CN114958697B

CN114958697B - Heavy metal tolerant ralstonia sp and application thereof

Info

Publication number: CN114958697B
Application number: CN202210849603.3A
Authority: CN
Inventors: 王承民; 季芳; 赵佳男; 王雪; 曹洪杨; 徐莉莉
Original assignee: Unibond Biotechnology Shanghai Co ltd; Institute of Zoology of Guangdong Academy of Sciences
Current assignee: Unibond Biotechnology Shanghai Co ltd; Institute of Zoology of Guangdong Academy of Sciences
Priority date: 2022-07-19
Filing date: 2022-07-19
Publication date: 2023-03-24
Anticipated expiration: 2042-07-19
Also published as: CN114958697A

Abstract

The application discloses a heavy metal resistant bacterium, which is Ralstoniapacikettii, is preserved in Guangdong province microbial strain preservation center (GDMCC), and has a preservation number of GDMCCNo:62443; wherein the heavy metal comprises Mn ²⁺ 、Cu ²⁺ 、Pb ²⁺ At least one of (1). The application also provides application of the heavy metal resistant bacteria in treatment of heavy metal polluted water or soil. The heavy metal resistant bacteria has good heavy metal removal capacity, can be used for treating the soil or water polluted by the heavy metal resistant bacteria, and has the advantages of low cost, high efficiency, no secondary pollution and the like.

Description

Heavy metal tolerant ralstonia sp and application thereof

Technical Field

The invention relates to the field of microorganisms, and more particularly to a heavy metal tolerant ralstonia species and uses thereof.

Background

Heavy metals have teratogenic, carcinogenic, mutagenic effects, and since heavy metals are not easily degraded by organisms, and can be transmitted and enriched through food chains, they can enter human body through various ways, and seriously harm human health. The heavy metal pollution of the water body mainly refers to the phenomenon that the concentration of metal ions such as copper, lead, zinc, nickel, manganese, cadmium, cobalt and the like in the water body exceeds a certain concentration standard, and becomes one of great hazards in the water body pollution.

Lead is absorbed by the digestive tract and then accumulated in the body. Mainly in the skeleton, and accumulates in small amounts in the liver, brain, kidney and blood, lead can cause damage to the hematopoietic system, causing anemia and hemolysis. Chronic lead poisoning nephropathy can be caused after long-term lead intake. Lead poisoning may also lead to stillbirth and abortion, carcinogenesis, and mutagenicity in humans. Nickel enters the body and accumulates in the spinal cord, brain and five zang organs, with the lung being the main factor. Nickel activates or inhibits a series of enzymes to cause stomatitis, gingivitis and acute gastroenteritis, and has damage to the heart muscle and liver. Cadmium can be enriched in living body and enter human body through food chain to cause chronic poisoning. The biological half-life of cadmium is 10-30 years, and most of the accumulated cadmium stays in the human body even if the contact is stopped. The kidney is the most important accumulation site and target organ of cadmium, and can seriously cause renal failure; the effects on the skeleton are osteomalacia and osteoporosis; causing severe damage to other body tissues.

Copper, zinc, cobalt and manganese are essential elements of animals and plants, but excessive copper can cause liver cirrhosis, diarrhea, dyskinesia and other problems. Excessive intake of heavy metals such as zinc, cobalt, manganese, etc. also causes the above problems.

Heavy metals in water are mostly from industrial fields including mining industry, metallurgy industry, mineral separation industry, tanning, painting and electroplating industry and the like. Untreated wastewater is discharged into lakes, rivers and the ocean for some reasons, causing heavy metal pollution of the water body. Heavy metals in water bodies can generate toxicity when the concentration of the heavy metals is very low, and have high harmfulness and difficult treatment. Heavy metal treatment mainly comprises physical, chemical and biological remediation. The microbial remediation method commonly used in bioremediation plays an important role in heavy metal pollution treatment due to the advantages of economic and ecological benefits and the like.

The microorganism restoration method is a process of adsorbing heavy gold of 23662by using microorganisms such as bacteria, fungi and the like, and has the main principle that heavy metal ions are reduced or adsorbed into mass precipitates by using microorganisms in a water body or domesticated high-efficiency microorganisms under a proper condition so as to reduce the content of the heavy gold of 23662in the water body.

