CN111826302B

CN111826302B - Iron oxidizing bacteria, and application thereof in arsenic-polluted soil remediation, product and method

Info

Publication number: CN111826302B
Application number: CN202010336688.6A
Authority: CN
Inventors: 吴川; 薛生国; 罗兴华; 钱子妍; 江星星; 吴凯凯
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2020-04-26
Filing date: 2020-04-26
Publication date: 2021-12-31
Anticipated expiration: 2040-04-26
Also published as: CN111826302A

Abstract

The invention discloses iron-oxidizing bacteria, which are applied to arsenic-polluted soil remediation. The repairing agent is added into the arsenic-polluted soil for incubation, so that the content of available arsenic in the soil can be effectively reduced. The iron-oxidizing bacteria provided by the invention can be applied to preparation of an arsenic-polluted soil remediation agent. Provides an optimized arsenic pollution repairing agent formula and improves the arsenic removal capability. Provides a method for restoring the arsenic-polluted soil, an optimized restoring condition and an improved soil restoring effect.

Description

Iron oxidizing bacteria, and application thereof in arsenic-polluted soil remediation, product and method

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an iron oxidizing bacterium, and application, a product and a method thereof.

Background

Arsenic (As) is a metalloid element with high toxicity and carcinogenicity. In an environmental medium, the biological agent has high mobility and biological enrichment capacity, seriously pollutes the environment and affects the human health. Rice is staple food of about 30 hundred million population, however, it is more easily enriched with arsenic than other grains, the enrichment efficiency is more than 10 times of other grains, and the eating of rice is always the main exposure way of arsenic. As (III) is one of the main forms of arsenic in rice soil. The arsenic pollution of rice soil can cause the accumulation of arsenic in rice grains, and the arsenic pollution has great threat to human health.

The biological mineral provides a new way for restoring the heavy metal pollution of the soil. Compared with the traditional method, the biological mineralization has the advantages of low cost, good long-acting property, small secondary pollution, weak environmental disturbance and the like. The product of the method generally has unique morphology and characteristics, such as nanoparticles, high specific surface area, high reactivity and the like, plays an important role in the morphological transformation process of heavy metal, and directly influences the toxicity and the mobility of heavy metal ions. Microorganisms play an important role in biological mineralization. Wherein iron oxidizing bacteria (FeOB) can induce to generate insoluble iron (hydrogen) oxygen mineral, AsO₄ ^3-And AsO₃ ^3-The plasma has stronger adsorption capacity, and arsenic adsorbed on the surface of the iron (hydrogen) oxygen mineral possibly enters mineral crystal lattices to form Fe-As secondary minerals, so that the iron (hydrogen) oxygen mineral is more stable; other heavy metal ions can be coupled to generate coprecipitation in the process of oxidizing ferrous iron by the iron oxidizing bacteria, and the bioavailability and the migration capability of the heavy metal ions can be reduced. In addition, the toxicity of the valence-variable metal ions such as arsenic can be reduced by the change of oxidation-reduction potential in the metabolic process of iron-oxidizing bacteria. Therefore, the biological mineralization of iron-oxidizing bacteria provides a potential solution for the remediation of arsenic-contaminated soil.

However, at present, few studies are made on the biological mineralization remediation of arsenic in soil, and the pollution condition of arsenic in rice soil cannot be effectively improved.

Disclosure of Invention

Aiming at the defects or improvement requirements of the prior art, the invention provides iron-oxidizing bacteria, an application, a product and a method thereof, and aims to remove effective arsenic in soil through the biological mineralization of the iron-oxidizing bacteria, thereby improving the arsenic pollution condition in the soil and realizing the remediation of arsenic-polluted soil, and thus solving the technical problems that the effective arsenic is difficult to remove and the arsenic is difficult to restore in the arsenic-polluted soil.

To achieve the above object, according to one aspect of the present invention, there is provided an iron-oxidizing bacterium, which is classified and named as ochrobacterum sp. eeelcw01, and which is deposited in the chinese culture collection center CCTCC at 3/11/2020 with the deposition number of CCTCC M2020053.

According to another aspect of the invention, the application of the iron oxidizing bacteria for repairing arsenic-polluted soil is provided.

According to another aspect of the invention, an arsenic pollution remediation agent is provided, which comprises the iron oxidizing bacteria provided by the invention, preferably a suspension of iron oxidizing bacteria with an OD600 value of 1.9-2.1.

