CN115710570A - Trichloroethylene degrading bacterium and application thereof - Google Patents
Trichloroethylene degrading bacterium and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of bioengineering, and provides a trichloroethylene degrading bacterium and application thereof. The trichloroethylene degrading bacteria belongs to gram-positive bacteria, is identified as bacillus subtilis by molecular level, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation number of CGMCC 24319, can grow by using trichloroethylene as a unique carbon source, does not need to depend on a co-metabolism substrate for degradation, can be directly degraded when being put into a sewage treatment system containing the trichloroethylene, and further prevents the trichloroethylene from entering soil or underground water environment to cause long-term pollution.
Description
Technical Field
The invention belongs to the field of bioengineering, and provides a trichloroethylene degrading bacterium and application thereof.
Background
Trichloroethylene (TCE) is a common chemical feedstock, but has become one of the common organic pollutants in environmental waters due to improper disposal and discharge. It has potential carcinogenic, teratogenic, mutagenic toxicity, and poses great threat to the ecological environment and human body. In atmospheric environment, TCE is degraded in one week, but the half life period in soil is about 0.5-1.5 years due to strong adsorption property and stability of TCE; the half life in groundwater is about 1-4.5 years. The conversion of TCE between atmospheric and surface water environments relies primarily on soil access to the bulk of groundwater, and therefore TCE, once in soil and groundwater environments, can cause long-term contamination of large areas. Therefore, it is very important to reduce TCE pollution in the above-ground environment timely and effectively.
The physical, chemical and biological methods can remove TCE. The physical and chemical methods such as adsorption, membrane separation and stripping are expensive, and only transfer the pollutants but not convert the pollutants into harmless products, which is easy to cause secondary pollution. The chemical methods currently studied are a TiO2 photocatalysis method, a zero-valent iron reduction method and a bimetallic reduction method. Chemical processes are relatively expensive and more cost effective processes are being sought. TCE is converted into a non-toxic substance through the action of microorganisms, is economical and effective, has no secondary pollution, and becomes a hot point of research in recent years.
Microorganisms are the main body of the TCE biodegradation process, and the metabolism of pollutants by microorganisms is the core of the microbial degradation process. The biological method has the advantages of high treatment efficiency, complete degradation and the like, does not cause secondary pollution, and is widely applied to the remediation and treatment engineering of the organic matters difficult to degrade. TCE is an organic matter with strong toxicity after all, most experiments prove that high-concentration TCE has strong toxicity to microorganisms, most aerobic bacteria in the research of trichloroethylene degrading bacteria are degraded by aerobic-co-metabolism, a growth substrate, namely a first substrate is used as an electron donor, microorganisms convert a non-growth substrate (trichloroethylene) by taking the first substrate as a carbon source and an energy source, the number of the TCE directly degrading bacteria separated at present is limited, the polluted site environment is complex, and the TCE can not be effectively removed by simply depending on the TCE directly degrading bacteria.
Therefore, it is especially important to isolate and breed strains with high tolerance and degradation ability for application in TCE bioremediation and bioaugmentation technology.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a trichloroethylene degrading bacterium with strong environmental adaptability so as to solve the problem that trichloroethylene is poor in biodegradability and difficult to degrade.
Firstly, the trichloroethylene degrading bacteria is obtained by collecting a sample from a sewage treatment system of a petrochemical sewage plant, enriching the sample, and further performing gradient enrichment, separation and purification on the sample in an inorganic salt culture medium taking trichloroethylene as a unique carbon source. Meanwhile, fujiwara test is further carried out, and the bacterium can grow by using trichloroethylene as a unique carbon source.
The trichloroethylene degrading bacteria is Bacillus subtilis, is preserved in China general microbiological culture Collection center (CGMCC), and has the preservation number of CGMCC No.24319.
The bacillus subtilis is gram-positive bacteria, the surface of a bacterial colony is wrinkled, rough and opaque, the bacterial colony is white or yellowish, the edge is irregular, and the bacillus subtilis is picked up and drawn.
