CN113913321A - Sodium benzoate degrading strain and application thereof - Google Patents
Sodium benzoate degrading strain and application thereof Download PDFInfo
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Abstract
The invention discloses a sodium benzoate degrading strain and application thereof, wherein the sodium benzoate degrading strain is named as (Desulfurobacterium ferrireducens) PLL0, is preserved in China general microbiological culture Collection center (CGMCC) of institute of microbiology, China academy of sciences, No. 3, West Lu No. 1, Beijing area, and is preserved in 13 months at 2021, and the preservation number is CGMCC No. 22520. The bacterium can degrade sodium benzoate by using ferric citrate as an electron acceptor in an anaerobic environment, and has wide application prospect in soil remediation. The sodium benzoate degrading strain PLL0 or the microbial agent containing the sodium benzoate degrading strain can be well applied to soil remediation, and the pollution of PAHs to soil is improved.
Description
Technical Field
The invention belongs to the technical field of microbial system classification and environmental biology, relates to a sodium benzoate degrading strain and application thereof, and particularly relates to a sodium benzoate degrading strain (desulfobacterium ferrarioides) PLL0 and application thereof.
Background
Polycyclic Aromatic Hydrocarbons (PAHs) are a general term for a class of persistent organic pollutants produced by incomplete combustion of organic matter at high temperatures. PAHs are produced in the processes of petroleum industrial production, petroleum commodity transportation, fossil fuel combustion, coal tar consumption and insufficient combustion of organic matters in automobile exhaust, and the domestic PAHs pollution is mainly mixed pollutants produced by fossil dye combustion and petroleum processing. In an anaerobic environment, degradation of polycyclic aromatic hydrocarbons is more difficult due to the limitation of microbial growth. Benzoic acid is an important intermediate for degrading polycyclic aromatic hydrocarbons, and the research on the degradation of benzoic acid has important significance on the research on the degradation of PAHs.
Benzoic acid, which is usually present as a benzoate, is an important model compound for studying the anaerobic degradation of PAHs. Benzoate is a white powder that is readily soluble in water, and water vapor has a particularly pungent odor. Excessive benzoic acid entering cells can affect the activity of the enzyme and the integrity of the cells, and the excessive benzoic acid entering the soil can inhibit the number of microorganisms to change the structure of a community. Benzoic acid can be subjected to open loop anaerobic degradation under the condition that nitrate, sulfate and Fe (III) are used as electron acceptors, microorganisms such as Azoarcus sp.CIB, Rhodopseudomonas palustris (Rhodopseudomonas palustris), Magnetospirillum Magnetoacticum MS-1 and Arch aeoglobus (Azoarcus evansii) which are separated can metabolize benzoic acid under anaerobic conditions, but the microorganism strains which can oxidize benzoic acid by using Fe (III) as the only electron acceptor are short at present.
Disclosure of Invention
The first purpose of the invention is to overcome the defects of the prior art and provide a strain for degrading sodium benzoate.
The invention also aims to provide application of the sodium benzoate degrading strain.
The purpose of the invention is realized by the following technical scheme:
a sodium benzoate degrading strain is named as (Desulbactrium ferrariucens) PLL0, is preserved in China general microbiological culture Collection center (CGMCC) of institute of microbiology, China institute of sciences, No. 3, West Lu 1 institute of microbiology, located in the sunward area of Beijing, 5.13 days in 2021, and has the preservation number of CGMCC No. 22520.
The invention obtains sodium benzoate degrading bacteria PLL0 by enriching and separating petroleum polluted soil collected from Qingcheng county of Qingyang city in Gansu China with benzoic acid-ferrihydrite culture medium.
