terms of NaCl, g/L) of 5, 15, 25, 35, 45, 55, 65, 75, 85 and 95, wherein the initial ammonia nitrogen and nitrate nitrogen addition amount is 70mg/L, shaking culturing for several days under the conditions of 150r/min-200r/min and 28-30 ℃, and screening heterotrophic nitrification aerobic denitrifying bacteria with the degradation effect on ammonia nitrogen and nitrate nitrogen not less than 90%, namely the salt-tolerant heterotrophic nitrification aerobic denitrifying bacteria with good degradation effect.
The heterotrophic nitrification aerobic denitrification bacteria immobilization method is characterized in that modified zeolite particles with the particle size of 3-8mm are selected as an immobilization carrier, and OD600=0.6-1.5 and the density of 1 × 10 are taken9~5×109Adding 50-100mL of CFU/mL bacterial liquid into 10-20mL of bacterial liquid per gram of modified zeolite, oscillating and adsorbing at the temperature of 28-30 ℃ and the rpm of 150-300 for 12-24h, removing the supernatant, and washing the modified zeolite with sterilized distilled water for several times to obtain the immobilized salt-tolerant heterotrophic nitrification aerobic denitrification bacterial zeolite.
The modified zeolite is characterized in that 5-10ml of phosphoric acid solution with pH =5 is added according to each gram of zeolite, and 2Oscillating at the speed of 150-300rpm for 30-90min at the temperature of 8-30 ℃, standing for 12-24h, and washing with sterilized distilled water for several times; then FeCl of 0.5-1mol/L is adopted3Soaking for 12-24h, rinsing with sterilized distilled water for several times to remove excessive FeCl3Sterilizing at 121 deg.C for 30min in autoclave to obtain modified zeolite.
The immobilized microbial inoculum package of the heterotrophic nitrification aerobic denitrifying bacteria is characterized in that the immobilized microbial inoculum package is covered at a sediment-water interface for removing sediment ammonia nitrogen; the fertilizer is piled or scattered in surface water bodies, aquaculture wastewater and large-area sea areas, is used for removing ammonia nitrogen and nitrate nitrogen, and can be activated for repeated use.
The salt-tolerant heterotrophic nitrification aerobic denitrification bacteria obtained by the method for acclimatizing the salt tolerance of the heterotrophic nitrification aerobic denitrification bacteria can be applied to the purification of fresh water, ammonia nitrogen and nitrate nitrogen polluted water in surface water, and can also be applied to the purification of culture wastewater and water polluted by ammonia nitrogen and nitrate nitrogen in large-area sea areas with salinity higher than 30.
The preparation method of the immobilized bacterial bag is characterized in that the immobilized salt-tolerant heterotrophic nitrification-aerobic denitrification bacterial zeolite prepared by the preparation method of the immobilized salt-tolerant heterotrophic nitrification-aerobic denitrification bacterial zeolite can be applied to purification of fresh water, surface water ammonia nitrogen and nitrate nitrogen polluted water, and can also be applied to purification of aquaculture wastewater and ammonia nitrogen and nitrate nitrogen polluted water under the condition that the salinity of a wide sea area is higher than 30.
The preparation method of the immobilized microbial inoculum bag of the heterotrophic nitrification aerobic denitrifying bacteria is characterized in that the salt-tolerant heterotrophic nitrification aerobic denitrifying bacteria are the domesticated Zoerella which has a salt-tolerant function and synchronously removes ammonia nitrogen and nitrate nitrogenZobellella sp.。
Obviously, the invention satisfactorily solves the problem that heterotrophic nitrification aerobic denitrifying bacteria lose the effect of the heterotrophic nitrification aerobic denitrifying bacteria in a high-salt environment; the prepared immobilized microbial inoculum bag is widely applied to shallow sea farms and culture ponds, effectively overcomes the defects that the existing free microbial inoculum is directly used, easily flows along with the sea tide, cannot be accurately positioned and has low utilization efficiency, greatly prolongs the sufficient retention time of the flowing culture wastewater and heterotrophic nitrification aerobic denitrifying bacteria, ensures the sufficient contact of the flowing culture wastewater and the heterotrophic nitrification aerobic denitrifying bacteria, and realizes the high-efficiency removal; and the activated and immobilized microbial inoculum bag can be repeatedly used, so that the cost is greatly reduced, the ecological safety of the water body in the mariculture area is protected, and the threat of foreign microorganisms to the quality of aquaculture products is avoided.
