CN115044492A

CN115044492A - Pseudomonas putida and application thereof

Info

Publication number: CN115044492A
Application number: CN202210143484.XA
Authority: CN
Inventors: 王娟; 李闻天; 翟雯航; 张钰; 曹一鸣; 曹永�; 胡雷雷
Original assignee: North China University of Water Resources and Electric Power
Current assignee: North China University of Water Resources and Electric Power
Priority date: 2022-02-16
Filing date: 2022-02-16
Publication date: 2022-09-13
Anticipated expiration: 2042-02-16
Also published as: CN115044492B

Abstract

The invention provides a Pseudomonas putida, which is classified and named Pseudomonas putida, YT-1, gram negative, and deposited in China general microbiological culture Collection center at 13 months 7 in 2021 with the deposition number: CGMCC No. 22872. The invention can realize the control of the migration and transformation process of arsenic in soil and the reduction of biological effectiveness, and utilizes the adsorption and tolerance of the pseudomonas putida to arsenic and the formation of a complex of iron (hydroxide) oxide and arsenic in soil to control the migration and transformation of arsenic in soil and reduce the biological effectiveness.

Description

Pseudomonas putida and application thereof

Technical Field

The invention belongs to the technical field of soil microorganism in-situ remediation treatment, and particularly relates to pseudomonas putida and application thereof.

Background

Arsenic (As) is a highly toxic metalloid element, which is classified As a class I carcinogen by the International cancer research organization and enters the terrestrial ecosystem by atmospheric sedimentation, wastewater discharge, and the application of fertilizers and pesticides containing arsenic. The paddy soil is the cultivated soil with the largest area in China, the spatial distribution of the concentration of heavy metal As in the soil in agricultural areas is in direct proportion to the carcinogenic risk, and arsenic in the polluted soil can enter human bodies through food chains to affect the public health.

At present, biological treatment modes for As pollution remediation in rice soil can be divided into two types: one is the removal of soil As by hyper-enriching plants. The principle is that As is extracted from soil through the metabolism of plants and stored in rhizome and leaves of the plants, and the absorption efficiency of the hyper-enrichment plants is improved through the means of inoculating arsenic redox flora, screening arsenic-resistant endophyte and the like. The other is to repair the arsenic contaminated soil by using microorganisms. The principle is that the function group on the surface of the microorganism and metal ions are subjected to complexation, coordination, ion exchange, biological adsorption and other reactions to achieve the effect of fixing heavy metals.

Under experimental conditions, the adsorption capacity of the soil microorganism in-situ remediation technology on arsenic is limited, and by utilizing the characteristics of large specific surface area, high surface charge and the like of iron-manganese (hydrogen) oxide, arsenic acid radicals in soil are adsorbed/non-specifically to form a complex, so that the effective state of the soil arsenic can be effectively reduced. Indigenous strains selected from rice soil having high tolerance and adsorption capacity to arsenic, in Fe (OH) ₃ Effectively reduces the biological effective state of the arsenic in the soil under the induction. Currently, Pseudomonas putida is adsorbed, Fe (OH) ₃ The application of inducing the migration capacity and the bioavailability of the arsenic in the soil is not reported.

Accordingly, there is a need to develop a pseudomonas putida strain and applications thereof to address the deficiencies of the prior art and to solve or mitigate one or more of the problems set forth above.

Disclosure of Invention

In view of the above, the invention provides a pseudomonas putida and an application thereof, which can realize the control of the migration and transformation process of arsenic in soil and the reduction of bioavailability, and utilize the adsorption and tolerance of the pseudomonas putida on arsenic and the formation of a complex of iron (hydrogen) oxide and arsenic in soil to control the migration and transformation of arsenic in soil and reduce the bioavailability.

In one aspect, the invention provides a strain of Pseudomonas putida classified and named Pseudomonas putida, YT-1, gram negative, deposited at the common microorganism center of the China Committee for culture Collection of microorganisms at 7/13/2021 (address: Beijing, Naja district, West Lu No. 1 of Beichen province No. 3), with the deposition number: CGMCC No. 22872.

The above aspects and any possible implementations further provide an implementation where the culture medium formulation used in the screening process for pseudomonas putida is a nutrient broth.

The above aspects and any possible implementation manners further provide a use of a strain of pseudomonas putida, including the pseudomonas putida, for remediating arsenic-contaminated paddy soil.

The above aspects and any possible implementation manner further provide an implementation manner, and the application method is specifically to use the pseudomonas putida to treat the paddy soil to be tested as arsenic pollution.

