CN117801979A - Staphylococcus epidermidis FHY-20 with dichloroethylene degradation capability and application thereof - Google Patents
Staphylococcus epidermidis FHY-20 with dichloroethylene degradation capability and application thereof Download PDFInfo
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Abstract
The invention discloses staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 with dichloroethylene degradation capability and application thereof, wherein staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 is preserved in China center for type culture collection, and addresses: chinese, university of armed chinese, postal code: 430072, deposit number: cctccc NO: m2023435, storage date 2023, month 03 and 30. The strain can degrade various common organic pollutants and has a certain practical significance for treating the organic pollutants of industrial pollution waste gas and wastewater.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to staphylococcus epidermidis FHY-20 with dichloroethylene degradation capability and application thereof.
Background
Dichloroethylene (DCE) is an important chemical raw material, is widely used as a solvent, a metal surface treatment agent, medicines and the like, and can enter the environment through the ways of industrial wastewater, exhaust emission, soil pollution, landfill sites and the like. DCE can enter the human body through skin absorption, inhalation, ingestion and other routes, and can cause serious damage to the central nervous system, liver, kidney, heart, respiratory system and the like. 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 degradation and purification of pollutants due to the advantages of high removal efficiency, low treatment cost, small secondary pollution and the like. The key point of biological treatment of DCE is to obtain the 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 staphylococcus epidermidis FHY-20 with dichloroethylene degradation capability and application thereof.
The aim of the invention is realized by the following technical scheme:
in a first aspect, the invention provides a strain of staphylococcus epidermidis with dichloroethylene degrading capability, the microorganism classification being named staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20, which has been deposited in China center for type culture Collection, at 2023, 03, 30: wu Changou Ipomoea of Wuhan, hubei province, china, university of Wuhan, 16 Lojia mountain road, postal code 430072; the preservation number is CCTCC NO: M2023435; the sequence of the 16S rRNA of FHY-20 is shown in SEQ ID NO. 1.
In a second aspect, the invention provides a bacterial suspension and a preparation method of the bacterial suspension, wherein the bacterial suspension takes staphylococcus epidermidis FHY-20 with dichloroethylene degradation capability as an active ingredient.
The bacterial suspension is prepared by solid culture, seed culture and liquid culture of staphylococcus epidermidis FHY-20 with dichloroethylene degradation capability.
The preparation method of the bacterial suspension comprises the following specific steps:
(1) Solid culture: inoculating staphylococcus epidermidis FHY-20 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 staphylococcus epidermidis FHY-20 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 1-30 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 0.88%, 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 1-30 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 staphylococcus epidermidis FHY-20 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: the strain FHY-20 can degrade DCE with initial concentration of 0-30 mg/L and finally degradeThe substance is CO 2 、H 2 O and cell biomass, average mineralization rate was 58.35%, average cell yield was 0.1977mg cells/mg DCE, and average chloride ion release rate was 85.44%. 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 common organic pollutants in the same industry, such as dichloromethane, dichloropropane, benzene, chlorobenzene, pentachlorobenzene, hexachlorobenzene and the like. The strain FHY-20 is taken from an exhaust gas treatment unit, has good degradation effect on chlorinated organics, 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 Staphylococcus epidermidis FHY-20;
FIG. 2 is a scanning electron microscope image of Staphylococcus epidermidis FHY-20;
FIG. 3 is a phylogenetic tree of Staphylococcus epidermidis FHY-20;
FIG. 4 is a graph showing the change of DCE concentration and cell biomass of Staphylococcus epidermidis FHY-20 with respect to degradation time at different concentrations of DCE, wherein FIG. 4 (a) is a graph showing the change of DCE concentration of Staphylococcus epidermidis FHY-20 with respect to degradation time at different concentrations of DCE, and FIG. 4 (b) is a graph showing the change of cell biomass of Staphylococcus epidermidis FHY-20 with respect to degradation time at different concentrations of DCE;
FIG. 5 shows the degradation of different concentrations of DCE by Staphylococcus epidermidis FHY-20 in CO 2 Yield of Cl - A graph of the change in the amount released and the cellular biomass;
FIG. 6 is a graph showing the degradation rate and the cell growth amount of Staphylococcus epidermidis FHY-20 on the industrial common organic pollutants, wherein FIG. 6 (a) is a graph showing the degradation rate and the cell growth amount of the industrial common organic pollutants at 24 hours, and FIG. 6 (b) is a graph showing 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 a staphylococcus epidermidis with dichloroethylene degradation capability, which is named as staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 by microorganism classification, and is preserved in China center for type culture collection (China center for type culture collection) for 30 days in 2023, wherein the preservation number is CCTCC NO: M2023435; the sequence of the 16S rRNA of FHY-20 is shown in SEQ ID NO. 1.
