CN115011529B

CN115011529B - Heavy metal resistant achromobacter xylosoxidans and application thereof

Info

Publication number: CN115011529B
Application number: CN202210850133.2A
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: 2022-12-30
Anticipated expiration: 2042-07-19
Also published as: CN115011529A

Abstract

The application discloses a heavy metal resistant bacterium, this heavy metal resistant bacterium is xylose oxidation achromobacter xylosoxidans, and this heavy metal resistant bacterium is deposited in Guangdong province microbial strain collection center (GDMCC), and the deposit number is GDMCCNo:62219; wherein the heavy metal comprises Mn ²⁺ 、Cu ²⁺ 、Pb ²⁺ 、Zn ²⁺ At least one of (a). 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 resistant achromobacter xylosoxidans and application thereof

Technical Field

The invention relates to the field of microorganisms, and more particularly relates to a heavy metal resistant achromobacter xylosoxidans and application thereof.

Background

Heavy metals have teratogenic, carcinogenic, mutagenic effects, because heavy metals are not easily degraded by organisms, and can be transmitted and enriched through food chains, can enter human bodies 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 lead, nickel, cadmium, copper, zinc, manganese, cobalt and the like in the water body exceeds a certain concentration standard, and becomes a great harm 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, among which the lung is the main one. 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. And 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 microbe repairing method is a process of adsorbing heavy metal in water by using microbes such as bacteria, fungi and the like, and the main principle is that heavy metal ions are reduced or adsorbed into mass precipitates through reduction reaction of the microbes under a proper condition by using the microbes or domesticated efficient microbes in the water, so that the content of the heavy metal in the water is reduced.

Disclosure of Invention

The application provides a heavy metal resistant bacterium, this heavy metal resistant bacterium is xylose oxidation achromobacter xylosoxidans, and heavy metal resistant bacterium deposits in Guangdong province microbial strain collection center (GDMCC), and the deposit number is GDMCC No:62219; wherein the heavy metal comprises Mn ²⁺ 、Cu ²⁺ 、Pb ²⁺ 、Zn 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⁺ 、Zn ²⁺ At least one of (a).

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 state of strain MPEB0008918 on MHA solid medium.

Figure 2 shows the morphological characteristics of strain MPEB0008918 under light microscopy.

FIG. 3 shows the growth state of strain MPEB0008918 at a lead ion concentration of 2500 mg/L.

FIG. 4 shows the growth state of the strain MPEB0008918 at a manganese ion concentration of 2000 mg/L.

FIG. 5 shows the growth state of strain MPEB0008918 at a zinc ion concentration of 600 mg/L.

FIG. 6 shows the growth state of the strain MPEB0008918 at a cobalt ion concentration of 200 mg/L.

FIG. 7 shows the growth state of strain MPEB0008918 at a cadmium ion concentration of 300 mg/L.

FIG. 8 shows the growth state of the strain MPEB0008918 at a copper ion concentration of 600 mg/L.

FIG. 9 shows the growth state of strain MPEB0008918 at a nickel ion concentration of 400 mg/L.

FIG. 10 shows a phylogenetic tree of strain MPEB000891816S rDNA sequence.

The heavy metal resistant bacteria MPEB0008918 is classified as Achromobacter xylosoxidans, is preserved in Guangdong province microorganism culture collection center (GDMCC) at 18 months 1 in 2022, and has the following preservation number: GDMCC No:62219; the preservation address is as follows: building No. 59, building No. 5 of the prefecture midroad No. 100 yard in Guangzhou city.

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 the strain belongs to Achromobacter xylosoxidans. In 2022, 18 d/1, in Guangdong province culture Collection of microorganisms (GDMCC), with the following preservation numbers: GDMCC No:62219. 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 Achromobacter xylosoxidans 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 (2 g/L beef powder, 1.5g/L soluble starch, 17.5g/L acid hydrolyzed casein, pH 7.4), placing the conical flask in a shaker at 35 ℃, culturing for 24h at 150RPM, taking 1ml of the conical flask, adding the conical flask into a solution with the manganese concentration of 200mg/L, culturing for 48h, and transferring the conical flask into a culture medium with the manganese concentration of 400mg/L to continue culturing for 48h. 20ul of the culture medium in the last solution was streaked on a solid medium containing 400mg/L of manganese. Monoclonals with different forms are selected and respectively inoculated into MHA (6 g/L of beef powder, 1.5g/L of soluble starch, 17.5g/L of acid hydrolyzed casein and 17g/L of agar, and pH is 7.3) without any metal ions for 24h of culture. Until the 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 bacterium MPEB0008918 is obtained by screening and is Achromobacter xylosoxidans.

Achromobacter xylosoxidans (Achromobacter xylosoxidans) are characterized as follows:

the form is as follows: the strain is cultured on an MHA plate at 35 ℃ for 24h, and the colony form on the MHA plate is gray-white, opaque, round, small, neat in edge and not viscous (figure 1); the colonies were tan in the lead ion containing medium. As shown in FIG. 2, the cells were observed by a microscope to have a short rod shape and blunt ends. The strain was gram-stained and showed gram-negative bacteria.

Metal tolerance: and (4) performing streak culture on the rejuvenated strains for 16h 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, the strain MBEP0008918 was able to grow on these plates.

FIG. 3 shows the growth of the strain MPEB0008918 at a lead ion concentration of 2500 mg/L. FIG. 4 shows the growth state of the strain MPEB0008918 at a manganese ion concentration of 2000 mg/L. FIG. 5 shows the growth state of strain MPEB0008918 at a zinc ion concentration of 600 mg/L. FIG. 6 shows the growth state of the strain MPEB0008918 at a cobalt ion concentration of 200 mg/L. FIG. 7 shows the growth state of strain MPEB0008918 at a cadmium ion concentration of 300 mg/L. FIG. 8 shows the growth state of the strain MPEB0008918 at a copper ion concentration of 600 mg/L. FIG. 9 shows the growth state of strain MPEB0008918 at a nickel ion concentration of 400 mg/L. As shown in fig. 3 to 9, it is considered that the strain is resistant to heavy metals at this concentration by plate experiments. 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 Achromobacter xylosoxidans (Achromobacter xylosoxidans).

Heavy metal removal characteristics:

the seed solution of MBEP0008918 was inoculated at 2% inoculum size in MHB medium and shake-cultured at 35 ℃ and 150RPM for 18h. Centrifuging at 8000r/min for 10min, collecting 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 pH6 and a temperature of 35 ℃ in an amount of 2.5g/L. After constant temperature oscillation at 150RPM 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 calculation method of the removal efficiency R of the strain to the heavy metals comprises the following steps:

whereinR 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 24h after the achromobacter xylosoxidans provided by the application is inoculated into MHB solution containing heavy metal with various concentrations is shown in Table 1:

TABLE 1

Thus, strain MBEP0008918 of the present application is Mn-rich ²⁺ 、Ni ²⁺ 、Cu ²⁺ 、Pb ²⁺ 、Zn ²⁺ 、 Co ²⁺ All showed strong tolerance. For Mn ²⁺ 、Cu ²⁺ 、Pb ²⁺ 、Zn 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 characterized in that the heavy metal resistant bacterium is achromobacter xylosoxidansAchromobacter xylosoxidansThe heavy metal resistant bacteria are preserved in Guangdong province microbial culture collection center (GDMCC), and the preservation number is GDMCC No:62219;

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