CN118146988B - Passivating heavy metal cadmium strain Cd04 and application thereof - Google Patents
Passivating heavy metal cadmium strain Cd04 and application thereof Download PDFInfo
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- 229910052793 cadmium Inorganic materials 0.000 title claims abstract description 54
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 23
- 241000715004 Chryseobacterium bernardetii Species 0.000 claims abstract description 8
- 238000002161 passivation Methods 0.000 claims abstract description 6
- 239000002689 soil Substances 0.000 abstract description 12
- 244000005700 microbiome Species 0.000 abstract description 8
- 108020004465 16S ribosomal RNA Proteins 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 5
- 238000012163 sequencing technique Methods 0.000 abstract description 5
- 238000004321 preservation Methods 0.000 abstract description 4
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 abstract description 3
- 241000193830 Bacillus <bacterium> Species 0.000 abstract description 3
- 230000000877 morphologic effect Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 12
- 230000000694 effects Effects 0.000 description 10
- 238000000034 method Methods 0.000 description 9
- 238000001179 sorption measurement Methods 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000006137 Luria-Bertani broth Substances 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000008055 phosphate buffer solution Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000002354 inductively-coupled plasma atomic emission spectroscopy Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- 206010028400 Mutagenic effect Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 231100000243 mutagenic effect Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- 238000000638 solvent extraction Methods 0.000 description 1
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- 231100000378 teratogenic Toxicity 0.000 description 1
- 230000003390 teratogenic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The invention belongs to the technical field of microbiology, and particularly relates to a passivation heavy metal cadmium strain Cd04 and application thereof. A passivating heavy metal cadmium strain Cd04, the taxonomical name of which is golden yellow bacillus Chryseobacterium bernardetii Cd04, is preserved in China Center for Type Culture Collection (CCTCC) in 10 months 30 of 2023, and the preservation number is CCTCC No: m20232084. The invention screens the cadmium-resistant strain, identifies the cadmium-resistant strain through morphological observation and 16S rRNA sequence sequencing, obtains the strain Chryseobacterium bernardetii Cd04 capable of passivating heavy metal cadmium, has stronger cadmium-resistant capability, and can provide strain resources for developing soil heavy metal pollution microorganism repair technology.
Description
Technical Field
The invention belongs to the technical field of microbiology, and particularly relates to a passivation heavy metal cadmium strain Cd04 and application thereof.
Background
Cadmium (Cd) is a heavy metal element with extremely strong biological toxicity, has strong chemical activity in the environment, large mobility and lasting toxicity, and Cd 2+ widely exists in soil and water and can enter human bodies along with food chains, so that a large amount of cadmium can accumulate in the bodies to have teratogenic, oncogenic and mutagenic effects.
The repairing method for the cadmium polluted soil comprises physical repairing, chemical repairing and biological repairing. Specifically, the chemical method includes: sedimentation and cementation; the physical method comprises the following steps: ion exchange, solvent extraction, membrane filtration and activated carbon adsorption; the biological cadmium removing method comprises the following steps: adsorption and precipitation. Wherein the bio-adsorption sequesters Cd 2+ by extracellular polysaccharides or bio-macromolecular substances such as oxalic acid and malic acid secreted by the cells. The biological precipitation is that the free Cd 2+ can form CdS or Cd (OH) 2 to be precipitated by the organism, so as to achieve the purpose of removing. Compared with physical and chemical methods, the biological removal method has the advantages of low price, environmental friendliness and the like, and is paid attention to. Research shows that the biological repair can effectively reduce the bioavailability of heavy metal cadmium, reduce the absorption of crops and ensure the safety of agricultural products.
In nature, microorganisms (bacteria, algae, yeast, etc.) participate in the conversion of heavy metals, or convert heavy metals into non-toxic or low-toxic forms, or fix them in biological cells by adsorption, thereby achieving the result of natural purification. The method has the advantages of multiple microorganism types, fast growth, fast metabolism and strong environmental adaptability, so that the utilization of the microorganism technology for treating the cadmium pollution in the environment is a hot spot for the current environmental pollution treatment research.
The inventor researches microorganisms in soil environment to screen out a strain with the function of passivating heavy metal cadmium, has strong cadmium resistance, can adsorb and remove cadmium in solution, and can provide strain resources for developing a soil heavy metal pollution microorganism repair technology.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Disclosure of Invention
The invention aims to provide a strain Cd04 for passivating heavy metal cadmium and application thereof.
The first object of the present invention is to provide a passivated heavy metal cadmium strain Cd04, which has a taxonomic name of golden yellow bacillus Chryseobacterium bernardetii Cd04, and is preserved in the China Center for Type Culture Collection (CCTCC) No: m20232084.
