CN103923839A - Identification identification and application of heavy metal tolerant penicillium in coal gangue - Google Patents
Identification identification and application of heavy metal tolerant penicillium in coal gangue Download PDFInfo
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- CN103923839A CN103923839A CN201310015361.9A CN201310015361A CN103923839A CN 103923839 A CN103923839 A CN 103923839A CN 201310015361 A CN201310015361 A CN 201310015361A CN 103923839 A CN103923839 A CN 103923839A
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- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 53
- 239000003245 coal Substances 0.000 title claims abstract description 29
- 241000228143 Penicillium Species 0.000 title abstract description 6
- 229910052793 cadmium Inorganic materials 0.000 claims abstract description 12
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000002689 soil Substances 0.000 claims abstract description 8
- 241000228168 Penicillium sp. Species 0.000 claims abstract description 4
- 230000001580 bacterial effect Effects 0.000 claims description 29
- 241000228150 Penicillium chrysogenum Species 0.000 claims description 12
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- 238000000034 method Methods 0.000 description 9
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a heavy metal cadmium tolerant penicillium BJKD4 (Penicillium sp.) screened from coal gangue in a mining area. The strain is preserved in China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC NO.6620. The Penicillium BJKD4 strain provided by the invention has strong tolerance to cadmium salts and compound pollution of heavy metals, and plays an important role in reclamation of coal gangue mountains and ecological restoration of soil heavy metal pollution.
Description
Technical field:
The present invention relates to administer the bioremediation technology field of heavy metal contamination, be specifically related to a kind of separation and application thereof of the Penicillium notatum of resistance to heavy metal cadmium.
Background technology:
High sulfur coal gangue can produce highly acid leaching liquor through illuvial clayization, cause heavy metal to be wherein leached out, these acid waste waters containing heavy metal are along Gangue runoff, to Soil Surrounding and water body migration, cause waste dump and Soil Surrounding and water body to be subject to severe acidification and heavy metal contamination, as cadmium, copper, lead, zinc etc.Some metal is the indispensable element of organism vital movement, but excessive heavy metal all exists potential toxic effect, and Berny P etc. has reported a series of serious harms that heavy metal causes the mankind, animal, plant and microorganism.Be widely used in the method such as physics or chemistry that heavy metal contamination is repaired, there are many shortcomings, somewhat expensive, in treating processes, cause secondary pollution to surrounding enviroment again to reagent adding in soil etc., the research such as Wang and chen shows that these methods all can not effectively reduce heavy metal toxicity when heavy metal contaminants content is during lower than 100mg/L in addition.And biological restoration is considered to a kind of clean, pollution-free and method that efficiency is high, be more and more subject to vast focus of attention, wherein microorganism to repair be main recovery technique.
If for the indigenous fungus and bacterium of microbial host of repairing heavy metal pollution, different microorganisms kind is also different to the patience of heavy metal contamination, be generally fungus G reatT.GreaT.GT bacterium > actinomycetes, again because fungi distributes very wide at occurring in nature, strong to adaptive capacity to environment, and many have heavy metal resistance and an accumulation ability, obtains extensive concern in biologic treating technique.Microorganism in contaminated soil often has higher heavy metal resistance, has adapted to local physical environment from the indigenous microorganism of zone of pollution separation screening, so effective repairing heavy metal pollution.Du Aixue etc. isolate the anti-Cu of a plant height from copper mining tailing soil
2+, Zn
2+and Cd
2+deng Penicillium notatum.The pseudomonas putida (Pseudomonas putida strain S4) that filters out high resistance copper in copper mine smelting waste water can effectively be removed the Cu in water body providing under nutritive medium condition
2+and Zn
2+.White-rot fungi has stronger removal effect to lead, and its maximal absorptive capacity can reach 108.4mg/g, and adsorption rate can reach 95%.From cadmium pollution soil, filter out 2 saccharomycetes, can reach respectively 79.85% and 89.04% to Cd2+ adsorption rate.Although multiple resistance to heavy metal fungi is separated, but because high sulfur coal gangue produces the strongly acid wastewater containing higher concentration heavy metal through illuvial clayization, cause most microorganisms to multiply, in addition, have no report for microorganism and the remediation microbial inoculum of administering coal gangue pollution.Therefore, in acid waste dump, screen resistance to heavy metal fungi, study patience and the enrichment mechanism of its heavy metal, the reparation tool of coal gangue hill heavy metal contamination is of great significance.
