CN114164139A

CN114164139A - Alkali-resistant chromium-resistant bacillus cereus and application thereof

Info

Publication number: CN114164139A
Application number: CN202111252960.3A
Authority: CN
Inventors: 程扬健; 余倩怡
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2021-10-27
Filing date: 2021-10-27
Publication date: 2022-03-11
Anticipated expiration: 2041-10-27
Also published as: CN114164139B

Abstract

The invention discloses an alkali-resistant chromium-resistant bacillus cereus FZUY-01 strain and application thereof, wherein the strain is classified and named as bacillus cereus (Bacillus cereus)Bacillus cereus) The strain is preserved in China general microbiological culture Collection center on 9/2/2021 with the preservation number: CGMCC NO. 23342. The strain can remove hexavalent chromium under an alkaline condition, and provides strain resources and a theoretical basis for microbial remediation of hexavalent chromium pollution.

Description

Alkali-resistant chromium-resistant bacillus cereus and application thereof

Technical Field

The invention belongs to the field of microbial application, and particularly relates to an alkali-resistant chromium-resistant bacillus cereus FZUY-01 and application thereof.

Technical Field

With the continuous development of the industry, China becomes a country with large production, use and demand of chromium salt, and has caused a great deal of serious pollution to soil and water in the process of producing and using chromium salt. Since the harm of heavy metal chromium to the ecological environment and human health is increasing, the problem of chromium pollution of soil, sediment, surface water and underground water is also receiving more and more attention. The chromium slag is industrial waste slag containing Cr (VI) generated in the production process of metal chromium and chromium salt, belongs to heavy metal dangerous solid waste, and mainly comes from the industries of chromium ore processing, electroplating, leather, metallurgy, chemical industry and the like. The chromium slag contains 1% -2% of Cr (VI), the land resource occupation is caused by the large stacking of the chromium slag, the soil and underground water in the stacking area are seriously polluted by the chromium slag, the total chromium content in the soil in the stacking area can exceed several times or even tens of times of the background value of the local soil, and the concentration of the Cr (VI) in the underground water can exceed the quality standard limit value by thousands of times.

One of the commonly used methods for treating heavy metal pollution at present is a chemical precipitation method, which comprises the steps of adding some reducing chemical substances under specific conditions to enable highly toxic heavy metal ions in a solution to perform a series of chemical reactions with the heavy metal ions so as to enable the heavy metal ions to be converted into insoluble or indissolvable compounds, and separating the indissolvable metal compounds from water by using methods such as centrifugation and filtration. In addition, the second method commonly used for treating heavy metal pollution is a physical adsorption method, which is a method for removing heavy metal ions from a water body by adsorbing and removing heavy metal ion pollutants in water by using a porous solid adsorbent and adsorbing one or more components in the pollutants on the surface of the solid under the action of molecular attraction or chemical bond on the surface of the solid adsorbent.

In summary, the main methods for treating heavy metal pollution at present are physical methods and chemical methods, and the traditional treatment methods have high operation cost, are only suitable for treating chromium-polluted sites with small area and high concentration, and are not suitable for treating chromium-polluted soil and water areas with large area and wide range. In recent decades, with the development of environmental biology, people have gradually recognized that the method not only can greatly reduce the cost, but also is suitable for real-time and in-situ treatment of a large amount of soil and water polluted by heavy metal chromium, because the growth period of microorganisms is short, the propagation speed is high, and the enrichment and removal capacity for heavy metal pollution with medium and low concentration is very good.

After consulting the relevant documents, the academia has found that the academia has certain basic research on the treatment of heavy metals by microorganisms in recent years, and has certain stage research on the interaction mechanism of microorganisms and heavy metals. However, for some extreme environments, such as the use of microorganisms for treating chromium-contaminated sites under alkaline conditions, research is less, and the application of microorganisms to practical engineering is more rare.

Disclosure of Invention

The invention aims to provide an alkali-resistant chromium-resistant bacillus cereus FZUY-01 strain and application thereof.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

in the present invention, the classification of Bacillus cereus strain FZUY-01 is named as Bacillus cereus ((B. cereus))Bacillus cereus) The strain is separated from the mud flat soil of the Ministry Bay of Jinjing township in Jinjiang, Jinjiang province, is preserved in the China general microbiological culture Collection center in 2021, 9 months and 2 days, and has the preservation number as follows: CGMCC NO.23342, the preservation address is: xilu No. 1 Hospital No.3, Beijing, Chaoyang, North.

