CN115180724A - Hydrogen autotrophic microorganism denitrification and uranium fixation domestication device and domestication method - Google Patents
Hydrogen autotrophic microorganism denitrification and uranium fixation domestication device and domestication method Download PDFInfo
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Abstract
The invention provides a device and a method for domesticating nitrogen and uranium by using a hydrogen autotrophic microorganism, and belongs to the technical field of water treatment systems. The invention relates to a hydrogen autotrophic microorganism denitrification uranium fixation domestication device, which comprises a hydrogen supply H 2 Component, H 2 A transport member and a biochemical culturing member, said H 2 The conveying component comprises a glass measuring cylinder and a plastic measuring cylinder which is arranged in the glass measuring cylinder in an inverted mode, and the bottoms of the plastic measuring cylinders are respectively connected with a power supply H through pipe fittings 2 Component and biochemical cultureAnd the oxygen-free solution is filled in the glass measuring cylinder. The device solves the problem of H 2 The method has the advantages of easy overflow, low utilization rate and certain danger, can efficiently and cleanly domesticate the hydrogenotrophic microorganisms for denitrification and uranium fixation, and can provide basic data and theoretical basis for removing oxidative pollutants such as nitrate, uranium and the like in polluted water by using the hydrogenotrophic microorganisms in nitrogen/phosphate fertilizer pollution in the aspect of agriculture or uranium pollution in the aspect of industry.
Description
Technical Field
The invention belongs to the technical field of water treatment systems, and particularly relates to a device and a method for domesticating nitrogen and uranium by using a hydrogen autotrophic microorganism.
Background
The phosphate fertilizer contains trace uranium (mainly existing in a U (VI) form), and researches show that the U (VI) in underground water is easy to form a complex with high solubility and mobility, so that the phosphate fertilizer has higher toxicity and huge harm. The main disease condition caused by nitrate is 'blue infant syndrome', and the harm of uranium to human bodies is reflected in toxicity in aspects of chemistry, radioactivity and the like.
The pollution remediation technology of nitrate/uranium in wastewater has been studied at home and abroad, and the method can be divided into a physical and chemical method, a chemical denitrification method and a biological nitrification method according to the pollutant removal mechanism. The traditional physical and chemical methods comprise an ion exchange method, a reverse osmosis method, an adsorption method, a modified bentonite method active metal reduction method and the like, and the methods have the advantages of low energy consumption, simple process and stable effect. In-situ permeable reactive barrier technology has been to adopt nanometer zero-valent iron (nZVI) as the main reducing material, the size of nZVI molecule is only 1-100 nm, and also has the excellent characteristics of strong reducibility, large specific surface area, strong migration and transformation performance, etc., and the slurry prepared by the method is directly injected into polluted groundwater, and can realize the rapid degradation of pollutants in the groundwater, but certain self limitations of nZVI also exist in practical application, such as: easy agglomeration, easy oxidation and passivation in air, poor electron selectivity, easy loss and difficult recovery, large sludge production and the like. In combination with these physicochemical processes, they all share several common features, namely that the nitrates and uranium that need to be removed are merely transferred or concentrated, rather than being converted to non-toxic and harmless nitrogen and precipitates for removal. Secondly, the subsequent treatment cost is relatively high, and the method is difficult to be practically applied to in-situ remediation of underground water.
Most of the groundwater remediation methods can only remove nitrogen or uranium, the target is single, the simulated wastewater components are relatively simple (the main pollutants are only nitrate or uranium), and the complex groundwater environment cannot be well simulated. Of course, there are also some physicochemical methods for purifying groundwater contaminated by both nitrate and uranium and recovering uranium therefrom, but each has certain drawbacks and research potential. Compared with bioremediation methods such as a microbial reduction method and the like, the bioremediation method is unique in each method due to the advantages of environmental friendliness, low cost and the like.
The removal of nitrate by the microbial reduction method mainly utilizes denitrification reaction, and can be divided into heterotrophic denitrification and autotrophic denitrification according to different carbon sources utilized by microbes. The heterotrophic denitrification method is to add organic carbon sources (acetic acid, methanol, ethanol, etc.) into the water body as nutrient substances of the heterotrophic denitrifying bacteria. Heterotrophic denitrification has the advantages of good denitrification effect, large water treatment amount and high efficiency, but the organic carbon source is insufficient, and the accumulation amount of nitrite is increased in the denitrification process; if the carbon source is excessively added, secondary pollution is easily caused, the treatment cost is increased by adding the organic matrix outside, and the added organic carbon source is difficult to be uniformly distributed in underground water. Autotrophic denitrification may be further classified into sulfur autotrophic denitrification and hydrogen autotrophic denitrification. The sulfur autotrophic denitrification process can generate a large amount of sulfate radicals to cause secondary pollution, and the sulfuric acid can consume the alkalinity in the reaction to cause water body sulfate pollution, so that the method is not suitable for removing underground water nitrate with high sulfate concentration. In addition, the sulfur autotrophic denitrification process generally requires the addition of limestone to provide an inorganic carbon source and provide alkalinity to maintain the pH of the aqueous environment.