The enrichment medium (benzoic acid-ferrihydrite medium) of the sodium benzoate degrading strain consists of the following components: each 1L of ultrapure water contains CaCl2·2H2O 0.04g、MgSO4·7H2O 0.1g、NaHCO3 1.8g、 Na2CO30.43g, 10mL of 100 XNB salt mixture, 10mL of 1 XNB mineral solution, 15mL of 1 XDL vitamin solution, 1mM Na2SeO41mL of solution, electron donor sodium benzoate and electron acceptor ferrihydrite; pH 6.8-7.2 (buffered with bicarbonate); wherein, in the benzoic acid-ferrihydrite culture medium, the concentration of sodium benzoate is 2mM, and the concentration of iron element in ferrihydrite is 50mM (concentration obtained by measuring iron element).
The solid medium was prepared by adding 1.5% (w/v) agar powder to the above benzoic acid-ferrihydrite medium.
Packaging benzoic acid-ferrihydrite culture medium into anaerobic tubes or bottles before use, and charging mixed gas (CO)2:N2Volume ratio 20:80) to remove dissolved oxygen. Sealing the mouth of the anaerobic tube with a rubber plug and an aluminum cap, and sterilizing at 121 deg.C for 20 min.
The seed culture medium (ethanol-ferric citrate culture medium) of the sodium benzoate degrading strain is prepared by the following method: weighing 13.7g of ferric citrate, pouring into 150mL of boiling ultrapure water, and stirring with a glass rod until the ferric citrate is completely dissolved to obtain a ferric citrate solution; adding 600mL of ultrapure water into the ferric citrate solution, cooling to room temperature, adjusting the pH to 6.0-6.5 by using a NaOH solution, and sequentially adding the following components: c6H12O63.6g, 1 XDL vitamin solution 10mL, 1 XDL mineral solution 10mL, NaHCO3 2.5g，KCl 0.6g，1mM Na2SeO41mL, and the volume was adjusted to 1L with ultrapure water.
The solid culture medium is prepared by adding 1.5% (w/v) agar powder into liquid culture medium.
Before use, the seed culture medium is subpackaged into anaerobic tubes or anaerobic bottles, and mixed gas (CO) is filled2:N2Volume ratio 20:80) to remove dissolved oxygen. Sealing the mouth of the anaerobic tube with a rubber plug and an aluminum cap, and sterilizing at 121 deg.C for 20 min. And when the temperature of the culture medium is reduced to room temperature, adding ethanol to ensure that the final ethanol concentration is 20mM, thus obtaining the ethanol-ferric citrate culture medium (seed culture medium).
The 100 × NB salt mixture consists of the following reagents: KH (Perkin Elmer)2PO4 42g/L、K2HPO4 22g/L、NH4Cl 20g/L, KCl 38g/L, NaCl 36g/L, and the balance of ultrapure water.
The DL vitamin solution consists of the following components: 0.002g/L of biotin, 0.005g/L, B-120.0001 g/L of pantothenic acid, 0.005g/L of p-aminobenzoic acid, 0.005g/L of lipoic acid, 0.005g/L of nicotinic acid, 0.005g/L of riboflavin, 0.01g/L of pyridoxine hydrochloride, 0.002g/L of folic acid, 0.005g/L of thiamine and the balance of ultrapure water.
The NB mineral solution consists of the following components: NTA free acid non-trisodium salt 2.14g/L, MnCl2·4H2O 0.1g/L、FeSO4·7H2O 0.3g/L、CoCl2·6H2O 0.17g/L、ZnSO4·7H2O 0.2g/L、CuCl2·2H2O 0.03g/L、AlK(SO4)2·12H2O 0.005g/L、H3BO3 0.005g/L、Na2MoO4·2H2O 0.09g/L、 NiSO4·6H2O 0.11g/L、Na2WO4·2H2O0.02 g/L, and the balance of ultrapure water.
The DL mineral solution consists of the following components: NTA free acid non-trisodium salt 1.5g/L, MgSO4 3.0g/L、 MnSO4·H2O 0.5g/L、NaCl 0.5g/L、FeSO4·7H2O 0.1g/L、CaCl2·2H2O 0.1g/L、 CoCl2·6H2O 0.1g/L、ZnCl2 0.13g/L、CuSO4·5H2O 0.01g/L、AlK(SO4)2·12H2O 0.01g/L、 H3BO3 0.01g/L、Na2MoO4·2H2O 0.025g/L、NiCl2·6H2O 0.024g/L、Na2WO4·2H2O0.025 g/L and the balance of ultrapure water.
