CN113980829B - Pseudomonas flavescens, culture method thereof, culture thereof, treatment agent and repair method - Google Patents

Abstract

The invention relates to the technical field of environmental pollution treatment, in particular to pseudomonas flavescens, a culture method thereof, a culture thereof, a treating agent and a repairing method. The preservation number of the pseudomonas flavescens is CGMCC 22624. The Pseudomonas flavescens can promote the conversion of nitrate in the soil and water environment, and simultaneously, the iron can be oxidized to generate biological iron mineral to adsorb heavy metal, so that the pollution of nitrate and heavy metal in the soil and water environment is reduced.

Description

Pseudomonas flavescens, culture method thereof, culture thereof, treatment agent and repair method

Technical Field

The invention relates to the technical field of environmental pollution treatment, in particular to pseudomonas flavescens, a culture method thereof, a culture thereof, a treating agent and a repairing method.

Background

Nitrate-dependent ferrous oxidizing bacteria are ubiquitous in the environment, and ferric minerals formed by the action of the microorganisms can provide a potential mechanism for fixing heavy metals such as arsenic through coprecipitation or physical embedding or active adsorption, so that the purification and repair of water and soil polluted by the heavy metals are realized. White silver city in Gansu province is a typical industrial and mining city, serious water and soil environmental pollution is caused to the surrounding environment by mining and smelting a large amount of ores for a long time, and accumulation of a large amount of nitrate in stable state is caused by excessive fertilization of farmlands in arid regions. Therefore, there is an urgent need for a microorganism that can promote nitrate conversion in a soil and water environment, and at the same time, can adsorb arsenic through bio-iron minerals generated by oxidation of iron, and can also perform nitrate reduction and ferrous oxidation processes in a deep anaerobic or anoxic environment of the soil and water, thereby reducing nitrate and arsenic pollution.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide Pseudomonas flavescens, a culture method thereof, a culture thereof, a treating agent and a repairing method. The embodiment of the invention provides a novel Pseudomonas flavescens which can promote the conversion of nitrate in a water and soil environment, and simultaneously, the biological iron ore generated by oxidation of iron can adsorb heavy metal, so that the pollution of nitrate and heavy metal in the water and soil environment is improved.

The invention is realized in the following way:

in a first aspect, the invention provides a Pseudomonas flavescens with a collection number of CGMCC 22624.

Specifically, the strain was stored in China general microbiological culture Collection center (CGMCC, no. 3 of North West Lu No. 1, the Korean area of Beijing, and the institute of microorganisms, 100101) of China academy of sciences) at 31 months of 2021, and was named after classification: pseudomonas flavescens Pseudomonas flavescens with a preservation number of CGMCC No.22624.

In a second aspect, the present invention provides a method for culturing Pseudomonas flavescens according to the previous embodiment, comprising: the Pseudomonas flavescens described in the previous embodiment was inoculated into a medium containing both nitrate and ferrous ions for cultivation.

In an alternative embodiment, the instant invention provides a method of culturing pseudomonas flavescens according to the previous embodiment, comprising: inoculating the Pseudomonas flavescens into the culture medium, performing aerobic culture, and performing anaerobic culture;

preferably, the inoculum size of the Pseudomonas flavescens is 1-10%.

In an alternative embodiment, the instant invention provides a method of culturing pseudomonas flavescens according to the preceding embodiment, the medium comprising a liquid medium;

preferably, the medium comprises nitrate and ferrous ion salts;

preferably, the medium further comprises: a carbon source, sulfate, nitrogen source, phosphorus source, and magnesium source;

preferably, the medium comprises: monopotassium phosphate, sodium nitrate, calcium chloride, magnesium sulfate, ammonium sulfate and ferric ammonium citrate.