The invention also aims to protect the application of the Bacillus subtilis in the aspect of degrading trichloroethylene. The specific application range comprises the biological strengthening treatment of waste water, waste gas and soil containing trichloroethylene.
The specific application method comprises the following steps: the bacillus subtilis or the microbial inoculum thereof is put into a treatment system, can effectively resist the impact of trichloroethylene pollutants, and can utilize organic matters in a biochemical system as nutrient substances for self growth to quickly degrade the organic matters. Preferably, the effective viable count of the initial bacillus subtilis of the treatment system is ensured to be 1-8 multiplied by 10 7 cfu/mL can enable the strain to adapt to the environment quickly, and the trichloroethylene is used as a carbon source for metabolic growth, so that the trichloroethylene degradation effect of the strain is effectively exerted.
Furthermore, the trichloroethylene degrading bacteria can be continuously cultured and applied in trichloroethylene sewage, and the degrading effect is stable.
As a further scheme, the invention provides a microbial inoculum of the trichloroethylene degrading bacteria, which is prepared by fermentation:
the fermentation medium comprises the following components in percentage by mass: 5.5-6.5% of soybean meal, 6-6.5% of corn starch, 0.3-0.5% of glucose, 0.3-0.5% of calcium carbonate, 0.1-0.5% of corn steep liquor dry powder, 0.1-0.3% of dipotassium phosphate, 0.05-0.15% of magnesium sulfate, 0.1-0.2% of sodium chloride, 0.01-0.03% of manganese sulfate and the balance of water, wherein the pH value is 7-7.5. Autoclaving at 121 deg.C for 30 min.
The trichloroethylene degrading bacteria is subjected to high-density fermentation culture for 48-72h at the temperature of 30-37 ℃, and after the fermentation is finished, a liquid microbial inoculum is obtained, wherein the effective viable count of the microbial inoculum is 5 multiplied by 10 8 ～2×10 10 cfu/mL. Can be further prepared into solid microbial inoculum and the like, and the preparation method adopts the conventional technology.
Further, the fermentation temperature is 33 + -1 deg.C, dissolved oxygen is about 30%, culturing for 48-72h, dissolved oxygen rises, pH drops, and fermentation is stopped.
Preferably, the liquid microbial inoculum obtained by the method is applied to a processing system containing trichloroethylene, the adding amount (calculated by volume) of the microbial inoculum is 1 per mill to 5 per mill of the volume of a biochemical system, and preferably, the biochemical system is at the temperature of 25 ℃ to 38 ℃, the pH value is 6.0 to 8.0, and the dissolved oxygen is more than or equal to 2mg/L.
Preferably, the initial concentration of the trichloroethylene in the biochemical system is below 900mg/L, and more preferably, the initial concentration of the trichloroethylene in the biochemical system is below 300 mg/L; most preferably, the initial concentration of trichloroethylene in the biochemical system is below 150 mg/L.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a degrading bacterium capable of directly using trichloroethylene as a unique carbon source, which can rapidly degrade the trichloroethylene under the condition of using the trichloroethylene as the unique carbon source and energy source, has the characteristics of simplicity and high efficiency in application, simultaneously has strong toxicity tolerance capacity to the trichloroethylene, has wide application range to conditions such as temperature, pH and the like, and has good application prospect in biological purification of industrial wastewater and waste gas.
Preservation information
Preservation time: 1 month 17 days 2022;
the name of the depository: china general microbiological culture Collection center;
the preservation number is: CGMCC No.24319;
the address of the depository: xilu No. 1 Hospital No. 3, beijing, chaoyang, beicheng;
classification nomenclature Bacillus subtilis.
Drawings
FIG. 1 comparative graph of biodegradation of Bacillus subtilis YJY22-10 on trichloroethylene at different temperatures in example 4;
FIG. 2 comparative graph of the biodegradation of B.subtilis YJY22-10 on trichloroethylene under different pH conditions in example 4;
FIG. 3 comparative graph of degradation of Bacillus subtilis YJY22-10 in example 4 to different initial concentrations of trichloroethylene.