The preparation method of the ferrihydrite comprises the following steps: taking 54g FeCl3·6H2Dissolving O in 300mL of ultrapure water, placing the solution on a magnetic stirrer, and stirring the solution until FeCl is formed3·6H2Dissolving O completely, dropwise adding 10M NaOH at the speed of 3s per drop under vigorous and uniform stirring until the pH is close to 2, changing to 5M NaOH until the pH is close to 5, then adding 1M NaOH until the pH is close to 7.0, stirring for 30min, checking whether the pH is stable, standing the suspension in a dark place after the pH is stable for aging for 2-4h, slightly reducing the pH at the moment, and adjusting the pH to 7.0 by using 1M NaOH again; washing the iron ore suspension with ultrapure water, centrifuging at 4 deg.C at 3000g for 20min, discarding supernatant, adding ultrapure water, stirring to resuspend iron gel, centrifuging again, discarding supernatant, and repeating the above washing steps for 8 times to remove Cl dissolved in the suspension-And (4) until the conductivity of the supernatant liquid tends to be stable, and after the supernatant liquid is poured for the last time, resuspending the ferrihydrite by using 200mL of ultrapure water to obtain the ferrihydrite.
The sodium benzoate degrading strain is strictly anaerobic gram-positive bacteria, the bacteria are in a micro-bent rod shape, and the micro-bent rod shape is free of flagella, pili and spores, and is about 0.5-0.7 microns wide and 1.8-3.5 microns long.
The single colony formed on the solid plate of the seed culture medium is milky, convex, round, regular in edge and smooth in surface, and the strain PLL0 has no catalase and oxidase activity. The growth temperature range of the strain PLL0 is 25-37 ℃, and the optimal growth temperature is 30 ℃. The strain can grow in the culture medium with NaCl concentration in the range of 0-3% (w/v), but can not grow in the culture medium with NaCl concentration of 2.5% (w/v) or more, and the optimal NaCl concentration for growth is 2% (w/v).
The microbial agent contains the sodium benzoate degrading strain.
The sodium benzoate degrading strain or the microbial agent containing the same can be coupled with the reduction oxidation Fe (II) of benzoate under the anaerobic condition, and can be used for environmental remediation.
The environmental remediation is preferably soil remediation.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention provides a sodium benzoate degrading strain PLL0, which can degrade sodium benzoate by using ferric citrate as an electron acceptor in an anaerobic environment and has wide application prospect in soil remediation.
(2) The sodium benzoate degrading strain PLL0 or the microbial agent containing the sodium benzoate degrading strain can be well applied to soil remediation, and the pollution of PAHs to soil is improved.
Drawings
Fig. 1 is a graph comparing the results of processing of ferrihydrite with the results of JADE6.0 in a PDF database.
FIG. 2 is a transmission electron micrograph of sodium benzoate degrading strain PLL 0.
FIG. 3 is a phylogenetic tree diagram of the 16sRNA gene of the sodium benzoate-degrading strain PLL 0.
FIG. 4 is a standard curve of benzoic acid.
FIG. 5 is a standard curve of Fe (II).
FIG. 6 is a graph showing the results of sodium benzoate degradation by the sodium benzoate degrading strain PLL0 using ferric citrate as the electron acceptor.
FIG. 7 is a diagram for predicting metabolic pathways for degrading benzoic acid by using a sodium benzoate degrading strain PLL0 under an anaerobic condition.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Preparation of culture Medium
The 100 × NB salt mixture consists of the following reagents: KH (Perkin Elmer)2PO4 42g/L、K2HPO4 22g/L、NH4Cl 20g/L, KCl 38g/L, NaCl 36g/L, and the balance of ultrapure water.
