CN115044492B

CN115044492B - Pseudomonas putida and application thereof

Info

Publication number: CN115044492B
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: 2023-06-23
Anticipated expiration: 2042-02-16
Also published as: CN115044492A

Abstract

The invention provides pseudomonas putida, which is classified and named as pseudomonas putida Pseudomonas putida, YT-1 and gram negative, and is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 7 and 13 days in 2021, wherein the preservation number is as follows: CGMCC No.22872. The invention can realize the control of arsenic migration and transformation process in soil and the reduction of biological effectiveness, and utilizes the pseudomonas putida to adsorb and tolerate arsenic and form a complex with iron (hydrogen) 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 in-situ remediation treatment of soil microorganisms, and particularly relates to pseudomonas putida and application thereof.

Background

Arsenic (As) is a highly toxic metalloid element, and international cancer research institutions classify As a class I carcinogen, and can enter the terrestrial ecosystem through atmospheric settling, wastewater discharge, and application of arsenic-containing fertilizers and pesticides, etc. The paddy soil is cultivated soil with the largest area in China, the spatial distribution of the concentration of heavy metal As in the soil in the agricultural area is in direct proportion to the cancerogenic risk, and arsenic in the polluted soil can enter human bodies through food chains to influence public health.

At present, biological treatment modes for repairing As pollution in paddy soil can be divided into two types: one is to use super-enriched plants to remove As from the soil. The principle is that As is extracted from soil through the metabolism of plants and stored in the roots, stems and leaves of the plants, and the absorption efficiency of the super-enriched plants is improved through means such As inoculating arsenic redox flora, screening arsenic-resistant endophytes 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 is subjected to complexation, coordination, ion exchange, biological adsorption and other reactions with metal ions, so as to achieve the effect of fixing heavy metals.

Under experimental conditions, the in-situ remediation technology of soil microorganisms has limited arsenic adsorption capacity, and the characteristics of large specific surface area, high surface charge and the like of the ferro-manganese (hydrogen) oxide are utilized to specifically adsorb/non-specifically adsorb arsenate in soil to form a complex, so that the effective state of the soil arsenic can be effectively reduced. Indigenous strains screened from rice soil and having higher tolerance and adsorption capacity to arsenic are selected from Fe (OH) ₃ Effectively reduces the biological effective state of the soil arsenic under induction. At present, pseudomonas putida is adsorbed, fe (OH) ₃ The application of inducing the reduction of the migration capacity and bioavailability of the arsenic in the soil is not reported.

Accordingly, there is a need to develop a pseudomonas putida strain and its use to address the deficiencies of the prior art, to solve or alleviate one or more of the problems described above.

Disclosure of Invention

In view of the above, the invention provides a pseudomonas putida strain and application thereof, which can realize the control of arsenic migration and transformation process in soil and the reduction of bioavailability, and utilize the pseudomonas putida strain to adsorb and endure arsenic and form a complex with 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 pseudomonas putida strain, which is classified and named as pseudomonas putida Pseudomonas putida, YT-1, gram negative, and is preserved in China general microbiological culture Collection center (address: north Chen Xi No. 1, 3 of the area of Chachiensis in Beijing, the area of Chachiensis), wherein the preservation number is: CGMCC No.22872.

In aspects and any one of the possible implementations described above, there is further provided an implementation in which the culture medium formulation used by the pseudomonas putida in the screening process is a nutrient broth culture medium.

In aspects and any possible implementation manner described above, there is further provided a use of a strain of pseudomonas putida, including the pseudomonas putida for repairing arsenic contaminated paddy soil.

In the aspects and any possible implementation manner as described above, further provided is an implementation manner, where the application method specifically uses the pseudomonas putida to treat the rice soil to be tested for arsenic pollution.

In the aspects and any possible implementation manner, there is further provided an implementation manner, where the application method further includes adding Fe (OH) during the treatment of the rice soil to be tested for arsenic pollution by using the pseudomonas putida ₃ Fix arsenic in soil and reduce 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 has stronger adsorption capacity to arsenic in bacterial cells;

(2) The culture condition of the strain is simple, the culture medium is easy to obtain, the strain is easy to store, and the strain has certain development potential for industrial application;

(3)Fe(OH) ₃ promote the fixation of pseudomonas putida to soil arsenic, reduce mobility and biological effectiveness, and provide an in-situ repair technology for environment-friendly arsenic-polluted paddy soil.

