CN114455717B

CN114455717B - Application of high-antimony-resistance enterobacterium Z1 in removal of antimony and arsenic in water body

Info

Publication number: CN114455717B
Application number: CN202111652461.3A
Authority: CN
Inventors: 史凯祥; 王革娇; 张玉潇; 李明顺
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2023-12-19
Anticipated expiration: 2041-12-30
Also published as: CN114455717A

Abstract

The invention belongs to the technical field of agricultural environmental microorganism application, and particularly relates to application of high-antimony-resistance enterobacteria Z1 in removing antimony and arsenic in water. The isolated enterobacteria Z1 are preserved in China Center for Type Culture Collection (CCTCC) NO: M2019147, have high antimony resistance, and have the capability of generating antimony trioxide sediment to remove antimony in polluted water; in a water body in which antimony and arsenic coexist, the separated enterobacteria Z1 can remove antimony and arsenic in the water body at the same time, and can be used as a novel microbial agent for repairing the water body polluted by antimony and arsenic.

Description

Application of high-antimony-resistance enterobacterium Z1 in removal of antimony and arsenic in water body

Technical Field

The invention belongs to the field of agricultural environmental microorganism application, and particularly relates to application of high-antimony-resistance enterobacteria Z1 in removing antimony and arsenic in water.

Background

Antimony (Sb) and arsenic (As) are located in the fifth main group of the periodic table of elements, two (class) heavy metals that are widely found in nature, and have many similar chemical situations. In nature, the main forms of arsenic are trivalent arsenic and pentavalent arsenic, and the main forms of antimony are trivalent antimony and pentavalent antimony, wherein the toxicity of trivalent arsenic and trivalent antimony is far greater than that of the corresponding pentavalent forms: pentavalent arsenic and pentavalent antimony. The sources of arsenic and antimony in the environment mainly comprise natural processes such as volcanic eruption, rock weathering and the like, and artificial factors such as the use of arsenic-or antimony-containing medicaments, preservatives, fireproof agents and the like. Currently, two hundred million people worldwide are suffering from arsenic contaminated groundwater, including regions of multiple countries such as bangladesh, brazil, canada, china, hungary, india, indonesia, mexico, the united states, and pakistan. Furthermore, according to recent reports by the U.S. geological survey 2021, approximately 80% of antimony production worldwide has been concentrated in china, russia and bolivia. The natural pollution environment is usually accompanied by arsenic and antimony, for example, areas such as China's cold water river are polluted by arsenic and chromium, once the arsenic and the antimony enter a human body through a food chain, the arsenic and the antimony can react with sulfhydryl groups in human tissues, and cell hypoxia is caused by inhibiting enzyme action and disturbing cell ion balance. These adverse effects ultimately lead to metabolic dysfunction and damage to the nervous system and vital organs.

Currently, the treatment methods for arsenic and antimony contamination include: physical repair, chemical repair, and biological repair. Physical repair methods include methods of changing soil, turning soil, and landfilling. The chemical remediation method aims at adding a curing agent and a stabilizing agent into arsenic and antimony polluted soil or water body, so that the release of arsenic and antimony in the soil is reduced. The bioremediation method is to absorb arsenic and antimony in soil or water body by the arsenic and antimony super-enrichment plants. As a novel green environmental restoration means, the microorganism can passivate soil or remove arsenic and antimony in water body through the metabolic process, so that the method is an effective and environment-friendly environmental restoration means. Compared with physical and chemical methods, the microbial remediation method has the advantages of low price, environmental friendliness and the like, and is paid attention to.

