CN105733966B - Radiation-resistant filamentous fungus M30 and application thereof in biological treatment for adsorbing cadmium - Google Patents
Radiation-resistant filamentous fungus M30 and application thereof in biological treatment for adsorbing cadmium Download PDFInfo
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- CN105733966B CN105733966B CN201610282197.1A CN201610282197A CN105733966B CN 105733966 B CN105733966 B CN 105733966B CN 201610282197 A CN201610282197 A CN 201610282197A CN 105733966 B CN105733966 B CN 105733966B
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Abstract
The invention discloses a radiation-resistant filamentous fungus M30 and application thereof in biological treatment for adsorbing cadmium, wherein a radiation-resistant Acremonium chrysosporium (which has high adsorption property on cadmium ions) is obtained by separating, purifying, screening and culturing a soil sample in a arid desert area around Apocynum venetum RoxbAcremonium sp.) M30CGMCC No.11007, the strain has obvious difference in physiological and biochemical characteristics and molecular level from common fungus strain, and the screened strain M30 has thallus adding amount of 5g/L, Cr2+For Cr, the solution is 100mg/L, pH6.5, temperature is 20 deg.C, and adsorption time is 1min2+Has the best adsorption effect, Cr2+The removal rate reaches more than 90 percent, is a typical new strain, and has practical significance for treating the cadmium pollution environment.
Description
Technical Field
The invention relates to the technical field of environmental biological treatment, in particular to a radiation-resistant filamentous fungus and application thereof in the aspect of adsorbing heavy metal cadmium.
Background
The global release of heavy metals into the environment is statistically up to several million tons per year and is also increasing year by year. In recent years, the heavy metal pollution events of soil in China are frequent, so that the heavy metal pollution events not only form serious threats to the quality of cultivated land and agricultural products, but also directly damage the health of people and influence the social stability.
Cadmium is a widely used heavy metal and also an environmental pollution element with extremely strong toxicity. The cadmium compound has high fat solubility, biological enrichment and toxicity, the accumulation of the cadmium compound in a human body can destroy the skeleton and hematopoietic system of the human body to cause anemia, kidney damage and the like, and the cadmium compound has high migration and is easy to be absorbed and accumulated by crops. The cadmium exceeding of the soil and the cadmium pollution cause cadmium rice events, which cause great social panic. Currently, 3.9 million tons of cadmium are released into the environment worldwide each year. The cultivated land area of China polluted by heavy metals such as cadmium is nearly 2.0 multiplied by 107Hectare.
Compared with the traditional method, the bioremediation method using the microorganisms has the advantages of wide material sources, high efficiency of treating heavy metal at low concentration (1-100 mg/L), large adsorption capacity, high speed, good selectivity, simple adsorption equipment, easy operation and the like, has obvious advantage of treating heavy metal pollution, and can effectively solve the problem of heavy metal pollution of water and soil. However, the existing separated heavy metal-resistant microorganisms have few populations and weak removal capability, and cannot meet the current situation of complicated pollution of the actual environment, so that the population diversity is urgently needed to be enriched, and mature treatment processes and methods are further researched and perfected to improve the removal capability of heavy metals.
The radiation-resistant microorganisms are extreme microorganism resources capable of living under high radiation dose, have strong tolerance and adsorbability to heavy metals, and therefore the adsorption capacity of the radiation-resistant microorganisms to the heavy metals needs to be further researched, resistant strains with relatively high adsorption capacity and stable performance are provided for the microorganisms to repair the heavy metal pollution, and the radiation-resistant microorganisms have practical significance for treating the cadmium pollution environment.
Disclosure of Invention
Aiming at the technical current situation that no relevant report about radiation-resistant filamentous fungi and application thereof for adsorbing heavy metal cadmium is found in the prior art and separated and screened strains have stronger tolerance and adsorbability to heavy metals, the invention aims to provide a resistant strain with relatively high adsorption capacity and stable performance for repairing cadmium pollution by microorganisms. The invention screens out a radiation-resistant Acremonium strictum with higher adsorption characteristic to cadmium ions by separating in a soil sampleAcremoniumsp.) M30CGMCC No.11007, the separated bacteria are used in biological treatment of cadmium polluted water and the application scheme of the bacteria is provided.
The invention adopts the main technical scheme that:
the soil sample is collected from arid desert areas around Xinjiang Apocynum by Xinjiang agricultural academy of sciences microbial application research, the collected soil sample is irradiated by a 5000 KGy cobalt source, a large amount of radiation-resistant fungus strains with good growth are screened and selected preferably by taking a PDA (personal digital assistant) culture medium added with streptomycin as a separation culture medium, and a fungus strain M30 with high adsorption property to cadmium ions is screened out. The fungus strain with the number of M30 separated and screened by the invention is used for carrying out biological treatment on cadmium-polluted water, and the addition amount of the fungus strain in thalli is 5g/L, Cr2+For Cr, the solution is 100mg/L, pH6.5, temperature is 20 deg.C, and adsorption time is 1min2+The removal rate reaches more than 90 percent, and for Cr2+Has the best adsorption effect.
