Citrobacter ivorii and application thereof in removing chloramphenicol and dissolving phosphorus and potassium
阅读说明：本技术 一株葡萄牙柠檬酸杆菌及其在去除氯霉素和溶磷解钾中的应用 (Citrobacter ivorii and application thereof in removing chloramphenicol and dissolving phosphorus and potassium ) 是由 李永涛 谭泽文 龚贝妮 杨秀月 刘怡灵 陈恋 于 2021-07-07 设计创作，主要内容包括：本发明中公开了一株葡萄牙柠檬酸杆菌及其在去除氯霉素和溶磷解钾中的应用。该葡萄牙柠檬酸杆菌的名称为Citrobacter portucalensis SZW2,保藏编号为GDMCC NO：61695,该菌株于2021年5月27日保藏于广州市先烈中路100号大院59号楼5楼的广东省微生物菌种保藏中心。本发明中的葡萄牙柠檬酸杆菌SZW2可通过共代谢方式对氯霉素进行去除,且可以分泌有机酸类物质降低体系中pH,从而溶解不可溶性磷和钾类物质,以能提高土壤有效磷和钾含量,因此,可将葡萄牙柠檬酸杆菌SZW2用于去除氯霉素或促植物生长方面。(The invention discloses a Citrobacter ivorum strain and application thereof in removing chloramphenicol and dissolving phosphorus and potassium. The Citrobacter canicola is named Citrobacter portucalensis SZW2, and the preservation number is GDMCC NO: 61695, said strain is preserved in 27 th.5.2021 in Guangzhou province of microbial culture collection center of No. 59 building 5 of Mieli Zhonglu No. 100 college in Guangzhou city. The citrobacter ivorangii SZW2 can remove chloramphenicol in a co-metabolism mode, and can secrete organic acid substances to reduce the pH value in a system, so that insoluble phosphorus and potassium substances are dissolved, and the content of available phosphorus and potassium in soil can be increased, therefore, the citrobacter ivorangii SZW2 can be used for removing chloramphenicol or promoting plant growth.)
1. A strain of Citrobacter ivorii is characterized in that: the name is Citrobacter portucalensis SZW2, the preservation number is GDMCC NO: 61695, said strain is preserved in 27 th.5.2021 in Guangzhou province of microbial culture collection center of No. 59 building 5 of Mieli Zhonglu No. 100 college in Guangzhou city.
2. The method for culturing the citrobacter ivorum of claim 1, comprising the following steps: inoculating Citrobacter viticola into a culture medium, and culturing at 28-37 ℃.
3. The method of claim 2, wherein:
the culture medium is one of LB culture medium, MSM culture medium containing chloramphenicol and peptone, NBRIP culture medium and potassium bacteria culture medium;
the concentration of chloramphenicol in the MSM culture medium containing chloramphenicol is 10 mg.L-1The following;
the concentration of chloramphenicol in the MSM culture medium containing chloramphenicol and peptone is 10 mg.L-1Hereinafter, the concentration of peptone is 10 g.L-1；
The formula of the NBRIP culture medium is as follows: glucose 10 g.L-1，Ca3(PO4)2 5.0g·L-1，MgCl2 5g·L-1，MgSO4·7H2O 0.25g·L-1，KCl 0.2g·L-1，(NH4)2SO4 0.1g·L-1Adjusting the pH value to 7.2;
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-1Adjusting the pH value to 6.8-7.0;
the cultivation is carried out in a shaking table at a rotating speed of 125-150 r.min-1；
The culture time is 18-48 h.
4. A Citrobacter viticola culture, characterized in that: inoculating the citrobacter ivorangii of claim 1 into a culture medium, and culturing at 28-37 ℃;
the culture medium is one of LB culture medium, MSM culture medium and MSM culture medium containing peptone.
5. The use of Citrobacter ivorum of claim 1 for removing or reducing chloramphenicol in an environment, characterized in that: the environment is a soil environment or a water body environment.
