CN112646753B - Klebsiella aerogenes and application thereof - Google Patents

Klebsiella aerogenes and application thereof Download PDF

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CN112646753B
CN112646753B CN202110082013.8A CN202110082013A CN112646753B CN 112646753 B CN112646753 B CN 112646753B CN 202110082013 A CN202110082013 A CN 202110082013A CN 112646753 B CN112646753 B CN 112646753B
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klebsiella aerogenes
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soil
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李永涛
龚贝妮
谭泽文
徐会娟
刘怡灵
陈恋
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South China Agricultural University
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Abstract

The invention discloses a Klebsiella aerogenes strain and application thereof. The strain is named as Klebsiella aerogenes S1 with the preservation number GDMCC NO: 61041, the strain is deposited in Guangdong province culture collection center of No. 59 building 5 building of Michelia media No. 100 college of Michelia Tokyo, Guangzhou, 6.3.6.8.2020. Experiments show that the Klebsiella aerogenes S1 can remove cadmium ions in soil and water in a co-metabolism mode, and can also reduce the pH value in a system so as to dissolve insoluble phosphorus and potassium substances, so that the Klebsiella aerogenes S1 has the potential of removing cadmium ions and improving the effective phosphorus and potassium contents in soil, and can be used for environmental pollution control and plant growth promotion.

Description

Klebsiella aerogenes and application thereof
Technical Field
The invention belongs to the technical field of inorganic pollutant microbial remediation, and particularly relates to a Klebsiella aerogenes strain and application thereof.
Background
Cadmium (Cd) is one of heavy metal elements with strong toxicity, is a non-essential element for the growth and development of organisms, and has toxic action on animals, plants and human bodies. Cadmium is widely distributed in nature and has small content, the circulation of cadmium is originally in a balanced state, and the cadmium does not cause pollution and harm to the environment; however, due to the intervention of human activities, the migration loss and local enrichment of cadmium and cadmium compounds are accelerated, so that serious ecological environment pollution is caused, and the human health is seriously threatened.
There are various ways in which cadmium enters the environment, with surface runoff, industrial and mining wastewater discharge being the predominant way. Cadmium pollution is different from organic pollution due to the structural characteristics of the cadmium pollution and cannot be decomposed and destroyed; and therefore only the way it exists and the transfer position can be changed. At present, the methods for treating heavy metal pollution at home and abroad mainly comprise a physical method, a chemical method and a bioremediation method. The physical and chemical method and the plant repairing method have the defects of large engineering quantity, high cost, long time consumption, serious secondary pollution and the like, and are gradually replaced by the microbial repairing method. The microbial remediation method has the main advantages of low cost, strong specificity, low cost, small secondary pollution and the like, and is widely applied to sewage treatment and soil pollution treatment.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a strain of klebsiella aerogenes.
The invention also aims to provide application of the klebsiella aerogenes in removing cadmium ions in soil and/or water.
Still another object of the present invention is to provide the use of said klebsiella aerogenes for the solubilization of insoluble phosphorus and/or potassium compounds.
The invention further aims to provide the application of the klebsiella aerogenes in the aspect of increasing available phosphorus and/or available potassium in soil.
The purpose of the invention is realized by the following technical scheme:
the Klebsiella aerogenes strain is named Klebsiella aerogenes S1, and the preservation number is GDMCC NO: 61041, the strain is preserved in Guangdong province microbial strain preservation center of No. 59 building 5 building of Michelia Tokyo No. 100 college of Michelia Tokyo, Guangzhou, 6.3.2020.
The 16S rDNA sequence of the Klebsiella aerogenes consists of 1416 basic groups (bp), and the nucleotide sequence is shown as SEQ ID NO. 1.
A method for culturing the Klebsiella aerogenes comprises the following specific steps: inoculating the Klebsiella aerogenes into a culture medium, and culturing at the temperature of 28-37 ℃.
