CN110804567B - Serratia marcescens capable of degrading chlorobenzene and application thereof - Google Patents
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Abstract
The invention discloses serratia marcescens capable of degrading chlorobenzene and application thereof, wherein the preservation number of the strain is CCTCC NO: m2019674, Serratia marcescens TF-1 of the invention can not only take chlorobenzene as the only carbon source and energy source and degrade the chlorobenzene, but also degrade the chlorobenzene by co-metabolism, can keep higher activity in poor-culture polluted sites and complex polluted sites, is suitable for the application in the fields of wastewater, drinking water and soil remediation, and is expected to make a new breakthrough in the engineering application field of chlorinated aromatic hydrocarbon biodegradation.
Description
Technical Field
The invention relates to the technical field of environmental biology, in particular to Serratia marcescens capable of degrading chlorobenzene, and also relates to application of Serratia marcescens capable of degrading chlorobenzene.
Background
Chlorobenzene is a compound which is artificially synthesized and has relatively stable chemical properties, and is widely used in the industries of medicines, pesticides, engineering plastics, family hygiene, material synthesis, dye industry and the like. But the chlorine atom in chlorobenzene has higher electronegativity, and can strongly attract electrons on a benzene ring, so that the benzene ring becomes an electron-phobic ring, and therefore, the chlorobenzene has quite stable chemical properties, is difficult to degrade, has high toxicity, has inhibiting and anesthetic effects on a central nervous system, and even is carcinogenic; generally, the waste water with high chloride ion content has high salinity. Along with the use of chlorobenzene in large quantities, a large amount of chlorobenzene exists in the environment, which poses serious threats to human health and ecological environment, and along with the increasing requirements on the ecological environment in the sustainable development in China and China, how to effectively eliminate the pollution becomes an important research content in the field of environmental protection.
From the viewpoint of solving large-area pollution, biodegradation is considered to be the most effective way to eliminate chlorobenzene-based pollutants because of its high efficiency and low cost. The biodegradation comprises two modes of anaerobic biodegradation and aerobic biodegradation. The anaerobic degradation is usually carried out by using dechlorination bacteria such as Hyphomicrobium, Dehalococcides and the like to reduce and dechlorinate high-chlorinated organic matters. Dechlorinated bacteria often form consortia with acetogenic bacteria (homoacetogens) and methanogens (methanogens), and in the presence of a carbon source and an exogenous electron donor, chlorohydrocarbons serve as electron acceptors to supply energy to anaerobic microorganisms while being degraded. However, anaerobic degradation has the defect of incomplete degradation, and dechlorinated products have larger biological toxicity and carcinogenicity. The aerobic degradation way can completely degrade the chlorohydrocarbon through hydroxylation or epoxidation, and has obvious advantages compared with the anaerobic biodegradation which is not completely degraded.
At present, the research on the degradation of chlorobenzene by using serratia marcescens is not reported in China. Currently, chlorobenzene can be degraded only in a single form such as co-metabolism or anaerobic dechlorination in functional strains, and the requirements on conditions are strict, so that the application of chlorobenzene aerobic oxidation in engineering is limited to a certain extent.
Disclosure of Invention
In view of the above, the invention aims to provide serratia marcescens capable of degrading chlorinated aromatic hydrocarbons, which can degrade chlorinated aromatic hydrocarbons, can be applied to the fields of wastewater, drinking water, soil remediation and the like, is particularly suitable for degradation of chlorinated aromatic hydrocarbon pollutants in wastewater and soil biological modification, and has a wide popularization significance.
In order to achieve the purpose, the invention provides the following technical scheme:
1. serratia marcescens (TF-1) capable of degrading chlorobenzene is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019674.
The Serratia marcescens capable of degrading chlorinated aromatic hydrocarbons is classified and named Serratia marcescens strain TF-1, is separated from polluted soil around a power plant of a Wu thousand cubic gas cabinet of an original Chongqing iron and steel company in the Chongqing Dadu district, and belongs to the Serratia marcescens. The diameter of the bacterial colony is 0.5-0.8 mu m, the length is 0.9-2.0 mu m, the end is round, the bacterial colony is facultative anaerobic, most of the bacterial colony is opaque and has a certain rainbow color; white, pink or red; gram staining was negative. The optimal growth temperature is 25-40 ℃, and the pH is 6.5-7.5; the homology with strains strain RJ-10-16, strain RJ-10-14, marcocens strain AG2105 and strain KUJM3 of Serratia sp. is 99%.
Preferably, the 16S rDNA sequence of the Serratia marcescens TF-1 is shown in SEQ ID NO. 3.
Preferably, the serratia marcescens TF-1 takes chlorobenzene as a sole carbon source.