The DL vitamin solution consists of the following components: 0.002g/L of biotin, 0.005g/L, B-120.0001 g/L of pantothenic acid, 0.005g/L of p-aminobenzoic acid, 0.005g/L of lipoic acid, 0.005g/L of nicotinic acid, 0.005g/L of riboflavin, 0.01g/L of pyridoxine hydrochloride, 0.002g/L of folic acid, 0.005g/L of thiamine and the balance of ultrapure water.
The NB mineral solution consists of the following components: NTA free acid non-trisodium salt 2.14g/L, MnCl2·4H2O 0.1g/L、FeSO4·7H2O 0.3g/L、CoCl2·6H2O 0.17g/L、ZnSO4·7H2O 0.2g/L、CuCl2·2H2O 0.03g/L、AlK(SO4)2·12H2O 0.005g/L、H3BO3 0.005g/L、Na2MoO4·2H2O 0.09g/L、 NiSO4·6H2O 0.11g/L、Na2WO4·2H2O0.02 g/L, and the balance of ultrapure water.
The DL mineral solution consists of the following components: NTA free acid non-trisodium salt 1.5g/L, MgSO4 3.0g/L、 MnSO4·H2O 0.5g/L、NaCl 0.5g/L、FeSO4·7H2O 0.1g/L、CaCl2·2H2O 0.1g/L、 CoCl2·6H2O 0.1g/L、ZnCl2 0.13g/L、CuSO4·5H2O 0.01g/L、AlK(SO4)2·12H2O 0.01g/L、 H3BO3 0.01g/L、Na2MoO4·2H2O 0.025g/L、NiCl2·6H2O 0.024g/L、Na2WO4·2H2O0.025 g/L and the balance of ultrapure water.
Preparing ferrihydrite: taking 54g FeCl3·6H2Dissolving O in 300mL of ultrapure water, placing the solution on a magnetic stirrer, and stirring the solution until FeCl is formed3·6H2Dissolving O completely, dropwise adding 10M NaOH at the speed of 3s per drop under vigorous and uniform stirring until the pH is close to 2, changing to 5M NaOH until the pH is close to 5, then adding 1M NaOH until the pH is close to 7.0, stirring for 30min, checking whether the pH is stable, standing the suspension in a dark place after the pH is stable for aging for 2-4h, slightly reducing the pH at the moment, and adjusting the pH to 7.0 by using 1M NaOH again; washing the iron ore suspension with ultrapure water, centrifuging at 4 deg.C at 3000g for 20min, discarding supernatant, adding ultrapure water, and stirringResuspending the iron gel, centrifuging again to remove the supernatant, and repeating the above washing step 8 times to remove the dissolved Cl in the suspension-Until the conductivity of the supernatant liquid tends to be stable, after the supernatant liquid is poured for the last time, 200mL of ultrapure water is used for resuspending ferrihydrite, namely ferrihydrite is obtained, and the ferrihydrite is placed in a refrigerator at 4 ℃ and is stored in a dark place.
And (3) characterization of mineral morphology of ferrihydrite:
and (3) placing the prepared suspension on a magnetic stirrer, stirring for 15min until the suspension is uniformly distributed, placing 2mL of ferrihydrite into a 10mL centrifuge tube, sealing the opening of the centrifuge tube with a sealing film, placing the centrifuge tube in a refrigerator at minus 80 ℃ for quick freezing for 2h, and then placing the centrifuge tube in a vacuum freeze dryer (opening the hydrazine in advance to be below minus 40 ℃) for freeze drying for 12 h. And (3) detecting the ore shape of the prepared ferrihydrite by adopting an X-ray diffractometer, wherein the specific operation and parameters are as follows: the sample to be detected is spread on the round plastic slide, and the sample is placed in the detection groove of the instrument after being compacted. Setting XRD instrument parameters: the included value is 0.02 degrees, the tube voltage is 40kV, the scanning interval is 2 theta (10-80 degrees), t is 0.2s, the total scanning time is about 20min, the FOM value is lower than 10 and the lower the value is, the higher the reliability of the ferrihydrite contained in the mineral component is.
