CN114395482B - Acid-producing bacteria JC-C and application thereof, and culture and identification method of acid-producing bacteria JC-C - Google Patents
Acid-producing bacteria JC-C and application thereof, and culture and identification method of acid-producing bacteria JC-C Download PDFInfo
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Abstract
The invention discloses an acidogenic bacterium JC-C, which is named as: penicillium chrysogenum (Penicillium chrysogenum) JC-C has been deposited in China general microbiological culture Collection center (CGMCC) with a deposit number of CGMCC NO:21423. the method can be applied to acid production, and the fermentation medium is utilized to perform constant-temperature shake culture under the conditions of 30 ℃ of temperature, 4 of pH value and 160rpm of rotating speed, so that the acid production efficiency is highest. The acidogenic bacteria can provide strain resources for biological acidogenesis.
Description
Technical Field
The invention belongs to the technical field of environmental microorganisms, relates to acid-producing bacteria JC-C and application thereof, and also relates to a culture and identification method of the acid-producing bacteria JC-C.
Background
Mining industry is generally used as an important local economic pillar of mining areas, and provides powerful support for local economic construction and social development; meanwhile, with development, transportation and smelting processing of mineral resources, the problem of soil pollution is increasingly serious, and the pollution hazard of heavy metal soil is greatest and the treatment is difficult, so that people are paid attention to the heavy metal soil. Heavy metals are usually present in a stable state in the environment, but due to mining production, heavy metals originally present in deep soil, mines are transported to the surface and with the diffusion of water circulation water into the entire ecological environment, the life health of local residents has been seriously threatened.
At present, the soil pollution restoration technology of mining areas is mainly a physical restoration technology and a chemical restoration technology. Along with the development of science and technology, the technology also becomes an important means for repairing microorganisms in mining area soil in recent years, and the main principle is that microorganisms with high heavy metal resistance are screened and extracted in heavy metal pollution areas and cultured, and heavy metal toxicity in the soil is reduced by utilizing manners of chelating, dissolving and the like of organic acids generated by life metabolic activity and metabolic secretion of the microorganisms. Compared with the traditional repair method, the new repair method has the characteristics of low operation cost, obvious effect, environmental friendliness and the like, and is increasingly valued and applied at home and abroad.
Disclosure of Invention
The invention aims to provide an acidogenic bacterium JC-C, application thereof and a culture and identification method of the acidogenic bacterium JC-C, and strain resources are provided for biological acidogenesis and improvement of the problem of heavy metal pollution soil in the surrounding area caused by random treatment of tailings.
The invention adopts the technical scheme that an acidogenic bacterium JC-C is named as: penicillium chrysogenum (Penicillium chrysogenum) has been deposited in China general microbiological culture Collection center (CGMCC) with a deposit number of CGMCC NO:21423.
the culture method of the acid-producing bacteria JC-C comprises the following steps:
step 1, separating and culturing strains
Collecting soil severely polluted by heavy metals as a sample;
preparation of a search medium: sucrose 30g, naNO 3 3.0g、K 2 HPO 4 1.0g、KCl 0.5g，FeSO 4 ·7H 2 Mixing 0.01g of O, 20.0g of agar and 1L of distilled water, sterilizing at high temperature of 121 ℃ for 20min, pouring a flat plate until the mixture is solidified, and preparing a sterile check culture medium for separation culture;
mixing the collected soil sample with sterile water to prepare soil suspension, carrying out gradient dilution on the soil suspension, coating the soil suspension on the surface of a culture medium, numbering, and culturing in a constant-temperature incubator at 30 ℃ for 3-5d;
step 2, identifying acid-producing strains
Preparing an acid production identification medium: adding 1.6% bromocresol purple into a solid culture medium, sterilizing at 121 ℃ for 20min, pouring the solid culture medium into a flat plate, and preparing a sterile acid-producing identification culture medium for later use after the solid culture medium is solidified, wherein the color of the sterile acid-producing identification culture medium is purple;
taking out the strain cultured in the step 1, observing the strain form, and selecting out bacterial colonies with different forms to culture in an acid-producing identification medium;
after 3-5d of culture, the culture medium turns from purple to yellow to indicate that the strain has acid production capability, if no change exists, the strain does not have acid production capability, and the strain with acid production capability is selected to be inoculated into an acid production identification culture medium for multiple times to obtain a purified single strain;
when adding heavy metals into the check culture medium, zn is added 2+ A concentration of more than 400mg/L, or Pb 2+ Concentration ofAbove 2000mg/L, or Cd 2+ When the concentration is higher than 200mg/L, the acid-producing bacteria JC-C can be severely inhibited.
