CN113577637B - Method for enhancing microbial degradation of polycyclic aromatic hydrocarbon and microbial agent - Google Patents

Method for enhancing microbial degradation of polycyclic aromatic hydrocarbon and microbial agent Download PDF

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CN113577637B
CN113577637B CN202111042043.2A CN202111042043A CN113577637B CN 113577637 B CN113577637 B CN 113577637B CN 202111042043 A CN202111042043 A CN 202111042043A CN 113577637 B CN113577637 B CN 113577637B
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mycobacterium
modified montmorillonite
polycyclic aromatic
aromatic hydrocarbon
njs
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CN113577637A (en
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王芳
王紫泉
曾军
生弘杰
相雷雷
卞永荣
蒋新
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Institute of Soil Science of CAS
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    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
    • A62D3/10—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by subjecting to electric or wave energy or particle or ionizing radiation
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/20—Organic substances
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2203/00—Aspects of processes for making harmful chemical substances harmless, or less harmful, by effecting chemical change in the substances
    • A62D2203/02—Combined processes involving two or more distinct steps covered by groups A62D3/10 - A62D3/40

Abstract

The invention discloses a method for strengthening microbial degradation of polycyclic aromatic hydrocarbon and a microbial agent, and belongs to the field of environmental pollution bioremediation. The strengthening method comprises the step of inoculating microorganisms to the cation modified montmorillonite; the microorganism comprises Mycobacterium sp.NJS-1. The aim of enhancing biodegradation of polycyclic aromatic hydrocarbon (pyrene) is achieved by utilizing different cations to modify montmorillonite to provide a microorganism growth medium and improve growth of mineral interface degrading bacteria and regulating key enzyme metabolic activity and bioelectrochemical activity among mineral-bacteria through surface cation influence, and the method has great application potential in environments such as mineral-microorganism combined remediation of polycyclic aromatic hydrocarbon polluted soil.

Description

Method for enhancing microbial degradation of polycyclic aromatic hydrocarbon and microbial agent
Technical Field
The invention relates to a method for strengthening microbial degradation of polycyclic aromatic hydrocarbon, which belongs to the technical field of biological treatment of environmental pollution and is mainly used for repairing the polycyclic aromatic hydrocarbon polluted environment such as wastewater, soil and the like.
Background
Polycyclic Aromatic Hydrocarbons (PAHs) are pollutants widely detected in soil, sediment, water and atmospheric environment, mainly derived from human activities, and include incomplete combustion of fossil fuels, industrial emissions, automobile exhaust gas, and the like. Polycyclic aromatic hydrocarbon pollution in the environment has carcinogenic, teratogenic and mutagenic effects and constitutes a potential risk to human health. Remediation of polycyclic aromatic hydrocarbon pollution is an important environmental problem which is beneficial to human health and sustainable development of an ecosystem.
Microbial remediation is a low-cost, beneficial and environmentally friendly remediation method. But the restoration method of directly adding degrading bacteria into the polluted soil and other environments has low degrading bacteria activity and undesirable degrading effect.
The prior art with the Chinese patent application publication number of CN104492804A discloses a system and a method for restoring polluted soil by ozone pretreatment and enhanced microbial degradation, aiming at the polycyclic aromatic hydrocarbon polluted soil, firstly ozone pretreatment is adopted, then a biosurfactant and a liquid culture medium are added into a reaction container, and combined degradation and periodic sampling determination are carried out until restoration is completed. The method adopts ozone pretreatment and biosurfactant solubilization technologies to strengthen the microbial degradation combined technology, overcomes the limitation of a single technology, is simple and convenient to operate, low in cost, short in operation period and good in removal effect, has the removal rate of polycyclic aromatic hydrocarbon of more than 93 percent, does not produce secondary pollution, greatly weakens the harm of polycyclic aromatic hydrocarbon polluted soil to human bodies and environment, and provides a new thought for soil remediation. The combination of ozone pretreatment, biosurfactant solubilization and indigenous microorganism degradation is fully utilized, so that various technologies complement each other, the ozone pretreatment improves the repair effect of rear-end biodegradation, the removal rate of polycyclic aromatic hydrocarbon reaches more than 93%, and the repair period is shortened. The method for carrying out ozone pretreatment on the polycyclic aromatic hydrocarbon polluted soil is easy to realize in a laboratory, but is difficult to be suitable for the in-situ treatment of the large-area polluted soil.