The invention particularly provides radiation-resistant Acremonium strictum (A)Acremonium sp.) A screening method of M30CGMCC No. 11007. Separating, screening and culturing in collected soil sample to obtain a batch of fungus microbial strains, screening a fungus strain M30 with high cadmium ion adsorption property, and performing microbiological classification and identification to obtain a strain belonging to Acremonium chrysosporium (A)Acremonium sp.) And (3) strain.
Specifically, the invention is characterized in that a collected soil sample is irradiated by a 5000 KGy cobalt source, separation, screening and culture are carried out, a strain with the number of M30 is screened out, and the strain belongs to Acremonium chrysosporium (Acremonium chrysosporium) through microbiological classification and identificationAcremonium sp.) And (3) strain. The strain has been deposited in the international collection of microorganisms under the Budapest treaty before the filing date: china general microbiological culture Collection center (CGMCC). Address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101. the preservation date is 2015, 7 months and 9 days, and the preservation number of the strains is CGMCC number 11007. Microbiologically identified as Acremonium cladosporioides (A), (B), (C), (Acremonium sp.) M30. The optimal growth conditions of the strain are as follows: the temperature is 25 ℃, the culture medium is a PDA culture medium (200 g/L of potato, 20g/L of glucose, 15g/L of agar and 1L of distilled water, the pH is natural), and the culture time is 5 d; after 5 days of culture, the colony on the surface of the PDA culture medium is circular, has the diameter of 7-12mm, is white, has a powdery middle part and a velvet edge, sometimes forms a coremium bunch and has a light yellowish-brown back surface. According to the morphological characteristics, the M30 strain is identified morphologically, physiologically and biochemically by referring to a fungal identification manual, the strain M30 is determined to be Acremonium cladosporioides by comparing fungal classification search tables and combining molecular biology sequencing and LSU sequence analysisAcremoniumsp.）。
The strain M30 is subjected to total DNA extraction and LSU gene PCR amplification product sequencing to obtain 825bp sequence, and is subjected to comparison and bisection by GenBank \ Blast homologous sequence ratioAnalysis, which is similar to Acremonium cladosporioides reported in the prior art (A)Acremoniumsp.) The homology is higher. Obtaining a standard strain LSU gene sequence from GenBank, carrying out homologous evolution analysis, carrying out CLUSTAL X multi-sequence comparison, and adopting an MEGA 5.0 software adjacency method (Neighbor Joining) to construct a phylogenetic tree, wherein the strain M30CGMCC No.11007 belongs to Acremonium fungi (A) (A.sp.sp.sp.sp.sp.sp.sp.Acremoniumsp.) Which is a strain of the genus and the genusAcremonium exuviarumUAMH9995T andAcremonium salmoneumCBS 721.71T forms a stable branch and has recent homologies of 98.5% and 97.5%, respectively, greater than 96% with all other models. Therefore, in view of the fact that the LSU highest homology among other species in the genus is often more than 99%, meanwhile, the strain M30CGMCC No.11007 has obvious differences in morphological aspect and physiological and biochemical characteristics, and strong tolerance capability and adsorption capability of heavy metal cadmium ions, and the M30 strain is a new strain which is tentatively named Acremonium (A) A (B)Acremoniumsp.)M30。
The molecular sequencing result shows that the LSU gene sequence of the radiation-resistant fungus M30CGMCC No.11007 is 825 bp. The invention provides a radiation-resistant Acremonium teres (A)Acremonium sp.) M30 has obvious physiological and biochemical characteristic difference and molecular level difference with common fungus strains, according to the analysis of the physiological and biochemical characteristic of the strains, the analysis of the molecular level and the comprehensive identification of systematic taxonomy, the strain numbered M30 has some common attributes compared with the common acremonium fungus strains, but according to the obvious physiological and biochemical characteristic difference and molecular level difference with the common acremonium fungus strains, the M30 strain is a typical new strain, has stronger tolerance and adsorption capacity to heavy metal cadmium ions, and is identified as radiation-resistant acremonium from the taxonomy (M30 strain) (the M30 strain is identified as radiation-resistant acremonium from the taxonomy)Acremonium sp.）。
The invention further provides radiation-resistant Acremonium teres (Acremonium sp.) M30CGMCC No. 11007.
1. The separation culture medium adopts: PDA culture medium, every 100ml culture medium adds 1% streptomycin solution 0.3ml, add cadmium ion solution at the same time, make its concentration reach 1500mg/L separately.