6. Use of the Citrobacter ivorum of claim 1 and/or the Citrobacter ivorum culture of claim 4 for promoting plant growth.
7. Use of at least one of Citrobacter ivorum according to claim 1 and a Citrobacter ivorum culture according to claim 4 for solubilizing insoluble phosphorus and/or potassium compounds.
8. Use according to claim 7, characterized in that:
the insoluble phosphorus compound is a phosphorus-containing mineral; further tricalcium phosphate;
the insoluble potassium compound is a potassium-containing mineral; further potassium feldspar.
9. Use of at least one of Citrobacter ivorum according to claim 1 and Citrobacter ivorum culture according to claim 4 for increasing available phosphorus and/or fast-acting potassium in soil.
10. A biological agent for dissolving insoluble phosphorus compounds, dissolving insoluble potassium compounds, and/or for removing or reducing chloramphenicol from an environment, characterized by: comprising the Citrobacter ivorum of claim 1;
the environment is a soil environment or a water body environment.
Technical Field
The invention belongs to the technical field of organic pollutant microbial remediation, and particularly relates to a Citrobacter vinaceus and application thereof in removal of chloramphenicol and dissolution of phosphorus and potassium.
Background
Chloramphenicol (CAP) is the first artificially synthesized antibiotic, and is a broad-spectrum antibiotic, which has inhibitory action on gram-positive and gram-negative bacteria, especially on typhoid bacillus and paratyphoid bacillus, and the action mechanism of the antibiotic is to inhibit the synthesis of mycoprotein. Because of having good antibacterial action and pharmacokinetic properties, the compound has been widely applied to the treatment of human and animal diseases, and especially plays an important role in the control and treatment of various infectious diseases of various poultry, livestock and aquatic products.
In recent years, unreasonable use of chloramphenicol causes drug residues in soil and accumulated poisoning of livestock and poultry antibiotics, which poses potential threats to the health of consumers. In addition, due to incomplete metabolism of antibiotic drugs, its original compounds or metabolites enter the environment through excreta such as feces and urine, and often maintain activity for years in the environment, affecting microorganisms and some animals and plants to different degrees. Therefore, the reduction of the residual quantity of chloramphenicol in the environment has important significance for human health, soil fertilizer efficiency, plant growth, environmental microbial diversity and ecological safety.
Chloramphenicol is stable in nature, is difficult to degrade, and is difficult to remove by a single physical or chemical method. The current common methods for degrading chloramphenicol are mainly physical or chemical methods such as a Fenton advanced oxidation method, a photocatalytic reduction degradation method, microwave radiation and the like, but have the defects of high treatment cost, harsh reaction conditions, high energy consumption, easy formation of secondary pollution and the like, and are not easy to widely popularize. The microbial degradation is used as an important way for the degradation of antibiotics in environmental soil, and has the advantages of low treatment cost, environmental friendliness, simple operation and the like. The method for screening out the high-efficiency chloramphenicol degrading bacteria by utilizing the original bacteria source in the environment is an important means for reducing or even eliminating the chloramphenicol pollution in the environment, and has wide development prospect.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a Citrobacter ivorum strain.
The invention also aims to provide application of the citrobacter ivorum in removing Chloramphenicol (CAP) in soil and/or water.
Still another object of the present invention is to provide the use of said Citrobacter ivorum for solubilizing insoluble phosphorus and/or potassium compounds.
Still another object of the present invention is to provide the use of said Citrobacter ivorum for increasing available phosphorus and/or available potassium in soil.
The purpose of the invention is realized by the following technical scheme:
the Citrobacter vittatus is named as Citrobacter vittatus (SZW 2) with the deposit number of GDMCC NO: 61695, said strain is preserved in 27 th.5.2021 in Guangzhou province of microbial culture collection center of No. 59 building 5 of Mieli Zhonglu No. 100 college in Guangzhou city.
