CN114410567B - Composite nutrition preparation and application thereof in improving capability of microorganisms in decomposing harmful substances - Google Patents
Composite nutrition preparation and application thereof in improving capability of microorganisms in decomposing harmful substances Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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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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- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
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- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
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- C02F2101/322—Volatile compounds, e.g. benzene
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- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
- C02F2101/327—Polyaromatic Hydrocarbons [PAH's]
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
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- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/40—Organic compounds containing sulfur
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of environmental biology, in particular to a compound nutritional preparation and application thereof in improving the capability of microorganisms in decomposing harmful substances. The compound nutritional formulation includes cinnamic acid, citric acid, and cyclodextrin: the mass ratio of the cinnamic acid to the citric acid to the cyclodextrin is 1: (8-30): (5-20). The invention provides a compound nutrition preparation aiming at microorganisms with the function of effectively decomposing harmful substances in a polluted site, the microorganisms can effectively decompose the harmful substances in the polluted site, such as polycyclic aromatic hydrocarbon, benzene series, methyl tertiary butyl ether and the like, and the degradation efficiency is further obviously improved under the condition of adding a compound nutrition package comprising cinnamic acid, citric acid and cyclodextrin, the compound nutrition preparation can be used for repairing and purifying the water and soil environment polluted by organic compound, has important significance for promoting sustainable development, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of environmental biology, in particular to a compound nutritional preparation and application thereof in improving the capability of microorganisms in decomposing harmful substances.
Background
Polycyclic Aromatic Hydrocarbons (PAHs) refer to organic compounds in which two or more benzene rings are connected in fused and non-fused forms. PAHs are ubiquitous in the environment, and because of the oncogenic, teratogenic and mutagenic properties of such compounds, they are potentially dangerous to both human health and the ecological environment. With the increasing content of Polycyclic Aromatic Hydrocarbons (PAHs) in the environment, purifying and repairing PAHs-polluted environments have become a hot spot of research.
Although PAHs can be degraded and transferred by chemical oxidation, photolysis, and volatilization, they are inefficient. Bioremediation is currently considered to be the most cost effective method of removing related harmful substances such as PAHs in the environment. In general, as the number of benzene rings of a polycyclic aromatic hydrocarbon increases, the biodegradation rate thereof decreases. Therefore, the low molecular weight polycyclic aromatic hydrocarbon can be degraded quickly in the environment, the time in the environment is shorter, and the high molecular weight polycyclic aromatic hydrocarbon has stronger adsorptivity due to lower water solubility, thereby reducing the bioavailability and being easy to exist in the environment for a long time. The research on microbial degradation of PAHs is mainly focused on screening, separation and purification of PAHs degrading microorganisms, and the degrading microorganisms which are separated at present mainly include Mycobacterium (Mycobacterium), rhodococcus (Rhodococcus), pseudomonas (Pseudomonas), sphingomonas (Sphingomonas), micrococcus (Micrococcus), sphingomonas (Novosphingans), aeromonas (Aeromonas), bacillus (Bacillus), nocardia (Nocardioides), marine bacterium (Marinobacter), vibrio (Vibrio), bayesiana (Beirnocardia), corynebacterium (Corynebacterium), cyanobacterium (Cyanobacter), and Cyclobacterium (Cyclococcus).
The benzene compounds are volatile monocyclic aromatic compounds, including benzene, toluene, ethylbenzene, xylene and the like, and are called BTEX for short, wherein the BTEX mainly exists in crude oil and petroleum products, is widely applied to industries of pesticides, chemical fiber textile, plastic chemical industry and the like, and is a toxic compound with wider distribution in the environment. Similar to polycyclic aromatic hydrocarbons, BTEX also has a "tri-effect" and has been identified as a strong carcinogen. The research and development of the technology for controlling the BTEX pollution in the waste water and the waste gas is very important and urgent.
At present, microbial degradation technology is one of the most effective methods for degrading benzene-based organics. One of the keys to the microbial treatment of toxic compounds such as BTEX in the environment is to obtain an excellent strain with the ability to degrade BTEX efficiently. A number of BTEX degrading bacteria have been isolated, mainly including Pseudomonas (Pseudomonas), rhodococcus (Rhodococcus), acinetobacter (Acinetobacter), nocardia (Nocardioides), flavobacterium (Flavobacterium), ralstonia (Ralstonia), alcaligenes (Alcaligenes) and Cladophyllalophora, etc. However, these existing degradation strains can degrade only one or two benzene compounds, the range of degradation substrates is limited, and the degradation efficiency of most strains needs to be further improved.