Staphylococcus epidermidis is a common strain in the environment, and no report of DCE degradation by staphylococcus epidermidis has been found through patent search and other related documents. The discovery of the degradation bacteria has important significance for purifying 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 staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20
1. Isolation and purification of staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20
Staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 is a gram-positive bacterium domesticated and separated from activated sludge of waste gas treatment devices of certain rubber plants in Zhejiang, and comprises the following specific steps:
(1) Sampling: taking multi-point samples from the activated sludge of the waste gas treatment device of a rubber plant in Zhejiang Taizhou as raw materials for screening staphylococcus epidermidis FHY-20 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-20.
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 7.0. 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 7.0.
2. Identification of Strain FHY-20
a. Physiological and biochemical characteristics of Strain FHY-20
Carrying out morphological observation and physiological and biochemical identification on the obtained strain FHY-20, 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 coccus, flagellum-free and gram-positive, spores can be generated, and the hydrolysis of starch and the positive of indole tests can be utilized by utilizing sucrose and trehalose. Strain FHY-20 is shown in figure 1; the form of the cells was observed under a scanning electron microscope as a coccus, as shown in FIG. 2.
b. 16S rRNA sequence analysis of Strain FHY-20
Strain FHY-20 was identified as Staphylococcus epidermidis by 16S rRNA sequence analysis and physiological and biochemical experiments. The sequencing results were:
ggacagtgcggcgtgctatacatgcaagtcgagcgaacagacgaggagcttgctcctctgacgttagcggcggacgggtgagtaacacgtggataacctacctataagactgggataacttcgggaaaccggagctaataccggataatatattgaaccgcatggttcaatagtgaaagacggttttgctgtcacttatagatggatccgcgccgcattagctagttggtaaggtaacggcttaccaaggcaacgatgcgtagccgacctgagagggtgatcggccacactggaactgagacacggtccagactcctacgggaggcagcagtagggaatcttccgcaatgggcgaaagcctgacggagcaacgccgcgtgagtgatgaaggtcttcggatcgtaaaactctgttattagggaagaacaaatgtgtaagtaactatgcacgtcttgacggtacctaatcagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttatccggaattattgggcgtaaagcgcgcgtaggcggttttttaagtctgatgtgaaagcccacggctcaaccgtggagggtcattggaaactggaaaacttgagtgcagaagaggaaagtggaattccatgtgtagcggtgaaatgcgcagagatatggaggaacaccagtggcgaaggcgactttctggtctgtaactgacgctgatgtgcgaaagcgtggggatcaaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgtttaggggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacgaccgcaaggttgaaactcaaaggaattgacggggacccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaccttaccaaatcttgacatcctctgaccctctagagatagagttttccccttcggggac
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-20 was tested, and the 16S rRNA sequence of strain FHY-20 was shown in SEQ ID NO. 1.
The 16S rRNA sequence of the strain FHY-20 is uploaded to a gene sequence in Genbank for homology comparison, and the homology of the strain is Staphylococcus epidermidis and Staphylococcus epidermidis NBRC 100911 is highest and reaches 99.79%. FIG. 3 is a phylogenetic tree of strain FHY-20.
