CN109504641B - Bacillus subtilis DDT98806 and application thereof - Google Patents
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Abstract
Bacillus subtilis DDT98806 which is preserved in China center for type culture Collection of Wuhan university in 2018, 10 and 23 months with the preservation number of CCTCC M2018706 and the preservation address of Wuhan university in Wuhan City, Hubei province. The application of the bacillus subtilis DDT98806 in preparing the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum and the application of the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum in repairing organic polluted soil containing dichlorodiphenyl trichloroethane, naphthalene, fluorene, phenanthrene, anthracene and fluoranthene. The biodegradation rate of the soil polluted by the polycyclic aromatic hydrocarbon and the dichlorodiphenyl trichloroethane is improved, and the repair period is obviously shortened.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus subtilis DDT98806 and application thereof.
Background
Polycyclic aromatic hydrocarbons are organic compounds composed of two or more benzene rings arranged in a linear, zigzag, or clustered form, are the carcinogens which have been found at the earliest and in the largest quantities, and are emitted to the atmosphere worldwide in about several tens of thousands of tons every year. Due to self hydrophobicity and low water solubility, the substances can be quickly deposited in the environment, and along with the increase of the number of benzene rings, the fat solubility is enhanced, the existence time in the environment is prolonged, and the genetic toxicity is increased. The DDT is a chlorinated derivative, belongs to a typical organochlorine pesticide, is mainly used as an insecticide, has a wide insecticidal spectrum and long residual effect, particularly has an obvious effect on cotton bollworms, armyworms and pink bollworms, and is widely used on cotton, fruits and vegetables. However, the snivel is not easily decomposed under natural conditions, has the residual period of 4-30 years in soil, is insoluble in water, is heat-resistant and acid-resistant, and has the characteristics of lipid solubility, biotoxicity, biological accumulation and the like, so that the snivel is forbidden in China for many years, but the snivel is widely existed in the environments such as soil and water systems, can be detected in the air, soil, rivers, underground water, plants and wild animals, and the residual concentration still remains high and far exceeds the corresponding national pollution standard.
At present, the treatment methods of polycyclic aromatic hydrocarbon pollution and DDT pollution reported at home and abroad comprise physical, chemical and biological methods. The physical and chemical treatment costs are high, secondary pollution is easy to cause, and the soil and the like are interfered and destroyed. The biological treatment technology has the advantages of low cost, high efficiency and generally little secondary pollution.
In the technical field of remediation of soil polluted by polycyclic aromatic hydrocarbon and dichlorodiphenyl trichloroethane organic matters, a technical problem which is urgently solved at present is to provide a microbial agent which has a good remediation effect and does not have secondary pollution.
Disclosure of Invention
The technical problem to be solved by the present invention is to overcome the disadvantages of the prior art and to provide a Bacillus subtilis DDT 98806.
The invention aims to solve another technical problem of providing a new application of the bacillus subtilis DDT 98806.
The technical scheme for solving the technical problems is as follows:
a bacillus subtilis DDT98806 is preserved in China type culture collection center of Wuhan university at 10 months and 23 days in 2018, the preservation number is CCTCCM 2018706, the preservation address is Wuhan university in Wuhan City, Hubei province, and the sequence is as follows:
the application of the bacillus subtilis DDT98806 in preparing a bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum is as follows: 1g of the solid complex microbial inoculum contains 1.5X 10 colonies9～4.5×109CFU Bacillus subtilis DDT98806 with colony number of 2.5 × 109～5.5×109CFU/g of Aspergillus terreus DDT 98801.
The bacillus subtilis DDT98806 bacterial colony is grey white, round, wrinkled and rough.
Aspergillus terreus (Aspergillus terreus) DDT98801, the preservation date is 11 months and 22 days in 2017, the preservation unit is China center for type culture Collection, the strain preservation number is CCTCC No: m2017710, deposited at Wuhan university, Wuhan city, Hubei, is disclosed in Chinese patent publication No. CN 108130281A.
The colony of the aspergillus terreus DDT98801 is earthy yellow, white at the edge, villous and slightly convex at the central part, fine radial furrows on the surface and dark yellow at the back. As the incubation time increased, the villous projections gradually flattened to form powdery spores. Conidiophores are short and colorless, the spore-forming structure is double-layered and grows on the upper half part of the hemispherical apical sac, the conidiophore heads begin to be spherical and then are in a compact cylindrical shape, and the conidiophore is spherical and has the diameter of 2-2.5 mu m.
Application of the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum in repairing contaminated soil containing dichlorodiphenyl trichloroethane. The using method comprises the following steps:
(1) pretreating the polluted soil
Breaking the mixed organic contaminated soil containing the dichlorodiphenyl trichloroethane, fully and uniformly mixing, and isolating the mixed organic contaminated soil containing the dichlorodiphenyl trichloroethane with the surrounding unpolluted soil by using a plastic film.
