CN107937321B - Bacillus subtilis and application thereof in degradation of polycyclic aromatic hydrocarbon pollutant phenanthrene - Google Patents
Bacillus subtilis and application thereof in degradation of polycyclic aromatic hydrocarbon pollutant phenanthrene Download PDFInfo
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- CN107937321B CN107937321B CN201810018261.4A CN201810018261A CN107937321B CN 107937321 B CN107937321 B CN 107937321B CN 201810018261 A CN201810018261 A CN 201810018261A CN 107937321 B CN107937321 B CN 107937321B
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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Abstract
The invention discloses bacillus subtilis and application thereof in degradation of phenanthrene as a polycyclic aromatic hydrocarbon pollutant, wherein the bacillus subtilis is classified and named as bacillus subtilis (Bacillus subtilis)Bacillus subtilis) ZL09-26 with the deposit number GDMCC No: 60293 the invention also provides an application of bacillus subtilis ZL09-26 in phenanthrene degradation, and provides a method for improving phenanthrene degradation efficiency by using small molecular organic acid. The bacillus subtilis ZL09-26 can realize degradation of polycyclic aromatic hydrocarbon pollutants (phenanthrene), and can improve the degradation efficiency of the bacillus subtilis ZL09-26 on the polycyclic aromatic hydrocarbon pollutants (phenanthrene) by using small-molecular organic acids (citric acid, oxalic acid and glutaric acid).
Description
Technical Field
The invention belongs to the technical field of microorganisms and environmental engineering, and particularly relates to bacillus subtilis and application thereof in degradation of phenanthrene as a polycyclic aromatic hydrocarbon pollutant.
Background
Polycyclic Aromatic Hydrocarbons (PAHs) are widely present in diesel exhaust gas, atmospheric particulates and soil in the industrial field, have harmful effects such as carcinogenesis, teratogenesis and mutagenesis, and are a worldwide research hotspot and difficult problem for removing polycyclic aromatic hydrocarbon pollutants for a long time. With the increasing development and demand for petroleum worldwide, more and more polycyclic aromatic hydrocarbons are released into the natural environment. The U.S. Environmental Protection Agency (EPA) lists 16 of PAHs as pollutants for preferential control in the environment, and phenanthrene, which is a persistent organic pollutant in the 16 PAHs, is often used as a model compound for research on biodegradation of PAHs.
The treatment method of polycyclic aromatic hydrocarbon pollution mainly comprises three main categories: physical, chemical and biological methods. Physical and chemical methods have relatively quick response, but have high cost and are easy to generate secondary pollution. The biological method has the advantages of low cost, high efficiency, small damage to the environment, no secondary pollution and the like, is favored, and has good application prospect. At present, although the research on the biodegradation of polycyclic aromatic hydrocarbons is more, the degradation efficiency is low, the speed is slow, and the like, so that the method for rapidly improving the biodegradation of PAHs is particularly urgent.
Small molecular organic acid is an important active substance in soil environment, and mainly comes from secretion of plant root systems, decomposition of animal and plant residues, secretion and synthesis of microorganisms, conversion of organic matters in soil and the like. The small molecular organic acid in the soil has important environmental effect, has certain influence on the migration, the transformation and the degradation of organic pollutants besides the influence on the migration, the toxicity and the biological effectiveness of heavy metals in the soil, and has different action mechanisms. Some of these organic acids have special functions, such as affecting the dissolution of minerals in soil, regulating plant nutrition; the fertilizer acts with pollutants in soil, and the stress effect of the pollutants is reduced; provides energy and carbon sources for the growth of the microorganisms, further maintains the activity of rhizosphere microorganisms, creates good external environment for the growth of roots and the like. Meanwhile, small molecular organic acids are secreted in the growth and metabolism process of microorganisms in soil, and the small molecular organic acids can influence the growth of plant rhizosphere to different degrees. Different soil organic matter contents and organic matter types have great influence on the generation, degradation and conversion of small molecular organic matters.