The culture medium is LB culture medium, MSM culture medium, and contains Cd2+The MSM medium of (1), containing Cd2+And peptone, one of MSM medium, PKO medium and potassium bacteria medium; preferably LB medium.
The Cd content2+In the MSM Medium of (1)2+Has a concentration of 10 to 80 mg.L-1。
The Cd content2+And Cd in MSM Medium of peptone2+Has a concentration of 10 to 80 mg.L-1The concentration of peptone is 10 g.L-1。
The formula of the PKO culture medium is as follows: glucose 10 g.L-1，(NH4)2SO4 0.5g·L-1，NaCl 0.2g·L-1，MgSO4·7H2O 0.1g·L-1，KCl 0.3g·L-1，MnSO4·4H2O 0.03g·L-1，FeSO40.003g·L-1，Ca3(PO4)2 5.0g·L-10.5 g.L of yeast powder-120.0 g.L agar powder-1，pH 6.8～7.0。
The potassium bacteria culture medium comprises the following formula: potassium feldspar 2.5 g.L-1，Na2HPO4 0.2g·L-1，MgSO4·7H2O 0.02g·L-1，NaCl 0.2g·L-1，CaCO3 5.0g·L-1，CaSO4·2H2O 0.1g·L-1Glucose 10 g.L-120.0 g.L of agar powder-1And adjusting the pH value to 6.8-7.0.
The preferable temperature of the culture is 28-30 ℃; more preferably 30 deg.c.
The culture time is 18-48 h; preferably 20-48 h.
The cultivation is carried out in a shaking table, and the rotating speed of the shaking table is 125-150 rpm.
The application of the Klebsiella aerogenes in removing cadmium ions (reducing the cadmium content in the environment) in soil and/or water body is provided.
The application of the Klebsiella aerogenes in removing cadmium ions in soil and/or water is that the Klebsiella aerogenes is added into the soil and/or water environment containing cadmium ions, the strain can resist the growth of heavy metal cadmium and can eliminate the cadmium ions in the soil or water so as to remove or reduce the effective concentration of the cadmium ions in the environment.
The concentration of cadmium ions in the water body containing the cadmium ions is 10-80 mg.L-1。
The cadmium ions are divalent cadmium ions (Cd)2+)。
The removal time is more than 6d (days).
The Klebsiella aerogenes is applied to the preparation of the cadmium ion passivator.
The cadmium ions are divalent cadmium ions (Cd)2+)。
The application of the Klebsiella aerogenes in dissolving insoluble phosphorus and/or potassium compounds is provided.
The application of the Klebsiella aerogenes in dissolving insoluble phosphorus and/or potassium compounds is to inoculate the Klebsiella aerogenes into a culture medium containing the insoluble phosphorus and/or potassium compounds and culture the Klebsiella aerogenes at the temperature of 28-37 ℃, and the Klebsiella aerogenes can reduce the pH value of a system to dissolve the insoluble phosphorus and/or potassium compounds.
The insoluble phosphorus compound is preferably tricalcium phosphate (Ca)3(PO4)2)。
The insoluble potassium compound comprises a potassium-containing mineral; preferably potassium feldspar (K)2O·Al2O3·6SiO2)。
The culture medium containing the insoluble phosphorus compound is preferably a PKO culture medium, and the formula is as follows: glucose 10 g.L-1，(NH4)2SO4 0.5g·L-1，NaCl 0.2g·L-1，MgSO4·7H2O 0.1g·L-1，KCl 0.3g·L-1，MnSO4·4H2O 0.03g·L-1，FeSO4 0.003g·L-1，Ca3(PO4)2 5.0g·L-10.5 g.L of yeast powder-120.0 g.L of agar powder-1，pH 6.8～7.0。
The culture medium containing the insoluble potassium compound is preferably a potassium bacteria culture medium, and the formula is as follows: potassium feldspar 2.5 g.L-1，Na2HPO4 0.2g·L-1，MgSO4·7H2O 0.02g·L-1，NaCl 0.2g·L-1，CaCO3 5.0g·L-1，CaSO4·2H2O 0.1g·L-1Glucose 10 g.L-120.0 g.L of agar powder-1And adjusting the pH value to 6.8-7.0.
