CN115232765B - Klebsiella WH-15 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses klebsiella WH-15 and application thereof. The strain is preserved in China Center for Type Culture Collection (CCTCC) No. M2022400. The strain has better adsorption effect on heavy metal lead, mercury and arsenic, and can be applied to bioremediation engineering of lead-polluted water bodies so as to promote lead immobilization and adsorption of mercury and arsenic and improve remediation efficiency.

Description

A kind of Klebsiella WH-15 and its application

technical field

The invention belongs to the technical field of microorganisms, and specifically discloses a Klebsiella WH-15 and an application thereof.

Background technique

Lead (Lead, Pb) is a non-degradable heavy metal, which mainly comes from mining, smelting and other industries. Its toxicity is related to the form of lead compounds, and the exchangeable state is the main cause of lead pollution in the environment. It makes it easier for Pb to be leached from the ore, which increases the threat to the environment. Pb mainly enters the human body through the respiratory tract and gastrointestinal tract. Long-term intake will cause serious damage to the body's blood system, nervous system, etc., and pose a serious threat to human health. Therefore, changing the existing form of Pb and reducing its mobility and bioavailability are of great significance to the control of environmental Pb pollution and the protection of human health.

Lead pollution control methods mainly include physical, chemical and biological methods. The physical and chemical methods commonly used in the treatment of lead pollution in the environment mainly include thermal desorption method, fixed stabilization technology, soil leaching method, electrokinetic remediation method, photocatalytic method, etc. Among them, physical methods and chemical methods have not become ideal lead pollution control measures due to their high price, insignificant effect, and secondary pollution. Microbial remediation is to reduce the exchangeable lead in the environment through the absorption, conversion or solidification of lead by microorganisms. The microbial method to remediate lead-contaminated soil has become a new way to treat lead pollution in situ because of its safety, environmental protection, and low cost. The key to the development of this technology is to obtain excellent and highly efficient lead-absorbing strains from the environment.

At present, researchers have isolated some high-efficiency lead-absorbing strains from the environment, mainly distributed in Pseudomonas, Alcaligenes and Bacillus. However, so far there are still only a few cases of microbial remediation of lead-contaminated sites. The main reason may be that the resources of highly efficient lead-absorbing strains are not abundant enough. Therefore, isolating strains from polluted environments is still an important means to obtain dominant bacterial species resources.

Contents of the invention

In view of the above technical problems, the present invention provides Klebsiella WH-15, which can effectively adsorb Pb ²⁺ under aerobic conditions, and the strain also has certain adsorption effects on heavy metal mercury and arsenic.

The first aspect of the present invention provides a kind of Klebsiella WH-15, preserved in China Center for Type Culture Collection, preservation number CCTCC NO.M2022400.

The second aspect of the present invention provides the use of the Klebsiella WH-15 in heavy metal adsorption.

Preferably, the heavy metal is one or more of arsenic, mercury and lead.

The third aspect of the present invention provides the fermentation method of the Klebsiella WH-15, comprising the following steps: inoculating the Klebsiella WH-15 in the LB liquid medium, at 30°C, 150r/h Min under culture 24h.

The fourth aspect of the present invention provides the fermentation liquid obtained according to the above fermentation method.

The fifth aspect of the present invention provides the use of the fermentation broth in heavy metal adsorption.

The sixth aspect of the present invention provides a microbial agent for heavy metal adsorption, which includes the Klebsiella WH-15 and/or the fermentation broth, and auxiliary materials.

Preferably, the adjuvant is a solid or liquid culture substrate for cultivating the Klebsiella WH-15.

Compared with prior art, the beneficial effects of the present invention are:

1, the present invention provides a kind of Klebsiella WH-15, after this bacterial strain acts on the water body polluted by lead, can obviously promote the ^Pb in the water body to be solidified by the bacterial strain, this bacterial strain has extremely good in processing by the lead polluted water body It has great application potential and can be applied to the bioremediation engineering of lead-polluted water bodies to promote the immobilization of lead and improve the efficiency of remediation.

2. The strain also has a certain adsorption effect on mercury and arsenic. When the mercury concentration is 200mg/L and 600mg/L, the adsorption efficiency in 24 hours is 54.8% and 7.4% respectively; when the arsenic concentration is 200mg/L and 600mg/L , 24h adsorption efficiencies were 20.9% and 18.8%.

Explanation of Biological Deposit Information:

Biological material: WH-15, taxonomic name: Klebsiella, Latin name Klebsiella grimontii, this strain was preserved in the China Center for Type Culture Collection on April 18, 2022, and the preservation number is: CCTCC NO.M2022400, Preservation address: Wuhan University, Wuhan, China.

Description of drawings

Fig. 1 is the Gram stain microscopic examination picture of the bacterial strain that the present invention obtains;

Fig. 2 is the colony morphology of the bacterial strain obtained by the present invention on the LB plate;

Fig. 3 is a phylogenetic tree of the strains obtained in the present invention.

Detailed ways

The present invention is further explained in conjunction with the embodiments and corresponding drawings, and the following embodiments are only for illustration purposes, and are not intended to limit the scope of the present invention.

