CN112391375A - Preparation method and application of biochar immobilized denitrifying bacteria for rapidly removing nitrate in water body - Google Patents
Preparation method and application of biochar immobilized denitrifying bacteria for rapidly removing nitrate in water body Download PDFInfo
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- 238000010008 shearing Methods 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
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- 150000002823 nitrates Chemical class 0.000 claims 8
- 239000001509 sodium citrate Substances 0.000 claims 1
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- 241000475042 Stolonifera Species 0.000 description 1
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- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 description 1
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/28—Treatment of water, waste water, or sewage by sorption
- C02F1/283—Treatment of water, waste water, or sewage by sorption using coal, charred products, or inorganic mixtures containing them
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/28—Anaerobic digestion processes
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/163—Nitrates
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Abstract
The invention discloses a method for quickly removing nitrate in water by using biochar immobilized denitrifying bacteria. The method comprises the following specific steps: firstly, screening high-efficiency denitrifying bacteria to obtain 1 strain of Pseudomonas stutzeri (Pseudomonas stutzeri) N3; and co-culturing the bacterial suspension of N3 and biochar, adsorbing and immobilizing, and washing with normal saline to obtain the biochar immobilized denitrifying bacteria. The method utilizes the biochar as the carrier to immobilize the denitrifying bacteria, not only has the advantages of wide material source, simple preparation, low cost, good treatment effect and the like, but also realizes the resource utilization of waste. According to the invention, the biological carbon is used for immobilizing the denitrifying bacteria, and the removal effect of the immobilized nitrate is obviously superior to that of the single biological carbon adsorption and denitrifying bacteria degradation, so that the method has an important significance for quickly removing the nitrate in the water body.
Description
Technical Field
The invention relates to a preparation method and application of biochar immobilized denitrifying bacteria for quickly removing nitrate in a water body, and belongs to the technical field of environmental bioremediation.
Background
In recent years, with frequent occurrence of eutrophication of lakes, people have come to recognize the importance of controlling nutrient salts such as nitrogen, phosphorus, etc. in water. Nitrate is the predominant form of nitrogen present in wastewater treatment plant effluent and is considered to be one of the major pollutants responsible for eutrophication. Large amounts of nitrate in lakes may cause eutrophication, while too high a nitrate content in drinking water may cause cancer and harm human health.
The denitrification can convert nitrate nitrogen into nitrogen to be removed from a water body under the action of microorganisms, has the advantages of low investment, good effect, no secondary pollution and the like, and is the most economic and effective denitrification mode at present. However, in the practical application process, the free bacteria often have certain limitations, such as poor environmental tolerance, easy loss of strains, and the like. The microorganism immobilization technology can effectively solve the problem of strain loss by immobilizing free microorganisms in or on a carrier material. Meanwhile, the technology also has the advantages of high microbial activity, good environmental tolerance, high treatment efficiency, easy separation of products and the like, and is widely applied to the treatment of polluted water at present.
As a novel functional material, the biochar has a developed pore structure and a huge specific surface area, and is rich in functional groups such as hydroxyl, carboxyl, lactone and the like, so that the biochar has good adsorption performance on various pollutants in the environment. Meanwhile, the developed microporous structure on the surface of the biochar provides sufficient growth space for microorganisms, so that the biochar is prevented from being interfered by external adverse environment; the biochar contains a large amount of nutrient elements and can be used for the growth of microorganisms, and the conditions are provided for the immobilization of the microorganisms. In addition, the biochar is an effective means for plant resource utilization, has the advantages of simple preparation process, easily available materials, low price, good treatment effect and the like, and is an ideal immobilized carrier. At present, the biochar immobilization technology has good application prospect in the aspect of polluted water treatment.
