CN117821280A - Bacillus brevis FHY-12 with dichloroethylene degradation capability and application thereof - Google Patents
Bacillus brevis FHY-12 with dichloroethylene degradation capability and application thereof Download PDFInfo
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Abstract
The invention discloses a soil bacillus brevis (Brevibacillus agri) FHY-12 with dichloroethylene degradation capability and application thereof, wherein the soil bacillus brevis (Brevibacillus agri) FHY-12 is preserved in China center for type culture collection, and addresses: chinese, university of armed chinese, postal code: 430072, deposit number: cctccc NO: m2023419, storage date 2023, month 03 and 28. The discovery of the strain is of great significance to the efficient purification of chlorinated organic pollutants such as DCE and the like in industrial wastewater and waste gas.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a bacillus brevis FHY-12 strain with dichloroethylene degradation capability and application thereof.
Background
Dichloroethylene (DCE) is an important chemical raw material, can be used as a solvent for paint, resin, wax, rubber and acetate fiber, can also be used as a dry cleaning agent, an insecticide, a bactericide, a low-temperature extractant, a refrigerant and the like, and is a common groundwater pollutant. DCE has a major hazard to human health, mainly affects the central nervous system, stimulates the eyes and upper respiratory tract, can damage the cornea and burn the skin, and can cause neurasthenia syndrome after long-term contact. The 10 th 2021 department of ecological environment incorporates DCE into the new pollutant remediation action program (solicitation manuscript), and therefore, the development of DCE removal technology is urgent.
The biological purification technology is widely applied to the treatment of pollutants due to the advantages of high removal efficiency, low treatment cost, no secondary pollution and the like. The key point of biological purification of DCE is to obtain a strain with the capability of efficiently degrading DCE. However, due to the extremely poor solubility of DCE and the high bond energy between carbon and chlorine atoms, only a small amount of DCE degrading bacteria were isolated, mainly including Pseudomonas (Pseudomonas), bacillus (Bacillus), fusobacterium (Fusobacterium), pandorea (Pandoraaea) and the like, and the degradation efficiency still has to be further improved. At present, the separation and screening of high-efficiency DCE degrading bacteria from the environment are still one of the important methods for eliminating chlorinated organic pollutants in the environment.
Disclosure of Invention
Aiming at overcoming the defects of the prior art, the invention provides the soil bacillus brevis FHY-12 with dichloroethylene degradation capability and the application thereof.
The aim of the invention is realized by the following technical scheme:
in a first aspect, the invention provides a strain of Brevibacillus tumefaciens with dichloroethylene degrading capability, the microorganism classification is named Brevibacillus tumefaciens (Brevibacillus agri) FHY-12, which has been deposited in China center for type culture Collection at 2023, 03, 28: the university of martial arts in China, postal code 430072; the preservation number is CCTCC NO: M2023419; the 16S rRNA sequence of FHY-12 is shown in SEQ ID NO. 1.
In a second aspect, the invention provides a bacterial suspension with soil bacillus brevis FHY-12 with dichloroethylene degradation capability as an active ingredient and a preparation method of the bacterial suspension.
The bacterial suspension is prepared by solid culture, seed culture and liquid culture of soil bacillus brevis FHY-12 with dichloroethylene degradation capability.
The preparation method of the bacterial suspension comprises the following specific steps:
(1) Solid culture: inoculating soil bacillus brevis FHY-12 with dichloroethylene degradation capability to a slant culture medium, and culturing for 2-4 days at 20-40 ℃ to obtain slant thalli; the final concentration composition of the slant culture medium is as follows: k (K) 2 HPO 4 1500mg/L，KH 2 PO 4 500mg/L NaCl 1000mg/L dichloroethylene 50mg/L MgSO 4 ·7H 2 200mg/L of O, water as solvent, pH value of 6.0-8.0 and 18-20 g/L of agar;
(2) Seed culture: bacterial colony is selected from the inclined plane thallus and inoculated to a seed culture medium, and the bacterial colony is cultured for 12 to 24 hours at the temperature of 20 to 40 ℃ to obtain seed liquid; the final concentration composition of the seed culture medium is as follows: 10g/L NaCl, 5g/L yeast extract powder, 10g/L peptone, water as solvent and pH value of 6.0-8.0;
(3) Liquid culture: inoculating the seed solution to a fermentation culture medium according to the inoculum size with the volume concentration of 1%, and culturing for 12-24 hours at the temperature of 20-40 ℃ to obtain a fermentation culture solution, namely a bacterial suspension; the final concentration composition of the fermentation medium is as follows: k (K) 2 HPO 4 1500mg/L，KH 2 PO 4 500mg/L NaCl 1000mg/L dichloroethylene 50mg/L MgSO 4 ·7H 2 200mg/L O, water as solvent and pH 6.0-8.0.