The 16S rDNA sequence of the Citrobacter vinaceus consists of 1430 bases (bp), and the nucleotide sequence is shown in SEQ ID NO. 1.
A method for culturing the Citrobacter ivorii comprises the following specific steps: inoculating the citrobacter vittae into a culture medium, and culturing at 28-37 ℃.
The culture medium is one of LB culture medium, MSM culture medium containing Chloramphenicol (CAP), MSM culture medium containing chloramphenicol and peptone, NBRIP culture medium and potassium bacteria culture medium; preferably LB medium.
The concentration of chloramphenicol in the MSM culture medium containing chloramphenicol is 10 mg.L-1The following; preferably 2.5 to 10 mg.L-1(ii) a More preferably 10 mg.L-1。
The concentration of chloramphenicol in the MSM culture medium containing chloramphenicol and peptone is 10 mg.L-1The following (preferably 2.5 to 10 mg. L)-1(ii) a More preferably 10 mg.L-1) The concentration of peptone is 10 g.L-1。
The formula of the NBRIP culture medium is as follows: glucose 10 g.L-1，Ca3(PO4)2 5.0g·L-1，MgCl25g·L-1，MgSO4·7H2O 0.25g·L-1，KCl 0.2g·L-1，(NH4)2SO4 0.1g·L-1The pH was adjusted to 7.2.
The potassium bacteria culture medium comprises the following formula: potassium feldspar (K)2O·Al2O3·6SiO2)2.5g·L-1，Na2HPO40.2g·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-1And adjusting the pH value to 6.8-7.0.
The cultivation is carried out in a shaking table at a rotating speed of 125-150 r.min-1。
The preferable temperature of the culture is 28-30 ℃; more preferably 30 deg.c.
The culture time is 18-48 h; preferably 18-24 h.
A Citrobacter ivorii culture is prepared by inoculating Citrobacter ivorii in culture medium, and culturing at 28-37 deg.C.
The culture medium is one of LB culture medium, MSM culture medium and MSM culture medium containing peptone; more preferably LB medium.
The concentration of peptone in the MSM medium containing peptone is preferably 10 g.L-1。
The cultivation is carried out in a shaking table at a rotating speed of 125-150 r.min-1。
The preferable temperature of the culture is 28-30 ℃; more preferably 30 deg.c.
The culture time is 18-48 h; preferably 18-24 h.
The application of the Citrobacter vinaceum in removing or reducing Chloramphenicol (CAP) in the environment is provided.
The environment comprises a soil environment and a water body environment.
The application of the citrobacter ivorangii in removing or reducing Chloramphenicol (CAP) in the environment is that the citrobacter ivorangii is added into the soil and/or water environment containing chloramphenicol, the strain can tolerate the growth of chloramphenicol, and can eliminate chloramphenicol in the soil or water.
The concentration of the chloramphenicol in the water body containing the chloramphenicol is 10 mg.L-1The following.
The removing time is more than 0.25 days; preferably for 6 days or more.
The application of the citrobacter ivorangii and/or citrobacter ivorangii culture in promoting plant growth.
The use of at least one of Citrobacter viniferum and Citrobacter viniferum cultures for solubilizing insoluble phosphorus and/or potassium compounds.
The application of at least one of the citrobacter vittaci and the citrobacter vittaci culture in dissolving insoluble phosphorus and/or potassium compounds is characterized in that the citrobacter vittaci is inoculated into a culture medium containing insoluble phosphorus and/or potassium compounds and cultured at the temperature of 28-37 ℃, and the citrobacter vittaci can reduce the pH of a system to dissolve the insoluble phosphorus and/or potassium compounds; or adding the Citrobacter ivorum culture into a culture medium containing insoluble phosphorus and/or potassium compounds to dissolve the insoluble phosphorus and/or potassium compounds.