Disclosure of Invention
The invention aims to provide a preparation method of biochar immobilized denitrifying bacteria aiming at the defects of free denitrifying bacteria in water treatment application, which realizes the resource utilization of plants and provides a new idea for removing nitrate in water in the future by combining biochar adsorption and the degradation of the denitrifying bacteria while improving the effect of quickly removing the nitrate in the water.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a preparation method of biochar immobilized denitrifying bacteria for quickly removing nitrate in a water body, which comprises the steps of firstly screening high-efficiency denitrifying bacteria to obtain 1 strain of Pseudomonas stutzeri N3; and co-culturing the bacterial suspension of N3 and biochar, adsorbing and immobilizing, and washing with normal saline to obtain the biochar immobilized denitrifying bacteria. The method comprises the following specific steps:
the method comprises the following steps: culturing denitrifying bacteria in a denitrifying culture medium at 30 ℃ and 160rpm, centrifuging after 24 hours, discarding supernatant, and repeatedly washing with sterile normal saline for 3 times to obtain denitrifying bacteria;
step two: resuspending the denitrifying bacteria in sterile physiological saline and adjusting OD600Obtaining a denitrifying bacteria suspension with the concentration of about 1;
step three: mixing the biochar with the denitrifying bacteria suspension, culturing for 24h in a denitrifying culture medium at 30 ℃ and 160rpm, and washing free bacteria on the surface of the biochar by using sterile normal saline to obtain the biochar immobilized denitrifying bacteria.
According to the present invention, preferably, the denitrifying bacteria is denitrifying bacteria N3, the denitrifying bacteria N3 is Pseudomonas stutzeri (Pseudomonas stutzeri), and the culture is preserved in the chinese typical culture collection center (CCTCC), the preservation address is: china, wuhan university, zip code: 430072, strain preservation number M2015027.
According to the invention, the formulation of the denitrification medium is as follows: NaNO30.30g of citric acid IIISodium 1.72g, KH2PO4 1.0g，FeSO4·7H2O 0.03g，CaCl2·2H2O 0.123g，MgSO4·7H2O 0.3g，H2O1000 ml, pH 7.0 or so.
According to the invention, the mixing ratio of the biochar to the denitrifying bacteria in the step three is preferably 1: 60 (w/v).
According to the present invention, the biochar involved in the third step is preferably pine tree charcoal, peanut shell charcoal, pennisetum stolonifera charcoal, or reed charcoal.
According to the present invention, preferably, the above biochar is prepared by the following method:
the method comprises the following steps: cleaning raw materials for preparing the biochar with clear water, shearing, and drying.
Step two: putting the biochar raw materials into a crucible, compacting and tightly covering, raising the temperature to 600 ℃ in a muffle furnace at a speed of 10 ℃/min, and carbonizing for 3 hours at constant temperature.
Step three: soaking the biochar in 1mol/L hydrochloric acid for 2h to remove surface ash, repeatedly washing with deionized water, and drying to constant weight.
Step four: and grinding and sieving the biochar, and keeping the biochar with the diameter of 0.5-1 mm.
The principle of the invention is that the biochar has a developed pore structure, large specific surface area and rich surface functional groups, thereby showing good adsorption effect on nitrate in water. The prepared biological carbon immobilized denitrifying bacteria are added into a high-concentration nitrate water body by taking the biological carbon as an immobilized carrier, so that the migration of nitrate to the biological carbon carrier is promoted, a large amount of denitrifying bacteria and nitrate are simultaneously enriched on the surface of the biological carbon, and the contact between the biological carbon immobilized denitrifying bacteria and the nitrate is increased; in addition, the biochar can accelerate the removal of nitrate by promoting the electron transfer action of microorganisms.
The invention has the beneficial effects that:
1. the denitrifying bacteria N3 are fixed on the biochar carrier and are used for removing nitrate in water, and the nitrate removing effect/removing efficiency is obviously improved compared with that of simple biochar adsorption and free bacteria degradation;
2. according to the invention, the biological carbon is used as a carrier to immobilize the denitrifying bacteria, the preparation material of the biological carbon has wide source, the preparation process is simple, the cost is low, and the problem of resource utilization of plants can be effectively solved. Meanwhile, the biochar has stable property, can play a role in carbon sequestration and emission reduction in the production and use processes, and has important effects on improving the greenhouse effect and slowing down global warming.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 is a scanning electron micrograph (10000 times magnification) of each of the biochar-immobilized denitrifying bacteria;
FIG. 2 shows the effect of free denitrifying bacteria N3 on nitrate removal;
FIG. 3 shows the effect of nitrate removal before and after pine shell carbon immobilization;
FIG. 4 shows the effect of removing nitrate before and after immobilization of peanut shell carbon;
FIG. 5 shows the effect of removing nitrate before and after immobilization of the carbon of the grass husk of Conyza sativa;
FIG. 6 shows the effect of nitrate removal before and after reed carbon immobilization.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the examples are only for illustrating the present invention, and the present invention is by no means limited thereto.