(2) Preparation of solid composite microbial inoculum of bacillus and aspergillus terreus
The number of colonies was 2.5X 109～5.5×109Preparation method of CFU/g aspergillus terreus DDT98801 microbial inoculumThe following were used:
the number of colonies prepared according to the conventional method was 1.5X 109～4.5×109CFU/g of a bacillus DDT98806 solid microbial inoculum; the number of colonies prepared was 2.5X 109～5.5×109The solid microbial inoculum of the aspergillus terreus DDT98801 in a ratio of CFU/g to the aspergillus terreus is 1: 1 mixing to prepare the bacillus and aspergillus terreus solid complex microbial inoculum.
The number of the above colonies was 2.5X 109～5.5×109The preparation method of the Aspergillus terreus microbial inoculum of CFU/g is as follows:
collecting urea 5g, yeast powder 2g, NaCl0.4g, KH2PO4 10g、MgSO4·7H2O 5g、FeSO4·7H2O 0.3g、ZnSO4·7H2O 0.3g、K2HPO4 5g、CaCl20.004g, adding distilled water, stirring for dissolving, adjusting the pH to 6.5, and fixing the volume to 1000mL by using distilled water to prepare an aspergillus terreus liquid culture medium; the concentration of the mixture is 2.0 to 5.0 multiplied by 10 by using sterilized distilled water6Adding 20mL of spore suspension into the Aspergillus terreus culture medium, and culturing at 28 deg.C and 160 rpm for 3 days;
uniformly mixing bran and turf according to a mass ratio of 3:7, adding a phosphoric acid buffer solution into the mixture for soaking, adjusting the pH to 6.0, sterilizing at 121 ℃ for 30 minutes, mixing the bran-turf and a liquid microbial inoculum according to a mass ratio of 10:1, adding the phosphoric acid buffer solution to ensure that the water content is 20%, and culturing at 28 ℃ for 8 days to prepare an aspergillus terreus DDT98801 solid composite microbial inoculum for later use;
adding distilled water into 5g of urea, 2g of yeast powder, 0.4g of NaCl, KH2PO 410 g, 4.7H 2O 5g of MgSO, 0.3g of 4.7H 2O 0.3, 0.3g of ZnSO 4.7H 2O 0.3, 0.005g of MnSO 4.H 2O 0.005, K2HPO 45 g, CaCl 20.004g, 17mL of ethanol, 50mL of glycerol and 802 mL of Tween, stirring and dissolving, adjusting the pH to 6.5, fixing the volume to 1000mL by using the distilled water, and preparing an Aspergillus terreus culture medium; prepared with distilled water to a concentration of 2.0-5.0 × 106spores/mL spore suspension, 20mL spore suspension was added to the Aspergillus terreus medium, and cultured at 28 ℃ and 160r/min for 3 days.
Uniformly mixing bran and turf according to a mass ratio of 3:7, adding a phosphoric acid buffer solution into the mixture for soaking, measuring a supernatant, adjusting the pH value to 6.0, sterilizing at 121 ℃ for 30 minutes, adding the cultured liquid microbial inoculum according to a mass ratio of the bran turf to the liquid microbial inoculum of 10:1, adding the phosphoric acid buffer solution to enable the water content of the liquid microbial inoculum to be 20%, and culturing at 28 ℃ for 8 days to prepare a solid composite microbial inoculum of bacillus subtilis DDT98806 and aspergillus terreus DDT98801 for later use.
(3) Bioremediation of contaminated soil containing dichlorodiphenyl trichloroethane
Respectively inoculating the bacillus subtilis and aspergillus terreus solid composite microbial inoculum into the soil pretreated in the step (1), wherein the inoculation amount of the bacillus subtilis and aspergillus terreus composite microbial inoculum is 25-75 g/kg, and then adding urea and K2HPO4/KH2PO4And organic fertilizer, so that the content of C in soil is as follows: n: the mass ratio of the P element is 100: 10:1, adjusting the pH value to 4.5-6.5, adding water until the water content of the soil reaches 15% -30%, uniformly mixing, covering the surface of the polluted soil with a plastic film, ventilating, maintaining the water content of the soil stable, and treating for 21-35 days at 25-30 ℃.
In the step (3) of repairing the contaminated soil containing the dichlorodiphenyl trichloroethane, the soil pretreated in the step (1) is respectively inoculated with the bacillus and aspergillus terreus solid composite microbial inoculum, the best inoculation amount of the bacillus and aspergillus terreus composite microbial inoculum is 50g/kg, and then urea and K are added2HPO4And organic fertilizer, so that the content of C in soil is as follows: n: the mass ratio of the P element is 100: 10:1, adding water until the water content of the soil is optimally 20%, uniformly mixing, adjusting the pH value to be 4.5-6.5, covering the surface of the polluted soil with a plastic film, ventilating, maintaining the water content of the soil stable, and treating for 21-35 days at 25-30 ℃.
