CN114317335B - Bacillus cereus for degrading dimethyl sulfide and application thereof - Google Patents
Bacillus cereus for degrading dimethyl sulfide and application thereof Download PDFInfo
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- CN114317335B CN114317335B CN202111554880.3A CN202111554880A CN114317335B CN 114317335 B CN114317335 B CN 114317335B CN 202111554880 A CN202111554880 A CN 202111554880A CN 114317335 B CN114317335 B CN 114317335B
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The invention discloses bacillus cereus for degrading dimethyl sulfide and application thereof. The Bacillus cereus GDUTAN18 strain is preserved in China center for type culture Collection (China center for type culture Collection) with the preservation time of 2021, 11 months and 22 days, and with the preservation address of China university of Wuhan and the preservation number of CCTCC NO: m20211472. The bacillus cereus GDUTAN18 not only can grow and reproduce with the dimethyl sulfide as an energy substance, but also has good dimethyl sulfide degradation effect, can be used for degrading the dimethyl sulfide in the environment, and is used for treating the malodorous waste gas of the dimethyl sulfide, treating the wastewater polluted by the dimethyl sulfide, repairing the soil polluted by the dimethyl sulfide and the like. The invention not only widens the application range of bacillus cereus, but also provides a useful bacterial source for the biodegradation of the dimethyl sulfide.
Description
Technical Field
The invention belongs to the technical field of microorganisms and environmental remediation application thereof. More particularly, relates to bacillus cereus for degrading dimethyl sulfide and application thereof.
Background
The volatile malodorous sulfide is mainly derived from the chemical production process, especially from the industries of pulping and papermaking, petrochemical industry, wastewater treatment and the like. Dimethyl sulfide is an important component in volatile malodorous sulfides, and is one of eight malodorous pollutants which are preferentially controlled in malodorous pollutant emission standards (GB 14554-1993) of China. The dimethyl sulfide has extremely low olfactory threshold, has rotten cabbage and rotten vegetable smell, can cause nausea, vomiting and inappetence of human bodies when being contacted with the dimethyl sulfide, can also cause stimulation effects on nose, respiratory tract and the like, causes discomfort of bodies such as cough, chest distress and the like, and even can cause paralysis effect on a central nervous system with high concentration of dimethyl sulfide, thereby seriously affecting the health of the bodies. In addition, the thermal decomposition of dimethyl sulfide produces toxic sulfide fumes, which can also be oxidized or photolyzed into aerosols, exacerbating ozone destruction.
With the development of industry and the rapid population growth, organic sulfides emitted by human factors are attracting great attention. Therefore, the search for a method that can effectively treat dimethyl sulfide has been the focus of research by environmental scientists. The biological method for degrading the dimethyl sulfide has the advantages of low cost, simple process, convenient operation, good treatment effect, no secondary pollution and the like, and becomes the preferred method for treating the dimethyl sulfide. However, few microorganisms can be used for degrading the dimethyl sulfide, which seriously affects the development and application of the biological method for degrading the dimethyl sulfide. Therefore, screening microorganisms with the ability to degrade dimethyl sulfide is of great importance for the effective treatment of dimethyl sulfide.
Bacillus is a common bacterium, and contains various types of bacillus subtilis, bacillus licheniformis, bacillus cereus and the like, and can secrete antibacterial substances to inhibit the growth of pathogenic bacteria and degrade toxic and harmful substances, so that the bacillus has wide application in industries, agriculture and the like. For example, chinese patent CN113621536a discloses a strain of paenibacillus polymyxa SP1, wherein the paenibacillus polymyxa SP1 can produce antimicrobial peptides, and both the supernatant and the precipitate after fermentation contain antimicrobial substances, thus having broad-spectrum antimicrobial effect, inhibiting pathogenic bacteria such as common livestock and poultry breeding pathogens, breeding insect silkworms and common human skin pathogenic bacteria, and reducing diseases of breeding insects such as silkworms; meanwhile, the strain can also produce lipase, can solve the problem of absorbing fat feed by young livestock and poultry, improves the utilization rate of the feed, improves the production performance and intestinal health of livestock and poultry, and provides a basis for developing novel feed additives. For another example, chinese patent CN113025535a discloses a bacillus GDUTAN11 capable of degrading benzene series, which has good degradation properties for benzene, toluene, meta-xylene and para-xylene. However, no report on bacillus with the capability of degrading the dimethyl sulfide exists at present, so that the bacillus is widely excavated and utilized, the application range of the bacillus is widened, bacteria with the capability of degrading the dimethyl sulfide can be obtained through screening, and a useful bacterial source is provided for biological degradation of the dimethyl sulfide.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the prior art that the variety and the quantity of the dimethyl sulfide degrading bacteria are small, and provides bacillus cereus for degrading dimethyl sulfide and application thereof.