Of course, it is not necessary for any of the products embodying the invention to achieve all of the technical effects described above at the same time.

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, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a colony morphology of Pseudomonas putida Pseudomonas putida YT-1 of the present invention, example 1;

FIG. 2 is a view showing the morphology of a 40X 10 strain of Pseudomonas putida Pseudomonas putida YT-1 gram-stained laser confocal microscope in example 1 of the present invention;

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

FIG. 4 is a diagram showing the detection of genome extraction electrophoresis in example 3 of the present invention;

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

FIG. 6 is a graph showing the percentage change in arsenic content and the cumulative rate of arsenic content in the strain of Pseudomonas putida Pseudomonas putida YT-1 of example 4 according to the present invention;

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

FIG. 8 is a Pseudomonas putida Pseudomonas putida YT-1 binding to Fe (OH) according to example 5 of the present invention ₃ Schematic diagram of the change of the existing form of the arsenic in the soil before and after the restoration.

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

Detailed Description

For a better understanding of the technical solution of the present invention, the following detailed description of the embodiments of the present invention refers to the accompanying drawings.

It should be understood that the described embodiments are merely some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the 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 this application 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 pseudomonas putida and application thereof, wherein the pseudomonas putida is classified and named as pseudomonas putida Pseudomonas putida, YT-1, and is gram negative, and the pseudomonas putida is preserved in China general microbiological culture Collection center (address: north Chen West road No. 1, no. 3 in the Korean region of Beijing) of the China general microbiological culture Collection center (2021, 7, 13 days), 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 an application of the pseudomonas putida, comprising the pseudomonas putida, wherein the pseudomonas putida is used for repairing the arsenic polluted paddy soil. Realizes the control of arsenic migration and transformation process in soil and the reduction of biological effectiveness. By utilizing the adsorption and tolerance of the pseudomonas putida to arsenic and the formation of a complex between iron (hydrogen) oxide and arsenic in soil, the migration and conversion of arsenic in the soil are controlled and the bioavailability is reduced. The application method comprises the pretreatment of the 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 (hydrogen) oxide ₃ Added into the reaction system.

Example 1

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

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

The specific method and process are described as follows: 1g of soil sample is weighed, added into 10mL of sterile water, shaken for 1h, kept stand for 1min, and then 1mL of soil suspension is sucked and inoculated into LB culture medium for 24h at 30 ℃. Transfer 1mL of culture to fresh arsenic (50 mg/LNa) ₂ HAsO ₄ ·7H ₂ O) culturing for 24 hours at 30 ℃ in 100mL of culture medium; separating bacteria by serial dilution coating method, selecting single colony, and collecting solid culture medium (100 mg/L Na ₂ HAsO ₄ ·7H ₂ And O) streaking and purifying, wherein the culture condition is 30 ℃, and a purified strain is obtained. The plate colony diagram of the colony morphology of pseudomonas putida Pseudomonas putida YT-1 is shown in figure 1, the morphology of the strain of pseudomonas putida Pseudomonas putida YT-1 when amplified by 40 multiplied by 10 under a laser confocal microscope is shown in figure 2, and the strain is identified as pseudomonas putida (Pseudomonas putida) by performing physiological and biochemical characteristic measurement and 16SrDNA sequence analysis through an API kit, namely Pseudomonas putidaYT-1 of the invention. The conditions for separating, purifying and culturing the strain and detecting the activity are nutrient broth culture medium (10 g peptone, 3g beef extract powder, 5g NaCl, 1000mL distilled water, 1mol/L NaOH for regulating the pH to 7.0); the culture temperature was 30 ℃.

Example 2

Arsenic tolerance performance test of Pseudomonas putida Pseudomonas putida YT-1

Inoculating Pseudomonas putida Pseudomonas putida YT-1 suspension into a suspension containing 40mg/L, 60 mg/L, 80mg/L and 100mg/L Na ₂ HAsO ₄ ·7H ₂ In LB medium of O, the cells were cultured at 30℃in a shaker at 180rpm, and three replicates were set in each group. Measurement of bacterial growth Curve Using spectrophotometry, different culture time OD was recorded at 600nm _600nm The results of the measurement of the growth curve of Pseudomonas putida YT-1 cultured at various concentrations of As (V) are shown in FIG. 3. In the figure, the abscissa represents the incubation time (h), the ordinate represents the optical density of bacteria measured at 600nm, and the curve represents the growth curve of Pseudomonas putida YT-1.