In an alternative embodiment, the instant invention provides a method of culturing pseudomonas flavescens according to the previous embodiment, further comprising: pre-treating the pseudomonas flavescens prior to inoculation;

preferably, the pretreatment comprises: inoculating the preserved pseudomonas flavescens into a broth culture medium for culture, and forming a bacterial suspension;

preferably, the OD of the bacterial suspension ₆₀₀ 0.8-1.2.

In a third aspect, the present invention provides a culture of Pseudomonas flavescens obtained by culturing the Pseudomonas flavescens according to any of the previous embodiments;

preferably, the culture comprises: the Pseudomonas flavescens converts nitrate and oxidizes ferrous ions to form iron minerals.

In a fourth aspect, the present invention provides a treatment agent for remediating soil pollution or water pollution, comprising the Pseudomonas flavescens of the previous embodiment and the Pseudomonas flavescens culture of the previous embodiment.

In a fifth aspect, the present invention provides a method of remediating soil or water pollution comprising: the contaminated soil or contaminated water is treated with at least one of the Pseudomonas flavescens of the previous embodiment, the Pseudomonas flavescens culture of the previous embodiment, and the treating agent pair of the previous embodiment.

In an alternative embodiment, the method comprises: mixing at least one of the Pseudomonas flavescens, the Pseudomonas flavescens culture and the treating agent with contaminated soil or contaminated water for cultivation, wherein the contaminated soil or the contaminated water contains nitrate and ferrous ions;

comprising the following steps: at least one of the Pseudomonas flavescens, the Pseudomonas flavescens culture and the treating agent, an auxiliary agent containing both nitrate and ferrous ions, and contaminated soil or contaminated water are mixed and cultured.

In alternative embodiments, the contaminated soil is heavy metal contaminated soil or nitrogen deficient soil;

the contaminated water is water contaminated with heavy metals.

The invention has the following beneficial effects: the Pseudomonas flavovii provided by the embodiment of the invention can convert nitrate in polluted water or soil, so that the content of nitrogen which can be absorbed and utilized in the soil is improved, and the restoration of the soil is promoted. Meanwhile, ferrous iron can be oxidized to form a biological iron mineral while nitric acid is converted, and the biological iron mineral can adsorb heavy metals in polluted water or soil, so that the polluted water or soil can be purified and repaired.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a surface morphology of a colony of Pseudomonas flavescens provided in Experimental example 1 of the present invention;

FIG. 2 is a phylogenetic tree diagram of the 16S rRNA gene of Pseudomonas flavescens provided in experimental example 1 of the present invention;

FIG. 3 is a graph showing the results of the reduction of nitrate and the oxidation of ferrous iron by Pseudomonas flavescens provided in Experimental example 2 of the present invention;

FIG. 4 is a graph showing the effect of Pseudomonas flavescens on purifying trivalent arsenic and total arsenic in a solution according to experimental example 3 of the present invention;

FIG. 5 shows the removal of trivalent arsenic by Pseudomonas flavescens under different oxygen conditions as provided in Experimental example 4 of the present invention;

FIG. 6 is a scanning electron micrograph of a culture of Pseudomonas flavescens provided in Experimental example 4 of the present invention;

FIG. 7 is an XPS diagram of a culture of Pseudomonas flavescens provided in Experimental example 4 of the present invention;

FIG. 8 is a graph showing the result of restoring arsenic-contaminated farmland soil by Pseudomonas flavescens, which is provided in Experimental 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 more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The embodiment of the invention provides a Pseudomonas flavescens (named as Pseudomonas LZU-3 hereinafter), which has a preservation number of CGMCC 22624.

The embodiment of the invention provides a method for culturing Pseudomonas flavescens, which comprises the following steps:

firstly, the preserved pseudomonas yellowing is inoculated into a broth culture medium for culture, so that the pseudomonas yellowing is rejuvenating and bacteria increasing, and the pseudomonas yellowing and products thereof with better performance are obtained.

Any broth medium and culture conditions that can satisfy the rejuvenation and enrichment effects of Pseudomonas flavescens are within the scope of the present invention.