2. Preparing a soil sample diluent: dissolving the soil sample irradiated by 5000 KGy cobalt source in a triangular flask containing sterile water according to a solid-to-liquid ratio of 1: 10, stirring with a glass rod, culturing at 28 deg.C under constant temperature oscillation (160r/min) for 30min, taking out the triangular flask, and standing on a horizontal desktop for 30 min. Taking 1mL of supernatant fluid, placing in a test tube containing 9mL of sterilized water, mixing well, and gradually diluting to 10 degrees by gradient dilution method-3。
3. Separation and purification: taking the stock solution 10-1、10-2、10-3Each 1mL of the diluted solution was coated with Cr2+The medium was applied to a plate of 1500mg/L medium immediately and uniformly with a glass spatula. The plate was placed upside down in a 30 ℃ incubator for 48 hours. After the bacterial colony grows out, selecting single bacterial colony hypha or spore with obviously different bacterial colony characteristics, and continuously separating and purifying by adopting a scribing method until pure culture is obtained.
Radiation-resistant Acremonium cladosporioides determined by culture screening (Acremonium sp.) M30CGMCC No.11007 has circular colony, 7-12mm diameter, white color, powder in the middle part, velvet edge, sometimes formed into sporophyte bunch, and yellowish-brown back.
Further, the invention provides a radiation-resistant Acremonium (A)Acremonium sp.) The M30CGMCC No.11007 is applied to the biological treatment of cadmium-polluted water. By researching the influence on the thallus adsorption under the conditions of different cadmium ion concentrations, temperatures, pH values, inoculation amounts and adsorption time, M30 on Cr is obtained2+The optimum adsorption condition of (2) is that the addition amount of the bacterial cells is 5g/L, Cr2+The solution is 100mg/L, pH6.5, temperature is 20 deg.C, and adsorption time is 1 min.
By implementing the specific technical indexes of the invention, the content of the invention is realized, and the following beneficial effects can be achieved:
(1) the invention provides a radiation-resistant Acremonium teres (A)Acremonium sp.) M30CGMCC No.11007 is a typical new strain and has the characteristics of simple culture condition and quick propagation.
(2) The separated and screened radiation-resistant Acremonium strain thallus with the number of M30 is prepared into a biological adsorbent, and the adding amount of the thallus is 5g/L, Cr2+The solution is 100mg/LpH6.5, temperature 20 deg.C, and adsorption time 1min2+The removal rate can reach more than 90 percent, and different metal ions adsorb Cr to thalli2+In a different way, K+、Na+、Pb2+、Al3+The plasma had little effect on the adsorption, while Cu2+、Mg2+、Fe3+、Ca2+For Cr2+Removal has a significant impact.
(3) In the process of treating the low-concentration cadmium-polluted wastewater, the method can utilize the growth of the thalli to adsorb cadmium ions, can also directly use the thalli to adsorb, and has the advantages of high removal rate, high reaction speed, no secondary pollution, simple and convenient operation and the like.
Drawings
FIG. 1 shows radiation-resistant Acremonium terrestris (Acremonium sp.) M30CGMCC No.11007 is colony and thallus photograph, where A is colony morphology, B is 10 times under-ocular thallus morphology and C is 20 times under-ocular thallus morphology.
FIG. 2 shows Acremonium radiodurans (A)Acremonium sp.) M30CGMCC No.11007 phylogenetic dendrogram.
FIG. 3 shows Acremonium radiodurans (A)Acremonium sp.) The detection result of the tolerance of M30CGMCC No.11007 to different metal ions.
FIG. 4 shows radiation-resistant Acremonium terricola (Acremonium sp.) Graph of the influence of the using amount of M30CGMCC No.11007 wet bacteria on the adsorption effect.
FIG. 5 shows radiation-resistant Acremonium terricola (Acremonium sp.) Graph of the influence of different cadmium ion concentrations of M30CGMCC No.11007 on the adsorption effect.
FIG. 6 shows radiation-resistant Acremonium terricola (Acremonium sp.) Graph of the effect of the pH value of M30CGMCC No.11007 solution on the adsorption effect.
FIG. 7 shows Acremonium radiodurans (A)Acremonium sp.) Influence graphs of different adsorption time and temperature of M30CGMCC No.11007 on adsorption effect.
FIG. 8 shows radiation-resistant Acremonium terricola (Acremonium sp.) Graph of influence of different metal ions of M30CGMCC No.11007 on adsorption effect。
Detailed Description
The present invention will be described below by way of examples, but the present invention is not limited to the following examples. All raw and auxiliary materials selected for use in the present invention, as well as methods for culturing the selected bacterial species, are well known and used in the art, and all percentages referred to herein are by weight unless otherwise indicated.