The inoculation amount of the Klebsiella aerogenes is 1-5% by volume percentage; preferably 2% by volume.
The temperature of the culture is preferably 28-30 ℃.
The culture time is more than 18 h; preferably 18h to 6 d.
The application of the Klebsiella aerogenes in increasing available phosphorus and/or available potassium in soil is provided.
The application of the Klebsiella aerogenes in increasing the available phosphorus and/or the available potassium in the soil is that the Klebsiella aerogenes is added into the soil, and the Klebsiella aerogenes can reduce the pH value of a system, so that insoluble phosphorus and/or potassium compounds in the soil are dissolved, the content of the available phosphorus and/or the available potassium in the soil is increased, and the plant growth is promoted.
The insoluble phosphorus compound is preferably tricalcium phosphate (Ca)3(PO4)2)。
The insoluble potassium compound comprises a potassium-containing mineral; preferably potassium feldspar (K)2O·Al2O3·6SiO2)。
A biological agent for removing cadmium ions (cadmium ion passivator) in the environment contains the Klebsiella aerogenes.
The cadmium ions are divalent cadmium ions (Cd)2+)。
The environment comprises a soil environment and a water body environment.
A biological agent for dissolving insoluble phosphorus and/or potassium compounds, which contains the Klebsiella aerogenes.
After the Klebsiella aerogenes S1 grows on the LB plate for 18-20 h, the bacterial plaque is beige, round and convex, smooth, moist and glossy, and the diameter of the bacterial colony is about 2-2.5 mm after the bacterial plaque is cultured for 24h at 37 ℃. Can be used in the concentration of cadmium ions of 10-80 mg.L-1Contains 10 g.L-1Peptone grew well in an inorganic salt liquid medium.
Compared with the prior art, the invention has the following advantages and effects:
(1) the inventionThe Klebsiella aerogenes S1 can remove cadmium ions through a co-metabolism mode, and the strain S1 is inoculated to the strain with the cadmium ion concentration of 10-80 mg.L-1Contains 10 g.L-1In an inorganic salt liquid culture medium of peptone, the removal efficiency after 6 days of shaking culture at 30 ℃, 150rpm is 43.15%, 42.77%, 30.93% and 40.95% respectively, which shows that the strain S1 has the performances of resisting the growth of heavy metal cadmium and removing cadmium ions, and can be applied to the removal of high-concentration cadmium ions in soil and water.
(2) The Klebsiella aerogenes S1 of the invention can reduce the pH value in the system (which may be secreted organic acid substances such as oxalic acid, formic acid, acetic acid, citric acid, succinic acid and the like to make the pH value in the system low), thereby dissolving insoluble phosphorus and potassium substances, inoculating the strain S1 in a PKO or potassium bacteria culture medium, culturing at the constant temperature of 30 ℃ for 48h, and carrying out phosphorus and potassium dissolving qualitative test: after the strain S1 is spotted on a PKO or potassium bacteria culture medium for 6 days, transparent circles appear around thalli, which shows that the strain S1 can be applied to cadmium removal in liquid and soil, has the potential of improving the content of available phosphorus and potassium in soil, and can be used for promoting plant growth.
Drawings
FIG. 1 is a colony morphology (dish diameter 9cm) of strain S1.
FIG. 2 is a phylogenetic tree of strain S1.
FIG. 3 shows that the strain S1 contains 10 g.L-1The effect of peptone on removal of cadmium ions in MSM medium is shown.