In order to improve the bioremediation efficiency of the polycyclic aromatic hydrocarbon-phenanthrene polluted soil, the application basic research needs to be started from the following 2 aspects: (1) the bioavailability of the polycyclic aromatic hydrocarbon (phenanthrene) is improved; (2) and searching feasible microbial energy sources. In the aspect of improving the bioavailability of polycyclic aromatic hydrocarbon-phenanthrene, researchers do much work, mainly adopt a mode of adding a surfactant, but few researchers take small-molecular organic acids as effective means for improving the degradation efficiency of polycyclic aromatic hydrocarbon (phenanthrene) for research. In view of the above, it is necessary to find a strain capable of degrading polycyclic aromatic hydrocarbons existing in natural environment and greatly improving degradation efficiency after adding small molecular acid.
Disclosure of Invention
The invention aims to provide bacillus subtilis for polycyclic aromatic hydrocarbon pollutants (phenanthrene).
The bacillus of the invention is from petroleum pollution area of Shengli oil field, and is classified and named as bacillus subtilis (B)Bacillus subtilis) ZL09-26 with the deposit number GDMCC No: 60293 deposited in the culture Collection of microorganisms of Guangdong province (GDMCC, address: No. 59, No. 5, of Mieli Zhou 100, Guangzhou city), the preservation date is: 12 months and 1 day 2017.
The bacillus ZL09-26 provided by the invention has the following characteristics:
1) colony characteristics: the colonies are round and white or yellowish.
2) The morphological characteristics of the cells are as follows: the cells are oval to cylindrical and slightly curved, and the size of the cells is about (0.7-0.8) × (2-3) μm; gram staining was positive. Without capsule, the perigenic flagellum can move.
The invention also aims to provide application of the bacillus subtilis ZL09-26 in degradation of polycyclic aromatic hydrocarbon pollutants.
The invention also provides a preferable technical scheme, and the degradation efficiency of the bacillus subtilis ZL09-26 on polycyclic aromatic hydrocarbon pollutants (phenanthrene) is improved by using small molecular organic acids (citric acid, oxalic acid and glutaric acid).
The bacillus subtilis ZL09-26 can realize degradation of polycyclic aromatic hydrocarbon pollutants (phenanthrene), and can adjust cell metabolism of the bacillus subtilis ZL09-26 by a culture method of adding small molecular organic acid, so that the degradation efficiency of the bacillus subtilis ZL09-26 on the polycyclic aromatic hydrocarbon pollutants (phenanthrene) is improved.
Drawings
FIG. 1 shows the optimum pH value of Bacillus subtilis ZL09-26 for degrading phenanthrene which is a polycyclic aromatic hydrocarbon pollutant;
FIG. 2 shows the optimum temperature for Bacillus subtilis ZL09-26 to degrade phenanthrene which is a polycyclic aromatic hydrocarbon pollutant;
FIG. 3 shows the optimal substrate concentration of Bacillus subtilis ZL09-26 for degrading phenanthrene, a polycyclic aromatic hydrocarbon pollutant;
FIG. 4 shows degradation of PAHs-phenanthrene with and without small organic acid;
the biological material of the invention is classified and named as bacillus subtilisBacillus subtilis) ZL09-26 with the deposit number GDMCC No: 60293 deposited in the culture collection of microorganisms of Guangdong province (GDMCC), with a date of 12 months and 1 day 2017 and a deposit address of: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Miehu 100, Mingzhou, Junior.
Detailed Description
The LB medium used in the examples had the following composition: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and distilled water to reach the constant volume of 1000 mL.
The inorganic salt culture medium comprises the following components: 1.0 g of ammonium nitrate, 0.5 g of potassium dihydrogen phosphate, 1.5 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.02g of anhydrous calcium chloride, 0.5 g of ferric trichloride and distilled water with the constant volume of 1000 mL.
Example 1
This example specifically illustrates a method for isolation, selection and culture of Bacillus subtilis ZL09-26, comprising the steps of:
(1) 5g of soil from the petroleum-contaminated area of the Shengli oil field was placed in 250 mL of Erlenmeyer flask containing Luria-Bertani (LB) medium and cultured at 37 ℃ and 180rpm for 10 to 12 hours. Then transferring 5mL of the culture solution into an inorganic salt culture medium containing 200mg/L phenanthrene, performing acclimation culture at 37 ℃ and 180rpm for four periods, wherein each period is 5d, and taking the culture solution as 10 after the enrichment culture is finished-2、10-3、10-4And (3) gradient dilution, namely respectively coating 0.1 mL of the diluted solution on a phenanthrene-containing inorganic salt culture medium plate, repeating each gradient for 3 times, and culturing in a constant-temperature incubator at 25 ℃ for 48 hours. After the colonies grow out, the colonies with different shapes, sizes, colors and the like are selected and streaked on corresponding plates respectively until no impurity colonies exist. The obtained purified strain was then inoculated on a beef extract peptone slant and stored in a refrigerator at 4 ℃ for further use.