Detailed Description
The above-described subject matter of the present invention is described in further detail below with reference to specific embodiments, but it should not be understood that the scope of the subject matter of the present invention is limited to the examples below. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention, and the following embodiments are all completed by adopting the conventional prior art except for the specific description.
The "dissolved oxygen" of the microbial inoculum fermentation process referred to in the following examples refers to air saturation (%), which is a common method for indicating the dissolved oxygen concentration in the fermentation industry.
The "dissolved oxygen" in the aeration process of sewage treatment in the following examples refers to the absolute concentration of dissolved oxygen in water, expressed in mg/L, and is a commonly used dissolved oxygen expression method for sewage treatment in the environmental protection industry.
Example 1 enrichment, screening and isolation of Trichloroethylene-degrading bacteria
Sludge near a sewage treatment system of a certain petrochemical and agro-chemical sewage treatment plant area is taken for enrichment and screening treatment, and trichloroethylene degrading bacteria are enriched by adopting a method for gradually increasing the concentration of trichloroethylene.
The specific operation method comprises the following steps: taking 10mL of an activated sludge sample, inoculating the activated sludge sample into 100mL of enrichment medium (the initial concentration of trichloroethylene is 10 mg/L), carrying out enrichment culture by shaking and culturing for 2-5d at 30 ℃ in a shaking table at 160r/min, sampling and detecting the content of the trichloroethylene in the system, taking 10mL of bacterial liquid out and inoculating the bacterial liquid into fresh 100mL of inorganic salt medium (the concentration of the trichloroethylene is 20 mg/L) when the content of the trichloroethylene is degraded to be lower than 1mg/L, repeating the steps, and gradually increasing the concentration of the trichloroethylene in the medium to 500mg/L by taking 50mg/L, 75 mg/L, 100mg/L, 125 mg/L, 150mg/L and 200mg/L as gradients.
And (3) coating the final enrichment culture solution on an inorganic salt culture medium plate taking trichloroethylene as a unique carbon source after gradient dilution, and culturing for 2-3d in an incubator at 30 ℃. The single colonies with different forms growing on the plate are respectively streaked on an inorganic salt culture medium plate for purification for at least 3 times, the single colonies after purification are selected and inoculated in 10mL of LB liquid culture medium, the single colonies are respectively inoculated in 100mL of inorganic salt culture medium containing 50mg/L of trichloroethylene in an inoculation amount of 2% (V/V) in the next day, the cultivation is carried out for 24h at 30 ℃ at 160r/min, and then the sampling is carried out to detect the content of the trichloroethylene by adopting gas chromatography.
10 strains of bacteria were isolated by this method, of which 45 was able to completely degrade 100mg/L of trichloroethylene in 24 hours and stored.
To further verify that the isolated species are subject to growth metabolism using trichloroethylene as the sole carbon and energy source, a Fujiwara test was performed to determine the concentration of free polychlorinated hydrocarbons in the system. TCE was treated with pyridine in an alkaline environment and the absorbance of the aqueous phase was then measured spectrophotometrically at 470nm, indicating Fujiwara test positive when a pink color developed.
The content of trichloroethylene in the inorganic salt culture medium is 100mg/L, the bacteria with the number of 45 which are screened by 2% (V/V) inoculation are used for the experimental group, sterile water with corresponding content is added into the control group, the samples are sampled and detected after culturing for 24 hours at the temperature of 30 ℃ and at the speed of 160r/min, the water phase detected after the treatment of the experimental group has no pink, and the control group is pink, which indicates positive. The experiment shows that the strain can perform growth and metabolism by taking trichloroethylene as a unique carbon source.