Summary of the invention:
The object of this invention is to provide a kind of bacterial classification that can tolerate heavy metal cadmium---Penicillium notatum BJKD4 (Penicillium sp.).
Bacterial strain provided by the present invention is Penicillium notatum BJKD4 bacterial strain, this bacterial strain was preserved in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 09 25th, 2012, Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number CGMCC NO.6620.
The Penicillium notatum of resistance to heavy metal cadmium of the present invention has following beneficial effect: the ability of tolerance heavy metal cadmium is strong, can tolerate 3.2g/L concentration of cadmium ions, and various heavy salt is had to stronger patience, in the heavy metal contamination that biological restoration is caused by acid coal gangue leaching, play an important role.
Brief description of the drawings:
Fig. 1: the colonial morphology of BJKD4 bacterial strain in solid Cha Shi substratum
Fig. 2: the conidiophore of BJKD4 bacterial strain under microscope
Fig. 3: the phyletic evolution of BJKD4 bacterial strain is grown parsing tree
Fig. 4: different concns CdCl
2coerce the impact on BJKD4 strain growth
Fig. 5: the impact of different heavy metals on BJKD4 strain growth
Fig. 6: the impact of different concns coal gangue leach liquor on BJKD4 growth
Embodiment
Describe the present invention in detail by specific embodiment below
Embodiment 1: the separation of Penicillium notatum of the present invention.
Get at random 12 sampling points from Huainan Xie Jiaji coal field and mix composition representative samples, carry out enrichment screening.5g spoil sample is joined in 45mL Ma Dingshi liquid nutrient medium, and 28 DEG C, 180r/min enrichment culture 48h, then, get 2mL enrichment bacterium liquid and be inoculated in the Cd containing 100mg/L
2+fresh culture in cultivate, after muddiness, get 1mL enrichment culture liquid and be inoculated in the Cd containing 200mg/L
2+liquid nutrient medium in cultivate, the rest may be inferred, improves step by step Cd
2+concentration, at 300-5000mg/L Cd
2+substratum in tame cultivation.Get 0.2mL1000mg/L Cd
2+bacterium liquid be coated on 5000mg/L Cd
2+solid medium on, be inverted in 28 DEG C of constant incubators and cultivate 120h, separate the bacterial strain of resistance to Cd.Be mould according to colonial morphology preliminary observation BJKD4 mono-clonal bacterial strain, be inoculated into 500～3500mg/L Cd
2+in Cha Shi substratum, domestication is cultivated, and carries out strain identification and the characteristic research of resistance to heavy metal.
Ma Dingshi substratum: glucose 10g, potassium primary phosphate 1g, peptone 5g, magnesium sulfate 0.5g, 1/3000 rose-bengal 100mL, adding distil water 800mL, pH nature.121 DEG C of sterilizing 30min, add 0.03% Streptomycin sulphate diluent 100mL before use, make in every milliliter of substratum containing Streptomycin sulphate 30 μ g.Solid medium is in this substratum, to add 15～20g agar.
Cha Shi substratum: SODIUMNITRATE 2g, dipotassium hydrogen phosphate 1g, Repone K 0.5g, magnesium sulfate 0.5g, ferrous sulfate 0.01g, sucrose 30g, agar 18-20g, deionized water 1000mL, pH nature.Heavy metal screening culture medium is the heavy metallic salt that adds different concns in this substratum.