Application of acid-producing bacteria JC-C in acid production, and acid production efficiency of acid-producing bacteria JC-C is determined by using fermentation medium, wherein the fermentation medium adopts sucrose 100.0g and NaNO 3 1.5 g，KH 2 PO 4 0.5 g，KCl0.025g，MgSO 4 ·7H 2 0.025g of O, 1.6g of yeast extract, adjusting the pH to 4, and sterilizing at 121 ℃ for 20min;
adding sterile water into cultured acid-producing bacteria JC-C culture medium, lightly scraping colony surface with sterile coating rod to obtain bacterial strain spores, observing spore number with blood cell counting plate, adding sterile water, diluting bacterial strain spores to obtain bacterial strain spores, and mixing with sterile water according to a ratio of 3.6X10 6 The fermentation medium was added at a ratio of 50ml to perform constant temperature shaking culture, and the culture was performed in a constant temperature shaking incubator at a temperature of 30℃and a rotation speed of 160 rpm.
An identification method of acid-producing bacteria JC-C, comprising the following steps: and (3) identifying strains and determining the content of organic acid in fermentation broth by using an 18S rRNA gene sequence.
The strain identification by using the 18S rRNA gene sequence is specifically as follows:
PCR amplification of the 18S rRNA Gene sequence of the target Strain:
PCR amplification was performed by fungal universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). Under the action of polymerase, the 18S rRNA gene sequence of the target strain is obtained through pre-denaturation, annealing, extension, 35 cycles and repair extension.
The determination of the organic acid content in the fermentation broth in the fermentation medium is specifically as follows:
first, a 0.1mol/L NaOH standard solution and a 1% phenolphthalein indicator were prepared. Then accurately sucking 10.0mL of sample fermentation broth, transferring to a 100mL volumetric flask, adding distilled water to 100mL scale, and shaking uniformly. Filtering with filter paper, accurately sucking 20mL of filtrate, placing into a 100mL triangular flask, adding 2 drops of 1% phenolphthalein, titrating with 0.1mol/LNaOH standard solution until the color of the primary color does not fade within 0.5min as an end point, recording the dosage of sodium hydroxide, and repeating for three times to obtain an average value. And the NaOH consumption of the blank was determined.
The beneficial effects of the invention are as follows:
heavy metal resistant effect of acid-producing bacteria JC-C:
and culturing fungi at 30 ℃ in a bacteria inoculating mode of a puncher in a checking culture medium added with heavy metals, and observing the strain inhibition rate. Inhibition (%) = (blank plate diameter-heavy metal plate diameter)/(blank diameter-3 mm). When heavy metal Zn is added 2+ A concentration of more than 400mg/L, or Pb 2+ Concentration higher than 2000mg/L, or Cd 2+ When the concentration is higher than 200mg/L, the acid-producing bacteria JC-C can be severely inhibited.
Acid producing ability of acid producing bacteria JC-C against heavy metal stress:
when heavy metal treatment is not carried out, the initial pH value of the culture solution of the acidogenic bacteria JC-C is 5.44, the pH value of the culture solution is reduced to the minimum value of 4.58 after the culture of the acidogenic bacteria JC-C is carried out for 15 hours, and the pH value of the strain culture solution is reduced by 0.86; under the stress of heavy metals, the pH value of the initial culture solution of the acidogenic bacteria JC-C is reduced and the change range of the pH is reduced along with the increase of the concentration of the heavy metals, and the pH of the culture solution tends to rise in the later period of fermentation. The pH value of the initial culture solution of the bacterial strain JC-C is reduced, the pH change amplitude is reduced, the time for reaching the lowest pH value is prolonged, and the pH of the culture solution tends to rise in the later period of fermentation. When the heavy metal Pb is stressed, the pH value of the culture solution is reduced by 0.23 after the strain is treated for 24 hours at 300 mg/LPb; when heavy metal Cd is stressed, the pH of the culture solution is reduced by 0.62 after 50mg/L Cd is treated on the strain for 15 hours; when the heavy metal Zn is stressed, the pH value of the culture solution is reduced by 1.1 after the strain is treated for 15h at 100mg/LZn, which shows that the Zn with low concentration 2+ The strain acid production is promoted, but the acid production capacity of the strain is reduced along with the increase of the heavy metal addition amount;
acid-producing bacteria JC-C still have acid-producing capacity under different heavy metal concentrations. The screening and identifying method of the acidogenic bacteria JC-C can provide strain resources for biological acidogenesis.
