CN113980830B - Pseudomonas stutzeri, culture thereof and application thereof - Google Patents
Pseudomonas stutzeri, culture thereof and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of water or soil remediation, in particular to pseudomonas stutzeri, a culture thereof and application thereof. The preservation number of the pseudomonas stutzeri is CGMCC No.22623. The pseudomonas stutzeri can effectively convert nitrate radicals, improve the content of nitrogen in soil and is beneficial to soil remediation. Meanwhile, the pseudomonas stutzeri can take nitrate as an electron acceptor under anaerobic or anoxic conditions to biologically oxidize ferrous iron through electron transfer, so that ferrous iron forms iron minerals to promote the utilization of iron, the formed iron minerals can adsorb heavy metals, and then the heavy metals in water or soil are removed to further repair the water or soil.
Description
Technical Field
The invention relates to the technical field of water or soil remediation, in particular to pseudomonas stutzeri, a culture thereof and application thereof.
Background
Denitrification is an important microbial driving process, and the denitrification process and the ferrous iron oxidation process not only affect the geochemical process of iron and nitrogen, but also have important influence on the environmental behaviors of other elements, such as carbon circulation and heavy metal conversion.
In natural environment, the chemical oxidation speed of ferrous iron by nitrate is very slow, and unless the mediation of microorganisms exists, under the action of the microorganisms, nitrate which is stably existed in soil in a large amount can be converted into nitrogen which is directly absorbed and utilized by plants, thereby promoting the circulation of nitrogen and increasing the agricultural benefit. Because divalent iron is coupled and oxidized simultaneously in the nitrate reduction process to generate iron hydroxide (oxide), and the biological iron mineral can promote the adsorption and coprecipitation of arsenic due to the characteristics of low crystallinity, large specific surface area and many active points, thereby reducing the risk to the environment. The silver city in Gansu province is a typical industrial and mining city, and the long-term exploitation and smelting of a large amount of ores cause serious water and soil environmental pollution to the surrounding environment, so that a bacterium meeting the following requirements is urgently needed: microbial-mediated biomineralization can be carried out in arid regions of agricultural land which lack divalent iron and nitrate radical, or nitrate reduction and divalent iron oxidation can be carried out in deep anaerobic or anoxic environments in water and soil, so that nitrate radical and arsenic pollution is reduced.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide pseudomonas stutzeri, a culture thereof, application thereof, an environment repairing agent and a water and soil environment repairing method. The embodiment of the invention provides a novel pseudomonas stutzeri, which can effectively convert nitrate radicals and improve the content of nitrogen in soil, and simultaneously can convert the nitrate radicals and oxidize ferrous iron to form iron minerals to promote the utilization of iron, and the formed iron minerals can adsorb heavy metals, and then remove the heavy metals in water or soil to further repair the water or soil.
The invention is realized in the following way:
in a first aspect, the invention provides pseudomonas stutzeri with the preservation number of CGMCC No.22623.
Specifically, the strain has been stored in the general microbiological culture collection center of China Committee for culture Collection of microorganisms (CGMCC, institute No. 3 of West Lu 1 of the Kyoho, beijing, tokyo, chaoyang, ministry of microbiology, 100101) at 31/05/2021, and is named after classification: pseudomonas stutzeri (Pseudomonas stutzeri) with a preservation number of CGMCC No.22623.
In a second aspect, the present invention provides a culture of Pseudomonas stutzeri, which is prepared by culturing Pseudomonas stutzeri according to the preceding embodiment.
In an alternative embodiment, the process of culturing the pseudomonas stutzeri comprises: inoculating the pseudomonas stutzeri into an ore-forming culture medium containing nitrate and ferrous ions for culture;
preferably, the process of culturing the pseudomonas stutzeri comprises: inoculating the pseudomonas stutzeri to the mineralizing culture medium, and then carrying out aerobic culture and then carrying out anoxic culture;
preferably, the process of culturing the pseudomonas stutzeri comprises: inoculating the pseudomonas stutzeri to the mineralization culture medium, then carrying out aerobic culture for 1-1.5 days at the temperature of 23-27 ℃, and then carrying out anoxic continuous culture at the temperature of 23-27 ℃.
