CN117866794A - Acidophilic and high-efficiency dichloroethylene degrading soil bacillus brevis FHY-13 and application thereof - Google Patents
Acidophilic and high-efficiency dichloroethylene degrading soil bacillus brevis FHY-13 and application thereof Download PDFInfo
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- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical group ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 title claims abstract description 135
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- 230000000593 degrading effect Effects 0.000 title claims abstract description 23
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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
- C12N1/205—Bacterial isolates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/34—Chemical or biological purification of waste gases
- B01D53/46—Removing components of defined structure
- B01D53/68—Halogens or halogen compounds
- B01D53/70—Organic halogen compounds
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- B01D—SEPARATION
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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Abstract
The invention discloses an acidophilic and high-efficiency dichloroethylene degrading soil bacillus brevis (Brevibacillus agri) FHY-13 and application thereof, wherein the soil bacillus brevis (Brevibacillus agri) FHY-13 is preserved in China center for type culture collection, and addresses: chinese, university of armed chinese, postal code: 430072, deposit number: cctccc NO: m2023420, 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 an acidophilic and high-efficiency dichloroethylene degrading soil Brevibacillus FHY-13 and application thereof.
Background
Dichloroethylene (DCE) is a common organic compound with volatility and solubility and is widely used in the fields of chemical industry, pharmacy, paint, solvent and the like. However, DCE is classified as one of environmental pollutants due to its high toxicity, which is harmful to human health by long-term exposure or contact, and the treatment technology of DCE is attracting attention of scholars.
The biological purification technology is widely applied to the field of pollutant treatment due to high removal efficiency, low treatment cost and small secondary pollution. 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 an acidophilic and high-efficiency dichloroethylene degrading soil bacillus brevis FHY-13 and application thereof.
The aim of the invention is realized by the following technical scheme:
in a first aspect, the invention provides an acidophilic, highly effective dichloroethylene degrading Bacillus brevis soil, the microorganism classification named Bacillus brevis soil (Brevibacillus agri) FHY-13, which has been deposited in China center for type culture Collection at the year 2023, month 28, at the address: the university of martial arts in China, postal code 430072; the preservation number is CCTCCNO: M2023420; the sequence of the 16S rRNA of FHY-13 is shown in SEQ ID NO. 1.
In a second aspect, the invention provides a bacterial suspension taking acidophilic and high-efficiency dichloroethylene-degrading soil bacillus brevis FHY-13 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-13 for acidophilic and high-efficiency degradation of dichloroethylene.
The preparation method of the bacterial suspension comprises the following specific steps:
(1) Solid culture: inoculating acidophilic and high-efficiency dichloroethylene degrading soil bacillus brevis FHY-13 onto 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 acidophilic and high-efficiency dichloroethylene degrading soil bacillus brevis FHY-13 as an active ingredient in dichloroethylene degrading 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 ultra-high25mg/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 6.0-8.0; 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 0.89%, 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 1-25 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 6.0; 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.
In a fourth aspect, the invention also provides an application of the bacterial suspension taking acidophilic and high-efficiency dichloroethylene-degrading soil bacillus brevis FHY-13 as an active ingredient in degrading dichloromethane, dichloropropane, benzene, chlorobenzene, pentachlorobenzene or hexachlorobenzene.