The bacillus cereus FZUY-01 is a gram-positive bacterium; the bacterial colony is white round and wax-like, the surface is wet, and the edge is smooth; the thallus is in the shape of a long straight rod, the length of the thallus is about 3 to 5 μm, and the width of the thallus is about 0.5 to 1 μm.

The invention also provides application of the bacillus cereus FZUY-01 in removing hexavalent chromium ions.

Further, the bacillus cereus FZUY-01 removes hexavalent chromium under alkaline conditions.

Further, the bacillus cereus FZUY-01 has an initial pH of 10.0 when removing hexavalent chromium.

The invention has the advantages that:

the bacillus cereus FZUY-01 is a novel microorganism, can effectively reduce Cr (VI) under an alkaline condition, has a reduction rate of 65.77% in 72 hours of 100mg/L Cr (VI), has high tolerance concentration to Cr (VI), has a good growth condition when the concentration of Cr (VI) reaches 1400 mg/L, can be used for chromium pollution treatment, and provides strain resources and a theoretical basis for the promotion of chromium pollution bioremediation.

Drawings

FIG. 1: the colony morphology and the morphology characteristics of the FZUY-01 strain.

FIG. 2: phylogenetic tree of strain FZUY-01.

FIG. 3: alkali resistance versus growth curve of FZUY-01 strain.

FIG. 4: tolerance of the strain FZUY-01 to various concentrations of Cr (VI).

FIG. 5: the reductibility of FZUY-01 strain to Cr (VI) at various concentrations.

FIG. 6: XRD phase analysis of the product of the interaction of strain FZUY-01 with Cr (VI).

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the following examples are only examples of the present invention and do not represent the scope of the present invention defined by the claims.

EXAMPLE 1 isolation of FZUY-01 Strain

Weighing 5g of soil taken from the mud flat of the Jing Town Bay of Jinjing town of Jinjiang, Fujian province, dissolving the soil in 45 mL of sterile deionized water, oscillating the soil in a constant temperature shaking table at 37 ℃ for 30 minutes, and then respectively preparing 10 parts of the soil by using the sterile deionized water by adopting a gradient dilution method^-1、10^-2、10^-3、 10^-4、10^-5、10^-6Diluted soil suspension. 0.1mL of each dilution of the soil suspension was applied to LB solid medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agar powder 20 g/L; pH = 7.2) in triplicate for each dilution, and cultured in an oven at 37 ℃ for 24-48 hours in an inverted manner. Selecting a single colony, performing streak purification culture in an LB solid culture medium (tryptone 10g/L, yeast extract powder 5g/L, sodium chloride 10g/L, agar powder 20g/L, pH = 7.2) for 3-5 generations to obtain a pure culture of the strain, and naming the strain as FZUY-01.

Example 2 identification of FZUY-01 Strain

(1) And (3) morphological identification:

as shown in FIG. 1a, colonies of the FZUY-01 strain appeared as white round drops of wax, with wet surface and smooth edges. Gram-stained positive bacteria, the shape of the bacteria under a scanning electron microscope is shown in figure 1b, the bacteria are long straight rod-shaped, the length is about 3-5 mu m, and the width is about 0.5-1 mu m.

(2) Molecular biological identification:

the genomic DNA of the FZUY-01 strain is extracted by a kit (purchased from the Producer Sangon Biotech), and the 16S rDNA gene PCR amplification is carried out by adopting a 16S rDNA universal primer and the genomic DNA of the FZUY-01 strain as a template. The primers used were as follows:

27F（SEQ ID NO.1）：5’-AGTTTGATCMTGGCTCAG-3’，

1492R（SEQ ID NO.2）：5’-GGTTACCTTGTTACGACTT-3’。

the PCR amplification product is subjected to 1% agarose electrophoresis, cut and recovered, and sent to the company Limited in Biotechnology engineering (Shanghai) for sequence determination. The sequencing result is shown in SEQ ID NO. 3.

The obtained sequence is inhttps://blast.ncbi.nlm.nih.gov/Blast.cgiHomology analysis and comparison are carried out on a website, a phylogenetic tree (figure 2) is constructed by utilizing MEGA 7.0 software, and the FZUY-01 strain is determined to be bacillus cereus (B.cereus)Bacillus cereus）。

Bacillus cereus (A), (B) and (C)Bacillus cereus) The preservation number of the FZUY-01 in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms is CGMCC No. 23342; the preservation date is 2021, 9 months and 2 days.