When the number of bacteria reaches logarithmic growth phase, centrifuging the culture solution, removing supernatant, adding sterile sodium chloride solution (e.g. 0.8% sterile sodium chloride solution (w/v)), centrifuging, and repeating the operation to obtain OD ₆₀₀ 0.8-1.2 bacterial suspension.

The rejuvenated pseudomonas yellowing is inoculated into a culture medium containing nitrate radical and ferrous ion for culture. That is, the bacterial suspension is inoculated into a medium containing both nitrate and ferrous ions for cultivation.

The culture medium is mainly characterized by simultaneously containing nitrate ions and ferrous ion salts, other components can be properly adjusted and selected according to the Pseudomonas flavescens, and the culture medium can be a solid culture medium or a liquid culture medium, but when the liquid culture medium is adopted in the embodiment of the invention, the culture conditions are easy to control, the growth of the Pseudomonas flavescens is easier to control, and the performance of the Pseudomonas flavescens is improved.

Further, the medium further comprises: a carbon source, sulfate, nitrogen source, phosphorus source, and magnesium source; for example, the medium includes: monopotassium phosphate, sodium nitrate, calcium chloride, magnesium sulfate, ammonium sulfate and ferric ammonium citrate. The culture medium is more beneficial to the growth of the pseudomonas flavescens and the improvement of the performance of the pseudomonas flavescens. Particularly, part of iron in the ammonium ferric citrate can be divalent under illumination or high temperature and high pressure, and the ammonium ferric citrate can be used as a carbon source and a source of ferrous ions, so that the additional addition of ferrous ions after the sterilization of the culture medium is avoided, the operation of technicians is facilitated, and the possibility of environmental pollution of the culture medium is reduced.

The above-mentioned potassium dihydrogen phosphate, sodium nitrate, calcium chloride, magnesium sulfate, ammonium sulfate, ferric ammonium citrate, etc. may be used in a proper ratio as long as it can ensure the growth of Pseudomonas flavescens, but preferably, the formulation is: 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 and 10g/L ferric ammonium citrate. The use of the above ratio can be advantageous in promoting the culture of Pseudomonas flavescens.

Meanwhile, the culture conditions in the culture medium can be properly adjusted according to the growth condition of the Pseudomonas flavescens. For example, anaerobic culture may be directly used after inoculation, or aerobic culture may be performed first after inoculation, and then anaerobic culture may be performed. Since anaerobic culture is directly adopted after inoculation, the condition that the pseudomonas flavescens grows slowly can be caused, and then the culture product is insufficient, and the subsequent polluted water or soil purifying effect can be reduced. The method comprises the steps of first aerobic culture, greatly increasing the quantity of the pseudomonas flavescens, and then anaerobic culture, so that the pseudomonas flavescens can fully take nitrate radical as an electron acceptor to carry out biological oxidation on ferrous iron through electron transfer, and form microbial iron minerals, thereby improving the purifying effect of polluted water or soil.

The aerobic culture is a culture in an unsealed atmosphere, and the anaerobic culture is a culture in a sealed atmosphere without additional introduction of oxygen. And the conditions for the aerobic culture and the anaerobic culture, such as culture temperature, time, etc., are known in the art.

Further, any inoculum size is acceptable as long as the normal growth of Pseudomonas flavescens can be ensured, the formation of culture products is ensured. Preferably, the inoculum size of the Pseudomonas flavescens is 1-10% _(V/V) For example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% and the like, and any value between 1% and 10% is advantageous for obtaining Pseudomonas flavescens and a culture by using the above inoculum size, and for improving the purification effect of the soil and water environment.

The embodiment of the invention also provides a Pseudomonas flavescens culture which is obtained by culturing the Pseudomonas flavescens according to any one of the previous embodiments;

preferably, the culture comprises: the Pseudomonas flavescens converts nitrate and oxidizes ferrous ions to form iron minerals.