In recent years, researchers have been attracting attention to a new technology for remediating oxidative contaminants such as nitrates and uranium in polluted groundwater using a suspension autotrophic microorganism or a biofilm microorganism, and purifying the polluted groundwater using H 2 As electron donor, CO 2 As an inorganic carbon source, carbonate in water is converted into nontoxic and harmless nitrogen, dissolved uranium is converted into precipitated uranium and recycled, and other oxidizing pollutants in water are reduced into a low-toxicity/nontoxic form. By H 2 As an electron donor, the method has the advantages of low cost, high substrate utilization rate, no need of subsequent treatment and the like, thereby having great prospect. But due to H 2 Solubility in water is low and there is a risk of explosion, so that the autotrophic denitrification of hydrogen is limited in remediation of contaminated groundwater. I.a. vasiliadou et al by continuously feeding H into a flask 2 The method for culturing the hydrogen autotrophic microorganisms is used for researching the denitrification dynamics of the hydrogen autotrophic microorganisms, and the result is influenced to a certain extent because the reactor cannot be closed all the time and the dissolved oxygen in the system exists excessively. Thus, how to retain the substrate H in the biochemical culture for a long time 2 Decrease the internal H of the system 2 Is increased by the overflow of 2 The utilization rate becomes the bottleneck of the application of the hydrogen autotrophic denitrification to the remediation of polluted underground water.
Disclosure of Invention
In view of the above, the present invention aims to provide a device and a method for acclimatizing hydrogen autotrophic microorganisms to remove nitrogen and fix uranium by adjusting H 2 A supply mode solves H 2 The method has the advantages of easy overflow, low utilization rate and certain danger, and can efficiently and cleanly domesticate the hydrogen autotrophic microorganisms for removing nitrogen and fixing uranium.
In order to achieve the above purpose, the invention provides the following technical scheme:
a device for domesticating solid uranium by denitrogenation of hydrogen autotrophic microorganisms, which comprises a hydrogen supply H 2 Component, H 2 A transport member and a biochemical culturing member, said H 2 The conveying component comprises a glass measuring cylinder and a plastic measuring cylinder which is inverted in the glass measuring cylinder, and the bottoms of the plastic measuring cylinders are respectively connected with a power supply H through pipe fittings 2 The device comprises a component and a biochemical culture component, wherein an oxygen-free solution is filled in a glass measuring cylinder.
Preferably, the catalyst also comprises CO 2 An aeration part for supplying CO 2 The aeration part is connected with the biochemical culture part through a pipe fitting.
Preferably, the biochemical culture part is filled with a microorganism domestication solution, which comprises a soil microorganism suspension, a microorganism nutrient solution, an inorganic carbon source and nitrate/uranium.
More preferably, the soil microorganism suspension is: mixing the soil and water, and standing to form a mud-water mixture at the position of 0-5cm of a solid-liquid critical plane; the microorganism nutrient solution is K 2 HPO 4 220mg/L、 KH 2 PO 4 260mg/L、MgSO 4 ·7H 2 O 500mg/L、NaCl 120mg/L、CaCl 2 140mg/L、 FeCl 3 ·6H 2 O240 mg/L aqueous solution.
Preferably, the apparatus further comprises a silent stirrer and a magnetic stirrer which are arranged at the bottom of the biochemical culture part.
The invention also provides application of the device for domesticating the denitrified and solid uranium of the hydrogenotrophic microorganism in the domestication of the denitrified and solid uranium of the hydrogenotrophic microorganism or the improvement of wastewater.
The invention also provides a domestication method of the device for removing nitrogen and fixing uranium by using the hydrogen autotrophic microorganisms, which comprises the following steps:
filling soil microorganism suspension, microorganism nutrient solution and inorganic carbon source into a biochemical culture bottle, and introducing H 2 And CO 2 (ii) a Adding nitrate into a biochemical culture bottle, and performing denitrification domestication to obtain the hydrogen autotrophic denitrification microorganism; or adding uranium solution, and performing solid uranium acclimation to obtain the hydrogen autotrophic solid uranium microorganisms; or carrying out denitrification domestication and then carrying out uranium fixation domestication to obtain the hydrogen autotrophic denitrification uranium fixation microorganism.
Preferably, the domestication process is carried out in a dark environment, the domestication temperature is 25 +/-1 ℃, the pH value of the microbial domestication liquid is 6-7, and the DO value is 0-2mg/L; the initial concentration of nitrate nitrogen in the denitrified and domesticated microorganism domestication liquid is more than or equal to 15mg-N/L; the initial concentration of hexavalent uranium in the uranium fixation domestication microorganism domestication liquid is more than or equal to 300ug/L.
Preferably, the detection is performed in the acclimation process: h 2 When the amount is less than or equal to 500ml, the H is supplemented 2 (ii) a CO supplementation at pH > 7 2 (ii) a Microbial acclimation liquid NO 3 - Concentration is less than or equal to 2mg-N/L, NO 2 - Supplementing nitrate when the concentration is less than or equal to 0.1mg-N/L; and (4) supplementing uranium solution when the concentration of hexavalent uranium in the microbial domestication solution is less than 50ug/L.