Application of a bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum in repairing soil containing naphthalene pollution. The application method is the same as the remediation method of the contaminated soil containing the dichlorodiphenyl trichloroethane.
The application of the composite microbial inoculum of the bacillus subtilis DDT98806 and the aspergillus terreus DDT98801 in repairing soil polluted by fluorene. The application method is the same as the remediation method of the contaminated soil containing the dichlorodiphenyl trichloroethane.
The application of the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum in repairing soil containing phenanthrene pollution. The application method is the same as the remediation method of the contaminated soil containing the dichlorodiphenyl trichloroethane.
The application of the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum in repairing soil containing anthracene pollution. The application method is the same as the remediation method of the contaminated soil containing the dichlorodiphenyl trichloroethane.
Application of bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum in remediation of soil polluted by fluoranthene. The application method is the same as the remediation method of the contaminated soil containing the dichlorodiphenyl trichloroethane.
The invention has the following beneficial effects:
the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum is adopted to carry out remediation experiments on DDT, naphthalene, fluorene, phenanthrene, anthracene and fluoranthene contaminated soil, and the experimental results show that: after 21-35 days, the degradation rate of the dichlorodiphenyl trichloroethane in the polluted soil is 80.99%, and the degradation rates of naphthalene, fluorene, phenanthrene, anthracene and fluoranthene are respectively 89.00%, 69.50%, 71.81%, 75.66% and 63.22%. The degradation rate of the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum on the polluted soil remediation is improved by 30 percent compared with the degradation rate of a single microbial inoculum on the polluted soil remediation.
Drawings
FIG. 1 is a graph showing the degradation of polycyclic aromatic hydrocarbons and dichlorodiphenyl trichloroethane in soil by Bacillus DDT98806 over time.
FIG. 2 is a graph of the degradation of polycyclic aromatic hydrocarbons and dichlorodiphenyl trichloroethane in soil by Aspergillus terreus DDT98801 over time.
FIG. 3 is a graph showing the degradation of polycyclic aromatic hydrocarbons and dichlorodiphenyl trichloroethane in soil by the composite microbial inoculum of bacillus DDT98806 and aspergillus terreus DDT 98801.
Detailed Description
The invention will be further described with reference to the following figures and examples, but the scope of protection of the invention is not limited to these examples.
Example 1
A strain of Bacillus subtilis DDT98806 is preserved in China center for type culture collection of Wuhan university in 2018, 10 months and 23 days, with the preservation number of CCTCCM 2018706 and the preservation address of Wuhan university in Wuhan City, Hubei province.
The extraction method of the bacillus subtilis DDT98806 comprises the following steps:
(1) enrichment of
Weighing a soil sample collected from a ditch of a pesticide factory in Jiaxing city of Zhejiang province, China, and adding the soil sample into an enrichment liquid culture medium, wherein the mass-to-volume ratio of the soil sample to the enrichment liquid culture medium is 1: culturing in a constant temperature shaking table at 37 ℃ at the speed of 10, 150r/min for 48 hours by oscillation to obtain a culture solution;
the enriched liquid culture medium is prepared by adding 15mg DDT into per liter of inorganic salt liquid culture medium containing (NH) per liter of distilled water4)2SO4 0.2g、KH2PO4 1.0g、K2HPO4 1.0g、CaCl2 0.08g、FeSO4 0.002g、MgSO40.4g, and adjusting the pH value to 7.0;
(2) separating and purifying
Heating the culture solution obtained in the step (1) in a water bath at 80 ℃ for 15 minutes, and performing gradient dilution by using sterile water to sequentially obtain dilutions 101、102、103、104、105、106Absorbing 100 mu L of bacterial suspensions with different dilution times by using a pipette, respectively coating the bacterial suspensions on an enrichment solid culture medium by using a coating rod, wherein the enrichment solid culture medium contains 20g/L of agar, each bacterial suspension with different dilution times is subjected to three repetitions, culturing the bacterial suspensions in a constant-temperature incubator at 37 ℃, selecting single bacterial colonies with rapid growth and single color by using an inoculating loop, streaking and culturing the single bacterial colonies in a beef extract peptone culture medium plate, culturing the single bacterial colonies in the constant-temperature incubator at 37 ℃ for 1-2 days, repeating the operation, repeatedly carrying out separation culture until purified bacterial strains are obtained, and combining the same bacterial strains according to the bacterial colony morphology, the growth speed and the microscopic morphology of the purified bacterial strains;
(3) re-screening of purified strain
And (3) streaking the strain purified in the step (2) on an inorganic salt solid culture medium containing 20mg/L of dichlorodiphenyl trichloroethane, culturing the strain in a constant temperature incubator at 37 ℃ for 1-2 days, selecting a single colony with good growth, and identifying and naming the single colony as bacillus subtilis DDT 98806.