Detailed Description
The present invention will be described in further detail by way of examples.
Example 1
Sampling sediments in a certain mariculture area, and performing ammonia nitrogen and nitrate nitrogen inhibition experiments on a sediment interface by detecting that the ammonia nitrogen concentration of the sediments reaches 238mg/L and the nitrate concentration is 254mg/L under the condition that the salinity is 35 per mill by adopting an immobilized microbial inoculum bag, wherein the method comprises the following steps:
firstly, screening out high-efficiency heterotrophic nitrification aerobic denitrifying bacteria: weighing 10g of seabed sediment by dry weight, adding the seabed sediment into a 250mL conical flask containing 100mL of enrichment medium, performing shake culture at the temperature of 28 ℃ at 150r/min for several days, taking supernatant into a separation medium, and performing shake culture for several days under the conditions. Then, taking 10mL of supernatant to a fresh separation culture medium, performing shake culture for several days at the temperature of 28 ℃ at the speed of 150r/min, repeating the step for 3 times to improve the concentration of the heterotrophic nitrification aerobic denitrifying bacteria, and finally obtaining the heterotrophic nitrification aerobic denitrifying bacteria enrichment liquid; the heterotrophic nitrification aerobic denitrifying bacteria enrichment solution is added according to the proportion of 10-1-10-7Diluting in gradient, spreading on solid culture medium, culturing at 28 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 strains of heterotrophic nitrification aerobic denitrifying bacteria
Then, carrying out salt tolerance domestication on heterotrophic nitrification aerobic denitrifying bacteria: selecting multiple pure strains of nitrifying aerobic denitrifying bacteria obtained by separation into an enrichment medium, and performing shake culture at 28 ℃ for several days at 150r/min to respectively obtainTo single strain enrichment solution. When the single strain enrichment solution enters a logarithmic growth period, sequentially inoculating the single strain enrichment solution and 5% of the enrichment culture medium in another enrichment culture medium with 100mL of salinity (in terms of NaCl, g/L) of 5, 15, 25, 35, 45, 55, 65, 75, 85 and 95, wherein the initial ammonia nitrogen and nitrate nitrogen addition amount is 70mg/L, performing shake culture at 150-200r/min and 28 ℃ for several days, and screening heterotrophic nitrification aerobic denitrifying bacteria with good ammonia nitrogen and nitrate nitrogen degradation effect, namely the salt-tolerant heterotrophic nitrification aerobic denitrifying bacteria. Selecting out bacteria with better performance, wherein the salt-tolerant heterotrophic nitrification aerobic denitrification bacteria are domesticated Zolbert bacillus (Zhabeurella)Zobellellasp.）。
Thirdly, modifying the zeolite: adding 100g zeolite into 500ml phosphoric acid solution (pH = 5), shaking at 150rpm for 30min at 30 deg.C, standing for 24 hr, rinsing with sterilized distilled water for several times to remove excessive phosphoric acid; then 1mol/L FeCl is adopted3Soaking zeolite for 24 hr, washing with sterilized distilled water for 5 times to remove excessive FeCl3Sterilizing at 121 deg.C for 30min in autoclave to obtain modified zeolite.
Carrying out microbial immobilization on the modified zeolite: selecting 5mm modified zeolite as immobilized carrier, and collecting 500mL bacterial solution (OD 600=1.5, 1.05 × 10)9CFU/mL), adding 30g (dry weight) of modified zeolite into the bacterial liquid, oscillating and adsorbing at 30 ℃ and 150rpm for 24h, discarding the supernatant, washing the modified zeolite with sterilized distilled water for 3 times to remove unadsorbed bacteria and obtain the immobilized salt-tolerant heterotrophic nitrification-aerobic denitrification bacterial zeolite.
Selecting corrosion-resistant and water-permeable non-woven geotextile to prepare a bag shape, paving 10g of immobilized salt-resistant heterotrophic nitrification and aerobic denitrification bacteria zeolite in the geotextile bag with the thickness of 10cm multiplied by 10cm to obtain an immobilized bacteria package of heterotrophic nitrification and aerobic denitrification bacteria, and covering the immobilized bacteria package on a sediment-water interface for removing sediment ammonia nitrogen.