The first embodiment is as follows: radiation-resistant Acremonium terricola (Acremonium sp.) Separation, screening and identification of M30CGMCC No.11007
1. Isolation and selection of bacterial species
Acremonium used in the present invention (A)Acremonium sp.) M30 is obtained by sampling soil collected from arid desert areas around Apocynum venetum by Xinjiang academy of agricultural sciences microorganism application research institute, irradiating with 5000 KGy cobalt source, separating fungi from soil layer by conventional plate culture method, purifying strains by plate marking method, optimizing and screening a batch of good-growing fungus strains by taking different culture temperatures, pH values and culture media as enrichment conditions, and preferably selecting a strain with the number of M30.
A separation step:
(1) separating a culture medium: PDA culture medium is adopted, 0.3ml of 1% streptomycin solution is added into each 100ml of culture medium, and cadmium ion solution is added simultaneously, so that the concentration of the solution reaches 1500mg/L respectively.
(2) Preparing a soil sample diluent: dissolving the soil sample irradiated by 5000 KGy cobalt source in a triangular flask containing sterile water according to a solid-to-liquid ratio of 1: 10, stirring with a glass rod, culturing at 28 deg.C under constant temperature oscillation (160r/min) for 30min, taking out the triangular flask, and standing on a horizontal desktop for 30 min. Taking 1mL of supernatant fluid, placing in a test tube containing 9mL of sterilized water, mixing well, and gradually diluting to 10 degrees by gradient dilution method-3。
(3) Separation and purification: taking the stock solution 10-1、10-2、10-3Each 1mL of the diluted solution was coated with Cr2+The medium was applied to a plate of 1500mg/L medium immediately and uniformly with a glass spatula. The plate was placed upside down in a 30 ℃ incubator for 48 hours. After the bacterial colony grows out, picking out the bacterial colonyAnd (4) obviously different single colony hypha or spores are marked, and separation and purification are continued by adopting a scribing method until pure culture is obtained.
2. Culture conditions of the strains
(1) The growth medium of strain No. M30 was PDA medium: 200g/L of potato, 20g/L of glucose, 15g/L of agar and 1L of distilled water, wherein the pH is natural, and the potato is cultured for 96 hours at 30 ℃.
(2) The strain with the number M30 can grow under the condition of 20-35 ℃, the optimal growth temperature is 25 ℃, and the culture time is 3-5 days.
(3) The growth pH of the strain numbered M30 was natural.
(4) Heavy metal tolerance test of strain No. M30.
Inoculating the activated strain into seed test tube containing Chachi liquid culture medium, culturing at 30 deg.C, shaking at 150rpm for 36 hr, and inoculating to Hg with different contents according to 2% inoculum size2+、Cu2+、Zn2+、Cd2+、 Pb2+、Cr2+The Chashi liquid culture medium is added into a triangular flask with the addition amount of 500ml, 80ml of Chashi liquid culture medium is filled into the triangular flask, the Chashi liquid culture medium is cultured at 25 ℃, the Chashi liquid culture medium is cultured for 96 hours by shaking at 180rpm, and the tolerance condition of strains is observed.
Radiation-resistant Acremonium terricola (Acremonium sp.) The results of the detection of the tolerance of M30 to different heavy metal ions are shown in FIG. 3. It can be seen from FIG. 3 that the strain M30 is tolerant to various metals, and is resistant to Pb2+、Zn2+Has the best tolerance capacity, wherein the tolerance concentration is 1900mg/L and 1700mg/L respectively, and Cr2+Next, the maximum tolerated concentration reached 1500 mg/L. Strain to Hg2+The tolerance is the worst, and the growth of the thallus is obviously inhibited when the concentration reaches 50 mg/L. The result shows that the strain is used for heavy metal ions Cr2+Has higher tolerance and good application prospect in heavy metal biological treatment.
Specifically, the invention is characterized in that a collected soil sample is irradiated by a 5000 KGy cobalt source, separation, screening and culture are carried out, a strain with the number of M30 is screened out, and the strain belongs to Acremonium chrysosporium (Acremonium chrysosporium) through microbiological classification and identificationAcremonium sp.) And (3) strain. The strain has been deposited in the international collection of microorganisms under the Budapest treaty before the filing date: china general microbiological culture Collection center (CGMCC). Address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101. the preservation date is 2015, 7 months and 9 days, and the preservation number of the strains is CGMCC number 11007. Microbiologically identified as Acremonium cladosporioides (A), (B), (C), (Acremonium sp.) M30. The optimal growth conditions of the strain are as follows: the temperature is 25 ℃, the culture medium is a PDA culture medium (200 g/L of potato, 20g/L of glucose, 15g/L of agar and 1L of distilled water, the pH is natural), and the culture time is 5 d; after 5 days of culture, the colony on the surface of the PDA culture medium is circular, has the diameter of 7-12mm, is white, has a powdery middle part and a velvet edge, sometimes forms a coremium bunch and has a light yellowish-brown back surface.