Alkali resistance and growth curves of the 3 FZUY-01 Strain

(1) Alkali resistance: the scale-up to OD was carried out with an inoculum size of 3% (v/v)₆₀₀FZUY-01 strain suspensions with a value of 0.6-0.7 were inoculated into 100mL of LB liquid medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10 g/L) with pH =8, 9, 10, 11, 12, respectively, and cultured at 37 ℃ at 160rpm for 60 hours, and then the OD of the culture broth was measured by sampling₆₀₀. As shown in FIG. 3a, the highest alkalinity tolerated by the FZUY-01 strain was pH =11, and the strain grew more than the strain grown under the condition of alkaline pH =10Good results are obtained.

(2) Growth curve: the scale-up to OD was carried out with an inoculum size of 3% (v/v)₆₀₀FZUY-01 strain suspension with a value of 0.6-0.7 was inoculated into 100mL of LB liquid medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10 g/L) with pH =10, cultured at 37 ℃ under 160rpm, sampled at different intervals, and the OD of the culture solution was measured₆₀₀. Using cultivation time as abscissa, OD₆₀₀Values are plotted as ordinate against the growth curve of the strain.

As shown in fig. 3b, at pH =10, FZUY-01 strain was in the growth-delayed phase at 0-12 h, in the logarithmic growth phase at 12-44 h, in the stationary phase at 44-48 h, and in the decline phase after 48 h.

Example 4 tolerance of FZUY-01 Strain to different concentrations of Cr (VI)

The scale-up to OD was carried out with an inoculum size of 3% (v/v)₆₀₀FZUY-01 strain suspension with a value of 0.6-0.7 was inoculated into 50mL of LB liquid medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10 g/L; pH = 7.2), and the mixture was subjected to amplification culture at 37 ℃ and 160rpm until the OD of the culture solution reached₆₀₀Is 0.6 to 0.7, and then 50mL of Cr (VI) with the concentration of 0mg/L, 100mg/L, 200mg/L, 300mg/L, 400mg/L, 500mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/L and 1000 mg/L is inoculated with the inoculation amount of 3 percent (v/v), 1100mg/L, 1200 mg/L, 1300 mg/L, 1400 mg/L, 1500mg/L, 1600 mg/L, 1700 mg/L, 1800 mg/L, 1900 mg/L, 2000 mg/L LB liquid medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10 g/L; pH = 10), at 37 ℃ under 160rpm for 72 h. And then centrifuging the culture solution for 5min under the condition of 6500 r/min, collecting thallus precipitates, freeze-drying, and weighing the weight of the thallus precipitates (namely the dry weight of the thallus), wherein the growth condition of the strain is reflected by the weight of the thallus precipitates.

As shown in FIG. 4, at pH =10, the growth of the strain FZUY-01 was better at a Cr (VI) concentration of 0-1400 mg/L, whereas at a Cr (VI) concentration of 1500mg/L, the growth of the strain FZUY-01 was severely inhibited and was poor. As can be seen, the strain FZUY-01 has better tolerance to Cr (VI) with the concentration of less than 1500 mg/L.

Example 5 reducibility and removal of Cr (VI) at various concentrations by FZUY-01 Strain

The scale-up to OD was carried out with an inoculum size of 3% (v/v)₆₀₀FZUY-01 strain suspension with a value of 0.6-0.7 was inoculated into 50mL of LB liquid medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10 g/L; pH = 7.2), and the mixture was subjected to amplification culture at 37 ℃ and 160rpm until the OD of the culture solution reached₆₀₀0.6-0.7, 50mL of LB liquid medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10 g/L; pH = 10) with Cr (VI) concentrations of 100mg/L, 200mg/L, 300mg/L, 400mg/L, respectively, was inoculated in an inoculum size of 3% (v/v), cultured at 37 ℃ and 160rpm for 72 hours, and then the culture was centrifuged at 6500rpm for 5min to collect the supernatant and the precipitate, respectively. The concentration of Cr (VI) and the total Cr concentration in the supernatant were determined by diphenylcarbodihydrazide spectrophotometry. The centrifuged precipitate was washed three times with deionized water, then freeze-dried under vacuum, and ground to powder, and subjected to X-ray diffraction analysis (XRD) on an X-ray diffractometer.