The above aspects and any possible implementation manners further provide an implementation manner, and the application method further comprises adding Fe (OH) into the Pseudomonas putida during the treatment of the paddy soil to be tested with arsenic pollution ₃ Fixing arsenic in soil and reducing the biological effective state of arsenic in soil.

Compared with the prior art, the invention can obtain the following technical effects:

(1) the pseudomonas putida is gram-negative bacteria, has good tolerance to high-concentration arsenic, and simultaneously has strong adsorption capacity to arsenic in a bacterial strain cell;

(2) the culture conditions of the strains are simple, the culture medium is easy to obtain, the strains are easy to store, and certain development potential is realized for industrial application;

(3)Fe(OH) ₃ promotes the fixation of the pseudomonas putida to the arsenic in the soil, reduces the mobility and the biological effectiveness, and provides an environment-friendly in-situ remediation technology of the arsenic-polluted rice soil.

Of course, it is not necessary for any product to achieve all of the above-described technical effects simultaneously in the practice of the invention.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the morphological characteristics of the colony of Pseudomonas putida YT-1 of Pseudomonas putida in example 1 of the present invention;

FIG. 2 is a diagram of the morphological observation of Pseudomonas putida YT-1 strain with a laser confocal microscope 40X 10 after gram staining in example 1 of the present invention;

FIG. 3 is a graph showing the growth of Pseudomonas putida YT-1 in culture at various concentrations of As (V) in example 2 of the present invention;

FIG. 4 is a diagram of the genomic extraction electrophoretic detection in example 3 of the present invention;

FIG. 5 is a phylogenetic tree of Pseudomonas putida YT-1, Pseudomonas putida, according to example 3 of the present invention, based on the results of 16S rRNA sequence alignment;

FIG. 6 shows the percentage change of arsenic content in the culture medium and the accumulation rate of arsenic content in the strain of Pseudomonas putida YT-1 of example 4 of the present invention;

FIG. 7 shows Pseudomonas putida YT-1 binding to Fe (OH) of Pseudomonas putida in example 5 of the present invention ₃ Schematic diagram of arsenic TCLP content change of soil before and after remediation;

FIG. 8 shows malodors of example 5 of the present inventionPseudomonas putida YT-1 in combination with Fe (OH) ₃ And (3) a schematic diagram of the existing form change of the arsenic in the soil before and after remediation.

Wherein, in fig. 4: marker is DL9000, bands from top to bottom are 9000bp, 5000bp, 3000bp, 2000bp, 1000bp and 500bp, the sample loading amount is 3uL, the bright band is 30ng/uL, and the rest bands are 10 ng/uL.

Detailed Description

For better understanding of the technical solutions of the present invention, the following detailed descriptions of the embodiments of the present invention are provided with reference to the accompanying drawings.

It should be understood that the described embodiments are only some embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The terminology used in the embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the examples of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The invention provides a Pseudomonas putida and application thereof, wherein the Pseudomonas putida is classified and named as Pseudomonas putida, YT-1, gram negative, preserved in China general microbiological culture Collection center (address: Beijing city, Shangyang district, North Cheng, Xilu No. 1 institute No. 3) on 7-13 days 2021, and the preservation number is: CGMCC No. 22872. The culture medium formula used in the screening process of the pseudomonas putida is a nutrient broth culture medium.

The invention also provides application of the pseudomonas putida, which comprises the pseudomonas putida used for repairing arsenic-polluted rice soil. Realize the control of the migration and transformation process of arsenic in soil and the reduction of bioavailability. The adsorption and tolerance of the pseudomonas putida to arsenic and the coordination compound formed by the iron (hydrogen) oxide and the arsenic in the soil are utilized to control the migration and transfer of the arsenic in the soilTo neutralize and reduce bioavailability. The application method comprises the pretreatment of a sample by using the pseudomonas putida under the culture condition of 30 ℃. The application method also comprises the step of artificially synthesizing Fe (OH) by using the iron (hydroxide) ₃ Adding into the reaction system.

Example 1

Separation and screening of Pseudomonas putida YT-1 with high arsenic tolerance

The strain of the invention belongs to the genus Pseudomonas putida, Pseudomonas putida YT-1. The strain is obtained by separating and screening heavy metal polluted farmland soil in Yingtan City, Jiangxi province, and is specifically obtained by plate streaking, separating, purifying and screening after inoculation and culture in an arsenic-containing culture medium.