Example 1: nitrate removal effect of pine tree charcoal immobilized denitrifying bacteria
Cleaning pine branches with clear water, cutting into small segments of about 5cm, and drying in an oven at 100 deg.C. And putting the dried pine branches into a crucible, compacting and tightly covering, wrapping with tinfoil to ensure that the pine branches are isolated from air, heating to 600 ℃ in a muffle furnace at a speed of 10 ℃/min, and carbonizing for 3h at constant temperature to prepare the pine charcoal. The prepared pine tree charcoal is soaked for 2 hours by 1mol/L hydrochloric acid to wash away surface ash, and is dried to constant weight after being repeatedly washed by deionized water. Grinding and sieving pine tree charcoal, and keeping biochar with the diameter of 0.5-1 mm.
Culturing denitrifying bacteria N3 in denitrifying culture medium at 30 deg.C and 160rpm, centrifuging after 24 hr, removing supernatant, and repeatedly washing with sterile normal saline for 3 times; resuspending denitrifying bacteria N3 in sterile physiological saline, and adjusting OD600Is about 1; 0.08g of pine tree charcoal and a suspension of denitrifying bacteria were mixed according to a ratio of 1: 60(w/v), culturing at 30 ℃ and 160rpm for 24 hours, and adsorbing and immobilizing the denitrifying bacteria. After immobilization is finished, standing the sample, carrying out solid-liquid separation, removing supernatant, and washing free bacteria on the surface of the pine tree charcoal by using sterile normal saline to obtain pine tree charcoal immobilized denitrifying bacteria; the existence of denitrifying bacteria can be clearly seen on the surface of pine tree charcoal, which is shown in FIG. 1 (a).
Adding immobilized denitrifying bacteria prepared from 0.08g pine tree charcoal into 20ml NO with concentration of 15mg/L3 -in-N solution, at 30 ℃ and 160rpm, NO at 8h, as shown in FIG. 33 --N removal 72.15%, average removal rate 1.35 mg/L/h; as a control, as shown in FIG. 2, the denitrifying bacteria NO was dissociated under the same conditions3 --N removal was 46.21%, average removal rate was 0.78 mg/L/h; while the removal rate of nitrate by simple pine charcoal adsorption is 6.67%, and the average removal rate is 0.13 mg/L/h. Compared with free bacteria and pure pine tree charcoal, the immobilized denitrifying bacteria can be used for NO3 -The removal rate of-N is faster and reaches 1.73 times and 10.38 times of that of free bacteria and pine charcoal only. Free bacterium pair NO after 24h3 -The removal rate of-N reaches 70.74%, the removal rate of pure pine charcoal is only 11.2%, and the removal rate of immobilized denitrifying bacteria reaches 81.06%, which are respectively improved by 10.32% and 69.86% compared with the adsorption of free bacteria and biochar.
Example 2: nitrate removal effect of peanut shell carbon immobilized denitrifying bacteria
Peanut shell charcoal was prepared from peanut shells as the starting material in the manner described in example 1.
Culturing denitrifying bacteria N3 in denitrifying culture medium at 30 deg.C and 160rpm, centrifuging after 24 hr, and discardingCleaning, and repeatedly washing with sterile normal saline for 3 times; resuspending denitrifying bacteria N3 in sterile physiological saline, and adjusting OD600Is about 1; mixing 0.08g of peanut shell carbon and denitrifying bacteria suspension according to the weight ratio of 1: 60(w/v), culturing at 30 ℃ and 160rpm for 24 hours, and adsorbing and immobilizing the denitrifying bacteria. And (3) after the adsorption and immobilization are finished, standing the sample, separating solid from liquid, removing supernatant, washing free bacteria on the surface of the peanut shell carbon by using sterile normal saline to obtain the peanut shell carbon immobilized denitrifying bacteria, which is shown in figure 1 (b).