Preferably, the denitrification or uranium sequestration is continued for 5 cycles or more.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a hydrogen autotrophic microorganism denitrification uranium fixation domestication device and a domestication method, which are characterized in that a specially-made H is adopted 2 The conveying member is connected to supply H 2 The components and the biochemical culture components have low manufacturing cost, good air tightness, no danger of easy explosion and easy domestication of the hydrogen autotrophic microorganisms. Timed reading H 2 The scale can calculate the H of the hydrogen autotrophic microorganism 2 Utilization rate, and timely supplement of H in inverted measuring cylinder 2 Can also ensure that sufficient H exists in the biochemical culture bottle 2 And (4) supplying.
The two processes of denitrification and uranium fixation are respectively subjected to alkali production and acid production reactions, and the fluctuation of pH in the system can be effectively reduced by treating the two pollutants simultaneously; the device and the domestication method provided by the invention can synchronously degrade nitrate and hexavalent uranium in water, and have good treatment effect and stable domestication effect; the method can provide a new repairing idea for underground water polluted by nitrogen/phosphate fertilizer application in the aspect of agriculture, and can provide basic data and theoretical basis for a process for removing oxidative pollutants such as nitrate, uranium and the like in polluted water by using the hydrogen autotrophic microorganisms in mines or certain nuclear power plants, scientific research and radiation institutions for acid leaching uranium.
Drawings
FIG. 1: the invention relates to a hydrogen autotrophic microorganism denitrification uranium fixation domestication device;
FIG. 2 is a schematic diagram: NO in acclimation culture stage provided in example 3 of the present invention 3 - And NO 2 - Detecting a concentration dynamic graph;
FIG. 3: the dynamic graph of the detection concentration of hexavalent uranium in the domestication and culture stage provided by the embodiment 3 of the invention.
Detailed Description
The invention provides a hydrogen autotrophic microorganism denitrification uranium fixation domestication device, which comprises a hydrogen supply deviceH 2 Component, H 2 A transport member and a biochemical culturing member, said H 2 The conveying component comprises a glass measuring cylinder and a plastic measuring cylinder which is arranged in the glass measuring cylinder in an inverted mode, and the bottoms of the plastic measuring cylinders are respectively connected with a power supply H through pipe fittings 2 The device comprises a component and a biochemical culture component, wherein an oxygen-free solution is filled in a glass measuring cylinder. The invention further preferably arranges two threaded ports on the inverted plastic measuring cylinder, wherein one of the threaded ports is inserted into the pneumatic joint to be used as an air injection port, and then the two ends of the pneumatic joint are connected with the silicone tube with proper length and the air supply H 2 The components are connected, and a water stop clip is arranged at the gas injection port; another threaded portThrough pipe fittingsIs connected with the biochemical culture part.
The invention passes through specially-made H 2 The conveying member is connected to supply H 2 The components and the biochemical culture components have low manufacturing cost, good air tightness, no explosive danger, easy domestication of the hydrogen autotrophic microorganisms and timing reading of H 2 The scale can calculate the H of the hydrogen autotrophic microorganisms 2 Utilization rate, and timely supplement of H in inverted measuring cylinder 2 Can also ensure that sufficient H exists in the biochemical culture bottle 2 And (4) supplying.
Preferred supply of H in the present invention 2 The component is a hydrogen cylinder; preferably, the connecting pipe fitting in the domesticating device is a silica gel/rubber pipeline; preferably the oxygen-free solution is a saturated sodium sulfite solution.
The domestication device of the invention also comprises CO 2 Aeration means of said CO 2 The aeration part is connected with the biochemical culture part through a pipe fitting. Introduction of CO 2 Not only provides inorganic carbon source for the microorganism in the system, but also can buffer and regulate the pH value in the system, and CO is introduced by regulating the content of sodium bicarbonate in the system 2 The initial pH value of the system can be stabilized at 6-7 after saturation, so that the acidic underground water environment polluted by nitrate/uranium can be well simulated, and the growth of the hydrogen autotrophic microorganisms is promoted.
The biochemical culture part is preferably a biochemical culture bottle. The invention further preferably arranges two screw-thread ports on the biochemical culture bottle, one of which is inserted into the pneumatic connector as a gas injection port, and then the two ends of the pneumatic connector are connected with a silicone tube with proper length and CO 2 The aeration part is connected, and a water stop clamp is arranged at a gas injection port; another threaded portPassing through pipe fittingAnd H 2 The inverted plastic measuring cylinder of the conveying component is connected. According to the invention, a layer of tinfoil paper is further preferably wrapped around the biochemical culture bottle, and the light-resistant environment is manufactured to prevent algae from growing; and more preferably, the tinfoil paper is provided with an observation port, so that the observation is convenient. The invention has no special limit on the material of the biochemical culture bottle and the glue for sealing the device, and can ensure the air tightness of the device.