The second purpose of the invention is to provide the application of the passivation heavy metal cadmium strain Cd04 in the passivation environment.
Compared with the prior art, the invention has the following beneficial technical effects:
The invention screens the cadmium-resistant strain, identifies the cadmium-resistant strain through morphological observation and 16S rRNA sequence sequencing, obtains the strain Chryseobacterium bernardetii Cd04 capable of passivating heavy metal cadmium, has strong cadmium-resistant capability, can adsorb and remove cadmium in solution, and can provide strain resources for developing soil heavy metal pollution microorganism repair technology.
Description of preservation information
The strain Cd04 of the heavy metal cadmium is passivated, the taxonomic name is golden yellow bacillus Chryseobacterium bernardetii, and the strain is preserved in China center for type culture Collection, with a preservation address: the preservation number of the Wuhan university in Wuhan, china is CCTCC No: m20232084.
Drawings
FIG. 1 is a morphological feature of a cadmium-resistant strain Cd04 of the invention;
FIG. 2 is a graph showing the effect of initial concentration of a solution on the adsorption effect of the Cd04 strain of the invention;
FIG. 3 shows the effect of the amount of bacterial liquid on the adsorption effect of the Cd 04-resistant strain of the invention.
Detailed Description
The invention will now be described in further detail with reference to specific examples, which are intended to illustrate, but not to limit, the invention.
Materials and reagents used in the examples described below, unless otherwise indicated, were all commercially available. The experimental methods used in the following examples are conventional methods unless otherwise specified.
Example 1: bacterial strain screening and identification
1. Soil sample collection
And collecting cadmium-polluted soil in 2023, 9 and 25 days. Taking 5-20cm of paddy field surface soil in a 50mL sterile centrifuge tube by adopting a five-point sampling method, and storing in a dry ice box for screening cadmium-resistant strains.
2. Culture medium
LB solid medium: 10.0g/L tryptone, 5.0g/L yeast extract, 10.0g/L sodium chloride and 15g/L agar.
LB broth: tryptone 10.0g/L, yeast extract 5.0g/L, sodium chloride 10.0g/L, pH 7.0+ -0.1 (25deg.C)
3. Strain isolation and purification
10.0G of fresh soil sample is weighed and placed in a 250mL triangular flask containing about 10 sterile glass beads, 90mL of Phosphate Buffer Solution (PBS) is added, and the mixture is uniformly oscillated for 30min at a temperature of 28 ℃ under a condition of 200 r/min. After settling for 15min, the supernatant was discarded, and 5mL of PBS was added to suspend the sediment. 1mL of the above-described soil suspension was aspirated into a 10mL centrifuge tube using a sterile pipette, and 9mL of sterile water was added to obtain a 10 -1 -fold dilution. Subsequently, 1mL of 10 -1 -fold dilution was pipetted into a tube containing 9mL of sterile water, and the sequential operations gave 10 -2、10-3、10-4 dilutions, respectively. And (3) respectively sucking 0.1mL of the diluent, coating on an LB solid culture medium, pouring the diluent into a 28 ℃ incubator for culturing for 1-3d, streaking and transferring single colony on a plate for more than 3 times, and purifying and preserving strains.
4. Screening of cadmium-resistant strains
The purified bacterial strains are respectively coated on LB solid culture media with the final concentration of Cd 2+ content of 0mg/L, 5mg/L, 10mg/L, 20mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L, 250mg/L, 300mg/L, 350mg/L and 400mg/L according to the bacterial liquid dilutions of 10 -2 and 10 -3, and after bacterial colonies grow out, the growth condition is observed.
A strain with high cadmium resistance is screened out and named Cd04. The cadmium resistance of strain Cd04 is shown in Table 1 below.
TABLE 1 growth of the Cd04 strain at various cadmium content concentrations
Note that: + represents growth; -indicating no growth
5. Morphology observation of cadmium-resistant strain Cd04
The cadmium-resistant strain Cd04 is inoculated on LB solid medium and cultured at 37 ℃, and the colony morphology is shown in figure 1.
As can be seen from FIG. 1, the cadmium-resistant strain Cd04 has yellow colonies, circles, bulges, smoothness and complete edges.
6. Identification of Strain 16S rRNA
Sequencing of cadmium-resistant strain Cd04 by 16S Sanger method: bacterial universal primers 27F and 1492R are selected by taking the extracted genome DNA as a template, and 16S rRNA gene amplification is carried out. Wherein, the primer sequences are shown in Table 2; the PCR reaction system is shown in Table 3;
Table 216S rRNA primers
TABLE 3 PCR reaction system
The PCR procedure was: 95 ℃ for 5min; cycling for 25 times at 95 ℃ for 30s,56 ℃ for 30s and 72 ℃ for 90 s; 72℃for 10min.