In a third aspect, the invention provides an application of bacterial suspension taking soil Brevibacillus FHY-12 with dichloroethylene degradation capability as an active ingredient in dichloroethylene degradation and a specific method thereof.
The specific method comprises the following steps: inoculating the bacterial suspension into a first DCE liquid selection culture medium with the salt concentration of 0-1%, and culturing the bacterial suspension at the temperature of 25-35 ℃ by taking dichloroethylene as a unique carbon source to degrade the dichloroethylene; the final concentration composition of the first DCE liquid selection medium is: DCE 1-12 mg/L, KH 2 PO 4 376mg/L，K 2 HPO 4 456mg/L，(NH 4 ) 2 SO 4 480mg/L，NaNO 3 680mg/L，Mg(NO 3 ) 2 250mg/L，CaCl 2 ·2H 2 O11mg/L, trace elements, water as solvent and pH 7.0-9.0; the final concentration composition of the microelements is as follows: mnCl 2 ·H 2 O 60mg/L，ZnCl 2 88mg/L，KI 10mg/L，NaMoO 4 ·2H 2 O 100mg/L，H 3 BO 3 50mg/L, water as solvent.
Further, the specific method comprises the following steps: inoculating the bacterial suspension into a first DCE liquid selection culture medium with the salt concentration of 0.72%, and culturing the bacterial suspension at 35 ℃ by taking dichloroethylene as a unique carbon source to degrade the dichloroethylene; the final concentration composition of the first DCE liquid selection medium is: DCE 1-12 mg/L, KH 2 PO 4 376mg/L，K 2 HPO 4 456mg/L，(NH 4 ) 2 SO 4 480mg/L，NaNO 3 680mg/L，Mg(NO 3 ) 2 250mg/L，CaCl 2 ·2H 2 O11mg/L, trace elements, water as solvent and pH 7.5; the final concentration composition of the microelements is as follows: mnCl 2 ·H 2 O 60mg/L，ZnCl 2 88mg/L，KI 10mg/L，NaMoO 4 ·2H 2 O 100mg/L，H 3 BO 3 50mg/L, water as solvent.
In a fourth aspect, the invention also provides an application of the bacterial suspension taking the soil bacillus brevis FHY-12 with dichloroethylene degradation capability as an active ingredient in degradation of dichloromethane, dichloropropane, benzene, chlorobenzene, pentachlorobenzene or hexachlorobenzene.
The beneficial effects of the invention are as follows: strain FHY-12 degrades DCE with concentration of 0-12 mg/L, and degradation product is CO 2 、H 2 O and cell biomass, average mineralization rate 42.27%, average chloride ion release rate 97.34%, average growth rate of cell biomass 0.0454OD 600 /mg DCE. The discovery of the degradation bacteria has important significance for the efficient biological purification of DCE in wastewater and waste gas in chemical synthesis and pharmaceutical industry. Meanwhile, the strain can degrade common organic pollutants in the same industry, such as dichloromethane, dichloropropane, benzene, chlorobenzene, pentachlorobenzene, hexachlorobenzene and the like. The strain FHY-12 is taken from an exhaust gas treatment unit, has good degradation effect on chlorinated organic pollutants, especially DCE, and can more completely convert the DCE into CO 2 、H 2 Harmless substances such as O and cell biomass; meanwhile, the strain can degrade common industrial organic pollutants such as halohydrocarbon, benzene, chlorobenzene and the like to different degrees, so that the strain has wide application prospect in biological purification of industrial waste gas and wastewater.
Drawings
FIG. 1 is a plate diagram of Brevibacillus lutes FHY-12;
FIG. 2 is a scanning electron microscope image of Brevibacillus soil FHY-12;
FIG. 3 is a phylogenetic tree diagram of Brevibacillus soil FHY-12;
FIG. 4 shows the DCE concentration and OD of Brevibacillus luteus FHY-12 at different concentrations of DCE 600 The degradation time is shown in FIG. 4 (a), in which the concentration of DCE of Bacillus brevis FHY-12 in different concentrations of DCE is shown in FIG. 4 (b), in which the OD of Bacillus brevis FHY-12 in different concentrations of DCE is shown in FIG. 4 (b) 600 A graph of change with degradation time;
FIG. 5 shows the degradation of CO by Brevibacillus soil FHY-12 in different concentrations of DCE 2 Yield of Cl - Release amount and OD 600 Is a variation graph of (2);
FIG. 6 shows the degradation rate and OD of Brevibacillus soil FHY-12 on industrial common organic pollutants 600 Wherein FIG. 6 (a) is the degradation rate and OD of the organic pollutants common to industry at 24 hours 600 FIG. 6 (b) is a graph showing degradation rate and OD of the organic pollutants at 72 hours 600 Is a graph of the experiment.