Example 2
The application of the bacillus subtilis DDT98806 in the embodiment 1 in preparing a bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum is as follows: 1g of the solid complex microbial inoculum contains 1.5X 10 colonies9～4.5×109CFU Bacillus subtilis DDT98806 with colony number of 2.5 × 109～5.5×109CFU/g of Aspergillus terreus DDT 98801. Aspergillus terreus (Aspergillus terreus) DDT98801, the preservation date is 11 months and 22 days in 2017, the preservation unit is China center for type culture Collection, the strain preservation number is CCTCC No: m2017710, deposited at Wuhan university, Wuhan city, Hubei, is disclosed in Chinese patent publication No. CN 108130281A.
The number of colonies prepared according to the conventional method was 1.5X 109～4.5×109CFU/g of a bacillus DDT98806 solid microbial inoculum; the number of colonies prepared was 2.5X 109～5.5×109CFU/g Aspergillus terreus DDT98801 solid microbial inoculum, wherein the mass ratio of a bacillus microbial inoculum to an Aspergillus terreus microbial inoculum is 1: 1 mixing to prepare the bacillus and aspergillus terreus solid complex microbial inoculum.
The number of colonies prepared was 2.5X 109～5.5×109The method of the CFU/g aspergillus terreus DDT98801 microbial inoculum is as follows:
collecting urea 5g, yeast powder 2g, NaCl0.4g, KH2PO4 10g、MgSO4·7H2O 5g、FeSO4·7H2O 0.3g、ZnSO4·7H2O 0.3g、K2HPO4 5g、CaCl20.004g, adding distilled water, stirring for dissolving, adjusting the pH to 6.5, and fixing the volume to 1000mL by using distilled water to prepare an aspergillus terreus liquid culture medium; the concentration of the mixture is 2.0 to 5.0 multiplied by 10 by using sterilized distilled water6Adding 20mL of spore suspension into the Aspergillus terreus culture medium, and culturing at 28 deg.C and 160 rpm for 3 days;
uniformly mixing bran and turf according to a mass ratio of 3:7, adding a phosphoric acid buffer solution into the mixture for soaking, adjusting the pH to 6.0, sterilizing at 121 ℃ for 30 minutes, mixing the bran-turf and a liquid microbial inoculum according to a mass ratio of 10:1, adding the phosphoric acid buffer solution to ensure that the water content is 20%, and culturing at 28 ℃ for 8 days to prepare an aspergillus terreus DDT98801 solid composite microbial inoculum for later use;
collecting urea 5g, yeast powder 2g, NaCl0.4g, KH2PO4 10g、MgSO4·7H2O 5g、FeSO4·7H2O 0.3g、ZnSO4·7H2O 0.3g、MnSO4·H2O 0.005g、K2HPO4 5g、CaCl20.004g, 17mL of ethanol, 50mL of glycerol and 802 mL of tween, adding distilled water, stirring and dissolving, adjusting the pH to 6.5, and diluting to 1000mL by using distilled water to prepare an aspergillus terreus culture medium; preparing with distilled water to a concentration of 2.0 × 106～5.0×106spores/mL of spore suspension, 20mL of spore suspension was added to the Aspergillus terreus medium and cultured at 28 ℃ at 160 rpm for 3 days.
Uniformly mixing bran and turf in a mass ratio of 3:7, adding a phosphoric acid buffer solution into the mixture for soaking, measuring a supernatant, adjusting the pH to 6.0, sterilizing at 121 ℃ for 30 minutes, mixing the bran turf and a liquid microbial inoculum in a mass ratio of 10:1, adding the phosphoric acid buffer solution to ensure that the water content is 20%, and culturing at 28 ℃ for 8 days to prepare a bacillus subtilis DDT98806 and aspergillus terreus DDT98801 solid composite microbial inoculum for later use.
Example 3
Application of the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum in repairing contaminated soil containing dichlorodiphenyl trichloroethane. The using method comprises the following steps:
(1) pretreating the polluted soil
Breaking the polluted soil containing the dichlorodiphenyl trichloroethane, fully and uniformly mixing, and isolating the polluted soil and the surrounding unpolluted soil by using a plastic film.
(2) Repairing contaminated soil containing dichlorodiphenyl trichloroethane
The method for preparing the bacillus and aspergillus terreus solid complex microbial inoculum is the same as the example 2.