Disclosure of Invention

The application provides a heavy metal resistant bacterium, which is Ralstonia pickettii, the heavy metal resistant bacterium is preserved in Guangdong province microbial strain preservation center (GDMCC), and the preservation number is GDMCC No:62443; wherein the heavy metal comprises Mn ²⁺ 、Cu ²⁺ 、Pb ²⁺ At least one of (1).

The application also provides application of the heavy metal resistant bacteria in treatment of heavy metal polluted water bodies or soil, wherein the heavy metal comprises Mn ²⁺ 、Cu ²⁺ 、Pb ²⁺ At least one of (1).

The heavy metal resistant bacteria has good heavy metal removal capacity, can be used for treating the soil or water polluted by the heavy metal resistant bacteria, and has the advantages of low cost, high efficiency, no secondary pollution and the like.

Drawings

FIG. 1 shows the growth status of strain MPEB0011838 on MHA solid medium.

Figure 2 shows the morphological characteristics of strain MPEB0011838 under light microscopy.

FIG. 3 shows the growth state of strain MPEB0011838 at a lead ion concentration of 2500 mg/L.

FIG. 4 shows the growth state of the strain MPEB0011838 at a manganese ion concentration of 2000 mg/L.

FIG. 5 shows the growth state of the strain MPEB0011838 at a zinc ion concentration of 600 mg/L.

FIG. 6 shows the growth of the strain MPEB0011838 at a cobalt ion concentration of 200 mg/L.

FIG. 7 shows the growth state of strain MPEB0011838 at a cadmium ion concentration of 300 mg/L.

FIG. 8 shows the growth of the strain MPEB0011838 at a copper ion concentration of 600 mg/L.

FIG. 9 shows the growth of the strain MPEB0011838 at a nickel ion concentration of 400 mg/L.

FIG. 10 shows a phylogenetic tree of the strain MPEB001183816S rDNA sequence.

The heavy metal resistant bacteria MPEB0011838 is classified as Ralstoniapacikettii, is preserved in Guangdong province microorganism culture collection center (GDMCC) at 28 months 4 in 2022, and has the following preservation number: GDMCC No:62443; the preservation address is as follows: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.

Detailed Description

The following examples are presented to enable those skilled in the art to more fully understand the present invention and are not intended to limit the invention in any way.

The application provides a heavy metal resistant bacterium, and a strain belongs to Ralstonia pickettii. Deposited in the Guangdong province culture Collection (GDMCC) at 28 th month 4 in 2022, wherein the deposition numbers are: GDMCC No:62443. the strain has the beneficial effects that the strain shows stronger heavy metal tolerance, has stronger removal effect on lead and manganese, and can be used for bioremediation of water bodies and soil polluted by heavy metals.

The Ralstonia pickettii provided by the invention is separated from soil around Guangxi manganese ore. The soil is from the vicinity of Guangxi manganese ore, the manganese content of the soil is 60-188g/kg, and the cobalt content of the soil is 100-200mg/kg. Adding 1mg of soil sample into a 250ml conical flask filled with 50ml of MHB (beef powder 2g/L, soluble starch 1.5g/L, acid hydrolyzed casein 17.5g/L, pH 7.4), placing the conical flask in a shaker at 35 ℃ and 150RPM for culturing for 24h, taking 1ml of the conical flask, adding the conical flask into a solution with the manganese concentration of 200mg/L for culturing for 48h, and transferring the conical flask into a culture medium with the manganese concentration of 400mg/L for culturing for further 48h. 20ul of the culture broth in the last solution was streaked on a solid medium containing 400mg/L of manganese. The monoclonals with different forms are selected and respectively inoculated into MHA (beef powder 6g/L, soluble starch 1.5g/L, acid hydrolyzed casein 17.5g/L, agar 17g/L, pH 7.3) without any metal ions for 24h of culture. Until pure strains with consistent colony characteristics are cultured. The isolated strains were subjected to various heavy metal MIC (minimum inhibitory concentration) assays. The assay method used 96-well plate screening. The method comprises subjecting the strain to MIC detection of heavy metal in 96-well plate with culture medium containing heavy metals such as manganese, nickel, copper, zinc, and cobalt at concentration of 100-2500mg/L, culturing at 35 deg.C for 48 hr, and repeating for 3 times. And selecting the strain with the highest tolerance to the heavy metals as a target strain, and performing strain identification. The heavy metal resistant strain MPEB0011838 is obtained by screening and is Ralstonia pickettii.