As shown in FIG. 1, it can be seen from FIG. 1 that the ferrihydrite crystal form is weaker, but the ferrihydrite crystal form is FeOOH (ferrihydrite chemical formula) with lower reliability, and the ferrihydrite prepared by the method can be used as a benzoic acid-ferrihydrite culture medium component.
(1) Enrichment medium (benzoic acid-ferrihydrite medium): each 1L of ultrapure water contains CaCl2·2H2O 0.04g、MgSO4·7H2O 0.1g、NaHCO3 1.8g、Na2CO30.43g, 10mL of 100 XNB salt mixture, 10mL of 1 XNB mineral solution, 15mL of 1 XDL vitamin solution, 1mM Na2SeO41mL of solution, electron donor sodium benzoate and electron acceptor ferrihydrite; pH 6.8-7.2 (buffered with bicarbonate); wherein, in the benzoic acid-ferrihydrite culture medium, the concentration of sodium benzoate is 2mM, and the concentration of iron element in ferrihydrite is 50mM (concentration obtained by measuring iron element).
The solid medium was prepared by adding 1.5(w/v) agar powder to the above benzoic acid-ferrihydrite medium.
Packaging benzoic acid-ferrihydrite culture medium into anaerobic tubes or bottles before use, and charging mixed gas (CO)2:N2Volume ratio 20:80) to remove dissolved oxygen. Sealing the mouth of the anaerobic tube with a rubber plug and an aluminum cap, and sterilizing at 121 deg.C for 20 min.
(3) Ethanol-ferric citrate medium (seed medium): weighing 13.7g of ferric citrate, pouring into 150mL of boiling ultrapure water, and stirring with a glass rod until the ferric citrate is completely dissolved to obtain a ferric citrate solution; adding 600mL of ultrapure water into the ferric citrate solution, cooling to room temperature, adjusting the pH to 6.0-6.5 by using a NaOH solution, and sequentially adding the following components: c6H12O63.6g, 1 XDL vitamin solution 10mL, 1 XDL mineral solution 10mL, NaHCO3 2.5g，KCl 0.6g，1mM Na2SeO41mL, and the volume was adjusted to 1L with ultrapure water.
The solid culture medium is prepared by adding 1.5% (w/v) agar powder into liquid culture medium.
Before use, the seed culture medium is subpackaged into anaerobic tubes or anaerobic bottles, and mixed gas (CO) is filled2:N2Volume ratio 20:80) to remove dissolved oxygen. Sealing the mouth of the anaerobic tube with a rubber plug and an aluminum cap, and sterilizing at 121 deg.C for 20 min. And when the temperature of the culture medium is reduced to room temperature, adding ethanol to ensure that the final ethanol concentration is 20mM, thus obtaining the ethanol-ferric citrate culture medium (seed culture medium).
(3) Benzoic acid-ferric citrate medium: the preparation method is basically consistent with the preparation method of the ethanol-ferric citrate culture medium, except that sodium benzoate is added before aeration sterilization so that the final concentration of benzoic acid in the culture medium is 2mM, and no additional ethanol is required.
Example 1: (Desulfurobacterium ferrarireducens) PLL0 isolation, screening and characterization
1. Screening of strains
Weighing 5g of soil sample polluted by petroleum collected from Qingcheng city county (elevation 1260m, east longitude 107.73 degrees and north latitude 36.14 degrees) in Gansu Qingyang city, China, fully grinding by using a mortar, adding 2g of ground soil into 50mL of fresh enrichment medium (namely benzoic acid)-ferrihydrite medium), incubated at 30 ℃ in an incubator protected from light, the change in ferrous iron is monitored, the color of ferrihydrite gradually darkens from deep red and finally turns black as the redox reaction proceeds, and fe (iii) is reduced to fe (ii). When the concentration of Fe (II) reaches about 20mM, extracting the enrichment solution with 10% (v/v) inoculation amount and transferring the enrichment solution into a fresh enrichment medium; the steps are repeated for four times to fully enrich the microbial flora of the ferric oxide coupling sodium benzoate oxidation and ferrihydrite reduction. The fifth enrichment broth was diluted with 0.9% physiological saline (10) in a gradient manner-1、10-2、10-3、10-4、10-5) 10 will be-3～10-5The diluted bacterial liquid is evenly coated on a solid culture medium, 3 bacterial liquids are respectively cultured in parallel at 30 ℃ in a dark place. Until a single colony with a diameter of about 1mm and a medium size grows on the plate, picking the dominant single colony by using an aseptic toothpick or an aseptic syringe needle into a PE tube containing 100 mu L of 0.9% (w/v) NaCl for heavy suspension, repeatedly blowing and beating the single colony to completely release the thalli, transferring the single colony into 10mL of fresh liquid enrichment medium at one time, and culturing the single colony at 30 ℃ in a dark place to obtain the sodium benzoate degradation strain PLL 0. The above separation and purification are all carried out in an anaerobic box.