The invention utilizes a heterotrophic nitrification aerobic denitrification bacteria immobilized microbial inoculum bag to carry out ammonia nitrogen and nitrate degradation tests on the culture water body deposition interface: 1 immobilized microbial inoculum package is placed in a 0.25 square reactor, and a removal experiment is carried out in an environment with the temperature of 25 ℃, and the result shows that: the immobilized microbial inoculum bag can obviously inhibit the generation of ammonia nitrogen and nitrate nitrogen in water within 9 days, and the control rate of the ammonia nitrogen reaches 73% +/-17%; the inhibition rate on nitrate nitrogen reaches 68% +/-6%.
Example 2
Sampling sediments in a certain mariculture area, and performing an experiment for removing ammonia nitrogen and nitrate nitrogen in a water body by detecting that the ammonia nitrogen concentration of the sediments reaches 46mg/L and the nitrate concentration is 39mg/L under the condition that the salinity is 35 per mill by adopting an immobilized microbial inoculum bag, wherein the experiment comprises the following steps:
firstly, screening out high-efficiency heterotrophic nitrification aerobic denitrifying bacteria: weighing 20g of seabed sediment by dry weight, adding the seabed sediment into a 250mL conical flask containing 100mL of enrichment medium, performing shake culture at 30 ℃ for several days at 200r/min, taking supernatant into an isolated culture medium, and performing shake culture for several days under the conditions. Then, taking 10mL of supernatant to a fresh separation culture medium, performing shake culture for several days at the temperature of 30 ℃ at 200r/min, repeating the step for 3 times to improve the concentration of the heterotrophic nitrification aerobic denitrifying bacteria, and finally obtaining the heterotrophic nitrification aerobic denitrifying bacteria enrichment solution; the heterotrophic nitrification aerobic denitrifying bacteria enrichment solution is added according to the proportion of 10-1-10-7Diluting in gradient, spreading on solid culture medium, culturing at 28 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 strains of heterotrophic nitrification aerobic denitrifying bacteria
Then, carrying out salt tolerance domestication on heterotrophic nitrification aerobic denitrifying bacteria: and (3) selecting a plurality of pure-breed nitrifying aerobic denitrifying bacteria obtained by separation into an enrichment medium, and performing shake culture for a plurality of days at the temperature of 30 ℃ at 200r/min to respectively obtain single-strain enrichment liquid. When the single strain enrichment solution enters a logarithmic growth phase, sequentially inoculating the single strain enrichment solution and 5% of enrichment culture medium in another 100mL of medium with salinity (in terms of NaCl, g/L) of 5, 15, 25, 35, 45, 55, 65, 75, 85 and 95 and initial ammonia nitrogen andin an enrichment culture medium with the nitrate nitrogen addition amount of 70mg/L, shake culturing for several days at the temperature of 28 ℃ at the speed of 150r/min-200r/min, and screening heterotrophic nitrification-aerobic denitrifying bacteria with good degradation effect on ammonia nitrogen and nitrate nitrogen, namely the salt-tolerant heterotrophic nitrification-aerobic denitrifying bacteria. Selecting out bacteria with better performance, wherein the salt-tolerant heterotrophic nitrification aerobic denitrification bacteria are domesticated Zolbert bacillus (Zhabeurella)Zobellellasp.）。
Thirdly, modifying the zeolite: adding 100g zeolite into 500ml phosphoric acid solution (pH = 5), shaking at 150rpm for 30min at 30 deg.C, standing for 24 hr, washing with sterilized distilled water for 5 times to remove excessive phosphoric acid; then 1mol/L FeCl is adopted3Soaking zeolite for 24 hr, rinsing with sterilized distilled water for several times to remove excessive FeCl3Sterilizing at 121 deg.C for 30min in autoclave to obtain modified zeolite.