By inoculating Pseudomonas flavescens into a culture medium containing both nitrate and ferrous ions, it was found that it is capable of converting nitrate ions and oxidizing ferrous ions to form iron minerals, which in turn adsorb heavy metals, then the Pseudomonas flavescens can be used for the conversion of nitrate in contaminated water or soil, or for the oxidation of ferrous ions in contaminated water or soil; or can be used for removing heavy metals in polluted water or soil, and also shows that the pseudomonas flavescens or the culture thereof can be used for treating agents for restoring soil pollution or water pollution. That is, the present invention provides a treatment agent for remediating soil pollution or water pollution, which comprises the above Pseudomonas flavescens or a culture thereof.

Further, the present invention provides a method of remediating soil pollution or water pollution, comprising: treating contaminated soil or contaminated water with at least one of the above Pseudomonas flavescens, the above Pseudomonas flavescens culture and the above treating agent pair. The contaminated soil may be nitrogen-deficient soil with high nitrate content, but less biological nitrogen is actually available to plants or natural environment, or may be soil contaminated by heavy metals such as arsenic, and the contaminated water may be sewage contaminated by heavy metals or water with exceeding nitrate content.

Further, the repairing method comprises the following steps: at least one of Pseudomonas flavescens, the Pseudomonas flavescens culture and the treating agent is mixed with contaminated soil or contaminated water for cultivation, wherein the contaminated soil or the contaminated water contains nitrate and ferrous ions. At this time, the polluted water or soil contains nitrate and ferrous ions, and then the Pseudomonas flavovii can directly utilize the nitrate and ferrous ions in the polluted water or soil to convert the nitrate and ferrous ions into iron minerals, so that heavy metals are removed.

If the contaminated soil or the contaminated water does not contain nitrate and ferrous ions or the content of nitrate and ferrous ions is low, so that the heavy metal purification effect is insufficient, an auxiliary reagent containing nitrate and ferrous ions can be additionally added. At this time, more iron minerals can be formed, and the heavy metal purifying effect of the contaminated soil or the contaminated water is further improved.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Experimental example 1

Acquisition and identification of Pseudomonas flavescens

Sample: silver municipal sewage irrigation farmland soil

Step 1, placing a sample in a sterile self-sealing bag, preserving at low temperature by using an ice bag, weighing 50g of the fresh sample in a sterile plastic box, sealing by using abrasive cloth, placing in an incubator, culturing at 25 ℃ for 7d, adding 10g/L ferric ammonium citrate and 0.5g/L sodium nitrate solution according to the frequency of 1 time per day, and keeping the water content of the soil at 70%. And adding sterile distilled water after 7d, standing for 30min, taking supernatant, putting into a sterile centrifuge tube, and continuously culturing for 7d under the same condition.

Step 2: shaking the above cultured bacterial liquid in shaking table to thoroughly shake, and gradually diluting with sterile distilled water to give a dilution gradient of 10 ¹ 、10 ² 、10 ³ 、10 ⁴ 、10 ⁵ Multiple times. 10 mu L of water samples with different dilutions are respectively coated on a selective culture medium, and the selective culture medium is placed in a incubator with constant temperature of 37 ℃ for culture until 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, 15g/L agar powder. Adjusting pH to 6.8-7.2 with 2mol/L sodium hydroxide solution, sterilizing at 121deg.C for 21min under damp-heat, cooling for a period of time, and pouring into disposable sterile plate.

Step 3: colonies which are white or yellow on the selective medium and accord with the characteristics of the iron-oxidizing bacteria are selected on a new plate, after 2d of culture, the white colonies are gradually converted into brown circular colonies with metallic luster, see figure 1, and the colonies are picked up for streaking to check whether other bacteria exist, and purified culture is carried out.

