CN107502568B - Bacillus pumilus and application thereof in cadmium pollution remediation - Google Patents
Bacillus pumilus and application thereof in cadmium pollution remediation Download PDFInfo
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- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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Abstract
The invention belongs to the technical field of microorganism application, and particularly relates to bacillus pumilus and application thereof in cadmium pollution remediation. The Bacillus pumilus is classified and named as Bacillus pumilus SD1-1, has a preservation number of CGMCC No.13628, has a preservation date of 2017, 01-19 months and a preservation unit of China general microbiological culture Collection center. Cadmium content of rice produced by cadmium-containing soil of the bacillus pumilus strain SD1-1 and cadmium-containing soil without any microorganism is detected. The result shows that the cadmium content of the cadmium-containing soil added with the strain SD1-1 is reduced by 13.8 percent compared with the cadmium-containing soil without microorganism.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to bacillus pumilus and application thereof in cadmium pollution remediation.
Background
At present, cadmium pollution treatment methods are classified into three categories: physical/chemical remediation techniques, bioremediation techniques, and agroecological remediation techniques, each have advantages and limitations. The microbial remediation technology is a relatively advantageous treatment method, and certain microorganisms (bacillus subtilis, photosynthetic bacteria, lactic acid bacteria, mold and the like) in the soil have the effects of adsorbing, precipitating, oxidizing, reducing, chelating, biomethylating, accumulating in cells, volatilizing and the like on heavy metals, so that the activity and toxicity of cadmium in the polluted soil can be reduced by culturing engineering bacteria and putting the microorganisms. For example, some strains separated from roots of elsholtzia, ciliate desert-grass and sedum alfredii hance can passivate and fix Cd in soil and reduce exchangeable state content of Cd in soil. The method has the main advantages of economy, small engineering quantity, simple operation, less investment, low cost, environmental protection, no influence on agricultural production, large-area popularization and application and the like.
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 skilled in the art.
Disclosure of Invention
Aiming at the condition that the paddy field is polluted by heavy metal cadmium, which widely exists at present, the invention provides the bacillus pumilus which can reduce the effective cadmium in the paddy field, is derived from organic fertilizer compost materials, and shows strong absorption capacity to the cadmium element in a liquid culture medium containing the cadmium element.
The technical scheme provided by the invention is as follows:
the Bacillus pumilus is classified and named as Bacillus pumilus SD1-1, has a preservation number of CGMCC No.13628, and is preserved in 2017, 01-19 months and the preservation unit is China general microbiological culture Collection center.
The strain has the following properties: the colony is white and flat, the diameter of the colony on an LB solid plate is 4mm-5 mm, and the thallus is straight or slightly bent rod-shaped bacteria and gram-positive bacteria.
The screening method of the cadmium-absorbing microorganism comprises the following steps:
(1) collecting samples: the samples are from a Dodele fertilizer factory (mushroom residue is used as a main raw material) and fertile soil samples;
(2) strain separation: preparing a separation solid culture medium, wherein each liter of the separation solid culture medium contains 10g of peptone, 5g of yeast extract, 10g of NCl and 15g of agar; pH 7.0; sterilizing at 121 deg.C for 20 min; cooling to 55-60 deg.C, and pouring; diluting the collected sample, coating the sample on a flat plate, culturing the sample at 37 ℃ for 1 to 3 days, selecting a single colony according to the growth condition of the colony, and transferring the single colony to a new solid separation culture medium;
(3) strain screening: preparing a screening liquid culture medium, wherein each liter of the screening liquid culture medium comprises: 10.0g of glucose, 8.0g of yeast extract, 8.0g of peptone, 3.0g of ammonium sulfate, 3.0g of monopotassium phosphate and 3.0mg of cadmium, and the volume of the glucose, the yeast extract, the peptone and the cadmium are complemented by distilled water; screening the culture medium with pH of 7.0, subpackaging 10 bottles with each bottle being 100mL, and sterilizing at 115 ℃ for 20 min; inoculating the single colony obtained by separating in the step 2 into a liquid screening culture medium, and culturing the bacteria at 37 ℃ and 180rpm for 1 day; centrifuging at 12000rpm for 5min to remove thallus, measuring the residual content of cadmium in the supernatant, and selecting the strain with the maximum reduction amplitude of the cadmium content for the next research; culturing fungus at 180rpm for 3 days, centrifuging at 12000rpm for 20min to remove thallus, measuring residual cadmium content in supernatant, and selecting strain with maximum cadmium content reduction for further research.