FIG. 4 is a phosphorus and potassium solubilizing qualitative test chart of strain S1 (transparent circles show that the strain has the function of solubilizing phosphorus and potassium) wherein A is a phosphorus solubilizing qualitative test of strain C273; and B is a qualitative test of potassium dissolution of the strain C273.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
Example 1 screening, isolation and purification and identification of Strain S1
Screening method of strain S1 capable of resisting cadmium and removing cadmium ions
1. Material preparation
Strain screening soil source: the soil samples were taken from the contaminated soil in the paddy field around Dabaoshan, Shaokuan, Guangdong province and sealed with sampling bags and brought back to the laboratory at 4 ℃ for storage and use.
LB culture medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of NaCl, 7.0-7.2 of pH, and constant volume of distilled water to 1L; the solid medium was supplemented with 18.0g of agar powder. Sterilizing at 121 deg.C for 15 min.
Inorganic salt medium (MSM medium): 5mL of phosphoric acid buffer solution (KH)2PO4 8.5g·L-1、K2HPO4·H2O 21.75g·L-1、Na2HPO4·12H2O 33.4g·L-1、NH4Cl 5.0g·L-1)，3.0mL22.5g·L-1MgSO (2) of4Solution (MgSO)4·7H2O 46.125g·L-1)，1.0mL 0.25g·L-1FeCl of3Solution (FeCl)3·6H2O 0.42g·L-1)，1.0mL 36.4g·L-1In (C) is2Solution (CaCl)2·2H2O 48.22g·L-1) 1.0mL of a solution of trace elements (MnSO)4·H2O 39.9mg·L-1，ZnSO4·H2O 42.8mg·L-1，(NH4)6Mo7O24·4H2O 34.7mg·L-1) Mixing, adjusting pH to 7.0-7.2, adding pure water to constant volume to 1L, and sterilizing at 121 deg.C for 15 min.
Containing 10 g.L-1MSM medium for peptone: adding 10.0g of tryptone into 1L of MSM medium, and sterilizing at 121 ℃ for 15min at the pH of 7.0-7.2.
2. Laboratory apparatus and device
Vertical pressure steam sterilizer (BL-50A, Shanghai Silicaceae, Inc.), portable pH meter (PHB-4, Shanghai precision science, Inc.), Centrifuge (Centrifuge 5810R), electric heat oven (DGG-9070A, Shanghai Senxin laboratories, Inc.), digital display constant temperature water bath (HH series, Changzhou national instruments manufacturing, Inc.), refrigerator (RCD-205AG7, Haixin electric appliance), biochemical incubator (PYX-208S-A, Keli apparatus), clean bench (SW-CJ-1F, Sujing Antai air technology, Inc.), voro mutex mixer (XW-80A, Shanghai Jingzike, Inc.), MyCycler PCR (BIO-Beijing, USA), electrophoresis apparatus (RAD DYY-6C, Hexagon, etc.), NaDro nucleic acid protein quantitative detector (German, Germany research, Japan, and the present, Gel imaging system (BIO-RAD, USA), floor type constant temperature oscillator (HZQ-211C).
3. Enrichment screening and separation purification of bacterial strain
(1) Isolation and purification of the strains
Weighing 1g of collected contaminated soil, adding into 50mL of the soil containing 0.5 mg.L-1 Cd2+And 10 g.L-1Domestication culture of peptone in MSM medium (i.e. CdCl)2Adding into a reactor containing 10 g.L-1Culturing Cd in MSM medium of peptone2+To a final concentration of 0.5 mg.L-1) Adding into a reactor containing 10 g.L-1Culturing Cd in MSM medium of peptone2+To a final concentration of 0.5 mg.L-1) After acclimatization for two days, the cells were inoculated with 2% (v/v) of inoculum size to 1 mg.L-1 Cd2+And 10 g.L-1MSM medium acclimatization of peptone, and then transfer-grafting to Cd according to the inoculation amount of 2% (v/v)2+The concentrations were 2.5, 5 and 10 mg.L-1 Cd2+Contains 10 g.L-1Acclimatization in MSM medium of peptone. After acclimatization and screening, diluting the bacterial liquid to 10 degrees with a culture medium6、107、108And 109Respectively taking 0.2mL of diluent, coating the diluent on an LB solid culture medium, and culturing at 30 ℃ until bacterial colonies are formed; then selecting the grown single colony, separating and purifying.