The insoluble phosphorus compound comprises a phosphorus-containing mineral; preferably tricalcium phosphate (Ca)3(PO4)2)。
The culture medium containing the insoluble phosphorus compound is preferably an NBRIP culture medium, and the formula is as follows: glucose 10 g.L-1，Ca3(PO4)2 5.0g·L-1，MgCl2 5g·L-1，MgSO4·7H2O 0.25g·L-1，KCl 0.2g·L-1，(NH4)2SO4 0.1g·L-1，pH 7.2。
The insoluble potassium compound comprises a potassium-containing mineral; preferably potassium feldspar (K)2O·Al2O3·6SiO2)。
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-1And adjusting the pH value to 6.8-7.0.
The inoculation amount of the citrobacter ivorangii is 1-5% by volume; preferably 2% by volume.
The temperature of the culture is preferably 28-30 ℃.
The culture time is more than 18 h; preferably 6d or more.
The application of at least one of the citrobacter vittaci and the citrobacter vittaci culture in increasing available phosphorus and/or available potassium (available potassium) in soil is provided.
In order to add the citrobacter ivorangii into the soil, the citrobacter ivorangii can reduce the pH of a system so as to dissolve insoluble phosphorus and/or potassium compounds in the soil, so that the content of available phosphorus and/or available potassium in the soil is increased (further the growth of plants is promoted); or adding the Citrobacter viniferum culture into soil to dissolve insoluble phosphorus and/or potassium compounds in the soil, so as to increase the content of available phosphorus and/or available potassium in the soil (further promote plant growth).
The insoluble phosphorus compound comprises a phosphorus-containing mineral; 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 dissolving an insoluble phosphorus compound, dissolving an insoluble potassium compound, and/or for removing or reducing chloramphenicol in the environment, comprising the above Citrobacter viticola.
The environment comprises a soil environment and a water body environment.
After the citrobacter vittatus SZW2 grows on an LB flat plate for 18-20 hours, the bacterial plaque is circular, the edge of the bacterial plaque is semitransparent, and the middle of the bacterial plaque is raised to be beige; after culturing at 37 ℃ for 18h, the diameter of the colony is about 1-2 mm. Can be used at the concentration of 10 mg.L of chloramphenicol-1Contains 10 g.L-1Peptone grew well in inorganic salt liquid medium (MSM).
Compared with the prior art, the invention has the following advantages and effects:
(1) the Citrobacter viniferum SZW2 can remove chloramphenicol by co-metabolism, and strain SZW2 is inoculated to chloramphenicol with concentration of 10 mg.L-1Contains 10 g.L-1In inorganic salt liquid culture medium of peptone, at 30 ℃ and 150 r.min-1Shake culture for 0, 0.25, 0.5, 1, 2, 3, 4, 5 and 6 days (d), the chloramphenicol removal efficiency was 0, 5.79, 16.73, 21.55, 29.66, 30.95, 31.98, 35.04 and 36.80%, respectively, indicating that the strain SZW2 is resistant to growth of chloramphenicol and removes chloramphenicolCan be used for removing low-concentration chloramphenicol in soil and water body so as to remove or reduce the residue of chloramphenicol in the environment.
(2) The citrobacter vittae SZW2 can secrete organic acid substances to reduce the pH value in a system, so that insoluble phosphorus and potassium substances are dissolved, the strain SZW2 is inoculated in an NBRIP or potassium-solubilizing bacteria culture medium and shake-cultured for 6 days, and then the effective phosphorus and potassium contents in the solution are measured, so that the following can be seen: the effective phosphorus and potassium contents in the added strain SZW2 solution are 251.41 and 7.10 mg.L-1While the phosphorus and potassium contents in the blank non-bacteria-adding solution are 4.6 and 4.7 mg.L-1(ii) a The phosphorus dissolving amount is obviously higher than that of a control without adding bacteria, and the potassium dissolving content is lower, so that the strain SZW2 can dissolve insoluble phosphorus and potassium compounds, has the potential of improving the effective phosphorus and potassium content of soil, and can be used for promoting the growth of plants.