Preferably, the arsenic pollution remediation agent comprises nitrate salt, and 0.8-3.0 mmol of NO is added to every 1mL of iron oxidizing bacteria suspension₃ ^-。

Preferably, the arsenic pollution repairing agent comprises ferrous salt, and 1.5-3.0 mmol of ferrous ions are added to every 1mL of iron-oxidizing bacteria.

Preferably, the arsenic pollution repairing agent comprises acetate, and 0.5-1.5 mmol of acetate ions are added to every 1mL of iron-oxidizing bacteria.

According to another aspect of the invention, the arsenic-polluted soil remediation method is provided, and the arsenic-polluted soil is added with the arsenic-polluted remediation agent provided by the invention for co-incubation.

Preferably, the arsenic-contaminated soil remediation method is implemented by adding 3-6 ml of ferrooxidans bacterial suspension to contaminated soil containing 1mg of effective arsenic.

Preferably, the arsenic-polluted soil remediation method is carried out by co-incubation under the oxygen partial pressure of 0.01-0.03 Pa.

Preferably, the arsenic-contaminated soil remediation method is carried out for a co-incubation time of 7 days or more, preferably for a co-incubation time of 14 days or more, and more preferably for a co-incubation time of 28 days or more.

In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:

according to the invention, a strain of iron-oxidizing bacteria is obtained by separating and enriching from rice soil, and is identified and functionally researched, so that the metabolic activity of the iron-oxidizing bacteria can be coupled with arsenic mineralization in the soil, and the content of effective arsenic in the soil can be effectively reduced. The iron oxidizing bacteria provided by the invention can be applied to preparation of a soil arsenic pollution repairing agent.

Furthermore, the invention researches the conversion way of nitrate driven by the iron-oxidizing bacteria, and then discovers the characteristics of the iron-oxidizing bacteria dependent on nitrate, and preferably selects a reaction substrate and a carbon source according to the Fe (II) coupled arsenic mineralization oxidation experiment driven by the iron-oxidizing bacteria, so that an optimized arsenic pollution repairing agent formula is provided, and the arsenic removal capacity is improved.

In addition, a soil culture test is carried out to verify the remediation capability of the bacterium on the effective arsenic pollution in the soil, so that a remediation method for the arsenic-polluted soil is provided, the remediation conditions are optimized, and the remediation effect of the soil is improved.

Drawings

FIG. 1 is a scanning electron microscope image of iron-oxidizing bacteria provided by the present invention;

FIG. 2 is a diagram showing the identification of phylogenetic tree of Ferro-oxidizing bacteria according to the present invention;

FIG. 3 is a graph showing the results of the reaction kinetics driven by iron-oxidizing bacteria provided in example 1;

FIG. 4 is a graph showing the results of arsenic mineralization driven by iron-oxidizing bacteria under different conditions as provided in example 2;

FIG. 5 is XRD diffractograms before and after biomineralization;

FIG. 6 is a crystal structure diagram of 3 minerals produced by driving iron-oxidizing bacteria provided by the present invention;

FIG. 7 is a graph showing the results of remediation of arsenic-contaminated soil in Chenzhou, Hunan by iron-oxidizing bacteria as provided in example 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The iron-oxidizing bacteria provided by the invention are screened from soil of a typical arsenic-polluted farmland in hibiscus region of Changsha, Hunan province, and the separation, enrichment and identification processes are as follows:

the invention adopts FeS as a substrate to provide ferrous ions, and adopts an improved Walf mineral culture medium Modified Wolfe's Minor Medium (MWMM) to separate microaerophilic iron-oxidizing microorganisms.

(1) Preparation of an improved Wolff mineral culture medium MWMM: adding 1g of NH₄Cl、0.2g MgSO₄·7H₂O、0.1g CaCl₂·2H₂O and 0.05g K₂HPO₄·3H₂O was added to 1L of water and stirred with a glass rod until fully dissolved.

(2) Preparation of FeS substrate: 300mL of deionized water is put into a constant-temperature water bath kettle and heated to 50 ℃, and 23.3g of FeSO is added respectively₄·7H₂O and 20g NaS.9H₂And O. Firstly, the weighed FeSO₄·7H₂Adding O into a beaker in a water bath, continuously stirring by a glass rod until the O is completely dissolved, and then adding NaS 9H₂And adding the O into the beaker while slowly stirring by using a glass rod, generating a black precipitate immediately after the O and the O react, and continuously stirring for 3-5 minutes by using the glass rod after the O and the O are completely added to ensure that the substances are completely dissolved and react fully. Pouring the generated black precipitate FeS into a brown narrow-mouth glass bottle, adding deionized water, preventing air oxidation, placing in a dark dry place for natural precipitation, placing for 4-5 hours, pouring out supernatant, adding deionized water into the bottle, and fully stirring with a glass rod. Washing FeS in the bottle by the method, repeating the operation for 5-9 times for removing excessive impurities and S in the precipitate^2-Until the pH of the FeS layer is neutral.