Example 3

Molecular and physiological biochemical identification of pseudomonas putida Pseudomonas putida YT-1

(1) This example uses the 16S rRNA sequence to test and analyze Pseudomonas putida Pseudomonas putida YT-1 of the present invention.

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

The 16S rRNA gene fragment of the selected strain was PCR amplified, cloned, and sequenced using the upstream primer 8F and the downstream primer 1492R of the 16S rRNA conserved sequence. The fragment of about 1500bp is obtained after sequencing, the strain belongs to Pseudomonas putida after sequencing, and 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 first 10 positions and species classification information with the highest similarity in the 16SrRNA gene sequencing BLAST comparison result of Pseudomonas putidaYT-1 are shown in Table 1.

TABLE 1 BLAST comparison results

The phylogenetic genus of Pseudomonas putida (Pseudomonas putida YT-1) strain is shown in FIG. 5, and the results of the genetic test indicate that the similarity with both Pseudomonas putida strain NBRC 14164 and Pseudomonas putida strain ATCC 12633 reaches 99.717 sp. (Pseudomonas putida) identified as Pseudomonas putida sp.

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

TABLE 2 physiological and biochemical characteristics of strains

Test items	Detection results
		Reduction of NO3 nitrate to nitrite	-
TRP indole	-
		GLU acidified glucose	+
ADH arginine double water medium enzyme	+
		URE urease	+
ESC beta-glucosidase	+
		GEL protease	+
PNPG beta-galactosidase	-
		GLU assimilates glucose	+
ARA assimilates arabinose	-
		MNE assimilation mannose	-
MAN assimilates mannitol	-
		NAG assimilation N-acetyl-glucosamine	-
MALs assimilate maltose	-
		GNT assimilates gluconate	+
CAP assimilation of capric acid	+
		ADI assimilation of adipic acid	-
MLT assimilation of malic acid	+
		CIT assimilation citric acid	+
PAC assimilation of phenylacetic acid	-

++, of the material; a positive reaction; -: negative reaction

Example 4

Pseudomonas putida Pseudomonas putida YT-1 to remove arsenic in culture medium and add Na into bioaccumulation LB culture medium ₂ HAsO ₄ ·7H ₂ O, the concentration is 100mg/L, and the sterile operation inoculation is carried out on 1%Pseudomonas putida YT-1 bacterial suspension after sterilization and coolingCulturing at 30deg.C in a shaker at 150rpm for 24 hr for bioaccumulation and arsenic removal. The method comprises collecting 50mL of culture solution every 24h, centrifuging at 10,000rpm for 10min, collecting cell bacterial precipitate, and adding 10mL of [ HCl: HNO ] ₃ (3:1)]After the cell sediment is dissolved, adopting AFS-230E double-channel hydride to generate atomic fluorescence to determine the content of bioaccumulation arsenic in the cells; after centrifugation, the supernatant was used to determine the arsenic removal amount, and the As (V) concentration in the cell pellet and the culture solution were measured, respectively, and the concentration curve was drawn, and the result was shown in FIG. 6. The unvaccinated strain contained 100mg/L Na ₂ HAsO ₄ ·7H ₂ LB medium of O was used as a control sample for non-biological removal. The percentage formula of arsenic removal is:

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

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

Example 5

Application demonstration of Pseudomonas putida Pseudomonas putida YT-1 in soil treatment of arsenic-polluted rice

Fe(OH) ₃ Is large in specific surface area and high in activity, and 38.07g FeCl is weighed ₃ ·6H ₂ O is stirred and dissolved in 1000mL deionized water, 1mol/L NaOH solution is added dropwise to adjust the pH value to 7.0-7.6, and the reddish brown suspended substance is Fe (OH) ₃ And (5) raw liquid.