(2) Strain screening
And (2) inoculating the purified strain obtained by separation and purification in the step (1) into an LB liquid culture medium for amplification culture for 20-24 h. After activation, the mixture is added to a mixture containing 10 mg.L according to the proportion of 2% (v/v)-1 Cd 2+10 g.L of-1Culturing in MSM culture medium of peptone at 150rpm and 30 deg.C for 6 days, and collecting the bacterial liquid. Centrifuging the bacterial liquid at 8000rpm for 10min, collecting supernatant, filtering with 0.22 μm filter membrane, and collecting filtrate such as Zheng 22531[1]The research method uses flame Atomic Absorption Spectrometry (AAS) to measure (specific references: Zheng22531, Nizong, Xiyuquan, and the like, evaluation of heavy metal pollution conditions and risks in rice soil and rice in Shaoguan industrial mining areas [ J]The report of agricultural environmental science 2018,37(5):915-2+Removal rate (final concentration/initial concentration x 100%). Screening to obtain Cd2+The strain with the removal efficiency higher than 30% was designated as strain S1.
Secondly, observing the morphological characteristics of bacterial colonies
The strain S1 grows faster on an LB culture medium and can grow at the temperature of 30-37 ℃. The bacterial plaque is beige, round and convex, smooth, moist and glossy, and the diameter of the bacterial colony is about 2-2.5 mm after the bacterial plaque is cultured for 24 hours at 37 ℃ (figure 1). Can be in Cd2+The concentration is 10-80 mg.L-1Contains 10 g.L-1Peptone grew well in MSM medium.
2.16S rDNA amplification
And (3) amplifying by using the extracted total DNA of the bacteria as a template and adopting a bacterial 16S rDNA universal primer, wherein the forward primer is 27 f: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer 1492 r: 5'-GGTTACCTTGTTACGACTT-3' the 16S rDNA gene sequence was amplified.
The total PCR reaction was 25 μ L: 2 mul of each of the upstream and downstream primers, 0.5 mul of template DNA, 12.5 mul of 2 XTaq PCR Master Mix, sterile ultra pure to 25 mul of total volume.
The PCR reaction program is: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 50s, annealing at 56 ℃ for 50s, extension at 72 ℃ for 50s, and 35 cycles; and finally, supplementary extension is carried out for 5min at 72 ℃. After the PCR was completed, the PCR product was detected by 1.0% agarose gel electrophoresis, and DL 2000Marker was selected. And observing under a gel imaging system, wherein a clear bright band appears in the middle range of the Marker 1000bp and 2000bp bands.
3.16 determination of the S rDNA sequence
The PCR amplified product was sequenced by Biotechnology of Boxing Ke, Beijing Rui (Guangzhou division). The 16S rDNA gene sequence of the obtained strain is as follows (SEQ ID NO. 1):
TTAGCTACCTACTTCTTTTGCACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACATACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATATGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGGACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTAACGTCAATCGCTAAGGTTATTAACCTTAACGCCTTCCTCCTCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCATTACCCCACCTACTAGCTAATCCCATCTGGGCACATCTGATGGCATGAGGCCCGAAGGTCCCCCACTTTGGTCTTGCGACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCGCTCGTCACCCGAGAGCAAGCTCTCTGGCTACCGCTCGA。
the above sequence consists of 1381 bases (bp).
The obtained 16S rDNA gene sequence is submitted to a National Center for Biological Information (NCBI) webpage for BLAST comparison, and is subjected to homology comparison analysis with the 16S rDNA gene of a relevant model strain in an LPSN database (http:// www.bacterio.net/index. html), a model strain sequence with higher homology is downloaded, and an amplification product sequence is subjected to BLAST comparison and homology analysis on a National Center for Biological Information (NCBI) website, and a system development tree is constructed by a Neightbour-Joining method by adopting Mega 6.0 software. Comparison of the 16S rDNA sequence revealed that the strain S1 has 99.86% homology with Klebsiella aerogenes KCTC 2190 (FIG. 2).