(3) Preparing an MD-TMS mineral solution: 0.5g of EDTA, 3.0g of MgSO₄·7H₂O、0.5g MnSO₄·H₂O、1.0g NaCl、0.1g FeSO₄·7H₂O、0.1g Co(NO₃)₂·6H₂O、0.1g CaCl₂、0.1g ZnSO₄·7H₂O、0.01g CuSO₄·5H₂O、0.01g AlK(SO₄)₂、0.01g H₃BO₃、0.01g Na₂MoO₄、0.01g Na₂WO₄、0.02g NiCl₂·6H₂O、0.001g NaSeO₃Add 1L of water and stir with a glass rod until fully dissolved.

(4) Preparation of agar gel gradient tube:

(4-1) preparing a semi-solid culture medium base solution, mixing the FeS sediment with the supernatant removed and the MWMM in a beaker according to the volume ratio of 1:1, adding 1% (wt/vol) agar, and placing the mixture in a high-temperature sterilization pot for sterilization (121 ℃, 25 min).

(4-2) preparation of the upper layer Medium, adding 0.15% (wt/vol) agar to MWMM, and placing in a high temperature sterilization pot for sterilization. Sodium bicarbonate (final concentration 5.0 mmol. multidot.L) was added^-1(ii) a Filter sterilization), MD-TMS mineral element solution and vitamin solution (1 muL. mL)^-1(ii) a Filter sterilization) (preparation of vitamin solution: 10mg vitamin B6; 5mg vitamin B2; 5mg vitamin B1; 5mg of nicotinic acid; 5mg vitamin B5; 0.1mg vitamin B12; 5mg of p-aminobenzoic acid; 5mg of lipoic acid in 1L of deionized water), followed by filtered sterilized CO₂The pH of the medium was adjusted to 6.2-6.4 with gas.

(4-3) placing the two beakers respectively filled with the semi-solid culture medium base solution and the upper layer solution on an ultra-clean workbench after ultraviolet sterilization, accurately transferring 2.5mL of the semi-solid culture medium base solution to the bottom of a 30-mL autoclaved serum bottle by using a liquid transfer gun (the gun head is autoclaved), and forming a black solid plug after cooling for about 10 minutes. And then, using a pipette to pipette 15mL of the upper layer solution and the upper part of the black solid plug, and forming a semi-solid culture layer after cooling for about 30 minutes to obtain the agar gel gradient tube.

(5) Preparation of bacterial suspension: mixing fresh rice soil and sterile ultrapure water according to a ratio of 1:1(wt/vol), and then vibrating to treat the mixture to obtain an inoculation liquid for enriching iron-oxidizing bacteria.

(6) Placing the mixed bacterial suspension and the prepared gel gradient tube on a superclean workbench, dipping a small amount of bacterial suspension by using an inoculating ring, and extending into FeS black solidThe inoculation was completed at the interface of the plug and the gel coating. After the bottle cap is screwed on, the inoculated gradient tube is placed in a constant temperature incubator at the temperature of 21 +/-1 ℃ and is protected from light for 7 to 10 days. When reddish brown iron oxide ring appears in the culture medium, subculturing for 3 times, and performing gradient dilution with dilution range of 10^-3～10^-8Extracting FeOB growing in the highest dilution concentration, sampling and diluting again, and carrying out continuous passage for 7-8 times to obtain the enriched iron-oxidizing bacteria.

The more passages, the longer the time for the red-brown iron oxide ring to appear. After several dilutions and subcultures, reddish-brown iron oxide rings were observed for at least 4-5 days, which were slightly lighter in color and narrower in width than before.

The ferrite rod-shaped has a diameter of 0.2 to 0.4 μm and a length of 2 to 3 μm, as observed by a Scanning Electron Microscope (SEM) at 25kV (FIG. 1).