Based on the sequencing results and the physiological and biochemical test results, it was determined that strain FHY-20 belongs to Staphylococcus epidermidis. Therefore, the strain FHY-20 is named staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 and is preserved in China Center for Type Culture Collection (CCTCC) NO: m2023435, storage date 2023, 30, 03, address: chinese, university of martial arts, postal code address: 430072.
example 2: preparation method of bacterial suspension containing staphylococcus epidermidis FHY-20 with dichloroethylene degradation capability as active ingredient
The preparation process of the bacterial suspension comprises the following steps:
(1) Solid culture: inoculating staphylococcus epidermidis FHY-20 with dichloroethylene degradation capability to slant culture medium, and culturing at 30deg.C for 3 days to obtain slant thallus; 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 into fermentation medium at a volume concentration of 1%, and heating at 30deg.CCulturing for 18h to obtain fermentation culture solution, namely 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 staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 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 staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 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 0.69mg/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-20 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＝-84.119+17.573*X1+3.449*X2+1.571*X3-0.891*X1*X1
-0.127*X1*X2-0.492*X1*X3-0.527*X2*X2+0.133*X2*X3；
-1.378*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 = 0.9932 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 staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20
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 DCE of staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 reaches the maximum value: the incubation temperature was 35 ℃, the pH of the broth was 7.5, and the salt concentration of the broth was 0.88%, at which time the degradation rate predicted by the predictive model was 0.863 mg/(L.times.h). The actual degradation rate of the strain FHY-20 on DCE under the culture conditions is 0.847 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 the pH value of 7.5, adding NaCl to enable the salt concentration to reach 0.88%, 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 was cooled, 3 serum bottles were taken and added to the bacterial suspension prepared in example 2 and having an initial biomass of 0.69mg/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 shaking table at 35℃for 24 hours, the residual DCE concentration was analyzed while biomass was measured to obtain the degradation rate of strain FHY-20.
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 0.88%, the actual degradation rate of staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 on DCE is 0.851, 0.867 and 0.823 mg/(L.times.h), the average degradation rate is 0.847 mg/(L.times.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 staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 for degrading DCE.
Example 4: degradation Performance detection of Staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 on DCE at 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 0.88%, the degradation performance of staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 on DCE with the concentration of 1-35 mg/L is examined. Experimental results show that the strain FHY-20 can completely degrade 1-30 mg/L DCE, and can not completely degrade DCE with initial concentration greater than 30 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 0.88% 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 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 0.69mg/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, shake culture is carried out at 35 ℃, the concentration and biomass of the residual DCE in the culture solution are measured at fixed time, and the degradation curve and biomass change curve of the strain FHY-20 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-20 was able to degrade DCE at an initial concentration of not more than 30mg/L, and its biomass increased significantly with increasing concentration in this range, indicating that low concentrations of DCE could be utilized by FHY-20 as a carbon source. When the initial DCE concentration is more than 30mg/L, the strain FHY-20 can only partially degrade DCE within 72 hours, and the degradation rate is slow, which is probably due to some toxic metabolites generated by the strain under the condition of high concentration of DCE, and has a certain inhibition effect on the activity of the strain.