According to the invention, preferably, the biochemical culture part is filled with microorganism domestication liquid, which comprises soil microorganism suspension liquid, microorganism nutrient solution, inorganic carbon source and nitrate/uranium.
It is further preferred according to the present invention that the soil microorganism suspension is: mixing the soil and water, and standing to form a muddy water mixture at the position of 0-5cm of a solid-liquid critical plane; more preferably, 30 to 60g of soil in the area without man-made interference is mixed with 1000ml of boiling cooling water, and after standing, the mud-water mixture at the position of 0 to 5cm of the solid-liquid critical surface is taken as inoculation liquid. As an embodiment, the muddy water mixture is left standing in a transparent container, which is beneficial to observing the solid-liquid critical surface.
According to the invention, the microorganism nutrient solution is K 2 HPO 4 220mg/L、KH 2 PO 4 260mg/L、MgSO 4 ·7H 2 O 500mg/L、NaCl 120mg/L、CaCl 2 140mg/L、FeCl 3 ·6H 2 O240 mg/L aqueous solution; more preferably, the nutrient solution is sterilized after the preparation. The sterilization method is not particularly limited, and the conventional sterilization method of the acclimatized culture medium well known to those skilled in the art can be adopted, and as an implementation mode, the culture medium is sterilized at the temperature of 121 ℃ for 30min under high pressure.
In a further preferred embodiment of the present invention, the inorganic carbon source is sodium bicarbonate; the nitrate is sodium nitrate; the uranium is a uranium solution, preferably hexavalent uranium consisting of U 3 O 8 Provided is a method.
In the invention, the preferable microbial domestication liquid accounts for 50-90% of the biochemical culture part container, wherein the soil microbial suspension accounts for 10-15%; further preferably, the microorganism domestication liquid is used for biochemical culture80% of the nutrient container, wherein the soil microorganism suspension is 10%. After the microbial domestication liquid is preferably obtained, the microbial domestication liquid is treated by CO 2 Aerating, removing dissolved oxygen in the bacterial liquid, and providing an inorganic carbon source as a pH buffering agent; further preferred is CO 2 Aerating, and adjusting the pH value of the microbial domestication solution to 6-7 together with sodium bicarbonate; more preferably to a pH of 6.2.
The acclimatization apparatus of the present invention preferably further comprises a silent stirrer and a magnetic stirrer provided at the bottom of the biochemical culture unit.
The invention also provides a domestication method of the device for removing nitrogen and fixing uranium by using the hydrogen autotrophic microorganisms, which comprises the following steps: filling soil microorganism suspension, microorganism nutrient solution and inorganic carbon source into a biochemical culture bottle, and introducing H 2 And CO 2 (ii) a Adding nitrate into a biochemical culture bottle, and performing denitrification domestication to obtain the hydrogen autotrophic denitrification microorganism; or adding uranium solution to carry out solid uranium domestication to obtain the hydrogen autotrophic solid uranium microorganism; or carrying out denitrification domestication and then carrying out uranium fixation domestication to obtain the photoautotrophic denitrification uranium fixation microorganism.
The domestication process is preferably carried out in a dark environment, the domestication temperature is 25 +/-1 ℃, the pH value of the microorganism domestication liquid is 6-7, and the DO value is 0-2mg/L; the initial concentration of nitrate nitrogen in the denitrified and domesticated microorganism domestication liquid is more than or equal to 15mg-N/L; the initial concentration of hexavalent uranium in the solid uranium domesticated microorganism domestication liquid is more than or equal to 300ug/L.
The rotation speed of the magnetic stirrer in the acclimatization process is further preferably 150r/min. Further preferably, before acclimatization and culture, a light-proof, anaerobic and acidic environment is manufactured, and the method for removing oxygen and adjusting pH is CO exposure 2 And (5) gas is used for 2min.
Detection in the preferred acclimatization Process of the invention, H 2 When the amount is less than or equal to 500ml, the H is supplemented 2 (ii) a CO supplementation at pH > 7 2 (ii) a Microorganism acclimatization liquid NO 3 - Concentration is less than or equal to 2mg-N/L, NO 2 - Supplementing nitrate when the concentration is less than or equal to 0.1mg-N/L; and (4) supplementing uranium solution when the concentration of hexavalent uranium in the microbial domestication solution is less than 50ug/L. It is further preferred that the hydrogen gas is supplied in an amount not exceeding the maximum scale of the measuring cylinder. Further preferably, after sampling and detecting, the sample is supplemented into a biochemical culture bottleA corresponding volume of nutrient solution.
The invention prefers NO in denitrification acclimation 3 - 、NO 2 - The detection frequency of the concentration and the concentration of the hexavalent uranium during uranium fixation domestication is 1 to 3 times per day, and 3 times per day is further preferable. More preferably, the bacterial fluid to be tested is filtered and sterilized by a 0.45um filter head before detection.