Detailed Description
For the purposes of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and examples, it being understood that the specific examples described herein are for the purpose of illustrating the present invention only, and not all the examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are within the scope of the present invention.
The following test methods are not explicitly described for specific experimental conditions, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The materials, reagents and the like used, unless otherwise specified, are those obtained commercially.
The invention provides a strain of soil bacillus brevis with dichloroethylene degradation capability, which is named as soil bacillus brevis (Brevibacillus agri) FHY-12 in the classification of microorganisms, and is preserved in China center for type culture collection (China center for type culture collection) on the year 03 and the month 28, wherein the preservation number is CCTCC NO: M2023419; the 16S rRNA sequence of FHY-12 is shown in SEQ ID NO. 1.
Brevibacillus soil is a common bacillus, and no report of degrading DCE by using the Brevibacillus soil has been found through searching patents and other related documents. The discovery of the degradation bacteria has important significance for the efficient purification of chlorinated organic pollutants such as DCE and the like in industrial wastewater and waste gas.
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
Example 1: isolation, purification and identification of Brevibacillus tumefaciens (Brevibacillus agri) FHY-12
1. Isolation and purification of Brevibacillus soil (Brevibacillus agri) FHY-12
The soil bacillus brevis (Brevibacillus agri) FHY-12 is a gram-positive bacterium domesticated and separated from activated sludge of a waste gas treatment device of a rubber plant in Zhejiang, and comprises the following specific steps:
(1) Sampling: taking multi-point samples from the activated sludge of a waste gas treatment device of a rubber plant in Zhejiang Taizhou as raw materials for screening soil Brevibacillus brevis FHY-12 with dichloroethylene degradation capability;
(2) Isolation of strains: taking a proper amount of activated sludge in the waste gas treatment device, flushing with distilled water for 5 times, and then performing air exposure for 24 hours to remove residual organic matters; preparing a second DCE liquid selection culture medium, carrying out directional domestication on activated sludge, adding domesticated sludge with the volume fraction of 5% into the second DCE liquid selection culture medium, carrying out shaking culture on a constant-temperature shaking table at 30 ℃ and 160rpm, transferring to a new second DCE liquid selection culture medium for shaking culture for 3 days according to the volume fraction of 10%, until single colonies appear, picking single colonies, accessing the single colonies into the second DCE solid selection culture medium, and obtaining single colonies with rapid growth, regular colonies and stable characters, namely the strain FHY-12.
In this example, the final concentration composition of the second DCE liquid selection medium is: DCE 50mg/L, KH 2 PO 4 376mg/L，K 2 HPO 4 456mg/L，(NH 4 ) 2 SO 4 480mg/L，NaNO 3 680mg/L，Mg(NO 3 ) 2 250mg/L，CaCl 2 ·2H 2 O11mg/L, trace elements, water as solvent and pH 7.5. The final concentration composition of the microelements is as follows: mnCl 2 ·H 2 O 60mg/L，ZnCl 2 88mg/L，KI 10mg/L，NaMoO 4 ·2H 2 O 100mg/L，H 3 BO 3 50mg/L, water as solvent.
The final concentration composition of the second DCE-fixed selection medium is: DCE 50mg/L, KH 2 PO 4 376mg/L，K 2 HPO 4 456mg/L，(NH 4 ) 2 SO 4 480mg/L，NaNO 3 680mg/L，Mg(NO 3 ) 2 250mg/L，CaCl 2 ·2H 2 O11mg/L, agar 20g/L, trace elements, water as solvent and pH 7.5.
2. Identification of Strain FHY-12
a. Physiological and biochemical characteristics of Strain FHY-12
Carrying out morphological observation and physiological and biochemical identification on the obtained strain FHY-12, wherein bacterial colonies are yellow, and the edges are neat, smooth and moist; the bacterial strain is observed under a scanning electron microscope to be in a form of bacillus, no flagellum, gram-positive staining, positive oxidase, starch hydrolysis and positive indole test, and sucrose and lactose can be utilized. Strain FHY-12 is shown in figure 1; the form of the cells was observed under a scanning electron microscope and was bacillus, as shown in fig. 2.
b. 16S rRNA sequence analysis of Strain FHY-12
Strain FHY-12 was identified as Brevibacillus agri by 16S rRNA sequence analysis and physiological and biochemical experiments.