In alternative embodiments, the pseudomonas stutzeri is inoculated at a rate of 1-10%;
preferably, the mineralizing medium is a liquid medium;
preferably, the mineralizing medium comprises nitrate and ferrous salts;
preferably, the mineralization medium further comprises a pH buffer, a nitrogen source, a carbon source, a magnesium source, a calcium source, and a sulfate;
preferably, the mineralizing medium comprises 0.3-0.7g/L potassium dihydrogen phosphate, 0.3-0.7g/L sodium nitrate, 0.3-0.7g/L calcium chloride, 0.3-0.7g/L magnesium sulfate, 0.3-0.7g/L ammonium sulfate and 8-12g/L ferric ammonium citrate.
In an alternative embodiment, the process of culturing the pseudomonas stutzeri comprises: activating the pseudomonas stutzeri to obtain a bacterial suspension, and then inoculating the bacterial suspension into an ore-forming culture medium for culture;
preferably, the activation treatment comprises: inoculating the pseudomonas stutzeri into a nutrient broth culture medium for culture, so that the number of bacteria of the pseudomonas stutzeri reaches the logarithmic growth phase, and then centrifuging to obtain a bacteria suspension;
preferably, the OD of the bacterial suspension 600 Is 0.8-1.2.
In a third aspect, the present invention provides the use of a culture of Pseudomonas stutzeri according to the preceding embodiment or of Pseudomonas stutzeri according to any of the preceding embodiments in at least one of the following situations;
(1) Nitrate conversion in water and soil environment; (2) oxidizing ferrous ions in a water and soil environment; and (3) arsenic remediation in the water and soil environment. The water and soil environment mentioned in the embodiment of the invention includes polluted water environment or polluted soil environment.
In a fourth aspect, the present invention provides an environmental remediation agent comprising a culture of pseudomonas stutzeri according to the preceding embodiment or pseudomonas stutzeri according to any one of the preceding embodiments.
In a fifth aspect, the present invention provides a method for remediating an aqueous or soil environment, comprising performing water or soil remediation using a culture of pseudomonas stutzeri according to the foregoing embodiment or pseudomonas stutzeri according to any one of the foregoing embodiments.
In an alternative embodiment, the method comprises the following steps: mixing said Pseudomonas stutzeri or a culture of Pseudomonas stutzeri with water or soil containing nitrate and ferrous ions;
preferably, it comprises: mixing the pseudomonas stutzeri, raw materials containing nitrate radical and ferrous ion and water or soil.
The invention has the following beneficial effects: the embodiment of the invention provides a novel pseudomonas stutzeri, which can effectively convert nitrate radicals, improve the content of nitrogen in soil and is beneficial to soil remediation. Meanwhile, the pseudomonas stutzeri can take nitrate radical as an electron acceptor under the anoxic (anaerobic or oxygen-free) condition to carry out biological oxidation on ferrous iron through electron transfer, so that ferrous iron forms iron minerals to promote the utilization of iron, the formed iron minerals can adsorb and coprecipitate heavy metals, and then the heavy metals in water or soil are removed to further repair the water or soil.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a surface morphology of a colony of Pseudomonas stutzeri provided in an embodiment of the present invention;
FIG. 2 is a tree diagram of the phylogenetic 16S rRNA gene of Pseudomonas stutzeri provided by an embodiment of the present invention;
FIG. 3 is a graph showing the results of the nitrate reduction and ferrous iron oxidation process by Pseudomonas stutzeri provided in example 2 of the present invention;
FIG. 4 is a graph showing the effect of Pseudomonas stutzeri on the removal of trivalent arsenic and total arsenic from a solution according to example 3 of the present invention;
FIG. 5 is a graph showing the effect of Pseudomonas stutzeri on the removal of trivalent arsenic from a solution under different oxygen conditions according to example 4 of the present invention;
FIG. 6 is a scanning electron micrograph of a culture of Pseudomonas stutzeri provided in example 4 of the present invention;
FIG. 7 is a graph comparing the X-ray diffraction pattern of a culture of Pseudomonas stutzeri provided in example 4 of the present invention with goethite and lepidocrocite standard cards;
FIG. 8 is a graph showing the results of remediation of arsenic-contaminated farmland soil by Pseudomonas stutzeri provided in example 5 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
This example provides Pseudomonas stutzeri (hereinafter, this Pseudomonas stutzeri is designated as Pseudomonas LZU-2) with a preservation number of CGMCC No.22623.