The beneficial effects of the invention are as follows: strain FHY-13 can degrade DCE with initial concentration of 0-25 mg/L and final degradation product is CO 2 、H 2 O and cell biomass, average mineralization rate was 61.12%, average yield coefficient was 0.1904mg cells/mg DCE, average chloride ion release rate was 84.70%. The discovery of the degradation bacteria has important significance for the efficient purification of DCE in wastewater and waste gas in chemical synthesis and pharmaceutical industry; the strain can degrade other similar strainsOrganic pollutants common in industry, especially dichloromethane, dichloropropane, benzene, chlorobenzene, pentachlorobenzene, hexachlorobenzene and the like; the strain FHY-13 is obtained from an exhaust gas treatment unit, has good degradation effect on chlorinated hydrocarbon compounds, 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 pollutants such as 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-13;
FIG. 2 is a scanning electron microscope image of Brevibacillus soil FHY-13;
FIG. 3 is a phylogenetic tree diagram of Brevibacillus soil FHY-13;
FIG. 4 is a graph showing the change of the DCE concentration and the cell biomass of the Brevibacillus luteus FHY-13 with respect to the degradation time under the different DCE concentrations, wherein FIG. 4 (a) is a graph showing the change of the DCE concentration of the Brevibacillus luteus FHY-13 with respect to the degradation time under the different DCE concentrations, and FIG. 4 (b) is a graph showing the change of the cell biomass of the Brevibacillus luteus FHY-13 with respect to the degradation time under the different DCE concentrations;
FIG. 5 shows the degradation of different concentrations of DCE by Bacillus brevis FHY-13 in CO 2 Yield of Cl - A graph of the change in the amount released and the cellular biomass;
FIG. 6 is an experimental view of the degradation rate and the cell growth amount of the soil Brevibacillus brevis FHY-13 on the industrial common organic pollutants, wherein FIG. 6 (a) is an experimental view of the degradation rate and the cell growth amount of the industrial common organic pollutants at 24 hours, and FIG. 6 (b) is an experimental view of the degradation rate and the cell growth amount of the industrial common organic pollutants at 72 hours.
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 an acidophilic and high-efficiency dichloroethylene degrading Bacillus brevis, which is named as Bacillus brevis (Brevibacillus agri) FHY-13 in the microorganism classification and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2023420 in the year 2023 and the month 28; the sequence of the 16S rRNA of FHY-13 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 hydrocarbon pollutants such as DCE and other hydrocarbon pollutants 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-13
1. Isolation and purification of Brevibacillus soil (Brevibacillus agri) FHY-13
The soil bacillus brevis (Brevibacillus agri) FHY-13 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: multi-point sampling is carried out on the activated sludge of an exhaust gas treatment device of a certain rubber plant in Zhejiang Taizhou, and the activated sludge is used as a raw material for screening the soil bacillus brevis FHY-13 for acidophilic and high-efficiency degradation of dichloroethylene;
(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-13.
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 6.0. 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.
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 6.0.
2. Identification of Strain FHY-13
a. Physiological and biochemical characteristics of Strain FHY-13
Carrying out morphological observation and physiological and biochemical identification on the obtained strain FHY-13, wherein bacterial colonies are yellow, and the edges are neat, smooth and moist; the bacterial strain is observed to be long bacillus under a scanning electron microscope, has no flagella, is gram-negative, has oxidase positive, has starch hydrolysis and indole test positive, and can utilize sucrose and lactose. Strain FHY-13 is shown in figure 1; the form of the cells was observed under a scanning electron microscope and was long bacillus, as shown in FIG. 2.
b. 16S rRNA sequence analysis of Strain FHY-13
Strain FHY-13 was identified as Brevibacillus agri by 16S rRNA sequence analysis and physiological and biochemical experiments.
The sequencing results were:
ggctgcgcatgctatacatgcagtcgagcgagtcccttcggaggctagcggcggacgggtgagtaacacgtaggcaacctgcctctcagactgggataacatagggaaacttatgctaataccggataggtttttggatcgcatgatctgaaaagaaaagatggcttttcgctatcactgggagatgggcctgcggcgcattagctagttggtggggtaacggcctaccaaggcgacgatgcgtagccgacctgagagggtgaccggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaattttccacaatggacgaaagtctgatggagcaacgccgcgtgaacgatgaaggtcttcggattgtaaagttctgttgtcagggacgaacacgtaccgttcgaatagggcggtaccttgacggtacctgacgagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggatttattgggcgtaaagcgcgcgcaggcggctatgtaagtctggtgttaaagcccggggctcaaccccggttcgcatcggaaactgtgtagcttgagtgcagaagaggaaagcggtattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcggctttctggtctgtaactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaggtgttgggggtttcaataccctcagtgccgcagctaacgcaataagcactccgcctggggagtacgctcgcaagagtgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcccgctgaccgctctggagacagagcttcccttcggggcagcggtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttatctttagttgccagcattcagttgggcactctagagagactgccgtcgacaagacggaggaaggcggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggttggtacaacgggatgctacctcgcgagaggacgccaatctcttaaaaccaatctcagttcggattgtaggctgcaactcgcctacatgaagtcggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgggagtttgcaacacccgaagtcggtgaggtaaccgcaaggagccagccgccgaagtgggagttgcg
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-13 was tested, and the 16S rRNA sequence of strain FHY-13 was shown in SEQ ID NO. 1.