The sequencing results were:
gccatgcgcgtgctatacatgcaagtcgagcgagtctcttcgggggctagcggcggacgggtgagtaacacgtaggcaacctgcctctcagactgggataacatagggaaacttatgctaataccggataggtttttggatcgcatgatctgaaaagaaaagatggcttttcgctatcactgggagatgggcctgcggcgcattagctagttggtggggtaacggcctaccaaggcgacgatgcgtagccgacctgagagggtgaccggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaattttccacaatggacgaaagtctgatggagcaacgccgcgtgaacgatgaaggtcttcggattgtaaagttctgttgtcagggacgaacacgtaccgttcgaatagggcggtaccttgacggtacctgacgagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggatttattgggcgtaaagcgcgcgcaggcggctatgtaagtctggtgttaaagcccggggctcaaccccggttcgcatcggaaactgtgtagcttgagtgcagaagaggaaagcggtattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcggctttctggtctgtaactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaggtgttgggggtttcaataccctcagtgccgcagctaacgcaataagcactccgcctggggagtacgctcgcaagagtgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcccgctgaccgctctggagacagagcttcccttcggggcagcggtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttatctttagttgccagcattcagttgggcactctagagagactgccgtcgacaagacggaggaaggcggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggttggtacaacgggatgctacctcgcgagaggacgccaatctcttaaaaccaatctcagttcggattgtaggctgcaactcgcctacatgaagtcggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgggagtttgcaacacccgaagtcggtgaggtaaccgcaaggagccagccgccgaagtggtcatttgg
bacterial DNA extraction kit (OMEGA, cat. No. D3350) was used to extract and purify the DNA of the strain, and the strain was stored at 4 ℃. The 16S rRNA was PCR amplified using bacterial universal primer 27F (forward primer 27F,5AGAGTTTGA TCC TGG CTC AG-3 ') and 1492R (reverse primer 1492R5-GGT TAC CTT GTT ACG ACT T-3').
PCR experiments were performed using the high-fidelity PCR polymerase product KOD OneTM PCR Master Mix (product number KMM-101) from TOYOBO Co. The reaction system is shown in Table 1:
table 1: reaction system
The 16S rRNA sequence of strain FHY-12 was tested, and the 16S rRNA sequence of strain FHY-12 was shown in SEQ ID NO. 1.
The 16S rRNA sequence of strain FHY-12 was uploaded to the gene sequence in Genbank for homology comparison, and found to be Brevibacillus agri, the highest homology with Brevibacillus agri DSM 6348, reaching 99.65%. FIG. 3 is a phylogenetic tree of strain FHY-12. Meanwhile, physiological and biochemical test results show that the strain FHY-12 is gram-positive in staining, oxidase-positive in hydrolyzing starch and indole test-positive in oxidizing enzyme, and sucrose and lactose can be utilized.
Based on the sequencing results and the physiological and biochemical test results, it was determined that strain FHY-12 belongs to Brevibacillus agri. Therefore, the strain FHY-12 is named as Brevibacillus soil (Brevibacillus agri) FHY-12, and is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m2023419, storage date 2023, month 03, 28, address: chinese, university of martial arts, postal code address: 430072.
example 2: preparation process of bacterial suspension with soil bacillus brevis FHY-12 with dichloroethylene degradation capability as active ingredient
The bacterial suspension is prepared by solid culture, seed culture and liquid culture of soil bacillus brevis FHY-12 with dichloroethylene degradation capability, and comprises the following specific steps:
(1) Solid culture: inoculating soil bacillus brevis FHY-12 with dichloroethylene degradation capability to a slant culture medium, and culturing for 3 days at 30 ℃ to obtain slant thalli; the final concentration composition of the slant culture medium is as follows: k (K) 2 HPO 4 1500mg/L，KH 2 PO 4 500mg/L NaCl 1000mg/L dichloroethylene 50mg/L MgSO 4 ·7H 2 200mg/L of O, water as solvent, pH value of 7.0 and 18g/L of agar;
(2) Seed culture: selecting bacterial colony from the inclined plane thallus, inoculating to a seed culture medium, and culturing at 30 ℃ for 18h to obtain seed liquid; the final concentration composition of the seed culture medium is as follows: 10g/L NaCl, 5g/L yeast extract powder, 10g/L peptone, water as solvent and 7.0 pH value;
(3) Liquid culture: inoculating the seed solution to a fermentation culture medium according to an inoculum size with the volume concentration of 1%, and culturing for 18 hours at the temperature of 30 ℃ to obtain a fermentation culture solution which is a bacterial suspension; the final concentration composition of the fermentation medium is as follows: k (K) 2 HPO 4 1500mg/L，KH 2 PO 4 500mg/L NaCl 1000mg/L dichloroethylene 50mg/L MgSO 4 ·7H 2 O200 mg/L, water as solvent and pH 7.0.