Methyl tert-butyl ether (MTBE) is a high-octane gasoline additive, can effectively improve the octane number of gasoline and obviously enhance the antiknock performance of the gasoline. However, with the widespread use of MTBE, its high water solubility, fluidity and durability, which makes it frequently detected in environmental media, especially groundwater around gas stations, it has been demonstrated that MTBE is carcinogenic and toxic to mammals and can induce diseases such as cancer and asthma. Therefore, the research and development of pollution control technology of MTBE in wastewater and soil is very important and urgent.
At present, microbial degradation technology is one of the most effective methods for degrading methyl tert-butyl ether, and a plurality of MTBE degrading bacteria have been isolated, mainly including Huang Qing cytophagy (Hydrogenophaga flava), xanthobacter sp, pseudomonas aeruginosa (Pseudomonas aeruginosa), methylibium petroleiphilum and the like. However, these existing degradation strains can degrade only one or two organic compounds, the range of degradation substrates is limited, and the degradation efficiency of most strains needs to be further improved.
As is clear from the above, most of the existing microbial agents for repairing the pollution of polycyclic aromatic hydrocarbon, benzene-based organic matter and MTBE are degraded by single bacteria or fungi and mixed bacteria aiming at single pollutants. In practice, however, contamination in environments such as soil or water and especially in industrial sites often exhibits significant combined contamination characteristics. Therefore, it is often difficult to use any one of the existing microbial agents alone. The strain capable of realizing high-efficiency degradation on PAHs, BTEX, MTBE and other pollutants is relatively fresh, and the main reason is that the metabolic pathways of different pollutants are often different, the degradation gene clusters and enzyme systems in the corresponding degradation strain are also quite different, and the strain with multiple sets of pollutant metabolic pathways is not more. In addition, even though the strain has the degradation potential of various pollutants, the degradation efficiency of the strain tends to be reduced along with the extension of time in the application process of the microbial inoculum, which indicates that the strain does not have the condition of continuous field planting after being in the environment for a period of time.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a compound nutritional preparation and application thereof in improving the capability of decomposing harmful substances by microorganisms.
In a first aspect, the present invention provides a complex nutritional formulation comprising cinnamic acid, citric acid, and cyclodextrin: the mass ratio of the cinnamic acid to the citric acid to the cyclodextrin is 1: (8-30): (5-20).
Further, the mass ratio of the cinnamic acid to the citric acid to the cyclodextrin is 1: (8-30): 15.
further, the method comprises the steps of: 20-100 mg/L of cinnamic acid, 200-800 mg/L of citric acid and 300-1500 mg/L of cyclodextrin.
The invention develops a nutrition activator capable of activating or enhancing the degradation capability of microorganism organic matters according to the characteristics of the microorganism, which can effectively improve the capability of decomposing harmful matters.
The invention further provides a preparation method of the compound nutritional preparation, which comprises the following steps:
mixing cinnamic acid, citric acid, cyclodextrin and water, and uniformly mixing at 25-30 ℃.
Further, the method comprises the steps of:
mixing water and cyclodextrin at 75-90 deg.c, mixing cinnamic acid at 50-60 deg.c, mixing citric acid at 25-30 deg.c, and mixing.
The invention further provides the application of the compound nutritional preparation in improving the capability of decomposing harmful substances of microorganisms.
Further, the harmful substances include:
one or more of polycyclic aromatic hydrocarbon, benzene series organic matter or methyl tertiary butyl ether.
Further, the polycyclic aromatic hydrocarbon comprises one or more of fluorene, naphthalene, anthracene, acenaphthylene, dibenzothiophene, carbazole, chrysene, phenanthrene, pyrene, fluoranthene, benzofluoranthene, benzoanthracene or benzopyrene; the benzene series organic matters comprise one or more of benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene.
Further, the microorganism includes one or more of mycobacterium, pseudomonas, bacillus, sphingolipid, paracoccus, rhodococcus, achromobacter, gordonia, methylobacterium, or microbacterium.
Further, the microorganism is mycobacterium 16F.
Mycobacterium 16F is a mycobacterium strain disclosed in China patent CN201210380304.6, and the preservation number is CGMCC No.6367.