Carrying out microbial immobilization on the modified zeolite: selecting 8mm modified zeolite as immobilized carrier, and collecting 200mL bacterial liquid (OD 600=1.5, 1.05 × 10)9CFU/mL), adding 20g (dry weight) of modified zeolite into the bacterial liquid, oscillating and adsorbing at 30 ℃ and 150rpm for 24h, discarding the supernatant, and washing the zeolite with sterilized distilled water for 3 times to remove unadsorbed bacteria to obtain the immobilized salt-tolerant heterotrophic nitrification-aerobic denitrification bacterial zeolite. 10g of immobilized salt-tolerant heterotrophic nitrification aerobic denitrification bacterial zeolite is flatly paved in a geotextile bag with the thickness of 10cm multiplied by 10cm to obtain an immobilized microbial agent bag, and the immobilized microbial agent bag is covered on a sediment-water interface and used for removing sediment ammonia nitrogen.
Performing ammonia nitrogen and nitrate degradation tests on the culture water body deposition interface: 1 immobilized microbial inoculum package is placed in a 100mL reactor, and a removal experiment is carried out in an environment with the temperature of 25 ℃, and the result shows that: the immobilized microbial inoculum bag can obviously remove ammonia nitrogen and nitrate in water within 24 hours, and the control rate of the ammonia nitrogen reaches 96% +/-3%; the inhibition rate on nitrate nitrogen reaches 92% +/-5%. Therefore, the method is expected to be widely applied to aquaculture water.
Claims (6)
1. An immobilized microbial agent bag for efficiently removing denitrifying bacteria, which is characterized in that the immobilized microbial agent bag is arranged on a corrosion-resistant and water-permeable geotextile bagA plurality of immobilized salt-tolerant heterotrophic nitrification aerobic denitrification bacteria zeolite particles are evenly spread and distributed in the inner layer, the particle size of the modified zeolite is between 3 and 8mm, and the density of bacteria in the modified zeolite is 1 multiplied by 109~5×109CFU/mL, the bacteria are acclimatized heterotrophic nitrification aerobic denitrification bacteria;
the immobilized bacteria package is prepared by the following method, firstly, high-efficiency heterotrophic nitrifying and aerobic denitrifying bacteria are screened out, and then the heterotrophic nitrifying and aerobic denitrifying bacteria are subjected to salt tolerance acclimation to obtain salt-tolerant heterotrophic nitrifying and aerobic denitrifying bacteria; further carrying out immobilization, wherein the immobilization takes modified zeolite as a carrier and adsorbs salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria to obtain immobilized salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria zeolite; finally, the immobilized salt-tolerant heterotrophic nitrification aerobic denitrification bacterial zeolite is flatly laid in a square geotextile bag, and an immobilized microbial agent bag of the heterotrophic nitrification aerobic denitrification bacteria is obtained;
the modified zeolite is added into 5-10ml of phosphoric acid solution with pH =5 according to per gram of zeolite, is vibrated for 30-90min at the temperature of 28-30 ℃ and at the speed of 150-300rpm, is kept stand for 12-24h, and is then washed by sterilized distilled water for several times; then FeCl of 0.5-1mol/L is adopted3Soaking for 12-24h, rinsing with sterilized distilled water for several times to remove excessive FeCl3Sterilizing at 121 deg.C for 30min in autoclave to obtain modified zeolite.