The specific method and process are described as follows: weighing 1g of soil sample, adding 10mL of sterile water, shaking for 1h, standing for 1min, sucking 1mL of soil suspension, inoculating into an LB culture medium, and culturing at 30 ℃ for 24 h. Transfer 1mL of culture to fresh arsenic (50 mg/LNa) ₂ HAsO ₄ ·7H ₂ O) culturing in 100mL of medium at 30 ℃ for 24 h; separating bacteria by serial dilution coating method, selecting single bacterial colony and solid culture medium (100mg/L Na) ₂ HAsO ₄ ·7H ₂ O) streak purification, and culturing at 30 ℃ to obtain a purified strain. The plate colony pattern of the colony morphology of Pseudomonas putida YT-1 is shown in figure 1, the morphology of the strain of Pseudomonas putida YT-1 under a laser confocal microscope at magnification of 40X 10 is shown in figure 2, and the strain is identified as Pseudomonas putida (Pseudomonas putida), namely the Pseudomonas putida YT-1 of the invention, by performing physiological and biochemical characterization determination and 16SrDNA sequence analysis through an API kit. The conditions of separation, purification and culture of the strain and the conditions of detection and viability are nutrient broth culture medium (10g of peptone, 3g of beef extract powder, 5g of NaCl, 1000mL of distilled water and pH value adjusted to 7.0 by 1mol/L of NaOH); the incubation temperature was 30 ℃.

Example 2

Arsenic tolerance test for Pseudomonas putida YT-1

Inoculating Pseudomonas putida YT-1 bacterial suspension to a culture medium containing 40mg/L, 60 mg/L, 80mg/L and 100mg/L Na ₂ HAsO ₄ ·7H ₂ O in LB medium, placed in a shaker at 180rpm30 ℃ for cultivation, each set of three replicates. The bacterial growth curve is measured by recording different culture time OD at 600nm by using a spectrophotometer method _600nm The results of the growth curve measurement of Pseudomonas putida YT-1 in culture at various concentrations of As (V) are shown in FIG. 3. In the figure, the cultivation time (h) is plotted on the abscissa, the optical density of the bacteria measured at 600nm is plotted on the ordinate, and the growth curve of Pseudomonas putida YT-1 is plotted.

Example 3

Physiological and biochemical identification of Pseudomonas putida YT-1 molecules

(1) This example uses the 16S rRNA sequence to perform a test analysis on Pseudomonas putida YT-1 of the present invention.

16S rRNA identification method: bacterial genome extraction, PCR amplification and sequencing of 16S V1-V9 region, extraction of genome DNA by proteinase K cleavage, and electrophoresis detection of 3uL as shown in FIG. 4.

The 16S rRNA gene segment of the screened strain is subjected to PCR amplification, cloning and sequencing by using an upstream primer 8F and a downstream primer 1492R of a 16S rRNA conserved sequence. Obtaining a fragment of about 1500bp after sequencing, obtaining the pseudomonas putida through sequencing, wherein the sequencing result of the amplified fragment is shown in a sequence table.

Wherein the upstream primer 8F is AGAGTTTGATCCTGGCTCAG;

the downstream primer 1492R is TACGGYTACCTTGTTAYGACTT.

The statistical results of the top 10 bits with the highest similarity and the species classification information in the comparison results of 16SrRNA gene sequencing BLAST of Pseudomonas putida PseudapatideYT-1 are shown in Table 1.

TABLE 1 BLAST comparison results

Phylogenetic genera of Pseudomonas putida (Pseudomonas putida YT-1) strains are shown in FIG. 5, and the results of gene tests show that the strains have 99.717% similarity to both Pseudomonas putida strain NBRC 14164 and Pseudomonas putida strain ATCC 12633, and are identified as Pseudomonas putida sp (Pseudomonas putida sp.)

(2) The physiological and biochemical properties of Pseudomonas putida YT-1 are shown in Table 2.