Preferably, the serratia marcescens TF-1 can tolerate salinity with the highest mass fraction of 7%.
Preferably, the salinity is measured as sodium chloride.
2. The serratia marcescens TF-1 is applied to degradation of chlorinated aromatic hydrocarbons.
Preferably, the chlorinated aromatic hydrocarbon is chlorobenzene.
Preferably, the Serratia marcescens TF-1 is applied to degrading chlorinated aromatic hydrocarbon by taking benzene, toluene, formaldehyde, phenol, methanol or ethanol as a substrate.
Preferably, the serratia marcescens TF-1 is applied to the degradation of chlorobenzene by taking the chlorobenzene as a unique carbon source.
The invention has the beneficial effects that: the Serratia marcescens strain TF-1 disclosed by the invention can take chlorobenzene as a unique carbon source and energy source and degrade the chlorobenzene, and can degrade the chlorobenzene by co-metabolism; and the culture medium required by the amplification culture has simple components and low cost. The method is particularly suitable for degrading chlorobenzene pollutants in the wastewater and biologically modifying soil, can be widely popularized, and is the only discovered serratia with chlorobenzene degradation function at present. The invention solves the problem that the degradation of chlorinated hydrocarbon pollutants by anaerobic and facultative anaerobic organisms in the prior art can not be engineered.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a growth curve of Serratia marcescens TF-1;
FIG. 2 is a phylogenetic tree of Serratia marcescens strain TF-1;
FIG. 3 is a graph showing the degradation effect of Serratia marcescens TF-1 on chlorobenzene of different concentrations;
FIG. 4 is a graph showing the degradation effect of Serratia marcescens strain TF-1 in chlorobenzene at different salinity.
Strain preservation
In the invention, Serratia marcescens (Serratia marcescens strain) TF-1 is a strain separated from polluted soil around a power plant of a Wuwangchi gas holder of the original Chongqing iron and steel member company in the Chongqing Daqiao district, and is delivered to the China center for type culture collection for collection, wherein the collection number is CCTCC NO: m2019674, the address is located at Wuhan university in Wuhan, China, the preservation date is 8/29 in 2019, and the classification is named as Serratia marcescens TF-1.
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
The experimental materials used in the present invention are as follows: the composition of the MSM medium was as follows (g/L): CaCl2 0.1g；MgSO4·7H2O 0.2g；NH4SO4 2.5g；KH2PO44.5g, NaCl 0.2g, chlorobenzene (60 mg/L)130mg/L, 200mg/L), 1L of distilled water.
The composition of LB medium was as follows (g/L): 5.0g of beef extract, 10g of peptone, 10g of NaCl and 1L of distilled water.
Wherein the MSM solid medium and the LB solid medium can be prepared by adding 20g/L agar to the liquid medium as described above.
Plate: 90mm glass petri dish.
The rest reagents are commercial analytical pure products.
Polluted soil around a power plant of Wu thousand cubic gas chambers of an original Chongqing iron and steel shares company in the Chongqing Dadu district is taken as polluted particles on a sieve with the size of 4mm below and a sieve with the size of 2mm above.
Example 1 enrichment and optimization of strains
1) Enrichment culture: weighing 10g of the contaminated particles, adding into a 250ml conical flask filled with 100ml of deionized water, and placing into a shaking table at 30 ℃ and 160 r/min. After shaking and fully mixing for 3h, taking 10ml of supernatant, inoculating the supernatant into a 250ml conical flask filled with 100ml of LB culture medium, putting the conical flask into a shaking table, setting the temperature of the conical flask to be 30 ℃, culturing for 72h at 160r/min, and storing the conical flask in a refrigerator at 4 ℃ to obtain enriched bacterial liquid.
2) And (3) pure bacteria separation: performing 10-fold serial dilution on the enriched bacteria liquid with cooled sterile distilled water to obtain 10-fold dilution-1、10-2The diluent (2). MSM solid medium culture was performed by decantation. Placing the MSM solid culture medium in a biochemical incubator, culturing at 30 ℃ for 3d, carrying out multiple passages on strains with good growth (forming single colony), and purifying to obtain pure bacteria.
3) Strain optimization: preparing bacterial suspension by using pure strains, and adding the bacterial suspension into a saline bottle filled with a certain amount of inorganic salt culture medium; after 60mg/L, 130mg/L and 200mg/L of chlorobenzene were added, the mixture was sealed with a stopper. Placing into shaking table, shaking and culturing at 30 deg.C and 160r/min, and detecting bacterial liquid concentration and chlorobenzene concentration every 24 hr.