Fourthly, identifying the strain S1 as a new functional strain
According to the colony morphological characteristics and molecular biological identification result of the strain S1, the strain S1 is named as Klebsiella aerogenes S1. The strain is preserved in Guangdong province microorganism culture collection center (GDMCC) with the preservation number of GDMCC NO: 61041, the strain is used 6/3/2020. The address of the preservation unit is No. 59 building No. 5 building of No. 100 college of Jifura Zhonglu, Guangzhou city.
At present, no document reports that the Klebsiella aerogenes (Klebsiella aerogenes) has the functions of resisting the growth of heavy metal cadmium and removing cadmium at home and abroad. Therefore, the Klebsiella aerogenes S1 is a strain capable of reducing Cd in the solution2+Novel strains of function.
Example 2 Strain S1 containing Cd2+Cd in inorganic salt culture medium of peptone2+Removal of
Streaking the strain S1 on LB plate, culturing at 30 deg.C for 20h, selecting single colony, inoculating in LB liquid culture medium, culturing at 30 deg.C for 150r min-1After culturing for 20h in a shaking table, inoculating the seeds to Cd according to the proportion of 2% (v/v)2+Concentrations of 10, 20, 40 and 80 mg.L-1Containing 10 g.L-1Culturing tryptone in MSM medium, and sampling after 6d (days); 3 replicates were set up with no addition of strain S1 as a Control (CK). Sample liquid at 80000 r.min-1Centrifuging for 10min, filtering the supernatant with 0.22 μm filter membrane, and measuring Cd by flame Atomic Absorption Spectrometry (AAS)2+The same procedure as in example 1.
Cd after 6 days of culture2+The removal efficiency is shown in fig. 3: the results show that Cd is obtained after adding the strain S12+The concentration decreased significantly (blank removal was 0 and therefore not shown in the figure). Cd [ Cd ]2+Are 10, 20, 40 and 80 mg.L-1Then, Cd2+The removal efficiencies were 43.15%, 42.77%, 30.93% and 40.95%, respectively.
EXAMPLE 3 qualitative tests for phosphorus and Potassium solubilization of Strain S1
Phosphorus and potassium solubilizing (Phosphate solubilizing bacteria) of strain S1 (by which is meant the conversion of insoluble phosphorus and potassium elements to soluble phosphorus and potassium elements) (specific references: Rawat, P., Das, S., Shankhdhar, D., et al, Phosphate-solubilizing microorganisms: Mechanism and the roll in Phosphate solubilization and uptake [ J ]. Journal of Soil Science and Plant Nutrition.2020, https:// doi.org/10.1007/S42729-020-: inoculating a strain S1 which is drawn on an LB solid culture medium to an LB liquid culture medium, shaking and culturing for 18-24 h at 125-150 rpm and 28-30 ℃ in a shaking table, then respectively dotting the strain on a PKO solid culture medium and a potassium bacteria solid culture medium, inoculating 2% of the strain on the PKO liquid culture medium and the potassium bacteria liquid culture medium, setting 3 repeated tests, placing the strain in a 30 ℃ incubator or the shaking table (125-150 rpm) for 6d, observing a transparent ring formed by hydrolysis of an insoluble phosphorus compound and a potassium mineral around a colony in the culture medium, observing the size of the transparent ring, taking a picture, and measuring the pH of the solution. Whether the strain has the effects of the indissolvable phosphorus compound and the potassium mineral or not is judged according to the hydrolysis cycle. Wherein,
PKO culture medium formula: glucose 10 g.L-1，(NH4)2SO4 0.5g·L-1，NaCl 0.2g·L-1，MgSO4·7H2O 0.1g·L-1，KCl 0.3g·L-1，MnSO4·4H2O 0.03g·L-1，FeSO4 0.003g·L-1，Ca3(PO4)25.0g·L-10.5 g.L of yeast powder-120.0 g.L of agar powder-1pH is 6.8-7.0 (agar powder is not added in a liquid culture medium);
the potassium bacteria culture medium formula comprises: potassium feldspar 2.5 g.L-1，Na2HPO4 0.2g·L-1，MgSO4·7H2O 0.02g·L-1，NaCl 0.2g·L-1，CaCO3 5.0g·L-1，CaSO4·2H2O 0.1g·L-1Glucose 10 g.L-120.0 g.L of agar powder-1And adjusting the pH value to 6.8-7.0 (agar powder is not added in the liquid culture medium).