The preparation method of the cadmium absorption strain SD1-1 solid powder comprises the following steps:
(1) preparing LB liquid culture medium, wherein each liter contains: 10.0g peptone, 5.0g yeast extract, 10.0g sodium chloride, pH 7.0; sterilizing at 121 deg.C for 20 min;
(2) preparing a seed solution: taking out the strain SD1-1 stored in a refrigerator at 4 ℃, selecting a bacterial colony to be inoculated in a prepared LB culture medium, and culturing at 37 ℃ and 180rpm for 16h for later use;
(3) preparing a solid fermentation medium, wherein each kilogram of the solid fermentation medium comprises: 400g of wheat bran, 100g of soybean meal and 500g of water. Mixing, wrapping with clean gauze, and sterilizing at 121 deg.C for 40 min. Cooling to 50-55 deg.C;
(4) inoculation: inoculating the seed solution prepared in the step 2 into the solid culture medium prepared in the step 3, uniformly stirring, and culturing for 2 days at 37 ℃;
(5) drying and crushing: drying the cultured strains in the step 4 in an oven at 45 ℃ or drying the strains in the sun until the water content is less than 20%; pulverizing with pulverizer for use.
The application method of the cadmium-treated microorganism bacillus pumilus SD1-1 solid powder comprises the following steps: preparing soil for cultivating paddy rice, taking a small pot (length, width, height, 30cm, 15cm and 10cm), and filling the soil for cultivating paddy rice; the small pots are numbered as 1,2 and 3; wherein, the No.1 pot is filled with original soil, the No. 2 pot is added with the cadmium standard solution in the original soil until the theoretical cadmium content in the soil is 0.5mg/kg, the No. 3 pot is added with the cadmium standard solution in the original soil until the theoretical cadmium content in the soil is 0.5mg/kg and 10g of powdery bacillus pumilus capable of absorbing cadmium elements; after uniform mixing, planting the cultivated rice seedlings, and adding 10g of bacillus pumilus SD1-1 solid powder into a No. 3 pot when the rice grows to the first tillering stage.
Compared with the prior art, the invention has the following beneficial effects:
(1) cadmium content of rice produced by cadmium-containing soil of the bacillus pumilus SD1-1 and cadmium-containing soil without any microorganism is detected. The detection method is used for measuring lead and cadmium in the rice according to the national standard NY/T1100-2006 about graphite furnace atomic absorption spectrometry. The detection result shows that the cadmium content of the cadmium-containing soil added with the strain SD1-1 is reduced by 13.8 percent compared with the cadmium-containing soil without the added microorganism to produce rice.
(2) Experimental results show that the Bacillus pumilus SD1-1, SD1-2, SD1-3, SD1-5, SD2-1, SD2-3 and SD2-4 separated at this time have the capacity of absorbing cadmium elements in a culture solution under liquid culture conditions. Observed from colony morphology, except that SD1-5 is filamentous fungi, the other strains are bacillus bacteria. Wherein the absorption efficiency of SD1-5 to the culture solution cadmium with cadmium content of 1.5mg/L is 65% when cultured at 37 ℃ and 180rpm for 3 days; the absorption efficiencies of SD1-1, SD1-2, SD1-3, SD2-1, SD2-3 and SD2-4 to cadmium element with the concentration of 1.5mg/L are respectively 100%, 77%, 78.3%, 79%, 88.7% and 80.9% when the cells are cultured for 1 day at 37 ℃ and 180 rpm.