In the invention, the denitrification or solid uranium acclimation lasts for more than 5 periods, and the acclimation lasts for 6-10 periods. The one cycle of the present invention refers to: denitrogenation and domestication, the nitrate nitrogen concentration in the biochemical culture bottle is more than or equal to 15mg-N/L and is changed into NO 3 - Concentration is less than or equal to 2mg-N/L, NO 2 - The concentration is less than or equal to 0.1mg-N/L; and (4) uranium fixation and domestication, wherein the concentration of hexavalent uranium in a biochemical culture bottle is more than or equal to 300ug/L and is changed into the concentration of hexavalent uranium less than 50ug/L.
When the invention prefers to acclimatize and culture the hydrogen autotrophic denitrification uranium fixation microorganisms, after denitrification culture acclimatization, part of samples with nitrite accumulation and low nitrate treatment efficiency are eliminated, the remaining samples with better effect are expanded and cultured, denitrification acclimatization culture is carried out again for 6-10 periods, and then uranium fixation acclimatization culture is carried out.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A device for domesticating solid uranium by denitrification of a hydrogen autotrophic microorganism is shown in figure 1 and comprises: 1 hydrogen bottle, 2 water stop clips, 3 pneumatic joints, 4 plastic measuring cylinders, 5 glass measuring cylinders, 6 magnetic stirrers, 7 silent stirrers, 8 observation ports, 9 biochemical culture bottles and 10 tinfoil paper.
Two suitable screw thread openings are drilled on a plastic bottle cap and a plastic measuring cylinder 4 of a biochemical culture bottle 9 by screw taps, a pneumatic connector 3 is inserted, then silicone tubes with suitable lengths are connected to two ends of the pneumatic connector 3, the biochemical culture bottle 9 and the inverted measuring cylinder 4 are wrapped by rubber hoses to be connected in a combined mode, the rubber hoses on a gas injection opening and a sampling opening are clamped by a water stop clamp 2, and the device is built. Each connector of the device is sealed by special glue, and the air tightness of the device is checked after 48 hours, so that the air tightness is goodCan be put into use after being used. During the experiment, after pouring nutrient solution, bacterial solution and nitrate/uranium mother liquor into the culture, put into a noiseless stirring arm 7, pour into a proper amount of saturated sodium sulfite solution in the glass graduated flask, open the stagnant water clamp 2 of gas injection mouth on the inversion graduated flask, invert plastics graduated flask 4 in glass graduated flask 5, discharge the air in the inversion plastics graduated flask 4, then let in H from the air inlet 2 The partially saturated sodium sulfite solution was discharged and the H on the inverted plastic graduated cylinder 4 was recorded 2 And (5) initial calibration. The biochemical culture bottle 9 is wrapped by the tin foil paper 10 to manufacture a light-shading environment, the tin foil paper 10 is provided with an observation port 8 which can be flexibly opened and closed so as to know the growth condition of microorganisms in the biochemical culture bottle 9 and the stirring condition of the silent stirrer 7, and finally the biochemical culture bottle 9 is placed on the magnetic stirrer 6 for stirring, and the device is placed under a constant temperature for culture.
Example 2
The working process of the device for domesticating the denitrogenated and uranium-fixed by the hydrogen autotrophic microorganisms in the embodiment 1 comprises the following steps:
firstly, carrying out experiment preparation work, washing a biochemical culture bottle 9 with clear water for 5 times, washing for 10min with an ultrasonic cleaner, washing with the clear water for 5 times and ultrapure water for 3 times, and finally placing the biochemical culture bottle in an ultraviolet disinfection cabinet for sterilization for 1h; the following experiments are all operated on a sterile table, nutrient solution required by the hydrogen autotrophic microorganisms is prepared and sterilized by referring to the composition of underground water, and 1L of nutrient solution is poured into a biochemical culture bottle 9 after the cover is opened; then, inoculating the hydrogen autotrophic microorganism muddy water suspension into the nutrient solution, and putting a silent stirrer; then using CO 2 Aerating the polluted water body for 10min, driving out dissolved oxygen in the water body, adjusting the pH value to subacidity of underground water, providing an inorganic carbon source for the water body, and screwing down a cover of a biochemical culture bottle 9 after the completion; and (3) preparing the pretreated nitrate/uranium mother liquor, sterilizing, and injecting the nitrate/uranium mother liquor from a sample injection port of a biochemical culture bottle 9 by using an injector to simulate polluted underground water.
Then, pouring nearly full saturated sodium sulfite solution into a glass measuring cylinder 5, putting an inverted plastic measuring cylinder 4, opening a gas injection port water stop clamp 2, discharging air in the cylinder, and introducing H 2 Discharging partial saturated sulfurous acidSodium solution, record inverted cylinder H 2 And (3) scaling, placing the culture bottle on a magnetic stirrer for stirring, and placing the device at a constant temperature for culturing. Taking out a certain volume of the sample from the sampling port of the biochemical culture bottle 9 by a regular syringe to measure the change of concentration parameters of oxidative pollutants such as nitrate, nitrite and uranium and the change of conventional water quality parameters such as pH and DO, regularly recording the scale of the inverted plastic measuring cylinder 4, and obtaining the period H 2 The amount of consumption of (c).