Example 3: response surface optimization experiment of soil Brevibacillus (Brevibacillus agri) FHY-12 for degrading DCE environmental factors
1. Response surface test design and acquisition of DCE degradation rate prediction model
The influence of 3 factors of pH, culture temperature and salt concentration of the culture solution on the DCE biodegradation effect is examined, three-factor three-level response surface experimental Design is carried out by using Design Expert software, and the degradation rate of the Bacillus brevis (Brevibacillus agri) FHY-12 on the DCE under different culture conditions is predicted.
Three-factor three-level test Design is carried out by using Design Expert software, wherein three factors are respectively X1: culture medium pH, X2: culture temperature and X3: salt concentration of the culture solution; code value: -1, 0, 1 correspond to broth ph=6, broth ph=7, broth ph=8 or broth temperature=20 ℃, broth temperature=30 ℃, broth temperature=40 ℃ or broth salt concentration=0%, broth salt concentration=0.5%, broth salt concentration=1.0%, respectively.
The design experiment response values and predicted values are shown in table 2.
Table 2: design of experimental response and prediction values
First DCE liquid selection media of different culture medium pH, different culture temperatures and different culture medium salt concentrations were prepared according to Table 2, sub-packaged in 250mL serum bottles with a liquid loading amount of 50mL and sterilized at 121℃for 20min. After the first DCE liquid selection medium was cooled, the initial OD prepared in example 2 was added separately 600 Bacterial suspensions of 0.05 were inoculated into first DCE liquid selection media of different broth pH, different broth temperature and different broth salt concentrations.
In this example, the first DCE liquid selection medium is DCE 10mg/L, KH 2 PO 4 376mg/L，K 2 HPO 4 456mg/L，(NH 4 ) 2 SO 4 480mg/L，NaNO 3 680mg/L，Mg(NO 3 ) 2 250mg/L，CaCl 2 ·2H 2 O11mg/L, trace elements and water as solvent; the final concentration composition of the microelements is as follows: mnCl 2 ·H 2 O 60mg/L，ZnCl 2 88mg/L，KI 10mg/L，NaMoO 4 ·2H 2 O 100mg/L，H 3 BO 3 50mg/L, water as solvent.
DCE is taken as the only carbon source, the initial concentration is 10mg/L, and the DCE is sealed and then placed into a shaking table for shake culture at different culture temperatures. Serum bottles containing the same culture broth were also taken, and after sterilization, DCE was added but no FHY-12 bacterial suspension was added as a blank. Detection of DCE concentration and OD after 24h of incubation 600 And (3) carrying out secondary multiple regression on the value by using Design Expert software to obtain a prediction model as follows:
wherein Y is the degradation rate of DCE, and the unit is mg/(L.times.h). Correlation coefficient R of prediction model 2 = 0.9986, it is shown that the degradation rate predicted by the prediction model has a better correlation with the actual degradation rate.
2. Optimal environmental factor combination for degrading DCE by using Brevibacillus soil (Brevibacillus agri) FHY-12
Analyzing the obtained prediction model by using Design Expert software to obtain an environmental factor combination when the degradation rate of the Bacillus brevis (Brevibacillus agri) FHY-12 to the DCE reaches the maximum value: the incubation temperature was 35 ℃, the broth pH was 7.5, and the broth salt concentration was 0.72%, at which time the degradation rate predicted by the predictive model was 0.553 mg/(l×h). The actual degradation rate of the strain FHY-12 on DCE under the culture conditions is 0.540 mg/(L.times.h), which is relatively close to the predicted value.