Drawings
FIG. 1 shows the colony morphology of Bacillus pumilus SD1-1 on LB plates;
FIG. 2 is a microscopic form of a cell of Bacillus pumilus SD 1-1;
FIG. 3 shows the sequence alignment of 16S rRNA of Bacillus pumilus SD1-1 and 16S rRNA of Bacillus pumilus 2-1 strain;
FIG. 4 is a real photographed image of Bacillus pumilus SD1-1 after solid fermentation, drying and pulverization.
Description of preservation information
Bacillus pumilus SD1-1 with the preservation number of CGMCC No.13628, the preservation date of 2017, 01-19 months, the preservation unit of China general microbial strain preservation management center, the preservation address of Beijing Kogyo No. 3 of Xilu No.1 of Chaoyang district, China academy of sciences biology institute.
Detailed Description
The following examples are given to better understand the present invention and are not intended to limit the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following examples were performed in triplicate and the results averaged.
The screening culture medium formula comprises: 10g of glucose, 8g of yeast extract, 8g of peptone and KH2PO43g、 (NH4)2SO43g, 1.5mL of Cd standard solution (1000. mu.g/mL); dissolved with 800mL of deionized water, then adjusted to pH 6.0 (adjusted with 1M HCl and 1M NaOH); using deionized water to fix the volume to 1L; sterilizing at 115 deg.C for 30 min.
And (3) manufacturing a cadmium standard curve: the cadmium standard curve is made according to the national standard GB7475-87 of the people's republic of China about the atomic absorption spectrometry of copper, zinc, lead and cadmium in water. The method specifically comprises the following steps: when preparing cadmium standard solutions with different concentrations, a screening culture medium (without cadmium) diluted by 5 times by distilled water is used as a solvent. 10mL of each of 0.05mg/L, 0.1mg/L, 0.3mg/L, 0.5mg/L and 1mg/L of cadmium standard solution was prepared. The prepared standard solution is measured by a flame atomic spectroscopy extensometer. Obtaining a standard curve of the light absorption value and the cadmium concentration, the function of the standard curveThe formula is that y is 0.3431x +0.0084, R20.9993. Where y is the light absorption value and x is the cadmium concentration (mg/L).
Example 1: screening and identification of cadmium-absorbing strains
First, obtaining of the Strain
1. Collecting strains: 100g of organic fertilizer raw material piles (mushroom residues) of a fertilizer factory were sampled for 9 months in 2015 and 100g of fertile soil samples of the fertilizer factory were sampled for Shuangdingzhendongdele fertilizer factory in Guangxi Nanning, China.
2. Separating and screening of cadmium absorbing strain
(1) Preparing a separation culture medium by using deionized water, wherein each liter of the separation culture medium contains 10g of peptone, 5g of yeast extract, 10g of NCl and 15g of agar; pH7.0; sterilizing at 121 deg.C for 20 min; cooling to 55-60 deg.C, and flattening.
(2) 10g of the sample was placed in a 250mL triangular flask, 90mL of sterile water was added and an appropriate amount of glass beads was added, and stirred on a magnetic stirrer for 30 min. The sample is mixed well with water to disperse the cells. And adding 1mL of the soil suspension into a finger-shaped bottle containing 9mL of sterile water, fully and uniformly mixing, taking out 1mL of the soil suspension, and adding into the finger-shaped bottle containing 9mL of sterile water, fully and uniformly mixing. By analogy, 10 is made-1、10-2、 10-3、10-4、10-5And the sample solutions of different dilutions were made. Get 10-3、10-4、10-5Each 100. mu.L of the 3 dilutions was plated on a separate medium and incubated at 37 ℃.
(3) And after 24h, observing the growth condition of bacterial colonies, picking single bacterial colonies, transferring the single bacterial colonies to a new screening culture medium, and streaking, separating and purifying.
(4) Preparing a screening culture medium (liquid) with the pH of 7.0, wherein the culture medium comprises the following components: 10g of glucose, 8g of yeast extract, 8g of peptone and KH2PO43g、(NH4)2SO43g, 1.5mL of Cd standard solution (1000. mu.g/mL); dissolved with 800mL of deionized water, then adjusted to pH7.0 (adjusted with 1M HCl and 1M NaOH); using deionized water to fix the volume to 1L; sterilizing at 115 deg.C for 30 min.