2. The immobilized bacteria package of claim 1, wherein the method for screening the high efficiency heterotrophic nitrification-aerobic denitrifying bacteria comprises the following steps:
(1) enrichment culture: weighing 10-20g of seabed sediment by dry weight, adding the seabed sediment into a 250mL conical flask containing 100mL of enrichment culture medium, performing shake culture for several days at the temperature of 28-30 ℃ at the speed of 150-; then taking 10mL of supernatant to a fresh separation culture medium, performing shake culture for several days at the temperature of 28-30 ℃ and at the speed of 150-200r/min, repeating the steps for 3 times to improve the concentration of the heterotrophic nitrification aerobic denitrifying bacteria, and finally obtaining the heterotrophic nitrification aerobic denitrification fine powderEnriching the bacteria; the enrichment medium comprises: 5g of NaCl; 5g of yeast extract; 10g of tryptone and 1000mL of distilled water, adjusting the pH to be 8.0, and sterilizing at 121 ℃ for 30 min; the separation culture medium comprises: 5g of NaCl, KH2PO4 1.5g，MgSO4﹒7H2O 0.01g，Na2HPO47.9g, sodium citrate 5.96g, NaNO30.4268g，NH40.2686g of Cl, 2mL of trace element solution, 1000mL of deionized water, pH =8.0 adjustment and sterilization at 121 ℃ for 30 min; the trace element solution comprises: ZnSO4 2.2g，CaCl2 5.5g，Na2EDTA 63.70g，MnCl2·4H2O 5.06g，FeSO4·7H2O 5.0g，CuSO4·5H2O 1.57g，Na2MoO4·4H2O 1.1g，CoCl2·6H2O1.61 g and deionized water 1000 mL;
(2) and (3) purification and separation: the heterotrophic nitrification aerobic denitrifying bacteria enrichment solution is added according to the proportion of 10-1-10-7Diluting in a gradient manner, coating the diluted solution on a solid culture medium, culturing for several days at the temperature of 28-30 ℃, selecting colonies with different forms on another solid culture medium after the colonies grow, repeatedly streaking until the generated colonies are single bacteria with different forms, and separating to obtain a plurality of pure heterotrophic nitrification aerobic denitrifying bacteria, wherein the solid culture medium is as follows: 5g of NaCl, KH2PO4 1.5g，MgSO4﹒7H2O 0.01g，Na2HPO47.9g, sodium citrate 5.96g, NaNO3 0.4268g，NH4Cl 0.2686g, trace element solution 2mL, agar 20g, distilled water 1000mL, pH =7.2 adjusted, sterilized at 121 ℃ for 30 min.
3. The immobilized microbial inoculum package of claim 1, wherein the method for acclimatizing the salt tolerance of heterotrophic nitrification-aerobic denitrifying bacteria comprises the steps of selecting a plurality of pure-breed aerobic nitrification-aerobic denitrifying bacteria obtained by separation into a fresh enrichment medium, performing shake culture at the temperature of between 28 and 30 ℃ for a plurality of days at the speed of between 150 and 200r/min to respectively obtain single-strain enrichment solutions, sequentially inoculating the single-strain enrichment solutions and 5 percent of the enrichment medium into another new enrichment medium with the salinity of 5, 15, 25, 35, 45, 55, 65, 75, 85 and 95 and the initial ammonia nitrogen and nitrate nitrogen addition amount of 70mg/L at the speed of between 150 and 200r/min for a plurality of days at the temperature of between 28 and 30 ℃ when the single-strain enrichment solutions enter a logarithmic growth period, screening heterotrophic nitrification aerobic denitrifying bacteria with the degradation effect on ammonia nitrogen and nitrate nitrogen not less than 90 percent, namely salt-tolerant heterotrophic nitrification aerobic denitrifying bacteria with good degradation effect; the salinity is measured by NaCl and is unit g/L.
4. The immobilized microbial agent package of claim 1, wherein the immobilization is carried out by selecting modified zeolite particles with a particle size of 3-8mm as the immobilized carrier, and taking OD600=0.6-1.5 and a density of 1 x 109~5×109Adding 50-100mL of CFU/mL bacterial liquid into 10-20mL of bacterial liquid per gram of modified zeolite, oscillating and adsorbing at the temperature of 28-30 ℃ and the rpm of 150-300 for 12-24h, discarding the supernatant, and washing the modified zeolite with sterilized distilled water for several times to obtain the immobilized salt-tolerant heterotrophic nitrification aerobic denitrification bacterial zeolite.
5. The use of the immobilized microbial inoculum package of claim 1, wherein the immobilized microbial inoculum package is covered at a sediment-water interface for sediment ammonia nitrogen removal; the fertilizer is thrown into surface water bodies, culture wastewater and large-area sea areas, is used for removing ammonia nitrogen and nitrate nitrogen, and can be activated for repeated use.