Drawings
FIG. 1 is a colony morphology map of strain SZW2 (dish diameter 9 cm).
FIG. 2 is a phylogenetic tree diagram of strain SZW 2.
FIG. 3 shows that the strain SZW2 contains 10 g.L-1Degradation concentration of chloramphenicol in peptone MSM medium and strain growth and pH change profile.
FIG. 4 is a graph showing the results of quantitative determination of potassium phosphate solubilizing of the strain SZW 2; wherein A is the quantitative determination result of available phosphorus; and B is the quantitative determination result of the quick-acting potassium.
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 SZW2
Screening method of chloramphenicol degrading strain SZW2
1. Material preparation
Strain screening sample sources: digging a dry ditch beside a pig farm in Longping town of Yongzhou city, Qing and Yuan province to obtain a earthworm, sealing the earthworm and surrounding soil samples by using a sampling bag, and immediately removing and digging after the earthworm is brought back to a laboratory at 4 ℃ to obtain intestinal contents.
LB culture medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of NaCl, 7.0-7.2 of pH, constant volume of distilled water to 1L, and 18.0g of agar powder added into a solid culture medium. Sterilizing at 121 deg.C for 15 min.
Inorganic salt medium (MSM): 5mL of phosphoric acid buffer solution (KH)2PO4 8.5g·L-1、K2HPO4·H2O 21.75g·L-1、Na2HPO4·12H2O 33.4g·L-1、NH4 Cl 5.0g·L-1)，3.0mL 22.5g·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 trace element solution (containing 39.9 mg. L)-1MnSO4·H2O，42.8mg·L-1ZnSO4·H2O，34.7mg·L-1(NH4)6Mo7O24·4H2O), mixing, adjusting the pH value to 7.0-7.2, fixing the volume to 1L by using pure water, and sterilizing for 15min at 121 ℃.
Containing 10 g.L-1MSM medium for peptone: adding 10.0g of tryptone into the MSM culture medium solution, adjusting the pH to 7.0-7.2, fixing the volume to 1L by using pure water, and sterilizing for 15min at 121 ℃.
2. Laboratory apparatus and device
Vertical pressure steam sterilization pan (BL-50A, Shanghai Silique industries, Ltd.), portable pH meter (PHB-4, Shanghai precision science, Ltd.), Centrifuge (Centrifuge 5810R), electric heating oven (DGG-9070A, Shanghai Senxin experiment apparatus, Ltd.), digital display constant temperature water bath pan (HH series, Changzhou national apparatus manufacturing Co., Ltd.), refrigerator (RCD-205AG7, Hai Xin electric appliance), biochemical incubator (PYX-208S-A, Keli apparatus), clean bench (SW-CJ-1F, Sujing Antai air technology, Ltd.), voro mutex mixer (XW-80A, Shanghai Jing industries, Ltd.), MyCycler PCR (BIO-RAD, USA), electrophoresis apparatus (DYY-6C, Bei six instrument works), NaDrno nucleic acid protein quantitative detector (German rmmo), Germany, Gel imaging system (BIO-RAD, USA), floor type constant temperature oscillator (HZQ-211C).
3. Enrichment screening, separation and purification of chloramphenicol degrading strain
(1) Isolation and purification of the strains
Digging a dry water channel beside a pig farm in Longping town of Yongzhou city, Qingyuan province to obtain an earthworm, and then carrying the earthworm back to a laboratory to be immediately disassembled to obtain intestinal contents. 0.2g of the contents was weighed and added to 10mL of the solution containing 0.5 mg. L-1Chloramphenicol (CAP) content of 10 g.L-1Acclimatizing and culturing peptone in MSM culture medium, inoculating to culture medium containing 1 mg.L at an inoculum size of 2% (v/v) after acclimatization for 2 days- 1CAP content of 10 g.L-1MSM medium acclimatization of peptone, followed by sequential 2% (v/v) transfer to CAP concentrations of 2.5, 5 and 10 mg. L-1CAP content of 10 g.L-1And (3) respectively acclimating peptone in MSM culture medium for 3 d. After acclimatization and screening, diluting the bacterial liquid to 10 degrees with a culture medium7Taking 0.2mL of diluent, coating the diluent on an LB solid culture medium, culturing at 30 ℃ until a colony is formed, then selecting a single colony grown out, separating and purifying.