Colony morphology characteristic: BJKD4 bacterial strain is cultivated (Fig. 1) after 14d in solid Cha Shi substratum, bacterium colony is felted, there is transudate on surface, well-regulated radial pleat splits, there is boss Bing Youji road concentric stria at center, microscopic examination has barrier film (Fig. 2) to mycelia, bear the conidiophore of Dan Sheng from aerial hyphae, conidiophore has diaphragm, top life be arranged in broom shape between branch, branch 1 time or repeatedly, conidium conspires to create unbranched chain, conidium is spherical in shape.
Embodiment 2: the 18S rDNA sequencing of Penicillium notatum BJKD4 bacterial strain of the present invention
The 18S rDNA sequential analysis of bacterial strain: adopt CTAB (cetyl trimethylammonium bromide) method to extract genome.Use amplimer: SSU817TTA GCA TGG AAT AAT RRAATA GGA and SSU1536AAT GCAATG CYC TAT CCC CA carry out polymerase chain reaction (PCR) DNA amplification.50 μ L reaction systems contain: the deionized water 42.25 μ L of 10*PCR damping fluid 5 μ L, 10mmol/L Dntp0.25L, 45pmol/ μ L primer 0.5 μ L, 2.5U/ μ L Taq enzyme 0.5 μ L, DNA profiling 1 μ L and sterilizing.PCR reaction conditions: 94 DEG C of sex change 1min, 50 DEG C of annealing 30s, 72 DEG C of extension 90s, 40 circulations, 72 DEG C of final 5min that extend.PCR reaction product 2 μ L are in 1% agargel electrophoresis.DNA sequence dna is measured by Beijing Hua Da genome company, use BLAST (
http:// www.ncbi.nlm.nih.gov/BLAST) program carries out DNA sequence dna comparison.
BLAST compare of analysis shows: the 18S rDNA sequence (gene order number of registration: HM439093) of BJKD4 is 99% with the homology of Penicillium (Penicilliumsp.), Eupenicillium sp (Eupenicillium sp.) and Ascomycetes (Ascomycetesp.), with MEGA5.0 software building 18S rDNA phylogenetic tree (Fig. 3).Combining form is observed and analysis of molecules, and we think that BJKD4 belongs to Penicillium (Penicilliumsp.).
Embodiment 3: the ability of the resistance to heavy metal of Penicillium notatum of the present invention is measured
1, the patience of BJKD4 bacterial strain to heavy metal cadmium: by the activation inoculation of 2 days to containing 0,1,3,9,18 or 36mmol/LCd
2+solid Cha Shi substratum in, cultivate 16d at 28 DEG C, when 3d, start to measure colony diameter.
Test result shows: BJKD4 bacterial strain is containing 0-36mmol/L Cd
2+cha Shi solid medium in the 16d that grows, its colony diameter increases along with the prolongation of cultivated days, along with Cd
2+the increase of concentration and reduce (Fig. 4).Without Cd
2+in substratum (contrast), the lag phase of colony growth is 0-4d, and logarithmic phase is 4-15d, when 16d, enters stationary phase; And interpolation heavy metal Cd
2+(1-36mmol/L) after, lag phase extends to 6d, then enters logarithmic phase, reaches stationary phase when 14-15d.Compared with the control, BJKD4 bacterial strain is at 1-36mmol/L Cd
2+while growing 5-16d on flat board, its colony diameter is significantly lower than contrast, and different concns Cd
2+process colony diameter size is had no significant effect, illustrate that BJKD4 is resistance to cadmium bacterium.
2, the patience of BJKD4 bacterial strain to different heavy metals: bacterial strain activates 2d in liquid Cha Shi substratum, gets 2 μ L bacterium liquid and is inoculated into and contains single different concns heavy metal Cd
2+(0,1,3,9,18 and 36mmol/L), Cu
2+(0,0.05,0.1,0.2,0.3 and 0.4mmol/L), Ni
2+(0,0.9,1.8,2.7,3.6 and 4.5mmol/L), Pb
2+(0,1,2,4,6 and 8mmol/L), Zn
2+(0,14,28,42,56 and 70mmol/L) or Mn
2+in the solid Cha Shi substratum of (0,20,40,60,80 and 100mmol/L), be inverted in constant incubator, measure colony diameter while cultivating 8d for 28 DEG C.