The specific experimental process is as follows:
taking 200mL of a first DCE liquid selection medium with the pH value of 7.5, adding NaCl to enable the salt concentration of the culture solution to reach 0.72%, adjusting the pH value of the culture solution to 7.5, subpackaging into 4 250mL serum bottles, and sterilizing at 121 ℃ for 20min. After the broth cooled, 3 serum bottles were taken and added to the initial OD prepared in example 2 600 0.05 bacterial suspension, with an initial concentration of 10mg/L DCE as the sole carbon source. Only 10mg/L of DCE was added to the remaining 1 serum bottles, and no bacterial suspension was added as a blank. Shaking culture on a constant temperature shaker at 35deg.C for 24 hr, and detecting DCE concentration and OD 600 Values.
Under the conditions that the culture temperature is 35 ℃, the pH of the culture solution is 7.5 and the salt concentration of the culture solution is 0.72%, the actual degradation rate of the soil bacillus brevis (Brevibacillus agri) FHY-12 on DCE is 0.537, 0.541 and 0.542 mg/(L.h), the average degradation rate is 0.540 mg/(L.h), and the average degradation rate is 0.553 mg/(L.h) close to the model predicted value, which shows that the environmental factor combination is the optimal environmental factor condition of the soil bacillus brevis (Brevibacillus agri) FHY-12 for degrading the DCE.
Example 4: degradation performance detection of Bacillus brevis (Brevibacillus agri) FHY-12 on DCE with different concentrations
Under the condition of optimal environmental factors, namely the culture temperature is 35 ℃, the pH of the culture solution is 7.5, and the salt concentration of the culture solution is 0.72%, the degradation performance of the Bacillus brevis (Brevibacillus agri) FHY-12 on DCE with the concentration of 1-18 mg/L is examined. Experimental results show that the strain FHY-12 can completely degrade 1-12 mg/L of DCE, but cannot completely degrade DCE with initial concentration of more than 12 mg/L.
The specific experimental process is as follows:
a third DCE liquid selection medium having a pH of 7.5 and a salt concentration of 0.72% was prepared, and 200mL of the third DCE liquid selection medium was filled into 250mL serum bottles, respectively, and sterilized at 121℃for 20 minutes. After the broth cooled, 8 of the serum bottles were taken and added to the initial OD prepared in example 2 600 0.05 in culture, 1, 3, 5, 8, 10, 12, 15 and 18mg/L of DCE were added simultaneously as the sole carbon source, and 8 serum bottles were used as blank controls (DCE alone and not culture). After deoxygenation and sealing of serum bottle, shake culture is carried out at 35 ℃ to determine the concentration and OD of DCE at regular time 600 Values.
In this example, the third DCE liquid selection medium is DCE 1-18 mg/L, KH 2 PO 4 376mg/L，K 2 HPO 4 456mg/L，(NH 4 ) 2 SO 4 480mg/L，NaNO 3 680mg/L，Mg(NO 3 ) 2 250mg/L，CaCl 2 ·2H 2 O11mg/L, trace elements and water as solvent; the final concentration composition of the microelements is as follows: mnCl 2 ·H 2 O 60mg/L，ZnCl 2 88mg/L，KI 10mg/L，NaMoO 4 ·2H 2 O 100mg/L，H 3 BO 3 50mg/L, water as solvent.
As shown in FIGS. 4 (a) and 4 (b), strain FHY-12 was able to completely degrade DCE at an initial concentration of less than 12mg/L, and in this range, as the DCE concentration increased, the cell biomass increased. When the initial DCE concentration is more than 12mg/L, the strain FHY-12 can only degrade a small part of DCE, and the DCE concentration is not obviously changed, so that the strain FHY-12 has serious toxic action on the strain FHY-12 when being exposed to high-concentration DCE, and the normal metabolic activity of cells is inhibited.
Example 5: analysis of mineralization Rate, chloride ion Release Rate and cell Mass growth Rate of DCE by Brevibacillus soil (Brevibacillus agri) FHY-12
Under the condition of optimal environmental factors, namely the culture temperature is 35 ℃, the pH of the culture solution is 7.5, and the salt concentration of the culture solution is 0.72%, the mineralization rate, the chloride ion release rate and the cell biomass growth rate of the Bacillus brevis (Brevibacillus agri) FHY-12 to DCE with the concentration of 1-12 mg/L are examined. Experimental results show that strain FHY-12 can convert DCE into CO 2 、H 2 O and cell biomass, average mineralization rate of 42.27%, average chloride ion release rate of 97.34%, average growth rate of 0.0454OD 600 /mg DCE。
The specific experimental process is as follows:
a first DCE liquid selection medium having a pH of 7.5 and a salt concentration of 0.72% was prepared, and 50mL of the first DCE liquid selection medium was filled into 250mL serum bottles, respectively, and sterilized at 121℃for 20 minutes. After the broth cooled, 12 of the serum bottles were taken and added to the initial OD prepared in example 2 600 0.05 bacterial suspension, 1, 3, 5, 8, 10 and 12mg/L DCE was added simultaneously as the sole carbon source (2 serum bottles per concentration), and 6 serum bottles were taken as blank (DCE alone and bacterial suspension was not added). After deoxidizing and sealing a serum bottle, shake culturing at 35 ℃ to determine the concentration of DCE, the concentration of chloride ions and OD at regular time 600 Value, CO 2 The concentration and fitting are carried out to obtain the average mineralization rate, the average chloride ion release rate and the average growth rate of the cell biomass.