Disclosure of Invention
1. Problems to be solved
Aiming at the technical problem of limited efficiency of microbial degradation of polycyclic aromatic hydrocarbon in the prior art, the invention provides a method for enhancing microbial degradation of polycyclic aromatic hydrocarbon, which utilizes cation modified montmorillonite to regulate the activity of key enzyme of microorganism and the bioelectrochemical process of mineral-microorganism interface, thereby enhancing the life activities of microorganism growth and metabolism and the like and further improving the degradation rate of pollutants.
2. Technical scheme
A method for strengthening the degradation of polycyclic aromatic hydrocarbon by microorganisms comprises the step of inoculating the microorganisms to cation modified montmorillonite; the microorganism comprises Mycobacterium sp.NJS-1, is preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 5 days in 2021, and has the address: the No. 3 Xilu Beijing, Chaoyang, Beijing area, has a preservation number of CGMCC 1.10964. The invention utilizes the cation modified montmorillonite to adjust the microorganism to provide a microorganism growth medium, and the surface cations influence the activity of key enzyme and the bioelectrochemical process of a mineral-microorganism interface, thereby enhancing the life activities of microorganism growth and metabolism and the like and further increasing the degradation rate of pollutants.
Preferably, the cation-modified montmorillonite comprises one or more of fe (iii) -modified montmorillonite, co (ii) -modified montmorillonite, na (i) -modified montmorillonite, and particularly preferably fe (iii) -modified montmorillonite.
Preferably, the polycyclic aromatic hydrocarbon includes one or more of pyrene, benzo [ a ] pyrene, phenanthrene, fluoranthene, anthracene.
Preferably, the method specifically comprises the following steps:
s1 culturing and propagating Mycobacterium sp.NJS-1 bacteria by adopting a sterilized nutrient broth culture medium, then cleaning the bacteria liquid by using a sterilized phosphate buffer solution with pH7.0, and adjusting the number of the bacteria or the density of the bacteria to reach a specific value;
s2, adding a sterilized inorganic salt culture medium into the cation modified montmorillonite, and then inoculating the Mycobacterium sp.NJS-1 bacterium obtained in the step S1;
s3, mixing and culturing the inoculated cation modified montmorillonite and pollutants containing polycyclic aromatic hydrocarbon under proper conditions to degrade the polycyclic aromatic hydrocarbon;
or
S1, adsorbing and fixing pollutants containing polycyclic aromatic hydrocarbons by using cation modified montmorillonite;
s2 culturing and propagating Mycobacterium sp.NJS-1 bacteria by adopting a sterilized nutrient broth culture medium, then cleaning the bacteria liquid by using a sterilized phosphate buffer solution with pH7.0, and adjusting the number of the bacteria or the density of the bacteria to reach a specific value;
s3 adding a sterilized inorganic salt culture medium into the cation modified montmorillonite adsorbed with the pollutants, and then inoculating the Mycobacterium sp.NJS-1 bacterium obtained in the step S2 to perform mixed culture under appropriate conditions to degrade the polycyclic aromatic hydrocarbon.
Preferably, the cation saturated modified montmorillonite is prepared by an ion exchange method; selecting montmorillonite K10, grinding and sieving, wherein the size of the sieved sieve is not less than 200 meshes.