(2) Strain screening
And (2) inoculating the strain separated and purified 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)-1CAP content of 10 g.L-1150 r.min in MSM culture medium of peptone-1Culturing at 30 deg.C in dark, collecting bacterial liquid after 6 days, and measuring CAP concentration. The determination method comprises the following steps: bacterial liquid 8000r min-1Centrifuging for 1min, collecting supernatant, filtering with 0.22 μm organic filter membrane, measuring filtrate with High Performance Liquid Chromatography (HPLC), and screening to obtain strain with CAP degradability. Wherein, HPLC conditions are as follows: the liquid chromatography column is CNW C18-WP (4.6X 250 mm)5 mu m), the phase A is methanol, the phase B is acetonitrile, and the volume ratio is V(A):V(B)50:50, flow rate 0.8 mL/min-1The column temperature is 30 ℃; the sample volume is 20 mu L; the ultraviolet detector detects a wavelength of 278 nm. Screening to obtain a strain capable of degrading CAP (after the CAP concentration is measured by HPLC, the CAP concentration is obviously reduced, and the concentration of a blank control group (CK) is basically unchanged, so as to judge whether the strain has the degrading capability), and naming the strain as SZW 2.
Secondly, observing the morphological characteristics of the bacterial colony
The strain SZW2 grows faster on an LB culture medium and can grow at the temperature of 28-37 ℃. The plaque was rounded with translucent edges and beige in the middle (fig. 1). Can be used at CAP concentration of 10 mg.L-1Contains 10 g.L-1Good growth in peptone.
2.16S rDNA amplification
And (3) amplifying by using the extracted total DNA of the strain SZW2 as a template and a bacterial 16S rDNA universal primer, wherein the forward primer is 27 f: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer 1492 r: 5'-GGTTACCTTGTTACGACTT-3' (Stackelbrandt et al, 1991) amplified the 16S rDNA gene sequence.
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, and sterilized ultrapure water to a total volume of 25 mul.
The PCR reaction program is: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 50 s; annealing at 56 ℃ for 50 s; extension at 72 ℃ for 50s, 35 cycles; and finally, supplementary extension is carried out for 5min at 72 ℃. The PCR product was then detected by electrophoresis on a 1.0% agarose gel (DL 2000 Marker was selected). And observing under a gel imaging system, wherein a remarkable 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 is sent to Beijing Rui Boxing scientific Biotechnology Limited company (Guangzhou division) for sequencing, and the 16S rDNA gene sequence of the obtained strain is as follows (SEQ ID NO. 1):
TCAAGTGGTAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACATACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATATGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTAACGTCAATTGCTGCGGTTATTAACCACAACACCTTCCTCCTCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCGTTACCCCACCTACTAGCTAATCCCATCTGGGCACATCCGATGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCGGGCAGTTTCCCAGACATTACTCACCCGTCCGCCACTCGTCACCCAAGGAGCAAGCTCCTCTGTGCTACCGTTCGACTGCAGGTAGTCATCCGGG。
the above sequence consists of 1430 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 SZW2 has 99.23% homology with Citrobacter viticola (Citrobacter portucalensis) (FIG. 2).