Drawings
FIG. 1 is a diagram of an acidogenic bacterium of the present invention;
FIG. 2 is a phylogenetic tree constructed by the acid-producing strain of the present invention based on the 18S rRNA gene sequence;
FIG. 3 is a graph showing the growth of acid-forming bacteria of the present invention;
FIG. 4 is a graph showing the pH change in the medium of the acid-forming bacteria of the present invention during various fermentation periods;
FIG. 5 shows the effect of heavy metals Zn, cd and Pb with different concentrations on the growth of acidogenic bacteria;
FIG. 6 is a graph showing the effect of different concentrations of heavy metal Pb on pH of acid forming bacteria in the present invention;
FIG. 7 is a graph showing the effect of heavy metal Cd of different concentrations on pH of acid forming bacteria in the present invention;
FIG. 8 shows the effect of different concentrations of heavy metal Zn on pH of acidogens in the present invention;
FIG. 9 is a graph showing the effect of different temperatures on pH of fermentation broth of acid-producing bacteria JC-C;
FIG. 10 is a graph showing the effect of different temperatures on NaOH consumption by acid forming bacteria JC-C fermentation broth;
FIG. 11 is a graph showing the effect of different initial pH on pH of fermentation broth of acid-producing bacteria JC-C;
FIG. 12 is a graph showing the effect of different initial pH values on NaOH consumption by an acid forming bacteria JC-C fermentation broth;
FIG. 13 is a graph showing the effect of different rotational speeds on pH of fermentation broth of acid-producing bacteria JC-C;
FIG. 14 is a graph showing the effect of different rotational speeds on NaOH consumption by acid forming bacteria JC-C fermentation broth.
Preservation information: the acidogenic bacteria are preserved in the China general microbiological culture Collection center (address: national institute of microbiology, national academy of sciences, postal code 100101) of China general microbiological culture Collection center (CGMCC NO:21423. the name is: penicillium chrysogenum (Penicillium chrysogenum) JC-C.
Detailed Description
The invention will be described in detail below with reference to the drawings and the detailed description.
An acidogenic bacterium JC-C, the strain was named: penicillium chrysogenum (Penicillium chrysogenum) JC-C is preserved in China general microbiological culture Collection center (CGMCC) in the year 2021, 01 and 04, and the preservation number is CGMCC NO:21423.
the culture method of the acid-producing bacteria JC-C comprises the following steps:
step 1, separating and culturing strains
Collecting soil severely polluted by heavy metals as a sample; the aim is to isolate therefrom microorganisms having acid-producing capacity.
Preparation of a search medium: sucrose 30g, naNO 3 3.0g、K 2 HPO 4 1.0g、KCl 0.5g，FeSO 4 ·7H 2 0.01g of O, 20.0g of agar and 1L of distilled water, and then pouring a flat plate until the O and the agar solidify to prepare a sterile separation culture medium for later use;
mixing the collected soil sample with sterile water to prepare a soil suspension, specifically: mixing the collected soil sample with sterile water to prepare soil suspension, carrying out gradient dilution on the soil suspension, coating the soil suspension on the surface of a culture medium, numbering, and culturing in a constant-temperature incubator at 30 ℃ for 3-5d. Different suction pipes are needed to be replaced for sucking the soil suspension with different gradients.