(7) Identification of iron-oxidizing bacteria:

(7-1) taxonomic identification: the isolated genomic DNA of Fereroxida was extracted using the bacterial DNA kit (Omega) according to the protocol of the instructions. Extracting the product as a template to carry out PCR amplification, wherein the amplification system is as follows: performing PCR amplification on the V4 hypervariable region of the bacterium by using a universal forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and a reverse primer 1492R (5'-TACGGTTACCTTGTTACGACTT-3'); amplification was performed on a PCR instrument with an amplification system of 50 μ Ι _ comprising: 25 μ L I52 XHF Master Mix, 2 μ L27F (10 μ M), 2 μ L1492R (10 μ M), 1 μ L Template, 20 μ L ddH 2O; the amplification conditions were: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 seconds, annealing at 54 ℃ for 10 seconds, and extension at 72 ℃ for 15 seconds, for a total of 35 cycles; finally, extension was carried out at 72 ℃ for two minutes to ensure the integrity of the amplification product. The PCR product was detected by 1.2% agarose gel electrophoresis, PCR amplification was observed and the gel was recovered in a centrifuge tube using TSINGKE DNA recovery purification kit (No. GE0101) for purification of the PCR product. And mixing the purified products and other substances in proportion, storing the mixture in a refrigerator at the temperature of-45 ℃ for detection, and performing sequencing analysis on the qualified products by using an Illumina Hi Seq2500 sequencing platform. To analyze sequence similarity, the obtained 16S rRNA gene sequences were aligned in the BLAST program and phylogenetic trees were constructed using MEGA 7.0, as shown in FIG. 2, and the genera of the colonies were identified by analysis using 1320 base pair selected gene sequences.

(7-2) Whole genome sequencing: extraction of genomic DNA was performed according to Wizard genomic DNA purification kit (Promega). The purified genomic DNA was quantified using a TBS-380 fluorometer. High quality DNA was used for library sequencing. Genome sequencing uses a combination of PacBio RS II single molecule real-time Sequencing (SMRT) and Illumina sequencing platforms. At least 1. mu.g of genomic DNA was taken, Covaris was used to fragment the genomic DNA and the DNA sample was cut into-400 bp fragments. Using NEXTflex^TMPreparing a library by a Rapid DNA-Seq kit. The method comprises the following specific steps: connection A&B, a joint; screening to remove the self-connecting fragments of the joint; agarose gel electrophoresis is carried out for fragment screening, and fragments with one end being an A joint and one end being a B joint are reserved; sodium hydroxide denaturation to generate single-stranded DNA fragments; bridge PCR amplification. At least 15. mu.g of genomic DNA was taken, processed into-10 kb fragments using G-tubes (Covaris, MA), then fragment purified according to PacBio instructions (Pacific Biosciences, Calif.), blunt-ended, and ligated at both ends with SMRT ball sequencing adaptors, respectively. The prepared library was paired-end sequenced (2X 150bp) on an Illumina HiSeq X Ten instrument. The method comprises the following specific steps: adding modified DNA polymerase and 4 kinds of fluorescent labeled dNTPs, and only doping a single base in each cycle; scanning the surface of the reaction plate by laser, and reading the nucleotide species polymerized by the first round of reaction of each template sequence; chemically cleaving the "fluorophore" and the "stop group" to restore the 3' terminal viscosity and continuing to polymerize a second nucleotide; counting the fluorescent signal result collected in each round, obtaining the sequence of the template DNA fragment, purifying the sequencing library for three times by using 0.45-volume Agencour AMPure XP beads (Beckman Coulter Genomics, MA) reagent, then annealing the single-chain loop of the library, combining the single-chain loop of the library with the fixed polymerase at the bottom of ZMW (zero-mode waveguiding holes), adding a sequencing reaction reagent, emitting corresponding light after each base pair synthesis and detecting, displaying a pulse peak when each base is synthesized, and matching with a high-resolution optical detectionAnd the detection system carries out real-time detection. Bioinformatic analysis was performed using data generated by the PacBio RS II and Illumina platforms. All analyses were performed on the I-Sanger cloud platform of the meiji organism (www.i-Sanger. The specific procedure is as follows: the initial raw data of genome assembly is stored in a fastq format, so that the subsequent assembly is more accurate, the quality of the original data is cut, reads with lower sequencing quality, higher N content ratio and smaller length after quality trimming are removed, and high-quality clean data is obtained. PacBio data assembly is carried out by utilizing canu and HGAP software, reads are assembled into contigs, and then, artificial judgment is carried out to form a ring, so that complete chromosome and plasmid genome are obtained. Finally, the assembly results are corrected using the illumina sequencing data.

The analysis of gene similarity between Blast and 16S rRNA showed that the iron-oxidizing bacterium belongs to the genus Ochrobactrum and is most similar to Ochrobactrum anthracropi ATCC 49188, and the sequence similarity between the 16S rRNA gene and the gene sequence is 99.85%. According to the results, the strain is named as Ochrobactrum sp.EEELCW01, and is preserved in China Center for Type Culture Collection (CCTCC) at 3-11 months in 2020 with the preservation number of CCTCC M2020053 at the preservation address of Wuhan university in China.