Adding 0.08g of immobilized denitrifying bacteria prepared from peanut shell carbon into 20ml of NO with the concentration of 15mg/L3 -in-N solution, at 30 ℃ and 160rpm, NO at 8h, as shown in FIG. 43 -The N removal rate reaches 65.53 percent, and the average removal rate is 1.23 mg/L/h; as a control, denitrifying bacteria NO was dissociated under the same conditions3 --N removal was 46.21%, average removal rate was 0.78 mg/L/h; while pure peanut shell charcoal is almost to NO3 -N has no removal effect. Peanut shell carbon immobilized denitrifying bacteria NO pair3 -The average removal rate of-N is 1.57 times that of the free bacteria, and is obviously higher than that of the free bacteria. But 24 hours later, the final NO of the peanut shell carbon immobilized denitrifying bacteria and the free denitrifying bacteria3 -The N removal rates are not very different and are all about 70 percent.
Example 3: removal effect of round coin grass carbon immobilized denitrifying bacteria on nitrate
The pennisetum purpureum biochar was prepared from pennisetum purpureum as a raw material in the manner described in example 1.
Culturing denitrifying bacteria N3 in denitrifying culture medium at 30 deg.C and 160rpm, centrifuging after 24 hr, removing supernatant, and repeatedly washing with sterile normal saline for 3 times; resuspending denitrifying bacteria N3 in sterile physiological saline, and adjusting OD600Is about 1; mixing 0.08g of the carbon paniculate and the denitrifying bacteria suspension according to the weight ratio of 1: 60(w/v), culturing at 30 deg.C and 160rpm for 24 hr, and adsorbing and fixing denitrifying bacteria. After the adsorption and immobilization are finished, standing the sample, separating solid from liquid, discarding the supernatant, and washing the round coin peat with sterile normal salineAfter surface dissociation of the bacteria, the sphagnum teres carbon immobilized denitrifying bacteria are obtained, which is shown in figure 1 (c).
Adding 0.08g of immobilized denitrifying bacteria prepared from the carbon of the pennisetum sinese Roxb into 20ml of NO with the concentration of 15mg/L3 -in-N solution, at 30 ℃ and 160rpm, NO at 8h, as shown in FIG. 53 -The N removal rate reaches 94.83 percent, and the average removal rate is 1.77 mg/L/h; as a control, free denitrifying bacteria NO was obtained under the same conditions3 -The N removal rate is 46.21 percent, the average removal rate is 0.78mg/L/h, and the simple Tunica chunka removal rate is 46.35 percent, and the average removal rate is 0.86 mg/L/h. Compared with free bacteria and pure Tunica terniflora charcoal, the immobilized denitrifying bacteria can be used for NO3 -The removal rate of-N is faster, and is 2.26 times and 2.05 times of that of the free bacteria and the turfgrass carbon respectively. Free bacterium pair NO after 24h3 -The removal rate of-N is 70.74%, the removal rate of pure Tunica chuangnensis charcoal is only 46.48%, and the removal rate of immobilized denitrifying bacteria reaches 100%, which is respectively increased by 29.26% and 53.52% compared with the adsorption of free bacteria and biochar.
Example 4: nitrate removal effect of reed carbon immobilized denitrifying bacteria
According to the mode described in the embodiment 1, the reed biochar is prepared by taking reed as a raw material.
Culturing denitrifying bacteria N3 in denitrifying culture medium at 30 deg.C and 160rpm, centrifuging after 24 hr, removing supernatant, and repeatedly washing with sterile normal saline for 3 times; resuspending denitrifying bacteria N3 in sterile physiological saline, and adjusting OD600Is about 1; mixing 0.08 reed carbon and denitrifying bacteria suspension according to the weight ratio of 1: 60(w/v), culturing at 30 deg.C and 160rpm for 24 hr, and adsorbing and fixing denitrifying bacteria. And (3) after the adsorption and immobilization are finished, standing the sample, separating solid from liquid, removing supernatant, washing free bacteria on the surface of the reed carbon by using sterile normal saline to obtain the reed carbon immobilized denitrifying bacteria, wherein the details are shown in figure 1 (d).
Adding 0.08g of immobilized denitrifying bacteria prepared from reed carbon into 20ml of NO with the concentration of 15mg/L3 -in-N solution, at 30 ℃ and 160rpm, NO at 8h, as shown in FIG. 63 -The N removal rate was 61.62%, the average removal rate was 1.15 mg/L/h; as a control, free denitrifying bacteria NO was obtained under the same conditions3 -The N removal rate is 46.21 percent, the average removal rate is 0.78mg/L/h, and the pure reed carbon removal rate is 31.30 percent, and the average removal rate is 0.59 mg/L/h. Compared with free bacteria and pure reed carbon, the immobilized denitrifying bacteria pair NO3 -The removal rate of-N is faster, 1.47 times and 1.94 times of the two. After 24h, the reed carbon immobilized denitrifying bacteria and the free bacteria pair NO3 -The N removal rate reaches about 70 percent, and the pure Tuochen turfgrass removal rate is only 47.68 percent.