Step 4: single colonies are picked up and inoculated into nutrient broth medium, placed at a constant temperature of 25 ℃ and subjected to shaking culture at 150rpm for 2d, 2mL of fermentation broth is taken in a sterile centrifuge tube and sent to the Seamaceae organism company for 16S rRNA gene sequence amplification and sequencing, and target fragment PCR amplification is carried out by using universal primers 27F and 1492R.

The resulting sequences were entered into the NCBI Blast program for alignment and sequences with homology exceeding 99% were downloaded as references. After all sequences were aligned using the BioEdit Clustal W program, phylogenetic trees were constructed according to the adjacency method using MEGA X software, and the results after construction are shown in fig. 2. As can be seen from FIG. 2, the affinity between the strain screened in the soil of the oasis farmland and Pseudomonas flavescens (Pseudomonas flavescens) is closest to 99.2%, and therefore, it is defined as Pseudomonas flavescens. The 16S rRNA sequence of Pseudomonas was submitted to GenBank under accession number: MW 774272, and meanwhile, the strain is preserved to the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms to obtain a preservation number CGMCC 22624.

The above experimental procedure was performed aseptically and three sets were repeated, with the results obtained in duplicate being substantially identical.

Experimental example 2

Reduction of nitrate by Pseudomonas flavescens and oxidation study of ferrous iron

The method comprises the steps of picking a small amount of pseudomonas flavescens from a plate for preserving strains to a nutrient broth culture medium for culturing, wherein the nutrient broth culture medium comprises the following components: 10.0g/L peptone, 3.0g/L beef extract, 5g/L sodium chloride, and pH was adjusted to 7.0 using 2mol/L hydrochloric acid. Activating thallus at 25deg.C for 16 hr at 150rpm to make the bacterial quantity reach logarithmic growth phase, centrifuging at 4000r/min for 10min, removing supernatant, adding 0.8% sterile sodium chloride solution (w/v), centrifuging, repeating the above steps for 2 times to obtain bacterial suspension OD ₆₀₀ 0.8-1.2.

The bacterial suspension was inoculated into a medium (the composition of the medium is substantially the same as that of the selection medium of example 1 except that the medium does not contain agar powder) at an inoculation ratio of 1% (v/v). The grouping is as follows: negative control group: (1) nitrate + ferrous; experimental group: (2) LZU-3+ nitrate + ferrous; cell culture was performed using a cell culture flask with a sealed lid, which was shake-cultured at 150rpm at 25℃for 1d with a sterile abrasive cloth seal (providing an aerobic environment), and then continuously at 25℃with a bottle cap seal (providing an anaerobic environment), all samples were set up in three parallel groups each according to days 0d, 1d, 2d, 3d, 4d, 5d, 7d and 11 d.

Centrifuging 4mL culture solution at 4000r/min for 10min according to days, filtering with 0.22 μm water-based filter membrane, diluting appropriately, measuring nitrate concentration in the liquid by electrode method, and measuring soluble Fe in the liquid at 510nm by ultraviolet spectrophotometry ²⁺ Concentration. The nitrite concentration of the nitrate reduced intermediate product was determined by ethylenediamine spectrophotometry at a wavelength of 540nm and the ammonium concentration was determined by Navier reagent spectrophotometry.

The detection results are shown in fig. 3, wherein a is a nitrate concentration chart, B is a nitrite concentration chart, C is an ammonium concentration chart, and D is a soluble ferrous iron concentration chart. As can be seen from fig. 3, the nitrate concentration in the experimental group (i.e., the group to which pseudomonas flavescens was added) was continuously decreased as the culture time was increased, reaching the minimum level after the fourth day, and remained substantially unchanged. At the same time, nitrite is detected in the first three days, further indicating that nitrate is converted, and the concentration of ammonium ions in the solution is higher than the initial value after 11 d. The decrease in ferrous iron over time in the experimental group suggests that under the action of Pseudomonas flavescens, ferrous ions are oxidized and combined to form amorphous nano-iron particles, so that the concentration of iron ions in the solution decreases. In the negative control group, i.e., the group without bacteria, the nitrate concentration remained unchanged, the ferrous iron decreased slightly, and no nitrite appeared. The Pseudomonas flavescens has the function of reducing ferrous iron oxidation by depending on nitrate. And the nitrate removal rate can reach 88.8 percent respectively.