The molecular sequencing result shows that the radiation-resistant Acremonium (A) and (B) areAcremonium sp.) The LSU gene sequence of M30CGMCC No.11007 is 825bp, and the radiation-resistant Acremonium cladi (A) provided by the inventionAcremonium sp.) M30CGMCC No.11007 has obvious physiological and biochemical characteristic difference and molecular level difference with common fungus strain, and the strain numbered M30 has obvious difference with common Acremonium fungus strain in physiological and biochemical characteristic analysis, molecular level analysis and comprehensive identification of systematic taxonomy, and has stronger tolerance and adsorption capacity to heavy metal cadmium ion compared with common fungus strain, but is identified as Acremonium according to taxonomy (A)Acremonium sp.）。
3. Physiological and biochemical identification of strain M30
Morphological characteristics: the bacterial strain M30 is separated and cultured, and the colony on PDA culture medium is round, has diameter of 7-12mm, white color, powdery middle part, velvet edge, sometimes formed into spore bundle, and light yellowish brown back. According to the primary identification of the conventional fungus identification method, the strain M30 is identified and determined to be Acremonium cladosporioides by the comparison of fungus classification search tables and the LSU sequence analysisAcremonium sp.) The colony and thallus morphology is shown in figure 1.
Physiological and biochemical characteristics: the strain M30 has good growth on PDA culture medium, and Biolog FF identification plate test shows that the strain M30 can utilize Tween 80, N-acetyl-beta-D-glucosamine, ribitol, apricot glycoside, L-arabinose, D-arabitol, arbutin, D-cellobiose, dextrin, erythritol, D-fructose, L-trehalose, D-galactose, gentiobiose, D-gluconic acid, D-glucosamine, a-D-glucose, D-glucuronic acid, glycerol, glycogen, M-cellulose, 2-keto-D-gluconic acid, lactulose, maltose, maltotriose, D-mannitol, D-mannose, D-melezitose, glucose, D-melibiose, a-methyl-D-galactoside, beta-methyl-D-glucoside, a-methyl-D-glucoside, beta-methyl-D-glucoside, 6-O-D-glucopyranosyl-D-fructofuranose, D-psicose, D-raffinose, L-rhamnose, salicin, sedoheptanan, D-sorbitol, L-sorbose, stachyose, sucrose, D-trehalose, turanose, xylitol, D-xylose, y-aminobutyric acid, bromosuccinic acid, fumaric acid, L-malic acid, quinic acid, D-glucaric acid, sebacic acid, succinic acid, L-alanine, L-propylaminoylglycine, beta-methyl-D-glucoside, beta-D-glucoside, 6-O-glucopyranosylglucosyld-fructofuranose, l-asparagine, L-aspartic acid, L-glutamic acid, ornithine, L-phenylalanine, proline, pyroglutamic acid, L-serine, 2-aminoethanol, putrescine and adenosine.
Combining the morphological characteristics, referring to 'fungus identification handbook' to carry out morphological, physiological and biochemical identification on the M30 strain, comparing with a fungus classification search table, combining with molecular biology sequencing and LSU sequence analysis to determine that the strain M30 is the Acremonium fungus (R) ((R))Acremonium sp.）。
By the above-mentioned radiation-resistant Acremonium terricolaAcremonium sp.) The strain morphology, culture characteristic observation and physiological and biochemical index measurement of M30CGMCC No.11007 are compared with common strains for 6 heavy metal ions Pb in comparison with the common strains through the tests of the strain morphology observation, the strain culture characteristic observation, the growth temperature measurement, the tolerance test and the like2+、Cd2+、Hg2+、Cu2+、Cr2 +、Zn2+All have higher tolerance characteristics according to the fungal identification handbookThe method comprises the steps of performing the steps that although the strain with the number M30 has some common attributes compared with the common acremonium fungus strain, the M30 strain is a typical new strain according to obvious physiological and biochemical characteristic difference and molecular level difference with the common acremonium fungus strain, preliminarily identifying the strain as one of the acremonium of Hypocreales of Ascomycota, and comprehensively identifying the strain with the number M30 as radiation-resistant acremonium from the aspect of strain classificationAcremonium sp.）。
Example two: radiation-resistant Acremonium terricola (Acremonium sp.) Molecular level identification of M30CGMCC No.11007
1. DNA extraction: the fungus extraction method comprises the following steps:
(1) 200mg of thallus, grinding with liquid nitrogen, adding 3ml of 3% CTAB extraction buffer solution, carrying out water bath at 65 ℃ for 45min, and centrifuging at 4 ℃ at 4000r/min for 20 min.
(2) The supernatant was transferred to a centrifuge tube, 4. mu.l of 10mg/ml protease was added thereto, and the mixture was incubated at 37 ℃ for 1 hour in a water bath.