A first object of the present invention is to provide a Bacillus cereus GDUTAN18 strain degrading dimethyl sulfide.
The second object of the invention is to provide the application of the Bacillus cereus (GDUTAN 18) strain or the culture solution thereof in the degradation environment of the methyl sulfide.
The third object of the present invention is to provide the use of the Bacillus cereus (GDUTAN 18) strain or a culture broth thereof for preparing a preparation for degrading dimethyl sulfide in an environment.
The fourth object of the invention is to provide the application of the Bacillus cereus (GDUTAN 18) strain or the culture solution thereof in the treatment of the malodorous waste gas of the dimethyl sulfide.
The fifth object of the invention is to provide the application of the Bacillus cereus (GDUTAN 18) strain or the culture solution thereof in the treatment of the wastewater polluted by the dimethyl sulfide.
The sixth object of the invention is to provide the application of the Bacillus cereus GDUTAN18 strain or the culture solution thereof in restoring the soil polluted by the dimethyl sulfide.
A seventh object of the present invention is to provide a formulation for degrading dimethyl sulfide in the environment.
An eighth object of the present invention is to provide a method for treating a dimethyl sulfide contaminant in an environment.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a Bacillus cereus (GDUTAN 18 strain for degrading dimethyl sulfide, which is preserved in China center for type culture Collection (China center for type culture collection) for 2021 at 11 months and 22 days, and has a preservation address of China university of Wuhan and a preservation number of CCTCC NO: m20211472.
The 16rDNA sequence of the Bacillus cereus GDUTAN18 strain is shown as SEQ ID NO. 1.
The Bacillus cereus (GDUTAN 18) strain of the present invention is screened from landfill leachate isolated from a landfill. The Bacillus cereus GDUTAN18 strain not only can grow and propagate in a culture medium which takes the methyl sulfide as a carbon source and an energy source substance, but also has good methyl sulfide degradation effect, the degradation efficiency of the methyl sulfide with the concentration less than or equal to 15mg/L is over 90 percent in 72 hours, and the strain can be used for biological degradation of the methyl sulfide. Thus, the present application protects the following applications of Bacillus cereus (GDUTAN 18 strain):
the invention discloses application of Bacillus cereus GDUTAN18 strain or culture bacterial liquid thereof in degrading dimethyl sulfide in environment.
The invention also discloses application of Bacillus cereus GDUTAN18 strain or culture solution thereof in preparation of preparation for degrading dimethyl sulfide in environment.
The invention also discloses application of Bacillus cereus GDUTAN18 strain or culture bacterial liquid thereof in the treatment of the malodorous waste gas of the dimethyl sulfide.
The invention also discloses application of Bacillus cereus GDUTAN18 strain or culture bacterial liquid thereof in treatment of wastewater polluted by dimethyl sulfide.
The invention also applies for protecting the application of Bacillus cereus GDUTAN18 strain or the culture bacterial liquid thereof in restoring the soil polluted by the dimethyl sulfide.
The invention also provides a preparation for degrading the dimethyl sulfide in the environment, which contains Bacillus cereus (GDUTAN 18 strain or culture broth thereof.
Specifically, the environment is the atmosphere, a body of water or soil.
The invention also provides a method for treating the pollution of the dimethyl sulfide in the environment, namely, the Bacillus cereus GDUTAN18 strain or the culture bacterial liquid thereof is applied to the environment polluted by the dimethyl sulfide such as the atmosphere, the water body or the soil.