Respectively inoculating bacillus and aspergillus terreus solid compounds into the soil pretreated in the step (1)The inoculation amount of the combined bacterial agent is 50g/kg, and then urea and K are added2HPO4/KH2PO4Buffer solution and organic fertilizer, so that the ratio of C: n: the mass ratio of the P element is 100: 10:1, adjusting the pH value to 4.5-6.5, adding water until the water content of the soil is 20%, uniformly mixing, covering the surface of the polluted soil with a plastic film, ventilating, maintaining the water content of the soil stable, treating for 35 days at 25-30 ℃, wherein the specific treatment time is selected within 21-35 days according to the amount of pollutants contained in the polluted soil.
The bacillus and aspergillus terreus solid composite microbial inoculum is adopted to repair the contaminated soil containing the dichlorodiphenyl trichloroethane, and the degradation rate is 80.99%.
Example 4
The method for preparing the bacillus and aspergillus terreus solid complex microbial inoculum is the same as the example 2.
Application of a bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum in repairing soil containing naphthalene pollution. The application method is the same as the remediation method of the contaminated soil containing the dichlorodiphenyl trichloroethane.
The solid composite microbial inoculum of bacillus and aspergillus terreus in the embodiment is adopted to repair the soil polluted by naphthalene, and the degradation rate is 89.00%.
Example 5
The method for preparing the bacillus and aspergillus terreus solid complex microbial inoculum is the same as the example 2.
The application of the composite microbial inoculum of the bacillus subtilis DDT98806 and the aspergillus terreus DDT98801 in repairing soil polluted by fluorene. The application method is the same as the remediation method of the contaminated soil containing the dichlorodiphenyl trichloroethane.
The solid composite microbial inoculum of bacillus and aspergillus terreus of the embodiment is adopted to repair the soil polluted by fluorene, and the degradation rate is 69.50%.
Example 6
The method for preparing the bacillus and aspergillus terreus solid complex microbial inoculum is the same as the example 2.
The application of the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum in repairing soil containing phenanthrene pollution. The application method is the same as the remediation method of the contaminated soil containing the dichlorodiphenyl trichloroethane.
The bacillus and aspergillus terreus solid complex microbial inoculum of the embodiment is adopted to repair the soil polluted by phenanthrene, and the degradation rate is 71.81%.
Example 7
The method for preparing the bacillus and aspergillus terreus solid complex microbial inoculum is the same as the example 2.
The application of the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum in repairing soil containing anthracene pollution. The application method is the same as the remediation method of the contaminated soil containing the dichlorodiphenyl trichloroethane.
The solid composite microbial inoculum of bacillus and aspergillus terreus of the embodiment is adopted to repair the soil polluted by anthracene, and the degradation rate is 75.66%.
Example 8
The method for preparing the bacillus and aspergillus terreus solid complex microbial inoculum is the same as the example 2.
Application of bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum in remediation of soil polluted by fluoranthene. The application method is the same as the remediation method of the contaminated soil containing the dichlorodiphenyl trichloroethane.
The bacillus and aspergillus terreus solid composite microbial inoculum of the embodiment is adopted to repair the fluoranthene-contaminated soil, and the degradation rate is 63.22%.
Example 9
The method for preparing the bacillus and aspergillus terreus solid complex microbial inoculum is the same as the example 2.
In the step (2) of repairing the contaminated soil with dichlorodiphenyl trichloroethane in the above examples 3-8, the soil pretreated in the step (1) is respectively inoculated with the bacillus and aspergillus terreus solid composite microbial inoculum, the inoculation amount of the bacillus and aspergillus terreus composite microbial inoculum is 25g/kg, and then urea and K are added2HPO4/K2HPO4Buffer solution and organic fertilizer, so that the ratio of C: n: the mass ratio of the P element is 100: 10:1, adjusting the pH value to 4.5-6.5, adding water until the water content of the soil reaches 15%, uniformly mixing, covering the surface of the polluted soil with a plastic film, ventilating, and maintaining the soilThe water content of the soil is stable, and the soil is treated for 21-35 days at 25-30 ℃.
The other steps were the same as in example 2.
Example 10
The method for preparing the bacillus and aspergillus terreus solid complex microbial inoculum is the same as the example 2.
In the step (2) of repairing the soil polluted by the dichlorodiphenyl trichloroethane and the polycyclic aromatic hydrocarbon in the above examples 3-8, the soil pretreated in the step (1) is respectively inoculated with the bacillus and aspergillus terreus solid composite microbial inoculum, the inoculation amount of the bacillus and aspergillus terreus composite microbial inoculum is 75g/kg, and then the urea and the K are added2HPO4And organic fertilizer, so that the content of C in soil is as follows: n: the mass ratio of the P element is 100: 10:1, adjusting the pH value to 4.5-6.5, adding water until the water content of the soil reaches 30%, uniformly mixing, covering the surface of the polluted soil with a plastic film, ventilating, maintaining the water content of the soil stable, and treating for 21-35 days at 25-30 ℃.