(3) Adding 800 μ l Tris saturated phenol, shaking up for 13000r/min, and centrifuging for 10 min; and taking the supernatant.
(4) Adding equal volume of chloroform/isoamyl alcohol, shaking up, centrifuging at 13000r/min for 10min, and taking the supernatant.
(5) 10mg/ml RNase was added and treated in a water bath at 37 ℃ overnight.
(6) Adding 800 μ l chloroform/isoamyl alcohol, shaking, centrifuging at 13000r/min for 10min, and collecting supernatant.
(7) Adding 600 μ l of isoamyl alcohol, precipitating at-20 deg.C for 30min, collecting precipitate, washing with 75% alcohol, and vacuum drying in ultra-clean bench.
(8) DNA was dissolved in 100. mu.l of TE and stored at-20 ℃ until use. Mu.l of RNase was added, water bath was carried out at 37 ℃ for 1h, 400. mu.l of chloroform/isoamyl alcohol (24: 1) was added, centrifugation was carried out at 12000r/min for 10min, and the procedure was repeated 2 times.
2. LSU gene amplification and sequencing, adopting a fungus LSU gene universal primer for amplification:
primer V9: 5'-TGCGTTGATTACGTCCCTGC-3'
RLR3R：5′-GGTCCGTGTTTCAAGAC-3'，
The PCR amplification reaction system is 50 mu L, and the reaction conditions are as follows: 5min at 95 ℃; 95 ℃ 45s, 56 ℃ 45s, 72 ℃ 60s, 35cycles, 72 ℃ 7 min. The amplification product (about 900bp), PCR amplification product with 1% agarose gel electrophoresis detection, amplification product sequencing, strain M30 LSU gene SEQUENCE determination, strain M30CGMCC No.11007 gene SEQUENCE is 825bp, see the attached gene SEQUENCE table SEQUENCE Listing.
3. LSU sequence alignment and phylogenetic analysis
The invention obtains 825bp sequence by extracting total DNA and sequencing the PCR amplification product of LSU gene, and the 825bp sequence is compared and analyzed with GenBank \ Blast homologous sequence and is compared with Acremonium cladosporioides (A), (B) and (B) reported in the prior artAcremonium sp.) The homology is higher. The standard strain LSU gene sequence is obtained from GenBank, and is subjected to homologous evolution analysis, CLUSTAL X is subjected to multiple sequence alignment, and the phylogenetic tree is constructed by adopting the Saitou and Nei adjacency method (Neighbor Joining) in MEGA 5.0 software, and the result is shown in the attached figure 2. As can be seen from the dendrogram, the radiation-resistant Acremonium strictum provided by the inventionAcremonium sp.) M30CGMCC No.11007 has obvious physiological and biochemical characteristic difference and molecular level difference with common fungus strain, and the strain numbered M30 has obvious difference with common Acremonium fungus strain in physiological and biochemical characteristic analysis, molecular level analysis and comprehensive identification of systematic taxonomy, and has stronger tolerance and adsorption capacity to heavy metal cadmium ion compared with common fungus strain, but is identified as Acremonium according to taxonomy (A)Acremonium sp.）。
Example three: radiation-resistant Acremonium terricola (Acremonium sp.) Preparation of M30CGMCC No.11007 thallus
Activated Acremonium radiodurans of the strains of the invention (Acremonium sp.) Inoculating M30CGMCC No.11007 in seed test tube containing 5ml Chashi liquid culture medium, culturing at 30 deg.C, shaking at 200rpm for 36 hr, inoculating in Chashi liquid culture medium at 2% inoculation amountA500 ml Erichschnikov flask was filled with 100ml of Chachi liquid medium, and the medium was cultured at 30 ℃ and shaking at 180rpm for 96 hours. The culture obtained by fermentation culture is centrifuged for 5min at 8000rpm, and wet thalli are collected for later use.
Example four: radiation-resistant Acremonium terricola (Acremonium sp.) Application of M30CGMCC No.11007 in biological treatment of adsorbing cadmium
Accurately weighing a certain amount of Acremonium chrysosporium provided in the above embodimentAcremonium sp.) Adding M30CGMCC No.11007 thallus to Cr in certain concentration and pH value2+Oscillating and adsorbing the solution for a certain time according to different test requirements, centrifuging at 8000rpm for 5min, diluting the supernatant with proper gradient, filtering with 45 um microporous membrane, measuring by inductively coupled plasma mass spectrometry (ICP-MS), and calculating the Cr of the thallus pair according to the following formula2+Adsorption rate and adsorption amount of (3).
Adsorption rate (%) = (Ci-Cj)/Ci × l 00%; adsorption amount (mg/g) = (Ci-Cj)/Cb;
in the formula: ci and Cj are respectively Cr2+Cb is the cell concentration.