The present invention provides a method for preparing biochar immobilized denitrifying bacteria, but the above examples are only used to illustrate the technical scheme of the present invention but not to limit, and the methods and ways for implementing the technical scheme are many, and it should be considered as the protection scope of the present invention for those skilled in the art to make equivalent changes and modifications according to the claims of the present invention.
Claims (8)
1. A preparation method of biochar immobilized denitrifying bacteria for quickly removing nitrate in a water body is characterized by comprising the following steps:
the method comprises the following steps: culturing the denitrifying bacteria in a denitrifying culture medium, centrifuging, removing supernatant, and washing with sterile normal saline to obtain denitrifying bacteria;
step two: resuspending the denitrifying bacteria obtained in the step one in sterile physiological saline, and adjusting OD600Then obtaining denitrifying bacteria suspension;
step three: and (3) mixing the biochar with the denitrifying bacteria suspension obtained in the second step, and washing free bacteria on the surface of the biochar by using sterile normal saline after culture to obtain the biochar immobilized denitrifying bacteria.
2. The method for preparing biochar immobilized denitrifying bacteria for rapidly removing nitrates from water bodies as claimed in claim 1, wherein the culturing and treating conditions of the denitrifying bacteria in the first step are as follows: denitrifying bacteria speciesCulturing in 15mg/L denitrifying culture medium at 30 deg.C and 160rpm, centrifuging after 24 hr, discarding supernatant, and repeatedly washing with sterile physiological saline for 3 times; adjusting OD as described in step two600Is 1; the culture conditions in the third step are as follows: culturing at 30 deg.C and 160rpm for 24 h.
3. The method for preparing the biochar immobilized denitrifying bacteria for rapidly removing the nitrates from the water body according to claim 1 or 2, wherein the denitrifying bacteria is denitrifying bacteria N3, denitrifying bacteria N3 is Pseudomonas stutzeri (Pseudomonas stutzeri), and the strain preservation number is M2015027.
4. The preparation method of the biochar immobilized denitrifying bacteria for rapidly removing the nitrates from the water body according to the claim 1 or 2, wherein the formula of the denitrifying culture medium is as follows: NaNO30.30g, trisodium citrate 1.72g, KH2PO41.0g，FeSO4·7H2O 0.03g，CaCl2·2H2O 0.123g，MgSO4·7H2O 0.3g，H2O1000 ml, pH 7.0.
5. The preparation method of the biochar immobilized denitrifying bacteria for rapidly removing the nitrates from the water body as claimed in claim 1, wherein the mixing ratio of the biochar to the denitrifying bacteria in step three is 1: 60 (w/v).
6. The preparation method of the biochar immobilized denitrifying bacteria for rapidly removing nitrates from water bodies according to claim 1 or 5, wherein the biochar immobilized denitrifying bacteria comprise: the biochar in the third step is one of pine tree carbon, peanut shell carbon, pennisetum purpureum carbon and reed carbon.
7. The preparation method of the biochar immobilized denitrifying bacteria for rapidly removing the nitrates from the water body as claimed in claim 6, wherein: the biochar is prepared by the following method:
the method comprises the following steps: cleaning raw materials for preparing the biochar with clear water, shearing, and drying;
step two: putting all the biochar raw materials in a crucible, compacting and tightly covering, raising the temperature to 600 ℃ in a muffle furnace at a speed of 10 ℃/min, and carbonizing for 3 hours at constant temperature;
step three: soaking the biochar in 1mol/L hydrochloric acid for 2 hours to remove surface ash, repeatedly washing with deionized water, and drying to constant weight;
step four: and grinding and sieving the biochar, and keeping the biochar with the diameter of 0.5-1 mm.
8. The application of the biochar immobilized denitrifying bacteria prepared by the preparation method of the biochar immobilized denitrifying bacteria for quickly removing the nitrates in the water body according to any one of claims 1 to 7 in denitrification of the water body with high-concentration nitrates.
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