This example also provides a culture of Pseudomonas stutzeri, which was prepared by culturing the Pseudomonas stutzeri. The specific culture process is as follows:
activating the pseudomonas stutzeri to obtain a bacterial suspension, specifically, picking a few bacteria from a flat plate for storing strains into a nutrient broth culture medium, culturing to enable the number of the bacteria of the pseudomonas stutzeri to reach a logarithmic growth phase, and then centrifuging to obtain the bacterial suspension; OD of bacterial suspension 600 Is 0.8-1.2。
Further, a few of the bacteria are picked from a plate storing the strain into a nutrient broth, the bacteria are activated by shaking at 23 to 27 ℃ (for example, at any value between 23 ℃ and 27 ℃ such as 23 ℃, 24 ℃,25 ℃, 26 ℃ and 27 ℃), at 120 to 170rpm (at any value between 120rpm and 170rpm such as 120rpm, 130rpm, 140rpm, 150rpm, 160rpm and 170 rpm) for 14 to 18h (at any value between 14 h 15h, 16h, 17h, 18h and 19h 14 to 18 h) to allow the number of the bacteria to reach the logarithmic phase, the bacteria are centrifuged at 3000 to 5000r/min (at any value between 3000r/min, 4500r/min and 5000r/min 3000 to 5000 r/min) for 5 to 15min (5 min, 6 min, 7 min, 8 min, 9 min, 10min, 11 min, 12 min, 13 min, 14 min and 15 min) by a centrifuge, the supernatant is removed, 0.8% sterile sodium chloride solution (OD/v) is added, and the above operation is repeated 2 times to prepare a suspension 600 Is 0.8-1.2. Wherein the nutrient broth medium comprises 10.0g/L peptone, 3.0g/L beef extract, 5g/L sodium chloride, and 2mol/L hydrochloric acid to adjust pH to 7.0.
It should be noted that (1) the above culture conditions, including the culture temperature, the rotation speed of the shaker, the culture time, the centrifugation time, the rotation speed, etc., are not limited to the above ranges, and other conditions in the prior art are also possible, and the above ranges including the point values are all examples of the preferable effects of the embodiments of the present invention.
(2) Other prior art nutrient broth cultures capable of activating the preserved strain are within the scope of the embodiments of the present invention. The components of the nutrient broth culture medium and the mixture ratio of the components are only examples of the embodiment with good effect.
The bacterial suspension is inoculated into an ore-forming culture medium for culture, and any inoculation amount can be used for inoculation, but the inoculation ratio of the pseudomonas stutzeri is 1-10% (V/V), for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and other values of 1-10%, so that a better culture effect can be achieved, and the pseudomonas stutzeri can be obtained.
Specifically, the culturing process comprises: inoculating the pseudomonas stutzeri into an ore-forming culture medium containing nitrate and ferrous ions for culture; wherein, the culture process can be aerobic culture and then anoxic culture, or continuous anoxic culture, only the growth rate of the pseudomonas stutzeri is slower during the continuous anoxic culture or the anaerobic culture, the required culture time is longer, the obtained culture product is less, the subsequent water or soil remediation effect is poorer, and the continuous aerobic culture oxygen and nitrate radical generate competition effect on an electron acceptor to inhibit the reduction process of nitrate radical. However, the prior aerobic culture followed by the anaerobic culture can enable the pseudomonas stutzeri to be rapidly formed, thereby being beneficial to improving the effect of the pseudomonas stutzeri.
Specifically, the Pseudomonas stutzeri is inoculated to the mineralization medium, and then is subjected to aerobic culture for 1 to 1.5 days under the condition of 23 to 27 ℃ (for example, any value between 23 and 27 ℃ such as 23 ℃, 24 ℃,25 ℃, 26 ℃ and 27 ℃), and then is subjected to anaerobic continuous culture under the condition of 23 to 27 ℃ (for example, any value between 23 and 27 ℃ such as 23 ℃, 24 ℃,25 ℃, 26 ℃ and 27 ℃). By adopting the method, the growth of the pseudomonas stutzeri in the mineralization culture medium can be further ensured, the conversion of nitrate is further facilitated, the ferrous iron is oxidized to form weak crystalline iron mineral, and the iron mineral can effectively remove arsenic.
It should be noted that, the aerobic culture mentioned in the embodiments of the present invention is performed in an unsealed atmosphere environment, and the anaerobic culture is performed in an anoxic or anaerobic environment, and is performed in a sealed environment without introducing additional oxygen.