As shown in FIG. 5, the removal amount of chloride ions and the concentration of DCE are in a linear relation, the fitting linear equation is y= 0.6918x, and the correlation coefficient R 2 = 0.9964, indicating that strain FHY-12 can produce 0.6918mg of chloride ion per 1mg of DCE, and can theoretically produce 0.7107mg of chloride ion per 1mg of DCE, thus chloride ion is flatThe release rate was 97.34%.
CO 2 The generation amount of (2) and the degradation amount of DCE are also in linear relation, the fitting linear equation is y= 0.3838x, and the correlation coefficient R 2 = 0.9859, demonstrating that strain FHY-12 fully mineralizes 1mg of DCE to produce 0.3838mg of CO 2 The method comprises the steps of carrying out a first treatment on the surface of the In theory complete oxidation of DCE to H 2 O and CO 2 Fully mineralizing 1mg of DCE can 0.9078mg of CO 2 The method comprises the steps of carrying out a first treatment on the surface of the Thus, the average mineralization rate of strain FHY-12 was 42.27%.
Strain FHY-12 is able to synthesize self-cellular material using organic carbon during degradation of DCE. Cell biomass (OD) 600 ) And the degradation amount of DCE is linear relation of y=0.0454x, and the correlation coefficient R 2 = 0.9703, indicating that strain FHY-12 degrades 1mg DCE, OD 600 The value may be raised by 0.0454.
Example 6: analysis of degradation Performance of Brevibacillus soil (Brevibacillus agri) FHY-12 on common organic pollutants in industry
Organic pollutants common in the industries of dichloromethane, dichloropropane, benzene, chlorobenzene, pentachlorobenzene and hexachlorobenzene are used as unique carbon sources respectively, and the degradation capability of the soil bacillus brevis (Brevibacillus agri) FHY-12 on the common organic pollutants in the industries is examined. Experimental results show that the strain FHY-12 can degrade the organic pollutants to different degrees, can completely degrade chlorobenzene, pentachlorobenzene and hexachlorobenzene in 24 hours, and can partially degrade dichloromethane, dichloropropane and benzene in 72 hours.
The specific experimental process is as follows:
a first DCE liquid selection medium having a pH of 7.5 and a salt concentration of 0.72% was prepared, and 50mL of the first DCE liquid selection medium was filled into 250mL serum bottles, respectively, and sterilized at 121℃for 20 minutes. 6 of these serum bottles were taken and added to the initial OD prepared in example 2 600 0.05, while using methylene chloride, dichloropropane, benzene, chlorobenzene, pentachlorobenzene or hexachlorobenzene as the only carbon source, respectively, at an initial concentration of 10mg/L, 6 serum bottles were used as blank (DCE alone and no bacterial suspension added). Shake culturing at 35deg.C after deoxygenating and sealing serum bottleDetecting the concentration of organic pollutants and determining the OD (optical density) at a time different from 24h and 72h 600 Values, the degradation rate and OD of different organic carbon sources are plotted 600 Is a change curve of (a).
As shown in FIG. 6 (a), strain FHY-12 was able to degrade chlorobenzene, pentachlorobenzene and hexachlorobenzene more completely when the cultivation time was 24 hours, the degradation rates reached 99.7%, 95.7% and 80.2%, respectively, while the degradation rates of the strain for methylene chloride, dichloropropane and benzene were only 19.2%, 16.8% and 25.2%, respectively. As shown in FIG. 6 (b), when the cultivation time reached 72 hours, the degradation rates of the strain FHY-12 for methylene chloride, dichloropropane, benzene, chlorobenzene, pentachlorobenzene and hexachlorobenzene reached 77.2%, 42.4%, 47.3%, 100% and 100%, respectively. The test results show that the soil bacillus brevis (Brevibacillus agri) FHY-12 can degrade various common organic pollutants, and has a certain guiding significance for the application of the strain in actual industrial pollution control.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather to enable any modification, equivalent replacement, improvement or the like to be made within the spirit and principles of the invention.