Inoculating M30 strain into liquid PDA culture medium without cadmium ion, culturing for 3-5 days, collecting mycelium, performing thallus adsorption test, and studying the influence on thallus adsorption under different cadmium ion concentrations, temperatures, pH values, inoculum sizes and adsorption time conditions.
(1) Different bacterial dosages for radiation-resistant Acremonium teres (Acremonium sp.) Influence of M30CGMCC No.11007 adsorption
At 50mg/L Cr2+Adding wet bacteria with different masses (0.05 g, 0.1g, 0.2g and 0.3 g) into the cadmium nitrate solution under the condition that the pH is naturally the initial condition, adsorbing for 1h, and measuring the Cr in the solution2+The concentration of Cr in the culture broth is calculated2+Solution removal rate and unit cell adsorption rate. The results are shown in FIG. 4, and Cr is added with the increase of the amount of the bacterial cells2+The removal rate is increased continuously, and when the bacterial load reaches 3g/L, Cr in the solution2+The removal rate is close to 100%. From the unit cell adsorption rate, the maximum cell adsorption rate can reach 14.73mg/g wet cells, and the cell shows stronger adsorption capacity, but the cell amount is dependent on the cell amountThe adsorption rate decreases rapidly with increasing, which may be related to adsorption equilibrium and non-adsorption saturation of excess bacteria. Therefore, the solution Cr is considered comprehensively2+The removal rate and the adsorption rate per cell, and the cell addition amount of 5g/L were selected for subsequent experiments.
(2) (ii) different cadmium ion concentrations for radiation-resistant Acremonium teresAcremonium sp.) Influence of M30CGMCC No.11007 adsorption
Weighing 0.1g of thallus, respectively placing in Cr with cadmium ion concentration of 50mg/L, 100mg/L, 150mg/L, 200mg/L2+In the solution, the influence of different cadmium solution concentrations on the adsorption of the bacteria and the Cr content in the solution were measured2+The effect of removal rate. The results are shown in FIG. 5, and it can be seen from FIG. 5 that: with Cr2+Increase in concentration of Cr in the strain2+The removal rate is kept at the maximum removal rate for a short time, after the removal rate is close to 100%, the removal rate is quickly reduced and then slowly reduced, the adsorption rate of unit thalli is gradually increased, and the maximum unit adsorption amount can reach 16.5 mg/g in an experimental range because the thalli are relatively supersaturated at the initial low concentration and along with Cr in a solution2+The supersaturated bacterial load is broken, and instead Cr is added2+The excess amount of (B) causes a decrease in the removal rate, but the adsorption of the cell units is not saturated, so that the adsorption still gradually increases. Comprehensive consideration of Cr solution in experiment2+The removal rate and the adsorption rate per cell were determined by selecting a cadmium ion concentration of 100 mg/L.
(3) (ii) Acremonium radiodurans at different pH values of the solutionsAcremonium sp.) Influence of M30CGMCC No.11007 adsorption
Weighing 0.1g of thallus, respectively placing in 10ml of Pb with cadmium ion concentration of 100mg/L and pH values of 3, 4, 5, 6, 7, 8 and 92+And (3) standing and adsorbing the solution for 3 hours, and then measuring the influence of different pH values on thallus adsorption. The results are shown in FIG. 6, and the pH value is increased, the bacteria can react with Cr in the solution2+The removal rate is gradually increased, and the strain pair pb is at the pH value of 72+The highest adsorption rate of the solution reaches 90 percent, but when the pH value is more than 5, the increase of the solution tends to be reduced, which is probably that H is in a high-acidity environment+And Cr2+There is a competitive concern. But too high a pH (greater than 8), Cr2+Will form Cr (OH)2Precipitation, namely new pollution, causes bacteria to be nonsensical in adsorption, so that when the pH value of the solution adopted in the test is 5-7, the adsorption capacity of the bacteria to cadmium ions is strongest, and the optimal pH value is selected to be 6.5.
(4) Irradiation-resistant Acremonium terricola (at different adsorption times and temperatures)Acremonium sp.) Influence of M30CGMCC No.11007 adsorption
The temperature and the adsorption time are important influence factors for most adsorption processes, and the Cr removal of thalli is determined by measuring different times and different temperatures2+The influence of (c). The results are shown in FIG. 7, in which Cr is adsorbed to the cells2+The process is extremely rapid, the maximum value is basically reached within 1min of adsorption, and the adsorption time is slightly reduced after the adsorption is continuously increased, which shows that the strain is applied to Cr within 1min2+The adsorption rate of (2) is highest. As shown in FIG. 7, different adsorption temperatures have little influence on the adsorption process, and may only slightly differ at a later stage due to adsorption thermodynamics or molecular diffusion, and the decrease at 20 ℃ is minimal, which indicates that the strain has Cr to Cr at 20 ℃2+The highest solution adsorption rate reaches 90 percent.