The chlorobenzene is detected by gas chromatography under the following chromatographic conditions: DB-23 elastic quartz capillary column (30m x 0.32mm x 0.25 μm). Injection port temperature 240 ℃, detector temperature 260 ℃, column temperature: raising the temperature to 140 ℃ at 90 ℃ (1.0min) and 10 ℃/min, and then raising the temperature to 200 ℃ (1.0min) at 20 ℃/min; carrier gas: nitrogen (99.999%), the flow rate of carrier gas in the column is 2.0mL/min, and split-flow sample injection is carried out, wherein the split-flow ratio is 10: 1; ECD current 0.5 nA; the flow rate of tail gas blowing is 40 mL/min.
Culturing the selected strain in LB medium at 30 deg.C and 160rpm, and determining OD of bacterial liquid600 nmValues, growth curves are shown in figure 1.
Example 2 identification of bacterial species
Taking 1-5ml of the bacterial culture solution, extracting genomic DNA by using a bacterial genomic DNA extraction kit, and then performing broad-spectrum amplification by using a 6SrRNA broad-spectrum amplification primer F27: 5'-agagtttgatcatggctcag-3' (SEQ ID NO.1) and R1492: 5'-tacggttaccttgttacgactt-3' (SEQ ID NO. 2).
The target fragment was amplified by PCR using QIAquick Genomic DNA Buffer Set. Mu.l of the DNA was subjected to 3% agarose gel electrophoresis, and the objective fragment was recovered from the gel and subjected to DNA sequencing, which was requested to be performed by Szechwan department of Onghama. DNA sequencing was carried out using Seq Forward, Seq Reverse, and Seq Internal as primers.
The identification result shows that: the gram-negative bacteria of the genus have the colony diameter of 0.5-0.8 mu m, the length of 0.9-2.0 mu m, round ends, generally circumferential flagella movement, facultative anaerobism, and most of the colonies are opaque and have a certain rainbow color; white, pink or red. The sequencing result of 16S rDNA of Serratia marcescens strain TF-1 shows that the base length is 1500bp (SEQ ID NO.3), and the base sequence is compared with the GenBank nucleic acid sequence database to find that the homology with the strains RJ-10-16, RJ-10-14, marcescens strain AG2105 and strain KUJM3 of Serratia marcescens sp is 100%. It is known from the phylogenetic tree of Serratia marcescens strain TF-1 (see FIG. 2) that Serratia sp. has a close relationship with Escherichia sp.
Example 3 Effect of Serratia marcescens TF-1 on the degradation of chlorobenzene on various substrates
Preparing benzene, toluene, formaldehyde, phenol, methanol and ethanol with concentration of 60mg/L, 40mg/L, 80ml/L and 80ml/L respectively, subpackaging in 100ml serum bottle containing 50ml MSM culture medium (wherein chlorobenzene concentration is 60mg/L), culturing the MSM based onSterilizing at 121 deg.C, cooling, adding 2.5ml Serratia TF-1 into the bottle with micropipette, covering with rubber plug, shaking, culturing in shaking table at 30 deg.C and 160r/min for 156 hr, measuring the content of chlorinated olefin by gas chromatography headspace to determine the degradation effect of chlorinated olefin, and measuring OD of the bacterial liquid600 nmThe value determines the growth of the cells.
TABLE 1 degradation Effect of Serratia marcescens strain TF-1 on chlorobenzene under different substrates
+: represents growth; ++: indicating significant growth;
as can be seen from Table 1, when Serratia marcescens strain TF-1 uses benzene and toluene as substrates, the removal rate of chlorobenzene is high, and when formaldehyde, phenol, methanol, ethanol and the like are used as substrates, the removal rate of chlorobenzene can still be kept high, and the growth condition is good.
Although the homology of the strain separated by the invention and strains RJ-10-16, RJ-10-14, marcocens strain AG2105 and strain KUJM3 of Serratia sp is 100% according to the sequencing result of 16S rDNA, the strain separated by the invention is found from the research later on, the strain separated by the invention has high tolerance to chloride ions and chlorobenzene and has new functions, so the strain is identified as a new species in Serratia sp.
Example 4 degradation Effect of Serratia marcescens TF-1 on chlorobenzene of various concentrations
Adding a certain amount of chlorobenzene into 100ml saline bottle containing 50ml MSM culture mediumThe chlorobenzene concentration was 60mg/L, 100mg/L, 150mg/L, 200mg/L, 300mg/L, 350mg/L, respectively. Adding 0.5ml Serratia marcescens bacterial liquid (OD)600nm0.12), covering a rubber plug, shaking uniformly, putting into a shaking table, culturing under the conditions of 30 ℃ and 160r/min, continuously culturing for 156 hours, measuring the chlorobenzene content by using gas chromatography, and determining the degradation effect of chlorobenzene. The chlorobenzene degradation effect of different concentrations is shown in figure 3, and the result shows that the Serratia marcescens Serratia marcocens strain TF-1 has better degradation effect on chlorobenzene with the concentration as high as 350 mg/L.