(5) Inoculating the bacteria obtained in the step (3) into a liquid culture medium. Culturing at 37 deg.C and 180rpm for 1 day and three days.
(6) Centrifuging the bacterial culture solution at 12000rpm for 5min, collecting supernatant, diluting with distilled water by 5 times, and determining the content of residual cadmium. Centrifuging the fungus culture solution at 12000rpm for 15min, collecting supernatant, diluting with distilled water by 5 times, and determining the content of residual cadmium. And (4) screening out the bacterial strain with the capacity of absorbing cadmium by measuring the content of the residual cadmium.
(7) Experimental results show that the SD1-1, SD1-2, SD1-3, SD1-5, SD2-1, SD2-3 and SD2-4 separated at this time have the capacity of absorbing cadmium elements in a culture solution under the liquid culture condition. Observed from colony morphology, except that SD1-5 is filamentous fungi, the other strains are bacillus bacteria. Wherein the absorption efficiency of SD1-5 to the culture solution cadmium with cadmium content of 1.5mg/L is 65% when cultured at 37 ℃ and 180rpm for 3 days; the absorption efficiencies of SD1-1, SD1-2, SD1-3, SD2-1, SD2-3 and SD2-4 to cadmium element with the concentration of 1.5mg/L are respectively 100%, 77%, 78.3%, 79%, 88.7% and 80.9% when the cells are cultured for 1 day at 37 ℃ and 180 rpm. The invention only takes the SD1-1 strain with the highest absorption efficiency as an object to research the species, the solid preparation method and the specific application on the rice polluted by cadmium.
Solid culture of SD1-1 strain
1. Preparing LB liquid culture medium, wherein each liter contains: 10.0g peptone, 5.0g yeast extract, 10.0g sodium chloride, pH 7.0; sterilizing at 121 deg.C for 20 min.
2. Preparing a seed solution: the strain SD1-1 preserved in a refrigerator at 4 ℃ is taken out, and a colony is selected and inoculated in a prepared LB culture medium and cultured for 16h at 37 ℃ and 180rpm for later use.
3. Preparing a solid fermentation medium, wherein each kilogram of the solid fermentation medium comprises: 400g of wheat bran, 100g of soybean meal and 500g of water. Mixing, wrapping with clean gauze, and sterilizing at 121 deg.C for 40 min. Cooling to 50-55 deg.C.
4. Inoculation: inoculating the seed solution prepared in the step 2 into the solid culture medium prepared in the step 3, uniformly stirring, and culturing for 2 days at 37 ℃.
5. Drying and crushing: drying the cultured strains in the step 4 in an oven at 45 ℃ or sun-drying until the water content is less than 20%; pulverizing with pulverizer for use.
Identification of the strains
As shown in FIG. 1, the morphology of the strain SD1-1 on LB medium showed white colonies after 24h of culture at 37 ℃, the surface of the colonies was smooth and opaque, and the edges of the colonies were irregular. The colony radius is about 2 mm.
As shown in FIG. 2, in the microscopic form of the strain SD1-1, it was observed that the single bacterial cells of SD1-1 were rod-like and single-grown under a 40-fold microscope.
The molecular identification result of the strain SD1-1 is shown in figure 4, the 16S rRNA is obtained by amplification of primers K1:5-aactgaagagtttgatcctggctc-3 and K2:5-tacggt taccttgttacgactt-3 by taking SD1-1 bacterial liquid cultured for 24h at 37 ℃ and 180rpm as a template, a 1382bp nucleotide sequence is obtained by sequencing, the sequence obtained by sequencing is shown as SEQ ID NO.1, and homology comparison analysis shows that the sequence has 100 percent of homology with the Bacillus pumilus CLB-416S rRNA sequence.
Application of strain SD1-1
1. Preparing cultivated rice soil: the small pots (length, width, height, 30cm, 15cm, 10cm) were filled with soil for growing rice. The small pots are numbered 1,2 and 3. Wherein, the No.1 pot is filled with original soil, and the No. 2 and No. 3 pots are added with cadmium standard solution into the original soil until the theoretical cadmium content in the soil is 0.5 mg/kg.