The iron-oxidizing bacteria provided by the invention can be applied to the preparation of a soil arsenic pollution repairing agent, and preferably, the iron-oxidizing bacteria suspension with the OD600 value of 1.9-2.1 is selected; when the nitrate is used in combination with nitrate, 0.8-3.0 mmol of NO is added to every 1mL of the ferrooxidans bacterial suspension₃ ^-. Preferably, substrate ferrous ions are added, and 1.5-3.0 mmol of ferrous ions are added to every 1mL of the ferroxida bacteria suspension. Preferably, acetate ions are added as a carbon source, and 0.5-1.5 mmol of acetate ions are added to every 1mL of the iron oxidizing bacteria suspension.

The invention provides a method for repairing arsenic-polluted soil, which comprises the following steps:

adding the arsenic pollution repairing agent provided by the invention into arsenic polluted soil for incubation, so that 3-6 ml of bacterial suspension is needed for 1mg/kg of effective arsenic; the co-incubation is preferably carried out in a microaerobic environment, in particular under conditions of an oxygen partial pressure of 0.01 to 0.03 Pa; the co-incubation time is 7 days or more, preferably 14 days or more, more preferably 28 days or more.

When the arsenic pollution repairing agent provided by the invention is incubated with arsenic polluted soil, an obvious arsenic mineralization effect appears in about 7 days, and the content of effective arsenic is reduced by 20.4-36.2% compared with that of a control group when the arsenic pollution repairing agent is measured in 28 days.

Experiments show that under the anoxic condition, Fe (II) oxide of iron-oxidizing bacteria can be promoted to be coprecipitated with soil arsenic, and a good arsenic mineralization effect is achieved, so that arsenic-polluted soil is repaired.

The following are examples:

example 1

Kinetics experiment of ferrous oxidation and nitrate reduction and conversion:

the iron-oxidizing bacteria provided by the invention are adopted to oxidize and couple Fe (II) with NO under the neutral pH microaerobic condition₃ ^-The process of reduction was simulated. A total of 5 treatment groups were set (table 1). Respectively, sterilization control group (NaN)₃+FeOB+Fe(II)+NO₃ ^-) (addition of NaN₃As a sterilizing agent), biological control group (Fe (II) + NO₃ ^-) Nitrate control (FeOB + Fe (II)), ferrous control (FeOB + NO)₃ ^-) And experimental group (FeOB + Fe (II) + NO₃ ^-). The whole experimental system was cultured in a 150ml anaerobic flask with a culture volume of 150 ml. The preparation method of the culture medium comprises the following steps: 100mL of KH solution containing 0.14g/L was prepared₂PO₄,0.2g/L NaCl,0.3g/L NH₄Cl,0.5g/L MgSO₄·H₂O,0.1g/L CaCl₂·2H₂A culture medium of O; adding 1mL/L vitamin solution; 1mL/L MD-TMS microelement solution (same as above); 24mM bicarbonate buffer system was added to the medium, pH adjusted to 6.8-7.2, 5mM sodium acetate was added as carbon source, 10mM NO₃ ^-10mM FeCl as sole electron acceptor₂The electron donor and the upper space are filled with nitrogen/carbon dioxide (80:20v/v) to ensure the oxygen environment for the growth of the iron-oxidizing bacteria.

Before adding bacteria to the culture medium, the bacteria were cultured in LB medium without Fe (II) added to the medium to logarithmic phase. The bacterial concentration was adjusted to an OD600 value of 1.9-2.1 (the concentration of the bacterial suspension of the ferrooxidans used in the examples of the present invention, in all cases) with a spectrophotometer, 4% (v/v) was added, the strain was then purged with nitrogen for 10min, and a rubber lid and an aluminum lid were rapidly pressed and placed in a constant temperature incubator (28 and). Each reaction was set up for 3 replicates.

TABLE 1

Experiments were performed on days with destructive sampling, testing for NO on days 0, 0.5, 2, 4, 6, 7 respectively₃ ^-，NO₂ ^-，N₂O，NH₄ ⁺，Fe²⁺Total Fe content. Before sampling test, the anaerobic bottle is shaken to uniformly mix solid-liquid substances in the bottle, and the anaerobic bottle is fully shaken to keep the mineral substances in a suspension state in the culture solution. Dilute 5ml of sample by pipetting and then apply ion chromatography (ICS-110Dionex, US) to NO₃ ^-、NO₂ ^-And carrying out quantitative detection. Using a gas chromatograph with electron capture detector (GCMS-QP2010ULTRA, Shimadzu, Japan) for N₂Carrying out quantitative detection on O; NH (NH)₄ ⁺The detection adopts a Nashiner reagent colorimetric method; the total iron and ferrous content is determined by phenanthroline colorimetry.