Test result shows: the inhibition difference (Fig. 5) of different heavy metals to BJKD4 strain growth, its colony diameter is along with the rising of heavy metal concentration reduces gradually.BJKD4 colony diameter is containing 0.05mmol/L Cu
2+in substratum, decline rapidly, 0.2mmol/L Cu
2+time bacterial strain can not grow; Cd
2+when concentration is 1mmol/L, colony diameter significantly reduces, afterwards along with the increase of Cd concentration (1-36mmol/L) slowly reduces; Along with Ni
2+(0-4.5mmol/L), Pb
2+(0-8mmol/L) and Zn
2+(0-70mmol/L) rising of concentration, colony diameter straight line declines, and heavy metal concentration is when identical, Pb
2+and Zn
2+while coercing, bacterial plaque diameter is greater than Ni
2+diameter while coercing; Be less than 20mmol/LMn
2+when (lower concentration), can promote BJKD4 strain growth, and be greater than 20mmol/L Mn
2+when (high density), its diameter is with Mn
2+concentration raises and reduces rapidly.50% place at y axle maximum growth rate makes sea line, and the concentration on its x axle corresponding to growth rate curve intersection point place is EC50.Relatively EC50 learns that different heavy metals are followed successively by the toxicity size of BJKD4: Cu
2+> Ni
2+> Cd
2+> Pb
2+or Zn
2+> Mn
2+.
3, the patience of BJKD4 bacterial strain to Compound Heavy Metals: according to single heavy metal (Cd
2+, Ni
2+, Zn
2+, Mn
2+) impact on strain growth, design L
25(5
4) orthogonal test, prepare the solid medium of different heavy-metal composite pollutions, inoculation 2 μ L bacterium liquid, cultivate 8d for 28 DEG C, measure colony diameter.Experiment in triplicate, is used SPSS software analysis.
Test result shows: BJKD4 bacterial strain at the colony diameter of heavy-metal composite pollution substratum growth 8d in table 1.BJKD4 colony diameter all increases under 4 heavy metal species combined pollution conditions; The size (table 1) of extreme difference from intuitive analysis table (R) finds out that different heavy metals are followed successively by the size of BJKD4 growth effect: Mn
2+> Cd
2+> Zn
2+> Ni
2+.Analyze known Cd from K value
2+and Mn
2+suppress strain growth, and Zn
2+show as lower concentration promoter action, Ni
2+k value under 5 levels is close.Variance analysis (table 2) shows: Cd
2+, Zn
2+and Mn
2+the growth of remarkably influenced BJKD4, Ni
2+there is no remarkable effect.In addition, concentration is 2.25mmol/L Cd
2+, 15mmol/L Zn
2+, 0-2.4mmol/LNi
2+with 80mmol/L Mn
2+deng Compound Heavy Metals, the restraining effect of BJKD4 growth is less than to other combinations, shows that BJKD4 bacterial strain can the combined pollution of resistance to various heavy.
Diameter and the intuitive analysis of table 1 orthogonal test BJKD4
The variance analysis of table 2 orthogonal test
Embodiment 4: the growth of bacterial strain of the present invention in different concns coal gangue leach liquor
The coal gangue powder 500g that got 1mm sieve, adds 2.5L water soaking, and every day, the pH value of leach liquor was measured in timing, reached stationary value pH2.72 when 7-10 d.To soak the coal gangue leach liquor suction filtration of 10d, supernatant liquor is pressed different volumes than dilution with Cha Shi substratum or deionized water respectively, obtains the nutrient solution of 40mL containing the coal gangue leach liquor of 0,1/4,1/3,1/2,3/4 or 1 (v/v).Inoculate respectively the bacterium liquid of 1ml activation 2d, cultivate the centrifugal collection thalline of 5d, after 80 DEG C of oven dry, survey dry weight.