The invention has the following beneficial effects:
in the invention, in the process of screening, separating and purifying microorganisms in landfill leachate by taking dimethyl sulfide as a carbon source and an energy source substance, a Bacillus cereus GDUTAN18 strain capable of degrading dimethyl sulfide is obtained. The bacillus cereus GDUTAN18 strain not only can grow and reproduce in a culture medium with dimethyl sulfide as a carbon source and an energy source substance, but also has a good dimethyl sulfide degradation effect, the degradation efficiency of the dimethyl sulfide with the concentration less than or equal to 15mg/L in 72 hours is over 90 percent, and the bacillus cereus GDUTAN18 strain can be used for degrading the dimethyl sulfide in the environment, treating the stink waste gas of the dimethyl sulfide, treating the waste water polluted by the dimethyl sulfide, repairing the soil polluted by the dimethyl sulfide and the like. The invention not only widens the application range of bacillus cereus, but also provides a useful bacterial source for the biodegradation of the dimethyl sulfide, and has important significance for promoting the development of the biodegradation of the dimethyl sulfide.
Drawings
FIG. 1 is a colony morphology of Bacillus cereus GDUTAN18 strain on NA medium.
FIG. 2 is a diagram showing the morphology of cells of Bacillus cereus GDUTAN18 under an electron scanning microscope.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 screening isolation of Bacillus cereus GDUTAN18 Strain
The Bacillus cereus GDUTAN18 strain is obtained by screening, separating and purifying landfill leachate when dimethyl sulfide is used as a carbon source and an energy source substance.
According to the invention, bacteria in landfill leachate are screened by adding dimethyl sulfide into an inorganic salt culture medium, and then the bacteria are domesticated by taking dimethyl sulfide as a domestication substrate, wherein the final concentration of the domestication substrate is sequentially 1mg/L, 5mg/L, 10mg/L, 15mg/L and 20mg/L.
The specific steps of domestication are as follows: firstly, 10mL of landfill leachate is added into an inorganic salt culture medium containing 1mg/L of dimethyl sulfide, after domestication is carried out for 5 days at 37 ℃, 10% of inoculation amount is transferred into the inorganic salt culture medium containing the dimethyl sulfide with the next concentration, domestication is carried out, and the concentration is gradually increased until the concentration of the dimethyl sulfide is increased to 20mg/L. After the acclimation is finished, diluting bacterial liquid obtained by culturing under the condition of different acclimation substrate concentrations by 10 -1 ～10 -7 And then respectively and uniformly coating 0.2mL of the obtained product in a solid culture medium with dimethyl sulfide as a carbon source, selecting single bacterial colonies with high growth speed and neat edges for enrichment culture, then carrying out a dimethyl sulfide degradation experiment, and storing bacterial colonies with the best degradation effect and carrying out strain identification.
EXAMPLE 2 identification of Bacillus cereus GDUTAN18 Strain
The identification process and the identification result of the Bacillus cereus GDUTAN18 strain are shown as follows:
1. colony morphology characterization
As shown in FIG. 1, the colony morphology of Bacillus cereus GDUTAN18 strain was observed after inoculating the strain on NA (nutrient agar) medium for 24 hours, and the colony morphology of Bacillus cereus GDUTAN18 strain was circular, milky white, opaque, and the colony diameter was 4.0-5.0 mm.
2. Characteristics of bacterial cell shape
As shown in FIG. 2, the strain GDUTAN18 was in the form of a short rod-like approximately spherical shape, and the cell size was (2.8-3.0) μm X (1.0-1.2) μm, and no flagellum was observed under SEM.
3. Principal physiological and biochemical characteristics
The main physiological and biochemical characteristics of Bacillus cereus GDUTAN18 strain are shown in Table 1.
TABLE 1 main physiological and biochemical characteristics of Bacillus cereus GDUTAN18 Strain
Note that: the reaction status is classified into positive and negative, positive shape is coded as "+", and negative shape is coded as "-".
As is clear from Table 1, bacillus cereus GDUTAN18 strain is a gram positive bacterium.