Mycobacterium 16F is isolated from heavily contaminated soil of Beijing coking plant, and is obtained by artificial enrichment culture, separation and purification, and the Mycobacterium is identified as Mycobacterium sp, and the strain is named 16F. Microbiological characteristics: gram stain positive, short rod shape, no spore, biological characteristics of positive contact enzyme and oxidase, fresh yellow bacterial colony, round shape, neat edge, smooth surface, more moist and opaque surface, obligate oxygen demand, good growth at 24 ℃ to 37 ℃, can grow by using maltose, acetic acid and gentiobiose carbon sources, has optimal growth pH of 6.5-7.55 and has resistance to 50mg/L ampicillin.
The functional properties of mycobacteria 16F are as follows: can grow and degrade with multiple polycyclic aromatic hydrocarbons as the only carbon source and energy under the aerobic condition. For example, when the initial concentrations of fluorene, pyrene, benzopyrene were 50mg/L, 100mg/L and 25mg/L, respectively, the degradation removal rates after 7 days, 5 days and 35 days were 91.3%, 92.9% and 36.8%, respectively, and it was also possible to use benzene, m-xylene, toluene, methyl t-butyl ether and other organic matters, etc., for benzene, 200mg/L of toluene and 200mg/L of xylene, respectively, the degradation removal rates after 5 days, 4 days and 7 days were: 99.9%,99.6% and 90%, and the degradation removal rate at 10 days for methyl tert-butyl ether with an initial concentration of 200mg/L was 75.6%.
In practical application, the bacterial suspension of the mycobacterium 16F can be added into a basic salt culture medium containing polycyclic aromatic hydrocarbon, monocyclic benzene series organic matter and methyl tertiary butyl ether, and shake cultivation is carried out at 30 ℃ under the condition of avoiding light at 150rpm, so as to degrade the substrate. Wherein the concentration of the bacterial suspension of the mycobacterium 16F is 3.5X10 8 ～4.5×10 8 CFU/mL, preferably 4.1X10 8 CFU/mL。
The basic salt culture medium comprises the following formula: 2.5g of ammonium sulfate, 0.23mg of ferrous chloride, 1.5g of disodium hydrogen phosphate, 1.36g of monopotassium phosphate, 0.25g of magnesium sulfate, 10.7mg of calcium chloride, 0.5g of sodium chloride, 0.005g of ferric sulfate, 1mL of trace metal liquid and 1mL of vitamin complex solution,adding water to 1000mL, and pH value is 7.2-7.4; wherein the trace molten metal comprises the following components: coCl 2 ·6H 2 0 35mg、CuCl 2 0.20mg,、H 3 BO 3 6.0mg、MnCl 2 ·4H 2 O 25mg、Na 2 MoO 4 ·2H 2 O 3.0mg、NiCl 2 ·2H 2 O2.0 mg and ZnCl 2 2.5mg, water to 1000mL; the vitamin composite solution comprises the following components: 1.0mg of vitamin B6, 0.5mg of vitamin H and 1.5mg of vitamin C are added with water to 1000mL.
The invention has the following beneficial effects:
the invention provides a compound nutritional preparation capable of effectively improving the capability of decomposing harmful substances by microorganisms, which can be applied to one or more of mycobacterium, pseudomonas, bacillus, sphingolipid bacteria, paracoccus, rhodococcus, leucobacter, gordonia, methylobacterium or microbacterium, especially aiming at mycobacterium 16F, can effectively improve the degradation efficiency of decomposing polycyclic aromatic hydrocarbon, benzene organic matters, methyl tertiary butyl ether and the like by the mycobacterium 16F, can be used for repairing and purifying the water and soil environment polluted by aromatic hydrocarbon organic compound, has important significance for promoting sustainable development, and has wide application prospect.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1
The invention screens and obtains a mycobacterium by the following modes, and the specific flow is as follows:
1. the soil sample severely contaminated in Beijing coke plant was collected, passed through an 8 mesh screen and immediately placed in a refrigerator at 4℃for storage.
2. 200. Mu.L of a mixed solution of acetone and pyrene at a concentration of 50g/L was added to an open glass bottle having a volume of 500mL, and the mixture was placed in a super clean bench for 4 hours to remove acetone, or the glass bottle was left open for 24 hours to air dry naturally.