Taking antimony-tolerant bacteria as an example, table 1 summarizes the antimony-tolerant bacteria and their application to antimony repair in recent years related patent documents. Deng Renjian et al report that a strain of Pseudomonas antimonide-arsenoxide (publication No. CN 111676165A) and a strain of highly resistant Proteus antimonide DSHN0704 (publication No. CN 108676763A) belong to Pseudomonas sp and Proteus kaposis respectively, and that the tolerance to antimony is 8000mg/L and 1000mg/L respectively; rong Qun et al (publication No. CN 112980714A) reported an antimony oxidizing bacterium having a high resistance to oxidation and its use, which is a Klebsiella aerogenes (Klebsiella aerogenes) having a resistance to antimony of 4800mg/L; peng Xiawei et al report that NXH1 (publication No. CN 106244501B) belongs to Acinetobacter (Acinetobacter sp.), NXH2 (publication No. CN 106244500B) belongs to Klebsiella sp, NXH3 (publication No. CN 106434446B) belongs to Stenotrophomonas sp, XKS1 (publication No. CN 106479914B) belongs to Pseudomonas sp, and the above strains have tolerance to antimony of 2900mg/L, 3300mg/L, 3200mg/L, respectively; lu Xiaolu et al (publication No. CN 112980714A) reported a Bao Xishi strain AS-1 which is resistant to high arsenic and antimony contamination and oxidizes As (III) and its use, which belongs to Bao Xishi strain (Bosea sp.) and has a resistance to antimony of 600mg/L. Furthermore, only two strains of the above antimony-tolerant bacteria were reported to have the ability to produce antimony precipitate, the yield of antimony precipitate by Proteus kaposis DSHN0704 (publication No. CN 108676763A) was 7.97mg/L, and the yield of trivalent antimony by Zymomonas clathratus (publication No. CN 111154681A) was about 48.7mg/L.

At present, there are few studies on the formation of antimony precipitate by bacteria, and bacteria of the genus enterobacteria have not been reported for their related functions. The strain in the patent application is a strain of bacterium in Enterobacter sp.Z1, the antimony tolerance effect of the bacterium is obviously higher than that of other reported antimony tolerance bacteria, the Enterobacter sp.Z1 has the function of generating antimony precipitate so as to remove antimony in a water body, and the bacterium has the function of generating arsenic precipitate under the condition of adding antimony. In the earlier study of the national emphasis laboratory of China agricultural university microbiology, where the applicant is located, the composite microbial inoculum of the enterobacteria Z1 and the Klebsiella Z2 is found to have the capability of removing high-nitrogen polluted wastewater (invention patent application publication No. CN 110157637A), but the patent document does not relate to the detection of the tolerance and the removal capability of antimony and arsenic. The results show that the enterobacteria Z1 have potential application value.

TABLE 1 bacterial tolerance to antimony and removal in related patents

Disclosure of Invention

The invention aims to overcome the defects of the prior art, screen a strain with high antimony tolerance and can efficiently remove antimony and arsenic in the environment, such as enterobacter Z1, and repair the pollution of antimony and arsenic in the environment by applying microorganisms.

The invention is realized by the following technical scheme:

enterobacter Z1 was isolated from Jingzhou, hubei, and the applicant designated the isolate as Enterobacter Z1, belonging to the genus Enterobacter (Enterobacter sp.) and was delivered to China Center for Type Culture Collection (CCTCC) with a accession number of CCTCC NO: M2019147 in 3 months 15 of 2019.

The high antimony tolerance E.coli Z1 and the antimony and arsenic removal protocol of the present invention are shown in FIG. 1. The E.coli Z1 was subjected to growth conditions in medium of different antimony concentrations (see later for details), thereby detecting the antimony tolerance concentration. Then culturing the enterobacteria Z1 in liquid added with antimony (arsenic) with different concentrations, centrifuging the culture solution after a certain period of time, collecting supernatant to detect the content of the antimony (arsenic), and finally identifying to obtain the bacteria Z1 with high antimony tolerance and capable of removing the antimony and the arsenic in the water, wherein the bacteria Z1 are named as enterobacteria (Enterobacter sp.) by the applicant.

The invention has the positive effects that:

(1) The microorganism has the advantages of low price and environmental friendliness in the aspect of heavy metal pollution remediation, and is favorable for sustainable agricultural environment

Development continues, however, development of efficient microbial preparations is relatively lacking. The strains screened by the invention have an antimony tolerance of 12100mg/L, compared with the antimony-tolerant strains reported in Table 1. The antimony tolerance of the strain of the invention is higher than other strains that have been reported to date.

