The present invention belongs to the technical field of bioremediation of environmental pollutants, in particular to the preparation of a microcapsule material for highly efficient degradation of polycyclic aromatic hydrocarbons and its application in the bioremediation of wastewater and the remediation of contaminated soil.
Polycyclic aromatic hydrocarbons, as a kind of common organic pollutants in the environment, usually refer to a class of fused ring compounds containing two or more benzene rings and arranged in a linear, angular or clustered manner, and they have bioaccumulation and environmental durability as well as carcinogenic, teratogenic and mutagenic effects (three inductions), posing a huge threat to the biosecurity and human health in nature.
Most of the polycyclic aromatic hydrocarbons in the environment are accumulated in soil. Where appropriate, they may migrate to other environmental media, expanding the range of pollution and changing the route of exposure. The polycyclic aromatic hydrocarbons in farmland, mostly caused by sewage irrigation, can be passed through crops in the form of a biological chain, and may cause damage to the human body after their accumulation therein. It is very important and urgent to rehabilitate the farmland contaminated with polycyclic aromatic hydrocarbons, especially to strengthen the rehabilitation of long-term contaminated soil.
In the polycyclic aromatic hydrocarbons that are common in the polluted environment and difficult to be degraded, since the ketone metabolites of pyrene are more toxic and mutagenic than their parents, pyrene is often used as an indicator to monitor the pollution of polycyclic aromatic hydrocarbons and a model molecule of biodegradation of other polycyclic aromatic hydrocarbons, thus becoming a representative of polycyclic aromatic hydrocarbons.
Using microbial treatment to solve the pollution problem of polycyclic aromatic hydrocarbons in the environment, having been widely accepted, has good effects, low cost, less secondary pollution and other advantages, and is thus a kind of bioremediation technology with low consumption, high efficiency and environmental safety. Currently, by artificial enrichment culture and other technologies, people have isolated many microorganisms that can degrade or transform some kind of polycyclic aromatic hydrocarbons. However, due to the vicious competition of indigenous bacteria or the change of environmental conditions, etc., free microorganisms are often difficult to adapt to the actual rehabilitation environment and cannot achieve the desired rehabilitative effects. Therefore, the development of biomaterials with high environmental adaptability and high efficiency of degrading polycyclic aromatic hydrocarbons has become one of the hot issues in the research on the abatement and remediation of polluted environment.
A purpose of the present invention is to provide a microcapsule material capable of reducing pollution containing polycyclic aromatic hydrocarbons, and a preparation method and application thereof.
In order to achieve the purpose of the present invention, the following technical solution is adopted:
A preparation method of a microcapsule material capable of reducing pollution containing polycyclic aromatic hydrocarbons is provided, comprising the following steps:
(1) culturing mycobacterium gilvum CP 13 in a bacteria culture liquid to obtain a bacterial broth, wherein the mycobacterium gilvum CP 13 was deposited in China General Microbiological Culture Collection Center (CGMCC) on Jul. 22, 2013 with a CGMCC number of CGMCC No. 7963; and
(2) applying a calcium alginate and chitosan to encapsulate the bacterial broth in a microcapsule through layer-by-layer self-assembly to produce a microcapsule material capable of reducing pollution containing polycyclic aromatic hydrocarbons.
The bacteria culture liquid is nutrient broth medium having the following composition and dosage: beef extract 3 g/L, peptone 10 g/L, NaCl 5 g/L, agar 20 g/L, and a pH adjusted to 7.0-7.2.
The step (2) of encapsulating the bacterial broth in a microcapsule comprises the following steps: adding chitosan to the bacterial broth so that chitosan is deposited on the surface of bacteria, and then adding calcium alginate solution so that calcium alginate is further deposited on the surface of chitosan; repeating the operations above 2-3 times so that the bacterial broth can be encapsulated in the microcapsule.
The concentrations of chitosan and calcium alginate are both 0.1-2.0 g/L.
The microcapsule material capable of reducing pollution containing polycyclic aromatic hydrocarbons prepared by the above method is applied to degradation of polycyclic aromatic hydrocarbons in water and soil.
The polycyclic aromatic hydrocarbons are pyrene.
The microcapsule material is added in water in an amount of 0.1 g of the microcapsule material per 20 ml of water and added in soil in an amount of 2.0 g of the microcapsule material per 400 g of soil.
The present invention has the following advantages compared to the prior art:
(1) The microcapsule material of the present invention has strong adaptability to harsh environments and can be adapted to a wide range of pH and low and high temperature environments. Having superior ability to remove polycyclic aromatic hydrocarbons, the microcapsule material is capable of removing more than 81% of high concentration of polycyclic aromatic hydrocarbon pyrene contained in vegetable soil (120 mg of polycyclic aromatic hydrocarbon pyrene per kilogram of vegetable soil) within 40 days at room temperature (20° C.±5° C.), and removing more than 80% of pyrene in water (up to 50 mg/L) within 7 days, with its degradation performance not affected in strong acidity and alkalinity (pH 3-10) and at high and low temperatures (10° C.-40° C.) and under other harsh conditions. It can be used for treatment of industrial wastewater containing polycyclic aromatic hydrocarbons and soil bioremediation.