3. Adding 200-300mL of basic salt culture medium into the open glass bottle in the step (2), stirring for 30min at the rotating speed of 120r/min, and then adding the soil in the step (1)50g of soil; then placing the mixture in a shaking table with the rotation speed of 120r/min and the temperature of 25-30 ℃ for stirring, enriching and domesticating culture for 14 days, uniformly shaking, uniformly sucking 10 milliliters of enrichment liquid, transferring the enrichment liquid into a new glass bottle, and continuously transferring the enrichment liquid for 5 times. The glass bottle is also added with pyrene with the same concentration and 200-300mL of basic salt culture medium, wherein the basic salt culture medium comprises the following components: 2.5g of ammonium sulfate, 0.28mg of ferrous sulfate, 1.5g of disodium hydrogen phosphate, 1.36g of monopotassium phosphate, 0.25g of magnesium sulfate, 10.7mg of calcium chloride, 0.5g of sodium chloride, 0.04 g of ferric chloride, 1.0mL of trace metal liquid and 1mL of vitamin complex solution are added with water to 1000mL, the pH is controlled to 7.2-7.4, and the mixture is used as a liquid culture medium for standby after shaking. Wherein the trace molten metal comprises the following components: coCl 2 ·6H 2 0 35mg,CuCl 2 0.20mg,H 3 BO 3 6.0mg,MnCl 2 ·4H 2 O 25mg,Na 2 MoO 4 ·2H 2 O 3.0mg,NiCl 2 ·2H 2 O 2.0mg，ZnCl 2 2.5mg, water to 1000mL; the vitamin composite solution comprises the following components: vitamin B6.0 mg, vitamin H0.5 mg, vitamin C1.5mg, and water to 1000mL.
4. 600mL of basic salt culture medium, 0.6mL of trace metal solution and 0.6mL of vitamin complex solution are added into a conical flask with the volume of 1000mL, and the mixture is uniformly shaken to be used as a liquid culture medium for standby.
5. 50mL of the liquid medium prepared in step (4) and 0.9g of agar were added to a 150mL Erlenmeyer flask, and then sterilized in an autoclave at 121℃for 20 minutes, and when cooled to 65-70℃at room temperature, poured into a 9cm diameter petri dish in an ultra clean bench, and the petri dish was left half-open in the ultra clean bench for 6 hours to remove excess water on the surface of the medium.
6. And (3) adding 0.04mL of acetone solution with the concentration of 50g/L pyrene into the culture dish in the step (5) in an ultra-clean workbench, tilting the culture dish back and forth, uniformly coating with a glass rod, and enabling the pyrene acetone solution to be uniformly distributed on the surface of a culture medium, wherein the culture dish is placed in the ultra-clean workbench for 12 hours with a half-open cover so as to remove the acetone on the surface of the culture medium.
7. Sucking 1mL of the polycyclic aromatic hydrocarbon enrichment solution obtained in the step 3 in an ultra-clean workbench, sequentially diluting to 10mL, 100mL and 1000mL by using sterile water, then respectively sucking 1.0mL from the enrichment solution, adding the two to the culture dish prepared in the step 6, and coating the culture dish by using a sterile glass rod to uniformly distribute the culture medium dilution solution inoculated with the mixed flora. The culture dish is placed in an incubator with the temperature of 30 ℃ for culture observation, and obvious colony appearance can be found after 7-15 days.
8. And (3) picking the bacterial colony in the culture dish in the step (7) in an ultra-clean workbench, and repeatedly separating, primarily separating and purifying to obtain pure bacterial strains which can degrade the polycyclic aromatic hydrocarbon.
9. Re-screening of polycyclic aromatic hydrocarbon degrading bacteria: inoculating the initially separated and purified strains into Luria-Bertani culture medium, and culturing to OD 600 About 0.6, centrifuging to remove supernatant, washing and suspending the cells with a basal salt liquid medium; the Luria-Bertani medium (hereinafter referred to as LB medium) has the following composition: 10g of tryptone, 5g of yeast extract, 5g of sodium chloride, adding distilled water to 1000mL, and sterilizing at 121 ℃ for 20min under moist heat with pH of 7.2-7.4. The basic salt medium composition is the same as the composition in step 3.
10. And (3) absorbing 1.0mL of the bacterial suspension in the step (9) and inoculating the bacterial suspension into a basic salt liquid culture medium with pyrene as a sole carbon source, wherein the basic salt liquid culture medium with pyrene as the sole carbon source comprises the following components: adding 50g/L pyrene acetone solution into a sterilization triangular flask, volatilizing the acetone solution completely in an ultra-clean workbench, and adding an inorganic salt liquid culture medium to make the final concentration of polycyclic aromatic hydrocarbon be 100mg/L; the strain is subjected to shake culture at 30 ℃ and 150rpm in a dark place for 10 days, no bacterial suspension is used as a control, and the degradation effect of the strain is measured by adopting GC-MS, so that degradation bacteria capable of degrading polycyclic aromatic hydrocarbon are re-screened out.