Preferably, the cation modified montmorillonite is prepared by an ion exchange method, and specifically comprises the following steps:
a) the solid-to-liquid ratio of the montmorillonite to the chloride solution of the metal cations is 1: 20;
b) the equivalent concentration of metal cations is 0.1N;
c) adopting an ion exchange method, wherein the exchange times are not less than 2;
d) the oscillation balance time of each exchange is not less than 2 h;
e) after the exchange is completed, the reaction solution is washed with ultrapure water for more than 4 times until no Cl exists-。
Preferably, the degrading bacteria are Mycobacterium sp.NJS-1, are separated from a farmland polluted by polycyclic aromatic hydrocarbon, can grow by taking pyrene as a unique carbon source, and can degrade benzo [ a ] pyrene, phenanthrene, fluoranthene, anthracene and the like.
Preferably, the condition for propagation of the degrading bacterium Mycobacterium sp.njs-1 comprises: aerobic culture, inoculating a proper amount of bacterial liquid in a sterilized nutrient broth culture medium, and culturing overnight at 28 ℃ and 160rpm in a dark condition; washed twice with sterile ph 7.00.1m phosphate buffer, and the density of the bacteria was adjusted to OD600 of 0.30.
Preferably, the solid-liquid mass ratio of the modified montmorillonite of the degradation system in the step S2 or S3 to the inorganic salt culture medium is 1 (20-100), and the OD600 of the strain Mycobacterium sp.NJS-1 after inoculation and dilution is not less than 0.03.
Preferably, the iron ion modified montmorillonite improves the bacterial density of Mycobacterium sp.NJS-1 strain by more than 3 times within 1-3 d compared with the original bacterial density without the addition of the iron ion modified montmorillonite.
A microbial agent comprises Mycobacterium sp.NJS-1, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC 1.10964; also comprises iron ion modified montmorillonite. The cation modified montmorillonite can remarkably promote the growth of degrading bacteria Mycobacterium sp.NJS-1, and improve the activity of key enzyme and the activity of bioelectrochemistry.
Preferably, the exchange rate of iron ions in the iron ion modified montmorillonite is in the range of 50-100%.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) the peculiar high specific surface area and pore structure of the montmorillonite can influence the distribution of microorganisms, regulate the activity of the microorganisms and relieve the stress pressure of adverse environments (heavy metals, organic pollutants and the like) on the microorganisms; the interaction of the montmorillonite and the microorganism can obtain better viability competitiveness and more effective microorganism activity, thereby strengthening the bioremediation effect on the target pollutant to a certain extent; the cation modified montmorillonite has hydrophilic characteristic and stronger ion-pi bond interaction, and is beneficial to the attachment and distribution of microorganisms and pollutants; the invention utilizes the cation modified montmorillonite to adjust the activity of the key enzyme of the microorganism and the bioelectrochemical process of the mineral-microorganism interface, and utilizes the cation fixed on the surface of the montmorillonite to adjust the electrochemical property of the mineral based on the interaction of the montmorillonite, the microorganism and the polycyclic aromatic hydrocarbon, thereby promoting the microorganism to grow on the surface of the mineral to form a biomembrane, improving the bioelectrochemical process among the microorganism, the mineral and the pollutant and achieving the purpose of strengthening the degradation of the polycyclic aromatic hydrocarbon by the microorganism;
(2) the iron ion modified montmorillonite has an outstanding effect on strengthening the microbial degradation of polycyclic aromatic hydrocarbon, probably because Fe is an auxiliary group of some redox proteins, regulates and controls enzyme activity and transmits electrons through the change of the valence state of Fe, and can be used as an extracellular electron acceptor to play an important role in the bioelectrochemical process of a microorganism-mineral interface; the fact that the iron ion modified montmorillonite has strong affinity to microorganisms and can promote the growth of the microorganisms can be known from the increase of the density of the bacteria; from the results of peroxidase activity, dioxygenase activity and electron transfer activity, it can be known that the iron ion modified montmorillonite can effectively strengthen microbial oxidoreductase and bioelectrochemical activity;
(3) the iron ion modified montmorillonite and the microorganism are co-cultured to prepare the microbial agent, the operation is simple, secondary pollution is avoided, the iron ion modified montmorillonite has a remarkable promotion effect on the biochemical process of the microorganism, and the microbial agent can be applied to in-situ bioremediation of soil, sewage and other environments.