After taking out a certain amount of bacteria liquid by an injector, the biochemical culture bottle is immediately supplemented with an equivalent amount of nutrient solution. When the measuring cylinder is inverted H 2 When the scale is low, the hydrogen tank 1 is used for filling a certain volume of H into the air inlet of the inverted plastic measuring cylinder 4 2 . And (3) taking out a certain amount of bacterial liquid in the biochemical culture bottle 9 again by using a sterilizing injector at certain intervals for measuring the change of concentration parameters of oxidative pollutants such as nitrate, nitrite and uranium and the change of conventional water quality parameters such as pH and DO.
The reduction condition of oxidative pollutants such as nitrate, uranium and the like can be measured by sampling at a sampling port at fixed time, and the change condition of other water quality parameters such as pH, DO, ORP and the like can also be measured, so that the growth condition of microorganisms can be inferred; when the growth environment of the hydrogen autotrophic microorganisms is severe or the hydrogen autotrophic microorganisms lack nutrients, pH buffer solution, nutrients and the like can be injected through the sampling port, so that the hydrogen autotrophic microorganisms in the biochemical culture bottle can grow and breed in a good environment.
Example 3
A domestication culture method of a hydrogen autotrophic microorganism for removing nitrogen and fixing uranium comprises the following steps:
(1) The method comprises the following steps of (1) establishing a synchronous denitrification and uranium fixation hydrogen autotrophic microorganism domestication device: the main bodies of the domestication device are a flowing liquid phase bottle, a 2000ml glass measuring cylinder and a 1000ml plastic measuring cylinder (as shown in figure 1), the container is respectively cleaned by clean water and ultrapure water for 5 times and 3 times, and then placed into an ultra-clean workbench for ultraviolet sterilization for 20-30 min;
(2) Preparing nutrient solution, putting the nutrient solution into a high-pressure steam sterilization pot at 121 ℃ for sterilization for 20-30 min, taking out the nutrient solution, putting the nutrient solution into a clean bench, and cooling to room temperature;
(3) Collecting a soil sample from an area without man-made interference, pouring 40g of the soil sample into a 1000ml beaker, and standing to form a solid-liquid critical surface;
(4) Extracting 1800ml of nutrient solution treated in the step (2) by using a sterile syringe, injecting the nutrient solution into the biochemical culture bottle treated in the step (1), extracting 200ml of muddy water mixture with the upper layer of 1-5 cm from the solid-liquid critical surface in the standing beaker in the step (3), and injecting the muddy water mixture into the biochemical culture bottle, wherein the inoculation amount is 10%, and the total volume of the mixed solution is kept at 2000ml; the nutrient solution preferably comprises the following components: k 2 HPO 4 220mg/L、KH 2 PO 4 260mg/L、 MgSO 4 ·7H 2 O 500mg/L、NaCl 120mg/L、CaCl 2 140mg/L、FeCl 3 ·6H 2 O240 mg/L, introducing CO into the biochemical culture flask 2 Providing a growing inorganic carbon source for autotrophic microorganisms, and adjusting the pH of the bacterial liquid to be about 6.2 together with the added sodium bicarbonate; adding a silent stirrer into a biochemical culture flask, and introducing CO 2 And (5) gas is used for 2min, pH is adjusted, and dissolved oxygen in the bacteria liquid and air on the upper layer of the biochemical culture bottle are discharged. Pouring 1500ml of saturated sodium sulfite solution into the glass measuring cylinder, opening a water stop clamp on the inverted measuring cylinder, and filling the inverted measuring cylinder with H not exceeding the maximum measuring range 2 Finally, placing the biochemical culture bottle on a magnetic stirrer, controlling the external temperature at 25 +/-1 ℃, and stirring at the rotating speed of 150 r/min; extracting 2ml of bacterial liquid every day, filtering the bacterial liquid by a 0.45um filter membrane, and determining NO in the bacterial liquid 3 - And NO 2 - Until NO 3 - And NO 2 - The content of the sodium nitrate is reduced to the lower limit of detection, a cycle is completed, and the sodium nitrate is supplemented to 15mg-N/L in time; taking 20ml of bacterial liquid one week for measuring pH, DO and other conventional water quality parameters, supplementing nutrient solution with corresponding volume into the biochemical culture bottle after sampling, and recording H every day 2 Timely supplement H into the inverted measuring cylinder 2 (ii) a To NO 3 - Is highly efficient and stable, and NO NO is generated during the treatment 2 - Accumulating to finish the denitrification and domestication process of the hydrogen autotrophic microorganisms; period NO 3 - And NO 2 - The dynamic graph of the detected concentration is shown in FIG. 2;
(5) Eliminate the effectExpanding four samples with relatively good effect for relatively poor samples, adding 500ml of stock solution, 1500ml of nutrient solution and 1.08g of sodium bicarbonate into each expanded culture sample, mixing, introducing CO 2 Culturing for 6-10 periods again until the denitrification effect is stable after 2min till saturation, and obtaining a hydrogen autotrophic microorganism sample with stable denitrification effect;