The other steps were the same as in example 2.
In order to verify the beneficial effects of the invention, the inventor uses the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 solid complex microbial inoculum (referred to as complex microbial inoculum for short) prepared in the embodiment 2 of the invention to carry out a comparative experiment with the bacillus subtilis DDT98806 (referred to as bacillus subtilis for short) and the aspergillus terreus DDT98801 (referred to as aspergillus terreus for short), and the experimental conditions are as follows:
1. the composite microbial inoculum, the bacillus subtilis and the aspergillus terreus are adopted for the degradation contrast experiment of soil containing dichlorodiphenyl trichloroethane and organic pollutants, the bacillus subtilis is used for experiment 1, the aspergillus terreus is used for experiment 2, the composite microbial inoculum is used for experiment 3, and no microbial inoculum is added, so that experiment 4 (a control group) is obtained.
Taking clean soil without organic pollutants, naturally drying in the shade, sieving by a 1mm sieve, and removing large substances in the soil. Sterilizing at 121 deg.C for 20 min, packaging 100g of the above sterilized soil in 250mL triangular flask, adding dichlorodiphenyl trichloroethane to make its concentration 20mg/kg, and adding polycyclic aromatic hydrocarbons naphthalene, fluorene, phenanthrene, anthracene, and fluoranthene to make its concentration 80 mg/kg. The experiment was carried out according to the procedure of example 2. The contents of dichlorodiphenyl trichloroethane and polycyclic aromatic hydrocarbon in each treatment group were determined by GC-MS. Wherein each group of experiments is repeated three times, the average value is taken, and the degradation rate is calculated according to the following formula:
the results of the experiments and calculations are shown in table 1.
TABLE 1 conditions of the treatment groups and their degradation efficiency
The experimental results show that: after 35 days of treatment, the bacillus subtilis DDT98806 microbial inoculum has the degradation efficiencies of 60.00 +/-2.5%, 40.22 +/-2.4%, 48.1 +/-1.0%, 50.11 +/-1.9% and 37.05 +/-1.0% on naphthalene, fluorene, phenanthrene, anthracene and fluoranthene in soil and 42.55 +/-1.9% on dichlorodiphenyl trichloroethane respectively. The degradation efficiency of the aspergillus terreus DDT98801 microbial inoculum to naphthalene, fluorene, phenanthrene, anthracene and fluoranthene in soil is respectively 70.00 +/-3.5%, 50.1 +/-2.9%, 53.5 +/-1.5%, 69.11 +/-3.6% and 52.05 +/-2.1%, and the degradation efficiency to dichlorodiphenyl trichloroethane is 60 +/-1.9%. The degradation efficiency of the composite microbial inoculum of the bacillus subtilis DDT98806 and the aspergillus terreus DDT98801 to naphthalene, fluorene, phenanthrene, anthracene and fluoranthene in soil is respectively 89.00%, 69.50%, 71.81%, 75.66% and 63.22%, and the degradation efficiency to dichlorodiphenyl trichloroethane is 80.99%. Compared with a single microbial inoculum, the compound microbial inoculum of aspergillus terreus DDT98801 and bacillus DDT98806 used in the invention has the advantage that the degradation efficiency of polycyclic aromatic hydrocarbon and dichlorodiphenyl trichloroethane is improved by 30%.
2. Influence of inoculation amount of composite microbial inoculum on degradation rate of soil containing pollutants
Different addition amounts of the bacillus subtilis and aspergillus terreus composite microbial inoculum adopted in the experiment are respectively experiment 1, experiment 2, experiment 3 and experiment 4, and the inoculation amounts of experiment 1, experiment 2, experiment 3 and experiment 4 are respectively 50, 40, 25 and 75, which is shown in table 2.
Taking clean soil without organic pollutants, naturally drying in the shade, sieving by a 1mm sieve, and removing large substances in the soil. Sterilizing at 121 deg.C for 20 min, packaging 100g of the above sterilized soil in a 250mL triangular flask, adding dichlorodiphenyl trichloroethane to make its concentration 20mg/kg, and adding naphthalene, fluorene, phenanthrene, anthracene, and fluoranthene to make its concentration 80 mg/kg. The experiment was carried out according to the procedure of example 2. The contents of dichlorodiphenyl trichloroethane and polycyclic aromatic hydrocarbon in each treatment group were determined by gas chromatography mass spectrometry. Wherein each group of experiments is repeated three times, the average value is taken, and the degradation rate is calculated according to the following formula:
the results of the experiments and calculations are shown in table 2.