4. Molecular characterization
Total DNA of the GDUTAN18 strain was extracted using a DNA extraction kit (cat# B610367-0250) from Biotechnology Co., ltd.) and the 16S rDNA of the GDUTAN18 strain was amplified with bacterial 16S rDNA universal primer (27F: 5'-AGTTT GATCMTGGCTCAG-3'; 14992R: 5 '-GGTTACCTTGTTACGACTT-3') and sequenced for comparison.
The 16S rDNA sequence of the Bacillus cereus GDUTAN18 strain is shown below (SEQ ID NO. 1):
GGCGGGCCGCTGCTATAATGCAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTGGAACCCGCCCGCTAAAGGGGGACAAAGGATGGGGGACGCCCC
sequencing results showed that the 16S rDNA sequence of the GDUTAN18 strain was 1467bp long, and compared with the registered gene sequence in Genbank, the 16S rDNA sequence of the GDUTAN18 strain was found to be most similar to Bacillus cereus strain T M4, with a similarity of 89%.
As can be seen from the comprehensive morphological characteristics, physiological and biochemical characteristics and molecular identification results, the GDUTAN18 strain is identified as Bacillus cereus, so the GDUTAN18 strain is named as Bacillus cereus, and is preserved in China center for type culture Collection with the preservation time of 2021 for 11 months and 22 days, and with the preservation address of China university of Wuhan and the preservation number of CCTCC NO: m20211472.
EXAMPLE 3 evaluation of degradation Properties of Bacillus cereus GDUTAN18 Strain on dimethyl sulfide
1. Preparing inorganic salt culture medium, packaging, adding 100mL inorganic salt culture medium into a headspace bottle, and sterilizing at 121deg.C under high pressure for 30min.
2. Enrichment culture of Bacillus cereus GDUTAN18 strain in broth culture medium (beef extract 3.0g/L, peptone 10.0g/L, naCl 5.0.0 g/L) for 24 hr, centrifuging, collecting thallus, and washing with PBS three times; then, 10% of the inoculation amount is used for inoculating bacillus cereus GDUTAN18 into a serum bottle filled with 100mL of inorganic salt culture medium, a certain amount of methyl sulfide is added to ensure that the final concentration of the methyl sulfide in the inorganic salt culture medium is 5mg/L, 8mg/L, 10mg/L, 15mg/L, 20mg/L and 25mg/L respectively, a capped and sealed shaking table is arranged at the constant temperature of 37 ℃ and the methyl sulfide degradation test is carried out by shaking for 72 hours at 140 r/min.
3. Detection of degradation Capacity of Bacillus cereus (GDUTAN 18 Strain to dimethyl sulfide)
After degradation in inorganic medium containing dimethyl sulfide for 72h, the serum bottle headspace was sampled, the concentration of dimethyl sulfide in the gas phase was determined using an Agilent GC 8850 gas chromatograph (Agilent DB SULFUR SCD capillary column, 60m,nt DB SULFUR S) containing Sulfur Chemiluminescence Detector (SCD), and the concentration in the liquid phase was detected using henry's law, thus calculating the degradation rate.
The detection conditions are as follows: the temperature of the sample inlet gasification chamber is 80 ℃, and the flow rate of oxidized gas of the detector is 50mL/min; the heater is 250 ℃; the burner is 850 ℃; heating program: the temperature was kept at 35℃for 3min, and then 10℃per min was raised to 200℃for 3min. And (3) adopting an Agilent 1mL airtight needle to sample, wherein the sample injection amount is 1mL, and the split ratio is 10:1.