Drawings
FIG. 1 is the dynamics curve (pyrene initial concentration 15mg/L) of pyrene degradation under the conditions of inoculation of microorganism (+) and no inoculation of microorganism (-) in the presence of different cation modified montmorillonites in example 1 of the present invention, and Mt is the treatment of unmodified montmorillonites under the condition of inoculation bacteria;
FIG. 2 is a graph showing the pyrene degradation rate (pyrene initial concentration 100mg/L) of microbes after culturing for different periods of time in the presence of different cation-modified montmorillonites in example 1 of the present invention, where Blank is a treatment without inoculated bacteria and without montmorillonites, and Mt is a treatment without modified montmorillonites and without inoculated bacteria;
FIG. 3 shows the degradation process of pyrene in the presence of different cation-modified montmorillonites in example 2 of the present invention: a: the density of bacteria changes; b: a change in peroxidase activity; c: a change in dioxygenase activity; d: the relationship between the bacterial density and the pyrene degradation rate; e: the relation between the peroxidase activity and the pyrene degradation rate; f: the relationship between dioxygenase activity and pyrene degradation rate;
FIG. 4 shows the change of electrochemical activity (a: electron transfer activity and b: electron transfer capacity) of microbes in the pyrene degradation process in the presence of different cation-modified montmorillonites in example 2 of the present invention and the relationship between the change and the pyrene degradation.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; as used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limit values of 1 to about 4.5, but also include individual numbers (such as 2,3, 4) and sub-ranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed to include all of the aforementioned values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or feature being described.
The following description of the embodiments of the present invention is provided in connection with the accompanying drawings and examples, but the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
By way of example, the experiments in the examples below were carried out using pyrene and Mycobacterium sp. The strain is a degradation bacterium taking pyrene as a unique carbon source, is preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 5 days of 2021, and has the address: beijing, Chaoyang district, Beichen Xilu No. 1 Hospital No. 3, with a collection number of CGMCC 1.10964, is a gram-positive bacterium.
Example 1
Preparation of cation modified montmorillonite
The preparation process of the cation modified montmorillonite comprises the following steps:
(1) pretreatment of montmorillonite
Grinding montmorillonite, and sieving with 200 mesh nylon sieve.
(2) Cation modified montmorillonite prepared by ion exchange method
Weighing 3.0g of montmorillonite, placing in a series of 100mL centrifuge tubes, adding 60mL of ultrapure water respectively, ultrasonically dispersing and cleaning twice, and then adding 60mL of 0.1N NaCl, CuCl and the like with equivalent concentration2，CoCl2，NiCl2，FeCl3After covering, oscillating and balancing for 2h at 25 ℃ and 160rpm, then centrifuging for 10min at 4000rpm, discarding the supernatant, respectively adding 60mL of 0.1N metal cation solution again, oscillating and balancing for 2h at 25 ℃ and 160rpm, so as to achieve the purpose that the montmorillonite is completely saturated by cations. Centrifuging the sample, removing supernatant, adding 60mL ultrapure water, shaking and cleaning for 5min, centrifuging at 4000rpm for 10min, removing supernatant, and repeating cleaning for 3 times to remove weakly-bound cations and Cl-Ions. Drying the sample at 60 ℃, grinding the sample through a 200-mesh nylon sieve to prepare different cation saturated modified montmorillonite, and storing the montmorillonite in a glass reagent bottle for later use. The cation content and exchange rate of the modified montmorillonite are shown in table 1.