Further, the mineralization culture medium is a liquid culture medium; the mineralization culture medium comprises nitrate and ferrous salt; specifically, the mineralization culture medium further comprises a pH buffer solution, a nitrogen source, a carbon source, a magnesium source, a calcium source and sulfate radicals; for example, the mineralizing medium includes 0.3-0.7g/L potassium dihydrogen phosphate, 0.3-0.7g/L sodium nitrate, 0.3-0.7g/L calcium chloride, 0.3-0.7g/L magnesium sulfate, 0.3-0.7g/L ammonium sulfate, 8-12g/L ferric ammonium citrate. The adoption of the mineralization culture medium can be more beneficial to the growth of pseudomonas stutzeri and the promotion of the conversion of nitrate and the adsorption of arsenic. Specifically, part of iron in the ammonium ferric citrate becomes divalent under illumination or high temperature and high pressure, and the ammonium ferric citrate can also provide divalent iron while being used as a carbon source provided by the growth of microorganisms, so that the process of adding the divalent iron for the second time after sterilization is avoided.
It should be noted that: (1) The mineralizing medium must contain nitrate ions and ferrous ions, other pH buffer solutions, sulfate ions, magnesium sources and the like can be adjusted according to actual conditions, and the components and the mixture ratio of the mineralizing medium are only examples of embodiments with better effects of the invention.
(2) The culture conditions for Pseudomonas stutzeri in the mineral-forming medium include, but are not limited to, culture temperature and culture time, and the like, and are not limited to the examples of the present invention, and the culture conditions described above are only examples of preferable effects.
Further, embodiments of the present invention also provide a use of the above-mentioned pseudomonas stutzeri or the above-mentioned culture of pseudomonas stutzeri in at least one of the following cases; (1) nitrate conversion in a water and soil environment; (2) oxidizing ferrous ions in a water and soil environment; and (3) arsenic remediation in the water and soil environment.
Further, the present invention provides an environmental remediation agent comprising the aforementioned Pseudomonas stutzeri or a culture of the aforementioned Pseudomonas stutzeri.
Further, the present invention provides a method for remediating an aqueous or soil environment, comprising remediating water or soil using the aforementioned Pseudomonas stutzeri or a culture of the aforementioned Pseudomonas stutzeri. The soil may be nitrogen deficient soil with a high nitrate content and low actual plant available nitrogen, or heavy metal contaminated soil, such as arsenic contaminated soil, and the water may also be heavy metal contaminated wastewater.
Specifically, the repair method comprises the following steps: mixing the pseudomonas stutzeri with water or soil containing nitrate radical and ferrous ion; at the moment, the pseudomonas stutzeri can convert nitrate in the soil and increase the content of available nitrogen in the soil. And simultaneously, ferrous ions are oxidized to form iron minerals, and then heavy metals in water or soil are adsorbed, so that the content of the heavy metals in the water or soil is reduced.
Generally, nitrate radical or ferrous iron in soil or water is not particularly sufficient, so that the remediation of water or soil by pseudomonas stutzeri, particularly the removal effect of heavy metals, is not particularly excellent, and then raw materials containing nitrate radical and ferrous iron ions can be additionally added, so that the formation of iron minerals by pseudomonas stutzeri can be accelerated, and the removal effect of heavy metals can be improved. For example, the pseudomonas stutzeri, the raw material containing nitrate ions and ferrous ions are mixed with soil or water. At the moment, pseudomonas stutzeri converts nitrate radicals and simultaneously oxidizes ferrous ions to form iron minerals, and then the iron minerals adsorb heavy metals in soil or water, so that damaged soil is repaired. The soil in this case may be simply damaged soil or damaged soil containing nitrate and ferrous ions.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Enrichment and screening of Pseudomonas stutzeri
Raw materials: silver city mine blowdown canal sediment; enrichment: collecting a sample, filling the sample into a sterile self-sealing bag, storing the sample at a low temperature by using an ice bag, taking the sample back to a laboratory, weighing 10g of fresh soil into a sterile conical flask, adding 90mL of mixed solution of 10g/L ferric ammonium citrate and 0.5g/L sodium nitrate, sealing the mixed solution by using abrasive cloth, putting the mixed solution into an incubator, culturing the mixed solution at 25 ℃ for 7d, standing the mixed solution for 30min, taking supernatant, putting the supernatant into a sterile centrifugal tube, putting the sterile centrifugal tube into the incubator, and continuously culturing the sterile centrifugal tube for 7d under the same condition.