Fourthly, the strain SZW2 is identified as a new functional strain
According to the colony morphological characteristics and molecular biological identification results of the strain SZW2, the strain SZW2 is named as Citrobacter viticola (Citrobacter portucalensis) SZW 2. The strain is preserved in Guangdong province microorganism culture collection center (GDMCC) with the preservation number of GDMCC NO: 61695, the strain is deposited at 27.5.5.2021 at the address of No. 59 Lou 5 of Dazhou college No. 100 of Jieli Zhonglu, Guangzhou City. The Citrobacter viticola (Citrobacter portucalensis) has the function of removing chloramphenicol, so the Citrobacter viticola SZW2 is a new strain capable of reducing the content of chloramphenicol in a solution.
EXAMPLE 2 Strain SZW2 Chloramphenicol removal assay
Streaking strain SZW2, culturing at 30 deg.C for 20 hr on LB plate, selecting single colony, inoculating into LB liquid culture medium, culturing at 30 deg.C and 150rpm in shaker for 20 hr, inoculating into chloramphenicol with concentration of 10 mg.L at 2% (v/v)-1Contains 10 g.L-1In inorganic salt liquid culture medium of peptone, at 30 deg.C and 150r min-1Shake-culturing for 0, 0.25, 0.5, 1, 2, 3, 4, 5 and 6 days (d), taking the strain SZW2 without addition as a blank Control (CK), and determining the removal amount and removal efficiency of chloramphenicol by the strain SZW2 by setting 3 repeated tests; the CAP content was determined by HPLC (same procedure as in example 1).
The removal efficiency of the strain SZW2 on chloramphenicol is 0, 5.79, 16.73, 21.55, 29.66, 30.95, 31.98, 35.04 and 36.80 percent respectively. The strain SZW2 contains 10 g.L-1The removal of chloramphenicol from peptone MSM medium, as well as strain growth and pH change are shown in fig. 3: the growth of SZW2 shows an S-shaped curve, and the maximum concentration of the thallus is OD at 2 days (d)6000.82; and the pH of the solution was gradually increased to 9.1 and maintained at pH 8.9, because when SZW2 was grown using peptone (containing a large amount of amino groups) as a nutrient, a large amount of amine-based compounds was produced, resulting in an increase in the pH of the solution. The results show that the strain SZW2 has the performance of resisting growth of chloramphenicol and removing chloramphenicol, and can be applied to the removal of chloramphenicol in soil and water.
EXAMPLE 3 qualitative test of Potassium solubilization by phosphorus-solubilizing Strain SZW2
Bacterial strains which dissolve Phosphate and potassium (Phosphate/potassium solubilizing bacteria, meaning the conversion of insoluble phosphorus and potassium species to soluble phosphorus and potassium) (specific references: Rawat, P., Das, S., Shankhdhar, D., et al, Phosphate-solubilizing microorganisms: Mechanism and the role in Phosphate solubilization and uptake [ J ]. Journal of Soil Science and Plant Nutrition.2020, https:// doi.org/10.1007/s 42729-020-:
inoculating the strain SZW2 into an LB liquid culture medium, and culturing at 125-150 r.min-1Shaking table shaking culture at 28-30 deg.C for 18-24 hr, inoculating 2% (v/v) in NBRIP or potassium-decomposing bacteria liquid culture medium, setting 3 repeated tests, and standing at 30 deg.C for 150r min-1Culturing in shaking table for 6d, and measuring the content of available phosphorus and available potassium (the available potassium is measured by flame photometer); wherein the content of available phosphorus and available potassium is detected by referring to the methods in the fifth chapter and the sixth chapter of books (Boston. soil agriculture analysis, 3 edition, China agriculture Press, 2000.) respectively;
NBRIP medium formula: glucose 10 g.L-1；Ca3(PO4)2 5.0g·L-1；MgCl2 5g·L-1；MgSO4·7H2O 0.25g·L-1；KCl 0.2g·L-1；(NH4)2SO4 0.1g·L-1；pH 7.2；
Potassium bacteria culture medium: potassium feldspar (K)2O·Al2O3·6SiO2)2.5g·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-1，pH6.8～7.0。
The results are shown in FIG. 4: it can be seen that the strain SZW2 can reduce the pH value in the system, thus insoluble phosphorus and potassium substances are dissolved, and the effective phosphorus (phosphorus) and quick-acting potassium (potassium) contents in the added strain SZW2 solution are 251.41 and 7.10 mg.L-1While the phosphorus and potassium content in the blank non-bacteria-adding solutionAre 4.6 and 4.7 mg.L-1Wherein the phosphorus dissolving amount (content of available phosphorus) is obviously higher than that of a control without bacteria, and the potassium dissolving amount (content of quick-acting potassium) is lower. The strain SZW2 can dissolve insoluble phosphorus and potassium compounds, has the potential of improving the effective phosphorus and potassium content of soil, and can be used for promoting the growth of plants.