(2) And observing morphological characteristics of colonies and cells, and performing a series of physiological and biochemical experiments.
The observation results and experimental results are as follows:
1) colony characteristics: the colonies are round and white or yellowish.
2) The morphological characteristics of the cells are as follows: the cells are oval to cylindrical and slightly curved, and the size of the cells is about (0.7-0.8) × (2-3) μm; gram staining was positive. Without capsule, the perigenic flagellum can move.
3) Physiological and biochemical characteristics: aerobic growth; the gelatin can be liquefied, the glycerol can be utilized, the contact enzyme reaction is positive, the nitrate can be reduced into nitrite, the gelatin can be liquefied, the mannose can be utilized for fermentation, the arabinose, the amygdalin and the mannitol are not utilized for fermentation, and the starch can be hydrolyzed.
(3) The bacillus strains determined after culture and screening are subjected to enrichment culture in an LB culture medium at 37 ℃ for 12h, and thenThen, DNA was extracted by referring to TAKARA genome extraction kit. The 16S rRNA gene is subjected to PCR amplification, and the sequencing of the PCR product is sent to the Jinzhi company for completion. Obtaining 16S rDNA sequence of bacillus strain by PCR, identifying the obtained bacillus as bacillus subtilis by sequence determination, BLAST homology comparison and evolution analysisBacillus subtilis. The Bacillus strain was named Bacillus subtilis ZL 09-26.
Example 2
In this example, the optimum growth conditions of Bacillus subtilis ZL09-26 were determined by the following steps:
(1) the strain ZL09-26 is inoculated into LB culture medium for enrichment culture for 10-12h, then is inoculated into inorganic salt culture medium containing 200mg/L phenanthrene, and is subjected to shaking culture for 50h under the conditions of 37 ℃, 180rmp and different pH values (6.0, 6.5, 7.0, 7.5 and 8.0).
(2) The strain ZL09-26 is inoculated into LB culture medium for enrichment culture for 10-12h, then is inoculated into inorganic salt culture medium containing 200mg/L phenanthrene, and is subjected to shaking culture for 50h under the conditions of pH 8.0, 180rmp and different temperatures (20 ℃, 33 ℃, 37 ℃, 40 ℃ and 45 ℃).
(3) The strain ZL09-26 is inoculated into an LB culture medium for enrichment culture for 10-12h, then is inoculated into an inorganic salt culture medium containing phenanthrene as a unique carbon source, and is subjected to shaking culture for 50h under the conditions of 37 ℃, 180rmp and different concentrations of phenanthrene (50 mg/L, 100mg/L, 200mg/L, 450mg/L and 800 mg/L).
The optimal pH, temperature and phenanthrene concentration of the strain are respectively 8.0, 37 ℃ and 200mg/L under the growth condition that phenanthrene is used as a unique carbon source.
Example 3
This example illustrates the use of Bacillus subtilis ZL09-26 of the present invention in degradation of polycyclic aromatic hydrocarbon contaminants, phenanthrene.
Inoculating the strain ZL09-26 into LB culture medium for enrichment culture for 10-12h, inoculating into inorganic salt culture medium containing 200mg/L phenanthrene as unique carbon source at the inoculation amount of 2%, culturing for 15d under the growth conditions of 37 ℃, 180rmp and pH of 8.0, and measuring the residual phenanthrene content every 5 d. The results are shown in FIG. 4, where the phenanthrene content decreased to less than 5% after 15 days.
Example 4
This example illustrates that small organic acids can increase the efficiency of Bacillus subtilis ZL09-26 in degrading polycyclic aromatic hydrocarbon pollutant, phenanthrene.