Screening: shaking the cultured bacteria liquid in shaking table, diluting with sterile distilled water step by step, shaking, and diluting with dilution gradient of stock solution respectively by 10 1 、10 2 、10 3 、10 4 、10 5 And (4) multiplying. 10 mu L of water samples with different dilutions are respectively coated on selective culture media and placed in a constant temperature 37 ℃ incubator for culture until bacterial colonies grow out. The selective medium components are: 0.5g/L potassium dihydrogen phosphate, 0.5g/L sodium nitrate, 0.2g/L calcium chloride, 0.5g/L magnesium sulfate, 0.5g/L ammonium sulfate, 10g/L ferric ammonium citrate and 15g/L agar powder. Adjusting pH to 6.8-7.2 with 2mol/L sodium hydroxide solution, performing damp heat sterilization at 121 deg.C for 21min, cooling to 50 deg.C, and pouring into disposable sterile plate.
Selecting white or yellow colony conforming to characteristics of iron-oxidizing bacteria on a selective culture medium on a new plate, gradually converting the white colony into brown round colony with metal luster after culturing for 2d, selecting colony and streaking to check whether other bacteria exist or not as shown in figure 1, and performing purification culture.
Inoculating a single colony into a selective culture solution without agar powder (the components of the selective culture solution are consistent with those of the selective culture medium and only do not contain agar), placing the selective culture solution into a shaking table at a constant temperature of 25 ℃ and 150rpm for culturing for 2d, taking 2mL of fermentation liquor, conveying the fermentation liquor into a sterile centrifuge tube, carrying out 16S rRNA gene sequence amplification and sequencing on the fermentation liquor, and carrying out PCR amplification on a target fragment by using universal primers 27F and 1492R.
The obtained sequences were input into the NCBI Blast program for alignment, and sequences with homology over 99% were downloaded as reference. After all sequences were compared with the BioEdit Clustal W program, phylogenetic trees were constructed using MEGA X software according to the neighbor joining method, and the results after construction are shown in FIG. 2. From the figure, it is known that the selected strain of the drainage ditch deposit in the arid area is the most similar to the pseudomonad schoenbergii (Pseudomonas stutzeri) in the hydrophilicity up to 99.79%, and therefore, it is defined as the pseudomonad schoenbergii, and the 16S rRNA sequence of the pseudomonad is submitted to GenBank under the accession number: pseudomonas LZU-2 MW 774271, which is submitted to the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms to obtain the preservation number of CGMCC No.22623.
The above experimental processes all adopt aseptic operation, and three groups are repeatedly made.
Example 2
Study of Pseudomonas LZU-2 on nitrate reduction and ferrous iron oxidation processes
The method comprises the following steps: selecting a few bacteria from a plate for storing the strains to a nutrient broth culture medium, wherein the nutrient broth culture medium comprises the following formula: 10.0g/L peptone, 3.0g/L beef extract, 5g/L sodium chloride, adjusted to pH 7.0 using 2mol/L hydrochloric acid. Activating thallus at 25 deg.C with shaking table at 150rpm for 16h to make the number of thallus reach logarithmic phase, centrifuging at 4000r/min for 10min, removing supernatant, adding 0.8% sterile sodium chloride solution (w/v), centrifuging, repeating the above operation for 2 times to obtain fine powderBacterial suspension OD 600 Is 0.8-1.2.
Inoculating the bacterial suspension into an mineralization culture medium (the composition of the mineralization culture medium is basically consistent with that of the selection culture medium in example 1, except that the mineralization culture medium does not contain agar powder), wherein the inoculation ratio is 1% (v/v), and two treatments are set, namely (1) nitrate and ferrous iron; (2) LZU-2+ nitrate + ferrous iron, culturing in a cell culture bottle with a sealing cover, sealing with sterile abrasive cloth, and culturing in an aerobic environment of a shaker at 25 deg.C and 150rpm for 1d to rapidly propagate the strain. The cells were then capped with a bottle cap and continuously incubated at 25 ℃ in an anaerobic or anoxic environment, with all samples being set according to days 0d, 1d, 2d, 3d, 4d, 5d, 7d and 11d, three replicates each.