Example 5 degradation Effect of Serratia marcescens TF-1 on chlorobenzene at different salinity
Adding 100mg/L chlorobenzene into 100ml saline bottle containing 50ml MSM culture medium with salinity of 1%, 2%, 3%, 4%, 5%, 6%, 7% (based on NaCl mass ratio), and adding 0.5ml Serratia marcescens bacterial liquid (OD)600nm0.12), covering a rubber plug, shaking uniformly, putting the mixture into a shaking table, culturing for 168 hours at the temperature of 30 ℃ and at the speed of 160r/min, measuring the chlorobenzene content by using gas chromatography, and determining the chlorobenzene degradation effect, wherein the chlorobenzene degradation effect under different salinity is shown in figure 4. The results show that the serratia marcescens TF-1 can bear salinity with the highest tolerance mass fraction of 7%.
In conclusion, the serratia marcescens screened from the polluted soil around the fifty thousand cubic gas chambers of the original Chongqing iron and steel member company power plant in Chongqing Dadu district has high tolerance to chloride ions and chlorobenzene, can be degraded by taking the chlorobenzene as a carbon source and an energy source and can also be degraded by co-metabolism, and can keep high activity in poor-culture polluted sites and complex polluted sites. The unique degradation characteristic of the serratia marcescens is expected to make a new breakthrough in the engineering application field of the biodegradation of the chlorinated hydrocarbon.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
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tagcgattcc gacttcatgg agtcgagttg cagactccaa tccggactac gacgtacttt 180
atgaggtccg cttgctctcg cgaggtcgct tctctttgta tacgccattg tagcacgtgt 240
gtagccctac tcgtaagggc catgatgact tgacgtcatc cccaccttcc tccagtttat 300
cactggcagt ctcctttgag ttcccggccg aaccgatggc aacaaaggat aagggttgcg 360
ctcgttgcgg gacttaaccc aacatttcac aacacgagct gacgacagcc atgcagcacc 420
tgtctcagag ttcccgaagg caccaaagca tctctgctaa gttctctgga tgtcaagagt 480
aggtaaggtt cttcgcgttg catcgaatta aaccacatgc tccaccgctt gtgcgggccc 540
ccgtcaattc atttgagttt taaccttgcg gccgtactcc ccaggcggtc gatttaacgc 600
gttagctccg gaagccacgc ctcaagggca caacctccaa atcgacatcg tttacagcgt 660
ggactaccag ggtatctaat cctgtttgct ccccacgctt tcgcacctga gcgtcagtct 720
tcgtccaggg ggccgccttc gccaccggta ttcctccaga tctctacgca tttcaccgct 780
acacctggaa ttctaccccc ctctacgaga ctctagcttg ccagtttcaa atgcagttcc 840
caggttgagc ccggggattt cacatctgac ttaacaaacc gcctgcgtgc gctttacgcc 900
cagtaattcc gattaacgct tgcaccctcc gtattaccgc ggctgctggc acggagttag 960
ccggtgcttc ttctgcgagt aacgtcaatt gatgaacgta ttaagttcac caccttcctc 1020
ctcgctgaaa gtgctttaca acccgaaggc cttcttcaca cacgcggcat ggctgcatca 1080
ggcttgcgcc cattgtgcaa tattccccac tgctgcctcc cgtaggagtc tggaccgtgt 1140
ctcagttcca gtgtggctgg tcatcctctc agaccagcta gggatcgtcg cctaggtgag 1200
ccattacccc acctactagc taatcccatc tgggcacatc tgatggcaag aggcccgaag 1260
gtccccctct ttggtcttgc gacgttatgc ggtattagct accgtttcca gtagttatcc 1320
ccctccatca ggcagtttcc cagacattac tcacccgtcc gccgctcgtc acc 1373
Claims (5)
1. Serratia marcescens capable of degrading chlorobenzeneSerratia marcescens strain）TF-1，The method is characterized in that: is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019674.
2. Use of Serratia marcescens TF-1 according to claim 1 for degrading chlorinated aromatic hydrocarbons.
3. Use according to claim 2, characterized in that: the chlorinated aromatic hydrocarbon is chlorobenzene.
4. Use according to claim 2 or 3, characterized in that: the serratia marcescens TF-1 is applied to degrading chlorinated aromatic hydrocarbon by taking benzene, toluene, formaldehyde, phenol, methanol or ethanol as a substrate.
5. Use according to claim 2, characterized in that: the serratia marcescens TF-1 is applied to degradation of chlorobenzene by taking the chlorobenzene as a unique carbon source.
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