(5) Radiation-resistant Acremonium terricola of different metal ion pairs (Acremonium sp.) Influence of M30CGMCC No.11007 adsorption
At pH6.5, Cr2+50mL of 200mg/L solution was added with 0.5g of wet cells and 200mg/L of different heavy metal ion solutions (Cu)2+、Mg2+、Fe3+、Ca2+、K+、Na+、Pb2+、Al3+) After 1h of adsorption, the Cr content in the solution was measured2+Concentration and calculating Cr in the solution2+And (4) removing rate. The results are shown in FIG. 8, and different metal ions adsorb Cr to the bacteria2+In a different way, K+、Na+、Pb2+、Al3+The plasma had little effect on the adsorption, while Cu2+、Mg2+、Fe3+、Ca2+For Cr2+The removal has a significant effect, which may be related to the selectivity of particular groups of the bacteria.
By combining the above steps, it can be seen that the cells are made into a bio-adsorbent, and the cellsThe adding amount is 5g/L, Cr2+The solution is 100mg/L, pH6.5, temperature is 20 deg.C, adsorption time is 1min, and its maximum solution Cr2+The removal rate can reach more than 90 percent, and different metal ions adsorb Cr to thalli2+In a different way, K+、Na+、Pb2+、Al3+The plasma had little effect on the adsorption, while Cu2+、Mg2+、Fe3+、Ca2+For Cr2+Removal has a significant impact.
Acremonium radiodurans provided by the series of examples above (Acremonium sp.) M30CGMCC No.11007 is a typical new strain with the advantages of simple culture condition and fast propagation, and the strain is radiation-resistant Acremonium terricolaAcremonium sp.) The M30CGMCC No.11007 is applied to the process of treating the low-concentration cadmium-polluted wastewater, can adsorb cadmium ions by using the growth of bacteria and can also directly adsorb the cadmium ions by using the bacteria, and has the advantages of high removal rate, high reaction speed, no secondary pollution, simple and convenient operation and the like.
The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.
SEQUENCE LISTING
<110> institute for microorganism application of Sinkiang academy of agricultural sciences
<120> radiation-resistant filamentous fungus M30 and application thereof in biological treatment for adsorbing cadmium
<130>2016
<160>1
<170>PatentIn version 3.3
<210>1
<211>825
<212>DNA
<213> Acremonium sp.M 30CGMCC No.11007
<221>LSU
<400>1
aacggcgagt gaagcggcaa cagctcaaat ttgaaatctt ggcctcgtgc ccgagttgta 60
atttgtagag gatgcttttg gcgacgcgac ttccgagttc cctggaacgg gacgccatag 120
agggtgagag ccccgtccgg tcgtgcgcct agcctctgta aagctccttc gacgagtcga 180
gtagtttggg aatgctgatc taaatgggag gtatacgtct tctaaagcta aataccggcc 240
agagaccgat agcgcacaag tagagtgatc gaaagatgaa aagcactttg aaaagagggt 300
taagtagtac gtgaaattgc tgaaagggaa gcgcttatga ccagacttgg gcgcggcgga 360
tcatccggcg ttctcgccgg tgcactccac cgccccaggc cagcatcagt tcgcgccggg 420
ggacaaaggc ttcgggaatg tggctgcctc gggagtgtta tagcccgatg cgtaatacct 480
ggcgcggact gaggtccgcg ctctgcaagg atgctggcgt aatggtcatc agtgacccgt 540
cttgaaacac ggacccaagg agtcgtcttc gtatgcgagt gttcgggtgt caaaccccta 600
cgcggaatga aagtgaacgt aggagagagc ttcggcgcat ctccgaccga tcctgatgtt 660
ctgggatgga tttgagtaag agcatacggg gccggacccg aaagaaggtg aactatgcct 720
gtgtagggtg aagccagagg aaactctggt ggaggctcgc agcggttctg acgtgcaaat 780
cgatcgtcaa acatgggcat gggggcgaaa gactaatcga acctt 825
Claims (1)
1. Radiation-resistant Acremonium (A) LeptospiraAcremonium sp.) M30, wherein said Acremonium radiodurans (A)Acremonium sp.) The preservation number of the strain of M30 is CGMCC No. 11007.
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| CN107325980B (en) * | 2017-06-12 | 2020-06-16 | 新疆农业科学院微生物应用研究所（中国新疆-亚美尼亚生物工程研究开发中心） | Radiation-resistant paenibacillus KH9 and application thereof in biological antitranspirant |
| CN110343620B (en) * | 2019-07-01 | 2020-08-14 | 福建农林大学 | Plant rhizosphere fungus F9 capable of adsorbing cadmium ions and application thereof |
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