(2) The maximum value of the removal amount of the stibium in the water body by the enterobacteria Z1 is 252.4mg/L, which is larger than the other two stibium removal strains reported at present: for example, the yield of antimony precipitate of the Proteus Carlsbergensis DSHN0704 (invention patent application publication No. CN 108676763A) is 7.97mg/L; the amount of trivalent antimony produced by the fermentation of Clavularia volvulus (invention patent application publication No. CN 111154681A) is about 48.7mg/L.

(3) In a water body containing antimony, the enterobacteria Z1 also have the capability of removing arsenic in the water body. At present, relatively few bacteria capable of removing antimony in a water body are provided, and bacteria capable of removing antimony and arsenic at the same time are not reported.

(4) The invention can enrich microorganism fixed antimony and arsenic resource library, has simple operation, does not need to add chemical reagent, and is expected to grow and remove antimony and arsenic in environments such as polluted water or soil.

Drawings

Fig. 1: general technical roadmap of the invention.

Fig. 2: scanning electron microscope pictures of the enterobacteria Z1 of the invention. Reference numerals illustrate: panel A in FIG. 2 is an electron micrograph of E.coli Z1 without antimony; FIG. 2B is an electron micrograph of Enterobacter Z1 at an antimony addition level of 121 mg/L; panel C in FIG. 2 is an electron micrograph of E.coli Z1 at an antimony addition level of 6050 mg/L.

Fig. 3: antimony and arsenic tolerance bar graph of enterobacteria Z1 of the present invention. Reference numerals illustrate: panel A in FIG. 3 is a bar graph of E.coli Z1 tolerance to antimony; panel B in FIG. 3 is a bar graph of E.coli Z1 tolerance to arsenic.

Fig. 4: growth curve of enterobacteria Z1 of the present invention at different antimony concentrations. Reference numerals illustrate: panel A in FIG. 4 shows the growth curve of E.coli Z1 without antimony; FIG. 4B is a graph showing the growth of E.coli Z1 at an antimony addition level of 121 mg/L; FIG. 4C is a graph showing the growth of E.coli Z1 at an antimony addition level of 1210 mg/L; panel D in FIG. 4 shows the growth curve of E.coli Z1 at an antimony addition level of 6050 mg/L.

Fig. 5: the enterobacteria Z1 of the invention generate a graph and a bar graph of antimony and arsenic precipitation. Reference numerals illustrate: FIG. 5A is a graph showing the amount of antimony precipitate produced by E.coli Z1 at an antimony addition level of 121 mg/L; FIG. 5B is a graph showing the amount of antimony precipitate produced by E.coli Z1 at an antimony addition level of 1210 mg/L; panel C in FIG. 5 shows the amount of arsenic precipitate produced by Enterobacter Z1 at an arsenic addition of 7.5 mg/L; FIG. 5D is a graph showing the amount of arsenic precipitate produced by Enterobacter Z1 at an arsenic addition amount of 75 mg/L.

Fig. 6: the result of the X-ray diffraction identification of the antimony precipitate generated by the enterobacteria Z1 is identified.

Detailed Description

Description of the sequence listing.

The sequence table SEQ ID NO. 1 is a 16S ribosomal RNA gene sequence of E.coli Z1.

Example 1: detection of tolerance of enterobacter Z1 to antimony and arsenic

The method for detecting the antimony tolerance comprises the following steps: the E.coli Z1 was picked up and inoculated into 5mL of LB liquid medium and shake-cultured overnight at 28℃in a shaker. The bacterial culture was transferred to 5mL of R2A medium (0.5 g/L glucose, 0.3g/L dipotassium hydrogen phosphate, 0.5g/L starch, 0.5g/L peptone, 0.024g/L magnesium sulfate, 0.3g/L sodium acetate, 0.5g/L yeast extract, 0.5g/L casein hydrolysate) at 1% by volume and cultured overnight with shaking in a shaker at 28 ℃. A bacterial (E.coli Z1) culture broth was transferred to 5mL of R2A medium at an inoculum size of 1% by volume, and 0mg/L, 121mg/L, 605mg/L, 1210mg/L, 4840mg/L, 6050mg/L, 12100mg/L of trivalent antimony was added to the medium, and the culture was shake-cultured in a shaker at 28℃for 24 hours.