The results are shown in FIG. 4: the strain shows hydrolysis loop on PKO culture medium and potassium-dissolving bacteria solid culture medium, and shows that the strain has dissolved insoluble phosphorus compound (Ca)3(PO4)2) Or potassium mineral (potassium feldspar), and the pH of the solution is reduced to about 4.3.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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<120> Klebsiella aerogenes and application thereof
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gtcgagttgc agactccaat ccggactacg acatacttta tgaggtccgc ttgctctcgc 180
gaggtcgctt ctctttgtat atgccattgt agcacgtgtg tagccctact cgtaagggcc 240
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aaccttgcgg ccgtactccc caggcggtcg acttaacgcg ttagctccgg aagccacgcc 600
tcaagggcac aacctccaag tcgacatcgt ttacggcgtg gactaccagg gtatctaatc 660
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gcaccctccg tattaccgcg gctgctggca cggagttagc cggtgcttct tctgcgagta 960
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Claims (10)

1. The Klebsiella aerogenes strain is characterized in that: the name is Klebsiella aerogenes S1 with the preservation number of GDMCC NO: 61041, the strain is preserved in Guangdong province microbial strain preservation center of No. 59 building 5 building of Michelia Tokyo No. 100 college of Michelia Tokyo, Guangzhou, 6.3.2020.
2. A method for culturing the Klebsiella aerogenes strain of claim 1, comprising the steps of: inoculating the Klebsiella aerogenes in a culture medium, and culturing at the temperature of 28-37 ℃.
3. The method of claim 2, wherein:
the culture medium is LB culture medium, MSM culture medium, and contains Cd2+The MSM medium of (1), containing Cd2+And peptone, one of MSM medium, PKO medium and potassium bacteria medium;
the Cd content2+The MSM medium contains Cd2+Has a concentration of 10 to 80 mg.L-1；
The Cd content2+And Cd in MSM Medium of peptone2+Has a concentration of 10 to 80 mg.L-1The concentration of peptone is 10 g.L-1；
The culture time is 18-48 h.
4. The use of the klebsiella aerogenes strain of claim 1 for removing cadmium ions from soil and/or water.
5. Use according to claim 4, characterized in that: adding the Klebsiella aerogenes into soil and/or water environment containing cadmium ions to remove or reduce the effective concentration of the cadmium ions in the environment;
the cadmium ions are divalent cadmium ions.
6. The use of the klebsiella aerogenes of claim 1 for preparing a cadmium ion inactivation agent.
7. Use of the klebsiella aerogenes strain of claim 1 for dissolving insoluble phosphorus and/or potassium compounds.
8. Use according to claim 7, characterized in that: inoculating the Klebsiella aerogenes into a culture medium containing insoluble phosphorus and/or potassium compounds, and culturing at the temperature of 28-37 ℃, wherein the Klebsiella aerogenes can reduce the pH value of a system to dissolve the insoluble phosphorus and/or potassium compounds;
the culture time is more than 18 h.
9. Use of the klebsiella aerogenes strain of claim 1 for increasing available phosphorus and/or available potassium in soil.
10. A biological agent for removing cadmium ions and dissolving insoluble phosphorus and/or potassium compounds in the environment, which is characterized in that: comprising the Klebsiella aerogenes bacterium of claim 1;
the environment is a soil environment and/or a water body environment.
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