Taking 0.5mL of diluted soil suspension; uniformly coating soil suspension on the surface of a checking culture medium by using a sterile coating rod, numbering, and culturing in a constant temperature incubator at 30 ℃ for 3-5d;
step 2, identifying acid-producing strains
Preparing an acid production identification medium: adding 1.6% bromocresol purple into a check culture medium, sterilizing at 121 ℃ for 20 minutes, pouring a flat plate, and preparing a sterile acid-producing identification culture medium for later use after the bromocresol purple is solidified, wherein the color of the sterile acid-producing identification culture medium is purple;
taking out the strain cultured in the step 1, observing the strain form, and selecting out bacterial colonies with different forms to be streaked and cultured in an acid-producing identification medium;
after 3-5d of culture, the acid production identification culture medium turns from purple to yellow to indicate that the strain has acid production capability, if no change exists, the strain does not have acid production capability, and the strain with acid production capability is selected to be streaked into the acid production identification culture medium for multiple times to obtain a purified single strain; after obtaining single acidogenic strain, the strain is preserved in a refrigerator at 4 ℃ for subsequent experiments.
Strain morphology observation and physiological biochemistry
The bacterial colony is circular, the surface of the bacterial colony is provided with radial lines, mycelia with neat edges are white velvet, and the center of the mycelia is provided with a few pale yellow exudates.
As shown in FIG. 2, the phylogenetic tree of acid-producing bacteria JC-C constructed based on the 18S rRNA gene sequence, profile obtaining step:
systematic evolution analysis: the sequencing result was compared with the registered gene sequence on GenBank, and the highest homology with Penicillium chrysogenum (Penicillium chrysogenum) was found to be 100%. And (3) performing multi-sequence alignment analysis by using MEGA 5 software, and constructing a phylogenetic tree by using a Neighbor-joining method. The acid-producing bacteria JC-C is determined to be penicillium chrysogenum (Penicillium chrysogenum) by combining physiological and biochemical characteristics and molecular biological characteristics.
As shown in FIG. 3, the acid-producing bacteria JC-C has a short growth delay period in the liquid culture medium, the logarithmic growth phase is 1.5-3.5d, and the acid-producing bacteria JC-C is in a stationary phase after being cultured for 3.5 d. After 3.5d growth, the biomass of the strain reaches 5.2 mg.multidot.mL -1 。
Use of acid-producing bacteria JC-C in acid production, wherein the spore content of acid-producing bacteria JC-C is 3.6X10 6 Adding 50ml fermentation medium for constant temperature shake culture, and generating acid with highest efficiency at 30deg.C, pH 4 and rotation speed of 160 rpm.
When heavy metal treatment is not carried out, the initial pH value of the culture solution of the acidogenic bacteria JC-C is 5.44, the pH value of the culture solution is reduced to the minimum value of 4.58 after the culture of the acidogenic bacteria JC-C is carried out for 15 hours, and the pH value of the strain culture solution is reduced by 0.86; under the stress of heavy metals, the pH value of the initial culture solution of the acidogenic bacteria JC-C is reduced and the change range of the pH is reduced along with the increase of the concentration of the heavy metals, and the pH of the culture solution tends to rise in the later period of fermentation. The pH value of the initial culture solution of the bacterial strain JC-C is reduced, the pH change amplitude is reduced, the time for reaching the lowest pH value is prolonged, and the pH of the culture solution tends to rise in the later period of fermentation. When the heavy metal Pb is stressed, the pH value of the culture solution is reduced by 0.23 after the strain is treated for 24 hours at 300 mg/LPb; when heavy metal Cd is stressed, the pH of the culture solution is reduced by 0.62 after 50mg/L Cd is treated on the strain for 15 hours; when the heavy metal Zn is stressed, the pH value of the culture solution is reduced by 1.1 after the strain is treated for 15h at 100mg/LZn, which shows that the Zn with low concentration 2+ The strain acid production is promoted, but the acid production capacity of the strain is reduced along with the increase of the heavy metal addition amount;
an identification method of acid-producing bacteria JC-C, comprising the following steps: and (3) determining the strain identification and the content of organic acid in fermentation broth of a fermentation medium by utilizing an 18S rRNA gene sequence.
The strain identification by using the 18S rRNA gene sequence is specifically as follows:
PCR amplification of the 18S rRNA Gene sequence of the target Strain:
PCR amplification was performed by fungal universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATC-3'). Under the action of polymerase, the 18SrRNA gene sequence of the target strain is obtained through pre-denaturation, annealing, extension, 35 cycles and repair extension.