The method for detecting arsenic tolerance comprises the following steps: the E.coli Z1 was picked up and inoculated into 5mL of LB liquid medium and shake-cultured overnight at 28℃in a shaker. The bacterial (E.coli Z1) culture broth was transferred to 5mL of LB medium at an inoculum size of 1% by volume, and 0mg/L, 75mg/L, 562.5mg/L, 750mg/L, 1125mg/L of trivalent arsenic was added to the medium, followed by shaking culture in a shaker at 28℃for 24 hours. Placing the culture solution into a shaking table at 28deg.C for shaking culture for 24 hr, and measuring OD of bacteria by spectrophotometry ₆₀₀ The absorbance value of the bacteria is obtained, and the bacterial growth is obtained.

Example 2: growth test of Enterobacter Z1 at different antimony concentrations

The E.coli Z1 was picked up and inoculated into 5mL of LB liquid medium and shake-cultured overnight at 28℃in a shaker. The bacterial (E.coli Z1) broth was transferred to 5mL of R2A medium at 1% by volume and incubated overnight with shaking in a shaker at 28 ℃. The bacterial (E.coli Z1) culture broth was transferred to 100mL of R2A medium at an inoculum size of 1% by volume, and 0mg/L, 121mg/L, 1210mg/L, 6050mg/L of trivalent antimony was added to the medium and cultured by shaking in a shaker at 28 ℃. 2mL of the bacterial liquid was taken out every 4 hours, and the OD of the bacteria was measured by a spectrophotometer ₆₀₀ And (5) the absorbance value is obtained, and then a bacterial growth curve is obtained. The samples were taken together for 24 hours.

Example 3: test of removal amount of antimony and arsenic by Enterobacter Z1

The antimony removal test method is as follows: the E.coli Z1 was picked up and inoculated into 5mL of LB liquid medium and shake-cultured overnight at 28℃in a shaker. The bacterial (E.coli Z1) broth was transferred to 5mL of R2A medium at 1% by volume and incubated overnight with shaking in a shaker at 28 ℃. The bacterial culture (E.coli Z1) was transferred to 5mL of R2A medium at an inoculum size of 1% by volume, and 121mg/L and 1210mg/L of trivalent antimony were added to the medium and cultured with shaking in a shaker at 28℃in 4 groups each. Taking out one group of bacterial solutions of different treatment groups every 24 hours, discarding the bacterial solutions, cleaning the inner wall of the shake flask with deionized water for 5 times, and placing the shake flask in an oven for treatment until no water drops exist in the shake flask. 0.35mL of concentrated hydrochloric acid was added to the dried flask to fully dissolve the adherent on the inner wall, and then the sample was fixed to 5mL by adding deionized water.

The arsenic removal test method is as follows: the E.coli Z1 was picked up and inoculated into 5mL of LB liquid medium and shake-cultured overnight at 28℃in a shaker. The bacterial culture (E.coli Z1) was transferred to 5mL of R2A medium at an inoculum size of 1% by volume and cultured overnight with shaking in a shaker at 28 ℃. The bacterial culture (E.coli Z1) was transferred to 5mL of R2A medium at an inoculum size of 1% by volume, and 7.5mg/L trivalent arsenic, 7.5mg/L trivalent arsenic+121 mg/L trivalent antimony, 75mg/L trivalent arsenic, 75mg/L trivalent arsenic+121 mg/L trivalent antimony were added to the medium, and the medium was shake-cultured in a shaker at 28℃with 4 groups of each treatment in parallel. Taking out one group of bacterial solutions of different treatment groups every 24 hours, discarding the bacterial solutions, cleaning the inner wall of the shake flask with deionized water for 5 times, and placing the shake flask in an oven for treatment until no water drops exist in the shake flask. 0.35mL of concentrated hydrochloric acid was added to the dried flask to fully dissolve the adherent on the inner wall, and then the sample was fixed to 5mL by adding deionized water. The antimony and arsenic content of the solution was determined by atomic fluorescence spectrometry.