Pseudomonas putida Pseudomonas putida YT-1 is selected to be inoculated into LB culture medium and cultured to the logarithmic phase. The enriched Pseudomonas putida and 10mL of freshly prepared Fe (OH) were subjected to a reaction ₃ The suspension was added to 50g of arsenic-contaminated soil samples, the soil moisture content was adjusted to 70% of the field maximum water holding capacity (Water Holding Capacity, WHC), and the soil was incubated in a constant temperature incubator at 30℃in the dark for 14d, while a set of samples without inoculating the strain was set as a control. Pseudomonas putida not only can adsorb As (V) by cells, but also can adsorb Fe (OH) with high activity ₃ Also has higher adsorption capacity, and induces iron oxide and As (V) in strainIs subjected to complex precipitation. As can be seen from FIG. 7, the leaching Toxicity (TCLP) of the solid waste after repair is obviously reduced, the heavy metal content extracted by TCLP is 4.16mg/kg when not repaired, and the heavy metal content extracted by TCLP after 14d of repair is 1.68mg/kg. As shown in FIG. 8, the contents of the weakly adsorbed arsenic F1 and the strongly adsorbed arsenic F2 in the restored soil are respectively reduced by 47.7% and 49.7%, and the content of the residual arsenic F5 is improved by 14.8%. Fe (OH) is not added ₃ After the soil sample is inoculated with the strain, the content of weak adsorption arsenic F1 is obviously increased, the adsorption effect of pseudomonas putida Pseudomonas putida YT-1 arsenic is stronger, and the binding capacity between As and a soil solid phase is weakened and released from the soil. When Fe (OH) is added ₃ And then the adsorption force of the strain on iron (hydrogen) oxide and arsenic is utilized, and the content of residual arsenic F5 is obviously improved.

Experimental results in the embodiment show that the pseudomonas putida Pseudomonas putida YT-1 screened by the invention has strong arsenic adsorption capacity, and can effectively reduce the content of weak adsorption state arsenic F1 and strong adsorption state arsenic F2 by combining iron (hydrogen) oxide, so that the mobility and biological effectiveness of arsenic in soil are weakened, and meanwhile, the efficiency of in-situ remediation of the arsenic in the soil by microorganisms can be improved by taking the iron (hydrogen) oxide as a good soil remediation agent.

The Pseudomonas putida and the application thereof provided by the embodiment of the application are described in detail above. The above description of embodiments is only for aiding in understanding the method of the present application and its core ideas; meanwhile, as those skilled in the art will have modifications in the specific embodiments and application scope in accordance with the ideas of the present application, the present description should not be construed as limiting the present application in view of the above.

Certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will appreciate that a hardware manufacturer may refer to the same component by different names. The description and claims do not take the form of an element differentiated by name, but rather by functionality. As referred to throughout the specification and claims, the terms "comprising," including, "and" includes "are intended to be interpreted as" including/comprising, but not limited to. By "substantially" is meant that within an acceptable error range, a person skilled in the art is able to solve the technical problem within a certain error range, substantially achieving the technical effect. The description hereinafter sets forth the preferred embodiment for carrying out the present application, but is not intended to limit the scope of the present application in general, for the purpose of illustrating the general principles of the present application. The scope of the present application is defined by 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 product 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 product or system. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a commodity or system comprising such elements.

It should be understood that the term "and/or" as used herein is merely one relationship describing the association of the associated objects, meaning that there may be three relationships, e.g., a and/or B, may represent: a exists alone, A and B exist together, and B exists alone. In addition, the character "/" herein generally indicates that the front and rear associated objects are an "or" relationship.

While the foregoing description illustrates and describes the preferred embodiments of the present application, it is to be understood that this application is not limited to the forms disclosed herein, but is not to be construed as an exclusive use of other embodiments, and is capable of many other combinations, modifications and environments, and adaptations within the scope of the teachings described herein, through the foregoing teachings or through the knowledge or skills of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the present invention are intended to be within the scope of the appended claims.

Sequence listing

<110> North China Water conservancy university of hydropower

<120> Pseudomonas putida strain and use 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 in that pseudomonas putida is named as pseudomonas putida, YT-1, gram negative and is preserved in the China general microbiological culture Collection center (China general microbiological culture Collection center) at the year 7 and 13 of 2021, and the preservation number is: cgmccno.22872.

2. The use of a strain of pseudomonas putida as claimed in claim 1 for the remediation of arsenic contaminated paddy soil.

3. The use according to claim 2, characterized in that it is the treatment of rice soil to be tested for arsenic contamination with pseudomonas putida.

4. The use according to claim 3, further comprising the addition of Fe (OH) during the treatment of the rice soil contaminated with arsenic to be tested with Pseudomonas putida ₃ Fixed soilArsenic in soil reduces the biological effective state of arsenic in soil.