(6) Injecting 0.6ml of 1g/L uranium standard solution from a sampling port by using a sterilization syringe to ensure that the initial concentration of hexavalent uranium in the biochemical culture bottle is 300ug/L, operating according to the culture condition of the step (4), extracting 2ml of bacterial solution from the uniformly shaken biochemical culture bottle every day, filtering the bacterial solution by using a 0.45um filter membrane, measuring the content of hexavalent uranium, completing a period when the content of hexavalent uranium in the bacterial solution is lower than 50ug/L, and adding the bacterial solution again to 300ug/L hexavalent uranium for continuous domestication; the dynamic graph of the detected concentration of hexavalent uranium is shown in figure 3;
(7) Repeating the step (6) for 6-10 times until the effect of treating hexavalent uranium in the bacterial liquid is efficient and stable, and a large amount of yellow biological flocculates are generated in the reactor at the moment, so that the acclimation process of the synchronous denitrification uranium fixation hydrogen autotrophic microorganisms is completed;
the biochemical culture bottle is a flowing liquid phase bottle; the injectors used in the steps are all aseptic injectors, and the operation is carried out on an aseptic bench; charged H 2 And CO 2 All are high purity gases with a purity of 99.99%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A device for domesticating solid uranium by denitrogenation of hydrogen autotrophic microorganisms, which comprises a hydrogen supply H 2 Component, H 2 A transport member and a biochemical culture member, wherein the H is 2 The conveying component comprises a glass measuring cylinder and a plastic measuring cylinder which is inverted in the glass measuring cylinder, and the bottoms of the plastic measuring cylinders are respectively connected with a power supply H through pipe fittings 2 The device comprises a component and a biochemical culture component, wherein an oxygen-free solution is filled in a glass measuring cylinder.
2. The device for domestication of uranium by denitrification with a hydrogen autotrophic microorganism according to claim 1, further comprising CO 2 Aeration means for supplying CO 2 The aeration part is connected with the biochemical culture part through a pipe fitting.
3. The device for acclimatizing hydrogen autotrophic microorganisms to remove nitrogen and uranium according to claim 1, wherein the biochemical culture part is filled with a microorganism acclimation solution comprising a soil microorganism suspension, a microorganism nutrient solution, an inorganic carbon source and nitrate/uranium.
4. The device for domesticating the hydrogen autotrophic microorganisms to denitrify and consolidate the uranium according to claim 3, wherein the soil microorganism suspension is: mixing the soil and water, and standing to form a muddy water mixture at the position of 0-5cm of a solid-liquid critical plane; the microorganism nutrient solution is K 2 HPO 4 220mg/L、KH 2 PO 4 260mg/L、MgSO 4 ·7H 2 O 500mg/L、NaCl 120mg/L、CaCl 2 140mg/L、FeCl 3 ·6H 2 O240 mg/L aqueous solution.
5. The device for domestication of nitrogen and uranium by using a hydrogen autotrophic microorganism according to claim 1, further comprising a silent stirrer and a magnetic stirrer disposed at the bottom of the biochemical culture part.
6. The use of the device for acclimatization of the denitrification and uranium fixation by the photoautotrophic microorganism of any one of claims 1 to 5 in acclimatization of the denitrification and uranium fixation by the photoautotrophic microorganism or wastewater improvement.
7. A domestication method of a device for removing nitrogen and fixing uranium by using the hydrogenotrophic microorganism according to any one of claims 1 to 5, comprising the following steps:
loading soil microbe suspension, microbe nutrient solution and inorganic carbon source into biochemical culture part, and introducing H 2 And CO 2 ；
Adding nitrate into the biochemical culture part, and performing denitrification domestication to obtain the hydrogen autotrophic denitrification microorganism;
or adding uranium solution, and performing solid uranium acclimation to obtain the hydrogen autotrophic solid uranium microorganisms;
or carrying out denitrification domestication and then carrying out uranium fixation domestication to obtain the photoautotrophic denitrification uranium fixation microorganism.
8. The domestication method of claim 7, wherein the domestication process is carried out in a light-shielded environment, the domestication temperature is 25 ± 1 ℃, the pH of the microbial domestication liquid is 6-7, and the DO value is 0-2mg/L; the initial concentration of nitrate nitrogen in the denitrified and domesticated microorganism domestication liquid is more than or equal to 15mg-N/L; the initial concentration of hexavalent uranium in the solid uranium domesticated microorganism domestication liquid is more than or equal to 300ug/L.
9. The acclimatization method according to claim 7, wherein detection is performed during the acclimatization process: h 2 When the amount is less than or equal to 500ml, the H is supplemented 2 (ii) a CO supplementation at pH > 7 2 (ii) a Microorganism acclimatization liquid NO 3 - The concentration is less than or equal to 2mg-N/L and NO 2 - Supplementing nitrate when the concentration is less than or equal to 0.1mg-N/L; and (3) supplementing uranium solution when the concentration of hexavalent uranium in the microbial acclimation solution is less than or equal to 50ug/L.