The characteristics of Ralstonia (Ralstoniapacketti) are described below:

the form is as follows: the strain is cultured on an MHA plate at 35 ℃ for 24h, and the colony morphology of the strain on the MHA plate is off-white and spherical, the surface is smooth and wet, and the edge is regular (figure 1); as shown in FIG. 2, the cells were short rod-shaped and had blunt ends when observed with a microscope (10X 40). The strain was gram stained and showed gram negative bacteria. In addition, urease was shown to be positive.

Metal tolerance: and (3) performing streak culture on the rejuvenated 16h strain on a heavy metal-containing plate. The heavy metal flat plates respectively contain 0.2g/L of cobalt, 0.6g/L of zinc, 2g/L of manganese, 2.5g/L of lead, 0.4g/L of nickel, 0.6g/L of copper and 0.3g/L of cadmium. After 24 hours of culture, strain MBEP0011838 was able to grow on these plates.

FIG. 3 shows the growth of the strain MPEB0011838 at a lead ion concentration of 2500 mg/L. FIG. 4 shows the growth state of the strain MPEB0011838 at a manganese ion concentration of 2000 mg/L. FIG. 5 shows the growth state of strain MPEB0011838 at a zinc ion concentration of 600 mg/L. FIG. 6 shows the growth of the strain MPEB0011838 at a cobalt ion concentration of 200 mg/L. FIG. 7 shows the growth state of strain MPEB0011838 at a cadmium ion concentration of 300 mg/L. FIG. 8 shows the growth of the strain MPEB0011838 at a copper ion concentration of 600 mg/L. FIG. 9 shows the growth of the strain MPEB0011838 at a nickel ion concentration of 400 mg/L. As shown in fig. 3 to 9, it can be considered that the strain is resistant to this concentration of heavy metal by the plate experiment. The tolerance of the strain to high-concentration heavy metal provides a foundation for the application of the strain in removing the heavy metal.

16s analysis: genomic DNA was extracted from pure cultures of the strains of the present application, amplified and sequenced using the general software 27F and 1492r, and further phylogenetic trees were constructed by MEGA software (figure 10). The results showed that the bacterium was Ralstonia pickettii.

Heavy metal removal characteristics:

the seed solution of MBEP0011838 was inoculated into MHB medium at an inoculum size of 2% and shake-cultured at 35 ℃ and 150RPM for 18h. Centrifuging at 8000r/min for 10min to collect thallus, washing thallus with ultrapure water for 3 times, and using wet thallus as biological adsorbent.

The wet cells were added to a heavy metal solution of known concentration at pH7.4 and 35 ℃ in an amount of 2.5g/L. After oscillating at constant temperature of 120RPM for a certain time, sampling, centrifuging at 8000r/min for 10min, measuring the concentration of residual heavy metal ions in the supernatant with a SpectrAA220 type atomic absorption spectrophotometer, and calculating the removal rate.

The method for calculating the removal efficiency R of the strain to the heavy metals comprises the following steps:

wherein R is the removal efficiency of heavy metals, C ₀ The initial concentration (mg/L) of the heavy metal, and the concentration (mg/L) of the heavy metal in the solution at the equilibrium of Ce.

The removal rate of 48h after inoculating the ralstonia provided by the application into MHB solution containing heavy metal with various concentrations is shown in Table 1:

TABLE 1

Thus, the strain of the present application MBEP0011838 is Mn-paired ²⁺ 、Cu ²⁺ 、Pb ²⁺ 、Zn ²⁺ All showed strong tolerance. For Mn ²⁺ 、Cu ²⁺ 、Pb ²⁺ All show stronger removal capability and can be used for restoring water bodies or soil polluted by the heavy metals.

Those skilled in the art will appreciate that the above embodiments are merely exemplary embodiments and that various changes, substitutions, and alterations can be made without departing from the spirit and scope of the invention.

Claims

1. The heavy metal resistant bacterium is Ralstonia picttii (Ralstonia) MPEB0011838, and is deposited in Guangdong province culture Collection (GDMCC) with the deposit number of GDMCC No:62443;

wherein the heavy metal comprises Mn ²⁺ 、Cu ²⁺ 、Pb ²⁺ At least one of (1).

2. The use of the heavy metal-resistant bacteria of claim 1 in the treatment of heavy metal contaminated water or soil, wherein the heavy metal comprises Mn ²⁺ 、Cu ²⁺ 、Pb ²⁺ At least one of (1).