Test result shows: coal gangue leach liquor pH is in 3 left and right, and water pH is that 6.8, Cha Shi medium pH is 7.2.When BJKD4 bacterial strain (Fig. 6) is grown in water-reducible a series of coal gangue leach liquors: its fresh weight is containing obviously not rising in the water of 1-1/4 coal gangue leach liquor, but higher than the biomass in water; Δ pH analyzes and shows that its Δ pH slowly raises in the water that contains 1-1/4 coal gangue leach liquor, and a little more than the Δ pH of water; Grow 5d in a series of coal gangue leach liquors of Cha Shi substratum dilution time, its fresh weight significantly raises along with the reduction of coal gangue leach liquor content (1-1/4), and the fresh weight in 1/2-3/4 coal gangue leach liquor is apparently higher than Cha Shi substratum.Obviously rise (consistent with the increase trend of thalline biomass) at the nutrient solution Δ pH that contains 1-1/2 coal gangue leach liquor, and in the substratum that contains 1/2-1/4 coal gangue leach liquor, reach maximum value Δ pH.Cultivate after 5d and connect bacterium, the Δ pH that does not contain the water of coal gangue leach liquor is 0.09, and the Δ pH that does not contain the Cha Shi substratum of coal gangue leach liquor is-1.98, illustrates that this bacterial strain has the ecological adaptation ability that regulates surrounding environment pH.Bacterial strain is after cultivating in the leach liquor of different methods dilution, effectively improve pH of leaching solution, containing in lower concentration coal gangue leach liquor, strain bio amount also increases compared with the control, illustrate and in coal gangue leach liquor, contain this strain growth desired nutritional, and thalline can improve acid leaching solution pH value.
Claims (4)
1. the Penicillium notatum of the resistance to heavy metal cadmium of a strain, is characterized in that, this bacterial strain belongs to Penicillium Penicillium sp., called after BJKD4; This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number CGMCC NO.6620.
2. the Penicillium notatum BJKD4 of resistance to heavy metal cadmium as claimed in claim 1, its physiology and morphology biochemical character:
A, BJKD4 bacterium colony are felted, and there is transudate on surface, and well-regulated radial pleat splits, and there is boss Bing Youji road concentric stria at center, has mycelia also to have mycelium pellet in liquid medium within, and nutrient solution is more transparent.
B, mycelia have barrier film, bear the conidiophore of birth from aerial hyphae, and conidiophore has diaphragm, top life be arranged in broom shape between branch, branch 1 time or repeatedly, conidium conspires to create unbranched chain, conidium is spherical in shape.
3. the Penicillium notatum BJKD4 of resistance to heavy metal cadmium as claimed in claim 1, is characterized in that, the 18S rDNA sequence of described bacterial strain is as shown in SEQID NO.1.
4. the Penicillium notatum BJKD4 of the resistance to heavy metal cadmium as described in claims 1-3, can be used for that coal gangue hill is reclaimed and the restoration of the ecosystem of heavy metal pollution of soil.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805021A (en) * | 2015-05-15 | 2015-07-29 | 湖南省农业生物技术研究中心 | Cadmium-tolerant penicillium and separation method thereof |
| CN105750326A (en) * | 2016-04-21 | 2016-07-13 | 绍兴文理学院 | Method for remedying cadmium-polluted soil |
| CN110951626A (en) * | 2019-12-27 | 2020-04-03 | 北京林业大学 | Penicillium oxalicum capable of decomposing coal gangue |
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2013
- 2013-01-16 CN CN201310015361.9A patent/CN103923839A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805021A (en) * | 2015-05-15 | 2015-07-29 | 湖南省农业生物技术研究中心 | Cadmium-tolerant penicillium and separation method thereof |
| CN105750326A (en) * | 2016-04-21 | 2016-07-13 | 绍兴文理学院 | Method for remedying cadmium-polluted soil |
| CN110951626A (en) * | 2019-12-27 | 2020-04-03 | 北京林业大学 | Penicillium oxalicum capable of decomposing coal gangue |
| CN110951626B (en) * | 2019-12-27 | 2021-05-25 | 北京林业大学 | Penicillium oxalicum capable of decomposing coal gangue |
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