TABLE 1 content (mg/kg) of metal cations in modified montmorillonite and exchange rate
Figure GDA0003533901020000051
Cation modified montmorillonite polluted by bi-or polycyclic aromatic hydrocarbon
The method for mixing the polycyclic aromatic hydrocarbon with the cation modified montmorillonite comprises the following steps:
weighing 0.2g of the cation modified montmorillonite in the first item, adding 0.4ml of pyrene solution (the concentration is 500mg/L or 5000mg/L) prepared by acetone into a 50ml glass centrifuge tube, shaking up gently, standing in a fume hood, and obtaining the polluted montmorillonite with the pyrene content of 1mg/g or 10mg/g after acetone is completely volatilized.
Thirdly, culturing and propagating degrading bacteria
Activating and propagating degrading bacteria, wherein the degrading bacteria is Mycobacterium sp.NJS-1, and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC 1.10964. The strain is separated and screened from a farmland polluted by polycyclic aromatic hydrocarbon, and can grow by taking pyrene as a unique carbon source and energy. The activation and propagation comprises the following steps:
thawing degradation bacteria stored in glycerol at-80 ℃, inoculating into a sterilized nutrient broth culture medium, inoculating 0.2mL of preservation solution into each 100mL of culture medium, then carrying out aerobic light-resistant culture at 28 ℃, 160rpm for overnight, taking out after the strain grows to the logarithmic phase, transferring into a sterilized centrifuge tube, centrifuging at 5000rpm to collect the lower layer of bacteria, then adopting sterilized phosphate buffer (pH7.0) clear liquid for 2 times, resuspending, adjusting the bacterial density to ensure that OD600 is 0.30, and the bacterial number is about 1.6 multiplied by 108CFU/mL。
Microbiological degradation experiment of cationic modified montmorillonite surface pyrene
The microbial degradation of pyrene on the surface of cation modified montmorillonite comprises the following steps:
adding a sterilized inorganic salt culture medium into the pyrene-polluted modified montmorillonite obtained in the second item in a 50mL glass centrifuge tube, then inoculating degrading bacteria in the third item, and enabling the bacterial density OD600 to be not less than 0.03 after inoculation. The mass ratio of the modified montmorillonite to the inorganic salt culture medium solution in the degradation system is 1:50(1: 20-1: 100). Then, the mixture was placed on a shaker at a constant temperature of 28 ℃ and 160rpm and was incubated in the dark. And (3) extracting by adopting an organic solvent after culturing for different time, measuring the content of the residual pyrene in the extracting solution by adopting a High Performance Liquid Chromatography (HPLC) method, drawing a degradation kinetic curve (figure 1) and calculating the pyrene degradation rate. As shown in Table 2, both the zero order kinetics and the first order kinetics can better characterize the pyrene degradation law. Pyrene (C)015mg/L), the degradation rate reaches more than 95% in 5 days, the treatment effect of the Fe (III) modified montmorillonite is optimal, the degradation rate reaches 93.6% after 3 days, and the degradation rate is higher than 95% after 5 days by the treatment of Na (I) and Co (II) modified montmorillonite. As shown in figure 2, when the pyrene concentration is increased to 100mg/L, the degradation rate of the Fe (III), Co (II), Na (I) modified montmorillonite is more than 95% within 16 days, and the degradation rate of the treatment without montmorillonite is only less than 60%. The above results illustrate that the cation modified montmorillonite (in particularIs Fe (III) modified montmorillonite), and can obviously enhance the degradation effect of pyrene microorganisms (Mycobacterium sp.NJS-1). However, some cations which are unfavorable for the growth and metabolism of microorganisms, such as cation modified montmorillonite, such as Cu (II), Ni (II) and the like, can inhibit the degradation of pyrene by the degrading bacteria.