The strain is identified as Mycobacterium sp, the invention names the strain as 16F, and the Mycobacterium 16F is a Mycobacterium strain disclosed in Chinese patent CN201210380304.6, and the preservation number is CGMCC No.6367.
Example 2
The embodiment provides a composite nutrition package BSL, which can be divided into three types, specifically as follows:
the proportion of the BSL-A components is that cinnamic acid: citric acid: cyclodextrin (1:8:15), the corresponding cinnamic acid concentration is 100mg/L, the citric acid concentration is 800mg/L, and the cyclodextrin concentration is 1500mg/L, and the preparation method comprises the following steps: heating purified water to 75-90 ℃, adding cyclodextrin into water, electrically and magnetically stirring for 30 minutes at 120r/min, keeping the temperature of 75-90 ℃ for standing for 10 minutes, adding cinnamic acid when the solution is cooled to 50-60 ℃, continuously electrically and magnetically stirring for 20 minutes at 120r/min, keeping the temperature of 50-60 ℃ for standing for 10 minutes, finally adding citric acid, and stirring for 15 minutes at 25-30 ℃ for use.
The proportion of the BSL-B components is that cinnamic acid: citric acid: cyclodextrin (1:20:15), the corresponding cinnamic acid concentration is 40mg/L, the citric acid concentration is 800mg/L, and the cyclodextrin concentration is 600mg/L, and the preparation method comprises the following steps: heating purified water to 75-90 ℃, adding cyclodextrin into water, electrically and magnetically stirring for 20 minutes at 100r/min, keeping the temperature of 75-90 ℃ for standing for 10 minutes, adding cinnamic acid when the solution is cooled to 50-60 ℃, continuously electrically and magnetically stirring for 20 minutes at 120r/min, keeping the temperature of 50-60 ℃ for standing for 10 minutes, finally adding citric acid, and stirring for 15 minutes at 25-30 ℃ to obtain the finished product.
The proportion of the BSL-C components is that cinnamic acid: citric acid: cyclodextrin (1:30:15), the corresponding cinnamic acid concentration is 20mg/L, the citric acid concentration is 600mg/L, and the cyclodextrin concentration is 300mg/L, and the preparation method comprises the following steps: heating purified water to 75-90 ℃, adding cyclodextrin into water, electrically and magnetically stirring for 15 minutes at 100r/min, keeping the temperature of 75-90 ℃ for standing for 10 minutes, adding cinnamic acid when the solution is cooled to 50-60 ℃, continuously electrically and magnetically stirring for 10 minutes at 120r/min, keeping the temperature of 50-60 ℃ for standing for 10 minutes, finally adding citric acid, and stirring for 15 minutes at 25-30 ℃ to obtain the finished product.
Example 3
This example demonstrates the effect of mycobacterium 16F on degrading methyl tert-butyl ether, as follows:
1. adding methyl tertiary butyl ether solution into a sterilization triangular flask, adding an inorganic salt liquid culture medium to ensure that the final concentration of the methyl tertiary butyl ether is 200mg/L, inoculating 1% of mycobacterium 16F bacterial suspension with volume content, and performing shake culture at 30 ℃ in a dark place at 150rpm, wherein the methyl tertiary butyl ether is detected by gas-mass spectrometry on line in 1 day, 2 days, 4 days, 6 days and 10 days respectively, and the mycobacterium 16F can degrade 75.6% of methyl tertiary butyl ether with initial concentration of 200mg/L within 10 days.
The preparation method of the bacterial suspension comprises the following steps: selecting single colony of mycobacterium 16F strain, inoculating into triangular flask containing LB culture medium, shake culturing at 30deg.C and 120rpm for 72-96 hr; then adjusted with sterile water to 4.1X10/mL of Mycobacterium 16F bacterial suspension 8 The bacterial suspensions mentioned in the examples below were prepared according to this method, unless otherwise specified, for CFU/mL of Mycobacterium 16F cells.