TABLE 2 influence of inoculum size of complex inoculum on degradation efficiency
The experimental results show that: when the inoculation amount of the composite microbial inoculum is 50g/kg, the degradation effect on organic pollutants in soil is the best.
In conclusion, the composite microbial inoculum of the bacillus subtilis DDT98806 and the aspergillus terreus DDT98801 has obviously improved degradation efficiency on organic polluted soil, and particularly has better degradation effect on the DDT which is difficult to degrade.
Sequence listing
<110> Jiaxing Yifang environmental science and technology Limited
<120> Bacillus subtilis DDT98806 and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1486
<212> DNA
<213> Bacillus subtilis
<400> 1
cccaagggtt tttcctatac tgcaagtcga gcggacagat gggagcttgc tccctgatgt 60
tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg 120
gaaaccgggg ctaataccgg atgcttgttt gaaccgcatg gttcaaacat aaaaggtggc 180
ttcggctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc 240
accaaggcaa cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360
cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa 420
gaacaagtac cgttcgaata gggcggtacc ttgacggtac ctaaccagaa agccacggct 480
aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg 540
cgtaaagggc tcgcaggcgg ttccttaagt ctgatgtgaa agcccccggc tcaaccgggg 600
agggtcattg gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag 660
cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta 720
actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac 780
gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg 840
cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg 900
ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960
gtcttgacat cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag 1020
gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080
gcaacccttg atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac 1140
aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200
acgtgctaca atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa 1260
atctgttctc agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt 1320
aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380
caccacgaga gtttgtaaca cccgaagtcg gtgaggtaac ctttatggag ccagccgccg 1440
aaggtgggac agatgattgg ggtgaagtcg aaaccaatga agcccg 1486
<210> 2
<211> 570
<212> DNA
<213> Aspergillus terreus (Aspergillus terreus)
<400> 2
gtggcatcga gtcggtctcg tggccacctc ccacccgtga ctattgtacc ttgttgcttc 60
ggcgggcccg ccagcttgct ggccgccggg gggcgtctcg cccccgggcc cgtgcccgcc 120
ggagacccca acatgaaccc tgttctgaaa gcttgcagtc tgagttgtga ttctttgcaa 180
tcagttaaaa ctttcaacaa tggatctctt ggttccggca tcgatgaaga acgcagcgaa 240
atgcgataac taatgtgaat tgcagaattc agtgaatcat cgagtctttg aacgcacatt 300
gcgccccctg gtattccggg gggcatgcct gtccgagcgt cattgctgcc ctcaagcccg 360
gcttgtgtgt tgggtcctcg tcccccggct cccgggggac gggcccgaaa ggcagcggcg 420
gcaccgcgtc cggtcctcga gcgtatgggg ctttgtcttc cgctctgtag gcccggccgg 480
cgcccgccga cgcatttttt tgcaacttgt ttttttccag gttgacctcg gatcaggtag 540
ggatacccgc tgaacttaag catatcaaaa 570
Claims (10)
1. Bacillus subtilis (B.subtilis)Bacillus subtilis) DDT98806, deposited in the China center for type culture Collection of Wuhan university at 23.10.2018, with the collection number of CCTCC NO: m2018706, the preservation address is Wuhan university in Wuhan city, Hubei province.
2. Use of Bacillus subtilis DDT98806 as claimed in claim 1 for the preparation of Bacillus subtilis DDT98806 and Aspergillus terreus (Aspergillus terreus)Aspergillus terreus) The application of the DDT98801 composite bacterial agent is characterized in that the composite bacterial agent is as follows: in 1g of solid composite microbial inoculum, the colony count of the raw material is 1.5 multiplied by 109～4.5×109CFU Bacillus subtilis DDT98806 with colony number of 2.5 × 109～5.5×109CFU/g of Aspergillus terreus DDT 98801.
3. The use of the composite microbial inoculum of the bacillus subtilis DDT98806 and the aspergillus terreus DDT98801 in the remediation of the soil polluted by the dichloro-diphenyl-trichloroethane.