As shown in FIG. 5, under initial Cr (VI) concentration conditions of 100mg/L, 200mg/L, 300mg/L and 400mg/L, after 72h, the FZUY-01 strain reduces the initial Cr (VI) concentration to 34.23mg/L, 118.38mg/L, 208.29mg/L and 291.36mg/L respectively, and the Cr (VI) reduction rates are 65.77%, 40.81%, 30.57% and 27.16% respectively; the total chromium concentration is reduced to 90.79mg/L, 184.28mg/L, 284.31mg/L and 386.58mg/L, and the total chromium adsorption rate is respectively as follows: 9.21%, 7.86%, 5.23%, 3.35%; the concentrations of cr (iii) in the supernatant were: 56.57mg/L, 65.90mg/L, 76.02mg/L and 95.22 mg/L.

As shown in FIG. 6, the phase of the product of the interaction of FZUY-01 with Cr (VI) is amorphous.

The above results show that the removal of hexavalent chromium is achieved by both the bioadsorption of part of the strain FZUY-01 and the reduction of trivalent chromium by the strain FZUY-01.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> Fuzhou university

<120> alkali-resistant chromium-resistant bacillus cereus and application thereof

<130>

<160> 3

<170> PatentIn version 3.3

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<213> Artificial sequence

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agtttgatcm tggctcag 18

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<213> Artificial sequence

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ggttaccttg ttacgactt 19

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<212> DNA

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tgctcaggat gaacgctggc ggcgtgccta atacatgcaa gtcgagcgaa tggattaaga 60

gcttgctctt atgaagttag cggcggacgg gtgagtaaca cgtgggtaac ctgcccataa 120

gactgggata actccgggaa accggggcta ataccggata acattttgaa ccgcatggtt 180

cgaaattgaa aggcggcttc ggctgtcact tatggatgga cccgcgtcgc attagctagt 240

tggtgaggta acggctcacc aaggcaacga tgcgtagccg acctgagagg gtgatcggcc 300

acactgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccgc 360

aatggacgaa agtctgacgg agcaacgccg cgtgagtgat gaaggctttc gggtcgtaaa 420

actctgttgt tagggaagaa caagtgctag ttgaataagc tggcaccttg acggtaccta 480

accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 540

tatccggaat tattgggcgt aaagcgcgcg caggtggttt cttaagtctg atgtgaaagc 600

ccacggctca accgtggagg gtcattggaa actgggagac ttgagtgcag aagaggaaag 660

tggaattcca tgtgtagcgg tgaaatgcgt agagatatgg aggaacacca gtggcgaagg 720

cgactttctg gtctgtaact gacactgagg cgcgaaagcg tggggagcaa acaggattag 780

ataccctggt agtccacgcc gtaaacgatg agtgctaagt gttagagggt ttccgccctt 840

tagtgctgaa gttaacgcat taagcactcc gcctggggag tacggccgca aggctgaaac 900

tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 960

gcgaagaacc ttaccaggtc ttgacatcct ctgacaaccc tagagatagg gcttctcctt 1020

cgggagcaga gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1080

taagtcccgc aacgagcgca acccttgatc ttagttgcca tcattcagtt gggcactcta 1140

aggtgactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct 1200

tatgacctgg gctacacacg tgctacaatg gacggtacaa agagctgcaa gaccgcgagg 1260

tggagctaat ctcataaaac cgttctcagt tcggattgta ggctgcaact cgcctacatg 1320

aagctggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt 1380

gtacacaccg cccgtcacac cacgagagtt tgtaacaccc gaagtcggtg gggtaacctt 1440

ttggaa 1446

Claims

1. An alkali-resistant chromium-resistant bacillus cereus FZUY-01 is characterized in that: the classification of the strain is named as bacillus cereus (B.) (Bacillus cereus) And the strain is preserved in China general microbiological culture Collection center on 2021, 9 months and 2 days, with the preservation addresses as follows: the Beijing West Lu No. 1 Hospital No.3 of Chaoyang district, the preservation number is: CGMCC NO. 23342.

2. The use of bacillus cereus FZUY-01 of claim 1 for removing hexavalent chromium.

3. The use of bacillus cereus FZUY-01 for the removal of hexavalent chromium according to claim 2, wherein: and removing hexavalent chromium from the bacillus cereus FZUY-01 under an alkaline condition.

4. The use of bacillus cereus FZUY-01 for the removal of hexavalent chromium according to claim 3, wherein: the initial pH of the Bacillus cereus FZUY-01 when removing hexavalent chromium is 10.0.