The determination of the organic acid content in the fermentation broth of the fermentation medium is specifically:
first, a 0.1mol/L NaOH standard solution and a 1% phenolphthalein indicator were prepared. Then accurately sucking 10.0mL of sample fermentation broth, transferring to a 100mL volumetric flask, adding distilled water to 100mL scale, and shaking uniformly. Filtering with filter paper, accurately sucking 20mL of filtrate, placing into a 100mL triangular flask, adding 2 drops of 1% phenolphthalein, titrating with 0.1mol/LNaOH standard solution until the color of the primary color does not fade within 0.5min as an end point, recording the dosage of sodium hydroxide, and repeating for three times to obtain an average value. And the NaOH consumption of the blank was determined.
Acid-producing bacteria JC-C tolerance to heavy metals Cd, pb and Zn:
by observing Cd in different concentrations of acid-producing bacteria JC-C 2+ 、Pb 2+ And Zn 2+ The colony diameter after 10d of culture on the plate was examined for tolerance to heavy metals of the two acidogenic fungi. With the increase of the concentration of heavy metal ions, the acid-producing bacteria JC-C is inhibited to different degrees, and Cd 2+ The bacteriostasis rate of the composition to the acidogenic bacteria JC-C reaches about 80 percent when the concentration is 200mg/L, and the composition remarkably inhibits the growth of fungi and Cd 2+ Completely inhibiting at concentration of 300-500 mg/L; in Pb 2+ The concentration of 200mg/L and 300mg/L has less effect of inhibiting the acid-producing bacteria JC-C, and the Pb is the 2+ The inhibition rate reaches more than 80% at the high concentration of 2000 mg/L; in Zn 2+ When the concentration is increased to 400mg/L, the antibacterial rate of the strain ZJ-I reaches 80%.
The pH value of the acid-producing bacteria JC-C in a fermentation medium without adding heavy metals is 5.44 initially, then the pH value is gradually reduced, and the pH value is increased after 15 hours. With the increase of the heavy metal concentration, the pH value of the initial culture solution of the bacterial strain JC-C is reduced, the pH change amplitude is reduced, the time for reaching the lowest pH value is prolonged, and the pH of the culture solution tends to rise in the later period of fermentation. When the heavy metal Pb is stressed, the pH value of the culture solution is reduced by 0.23 after the strain is treated for 24 hours at 300 mg/LPb; when heavy metal Cd is stressed, the pH of the culture solution is reduced by 0.62 after 50mg/L Cd is treated on the strain for 15 hours; when the heavy metal Zn is stressed, the pH value of the culture solution is reduced by 1.1 after the strain is treated for 15 hours at 100 mg/LZn.
Illustrating the heavy metal resistant effect Pb of the acid-producing bacteria JC-C 2+ >Zn 2+ >Cd 2+ . Acid-producing bacteria JC-C has acid-producing capacity when resisting different heavy metals.
And analyzing the influence conditions of different temperatures, the pH value of the initial fermentation liquid and the rotating speed on the fermentation acid production of the strain by adopting a single-factor experimental method.
The fermentation medium adopts 100.0g of sucrose and NaNO 3 1.5 g，KH 2 PO 4 0.5 g，MgSO 4 ·7H 2 0.025g of O, 0.025g of KCl, 1.6g of yeast extract, 1L of distilled water, adjusting the pH to 5, and sterilizing at 121 ℃ for 20min; according to 3.6X10 6 Adding the mixture into a fermentation medium according to the proportion of 50ml, and culturing at 30 ℃ in a constant-temperature shaking incubator at 160 rpm;
1. temperature (temperature)
The temperature gradient was set at 25 ℃, 28 ℃, 30 ℃, 32 ℃ and 35 ℃, and the pH change in the fermentation broth and the consumption of NaOH in the fermentation broth at different temperatures were measured to determine the optimal acid production temperature conditions.
As shown in FIGS. 9 to 10, the pH of the fermentation broth was most lowered and the amount of NaOH consumed was the greatest when the culture temperature was 30℃and therefore the acid-producing bacteria JC-C had the strongest acid-producing ability when the culture temperature reached 30 ℃.