2. Adding strains: 10g of the prepared SD1-1 strain (powder) is added into a No. 3 basin and stirred uniformly.
3. Planting rice: transplanting the pre-cultivated rice seedlings into No.1, 2 and 3 pots, and watering regularly.
4. Adding the strains for 2 times: after the rice seedlings grow to the first tillering stage, adding 10g of SD1-1 bacterial powder to the No. 3 pot to the roots of the rice.
Fifth, cadmium-containing rice sample treatment
And after the rice is ripe, collecting the rice, and respectively shelling the rice collected in the No.1 basin, the No. 2 basin and the No. 3 basin. The preparation of the cadmium-containing rice sample is carried out according to a method of national standard NY/T1100-2006 graphite furnace atomic absorption spectrometry for measuring lead and cadmium in rice, and the specific operation is as follows:
1. preparing 0.5mol/L nitric acid solution: and (6) sucking. 3.2mL of nitric acid (ρ 1.42g/mL) was added to 50mL of water and diluted to 100 mL.
2. Preparing 100mg/L cadmium standard stock solution: accurately weighing 0.1000g of spectral metal cadmium in a 50mL beaker, adding 20mL of dilute nitric acid (1+5), slightly heating for dissolution, cooling, transferring to 1000mL, adding water to a constant volume until the volume reaches a marked line, and shaking up.
3. Preparing 500 mu g/L cadmium standard solution: and (3) diluting the cadmium standard stock solution to a target concentration step by using the nitric acid solution obtained in the step (1) before use.
4. Preparing a mixed acid solution: nitric acid (ρ 1.42 g/mL): perchloric acid (rho 1.68g/mL) 4:1
5. Sample digestion: the rice collected in No.1, 2,3 pots was weighed 30g each. After grinding until all the particles pass through a nylon sieve with the aperture of 0.25mm, 1g of the sample (accurate to 0.001g) is weighed into a 100mL conical flask, 10mL of mixed acid and a plurality of glass beads are added, and the mixture is shaken up and placed overnight. Adding a small funnel, slowly heating on an electric heating plate, cooling slightly if it turns brown-black, adding small amount of nitric acid, and heating until white smoke appears, and the digestive juice is colorless and transparent or slightly yellow. Taking down and cooling, quantitatively transferring by using water, fixing the volume to 25mL, and mixing for testing. And simultaneously performing a blank test.
Sixthly, measuring the cadmium content in the rice
1. Drawing a standard curve: accurately measuring 0.00mL, 5.0mL, 10.0mL, 20.0mL, 30.0mL and 40.0mL of cadmium standard use solution in 6 volumetric flasks with 50mL, and metering the volume to the scale by using a nitric acid solution. The cadmium concentration is 0.0. mu.g/L, 50.0. mu.g/L, 100.0. mu.g/L, 200.0. mu.g/L, 300.0. mu.g/L, 400.0. mu.g/L. And sequentially measuring the cadmium standard sample by using a flame atomic spectrophotometer. The standard curve of cadmium measured by the test is 0.3543x +0.0118, R2＝0.9985。
2. And (3) sequentially measuring the prepared rice sample by using a flame atomic spectrophotometer, wherein the measurement result shows that: the cadmium concentration of the No.1 basin rice is 63.5 mug/L, the cadmium concentration of the No. 2 basin rice is 176.5 mug/L, and the cadmium concentration of the No. 3 basin rice is 204.75/L. The cadmium content of the No. 2 basin is 13.8 percent less than that of the No. 3 basin.
The foregoing description of specific exemplary embodiments of the invention has been presented for the purposes of illustration and descriptionThe purpose is. 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 certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and 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.