And (3) carrying out anaerobic culture on the sodium benzoate degrading strain PLL0 for 14 days at the temperature of 30 ℃ in a dark place, wherein the bacterial colony of the sodium benzoate degrading strain PLL0 on the solid plate of the seed culture medium is milky, convex, round, regular in edge and smooth in surface. The gram staining result is positive, and the thalli are observed under an optical microscope to be in a long rod shape. Further observation of cell morphology by transmission electron microscopy revealed that the cells were in the form of microblogs, without flagella and pili, without spores, about 0.5-0.7 μm wide and 1.8-3.5 μm long (see FIG. 2).
Strain PLL0 has no catalase and oxidase activity. The growth temperature range of the strain PLL0 is 25-37 ℃, and the optimal growth temperature is 30 ℃. The strain can grow in the culture medium with NaCl concentration in the range of 0-3% (w/v), but can not grow in the culture medium with NaCl concentration of 2.5% (w/v) or more, and the optimal NaCl concentration for growth is 2% (w/v).
2. Phylogenetic analysis based on 16S rRNA genes
Preparation of PLL0 cells at late log phase: and (3) monitoring the reduction condition of the ferric citrate by using a phenazine method, and obtaining a growth curve of the ferric citrate. The thalli in the late log phase were collected for subsequent experiments. Inoculating the thallus PLL0 into a seed culture medium according to the inoculation amount of 10%, and culturing for seven days under the conditions of light-shielding and anaerobic conditions at the temperature of 30 ℃, thus obtaining the thallus PLL0 at the late logarithmic phase.
(1) PLL0 bacteria were collected at late logarithmic phase and washed once or twice with physiological saline to remove the influence of iron on DNA extraction. Genomic DNA extraction was then performed using the Tiangen bacterial genomic DNA extraction kit DP302-02 (purchased from Tiangen Biochemical technology, Beijing, Ltd.). A PCR product was obtained by running the PCR program using 27F and 1492R (synthesized in Beijing Huada Gene science, Ltd.) as universal primers for amplifying the 16S rRNA gene, and the length of the PCR product was estimated to be about 1500 bp.
(2) TA cloning sequencing of 16S rRNA gene
a. Preparing agarose gel (1%, w/v) containing GelRed dye;
b. adding the PCR product and DL 2000DNA marker into sample application hole of agarose gel;
c. connecting an electrophoresis apparatus, setting the voltage to be 150V, and operating for 25 min;
d. cutting and recycling the rubber: after the electrophoresis was completed, the DNA product was recovered using a Gel recovery kit (Takara MiniBEST Agarose Gel DNA Extraction kit);
e. constructing and transforming recombinant plasmids: using TA cloning kit (Takara pMD)TM19-T Vector Cloning Kit, available from TAKARA of Japan) was used to mix the DNA recovered product with pMDTMConnecting the 19-T plasmids to form recombinant plasmids, and transferring the recombinant plasmids into escherichia coli DH5 alpha competent cells by a heat shock method (42 ℃ water bath heat shock for 45s, and rapid transfer to ice and standing for 2 min);
f. screening and obtaining a positive clone containing a recombinant plasmid by using an LB solid plate (containing 50mg/L ampicillin and precoated with x-Gal and IPTG) according to a blue-white spot principle;
g. picking a single colony of the positive clone containing the recombinant plasmid to an LB liquid culture medium containing 50mg/L ampicillin, and culturing overnight; the positive clones were verified using the bacterial suspension as a template and universal primers M13F and M13R (synthesized in Beijing Huada Gene science, Ltd.). The positive clone culture medium was sent to Guangzhou branch of Beijing Liu He Hua Dageney science and technology Co.