The inorganic salt culture medium contains the following components in each 1L: k 2 HPO 4 1g，KH 2 PO 4 1g，NaCl 0.5g，NH 4 Cl 1g，MgSO 4 ·7H 2 O 0.2g，CaCl 2 15mg，FeSO 4 ·7H 2 O 2mg，CuSO 4 ·5H 2 O 0.4mg，KI 1mg，MnSO 4 ·H 2 O 4mg，H 3 BO 3 5mg，CoCl 2 ·6H2O 1mg，Na 2 MoO 4 ·2H 2 O 2mg，NiCl 2 ·6H 2 O2mg, pH 7.0 + -0.2, sterilizing, naturally adjusting pH, preparing with distilled water, sterilizing at 121 deg.C for 20min, adding agar 20g into solid culture medium, and adding trichloroethylene before use.
Adding trichloroethylene into the solid culture medium, attaching a piece of sterile filter paper on a cover of a culture dish, dripping the trichloroethylene on the filter paper, and culturing the thallus by using the vapor of the trichloroethylene.
The enrichment medium is prepared by adding water to 1.0L according to 10.0g of peptone, 5.0g of yeast powder and 10.0g of sodium chloride, adjusting pH value to 7.0, adding 20.0g of agar into the solid medium, and sterilizing at 121 deg.C for 20min.
Example 2 identification of the species of the trichloroethylene-degrading bacteria and the characteristics of the colonies
And (3) strain identification: the trichloroethylene degrading bacteria numbered 45 and obtained in the example 1 are subjected to 16S rDNA identification, an NCBI database compares the 16S rDNA sequence, and the identification result on the molecular level shows that the bacillus belongs to the bacillus subtilis, and the nucleotide sequence of the 16S rDNA is shown as SEQ ID in a sequence table.
The trichloroethylene degrading bacteria bacillus subtilis is gram-positive bacteria, the surface of a bacterial colony is wrinkled, rough and opaque, the bacterial colony is white or yellowish, the edge is irregular, and a drawn wire is picked up. It is named YJY22-10 and is biologically preserved with the biological preservation number of CGMCC No.24319
EXAMPLE 3 preparation of trichloroethylene-degrading microbial inoculum
The strain is used for preparing the trichloroethylene degrading microbial inoculum, and the specific preparation method comprises the following steps:
firstly, transferring a test tube slant strain stored on a nutrient agar culture medium at 4 ℃ to a room temperature (20-25 ℃) condition for activation for 4-8 h, wherein the culture medium does not need to be additionally supplemented during activation;
secondly, inoculating the single colony obtained in the first step into an LB liquid culture medium (100 ml), culturing overnight at 35 ℃ and 185r/min to obtain a first-stage seed solution, inoculating the obtained first-stage seed solution into the LB liquid culture medium according to 10% (V/V), culturing for 16h under the same condition to obtain a second-stage seed solution, and storing at 4 ℃;
and thirdly, inoculating the secondary seed solution obtained in the second step into a fermentation culture medium according to 1 per mill (V/V) for fermentation culture, controlling the fermentation temperature to be 33 +/-1 ℃, controlling the dissolved oxygen to be about 30 percent, culturing for 20-30h, raising the dissolved oxygen, reducing the pH, and stopping fermentation to obtain the trichloroethylene degrading microbial inoculum.
The viable count of the trichloroethylene degrading microbial inoculum obtained in the embodiment is 1.3 multiplied by 10 10 cfu/mL。
The fermentation medium comprises the following components in percentage by mass: 6% of soybean meal, 6% of corn starch, 0.5% of glucose, 0.3% of calcium carbonate, 0.1% of corn steep liquor dry powder, 0.2% of dipotassium phosphate, 0.1% of magnesium sulfate, 0.1% of sodium chloride, 0.02% of manganese sulfate, 0.1% of polyether defoamer and 7-7.5% of pH; autoclaving at 121 deg.C for 30 min.
The LB culture medium is prepared from peptone 10.0g, yeast powder 5.0g, sodium chloride 10.0g, water to 1.0L, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20min.
Example 4 detection of degradation characteristics of Trichloroethylene-degrading bacterium
And (3) inoculating 2mL of the trichloroethylene degrading microbial inoculum obtained in the example 3 into 50mL of inorganic salt culture medium, wherein trichloroethylene is used as the only carbon source and energy source for detecting the degrading capability. In order to reduce the volatilization of trichloroethylene and ensure the dissolved oxygen amount of the strains, the following degradation experiments are carried out in a glass bottle with a frosted opening of 250 mL.