6. The application of the heterotrophic nitrification-aerobic denitrifying bacteria with domesticated salt tolerance is characterized in that the heterotrophic nitrification-aerobic denitrifying bacteria with domesticated salt tolerance are prepared by the following method:
firstly, screening out high-efficiency heterotrophic nitrification aerobic denitrifying bacteria, and then carrying out salt tolerance domestication on the heterotrophic nitrification aerobic denitrifying bacteria to obtain salt tolerance heterotrophic nitrification aerobic denitrifying bacteria;
the screening of the high-efficiency heterotrophic nitrification aerobic denitrification bacteria comprises the following steps:
(1) enrichment culture: weighing 10-20g of seabed sediment by dry weight, and adding into a culture medium containing 100mL of enrichment mediumPerforming shake culture in a 250mL conical flask at the temperature of 28-30 ℃ at the speed of 150-; then, taking 10mL of supernatant to a fresh separation culture medium, performing shake culture for several days at the temperature of 28-30 ℃ at the speed of 150-200r/min, repeating the steps for 3 times to improve the concentration of the heterotrophic nitrification aerobic denitrifying bacteria, and finally obtaining the heterotrophic nitrification aerobic denitrifying bacteria enrichment solution; the enrichment medium comprises: 5g of NaCl; 5g of yeast extract; 10g of tryptone and 1000mL of distilled water, adjusting the pH to be 8.0, and sterilizing at 121 ℃ for 30 min; the separation culture medium comprises: 5g of NaCl, KH2PO4 1.5g，MgSO4﹒7H2O 0.01g，Na2HPO47.9g, sodium citrate 5.96g, NaNO30.4268g，NH40.2686g of Cl, 2mL of trace element solution, 1000mL of deionized water, pH =8.0 adjustment and sterilization at 121 ℃ for 30 min; the trace element solution comprises: ZnSO4 2.2g，CaCl2 5.5g，Na2EDTA 63.70g，MnCl2·4H2O 5.06g，FeSO4·7H2O 5.0g，CuSO4·5H2O 1.57g，Na2MoO4·4H2O 1.1g，CoCl2·6H2O1.61 g and deionized water 1000 mL;
(2) and (3) purification and separation: the heterotrophic nitrification aerobic denitrifying bacteria enrichment solution is added according to the proportion of 10-1-10-7Diluting in a gradient manner, coating the diluted solution on a solid culture medium, culturing for several days at the temperature of 28-30 ℃, selecting colonies with different forms on another solid culture medium after the colonies grow, repeatedly streaking until the generated colonies are single bacteria with different forms, and separating to obtain a plurality of pure heterotrophic nitrification aerobic denitrifying bacteria, wherein the solid culture medium is as follows: 5g of NaCl, KH2PO4 1.5g，MgSO4﹒7H2O 0.01g，Na2HPO47.9g, sodium citrate 5.96g, NaNO3 0.4268g，NH40.2686g of Cl, 2mL of trace element solution, 20g of agar and 1000mL of distilled water, adjusting the pH =7.2, and sterilizing at 121 ℃ for 30 min;
the heterotrophic nitrification aerobic denitrification bacteria are domesticated with salt tolerance as follows:
(3) selecting a plurality of pure strains of the nitrifying aerobic denitrifying bacteria separated in the step (2) into a fresh enrichment culture medium, shake culturing at 150-200r/min at 28-30 deg.C for several days to obtain single strain enrichment solutions, respectively, allowing the single strain enrichment solutions to enter logarithmic growth phase, sequentially inoculating the single strain enrichment solution and 5 percent of the enrichment culture medium in another 100mL of new enrichment culture medium with salinity of 5, 15, 25, 35, 45, 55, 65, 75, 85 and 95 respectively and initial ammonia nitrogen and nitrate nitrogen addition amount of 70mg/L, shake culturing for several days at the temperature of 28-30 ℃ at the speed of 150-200r/min, and screening heterotrophic nitrification aerobic denitrifying bacteria with the degradation effect on ammonia nitrogen and nitrate nitrogen not less than 90%, namely salt-tolerant heterotrophic nitrification aerobic denitrifying bacteria with good degradation effect;
the heterotrophic nitrification aerobic denitrifying bacteria domesticated in a salt tolerance manner are applied as follows:
the obtained salt-tolerant heterotrophic nitrification aerobic denitrification bacteria are applied to the purification of fresh water, surface water ammonia nitrogen and nitrate nitrogen polluted water, and are applied to the purification of aquaculture wastewater and large-area water polluted by ammonia nitrogen and nitrate nitrogen under the condition that the salinity of the sea area is higher than 30.
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