Example 4: x-ray diffraction identification of enterobacter Z1 precipitate

The E.coli Z1 was picked up and inoculated into 5mL of LB liquid medium and shake-cultured overnight at 28℃in a shaker. The bacterial culture (E.coli Z1) was transferred to 10mL of R2A medium at an inoculum size of 1% by volume and cultured overnight with shaking in a shaker at 28 ℃. The bacterial culture (E.coli Z1) was transferred to 800mL of R2A medium at an inoculum size of 1% by volume, 1210mg/L of trivalent antimony was added to the medium, and the medium was shake-cultured at 28℃for 24 hours. After 24 hours, the bacterial liquid is discarded, the inner wall of the shake flask is washed for 5 times, and white sediment adhered to the inner wall of the shake flask is scraped into water after a certain volume of deionized water is added. The liquid mixed with the white precipitate was placed in a 10mL centrifuge tube, the precipitate was collected by centrifugation at 6000rpm, and the precipitate was freeze-dried in a freeze-dryer for 12 hours. The dried precipitate was subjected to characterization by X-ray diffraction.

As can be seen from the antimony and arsenic tolerance bar graph of the bacteria in FIG. 3, the same amount of growth as in the absence of antimony can be achieved after 24 hours of cultivation by inoculating E.coli Z1 in a medium containing 121-1210mg/L of trivalent antimony R2A. At a trivalent antimony concentration of 4840-12100mg/L, E.coli Z1 can still grow to half the growth without adding antimony after 24 hours. As can be seen from the electron microscope photograph of the enterobacteria Z1 in FIG. 2 under the conditions of no antimony, 121mg/L of antimony and 6050mg/L of antimony, the cell morphology of the bacteria is consistent with that under the conditions of no antimony even under the conditions that the trivalent antimony concentration is as high as 6050 mg/L. The results show that the enterobacteria Z1 are bacteria with extremely high antimony tolerance, and the antimony tolerance concentration is 12100mg/L.

As can be seen from the growth curve graph of the bacteria in FIG. 4 under the conditions of no antimony, 121mg/L of trivalent antimony, 1210mg/L of trivalent antimony and 6050mg/L of trivalent antimony, the growth of the enterobacteria Z1 in the logarithmic growth phase is slowed down to a certain extent under the conditions of 121mg/L and 1210mg/L of trivalent antimony, but the bacteria still can reach the stationary phase after 12-16 hours, and the biomass is the same as that under the condition of no antimony. Under the condition of 6050mg/L of trivalent antimony, the biomass of the enterobacteria Z1 after 24 hours is lower than that of the three conditions, however, the growth of the enterobacteria Z1 can still reach OD ₆₀₀ The value is about 0.4-0.5. The results dynamically monitor the growth state of the enterobacteria Z1 under different trivalent antimony conditions, and again prove that the enterobacteria Z1 has high antimony tolerance.

As can be seen from the graph and bar graph of bacterial removal of antimony and arsenic in FIG. 5, the amount of antimony removed by E.coli Z1 increased with time at a trivalent antimony content of 121mg/L, and the amounts of antimony removed in days 1 to 4 were respectively: 5.5mg/L, 37.8mg/L, 24.1mg/L, 40.5mg/L, significantly higher than the control group without bacteria (E.coli Z1). After the trivalent antimony concentration is increased to 1210mg/L, the removal amount of antimony by the enterobacteria Z1 is obviously increased, and the removal amount of antimony in days 1 to 4 is as follows: 145.5mg/L, 209.3mg/L, 252.4mg/L, 245.9mg/L, significantly higher than the control group without bacteria (E.coli Z1). The above results indicate that enterobacteria Z1 have an antimony removal capacity and that the amount of antimony removed increases significantly with increasing initial trivalent antimony concentration. In addition, under the condition of containing only trivalent arsenic, the enterobacteria Z1 do not have the ability to remove arsenic, whereas under the condition of having both trivalent antimony and trivalent arsenic, the enterobacteria Z1 exhibit the ability to remove trivalent arsenic. Wherein the removal amount of trivalent arsenic by the enterobacteria Z1 is 0.3mg/L under the condition that the addition amount of trivalent arsenic is 7.5mg/L, and the removal amount of trivalent arsenic by the enterobacteria Z1 is 0.8mg/L under the condition that the addition amount of trivalent arsenic is 75 mg/L. The above results indicate that enterobacteria Z1 have a synergistic ability to remove antimony and arsenic, and that the amount of antimony removed increases significantly as the initial trivalent antimony or trivalent arsenic concentration increases.