The above experimental procedure was performed aseptically and three sets were repeated, with the results obtained in duplicate being substantially identical.

Experimental example 3

Study of Pseudomonas flavescens on purification of arsenic in aqueous solutions

The steps are as follows: the components of the medium and the ratio thereof, the formation process of bacterial suspension, the inoculation amount and the like were the same as those of example 2. By means of As ₂ O ₃ Arsenic mother liquor was prepared at a concentration of 1000 mg/L. After the medium was wet sterilized to room temperature, the arsenic mother liquor was filtered using a 0.22 μm filter membrane, and added to the medium so that the initial trivalent arsenic concentration of the solution was 3.3mg/L. Experimental grouping: a group of negative control groups: no bacteria were added and noted as nitrate + ferrous + trivalent arsenic, experimental group: LZU-3 was inoculated and designated LZU-3+nitrate+ferrous+trivalent arsenic. Then, the culture was performed for 1d in an aerobic environment at 25℃and 150rpm with a sterile abrasive cloth, and then, the culture was performed continuously at 25℃with a bottle cap, and anaerobic or anoxic environments were provided, all samples were set in accordance with days 0d, 1d, 2d, 3d, 4d, 5d, 7d and 11d, and three samples were set in parallel. Centrifuging 4mL of culture solution at 4000r/min according to days, filtering with 0.22 μm water-based filter membrane, measuring the concentration of the solution in the solution by using 2% potassium borohydride (w/v) as reducer and 7% hydrochloric acid (v/v) as carrierThe trivalent arsenic concentration of (2) can be reduced to trivalent by adding thiourea into the solution, and the total arsenic concentration can be measured.

As shown in fig. 4, the arsenic concentration was substantially unchanged during the first day of aerobic culture as the culture time increased, and the trivalent and total arsenic concentrations decreased rapidly from the end of the second day of anaerobic culture to a limit (revised GB 5749) at which the trivalent and total arsenic concentrations were below 10 μg/L of the sanitary standard for drinking water after the eleventh day, as: ferrous iron is oxidized and forms iron minerals under the action of the pseudomonas flavescens, then the iron minerals adsorb and coprecipitate arsenic, so that the arsenic concentration in the solution is reduced, the trivalent arsenic and the total arsenic concentration in a negative control group are increased along with the time, the condition that the pseudomonas flavescens can convert nitrate radical and oxidize ferrous iron, then adsorb arsenic, finally realize purification of arsenic, and the method can be used for purifying water or soil polluted by heavy metals.

The above experimental procedure was performed aseptically and three sets were repeated, with the results obtained in duplicate being substantially identical.

Experimental example 4

Characterization of the culture product of Pseudomonas flavescens

The steps are as follows: the components of the medium and the ratio thereof, the formation process of bacterial suspension, the inoculation amount and the like were the same as those of example 2. Experimental grouping: experimental group: the culture procedure and conditions were the same as in example 2; control group 1: culturing was performed only under aerobic conditions (i.e., shaking table aerobic environment culture at 25 ℃ C., 150rpm under sterile abrasive cloth seal of example 2); control group 2: culturing under anaerobic condition (i.e. sealing with bottle cap in example 2, providing anaerobic or anoxic environment at 25deg.C for continuous culture), culturing the above 3 groups for the same time, centrifuging the culture solution after 7d, filtering to determine trivalent arsenic concentration, and lyophilizing to obtain solid culture product.