The reaction kinetics for the different treatment groups are shown in figure 3. In NO₃ ^-+ Fe (II) and FeOB + Fe (II) + NO₃ ^-+NaN₃In the treatment, almost NO NO is present₃ ^-Reduction took place, the content of which decreased by about 2mM within 7 days; in FeOB + Fe (II) + NO₃ ^-And FeOB + NO₃ ^-In the treatment, NO₃ ^-The reduction (2) was vigorously reacted at a rate of about 2.1mM/d and 3.32mM/d in the first 2 days, and the reaction rate was relatively slow in 2 to 7 days. The above results confirm that FeOB promotes NO₃ ^-Whereas the inactive FeOB does not have this promoting effect. NO₂ ^-Is the first intermediate of both the denitrification and dissimilatory nitration pathways. On day 0.5, FeOB + Fe (II) + NO₃ ^-And FeOB + NO₃ ^-About 0.09mM and 0.15mM NO were detected in the treatment, respectively₂ ^-The product was not detected in the other treatments. NO₂ ^-The content showed a tendency to increase in the first few days and gradually decrease with the lapse of time, and the reason for this change was that it was further reacted as an intermediate. In the presence or absence of Fe (II) and NO₂ ^-Found that NO is present in the presence of Fe (II)₂ ^-Low accumulation of (b), indicating that the presence of Fe (II) favors NO₂ ^-To promote NO₃ ^-The reduction reaction of (2) continues.

During the course of denitrification and dissimilatory nitration, NO₂ ^-Can be further reacted to N₂O or NH₄ ⁺. In FeOB + Fe (II) + NO₃ ^-And FeOB + NO₃ ^-In the process, N₂The O content tends to rise over the first few days. Then, N₂The O content also gradually decreases with time. N is a radical of₂O is also an intermediate product of the reduction reaction, and the reduction of the content thereof is also caused by the further progress of the reaction. In NO₃ ^-+ Fe (II) and FeOB + Fe (II) + NO₃ ^-+NaN₃In the treatment, NO₃ ^-Although very weak, there is almost NO NO₂ ^-Or N₂O is detected, and NH₄ ⁺It was observed that this may be due to a fast reaction of the dissimilation. In NO₃ ^-+ Fe (II) and FeOB + Fe (II) + NO₃ ^-+NaN₃In the treatment, NO₃ ^-The reduction reaction of (2) is very weak, but almost NO NO is present₂ ^-Or N₂O is detected but NH is detected₄ ⁺However, NH₄ ⁺Is much lower than the content of the denitrification product. The results show that the iron-oxidizing bacteria are NO-pair₃ ^-The reduction pathway of (A) is mainly denitrification, and only a small amount of dissimilatory nitrification is involved.

In FeOB + Fe (II), NO₃ ^-+ Fe (II) and FeOB + Fe (II) + NO₃ ^-+NaN₃In the treatment, the oxidation rate of Fe (II) was slow and not completely oxidized within 7 days. However, in FeOB + NO₃ ^-In the + Fe (II) treatment, Fe (II) was almost completely oxidized (98%) at an oxidation rate of about 0.99 mM. d in two days^-1. The above phenomena indicate FeOB and NO₃ ^-Plays an important role in the fe (ii) oxidation process, and the reaction is driven by both chemical and biological oxidation. The total iron content in the solution gradually decreases with time, in FeOB + NO₃ ^-In the + Fe (II) treatment, the total iron content in the solution was reduced to 0.07mM on day 7. This result indicates that the iron in the solution is converted to other forms, adsorbed or forms iron minerals. In addition, FeOB and NO₃ ^-The presence of (a) accelerates the process of converting the iron in solution to other forms.

The above experiments show that when FeOB and NO are used₃ ^-When the arsenic pollution repairing agent is added as an auxiliary material (preferably, the adding proportion is 0.8-3.0 mmol of NO added to 1mL of the ferroxida bacteria suspension₃ ^-1.5-3.0 mmol of ferrous ions are added to every 1mL of iron-oxidizing bacteria), and the iron-oxidizing bacteria provided by the invention can be activated, so that the arsenic mineralization efficiency is improved.