Centrifuging 4mL culture solution at 4000r/min for 10min, filtering with 0.22 μm water system filter membrane, diluting, measuring nitrate concentration in the solution by electrode method, and measuring soluble Fe in the solution at 510nm by ultraviolet spectrophotometry 2+ Concentration, adding hydroxylamine hydrochloride to make Fe 3+ Reduction to Fe 2+ The total iron concentration was determined. The nitrite concentration of the intermediate nitrate was determined by ethylenediamine spectrophotometry at a wavelength of 540 nm.
The results of the experiment are shown in fig. 3, in which a in fig. 3 is a nitrate concentration graph, B in fig. 3 is a nitrite concentration graph, C in fig. 3 is a soluble ferrous concentration graph, and D in fig. 3 is a soluble total iron concentration graph.
As can be seen from FIG. 3, the nitrate concentration in the test with the addition of bacteria was continuously decreased with the increase of the culture time, and remained almost unchanged until the fourth day and reached the minimum level, while the occurrence of nitrite was detected in the first three days, and the ferrous iron and total iron concentrations were also continuously decreased with the number of days, which indicates that the iron ion concentration in the solution was decreased due to the binding of iron ions into amorphous nano-iron particles by the action of the microorganisms. In the control group without bacteria, the nitrate concentration remained unchanged, and although there was a slight decrease in ferrous iron, the total iron concentration remained unchanged and no nitrite was present. The pseudomonas LZU-2 has the function of reducing ferrous iron oxidation depending on nitrate. And the removal rate of nitrate radical can respectively reach: 94.4 percent.
All the experiments were performed under sterile conditions, and 3 replicate experiments were performed per aliquot.
Example 3
Research on removal effect of pseudomonas LZU-2 on trivalent arsenic in solution
The method comprises the following steps: the preparation of the mineralizing medium, preparation of the bacterial suspension and the inoculation amount were the same as in example 2. Using As 2 O 3 The preparation of the drug is 1000mg/L of trivalent arsenic mother solution. After the mineralization culture medium is subjected to wet sterilization and is returned to the room temperature, trivalent arsenic mother liquor is filtered by using a filter membrane of 0.22 mu m and is added into the culture medium, so that the initial trivalent arsenic concentration of the solution is 3.3mg/L. The bacteria are divided into four groups, one group is not added with bacteria, namely nitrate + ferrous iron + trivalent arsenic, the other group is inoculated with LZU-2, namely LZU-2+ nitrate + ferrous iron + trivalent arsenic, the other group is inoculated with LZU-2 without nitrate, namely LZU-2+ ferrous iron + trivalent arsenic, and the other group is inoculated with LZU-2 without iron (sodium citrate is used for replacing ferric ammonium citrate), namely LZU-2+ nitrate + trivalent arsenic. Then sealing with sterile abrasive cloth at 25 deg.C and 150rpm in aerobic environment for 1d, sealing with bottle cap, providing anaerobic or anoxic environment, and continuously culturing at 25 deg.C, wherein all samples are arranged according to days 0d, 1d, 2d, 3d, 4d, 5d, 7d and 11d, and each sample is arranged in three parallel. Taking 4mL of culture solution according to days, centrifuging at 4000r/min, filtering by using a 0.22-micron water-system filter membrane, taking 2% potassium borohydride (w/v) as a reducing agent, taking 7% hydrochloric acid (v/v) as a carrier, measuring the concentration of trivalent arsenic in the solution by using hydride atomic fluorescence, and adding thiourea into the solution to reduce pentavalent arsenic into trivalent arsenic to measure the total arsenic concentration.
As shown in the experimental result of FIG. 4, the arsenic concentration is basically unchanged in the aerobic environment of the first day as the culture time increases, the trivalent arsenic and total arsenic concentration rapidly decrease from the anaerobic or anoxic environment of the second day, and the trivalent arsenic and total arsenic concentration is lower than the limit of 10 mug/L of sanitary standard of domestic drinking water (revised version of GB 5749) after the eleventh day, which is mainly caused by arsenic concentration decrease due to the adsorption and coprecipitation of iron minerals generated under the action of microorganisms. For the non-inoculated test, trivalent arsenic and total arsenic concentrations remained unchanged with increasing time. In the control group without nitrate, the removal of trivalent arsenic is slow, and in the control group without iron, the concentration of trivalent arsenic is almost unchanged, which indicates that the adsorption of trivalent arsenic by bacteria is negligible.
All the experiments were performed under sterile conditions, and 3 replicate experiments were performed per aliquot.