4. The use of the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum for repairing contaminated soil containing dichlorodiphenyl trichloroethane according to the claim 3, characterized in that the use method thereof comprises the following steps:
(1) pretreating the polluted soil
Breaking the mixed organic contaminated soil containing the dichlorodiphenyl trichloroethane, fully and uniformly mixing, and isolating the mixed organic contaminated soil containing the dichlorodiphenyl trichloroethane with the unpolluted soil around by using a plastic film;
(2) preparation of solid composite microbial inoculum of bacillus and aspergillus terreus
The number of the above colonies was 2.5X 109～5.5×109The preparation method of the Aspergillus terreus microbial inoculum of CFU/g is as follows:
collecting urea 5g, yeast powder 2g, NaCl0.4g, KH2PO4 10 g、MgSO4•7H2O 5g、FeSO4·7H2O 0.3 g、ZnSO4·7H2O 0.3 g、K2HPO4 5 g、CaCl20.004g, adding distilled water, stirring for dissolving, adjusting the pH to 6.5, and fixing the volume to 1000mL by using distilled water to prepare an aspergillus terreus liquid culture medium; the concentration of the mixture is 2.0 to 5.0 multiplied by 10 by using sterilized distilled water6Adding 20mL of spore suspension into the Aspergillus terreus culture medium, and culturing at 28 deg.C and 160 rpm for 3 days;
uniformly mixing bran and turf according to a mass ratio of 3:7, adding a phosphoric acid buffer solution into the mixture for soaking, adjusting the pH to 6.0, sterilizing at 121 ℃ for 30 minutes, mixing the bran-turf and a liquid microbial inoculum according to a mass ratio of 10:1, adding the phosphoric acid buffer solution to ensure that the water content is 20%, and culturing at 28 ℃ for 8 days to prepare an aspergillus terreus DDT98801 solid composite microbial inoculum for later use;
collecting urea 5g, yeast powder 2g, NaCl0.4g, KH2PO4 10 g、MgSO4•7H2O 5g、FeSO4·7H2O 0.3 g、ZnSO4·7H2O 0.3 g、MnSO4·H2O 0.005 g、K2HPO4 5 g、CaCl20.004g, 17mL of ethanol, 50mL of glycerol and 802 mL of tween, adding distilled water, stirring and dissolving, adjusting the pH to 6.5, and diluting to 1000mL by using distilled water to prepare an aspergillus terreus culture medium; the concentration of the mixture is 2.0 to 5.0 multiplied by 10 by using distilled water6Adding 20mL of spore suspension into an Aspergillus terreus culture medium, and culturing at 28 ℃ and 160r/min for 3 days;
uniformly mixing bran and turf in a mass ratio of 3:7, adding a phosphate buffer solution into the mixture for soaking, measuring a supernatant, adjusting the pH to 6.0, sterilizing at 121 ℃ for 30 minutes, mixing the turf of the bran and a liquid microbial inoculum in a mass ratio of 10:1, adding the phosphate buffer solution to ensure that the water content is 20 percent, and culturing at 28 ℃ for 8 days to prepare a solid composite microbial inoculum of bacillus subtilis DDT98806 and aspergillus terreus DDT98801 for later use;
(3) bioremediation of DDT contaminated soil
Inoculating a bacillus and aspergillus terreus solid composite microbial inoculum into the soil pretreated in the step (1), wherein the inoculation amount of the bacillus and aspergillus terreus composite microbial inoculum is 25-75 g/kg, and then adding urea and K2HPO4∕KH2PO4 buffer solution and organic fertilizer, so that the ratio of C: n: the mass ratio of the P element is 100: 10:1, adding water until the water content of the soil reaches 15% -30% and the pH is 4.5-6.5, mixing uniformly, covering the surface of the polluted soil with a plastic film, ventilating, maintaining the water content of the soil stable, and treating for 21-35 days at 25-30 ℃.
5. The use of the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum according to claim 4 in repairing contaminated soil containing dichlorodiphenyl trichloroethane, wherein the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum comprises the following components in percentage by weight: in pairIn the step (3) of repairing contaminated soil containing dichlorodiphenyl trichloroethane, the soil pretreated in the step (1) is respectively inoculated with a bacillus and aspergillus terreus solid composite microbial inoculum, the inoculation amount of the bacillus and aspergillus terreus composite microbial inoculum is 50g/kg, and then urea and K are added2HPO4And organic fertilizer, so that the content of C in soil is as follows: n: the mass ratio of the P element is 100: 10:1, adding water until the water content of the soil reaches 20% and uniformly mixing, covering the surface of the polluted soil with a plastic film, ventilating, maintaining the water content of the soil stable, and treating for 21-35 days at 25-30 ℃.
6. The use of the composite microbial inoculum of bacillus subtilis DDT98806 and aspergillus terreus DDT98801 in claim 2 for remediating soil polluted by naphthalene.
7. The use of the composite microbial inoculum of bacillus subtilis DDT98806 and aspergillus terreus DDT98801 in claim 2 for remediating fluorene-contaminated soil.
8. The use of the composite microbial inoculum of bacillus subtilis DDT98806 and aspergillus terreus DDT98801 in claim 2 for remediating phenanthrene-contaminated soil.
9. The use of the composite microbial inoculum of bacillus subtilis DDT98806 and aspergillus terreus DDT98801 in claim 2 for remediating the soil polluted by anthracene.
10. The use of the bacillus subtilis DDT98806 and aspergillus terreus DDT98801 composite microbial inoculum in claim 2 for remediating fluoranthene-contaminated soil.
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