Example 2

Role of iron-oxidizing bacteria in batch experiments of fe (ii) biomineralization and influence of culture conditions:

effect of solution substrate on biomineralization: to 150mL of medium was added various substrates (sterile control group, +10mM sodium nitrate, +5mM sodium acetate, +10mM sodium nitrate +5mM sodium acetate), and sodium arsenite was added to provide As³⁺To a concentration of 500. mu. mol. L^-1And the pH was adjusted to around 7.0, then 3mL of iron oxidizing bacteria suspension was added.

Effect of solution microorganism concentration on biomineralization: adding different volumes (0, 3, 6 and 12mL) of iron-oxidizing bacteria suspension into each group, culturing at 28 deg.C for one week, centrifuging to collect the generated mineral precipitate, washing with deionized water twice, oven drying at 50 deg.C, and weighing.

Putting all solutions and instruments required by the experiment into a vacuum anaerobic box, spraying ethanol solution for disinfection, sealing the box body, opening an ultraviolet lamp for sterilization, and filling N₂About 30min to make the vacuum anaerobic tankThe oxygen environment required by the microorganism is achieved, namely the oxygen partial pressure is 0.01-0.03 Pa. All experiments were performed in triplicate, and at regular intervals (0d, 0.5d, 1d, 2d, 4d, 6d and 12d), 0.5mL of each supernatant was taken and stored by adding 3 drops of 12mol/L hydrochloric acid, and the total As (hydride generation atomic fluorescence spectroscopy), total Fe, Fe in solution were measured²⁺Content (phenanthroline colorimetry), and the resulting mineral precipitate was characterized by X-ray diffractometry (XRD) (TTR III, Rigaku Corporation, Japan).

NO₃ ^-Reduction-coupled Fe (II) oxidation can convert Fe in solution²⁺Further influencing the morphological transformation of arsenic (fig. 4). For all treatments with FeOB added suspensions, Fe in solution²⁺And total iron dropped significantly at a very high rate over 0.5 days, after which the reaction rate was very slow. This is because Fe (iii) produced during the oxidation of ferrous iron forms a shell on the cell surface, and particularly after the ferrous iron is completely oxidized, the microorganism surrounded by the shell not only has reduced metabolic activity but also may cause death of the strain, thereby affecting the efficiency of the microorganism in reducing other metabolites. At the same time, the total arsenic in the solution was largely removed in 0.5 days in the treatment with the addition of sodium arsenite, which was also the result of Fe²⁺Consistent with the change in total Fe. In the control group, total arsenic in the solution was not changed. Indicating that total arsenic in the solution could not be removed without the presence of ferrooxidans, which also confirms the role of ferrooxidans in arsenic contamination remediation. Under the treatment of adding different carbon sources and nitrogen sources, NaNO can be found₃And CH₃The combined effect of COONa is superior to that of either one added alone, the effect of treatment with only addition of carbon source is the worst, and the difference in effect exhibited at different microbial concentrations is large. While in the group where the nitrogen source was added, this difference was not very significant. The above results indicate that the iron-oxidizing bacteria are nitrate-dependent bacteria. And when the content of the added iron-oxidizing bacteria is more than 4 percent (3 mL of bacterial suspension is added to 1mg of effective arsenic), the best effect is shown on the removal rate of the total arsenic, and the total arsenic content can be completely removed within 0.5 day.

The resulting mineral was characterized by XRD (fig. 5). The results show that the minerals in lepidocrocite, arsenopyrite and clinicosaxite 3 are formed after the treatment by the iron oxidizing bacteria, and the diffraction peak of the iron mineral which is not treated by the iron oxidizing bacteria is not obvious after the treatment by the 8 percent iron oxidizing bacteria. Crystal structure diagrams of 3 minerals were constructed using Diamond software (fig. 6).

Through the above 2 experiments, it was found that the iron-oxidizing bacteria were nitrate-dependent bacteria: 0.8-3.0 mmol of NO is correspondingly needed for every 1mL of the ferroxida bacterial suspension₃ ^-(ii) a The presence of Fe (II) promotes NO₃ ^-The reduction reaction of (1) is continued to promote biological mineralization and further influence the conversion of arsenic form, and 1mL of the ferroxidans correspondingly contain 1.5-3.0 mmol of ferrous ions. The acetate provides a carbon source for the growth of the iron-oxidizing bacteria and promotes the reduction reaction of NO3 < - >, and 1mL of the iron-oxidizing bacteria correspondingly contain 0.5-1.5 mmol of acetate. The iron-oxidizing bacteria are microaerophilic bacteria, and the incubation is carried out under the condition of maintaining the oxygen partial pressure of 0.01-0.03 Pa.