2. Adding methyl tertiary butyl ether solution into a sterilization triangular flask, adding an inorganic salt liquid culture medium to ensure that the final concentration of the methyl tertiary butyl ether is 200mg/L, inoculating 1% of mycobacteria 16F bacterial suspension with volume content, simultaneously adding a compound nutrition bag BSL-C of example 2 as a treatment group, wherein a negative control group is a methyl tertiary butyl ether control group only inoculated with thalli, and simultaneously selecting conventional nutrient components such as yeast extract, peptone and glucose as a nutrition control group, wherein the concentration of the added yeast extract of the nutrition control group is 20mg/L, the concentration of the added glucose is 600mg/L, the concentration of the peptone is 300mg/L, the temperature of 30 ℃, and the light-shielding culture is 150rpm, and the degradation rate of the strain 16F is improved by about 23.3% compared with the efficiency of the negative control group only inoculated with thalli and the nutrition bag, and the degradation rate of the strain 16F is improved by 81.17% compared with the shake table nutrition bag degradation rate of the same period after adding the compound nutrition bag BSL-C for 10 days through gas-mass spectrum online detection.
Example 4
This example demonstrates the effect of mycobacteria 16F on degrading benzopyrene, specifically as follows:
1. adding 5g/L acetone solution of benzopyrene into a sterilization triangular flask, volatilizing the acetone solution completely in an ultra-clean workbench, adding an inorganic salt liquid culture medium to ensure that the final concentration of benzopyrene is 25mg/L, inoculating 2% of mycobacteria 16F bacterial suspension, culturing in a light-proof shaking table at 30 ℃ at 150rpm under the condition of no inoculation of a blank control of bacterial cells, and detecting on line by using gas phase-mass spectrometry for 10 days, 15 days, 20 days, 25 days, 30 days and 35 days respectively. The degradation rate of the strain 16F on benzopyrene with the initial concentration of 25mg/L reaches 36.8% by 35 days, which indicates that the strain can degrade the benzopyrene more quickly but cannot degrade the benzopyrene completely.
2. Adding 5g/L benzopyrene acetone solution into a sterilization triangular flask, volatilizing the acetone solution completely in an ultra-clean workbench, adding an inorganic salt liquid culture medium to ensure that the final concentration of polycyclic aromatic hydrocarbon is 25mg/L, inoculating 2% of mycobacteria 16F bacterial suspension, simultaneously adding a composite nutrition bag BSL-A of the embodiment 2 as a treatment group, wherein a negative control group is a benzopyrene control group only inoculated with thalli, and simultaneously selecting conventional nutritional components such as yeast extract, peptone and glucose as a nutritional control group, wherein the concentration of the yeast extract added in the nutritional control group is 100mg/L, the concentration of glucose is 800mg/L, the concentration of peptone is 1500mg/L, culturing at 30 ℃ in a dark place at 150rpm, and detecting by gas phase-mass spectrometry on a shaking table for 10 days, 15 days, 20 days, 25 days, 30 days and 35 days respectively. After the bacterial strain 16F is added into the composite nutrition package BSL-A, 71.9% of benzopyrene with the initial concentration of 25mg/L can be degraded within 35 days, the efficiency is improved by about 35.1% compared with a negative control group which is only added with thalli and not added with the nutrition package, the contemporaneous degradation rate of the nutrition control group is 51.4%, and the degradation efficiency of the composite nutrition package treatment group is improved by 20.5% compared with the nutrition control group.
Example 5
This example demonstrates the effect of mycobacteria 16F on degrading methyl tert-butyl ether in aged soil, as follows:
1. taking methyl tertiary butyl ether aging soil near a gas station, sieving with a 100-mesh sieve, fully and uniformly mixing, dividing into 15 equal parts, naturally airing each part by 200g until the weight is nearly constant, randomly taking 5 parts of the methyl tertiary butyl ether aging soil, marking the mixture as a treatment group 1-5, marking the rest 10 parts of the methyl tertiary butyl ether aging soil as a negative control group 1-5 and a nutrition control group 6-10 respectively, wherein the negative control group is a control group added with sterile water only, the nutrition control group is added yeast extract with the concentration of 20mg/L, glucose with the concentration of 600mg/L and peptone with the concentration of 300mg/L, and repeating all experiments for five times. Uniformly spreading aged soil in 1500mL rectangular aluminum box, and spraying bacterial suspension of strain 16F when soil sample covers the bottom of aluminum box60mL (prepared bacterial suspension with bacterial content of 4.1X10) 8 CFU/mL) spray pot, spraying soil sample in the box, then repeating the soil spraying operation until the soil sample and the bacterial suspension are completely and uniformly mixed, placing the aluminum box cover in a dark place after the aluminum box cover is covered, and spraying 60mL of clear water for comparison.