The 16S rRNA sequence of strain FHY-13 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 99.72%. FIG. 3 is a phylogenetic tree of strain FHY-13.
Based on the sequencing results and the physiological and biochemical test results, it was determined that strain FHY-13 belongs to Brevibacillus agri. Therefore, the strain FHY-13 is named as Brevibacillus soil (Brevibacillus agri) FHY-13 and is preserved in China Center for Type Culture Collection (CCTCC) NO: m2023420, storage date 2023, month 03, 28, address: chinese, university of martial arts, postal code address: 430072.
example 2: preparation process of bacterial suspension with acidophilic and high-efficiency dichloroethylene degrading soil bacillus brevis FHY-13 as active component
The preparation process of the bacterial suspension comprises the following steps:
(1) Solid culture: inoculating acidophilic and high-efficiency dichloroethylene degrading soil bacillus brevis FHY-13 to a slant culture medium, and culturing at 30 ℃ for 3 days 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-13 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-13 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 bacterial suspension prepared by the method of example 2 and having an initial biomass of 0.75mg/L was added to the first DCE liquid selection medium, respectively, and inoculated into different medium pH, different culture temperatures and different medium salt concentrations.
In this example, the first DCE liquid selection medium is DCE 20mg/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 20mg/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-13 bacterial suspension was added as a blank. After 24 hours of incubation, the culture broth was analyzed for residual concentration of DCE while biomass was measured. And (3) carrying out secondary multiple regression on the degradation rate by using Design Expert software according to the measured degradation rate, and fitting to obtain a prediction model as follows:
Y＝-73.008+14.117*X1+2.973*X2+1.817*X3-0.491*X1*X1
-0.092*X1*X2-0.383*X1*X3-0.440*X2*X2+0.194*X2*X3；
-5.167*X3*X3
wherein Y is the degradation rate of DCE, and the unit is mg/(L.times.h).
Correlation coefficient R of prediction model 2 The = 0.9937 shows that the degradation rate predicted by the prediction model has a better correlation with the actual degradation rate, and can be used for predicting the degradation rate of the strain on DCE under different culture conditions.
2. Optimal environmental factor for degrading DCE by using Brevibacillus soil (Brevibacillus agri) FHY-13
Analyzing the obtained prediction model by using Design Expert software to make the first-order partial derivative of the model zero so as to obtain an environmental factor combination when the degradation rate of the DCE of the soil bacillus brevis (Brevibacillus agri) FHY-13 reaches the maximum value: the incubation temperature was 35 ℃, the pH of the broth was 6.0, and the salt concentration of the broth was 0.89%, at which time the degradation rate predicted by the predictive model was 0.579 mg/(L.multidot.h). The actual degradation rate of the strain FHY-13 on DCE under the culture conditions is 0.568 mg/(L.times.h), which is relatively close to the predicted value.
The specific experimental process is as follows:
taking 800mL of a first DCE liquid selection medium with pH of 6.0, adding NaCl to enable the salt concentration of the culture solution to reach 0.89%, adjusting the pH of the culture solution to 6.0, subpackaging into 4 250mL serum bottles, and sterilizing at 121 ℃ for 20min. After the broth was cooled, 3 serum bottles were taken and added to the bacterial suspension prepared in example 2 and having an initial biomass of 0.75mg/L, and DCE having an initial concentration of 20mg/L was used as the sole carbon source. Only 20mg/L of DCE was added to the remaining 1 serum bottles, and no bacterial suspension was added as a blank. After shaking culture on a constant temperature shaker at 35℃for 24 hours, the residual DCE concentration was analyzed and biomass was determined.