10. The acclimation method according to claim 7, wherein the denitrification or uranium sequestration lasts for 5 cycles or more.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116493395A (en) * | 2023-03-30 | 2023-07-28 | 中核第四研究设计工程有限公司 | Uranium tailings microorganism treatment device |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5324491A (en) * | 1992-04-03 | 1994-06-28 | The United States Of America As Represented By The Secretary Of The Interior | Enzymatic reduction and precipitation of uranium |
| WO1998049106A1 (en) * | 1997-04-25 | 1998-11-05 | The University Of Iowa Research Foundation | Fe(o)-based bioremediation of aquifers contaminated with mixed wastes |
| JP2002143888A (en) * | 2000-11-09 | 2002-05-21 | Kurita Water Ind Ltd | Method for accumulating autotrophic denitrifying microorganism and biological method for removing nitrogen |
| CN201883094U (en) * | 2010-11-29 | 2011-06-29 | 山东建筑大学 | Determination device for performance of reducing oxidative pollutant in water by hydrogen autotrophic microorganism |
| CN103093847A (en) * | 2013-02-07 | 2013-05-08 | 南华大学 | Method of using native function microflora to in-situ purify ground water polluted by uranium |
| CN103359894A (en) * | 2013-08-06 | 2013-10-23 | 山东建筑大学 | Groundwater microbial denitrification system |
| WO2014082347A1 (en) * | 2012-11-27 | 2014-06-05 | 中国科学院沈阳应用生态研究所 | Method for processing drinking water contaminated by nitrates using two-stage modified active sludge and device therefor |
| CN107988133A (en) * | 2018-01-10 | 2018-05-04 | 桂林理工大学 | A kind of domestication and culture method of hydrogen autotrophic denitrification bacterium |
| CN108913540A (en) * | 2018-05-15 | 2018-11-30 | 西安建筑科技大学 | A kind of test micro anaerobic fermentation gas collecting apparatus and method |
| CN110328227A (en) * | 2019-03-22 | 2019-10-15 | 绍兴文理学院 | A kind of uranium contaminated soil autotrophy restorative procedure using siderite as electron donor |
-
2022
- 2022-06-15 CN CN202210675636.0A patent/CN115180724A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5324491A (en) * | 1992-04-03 | 1994-06-28 | The United States Of America As Represented By The Secretary Of The Interior | Enzymatic reduction and precipitation of uranium |
| WO1998049106A1 (en) * | 1997-04-25 | 1998-11-05 | The University Of Iowa Research Foundation | Fe(o)-based bioremediation of aquifers contaminated with mixed wastes |
| JP2002143888A (en) * | 2000-11-09 | 2002-05-21 | Kurita Water Ind Ltd | Method for accumulating autotrophic denitrifying microorganism and biological method for removing nitrogen |
| CN201883094U (en) * | 2010-11-29 | 2011-06-29 | 山东建筑大学 | Determination device for performance of reducing oxidative pollutant in water by hydrogen autotrophic microorganism |
| WO2014082347A1 (en) * | 2012-11-27 | 2014-06-05 | 中国科学院沈阳应用生态研究所 | Method for processing drinking water contaminated by nitrates using two-stage modified active sludge and device therefor |
| CN103093847A (en) * | 2013-02-07 | 2013-05-08 | 南华大学 | Method of using native function microflora to in-situ purify ground water polluted by uranium |
| CN103359894A (en) * | 2013-08-06 | 2013-10-23 | 山东建筑大学 | Groundwater microbial denitrification system |
| CN107988133A (en) * | 2018-01-10 | 2018-05-04 | 桂林理工大学 | A kind of domestication and culture method of hydrogen autotrophic denitrification bacterium |
| CN108913540A (en) * | 2018-05-15 | 2018-11-30 | 西安建筑科技大学 | A kind of test micro anaerobic fermentation gas collecting apparatus and method |
| CN110328227A (en) * | 2019-03-22 | 2019-10-15 | 绍兴文理学院 | A kind of uranium contaminated soil autotrophy restorative procedure using siderite as electron donor |
Non-Patent Citations (4)
| Title |
|---|
| 徐思雯等: "氢自养还原菌同步去除水中Cr(Ⅵ)和NO3-", 中国环境科学, vol. 40, no. 01, pages 31 - 33 * |
| 杨升等;: "某尾矿库附近各土壤层对U(Ⅵ)的吸附特性", 环境工程学报, vol. 13, no. 10 * |
| 王豫琪等: "基于碳酸氢钠为碳源的氢自养反硝化去除地下水中硝酸盐研究", 农业环境科学学报, vol. 30, no. 03, pages 564 - 572 * |
| 白也;张多英;曾伟民;赵丹丹;张彦龙;张淑梅;: "反硝化菌在地下水中除硝酸盐的应用进展", 生物技术, no. 04, pages 403 - 409 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116493395A (en) * | 2023-03-30 | 2023-07-28 | 中核第四研究设计工程有限公司 | Uranium tailings microorganism treatment device |
| CN116493395B (en) * | 2023-03-30 | 2023-11-07 | 中核第四研究设计工程有限公司 | Uranium tailings microorganism treatment device |
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