Inoculating strain ZL09-26 into LB culture medium, enrichment culturing for 10-12h, inoculating into inorganic salt culture medium containing 200mg/L phenanthrene and 100mg/L small molecular organic acid (citric acid, oxalic acid, glutaric acid) at an inoculation amount of 2%, culturing for 15d under the growth conditions of 37 ℃, 180rmp and pH of 8.0, and measuring the residual phenanthrene content every 5 d.
The result is shown in fig. 4, compared with the degradation condition in the culture medium without inoculating and adding the small-molecular organic acid, the degradation efficiency of the polycyclic aromatic hydrocarbon pollutant phenanthrene is obviously improved by adding the small-molecular organic acid, wherein the degradation efficiency of the polycyclic aromatic hydrocarbon pollutant phenanthrene by the bacillus is especially obviously improved by citric acid.
Claims (9)
1. The bacillus subtilis is characterized by being classified and named as bacillus subtilis (A)Bacillus subtilis) ZL09-26 with the deposit number GDMCC No: 60293.
2. the method for culturing Bacillus subtilis according to claim 1, wherein Bacillus subtilis ZL09-26 is inoculated in an inorganic salt medium containing 200mg/L phenanthrene and aerobically cultured at 37 ℃ and pH 8.0.
3. The culture method according to claim 2, wherein the inorganic salt medium comprises the following components: 1.0 g of ammonium nitrate, 0.5 g of potassium dihydrogen phosphate, 1.5 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.02g of anhydrous calcium chloride, 0.5 g of ferric trichloride and distilled water with the constant volume of 1000 mL.
4. The use of the bacillus subtilis of claim 1 for degrading phenanthrene which is a polycyclic aromatic hydrocarbon pollutant.
5. The use of claim 4, wherein the inorganic salt medium is added with a small organic acid selected from one of citric acid, oxalic acid and glutaric acid.
6. Use according to claim 4, characterized in that citric acid is added to the mineral salts medium.
7. The use as claimed in claim 4, wherein Bacillus subtilis ZL09-26 is inoculated into LB culture medium for enrichment culture for 10-12h, inoculated into inorganic salt culture medium containing 200mg/L phenanthrene and 100mg/L small molecular organic acid at an inoculum size of 2%, and cultured under the growth conditions of 37 ℃, 180rmp and pH 8.0; the small molecular organic acid is selected from one of citric acid, oxalic acid and glutaric acid.
8. The use according to claim 7, wherein the LB medium consists of: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and distilled water to reach the constant volume of 1000 mL.
9. The use according to any one of claims 5 to 7, wherein the mineral salts medium comprises: 1.0 g of ammonium nitrate, 0.5 g of potassium dihydrogen phosphate, 1.5 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.02g of anhydrous calcium chloride, 0.5 g of ferric trichloride and distilled water with the constant volume of 1000 mL.
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| CN109504641B (en) * | 2018-12-31 | 2022-04-01 | 嘉兴益方环境科技有限公司 | Bacillus subtilis DDT98806 and application thereof |
| CN111748483B (en) * | 2019-03-29 | 2023-01-24 | 中国科学院沈阳应用生态研究所 | Bacillus for degrading petroleum hydrocarbon and application thereof |
| CN111411103B (en) * | 2020-03-25 | 2022-12-27 | 南京师范大学 | Microorganism immobilization carrier prepared based on vinegar residue waste and preparation method and application thereof |
| CN114621907A (en) * | 2022-04-01 | 2022-06-14 | 南京师范大学 | Accelerating agent for microbial degradation of polycyclic aromatic hydrocarbon, application thereof and method for microbial degradation of polycyclic aromatic hydrocarbon pollutants |
| CN116987601B (en) * | 2023-09-27 | 2023-12-15 | 菏泽学院 | Microbial preparation and application thereof in polycyclic aromatic hydrocarbon degradation |
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Inventor after: Huang He Inventor after: Zhang Lei Inventor after: Yu Yadong Inventor after: Wang Weidong Inventor after: Zhang Zhiping Inventor after: Guan Yongxiang Inventor after: Feng Yangyang Inventor after: Feng Yun Inventor after: Zuo Wenlu Inventor before: Zhang Lei Inventor before: Yu Yadong Inventor before: Huang He Inventor before: Wang Weidong Inventor before: Zhang Zhiping Inventor before: Guan Yongxiang Inventor before: Feng Yangyang Inventor before: Feng Yun Inventor before: Zuo Wenlu |
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