Claims (7)
1. A strain of soil bacillus brevis with dichloroethylene degradation capability is characterized in that the strain is named as soil bacillus brevis Brevibacillus agri FHY-12 in a microorganism classification, and is preserved in China Center for Type Culture Collection (CCTCC) in the year 2023, the preservation number is M2023419; the 16S rRNA sequence of FHY-12 is shown in SEQ ID NO. 1.
2. A bacterial suspension taking the soil bacillus brevis as an active ingredient, which is characterized in that the bacterial suspension is prepared by solid culture, seed culture and liquid culture of soil bacillus brevis FHY-12 with dichloroethylene degradation capability.
3. A method of preparing a bacterial suspension according to claim 2, comprising the specific steps of:
(1) Solid culture: inoculating soil bacillus brevis FHY-12 with dichloroethylene degradation capability to a slant culture medium, and culturing for 2-4 days at 20-40 ℃ to obtain slant thalli; the final concentration composition of the slant culture medium is as follows: k (K) 2 HPO 4 1500mg/L，KH 2 PO 4 500mg/L NaCl 1000mg/L dichloroethylene 50mg/L MgSO 4 ·7H 2 200mg/L of O, water as solvent, pH value of 6.0-8.0 and 18-20 g/L of agar;
(2) Seed culture: bacterial colony is selected from the inclined plane thallus and inoculated to a seed culture medium, and the bacterial colony is cultured for 12 to 24 hours at the temperature of 20 to 40 ℃ to obtain seed liquid; the final concentration composition of the seed culture medium is as follows: 10g/L NaCl, 5g/L yeast extract powder, 10g/L peptone, water as solvent and pH value of 6.0-8.0;
(3) Liquid culture: inoculating the seed solution to a fermentation culture medium according to the inoculum size with the volume concentration of 1%, and culturing for 12-24 hours at the temperature of 20-40 ℃ to obtain a fermentation culture solution, namely a bacterial suspension; the final concentration composition of the fermentation medium is as follows: k (K) 2 HPO 4 1500mg/L，KH 2 PO 4 500mg/L NaCl 1000mg/L dichloroethylene 50mg/L MgSO 4 ·7H 2 200mg/L O, water as solvent and pH 6.0-8.0.
4. Use of the bacterial suspension of claim 2 for degrading dichloroethylene.
5. The use according to claim 4, characterized in that it is in particular: inoculating the bacterial suspension into a first DCE liquid selection culture medium with the salt concentration of 0-1%, and culturing the bacterial suspension at the temperature of 25-35 ℃ by taking dichloroethylene as a unique carbon source to degrade the dichloroethylene; the final concentration composition of the first DCE liquid selection medium is: DCE 1-12 mg/L, KH 2 PO 4 376mg/L，K 2 HPO 4 456mg/L，(NH 4 ) 2 SO 4 480mg/L，NaNO 3 680mg/L，Mg(NO 3 ) 2 250mg/L，CaCl 2 ·2H 2 O11mg/L, trace elements, water as solvent and pH 7.0-9.0; final concentration of the trace elementsThe composition is as follows: mnCl 2 ·H 2 O 60mg/L，ZnCl 2 88mg/L，KI 10mg/L，NaMoO 4 ·2H 2 O 100mg/L，H 3 BO 3 50mg/L, water as solvent.
6. The use according to claim 4, characterized in that it is in particular: inoculating the bacterial suspension into a first DCE liquid selection culture medium with the salt concentration of 0.72%, and culturing the bacterial suspension at 35 ℃ by taking dichloroethylene as a unique carbon source to degrade the dichloroethylene; the final concentration composition of the first DCE liquid selection medium is: DCE 1-12 mg/L, KH 2 PO 4 376mg/L，K 2 HPO 4 456mg/L，(NH 4 ) 2 SO 4 480mg/L，NaNO 3 680mg/L，Mg(NO 3 ) 2 250mg/L，CaCl 2 ·2H 2 O11mg/L, trace elements, water as solvent and pH 7.5; the final concentration composition of the microelements is as follows: mnCl 2 ·H 2 O 60mg/L，ZnCl 2 88mg/L，KI 10mg/L，NaMoO 4 ·2H 2 O 100mg/L，H 3 BO 3 50mg/L, water as solvent.
7. Use of a bacterial suspension according to claim 2 for the degradation of methylene chloride, dichloropropane, benzene, chlorobenzene, pentachlorobenzene or hexachlorobenzene.
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