As can be seen from FIG. 5, when Pseudomonas flavescens, nitrate and ferrous ions are present simultaneously and the Pseudomonas flavescens is rapidly propagated in the aerobic or aerobic condition before the anaerobic condition, a great amount of iron mineral is generated by reducing the competition of oxygen and nitrate as electron acceptors, and if only the aerobic or anaerobic environment is used, the iron mineral is formed, and the yield of the iron mineral is low, so that the removal of trivalent arsenic is affected. The facultative anaerobe can reach ideal effect through aerobic treatment and anaerobic treatment, avoids the need of adding vitamins and microelements to promote the expression of microbial functions in complete anaerobe, and simplifies experimental flow and cost.

The solid culture was identified by Scanning Electron Microscope (SEM) and X-ray photoelectron spectroscopy (XPS), and As shown in FIGS. 6-7, the solid culture formed by the culture method according to the embodiment of the invention comprises irregular and small-particle iron minerals, and the XPS analysis of the culture is performed to obtain As 3d in the spectra of As 3d and Fe 2p _5/2 The orbital binding energy of 45.0-45.2eV and 44.2-44.4eV correspond to As (V) and As (III), respectively, indicating that the bacterium can oxidize part of trivalent arsenic into pentavalent arsenic. And Fe 2p _1/2 And 2p _3/2 The binding energy corresponds to Fe (III), indicating the ferric iron mineral produced by the bacterium.

The above experimental procedure was performed aseptically and three sets were repeated, with the results obtained in duplicate being substantially identical.

Experimental example 5

The farmland soil irrigated and polluted by the big ditch sewage of the east of silver is collected, and the components and the contents of each element of the farmland soil are shown in table 1. Then, collecting a sample, naturally air-drying and grinding, sieving with a 2mm nylon sieve, weighing 20.0g, putting into a 50mL centrifuge tube, adding deionized water to maintain 60% of water content, and standing in a dark place for balancing for 14d. Experimental grouping: (1) negative control group: deionized water alone (noted CK 1); (2) positive control group 1: and (3) adding a repairing liquid: 10g/L ferric ammonium citrate and 0.5g/L sodium nitrate solution (denoted CK 2); (3) positive control group 2: pseudomonas LZU-3 (denoted as LZU-3) was added at a concentration of OD ₆₀₀ =0.1-0.5; (4) experimental group: adding Pseudomonas LZU-3, 10g/L ferric ammonium citrate and 0.5g/L sodium nitrate solution (marked as LZU-3+), and the concentration of the Pseudomonas LZU-3 is OD ₆₀₀ =0.1-0.5. The repairing agent has the addition sequence, and specifically comprises the following components: 6mL of the mixed solution of ferric ammonium citrate and bacteria is sucked by using a sterile needle tube, and the mixed solution is slowly injected into soil to keep 30 percentThe water content is increased, the bottle cap is put into a shaking table to be fully shaken, then the bottle cap is opened for aerobic culture for 1d, then the bottle cap is covered after adding sodium nitrate solution, the bottle cap is stood for repairing for 15d at the room temperature of 18-22.5 ℃, then the soil is naturally air-dried, a nylon sieve with the thickness of 0.149mm is used for weighing 1.0000g of the soil, and the repairing effect of arsenic is measured by using Tessier five steps of continuous extraction. The ammonium nitrogen in soil is measured by using an indophenol blue colorimetric method, the nitrate nitrogen is measured by using a hydrazine sulfate method, the organic matters are measured by using a potassium dichromate-sulfuric acid external heating method, the effective potassium is extracted by using 1mol/L ammonium acetate, and the iron in soil is extracted by using 0.5 mol/L. The results are shown in fig. 8 and table 2.