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
<110> southern China university of agriculture
<120> Citrobacter viticola and application thereof in removal of chloramphenicol and phosphorus-dissolved potassium
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1430
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 16S rDNA gene sequence
<400> 1
tcaagtggta gcgccctccc gaaggttaag ctacctactt cttttgcaac ccactcccat 60
ggtgtgacgg gcggtgtgta caaggcccgg gaacgtattc accgtggcat tctgatccac 120
gattactagc gattccgact tcatggagtc gagttgcaga ctccaatccg gactacgaca 180
tactttatga ggtccgcttg ctctcgcgag gtcgcttctc tttgtatatg ccattgtagc 240
acgtgtgtag ccctactcgt aagggccatg atgacttgac gtcatcccca ccttcctcca 300
gtttatcact ggcagtctcc tttgagttcc cggccgaacc gctggcaaca aaggataagg 360
gttgcgctcg ttgcgggact taacccaaca tttcacaaca cgagctgacg acagccatgc 420
agcacctgtc tcagagttcc cgaaggcacc aaagcatctc tgctaagttc tctggatgtc 480
aagagtaggt aaggttcttc gcgttgcatc gaattaaacc acatgctcca ccgcttgtgc 540
gggcccccgt caattcattt gagttttaac cttgcggccg tactccccag gcggtcgact 600
taacgcgtta gctccggaag ccacgcctca agggcacaac ctccaagtcg acatcgttta 660
cggcgtggac taccagggta tctaatcctg tttgctcccc acgctttcgc acctgagcgt 720
cagtctttgt ccagggggcc gccttcgcca ccggtattcc tccagatctc tacgcatttc 780
accgctacac ctggaattct acccccctct acaagactct agcctgccag tttcggatgc 840
agttcccagg ttgagcccgg ggatttcaca tccgacttga cagaccgcct gcgtgcgctt 900
tacgcccagt aattccgatt aacgcttgca ccctccgtat taccgcggct gctggcacgg 960
agttagccgg tgcttcttct gcgagtaacg tcaattgctg cggttattaa ccacaacacc 1020
ttcctcctcg ctgaaagtac tttacaaccc gaaggccttc ttcatacacg cggcatggct 1080
gcatcaggct tgcgcccatt gtgcaatatt ccccactgct gcctcccgta ggagtctgga 1140
ccgtgtctca gttccagtgt ggctggtcat cctctcagac cagctaggga tcgtcgccta 1200
ggtgagccgt taccccacct actagctaat cccatctggg cacatccgat ggcaagaggc 1260
ccgaaggtcc ccctctttgg tcttgcgacg ttatgcggta ttagctaccg tttccagtag 1320
ttatccccct ccatcgggca gtttcccaga cattactcac ccgtccgcca ctcgtcaccc 1380
aaggagcaag ctcctctgtg ctaccgttcg actgcaggta gtcatccggg 1430
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Forward primer 27f
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> reverse primer 1492r
<400> 3
ggttaccttg ttacgactt 19
- 上一篇：一种医用注射器针头装配设备
- 下一篇：一株鞘氨醇菌属多环芳烃降解菌株JT-M9-H