2. Preparation of bacterial suspension of strain 16F: single colonies of strain 16F were cultured in LB liquid test tubes for 6 days, and transferred into Erlenmeyer flasks of liquid LB medium at a volume ratio of 1%, and shake cultured at 30℃and 150rpm for 3-4 days. Timing OD 600 After the strain 16F grows to the logarithmic growth phase, the strain is harvested by centrifugation at 8000rpm for 10min, washed twice with sterile water and suspended with basic salt culture medium to obtain the suspension of the strain with the bacterial content of 4.10 multiplied by 10 8 CFU/mL。
3. The treatment groups 1-5 and the nutrition control groups 6-10 after 14 days are intensively sprayed once by using the degrading bacteria mycobacterium 16F, the using amount is 30mL, the treatment groups 1-5 are added with 30mL of the compound nutrition package BSL-C solution, the nutrition control groups 6-10 are sprayed by using the same amount of nutrient solution (the concentration of the nutrient solution components and the concentration are 20mg/L of yeast extract, the concentration of glucose is 600mg/L, and the concentration of peptone is 300 mg/L). The negative control groups 1-5 were sprayed with an equal amount of sterile water.
4. And (3) periodically sampling each aluminum box for 0, 7, 14, 21, 28 and 35 days, wherein the sampling method is S-shaped 25-point sampling, uniformly mixing after sampling, and detecting the degradation effect through GC-MS after ASE350 accelerated extraction.
Before restoration, the content of methyl tertiary butyl ether in the tested soil is 167.5mg/kg, and after 35 days of treatment, the content of methyl tertiary butyl ether in the bacteria-adding and compound nutrition package treatment group is 43.68mg/kg, the content of methyl tertiary butyl ether in the bacteria-adding and nutrition control group is 87.1mg/kg, the total removal rate of the compound nutrition package treatment group reaches 73.9%, and compared with the degradation efficiency of the negative control group and the nutrition control group, the degradation efficiency of the compound nutrition package treatment group is respectively improved by 61.1% and 25.9%.
Example 6
The embodiment provides an application of the mycobacterium 16F in an experiment for repairing actual PAHs contaminated soil, and particularly provides a capability of the mycobacterium 16F for repairing the actual PAHs contaminated soil (abbreviated as aged soil) based on the embodiment 4, wherein the experiment comprises the following specific steps:
1. test soil: the soil (0-25 cm) on the surface layer of a certain Polycyclic Aromatic Hydrocarbon (PAHs) high risk area from Beijing coking plant is naturally air-dried and screened by an 8-mesh screen (3 mm screen), and the water content of the soil is regulated to be about 35% for test. The aging soil polluted by the polycyclic aromatic hydrocarbon is fully and evenly mixed and then divided into 15 equal parts, each 400g of the aging soil is randomly taken, 5 parts of the aging soil are recorded as a compound nutrition package treatment group 1-5, the remaining 10 parts are respectively recorded as a negative control group 1-5 and a nutrition control group 6-10, the negative control group is a control group only added with sterile water, the nutrition control group is a control group added with yeast extract with the concentration of 100mg/L, the glucose concentration of 800mg/L and the peptone concentration of 1500mg/L, and all experiments are repeated for five times. Uniformly dispersing aged soil in 1500mL rectangular aluminum box, and spraying 60mL bacterial suspension of strain 16F from treatment group 1-5 and nutritional control group 6-10 (prepared bacterial suspension with bacterial content of 4.1X10) when soil sample covers the bottom of aluminum box 8 CFU/mL) spray pot, spraying soil sample in the box, then repeating the soil spraying operation until the soil sample and the bacterial suspension are completely and uniformly mixed, placing the box cover in a dark place after the cover of the aluminum box is well covered, and spraying 60mL of clean water in a negative control group.
2. Preparation of bacterial suspension of strain 16F: single colonies of strain 16F were cultured in LB liquid test tubes for 6 days, and transferred into Erlenmeyer flasks of liquid LB medium at a volume ratio of 1%, and shake cultured at 30℃and 150rpm for 3-4 days. Timing OD 600 After the strain 16F grows to the logarithmic growth phase, the strain is harvested by centrifugation at 8000rpm for 10min, washed twice with sterile water and suspended with basic salt culture medium to obtain the suspension of the strain with the bacterial content of 4.10 multiplied by 10 8 CFU/mL。
3. After 28 days, the negative control group 1-5 and the nutrition control group 6-10 are intensively sprayed with the degrading bacterial agent mycobacterium 16F once, the using amount is 60mL, wherein the compound nutrition package treatment group 1-5 is added with the mycobacterium 16F bacterial agent, 60mL of compound nutrition package BSL-A solution (cinnamic acid: citric acid: cyclodextrin 1:8:15, the concentration of the corresponding cinnamic acid is 100mg/L, the concentration of the citric acid is 800mg/L, and the concentration of the cyclodextrin is 1500 mg/L) is simultaneously added, the nutrition control group is added with 60mL of nutrient solution (the nutrient solution comprises yeast extract with the concentration of 100mg/L, the glucose concentration of 800mg/L and the peptone concentration of 1500 mg/L), and the negative control group 1-5 is sprayed with sterile water with the same volume.