TABLE 2 kinetics parameters of microbial degradation of pyrene on surface of cation-modified montmorillonite (C)0＝15mg/L)
Figure GDA0003533901020000061
Example 2
Research on promoting microbial growth by cation modified montmorillonite
The research on promoting the growth of microorganisms by using cation modified montmorillonite comprises the following steps:
method for constructing microorganism-mineral (Mycobacterium sp.NJS-1-cation modified montmorillonite) to degrade pyrene (C)015mg/L), the growth of the microorganisms is analyzed on the 1 st day and the 3 rd day of culture by a plate counting method, and the specific process is as follows: sucking 0.1mL of culture solution into 9mL of sterile water, shaking up, diluting the sample by stepwise dilution method to 104And 105And (3) respectively sucking 0.1mL of diluted sample, inoculating the diluted sample to a sterile nutrient broth solid culture medium, inverting the uniformly coated culture dish, culturing for 48 hours in an incubator at 28 ℃, and counting formed colonies to obtain the change of the bacterial growth density. The results are as follows (fig. 3 a): after 1 day and 3 days of culture, compared with the density of the original bacteria without the addition of the iron ion modified montmorillonite, the CFU (carbon fiber unit) of the cation modified montmorillonite is increased by 3-4 times and 8-10 times respectively compared with the CFU of the cation modified montmorillonite when inoculated, and is closely related to the degradation rate of pyrene (figure 3d), which shows that the cation modified montmorillonite promotes the growth of degradation bacteria, wherein the strengthening treatment effect of the Fe (III) modified montmorillonite is the best.
Research on strengthening activity of microbial degrading enzyme by cation modified montmorillonite
The study on the activity of the cation modified montmorillonite reinforced microbial degrading enzyme comprises the following steps:
pyrene (C) degradation by constructing microorganism-mineral (Mycobacterium sp.NJS-1-cation modified montmorillonite) by the method of example 1015mg/L), dioxygenase activity and electron transport activity and capacity were determined on days 1, 3 and 5 of culture by the following methods:
(1) peroxidase activity: the specific process is as follows: 0.2mL of the sample was aspirated, and 2mL of a sample containing 5mM dihydroxyphenylalanine (L-DOPA, pH7.0 in PBS buffer) and 0.1% H were added2O2After incubation at 28 ℃ for 1.5 hours, the amount of product formed (. epsilon.) was measured spectrophotometrically at a wavelength of 460nm460＝37000M-1cm-1). The results show (fig. 3 b): the peroxidase activity of different cation modified montmorillonite shows different change rules along with pyrene degradation, and the peroxidase activity of Fe (III) modified montmorillonite is always in a higher level.
(2) Dioxygenase activity ( Catechol 2,3 dioxygenase cathechol 2,3-dioxygenase, C23O): the specific process is as follows: 0.2mL of the sample solution was aspirated, 1mL of a sample solution containing 0.5mM of catechol (prepared in PBS buffer, pH7.0) was added, and after incubation at 28 ℃ for 4 hours, the amount of the product formed (. epsilon.) was measured spectrophotometrically at a wavelength of 375nm375＝36000M-1cm-1). The results show (fig. 3 c): with the gradual degradation of pyrene, the activity of dioxygenase was increased, and the activity of enzyme reached a maximum at day 5 of degradation. Wherein the treated dioxygenase activity was maximal for Fe (III) and Na (I) modified montmorillonite.
Through correlation analysis, the significant correlation relationship between the activity of bacterial peroxidase and the degradation rate of pyrene (figure 3e) and the activity of dioxygenase and the degradation rate of pyrene (figure 3f) in the degradation process of pyrene can be found, which indicates that the improvement of the activity of key enzyme is a main way for enhancing the degradation of pyrene by using cation modified montmorillonite.