The results are shown in Table 2:
TABLE 2 degradation rates of various initial concentrations of dimethyl sulfide
As shown in Table 2, the Bacillus cereus GDUTAN18 strain has good dimethyl sulfide degradation effect, and the degradation efficiency of the strain to the dimethyl sulfide with the concentration less than or equal to 15mg/L in 72 hours is over 90 percent, so that the strain can be used for degrading the dimethyl sulfide in the environment, treating the malodorous waste gas of the dimethyl sulfide, treating the waste water polluted by the dimethyl sulfide and repairing the soil polluted by the dimethyl sulfide.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> university of Guangdong Industrial science
<120> Bacillus cereus for degrading dimethyl sulfide and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1467
<212> DNA
<213> Bacillus cereus (Bacillus cereus)
<400> 1
ggcgggccgc tgctataatg cagtcgagcg aatggattaa gagcttgctc ttatgaagtt 60
agcggcggac gggtgagtaa cacgtgggta acctgcccat aagactggga taactccggg 120
aaaccggggc taataccgga taacattttg aaccgcatgg ttcgaaattg aaaggcggct 180
tcggctgtca cttatggatg gacccgcgtc gcattagcta gttggtgagg taacggctca 240
ccaaggcaac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300
ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360
ggagcaacgc cgcgtgagtg atgaaggctt tcgggtcgta aaactctgtt gttagggaag 420
aacaagtgct agttgaataa gctggcacct tgacggtacc taaccagaaa gccacggcta 480
actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttatccgga attattgggc 540
gtaaagcgcg cgcaggtggt ttcttaagtc tgatgtgaaa gcccacggct caaccgtgga 600
gggtcattgg aaactgggag acttgagtgc agaagaggaa agtggaattc catgtgtagc 660
ggtgaaatgc gtagagatat ggaggaacac cagtggcgaa ggcgactttc tggtctgtaa 720
ctgacactga ggcgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgagtgctaa gtgttagagg gtttccgccc tttagtgctg aagttaacgc 840
attaagcact ccgcctgggg agtacggccg caaggctgaa actcaaagga attgacgggg 900
gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960
tcttgacatc ctctgacaac cctagagata gggcttctcc ttcgggagca gagtgacagg 1020
tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080
caacccttga tcttagttgc catcattaag ttgggcactc taaggtgact gccggtgaca 1140
aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca 1200
cgtgctacaa tggacggtac aaagagctgc aagaccgcga ggtggagcta atctcataaa 1260
accgttctca gttcggattg taggctgcaa ctcgcctaca tgaagctgga atcgctagta 1320
atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380
accacgagag tttgtaacac ccgaagtcgg tggggtaacc ttttggaacc cgcccgctaa 1440
agggggacaa aggatggggg acgcccc 1467
Claims (10)
1. Bacillus cereus capable of degrading dimethyl sulfideBacillus cereus) The GDUTAN18 strain is characterized in that the GDUTAN18 strain is preserved in China center for type culture Collection (CCTCC NO) with the preservation time of 2021, 11 months and 22 days: m20211472.
2. The bacillus cereus GDUTAN18 strain of claim 1, wherein the 16rDNA sequence of the strain is shown in SEQ ID No. 1.
3. The bacillus cereus GDUTAN18 strain or the application of culture broth thereof in degrading dimethyl sulfide in environment in claim 1.
4. Use of the bacillus cereus GDUTAN18 strain or a culture broth thereof of claim 1 for preparing a preparation for degrading dimethyl sulfide in an environment.
5. The bacillus cereus GDUTAN18 strain or the application of a culture solution thereof in the treatment of the malodorous waste gas of the dimethyl sulfide.
6. The bacillus cereus GDUTAN18 strain or the application of a culture broth thereof in the treatment of wastewater polluted by dimethyl sulfide according to claim 1.
7. The application of bacillus cereus GDUTAN18 strain or a culture broth thereof in restoring soil polluted by dimethyl sulfide as claimed in claim 1.
8. A formulation for degrading dimethyl sulfide in an environment, wherein the formulation comprises the bacillus cereus GDUTAN18 strain or a culture broth thereof according to claim 1.
9. The formulation of claim 8, wherein the environment is the atmosphere, a body of water, or soil.
10. A method for treating the pollution of the dimethyl sulfide in the environment, which is characterized in that the bacillus cereus GDUTAN18 strain or the culture solution thereof in the environment polluted by the dimethyl sulfide such as the atmosphere, water or soil is applied.
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| 生物滴滤和过滤技术降解含硫恶臭有机废气的研究;陈雪泉;中国硕士论文电子期刊(第11期);第95-104页 * |
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