Example 4
Study of Pseudomonas LZU-2 cultures
The preparation of the mineralizing culture medium, the preparation of the bacterial suspension and the inoculation amount are the same as those of the embodiment 2, 3 groups are provided, one group is the same as the embodiment 2 in culture condition, one group is only cultured under aerobic condition (namely the sterile abrasive cloth of the embodiment 2 is sealed and cultured in aerobic environment of a shaker at 25 ℃ and 150 rpm), one group is only cultured under anoxic condition (namely the sterile abrasive cloth of the embodiment 2 is sealed and cultured continuously at 25 ℃ by providing anaerobic or anoxic environment by using a bottle cap) for 3 groups, the initial trivalent arsenic concentration is the same, then the culture solution of the 3 groups is respectively centrifuged and filtered at 0d, 1d, 4d and 8d to determine the trivalent arsenic concentration in the solution, and the precipitate is collected and lyophilized in vacuum to prepare a powder sample.
As can be seen from fig. 5, a large amount of iron minerals are generated only when the bacteria, nitrate and ferrous iron are present simultaneously and the bacteria are rapidly propagated under the aerobic condition, and then the competition between oxygen and nitrate as an electron acceptor is reduced under the anaerobic or anoxic condition, and if the bacteria are cultured in the aerobic environment or the anaerobic environment, the iron minerals are formed only with low yield and no obvious removal effect on trivalent arsenic.
The powder is characterized by Scanning Electron Microscope (SEM) and X-ray diffraction (XRD), and referring to figures 6-7, it can be seen that the powder formed by the culture method provided by the embodiment of the invention generates porous and small-particle iron mineral, the XRD spectrum peak is few and wide, and the powder is judged to be weak crystalline iron mineral generated by pseudomonas LZU-2.
Example 5
Collecting farmland soil polluted by industrial and mining sewage irrigation of silver market, wherein the total arsenic concentration in the farmland soil is 122.62mg/kg, the pH value is 7.78, the nitrate nitrogen content is 14.29mg/kg, the ammonium nitrogen content is 7.16mg/kg, and the organic matter content is 17.93g/kg. The sample is naturally air-dried and ground by a nylon sieve of 2mm, 100.0g of the sample is weighed and put into a plastic box, and deionized water is added for preservationKeeping the water content at 60%, and standing in dark for balancing for 14 days. Experimental grouping: (1) negative control group: adding only deionized water (noted as CK 1); (2) positive control group 1: adding a repairing liquid: 10g/L ferric ammonium citrate and 0.5g/L sodium nitrate solution (recorded as CK 2); (3) Positive control group 2: pseudomonas LZU-2 (denoted as LZU-2) was added to the mixture at OD 600 =0.1-0.5; (4) experimental group: pseudomonas LZU-2, 10g/L ferric ammonium citrate and 0.5g/L sodium nitrate solution (noted as LZU-2 +) were added, and the concentration of Pseudomonas LZU-2 was OD 600 And (5) =0.1-0.5. And (3) absorbing 10mL of corresponding liquid of the corresponding group by using a sterile needle tube every 5 days, inserting the corresponding liquid into deep soil for slow injection, adding deionized water to keep the water content at 30%, simulating daytime illumination by using a fluorescent lamp, repairing the soil at 22.5 ℃ for 15 days, naturally air-drying the soil, sieving the soil by using a 0.149mm nylon sieve, weighing 1.0000g of soil, and continuously extracting and determining the repairing effect of arsenic by using five steps of a Tessier. The soil ammonium nitrogen is measured by an indophenol blue colorimetric method, the nitrate nitrogen is measured by a hydrazine sulfate method, the organic matter is measured by a potassium dichromate-sulfuric acid external heating method, the effective potassium is extracted by 1mol/L ammonium acetate, and the iron in the soil is extracted by 0.5 mol/L.
See figure 8 and the following table:
according to the graph 8 and the table above, the pseudomonas LZU-2 which is added singly has a certain repairing effect on the polluted soil, but the repairing effect is general, and the mixture of the raw material containing nitrate ions and ferrous ions and the pseudomonas LZU-2 has the best repairing effect, so that the arsenic in exchangeable state and carbonate combined state can be remarkably reduced, and the arsenic is converted into the arsenic in residue state with less toxicity and stronger stability. After being repaired by adding the microbial inoculum, the transformation of nitrate nitrogen and ammonium nitrogen in soil can be promoted, and the physical and chemical properties of the soil can be improved.