And (3) carrying out 16S rRNA sequencing on the purified PCR product, wherein the sequencing result is shown in SEQ ID No.3. The 16S rRNA gene sequence amplified by the primer is compared with NCBI database, species information of similar sequences is obtained, evolutionary tree construction is constructed, the species information is judged in an auxiliary way by means of homology comparison, cd04 is determined to be Flavobacterium aureum Chryseobacterium bernardetii, and the Cd04 is named Chryseobacterium bernardetii Cd.
Example 2: cadmium removal test of strain Cd04
1. Preparation of bacterial liquid
The strain Cd04 was inoculated in LB broth, cultured in a 180r/min shaker at 28℃to the logarithmic phase (about 12 h), centrifuged at 4℃for 10min, the pellet was washed with sterilized deionized water, resuspended and centrifuged 3 times, and finally the bacteria were resuspended in 0.9% sodium chloride (sterile physiological saline) solution for further use, after which the OD 600 =1.
2. Effect of initial concentration of solution on adsorption Effect
LB broth media with final concentrations of 6.10mg/L, 15.25mg/L, 30.50mg/L, 61.0mg/L and 91.50mg/L of Cd 2+ were prepared, 50mL each, 3 replicates per treatment. After sterilization, 1% of bacterial solution is added, and the mixture is cultured for 24 hours at 28 ℃ in a biochemical incubator, and then centrifuged for 5 minutes at 12000r/min, and the supernatant is filtered by a 0.45 mu m filter membrane and then the concentration of the heavy metal solution is measured by ICP-OES. The results are shown in FIG. 2.
As can be seen from FIG. 2, the cadmium removal efficiency of the cadmium-resistant strain Cd04 to the solution is 38.48-60.66% in different initial cadmium concentration ranges; wherein, when the initial cadmium concentration is 91.50mg/L, the cadmium removal efficiency of Cd04 in the solution is highest.
Removal efficiency = (initial solution cadmium concentration-treated solution cadmium solubility)/initial solution cadmium concentration x 100%
3. Influence of bacterial liquid dosage on adsorption effect
50ML of LB broth medium with a final concentration of 6.10mg/L of Cd 2+ was prepared, and after sterilization, the bacterial solutions were inoculated into 50mL of LB broth medium at 1%, 2.5%, 5%, 10%, 15% and 20% inoculum sizes. Culturing at 28deg.C in a biochemical incubator for 24 hr, centrifuging at 12000r/min for 5min, filtering the supernatant with 0.45 μm filter membrane, and measuring heavy metal solution concentration with ICP-OES. The results are shown in FIG. 3.
As can be seen from FIG. 3, the cadmium removal efficiency of the cadmium-resistant strain Cd04 against the solution is 34.71-44.27% when the bacterial liquid input amount is 2.5-15%, wherein the cadmium removal effect of the cadmium-resistant strain Cd04 against the cadmium is best when the bacterial liquid input amount is 5.0%.
In conclusion, the cadmium-resistant strain Cd04 has strong cadmium-resistant capability, and can provide strain resources for developing soil heavy metal pollution microorganism repair technology.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (2)
1. The strain Cd04 is characterized in that the strain Cd04 is characterized by being preserved in China Center for Type Culture Collection (CCTCC) with a taxonomic name of Chryseobacterium bernardetii in 10 months of 2023: m20232084.
2. The application of the strain Cd04 in passivation of heavy metal cadmium in passivation environment.
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| CN111925956B (en) * | 2020-07-06 | 2022-04-05 | 南京农业大学 | Geobacillus altivelis with functions of producing alkali and passivating heavy metal cadmium and application thereof |
| CN114196591B (en) * | 2021-12-24 | 2023-07-14 | 康生元(肇庆)生物科技有限公司 | Pseudomonas flavescens strain KY592 with passivation effect on heavy metal cadmium and application thereof |
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| CN118028164A (en) * | 2024-02-28 | 2024-05-14 | 广西壮族自治区农业科学院 | Passivating heavy metal cadmium strain Cd05 and application thereof |
| CN118374395A (en) * | 2024-04-24 | 2024-07-23 | 广西壮族自治区农业科学院 | Passivation heavy metal arsenic strain As05 and application thereof |
| CN118389361A (en) * | 2024-05-22 | 2024-07-26 | 广西壮族自治区农业科学院 | Passivation heavy metal arsenic strain As01 and application thereof |
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