SEQUENCE LISTING
<110> Guangxi Doudle Biotechnology Ltd
<120> Bacillus pumilus and application thereof in cadmium pollution remediation
<130>ZYWS
<160>1
<170>PatentIn version 3.3
<210>1
<211>1382
<212>DNA
<213>Bacillus pumilus
<400>1
gaagggagct tgctcccgga tgttagcggc ggacgggtga gtaacacgtg ggtaacctgc 60
ctgtaagact gggataactc cgggaaaccg gagctaatac cggatagttc cttgaaccgc 120
atggttcaag gatgaaagac ggtttcggct gtcacttaca gatggacccg cggcgcatta 180
gctagttggt ggggtaatgg ctcaccaagg cgacgatgcg tagccgacct gagagggtga 240
tcggccacac tgggactgag acacggccca gactcctacg ggaggcagca gtagggaatc 300
ttccgcaatg gacgaaagtc tgacggagca acgccgcgtg agtgatgaag gttttcggat 360
cgtaaagctc tgttgttagg gaagaacaag tgcgagagta actgctcgca ccttgacggt 420
acctaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca 480
agcgttgtcc ggaattattg ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg 540
aaagcccccg gctcaaccgg ggagggtcat tggaaactgg gaaacttgag tgcagaagag 600
gagagtggaa ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc 660
gaaggcgact ctctggtctg taactgacgc tgaggagcga aagcgtgggg agcgaacagg 720
attagatacc ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag ggggtttccg 780
ccccttagtg ctgcagctaa cgcattaagc actccgcctg gggagtacgg tcgcaagact 840
gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa 900
gcaacgcgaa gaaccttacc aggtcttgac atcctctgac aaccctagag atagggcttt 960
cccttcgggg acagagtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt 1020
tgggttaagt cccgcaacga gcgcaaccct tgatcttagt tgccagcatt cagttgggca 1080
ctctaaggtg actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg 1140
ccccttatga cctgggctac acacgtgcta caatggacag aacaaagggc tgcaagaccg 1200
caaggtttag ccaatcccat aaatctgttc tcagttcgga tcgcagtctg caactcgact 1260
gcgtgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg 1320
gccttgtaca caccgcccgt cacaccacga gagttgcaac acccgaagtc ggtgaggtaa 1380
Claims (2)
1. The Bacillus pumilus is characterized by being named as Bacillus pumilus SD1-1 in a classification manner, the preservation number is CGMCC No.13628, the preservation date is 2017, 01-19 months and the preservation unit is China general microbiological culture preservation management center;
the culture method of the solid powder of the SD1-1 strain comprises the following steps:
(1) preparing LB liquid culture medium, wherein each liter contains: 10.0g peptone, 5.0g yeast extract, 10.0g sodium chloride, pH 7.0; sterilizing at 121 deg.C for 20 min;
(2) preparing a seed solution: taking out the strain SD1-1 stored in a refrigerator at 4 ℃, selecting a bacterial colony to be inoculated in a prepared LB culture medium, and culturing at 37 ℃ and 180rpm for 16h for later use;
(3) preparing a solid fermentation medium, wherein each kilogram of the solid fermentation medium comprises: 400g of wheat bran, 100g of soybean meal and 500g of water; mixing, wrapping with clean gauze, and sterilizing at 121 deg.C for 40 min; cooling to 50-55 deg.C;
(4) inoculation: inoculating the seed liquid prepared in the step (2) into the solid fermentation culture medium prepared in the step (3), uniformly stirring, and culturing for 2 days at 37 ℃;
(5) drying and crushing: drying the cultured strains in the step (4) in an oven at 45 ℃ or sun-drying until the water content is less than 20%; pulverizing with pulverizer for use.
2. The use of Bacillus pumilus of claim 1 for cadmium contamination remediation.
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| Title |
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| Assessment and characterization of heavy metal resistance in Palk Bay sediment bacteria;Chari Nithya et al.;《Marine environmental research》;20110213;第71卷(第4期);摘要 * |
| GFP标记短小芽孢杆菌LYMC-3在马尾松体内的定殖;李亮亮等;《华中农业大学学报》;20161130;第35卷(第6期);第68-73页 * |
| Isolation of heterotrophic bacteria from palk bay sediments showing heavy metal tolerance and antibiotic production;Chari Nithya et al.;《Microbiological Research》;20091216;第165卷(第7期);第578-593页 * |
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