Under the conditions that the culture temperature is 35 ℃, the pH of the culture solution is 6.0 and the salt concentration of the culture solution is 0.89%, the actual degradation rate of the soil bacillus brevis (Brevibacillus agri) FHY-13 on DCE is 0.577, 0.563 and 0.564 mg/(L.multidot.h), the average degradation rate is 0.568 mg/(L.multidot.h), and the average degradation rate is close to a model predicted value, so that the environmental factor combination is the optimal environmental factor condition of the soil bacillus brevis (Brevibacillus agri) FHY-13 for degrading DCE.
Example 4: degradation performance detection of Bacillus brevis (Brevibacillus agri) FHY-13 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 6.0, and the salt concentration of the culture solution is 0.89%, the degradation performance of the Bacillus brevis (Brevibacillus agri) FHY-13 on DCE with the concentration of 1-35 mg/L is examined. Experimental results show that the strain FHY-13 can completely degrade 1-25 mg/L DCE and cannot degrade DCE with initial concentration greater than 25 mg/L.
The specific experimental process is as follows:
a third DCE liquid selection medium with a pH of 6.0 and a salt concentration of 0.89% was prepared, and 200mL of the third DCE liquid selection medium was filled into 250mL serum bottles, respectively, and sterilized at 121℃for 20min. After the broth was cooled, 8 of the serum bottles were taken and added with the bacterial suspension prepared in example 2 and having an initial biomass of 0.75mg/L, while 1, 5, 10, 15, 20, 25, 30 and 35g/L of DCE were added as the sole carbon source, respectively, and 8 serum bottles were taken as blank controls (DCE alone and bacterial suspension alone were added). After the serum bottle is deoxidized and sealed, the serum bottle is subjected to shaking culture at 35 ℃, the concentration and biomass of the residual DCE in the culture solution are measured at fixed time, and the degradation curve and the biomass change curve of the strain FHY-13 for different initial concentrations of DCE are drawn.
In this example, the third DCE liquid selection medium is DCE 1-35 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 FIG. 4 (a) and FIG. 4 (b), strain FHY-13 was able to degrade an initial concentration of less than 25 mg.L -1 And in this range a more pronounced lag phase occurs with increasing concentration. When the initial DCE concentration was 30mg.L -1 When the strain FHY-13 only has a degradation effect on DCE in the first 72 hours, the concentration of residual DCE in the liquid phase is not changed obviously, and the degradation effect is probably caused by some toxic metabolites generated by the strain under the condition of high concentration of DCE, so that the activity of the strain is inhibited seriously.
Example 5: mineralization rate, chloride ion release rate and yield coefficient analysis of DCE by using Brevibacillus soil (Brevibacillus agri) FHY-13
Under the condition of optimal environmental factors, namely the culture temperature is 35 ℃, the pH of the culture solution is 6.0, and the salt concentration of the culture solution is 0.89%, the mineralization rate, the chloride ion release rate and the yield coefficient of the Bacillus brevis (Brevibacillus agri) FHY-13 to DCE with the concentration of 1-25 mg/L are examined. Experimental results show that strain FHY-13 can convert DCE into CO 2 、H 2 O and cell biomass, average mineralization rate of 61.12%, average chloride ion release rate of 84.70% and average yield coefficient of 0.1904mg cells/mg DCE.
The specific experimental process is as follows:
a first DCE liquid selection medium having a pH of 6.0 and a salt concentration of 0.89% 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 culture broth had cooled, 12 of the serum flasks were taken and added to the bacterial suspension prepared in example 2 with an initial biomass of 0.75mg/L, while 1, 5, 10, 15, 20 and 25mg/L of DCE were added as the sole carbon source (2 serum flasks per concentration), respectively, and 6 serum flasks were taken as blank (DCE alone and bacterial suspension alone). After deoxidizing and sealing a serum bottle, carrying out shaking culture at 35 ℃ and measuring DCE concentration, chloride ion concentration, cell biomass and CO at fixed time 2 Concentration. Drawing mineralization and dechlorination curves of the strain FHY-13 on different DCE concentrations, fitting to obtain average mineralization rate and average chloride ion release rate, and drawing cell biomass, chloride ion release amount and CO by combining cell proliferation amount 2 The average cell yield was calculated from the relationship between the amounts produced.