1. Biodegradation characteristics of bacillus subtilis YJY22-10 on trichloroethylene at different temperatures
Experiments on the biodegradation characteristics of the bacillus subtilis YJY22-10 to trichloroethylene at different temperatures find that the bacillus subtilis YJY22-10 has better degradation capability to trichloroethylene at the temperature of more than 30 ℃, and the degradation capability of the bacillus subtilis YJY22-10 at the temperature of 33 ℃ is optimal in practical application, and the specific implementation steps are as follows:
setting 8 experimental groups, repeating 3 groups, wherein the culture medium is an inorganic salt culture medium (liquid), adding trichloroethylene according to 150mg/L, enabling the pH to be natural, setting different culture temperatures after inoculation, setting the experimental temperatures to be 20-40 ℃ (20 ℃,25 ℃,28 ℃,30 ℃,33 ℃,35 ℃,38 ℃ and 40 ℃) respectively, placing the experimental temperatures in a shaking table for shaking culture at 160r/min, sampling after 24 hours, and carrying out headspace gas phase detection to obtain the optimal action temperature of 25-40 ℃, and detailed shown in figure 1;
2. biodegradation characteristic of bacillus subtilis YJY22-10 on trichloroethylene under different pH conditions
Setting 7 experimental groups, wherein each group is repeated for 3 times, the culture medium adopts an inorganic salt culture medium, trichloroethylene is added according to 150mg/L, different pH values are respectively set, the experimental range of the pH value is set to be 4-10 (respectively set to be 4,5,6,7,8,9,10), after inoculation, shaking culture is carried out in a shaking table at 30 ℃ and 160r/min, sampling extraction is carried out after 24 hours for detection, the adaptable range is wide, the optimal application pH value is 6.5-8.0, and detailed pictures are shown in figure 2.
3. Degradation condition of bacillus subtilis YJY22-10 on trichloroethylene with different initial concentrations
Setting 5 experimental groups in total, wherein each group is provided with three repetitions, adding 50mL of inorganic salt culture medium into a ground triangular flask, taking trichloroethylene as a unique carbon source, the initial concentration of the trichloroethylene of each experimental group is respectively 146, 293, 586, 879 and 1172mg/L, culturing at 30 ℃ and 160r/min, sampling after 24 hours, and detecting the residual amount of the trichloroethylene, wherein when the initial concentration is less than 900mg/L, the degradation rate of the trichloroethylene in 24 hours can reach more than 80%; when the initial concentration is below 150mg/L, the degradation rate of the nano-silver particles in 24 hours can reach about 100 percent. The bacterial strain is proved to have strong toxicity tolerance to trichloroethylene, and the detail is shown in figure 3.
Example 5 simulation application of trichloroethylene-degrading bacteria in trichloroethylene sewage treatment
A trichloroethylene-degrading bacterial agent was prepared by the method of example 3, the culture temperature was 33. + -. 1 ℃ and the dissolved oxygen was controlled to 30%, and the viable count of the obtained bacterial agent was 5X 10 8 cfu/mL。
Simulation application: the water quality source for treatment is water inlet of an aerobic section of a pesticide sewage treatment plant, the treatment capacity is 5L, the addition amount of a trichloroethylene degrading microbial inoculum is 0.3 percent (V/V), the dissolved oxygen is more than 2mg/L by aeration treatment at room temperature, the pH value of the sewage is 6.0-8.0, the COD of the original sewage is about 4000mg/L, the ammonia nitrogen is about 380mg/L, the organic amine is about 200mg/L, the concentration of the artificially added trichloroethylene is 100mg/L, the trichloroethylene content of the sewage is less than 1mg/L after 24 hours, and the degradation rate is more than 99.9 percent.