TABLE 1 elemental and content in farmland soil

TABLE 2 influence on physicochemical Properties of soil under different repair treatments

As can be seen from fig. 8 and table 2, the contaminated soil also contains a certain amount of nitrate and ferrous ions, so that when only pseudomonas flavescens is added, it is also possible to adsorb arsenic in the soil by utilizing nitrate and ferrous ions in the contaminated soil and then forming iron minerals, and therefore, only pseudomonas flavescens is added to have a certain purifying effect on the contaminated soil, but the purifying effect is limited. The mixed auxiliary agent containing nitrate and ferrous ions and pseudomonas LZU-3 are more excellent in restoring polluted soil, and arsenic in exchangeable state and carbonate combined state can be obviously reduced, so that the arsenic is converted into arsenic in a residue state with smaller toxicity and stronger stability. It can be seen that the dissolved salt ions in the restored soil are obviously reduced, and the restored soil has a promoting effect on the conversion of nitrogen.

The above experimental procedure was performed aseptically and three sets were repeated, with the results obtained in duplicate being substantially identical.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (16)

1. Pseudomonas flavescens @Pseudomonas flavescens) The method is characterized in that the preservation number is CGMCC NO.22624.

2. A method of culturing pseudomonas flavescens as claimed in claim 1, comprising: the Pseudomonas flavescens according to claim 1 is inoculated into a medium containing both nitrate and ferrous ions for cultivation.

3. The method for culturing Pseudomonas flavescens according to claim 2, comprising: inoculating the Pseudomonas flavescens into the culture medium, performing aerobic culture, and performing anaerobic culture; wherein the inoculation amount of the pseudomonas yellowing is 1-10%.

4. A method of culturing pseudomonas yellowing according to claim 2 or 3, wherein the culture medium comprises a liquid culture medium.

5. A method of culturing pseudomonas yellowing according to claim 2 or 3, wherein the culture medium comprises a nitrate salt and a ferrous ion salt.

6. The method for culturing pseudomonas flavescens according to claim 5, wherein the medium further comprises: a carbon source, sulfate, nitrogen source, phosphorus source, and magnesium source.

7. A method of culturing pseudomonas flavescens according to claim 2 or 3, wherein the culture medium comprises: monopotassium phosphate, sodium nitrate, calcium chloride, magnesium sulfate, ammonium sulfate and ferric ammonium citrate.

8. The method for culturing pseudomonas flavescens according to claim 2, wherein the method further comprises: the Pseudomonas flavovii was pretreated prior to inoculation.

9. The method for culturing Pseudomonas flavescens according to claim 8, wherein the pretreatment comprises: the preserved Pseudomonas flavescens is inoculated into a broth medium for culture, and a bacterial suspension is formed.

10. The method for culturing Pseudomonas flavescens according to claim 9, wherein the OD of the bacterial suspension is the OD of the bacterial suspension ₆₀₀ 0.8-1.2.

11. A culture of pseudomonas flavescens, characterized in that it is cultivated by the method of cultivating pseudomonas flavescens according to any of claims 2-10.

12. The culture of pseudomonas flavescens according to claim 11, wherein the culture comprises: the Pseudomonas flavescens converts nitrate and oxidizes ferrous ions to form iron minerals.

13. A treatment agent for remediating soil pollution or water pollution, characterized in that it comprises the pseudomonas flavescens strain of claim 1 or the pseudomonas flavescens culture of claim 11.

14. A method of remediating soil or water pollution comprising: treatment of contaminated soil or contaminated water with at least one of the Pseudomonas flavescens of claim 1, the Pseudomonas flavescens culture of claim 11 and the treatment agent of claim 13.

15. A method of remediating soil or water contamination as recited in claim 14, comprising: mixing at least one of the Pseudomonas flavescens, the Pseudomonas flavescens culture and the treating agent with contaminated soil or contaminated water for cultivation, wherein the contaminated soil or the contaminated water contains nitrate and ferrous ions;

comprising the following steps: at least one of the Pseudomonas flavescens, the Pseudomonas flavescens culture and the treating agent, an auxiliary agent containing both nitrate and ferrous ions, and contaminated soil or contaminated water are mixed and cultured.

16. The method of remediating soil or water pollution of claim 14, wherein the contaminated soil is heavy metal contaminated soil or nitrogen deficient soil;

the contaminated water is water contaminated with heavy metals.

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