(2) The microcapsule material of the present invention consists of an environmentally safe bacterium mycobacterium gilvum CP13 that degrades a variety of tricyclic polycyclic aromatic hydrocarbons and tetracyclic polycyclic aromatic hydrocarbons. The microcapsule material is capable of removing a variety of polycyclic aromatic hydrocarbons in soil, suitable for southern rice field soil, vegetable soil, red soil and other soils, with the significant effect of remediating soil contaminated by polycyclic aromatic hydrocarbons.
The mycobacterium gilvum CP13 provided by the present invention was deposited on Jul. 22, 2013 in China General Microbiological Culture Collection Center (CGMCC) (located in Institute of Microbiology, Chinese Academy of Sciences, No. 3, 1 Beichen West Road, Chaoyang District, Beijing, P.R. China) with a CGMCC number of CGMCC No. 7963. This strain has been disclosed in a patent with a publication number CN102824897A and belongs to the prior art.
) the microcapsule material of the present invention.
The present invention will be further described below in detail with reference to specific examples; however, the embodiments of the present invention are not limited thereto, and process parameters not specifically given can be determined by referring to conventional techniques.
Preparation of the microcapsule material of the present invention capable of degrading polycyclic aromatic hydrocarbons
A method for culturing mycobacterium gilvum CP13 strain of the present invention is as follows:
Respectively performing streak inoculation of the CP13 strains in beef juice peptone medium, and culturing at 28° C.-30° C. for 48 h; then respectively inoculating them into a 500 mL flask, and culturing at 30° C. and 150 r/min for 12 h; and finally inoculating them into a 5 L seed tank in accordance with an inoculum concentration of 5%, and culturing at 180 r/min, pH 7.5 and a ventilatory capacity of 5 L/min for 24 h.
Composition and dosage of the nutrient broth medium are as follows (g/L): Beef extract 3, peptone 10, NaCl 5, agar 20, and a pH adjusted to 7.0-7.2.
The specific steps are as follows: (1) Culturing mycobacterium gilvum CP 13 in a bacteria culture liquid to obtain a bacterial broth, (2) adding 20 ml of 0.2 g/L polycation (chitosan) to the bacterial broth so that polycation is deposited on the surface of bacteria, and then adding the same volume and concentration of polyanion (calcium alginate) solution so that polyanion is further deposited on the surface of the chitosan. Repeating the operations above 2-3 times so that the bacterial broth can be encapsulated in the microcapsule. The bacteria-immobilized microcapsules are thus prepared. They are sealed in sterile bags and stored in a refrigerator at 4° C.
Inoculating the microcapsule material prepared in Example 1 at a ratio of 0.1 g/20 ml to a MSM culture solution with the concentration of pyrene at 10 mg/L; taking a pyrene-containing MSM medium without the microcapsule material as a blank and placing it in a shaker at 30° C., culturing in darkness while shaking at 150 r/min, and then determining the residual amount of pyrene in the culture solution. The formulation of the MSM culture solution: 100 mg/L (NH4)2SO4, 20 mg/L MgSO4·7H2O, 10 mg/L CaCl2·2H2O; trace elements: 1.2 mg/L FeSO4·7H2O, 0.3 mg/L MnSO4·H2O, 0.3 mg/L ZnSO4·7H2O, 0.1 mg/L CoSO4·7H2O, 0.1 mg/L (NH4)6Mo7O24·4H2O; phosphate buffer solution: 2.5 g/L K2HPO3, 0.77 g/L KH2PO3, pH 7.2-7.4.
As can be seen from
Inoculating the microcapsule material prepared in Example 1 at a ratio of 0.1 g/20 ml to a MSM culture solution with the concentration of pyrene at 10 mg/L; taking a pyrene-containing MSM medium without the microcapsule material as a blank and free bacteria as a control and placing them in a shaker at 30° C., culturing in darkness while shaking at 150 r/min, and then determining the residual amount of pyrene in the culture solution. As can be seen from
This example shows that the microcapsule material of the present invention capable of degrading polycyclic aromatic hydrocarbons can degrade pyrene well in a wide pH range of acidic, neutral and alkaline conditions and at 10° C.-40° C., ensuring its application at different pHs and in low and high temperature environments.
Experiment on remediation of pyrene contaminated soil by the microcapsule material of the present invention
Adding 400 g of not sterile soil, to which pyrene has been added at 120 mg of pyrene per kilogram of soil, to a 500 ml beaker, and adding 2.0 g of the microcapsule material of Example 1 to the soil so that the initial inoculum is 107 CFU per gram of soil. Using free CP13 as a control at the same amount of 107 CFU per gram of soil, and incubating in an incubator at 30° C. Sampling to determine the residual amount of pyrene in the soil after culturing for 6 d, 10 d, 20 d, 40 d.
As indicated by the results in
Number | Date | Country | Kind |
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201510571244.X | Sep 2015 | CN | national |
Filing Document | Filing Date | Country | Kind |
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PCT/CN2015/100076 | 12/31/2015 | WO | 00 |