4. Each aluminum box is sampled periodically, namely 0, 7, 14, 21, 28, 35, 42, 49 and 56 days, the sampling method is S-shaped 25-point sampling, the sampling is uniformly mixed, and the degradation effect is detected by GC-MS after ASE350 accelerated extraction.
Before restoration, the total content of benzopyrene components of persistent organic pollutants in the tested soil is 31.58mg/kg, and after 56 days of treatment, the benzopyrene content of a nutrient control group added with bacteria is 12.94mg/kg, the benzopyrene content of a treatment group added with bacteria and a compound nutrition packet is 8.89mg/kg, the clear water control without nutrition addition is 23.62mg/kg, the removal rate of the treatment group added with bacteria and the compound nutrition packet reaches 71.84%, the removal rate of the nutrient control group added with bacteria is 52.69%, and the restoration efficiency of the treatment group added with bacteria and the compound nutrition packet is improved by about 19.15% compared with that of the nutrient control group.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (6)
1. Use of a compound nutritional formulation for increasing the ability of microorganisms to break down harmful substances; the compound nutritional formulation includes cinnamic acid, citric acid, and cyclodextrin: the mass ratio of the cinnamic acid to the citric acid to the cyclodextrin is 1: (8-30): (5-20);
the harmful substances include: one or more of a polycyclic aromatic hydrocarbon or methyl tertiary butyl ether;
the microorganism is mycobacterium 16F, and the preservation number is CGMCC No.6367.
2. The use according to claim 1, wherein the mass ratio of cinnamic acid, citric acid and cyclodextrin is 1: (8-30): 15.
3. the use according to claim 1, wherein the complex nutritional formulation comprises: 20-100 mg/L of cinnamic acid, 200-800 mg/L of citric acid and 300-1500 mg/L of cyclodextrin.
4. The use according to claim 1, wherein the method of preparing the complex nutritional formulation comprises:
mixing cinnamic acid, citric acid, cyclodextrin and water, and uniformly mixing at 25-30 ℃.
5. The use according to claim 4, wherein the method of preparing the complex nutritional formulation comprises:
mixing water and cyclodextrin at 75-90 ℃, further mixing cinnamic acid at 50-60 ℃, and further mixing citric acid at 25-30 ℃ and uniformly mixing.
6. The use according to claim 1, wherein the polycyclic aromatic hydrocarbon comprises one or more of fluorene, naphthalene, anthracene, acenaphthylene, carbazole, chrysene, phenanthrene, pyrene, fluoranthene, benzofluoranthene, benzoanthracene, or benzopyrene.
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| CN102458160A (en) * | 2009-06-19 | 2012-05-16 | 百事可乐公司 | Beverage preservative system containing pimaricin - cyclodextrin complex |
| CN102899271A (en) * | 2012-09-29 | 2013-01-30 | 轻工业环境保护研究所 | Mycobacterium 16F for efficiently degrading polycyclic aromatic hydrocarbons and benzene organic matters and application thereof |
| JP2013245205A (en) * | 2012-05-28 | 2013-12-09 | Fujifilm Corp | Skin care composition and cosmetic |
| CN105767146A (en) * | 2014-12-17 | 2016-07-20 | 王志虹 | Kiwi fruit fresh-keeping agent and preparation method thereof |
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| CN102458160A (en) * | 2009-06-19 | 2012-05-16 | 百事可乐公司 | Beverage preservative system containing pimaricin - cyclodextrin complex |
| JP2013245205A (en) * | 2012-05-28 | 2013-12-09 | Fujifilm Corp | Skin care composition and cosmetic |
| CN102899271A (en) * | 2012-09-29 | 2013-01-30 | 轻工业环境保护研究所 | Mycobacterium 16F for efficiently degrading polycyclic aromatic hydrocarbons and benzene organic matters and application thereof |
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