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= 0.6021x, and the correlation coefficient R 2 = 0.9910, indicating that strain FHY-13 can produce 0.6021mg of chloride ions per 1mg of DCE, and can theoretically produce 0.7107mg of chloride ions per 1mg of DCE, so that the average release rate of chloride ions is 84.70%.
CO 2 Is also linear with the degradation of DCE, which is to be calculatedThe straight line equation is y= 0.5549x, and the correlation coefficient R 2 = 0.9946, demonstrating that strain FHY-13 fully mineralizes 1mg of DCE to produce 0.5549mg 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 the strain FHY-13 was 61.12%.
Strain FHY-13 is able to synthesize self-cellular material using organic carbon during degradation of DCE. The linear relation between the cell proliferation amount and the DCE degradation amount is y= 0.1904x, and the correlation coefficient R 2 = 0.9886, indicating that strain FHY-13 is able to synthesize 0.1904mg of autologous cell biomass per degradation of 1mg DCE.
Example 6: analysis of degradation Performance of Brevibacillus soil (Brevibacillus agri) FHY-13 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-13 on the common organic pollutants in the industries is examined. Experimental results show that the strain FHY-13 can degrade the organic pollutants to different degrees, can completely degrade chlorobenzene, pentachlorobenzene and hexachlorobenzene in 24 hours, and can 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 6.0 and a salt concentration of 0.89% 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 the serum bottles were added to the bacterial suspension prepared in example 2 and having an initial biomass of 0.75mg/L, while methylene chloride, dichloropropane, benzene, chlorobenzene, pentachlorobenzene or hexachlorobenzene each having an initial concentration of 10mg/L was used as the sole carbon source, and 6 serum bottles were used as a blank (DCE alone without the bacterial suspension). After the serum bottle is deoxidized and sealed, the serum bottle is cultured in an oscillating way at 35 ℃, the concentration of the residual organic matters in the liquid phase is respectively analyzed for 24 hours and 72 hours, the corresponding biomass is measured, and the degradation rate of different organic carbon sources and the cell growth of the strain FHY-13 are drawn.
As shown in FIG. 6 (a), strain FHY-13 was able to degrade most of chlorobenzene, pentachlorobenzene and hexachlorobenzene to 91.5%, 83.3% and 76.7% respectively, while strain FHY-13 had only 14.4%, 17.3% and 32.8% degradation to methylene chloride, dichloropropane and benzene, respectively, when the culture time was 24 hours. As shown in FIG. 6 (b), when the cultivation time was 72 hours, the degradation rates of the strain FHY-13 for methylene chloride, dichloropropane, benzene, chlorobenzene, pentachlorobenzene and hexachlorobenzene were 78.9%, 45.7%, 59.5%, 100% and 100%, respectively. The test results show that the strain (Brevibacillus agri) FHY-13 can degrade various common organic pollutants, and has 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. The acidophilic and high-efficiency dichloroethylene degrading soil bacillus brevis is characterized in that the microorganism classification is named as soil bacillus brevis Brevibacillus agri FHY-13, and the microorganism classification is preserved in China Center for Type Culture Collection (CCTCC) at the year 2023, the month 28, and the preservation number is M2023420; the sequence of the 16S rRNA of FHY-13 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 acidophilic soil bacillus brevis FHY-13 for efficiently degrading dichloroethylene.
3. A method of preparing a bacterial suspension according to claim 2, comprising the specific steps of:
(1) Solid culture: inoculating acidophilic and high efficiency degrading dichloroethylene soil Bacillus brevis FHY-13 onto slant culture medium, and culturing at 20-40 deg.c for 2-4 days to obtainObtaining 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 1-25 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 6.0-8.0; 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, the solvent is water.
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 0.89%, 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 1-25 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 6.0; 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.
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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