Example 5: analysis of mineralization rate, chloride Release Rate and cell yield of DCE by Staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20
Under the condition of optimal environmental factors, namely the culture temperature is 35 ℃, the pH value of the culture solution is 7.5, and the salt concentration of the culture solution is 0.88 percent, the mineralization rate and chlorine of staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 on DCE with the concentration of 1-30 mg/L are examinedIon release rate and average yield coefficient. Experimental results show that strain FHY-20 can convert DCE into CO 2 、H 2 O and cell biomass, average mineralization rate was 58.35%, average chloride ion release rate was 85.44% and average cell yield was 0.1977mg cells/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 0.88% 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 charged with 0.69mg/L of the initial biomass bacterial suspension prepared by the method of example 2, while 5, 10, 15, 20, 25 and 30mg/L of DCE were each added as the sole carbon source (2 serum flasks per concentration), and 6 serum flasks were used as the blank (DCE alone without bacterial suspension). After deoxidizing and sealing a serum bottle, shake culturing at 35 ℃ and measuring DCE concentration, chloride ion concentration, cell biomass and CO in the culture solution at fixed time 2 Concentration. Drawing mineralization and dechlorination curves of the strain FHY-20 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= 0.6072x, and the correlation coefficient R 2 = 0.9936, indicating that strain FHY-20 can produce 06072mg of chloride ions per 1mg of DCE, and can theoretically produce 0.7107mg of chloride ions per 1mg of DCE, so that the average chloride ion release rate is 85.44%.
CO 2 The generation amount of (2) and the degradation amount of DCE are also in linear relation, the fitting linear equation is y= 0.5297x, and the correlation coefficient R 2 = 0.9971, demonstrating that strain FHY-20 fully mineralizes 1mg of DCE to produce 0.5297mg 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-20 was 58.35％。
Strain FHY-20 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= 0.1977x, and the correlation coefficient R 2 = 0.9857, indicating that strain FHY-20 is able to synthesize 0.1977mg of autologous cell biomass per degradation of 1mg DCE.
Example 6: analysis of degradation Property of Staphylococcus epidermidis (Staphylococcus epidermidis) FHY-20 on Industrial common organic pollutants
The degradation capability of staphylococcus epidermidis (Staphylococcus epidermidis) on organic pollutants common in the industries of methylene dichloride, dichloropropane, benzene, chlorobenzene, pentachlorobenzene and hexachlorobenzene are respectively taken as the only carbon source. Experimental results show that the strain FHY-20 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 48 hours. The specific embodiment is as follows:
the specific experimental process is as follows:
a first DCE liquid selection medium having a pH of 7.5 and a salt concentration of 0.88% 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 0.69mg/L, while methylene chloride, dichloropropane, benzene, chlorobenzene, pentachlorobenzene or hexachlorobenzene each having an initial concentration of 20mg/L was used as the sole carbon source, and 6 of the 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 pollutants and the cell growth of the strain FHY-20 are drawn.
As shown in FIG. 6 (a), when the cultivation time was 24 hours, the strain FHY-20 was able to completely degrade chlorobenzene, pentachlorobenzene and hexachlorobenzene, the degradation rates were all 100%, and at this time the degradation rates of the strain for methylene chloride, dichloropropane and benzene were 22.3%, 18.1% and 30.6%. As shown in FIG. 6 (b), when the cultivation time was 48 hours, the degradation rates of the strain FHY-20 for methylene chloride, dichloropropane and benzene reached 80.7%, 63.9% and 90.1%, respectively. The test results show that the strain (Staphylococcus epidermidis) FHY-20 can degrade various common organic pollutants, and has a certain practical significance for treating the organic pollutants of industrial pollution waste gas and wastewater.
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 staphylococcus epidermidis with dichloroethylene degrading capacity is characterized in that the microorganisms are named as staphylococcus epidermidis Staphylococcus epidermidis FHY-20 and are preserved in China Center for Type Culture Collection (CCTCC) in the year 2023 and the month 30, and the preservation number is M2023435; the sequence of the 16S rRNA of FHY-20 is shown in SEQ ID NO. 1.
2. A bacterial suspension containing staphylococcus epidermidis as the active ingredient, which is characterized in that the bacterial suspension is prepared by solid culture, seed culture and liquid culture of staphylococcus epidermidis FHY-20 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 staphylococcus epidermidis FHY-20 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 1-30 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.
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 0.88%, and culturing the bacterial suspension at 35 ℃ by taking dichloroethylene as a unique carbon source to degrade the dichloroethylene; the saidThe final concentration composition of the first DCE liquid selection medium was: DCE 1-30 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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