(III) research on bioelectrochemical activity of microorganisms reinforced by cation modified montmorillonite
The study on the bioelectrochemical activity of the microorganisms reinforced by the cation modified montmorillonite comprises the following steps:
construction of a microorganism-mineral Using the method of example 1Pyrene (C) degraded by substance (Mycobacterium sp.NJS-1-cation modified montmorillonite)015mg/L), dioxygenase activity and electron transport activity and capacity were determined on days 1, 3 and 5 of culture by the following methods:
(1) electron Transport System Activity (ETSA): 0.5mL of the sample solution was aspirated, and 0.5mL of PBS buffer (pH7.0) and 0.5mL of iodonitrotetrazole violet (INT, 0.1%) were added, followed by incubation at 30 ℃ in the dark. After the incubation was completed, 5mL of methanol was added to terminate the reaction, the product formazan (INTF) was extracted, and then the absorbance of the product was measured at 490nm using a spectrophotometer, and the product INTF was calculated according to a standard curve. The electron transfer activity is expressed as the INTF produced per unit time. The results show (fig. 4a), the electron transfer activity of na (i) modified montmorillonite treatment increases with pyrene degradation, while Fe (iii) modified montmorillonite treatment has a decreasing trend, and in general, the electron transfer activity of Fe (iii) modified montmorillonite is higher than that of na (i) modified montmorillonite, which is probably because Fe is a prosthetic group of some redox proteins, regulates the enzyme activity and transfers electrons through the change of its valence state, and can be used as an extracellular electron acceptor to play an important role in the bioelectrochemical process of a microorganism-mineral interface, so that the degradation speed of pyrene at the interface of iron ion modified montmorillonite is faster.
(2) Electron transfer capacity: the sample current time (i-t) curve was recorded using an electrochemical workstation and the electron donating ability (EDC) of the sample was measured. The specific process is as follows: 9mL of PBS buffer (pH 7) and 0.1M KCl were added to the cell to equilibrate the desired redox potential to 0.61V, followed by 0.1mL of the electron transfer medium ABTS (10mM) to give rise to an oxidation peak current, and once a constant background current was again reached, 0.1mL of sample solution was added to the cell to give rise to a sample oxidation peak current.
The electron supply capacity of the sample was calculated by the following formula:
Figure GDA0003533901020000081
where V is the sample volume, Iox is the oxidation current after baseline correction, and F is the faraday constant (F-96485C mol)-1) EDC in μmol e-mL-1. The results show (fig. 4 b): the cation modified montmorillonite-microbial degradation system has higher electron supply capacity, the electron supply capacity is firstly increased and then reduced along with the degradation of pyrene, the electron supply capacity of the Fe (III) modified montmorillonite is slightly higher than that of the Na (I) modified montmorillonite at the initial stage, but the electron supply capacity is opposite at the later stage. The results show that as pyrene degrades, the electron donating ability of the degradation system increases and then decreases. According to the results, the cation modified montmorillonite can enhance pyrene degradation by regulating degrading enzyme activity and bioelectrochemical activity.
The results show that the metal cation modified montmorillonite such as Fe (III), Co (II), Na (I) and the like can promote the bioelectrochemical processes such as electron transfer between minerals and microorganisms by promoting the growth of microorganisms and the metabolic activity of key enzymes, so as to further strengthen the degradation of polycyclic aromatic hydrocarbon (pyrene), wherein the Fe (III) modified montmorillonite has the best strengthening effect and has better application potential in soil pollution remediation.
In some embodiments, the cation-modified montmorillonite enhances microbial degradation by the following method:
s1 culturing and propagating Mycobacterium sp.NJS-1 bacteria by adopting a sterilized nutrient broth culture medium until the number of bacteria or the density of the bacteria reaches a specific value; s2 adding a sterilized inorganic salt culture medium to the cation-modified montmorillonite, and then inoculating the Mycobacterium sp.NJS-1 bacterium of step S1; s3, adding the inoculated cation modified montmorillonite into a polluted solution containing polycyclic aromatic hydrocarbon for mixed culture to degrade polycyclic aromatic hydrocarbon, such as pyrene, and compared with the single Mycobacterium sp.NJS-1 bacterium, the montmorillonite is directly used for degrading pyrene and has higher degradation rate; compared with the method that pyrene is degraded after unmodified montmorillonite and Mycobacterium sp.NJS-1 bacteria are mixed, the method has higher degradation rate.