TABLE 2 physiological and biochemical characteristics of the strains

Test items	Detection results
		Reduction of NO3 nitrate to nitrite	-
TRP indoles	-
		GLU acidified glucose	+
ADH arginine double-water enzyme	+
		URE urease	+
ESC beta-glucosidase	+
		GEL protease	+
PNPG beta-galactosidase	-
		GLU assimilation of glucose	+
ARA assimilation of arabinose	-
		MNE assimilation mannose	-
MAN assimilation of mannitol	-
		NAG-assimilating N-acetyl-glucosamine	-
MAL assimilation maltose	-
		Gnt assimilation gluconate	+
CAP assimilation of capric acid	+
		ADI assimilation of adipic acid	-
MLT assimilation malic acid	+
		CIT assimilation citric acid	+
PAC (poly aluminum chloride) assimilated phenylacetic acid	-

+; positive reaction; -: negative reaction

Example 4

Na is added into LB culture medium for removing arsenic in the culture medium and accumulating organisms by Pseudomonas putida YT-1 ₂ HAsO ₄ ·7H ₂ O, the concentration is 100mg/L, 1% Pseudomonas putida YT-1 bacterial suspension is inoculated in an aseptic operation after sterilization and cooling, the mixture is placed in a shaking table at 150rpm and 30 ℃ for culture, and biological accumulation and arsenic removal measurement are carried out every 24 h. The specific method comprises collecting 50mL culture solution every 24h, centrifuging at 10,000rpm for 10min, collecting cell thallus precipitate, and adding 10mL [ HCl: HNO ] ₃ (3:1)]After the cell precipitate is dissolved, AFS-230E double-channel hydride is adopted to generate atomic fluorescence to determine the content of the biologically accumulated arsenic in the cell; the culture supernatant after centrifugation was used to measure the amount of arsenic removed, and the As (V) concentration in the cell pellet and the culture broth, respectively, was measured to prepare concentration curves, and the results are shown in FIG. 6. The non-inoculated strain contains 100mg/L Na ₂ HAsO ₄ ·7H ₂ LB medium for O was used as a non-bioremediation control sample. The percentage arsenic removal formula is:

initial arsenic content of IC initial arsenate concentration (mg/L)

FC final arsenate concentration (mg/L) final arsenic content

Example 5

Demonstration of the application of Pseudomonas putida YT-1 in soil treatment of arsenic-contaminated rice

Fe(OH) ₃ Has large specific surface area and high activity, 38.07g FeCl is weighed ₃ ·6H ₂ O is stirred and dissolved in 1000mL of deionized water, 1mol/L NaOH solution is dripped to adjust the pH value to 7.0-7.6, and a reddish brown suspended substance, namely Fe (OH) ₃ And (4) stock solution.

Pseudomonas putida YT-1 is inoculated to LB culture medium and cultured to logarithmic phase. The enriched pseudomonas putida and 10mL of newly prepared Fe (OH) ₃ Adding the suspension into 50g of arsenic-contaminated soil sample, adjusting the Water content of the soil to 70% of the maximum Water Holding Capacity (WHC) in the field, placing the soil in a constant-temperature incubator for dark culture at 30 ℃ for 14d, and setting a group of samples without inoculated strains as a control. Pseudomonas putida can adsorb As (V) and highly active Fe (OH) by cells ₃ Also has higher adsorption capacity, and induces iron oxide and As (V) to generate complex precipitation under the action of the bacterial strain. As can be seen from FIG. 7, the leaching Toxicity (TCLP) of the repaired solid waste is obviously reduced, the content of heavy metal extracted by TCLP is 4.16mg/kg when the solid waste is not repaired, and the content of heavy metal extracted by TCLP is 1.68mg/kg after the solid waste is repaired for 14 days. As shown in FIG. 8, the contents of weakly adsorbed arsenic F1 and strongly adsorbed arsenic F2 in the repaired soil are reduced by 47.7% and 49.7%, respectively, and the content of residual arsenic F5 is increased by 14.8%. No addition of Fe (OH) ₃ After the soil sample is inoculated with the strain, the content of weakly adsorbed arsenic F1 is obviously increased, the adsorption effect of Pseudomonas putida YT-1 arsenic is stronger, the binding capacity between As and the soil solid phase is weakened, and the arsenic is released from the soil. When Fe (OH) is added ₃ And then the content of the residue arsenic F5 is obviously improved by utilizing the adsorption force of the strain on iron (hydroxide) and arsenic.

The experimental results of the above examples show that the screened Pseudomonas putida YT-1 has strong arsenic adsorption capacity, the contents of weakly adsorbed arsenic F1 and strongly adsorbed arsenic F2 can be effectively reduced by combining iron (hydrogen) oxide, the migration and biological effectiveness of arsenic in soil are weakened, and meanwhile, the iron (hydrogen) oxide serving as a good soil remediation agent can improve the efficiency of in-situ remediation of soil arsenic by microorganisms.