The above experiments were performed in 3 replicates per treatment.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (19)
1. Pseudomonas stutzeri (A)Pseudomonas stutzeri) The preservation number is CGMCC NO.22623.
2. A culture of Pseudomonas stutzeri, which is prepared by culturing the Pseudomonas stutzeri of claim 1.
3. The culture of Pseudomonas stutzeri according to claim 2, wherein the process of culturing the Pseudomonas stutzeri comprises: inoculating the pseudomonas stutzeri into an ore-forming culture medium containing nitrate and ferrous ions for culture.
4. The culture of Pseudomonas stutzeri according to claim 3, wherein the process of culturing the Pseudomonas stutzeri comprises: inoculating the pseudomonas stutzeri to the mineralization culture medium, and then carrying out aerobic culture and then carrying out anoxic culture.
5. The culture of Pseudomonas stutzeri according to claim 3, wherein the process of culturing the Pseudomonas stutzeri comprises: inoculating the pseudomonas stutzeri to the mineralization culture medium, and then carrying out aerobic culture for 1-1.5 days at the temperature of 23-27 ℃, and then carrying out anoxic continuous culture for 7-10 days at the temperature of 23-27 ℃.
6. Culture of Pseudomonas stutzeri according to any of claims 3 to 5, characterized in that the inoculation proportion of Pseudomonas stutzeri is between 1 and 10%.
7. The culture of Pseudomonas stutzeri according to any one of claims 3 to 5, wherein the mineralizing medium is a liquid medium.
8. The culture of Pseudomonas stutzeri according to any of claims 3 to 5, wherein the mineralizing medium comprises nitrate and ferrous salts.
9. The culture of Pseudomonas stutzeri according to claim 8, wherein the mineralizing medium further comprises a pH buffer, a nitrogen source, a carbon source, a magnesium source, a calcium source, and a sulfate.
10. The culture of Pseudomonas stutzeri according to any one of claims 3 to 5, wherein the mineralizing medium comprises 0.3 to 0.7g/L potassium dihydrogen phosphate, 0.3 to 0.7g/L sodium nitrate, 0.3 to 0.7g/L calcium chloride, 0.3 to 0.7g/L magnesium sulfate, 0.3 to 0.7g/L ammonium sulfate and 5 to 25g/L ferric ammonium citrate.
11. The culture of Pseudomonas stutzeri according to any one of claims 3 to 5, wherein the process of culturing the Pseudomonas stutzeri comprises: activating the pseudomonas stutzeri to obtain a bacterial suspension, and then inoculating the bacterial suspension into an ore-forming culture medium for culture.
12. The culture of pseudomonas stutzeri according to claim 11, wherein the activation treatment comprises: inoculating the pseudomonas stutzeri into a nutrient broth culture medium for culture, so that the number of bacteria of the pseudomonas stutzeri reaches an logarithmic growth phase, and then centrifuging to obtain a bacterial suspension.
13. The culture of Pseudomonas stutzeri according to claim 11, wherein the OD of the bacterial suspension 600 Is 0.8-1.2.
14. Use of a culture of pseudomonas stutzeri according to claim 1 or pseudomonas stutzeri according to any one of claims 2 to 13 in at least one of;
(1) Nitrate conversion in water and soil environment; (2) oxidizing ferrous ions in a water and soil environment; and (3) arsenic remediation in the water and soil environment.
15. An environmental remediation agent comprising a culture of pseudomonas stutzeri according to claim 1 or pseudomonas stutzeri according to any one of claims 2 to 13.
16. A method for remediating an aqueous or soil environment, comprising using a culture of Pseudomonas stutzeri according to claim 1 or Pseudomonas stutzeri according to any one of claims 2 to 13 for remediation of water or soil.
17. A method of remediating an aquatic and soil environment as recited in claim 16, comprising: mixing said Pseudomonas stutzeri or a culture of said Pseudomonas stutzeri with water or soil containing nitrate and ferrous ions.
18. A method of remediating an aquatic and soil environment as recited in claim 16, comprising: mixing the pseudomonas stutzeri and raw materials containing nitrate radical and ferrous ion, and then mixing with water or soil.
19. The method for remediating an aquatic-soil environment as recited in claim 17 or 18, wherein the water is heavy metal contaminated water; the soil is heavy metal contaminated soil and/or soil lacking nitrate nitrogen.
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