In some embodiments, iron ion modified montmorillonite prepared in different batches is used for enhancing the microbial degradation, wherein the exchange rate of iron ions is within a range of 50-100%, and the degradation rate is higher compared with that of single Mycobacterium sp.NJS-1 bacterium which is directly used for degrading pyrene; and has higher degradation rate compared with the degradation of pyrene after mixing unmodified montmorillonite and Mycobacterium sp.NJS-1 bacteria.
The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.

Claims (2)

1. A method for strengthening the microbial degradation of polycyclic aromatic hydrocarbon is characterized by comprising the steps of inoculating microorganisms to cation modified montmorillonite; the microorganism is Mycobacterium (Mycobacterium sp.) NJS-1, and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC 1.10964;
the cation modified montmorillonite comprises one of Fe (III), Co (II) or Na (I) modified montmorillonite;
the polycyclic aromatic hydrocarbon is pyrene;
the method comprises the following steps:
s1 culturing and propagating Mycobacterium (Mycobacterium sp.) NJS-1 by adopting a sterilized nutrient broth culture medium, and then adjusting the density of the strain to OD600 of 0.30;
s2, adding a sterile inorganic salt culture medium into the cation modified montmorillonite, wherein the solid-liquid mass ratio of the modified montmorillonite to the inorganic salt culture medium is 1: 20-1: 100; then inoculating the Mycobacterium (Mycobacterium sp.) NJS-1 of step S1, wherein the OD600 of the Mycobacterium (Mycobacterium sp.) NJS-1 strain is not less than 0.03 after inoculation and dilution;
s3, carrying out mixed culture on the inoculated cation modified montmorillonite and the pollutant containing polycyclic aromatic hydrocarbon under a proper condition to degrade the polycyclic aromatic hydrocarbon;
or
S1, adsorbing and fixing pollutants containing polycyclic aromatic hydrocarbons by using cation modified montmorillonite;
s2 culturing and propagating Mycobacterium (Mycobacterium sp.) NJS-1 by adopting a sterilized nutrient broth culture medium, and adjusting the density of the strain to OD600 to 0.30;
s3, adding a sterile inorganic salt culture medium into the cation modified montmorillonite adsorbed with the pollutants, wherein the solid-liquid mass ratio of the modified montmorillonite to the inorganic salt culture medium is 1: 20-1: 100; then inoculating the Mycobacterium (Mycobacterium sp.) NJS-1 of step S2, wherein the OD600 of the Mycobacterium (Mycobacterium sp.) NJS-1 strain is not less than 0.03 after inoculation and dilution; mixed culture is carried out under proper conditions, and polycyclic aromatic hydrocarbon is degraded;
the cation modified montmorillonite is prepared by an ion exchange method, and specifically comprises the following steps:
a) the solid-to-liquid ratio of the montmorillonite to the chloride solution of the metal cations is 1: 20;
b) the equivalent concentration of metal cations is 0.1N;
c) adopting an ion exchange method, wherein the exchange times are not less than 2;
d) the oscillation balance time of each exchange is not less than 2 h;
e) after the exchange is completed, the reaction solution is washed with ultrapure water for more than 4 times until no Cl exists-。
2. The method for enhancing microbial degradation of polycyclic aromatic hydrocarbons according to claim 1, wherein the mycobacteria (Mycobacterium sp.) NJS-1 propagation conditions comprise: aerobic culture, inoculating a proper amount of bacterial liquid in a sterilized nutrient broth culture medium, and culturing overnight at 28 ℃ and 160rpm in a dark condition; the cells were washed twice with sterile pH7.00.1M phosphate buffer to adjust cell density.
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