The pseudomonas putida and the application thereof provided by the embodiment of the application are described in detail above. The above description of the embodiments is only for the purpose of helping to understand the method of the present application and its core ideas; meanwhile, for a person skilled in the art, according to the idea of the present application, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present application.

As used in the specification and claims, certain terms are used to refer to particular components. As one skilled in the art will appreciate, manufacturers may refer to a component by different names. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, that a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

It should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of additional like elements in the article of commerce or system in which the element is comprised.

It should be understood that the term "and/or" as used herein is merely one type of association that describes an associated object, meaning that three relationships may exist, e.g., a and/or B may mean: a exists alone, A and B exist simultaneously, and B exists alone. In addition, the character "/" herein generally indicates that the former and latter associated objects are in an "or" relationship.

The foregoing description shows and describes several preferred embodiments of the present application, but as aforementioned, it is to be understood that the application is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the application as described herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the application, which is to be protected by the claims appended hereto.

Sequence listing

<110> North China university of water conservancy and hydropower

<120> Pseudomonas putida strain and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1410

<212> DNA

<213> pseudomonas sp.

<400> 1

ccatgcagtc gagcggatga gaagagcttg ctcttcgatt cagcggcgga cgggtgagta 60

atgcctagga atctgcctgg tagtggggga caacgtttcg aaaggaacgc taataccgca 120

tacgtcctac gggagaaagc aggggacctt cgggccttgc gctatcagat gagcctaggt 180

cggattagct agttggtgag gtaatggctc accaaggcga cgatccgtaa ctggtctgag 240

aggatgatca gtcacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg 300

gggaatattg gacaatgggc gaaagcctga tccagccatg ccgcgtgtgt gaagaaggtc 360

ttcggattgt aaagcacttt aagttgggag gaagggcatt aacctaatac gttagtgttt 420

tgacgttacc gacagaataa gcaccggcta actctgtgcc agcagccgcg gtaatacaga 480

gggtgcaagc gttaatcgga attactgggc gtaaagcgcg cgtaggtggt ttgttaagtt 540

ggatgtgaaa gccccgggct caacctggga actgcatcca aaactggcaa gctagagtac 600

ggtagagggt ggtggaattt cctgtgtagc ggtgaaatgc gtagatatag gaaggaacac 660

cagtggcgaa ggcgaccacc tggactgata ctgacactga ggtgcgaaag cgtggggagc 720

aaacaggatt agataccctg gtagtccacg ccgtaaacga tgtcaactag ccgttggaat 780

ccttgagatt ttagtggcgc agctaacgca ttaagttgac cgcctggggg agtacggccg 840

caaggttaaa actcaaatga attgacgggg gcccgcacaa gcggtggagc atgtggttta 900

attcgaagca acgcgaagaa ccttaccagg ccttgacatg cagagaactt tccagagatg 960

gattggtgcc ttcgggaact ctgacacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt 1020

gagatgttgg gttaagtccc gtaacgagcg caacccttgt ccttagttac cagcacgtaa 1080

tggtgggcac tctaaggaga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa 1140

gtcatcatgg cccttacggc ctgggctaca cacgtgctac aatggtcggt acagagggtt 1200

gccaagccgc gaggtggagc taatctcaca aaaccgatcg tagtccggat cgcagtctgc 1260

aactcgactg cgtgaagtcg gaatcgctag taatcgcgaa tcagaatgtc gcggtgaata 1320

cgttcccggg ccttgtacac accgcccgtc acaccatggg agtgggttgc accagaagta 1380

gctagtctaa ccttcgggag gacggtacca 1410

Claims

1. The Pseudomonas putida is characterized by being classified and named as Pseudomonas putida, YT-1, gram negative, deposited at China general microbiological culture Collection center at 7-13 months 2021 with the deposition number: CGMCC No. 22872.

2. The Pseudomonas putida according to claim 1, wherein the culture medium formulation used in the screening process for Pseudomonas putida is a nutrient broth culture medium.

3. Use of a strain of pseudomonas putida comprising pseudomonas putida according to any one of claims 1 to 2 for the remediation of arsenic-contaminated paddy soil.

4. The use according to claim 3, characterized in that it consists in treating the paddy soil contaminated with arsenic to be tested with said Pseudomonas putida.

5. The Pseudomonas putida according to claim 4, wherein the application method further comprises adding Fe (OH) during the treatment of the paddy soil to be tested with arsenic contamination by using the Pseudomonas putida ₃ Fixing arsenic in soil and reducing the biological effective state of arsenic in soil.