Example 1

Screening of Strains with High Efficiency Adsorbing Lead

The present invention screens bacterial strains that efficiently adsorb lead from the environment through enrichment and domestication, separation and purification, and adsorption experiments. The specific screening process is as follows:

1. Enrichment and domestication

Collect long-term lead-contaminated soil from around a lead-zinc mine in Weining, Guizhou Province;

Preparation of LB medium: Weigh 10g of peptone, 5g of yeast powder, 10g of sodium chloride, and 1L of distilled water, mix them, adjust the pH to 7.0, put 50mL of LB medium into a 150mL Erlenmeyer flask, and autoclave at 121°C for 20min reserve;

Take 2g of lead-contaminated soil sample and inoculate it into 50mL LB medium, add a few glass beads, shake and culture at 30°C and 150r/min for 2 days, let it stand still, take 5mL culture solution and transfer it to fresh LB medium in the same Cultivate again under the same conditions, take 5mL of the culture solution and inoculate it into the sterilized LB medium containing Pb ²⁺ 100mg/L. , indicating that the bacterial abundance in the medium is high, and the strains can be isolated.

2. Separation and purification

Take 1mL of the enriched bacteria solution and dilute it to 10 ^-5 with sterile water, spread 1mL of the diluted solution on an LB agar plate containing 100mg/L of Pb ²⁺ . Place the plate upside down in a constant temperature incubator at 30°C, observe the growth of the colonies, select the colonies with different shapes and sizes and grow well, and perform repeated streaking and purification until pure species are obtained. This step can obtain strains with the potential to tolerate various heavy metals.

3. Re-screening

The purified primary screened strains were picked and cultured in 50mL LB medium respectively, cultured on a shaker at 30°C and 150r/min for 24 hours, transferred once, and cultured for another 24 hours as seed liquid for later use;

Inoculate the seed solution of the primary screened strains into LB medium with a Pb ²⁺ concentration of 500 mg/L, culture for 24 hours under the same conditions, and measure the optical density and lead residual concentration of the bacterial suspension at a wavelength of 600 nm. According to the OD ₆₀₀ to judge the tolerance of the strain, and calculate the adsorption efficiency according to the residual concentration of lead. Among the re-screened strains, the OD ₆₀₀ value of strain WH-15 was 5.335, and the adsorption efficiency to 500mg/L lead was as high as 99%, which indicated that strain WH-15 was a good lead-adsorbing bacteria.

4. Identification

16S rDNA analysis was amplified with bacterial universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5′-GGTTACCTTGTTACGACTT-3), and the sequence length was 1402bp. The strain is a Gram-negative bacterium, and its morphological characteristics are as follows: the bacterial body is short rod-shaped (see Figure 1), and its single colony is small and raised on the LB plate, which is light yellow, with a moist and smooth surface, opaque, and complete edges. The texture is viscous, easy to provoke, and can produce pigments to turn the surrounding medium yellow (see Figure 2). The phylogenetic tree of strain WH1 is shown in Figure 3.

16S rDNA sequence of strain WH-15:

AGTCGAACGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGTAACG GCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAATAAGGTTAATAACCTTGTCCAT TGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTGGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCG GTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGACTTGGAGGTTGTTCCCTTGAGGAGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGT TAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCT TTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAAT CGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCT

According to the comparison of the 16S rDNA sequence results in the NCBI database, the strain WH15 was identified as Klebsiellagrimontii, and its Chinese name was Klebsiella.

Example 2

Adsorption of lead, mercury and arsenic by strain WH-15

Prepare 20 mL of LB medium with Pb ²⁺ concentrations of 100 mg/L, 200 mg/L, 500 mg/L, 1000 mg/L and 2000 mg/L, mercury and arsenic concentrations of 200 and 600 μg/L respectively, and add 0.5 mL of seed solution , cultured at 150r/min, 30℃ constant temperature, lead cultured for 24h, mercury and arsenic cultured for 12h and 24h respectively, centrifuged at 8000r/min for 5min, collected the supernatant, and separately determined Pb ²⁺ , According to the content of mercury and arsenic, the adsorption capacity of the strain to heavy metals was judged according to the adsorption efficiency. The results are shown in Table 1, Table 2 and Table 3. Strain WH-15 has high tolerance and adsorption efficiency to lead, and also has certain adsorption efficiency to mercury and arsenic.

Table 1 The adsorption effect of strain WH-15 on different concentrations of Pb ²⁺

Initial concentration (mg/L) Final concentration (mg/L) Adsorption efficiency (%) 100 3.75 96.25 200 5.07 97.46 300 3.20 98.93 500 1.33 99.73 1000 3.60 99.64 2000 408.18 82.06

Table 2 The adsorption effect of strain WH-15 on mercury Unit: %

Table 3 The adsorption effect of strain WH-15 on arsenic Unit: %

The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: the specific implementation of the present invention can still be carried out Modification or equivalent replacement of some technical features; without departing from the spirit of the technical solution of the present invention, all of them shall be included in the scope of the technical solution claimed in the present invention.

Claims (8)

1. A Klebsiella WH-15, characterized in that the bacterial strain is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO.M2022400. 2. the purposes of Klebsiella WH-15 described in claim 1 in heavy metal adsorption. 3. The use according to claim 2, characterized in that the heavy metal is one or more of arsenic, mercury and lead. 4. a fermentation method of Klebsiella WH-15 described in claim 1, is characterized in that, comprises the following steps: described Klebsiella WH-15 is inoculated in LB liquid medium, at 30 Cultivate at 150r/min for 24h. 5. A fermented liquid obtained by the fermentation method according to claim 4. 6. the purposes of fermented liquid described in claim 5 in heavy metal adsorption. 7. A microbial agent for heavy metal adsorption, characterized in that it comprises Klebsiella WH-15 according to claim 1 and/or the fermentation broth according to claim 5, and auxiliary materials. 8. microbial bacterial agent according to claim 7, is characterized in that, described adjuvant is the solid or liquid culture substrate that is used to cultivate described Klebsiella WH-15.

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