FIG. 6 shows the results of X-ray diffraction identification of the antimony precipitate produced by the E.coli Z1 of the present invention, and shows that the precipitate produced by the E.coli Z1 is antimony trioxide, which indicates that the bacteria converts soluble trivalent antimony into insoluble antimony trioxide by in vivo metabolism, and finally realizes the removal of antimony.

In conclusion, the enterobacteria Z1 of the invention has extremely high tolerance to antimony and can remove antimony and arsenic in water, so that the enterobacteria Z1 of the invention has great application potential in the restoration of antimony and arsenic polluted environments (such as water).

Sequence listing

<110> university of agriculture in China

<120> application of high antimony-resistant enterobacterium Z1 in removing antimony and arsenic in water body

<141> 2021-12-30

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gtcgaacggt agcacagaga gcttgctctc gggtgacgag tggcggacgg gtgagtaatg 120

tctgggaaac tgcctgatgg agggggataa ctactggaaa cggtagctaa taccgcataa 180

cgtcgcaaga ccaaagaggg ggaccttcgg gcctcttgcc atcagatgtg cccagatggg 240

attagctagt aggtggggta acggctcacc taggcgacga tccctagctg gtctgagagg 300

atgaccagcc acactggaac tgagacacgg tccagactcc tacgggaggc agcagtgggg 360

aatattgcac aatgggcgca agcctgatgc agccatgccg cgtgtatgaa gaaggccttc 420

gggttgtaaa gtactttcag cggggaggaa ggtgttgtgg ttaataaccg cagcaattga 480

cgttacccgc agaagaagca ccggctaact ccgtgccagc agccgcggta atacggaggg 540

tgcaagcgtt aatcggaatt actgggcgta aagcgcacgc aggcggtctg tcaagtcgga 600

tgtgaaatcc ccgggctcaa cctgggaact gcattcgaaa ctggcaggct agagtcttgt 660

agaggggggt agaattccag gtgtagcggt gaaatgcgta gagatctgga ggaataccgg 720

tggcgaaggc ggccccctgg acaaagactg acgctcaggt gcgaaagcgt ggggagcaaa 780

caggattaga taccctggta gtccacgccg taaacgatgt cgacttggag gttgtgccct 840

tgaggcgtgg cttccggagc taacgcgtta agtcgaccgc ctggggagta cggccgcaag 900

gttaaaactc aaatgaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960

gatgcaacgc gaagaacctt acctactctt gacatccaga gaacttagca gagatgcttt 1020

ggtgccttcg ggaactctga gacaggtgct gcatggctgt cgtcagctcg tgttgtgaaa 1080

tgttgggtta agtcccgcaa cgagcgcaac ccttatcctt tgttgccagc ggtccggccg 1140

ggaactcaaa ggagactgcc agtgataaac tggaggaagg tggggatgac gtcaagtcat 1200

catggccctt acgagtaggg ctacacacgt gctacaatgg cgcatacaaa gagaagcgaa 1260

ctcgcgagag caagcggacc tcataaagtg cgtcgtagtc cggattggag tctgcaactc 1320

gactccatga agtcggaatc gctagtaatc gtagatcaga atgctacggt gaatacgttc 1380

ccgggccttg tacacaccgc ccgtcacacc atgggagtgg gttgcaaaag aagtaggtag 1440

cttaaccttc gggagggcgc ttaccacttt gtgattcatg actggggtga agtcgtaaca 1500

aggtaaccgt aggggaacct gcggttggat cacctcctta 1540

Claims

1. The application of the separated enterobacteria Z1 (Enterobacter sp.) in removing antimony and arsenic pollution in water is characterized in that the enterobacteria Z1 are preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2019147.

2. Use of an isolated enterobacter Z1 (Enterobacter sp.) according to claim 1 for the removal of antimony and arsenic contamination in a body of water, including the use in the preparation of a bacterial agent for the removal of antimony and arsenic.

3. Use of an enterobacter Z1 (Enterobacter sp.) inoculant according to claim 2 for the removal of antimony and arsenic pollution from a body of water, wherein the use further comprises the use for the remediation of antimony and arsenic pollution in soil.