Example 3

Iron-oxidizing bacteria are added into arsenic-polluted soil in Chenzhou, Hunan, and 3ml of bacterial suspension is added for 1mg of available arsenic. Four experimental groups of nitrogen addition, nitrogen-free control, bacteria-free and bacteria-free blank are set (according to the fertilizer application amount per kilogram of soil, NaNO: the₃0.85g,). And (3) using sterile water containing 1mL/L of trace elements to soak the soil, maintaining the aerobic reduction condition required by the iron-oxidizing bacteria, using dilute HCl or NaOH to adjust the soil solution to be neutral, and balancing for about one week. Culturing for 30 days, sampling once a day in the previous week, sampling once a week later, placing the sample in an oven for drying, grinding, sieving with a 100-mesh sieve, sealing and storing the screened soil powder, and performing subsequent determination.

The pH value of the soil is measured periodically, and 20ml of 0.5mol/L NaHCO is added into a sample₃The arsenic content in the active form was measured by hydride generation atomic fluorescence spectroscopy (HG-AFS) after centrifugation, and the results are shown in FIG. 7.

Compared with the available arsenic concentration (1.32mg/kg) in the original soil blank control group, the available arsenic content of the first sampling (day 0) is obviously reduced. Wherein the concentration of the nitrogen-free sterile group is 0.84mg/kg and is reduced by about 36%, the concentration of the nitrogen-free sterile group is 0.84mg/kg and is reduced by about 37%, the concentration of the nitrogen-containing sterile group is 1.16mg/kg and is reduced by about 13.1%, and the concentration of the nitrogen-containing sterile group is 0.82mg/kg and is reduced by about 38.4%. Along with the increase of the culture time, the effective arsenic content of the nitrogen-free sterile group is increased to 1.22mg/kg and the content of the nitrogen-containing sterile group is increased to 1.35mg/kg and exceeds that of the original soil control group when the last sampling is carried out, the effective arsenic content of the nitrogen-free sterile group and the effective arsenic content of the nitrogen-containing sterile group are both increased and kept stable, and then are reduced to some extent, and the effective arsenic content of the nitrogen-free sterile group is changed to 1.05mg/kg and the effective arsenic content of the nitrogen-containing sterile group is changed to 0.84mg/kg when the last sampling is carried out, and the effective arsenic content is obviously reduced compared with that of the original soil control group. Through calculation, the concentration of the available arsenic of the nitrogen-free bacterium group is reduced by about 20.4 percent, while the concentration of the available arsenic of the nitrogen-containing bacterium group is reduced by about 36.2 percent, so that the reduction range of the concentration of the available arsenic of the nitrogen-containing bacterium group is much higher than that of the nitrogen-free bacterium group, and the nitrogen-containing bacterium group has certain long-acting property for fixing the arsenic in the polluted soil.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. An iron oxidizing bacterium, characterized in that it is classified and named as (A)Ochrobactrum sp.）EEELCW01And is preserved in China Center for Type Culture Collection (CCTCC) at 11 months 3 in 2020 with the preservation number of CCTCC M2020053.

2. Use of iron oxidizing bacteria according to claim 1 for remediation of arsenic contaminated soil.

3. An arsenic contamination remediation agent comprising OD₆₀₀The bacterial suspension of the iron oxidizing bacteria, nitrate salt and ferrous salt of claim 1 with the value of 1.9-2.1, wherein 0.8-3.0 mmol of NO is added to every 1mL of bacterial suspension of the iron oxidizing bacteria₃ ^-And 1.5 to 3.0mmol of ferrous ions.

4. The arsenic pollution remediation agent of claim 3 comprising acetate, wherein 0.5 to 1.5mmol of acetate ions are added per 1mL of iron-oxidizing bacteria.

5. A method for remediating arsenic-contaminated soil, which comprises adding the arsenic-contaminated remediation agent as defined in claim 3 or 4 to arsenic-contaminated soil and incubating the mixture.

6. The method for remediating arsenic-contaminated soil as claimed in claim 5, wherein 3 to 6ml of the suspension of the iron oxidizing bacteria is added per 1mg of the arsenic-contaminated soil containing arsenic in an effective state.

7. The method for remediating arsenic-contaminated soil as claimed in claim 5, wherein the co-incubation is performed at an oxygen partial pressure of 0.01 to 0.03 Pa.

8. The method for remediating arsenic-contaminated soil as claimed in claim 7, wherein the co-incubation time is 7 days or more.

9. The method for remediating arsenic-contaminated soil as claimed in claim 8, wherein the co-incubation period is 14 days or more.

10. The method for remediating arsenic-contaminated soil as claimed in claim 9, wherein the co-incubation time is 28 days or more.