Example 6 application of Trichloroethylene degrading bacteria in Trichloroethylene Sewage treatment
A trichloroethylene-degrading bacterial agent was prepared by the method of example 3, the culture temperature was 35. + -.1 ℃ and the dissolved oxygen was controlled to 30%, and the number of viable bacteria of the obtained bacterial agent was 1.5X 10 10 cfu/mL。
The water quality source for treatment is water inlet of an aerobic pool of a petrochemical sewage treatment plant, the treatment capacity is 10L, the addition amount of a trichloroethylene degrading microbial inoculum is 0.2% (V/V), aeration treatment is carried out at room temperature to ensure that the dissolved oxygen is more than 2mg/L, the pH of sewage is 6.0-8.0, the concentration of trichloroethylene is less than 5mg/L, the COD is about 2200mg/L, the concentration of volatile phenol is about 240mg/L, the ammonia nitrogen is about 100mg/L, and the sulfide is about 10mg/L, water inlet and outlet are carried out once every 24 hours, the concentration of trichloroethylene in the inlet water and the outlet water is detected, the system is continuously used, and the bacteria has continuous degradation capacity according to the tracking condition.
Claims (10)
1. A trichloroethylene degrading bacterium is characterized in that the strain is Bacillus subtilis and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24319.
2. The trichloroethylene degrading bacterium of claim 1 wherein the strain is capable of growth and metabolism using trichloroethylene as the sole carbon source.
3. The application of the bacillus subtilis in the aspect of degrading the trichloroethylene is characterized in that the bacillus subtilis is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.24319.
4. The application of the bacillus subtilis in degrading the trichloroethylene according to the claim 3, wherein the specific application range comprises: the biological strengthening treatment of waste water, waste gas and soil containing trichloroethylene.
5. The application of the bacillus subtilis in degrading trichloroethylene according to claim 3, which is characterized in that the specific application method comprises the following steps: the bacillus subtilis or the microbial inoculum thereof is put into a treatment system to ensure that the effective viable count of the bacillus subtilis at the initial stage of the treatment system is 1-8 multiplied by 10 7 cfu/mL。
6. The use of Bacillus subtilis according to claim 3 for the degradation of trichloroethylene in a continuous culture in trichloroethylene-containing wastewater.
7. A trichloroethylene degrading microbial inoculum is characterized by being prepared by fermentation:
the fermentation medium comprises the following components in percentage by mass: 5.5-6.5% of soybean meal, 6-6.5% of corn starch, 0.3-0.5% of glucose, 0.3-0.5% of calcium carbonate, 0.1-0.5% of corn steep liquor dry powder, 0.1-0.3% of dipotassium phosphate, 0.05-0.15% of magnesium sulfate, 0.1-0.2% of sodium chloride, 0.01-0.03% of manganese sulfate and the balance of water, wherein the pH value is 7-7.5; sterilizing at 121 deg.C under high pressure for 30 min;
performing high-density fermentation culture on trichloroethylene degrading bacteria at 30-37 deg.C for 48-72 hr to obtain liquid microbial inoculum with effective viable count of 5 × 10 8 ～2×10 10 cfu/mL；
The trichloroethylene degrading bacteria are Bacillus subtilis and are preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24319.
8. The trichloroethylene degrading bacterial agent of claim 7, wherein the fermentation temperature is 33 ± 1 ℃, the dissolved oxygen is about 30%, the cultivation is carried out for 48-72 hours, the dissolved oxygen is increased, the pH is reduced, and the fermentation is stopped.
9. The use method of the trichloroethylene degrading microbial inoculum according to claim 7, characterized in that the obtained liquid microbial inoculum is applied to a processing system containing trichloroethylene, the adding amount of the microbial inoculum is 1 per mill to 5 per mill of the volume of a biochemical system, the conditions of the biochemical system are that the temperature is 25 ℃ to 38 ℃, the pH value is 6.0 to 8.0, and the dissolved oxygen is more than or equal to 2mg/L.
10. The method of claim 9, wherein the initial concentration of trichloroethylene in the biochemical system is less than 900 mg/L.
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