2.pH
The initial fermentation liquor pH is set to be 4, 5, 6, 7 and 8, and the pH change in the fermentation liquor and the consumption of the fermentation liquor NaOH under different initial fermentation liquor pH are respectively measured to determine the optimal pH condition of the initial fermentation liquor for acid production.
As shown in FIGS. 11-12, the pH of the fermentation broth was most lowered when the pH of the initial fermentation broth was 4, and the amount of NaOH consumed was the greatest, so that the acid-producing bacteria JC-C had the strongest acid-producing ability when the pH of the initial fermentation broth was 4.
3. Rotational speed
The rotation speed gradient was set to 100rpm, 120rpm, 140rpm, 160rpm and 180rpm, and the pH change in the fermentation broth and the consumption of NaOH in the fermentation broth at different rotation speeds were measured to determine the optimal rotation speed conditions for acid production.
As shown in FIGS. 13 to 14, the pH of the fermentation broth was most lowered and the amount of NaOH consumed was the greatest when the rotation speed of the culture was 160rpm, so that the acid-producing bacteria JC-C had the greatest acid-producing ability when the rotation speed of the initial fermentation broth was 160 rpm.
Sequence listing
<110> Western Annulus theory school
<120> an acidogenic bacterium JC-C, use thereof and method for culturing and identifying acidogenic bacterium JC-C
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agaacgcagc gaaatgcgat aactaatgtg aattgcagaa ttcagtgaat catcgagtct 300
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ggtagggata cccgctgaac ttaagcatat c 571
Claims (3)
1. An acidogenic bacterium JC-C, characterized in that the strain is named: penicillium chrysogenum JC-C is preserved in China general microbiological culture Collection center (CGMCC) in the year 2021, 01 and 04, with the preservation number of CGMCC NO:21423.
2. use of an acidogenic bacterium JC-C according to claim 1 in acidogenesis.
3. The use according to claim 2, wherein the acid production efficiency of acid-producing bacteria JC-C is determined using a fermentation medium using sucrose 100.0g, nano 3 1.5g，KH 2 PO 4 0.5g，KCl0.025g，MgSO 4 ·7H 2 0.025g of O, 1.6g of yeast extract, adjusting the pH to 4, and sterilizing at 121 ℃ for 20min;
acid-producing bacteria JC-C bacterial liquid is mixed according to 3.6X10 6 The fermentation medium was added in a ratio of 50ml, and the culture was carried out in a constant temperature shaking incubator at a temperature of 30℃and a rotation speed of 160 rpm.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103627642A (en) * | 2013-12-16 | 2014-03-12 | 华中农业大学 | Penicillium chrysogenum CH03 strain and application thereof in restoring heavy metal pollution in soil |
| CN104862233A (en) * | 2014-02-25 | 2015-08-26 | 上海医药工业研究院 | Penicillium chrysogenum and application thereof |
| CN108165498A (en) * | 2018-01-23 | 2018-06-15 | 浙江师范大学 | The Penicillium griseofulvum Pg-35 bacterial strains and its ferment filtrate of antagonism rice leaf spot bacteria and the application in the anti-smelting of plant disease |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103627642A (en) * | 2013-12-16 | 2014-03-12 | 华中农业大学 | Penicillium chrysogenum CH03 strain and application thereof in restoring heavy metal pollution in soil |
| CN104862233A (en) * | 2014-02-25 | 2015-08-26 | 上海医药工业研究院 | Penicillium chrysogenum and application thereof |
| CN108165498A (en) * | 2018-01-23 | 2018-06-15 | 浙江师范大学 | The Penicillium griseofulvum Pg-35 bacterial strains and its ferment filtrate of antagonism rice leaf spot bacteria and the application in the anti-smelting of plant disease |
Non-Patent Citations (1)
| Title |
|---|
| 王雪莹等.邓新辉 ; 彭扶风 ; 陈韵声 ; 何权运 ; 孔乐群 ; 李贝贝 ; 张霞 ; 具重金属抗性产酸菌的分离及生物学特性研究.微生物学通报.2020,第47卷(第10期),3226-3236. * |
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