(3) Phylogenetic analysis
Calculating sequence similarity of 16S rRNA gene of sodium benzoate degrading strain PLL0 with other strains by EzBiocloud platform, comparing 16S rRNA gene sequence of all effective strains of Desutobacterium with 16S rRNA gene sequence of PLL0 strain by ClustalW algorithm, constructing phylogenetic tree of strain PLL0 based on MEGA X maximum likelihood estimation method and adjacent method, and constructing phylogenetic tree of sodium benzoate degrading strain PLL0 by mesophilic helicobacter (Heliobacterium modectium) ATCC 51547TIs the outer group.
The length of the 16S rRNA gene sequence of the strain PLL0 is 1459bp, and the sequence is shown as SEQ ID NO. 1. The resulting sequences were aligned to the relevant 16S rRNA sequences on Genebank. It was found that the most similar to the 16S rRNA gene sequence of the sodium benzoate-degrading strain PLL0 was the aromatic desulfurization bacterium (D. aromatic carboxyrans) UKTLT(95.4%) similarity of the 16S rRNA gene sequence to all other known strains of the genus Desulfurobacterium was less than 94.0% (94.0-92.3%). Phylogenetic trees were constructed based on the 16S rRNA gene sequence, as shown in figure 3.
Sodium benzoate degrading strain PLL0 and aromatic desulfurization bacteria (D.aromatic cvorans) UKTLTA cluster was formed, indicating that the two bacteria had the highest homology. Strain PLL0 and strain UKTLTClusters formed are distant from other species of the genus desulfobacterium (desulfobacterium); for strain UKTLTThe sequence of the 16S rRNA gene is analyzed, and the similarity of the sequence with aromatic desulfurization bacteria (D.aromaticivorans) is the highest, but only 94.0 percent, and the similarity with other known strains of desulfurization bacteria (Desulibacter) is lower than 93.7 percent and is far lower than the threshold value for establishing new species (98.7 percent). The phylogenetic tree and 16S rRNA gene sequence similarity analysis result shows that the strain PLL0 and the strain UKTL have the possibility of establishing a new genus.
To further determine the taxonomic status of strain PLL0, we analyzed the A between the genome of strain PLL0 and its related speciesNI and dDDH, strain PLL0 and strain Desulfurobacterium (D.chlororespirans) Co23TThe dDDH value was 12.6% and the ANI value was 67.6%. The values of dDDH and ANI are well below the thresholds for establishing new species (dDDH)<70％，ANI<95%), indicating that the strain PLL0 may represent a new genus and species different from the genus Desulfurobacterium. Due to the current strain UKTLTThe present invention temporarily assigned strain PLL0 to the genus desulfobacterium (desulfobacterium) and was named (desulfobacterium ferriducens) PLL 0.
The sodium benzoate degrading strain is named as (Desulfurobacterium ferrireducens) PLL0, is preserved in China general microbiological culture Collection center (CGMCC) of China institute of microbiology, No. 3, West Lu No. 1 institute of microbiology, located in the sunward area of Beijing, 5.13 days in 2021, and has the preservation number of CGMCC No. 22520.
Example 2: application of sodium benzoate degrading strain in degrading sodium benzoate under anaerobic condition
Preparation of sodium benzoate degrading strain PLL0 seed liquid: and monitoring the reduction condition of ferric citrate by using a phenazine method to obtain a growth curve of PLL0 in a seed culture medium, wherein the sodium benzoate degrading strain PLL0 bacterial liquid in the logarithmic phase is the seed liquid. Inoculating strain PLL0 into seed culture medium according to the inoculum size of 10% (v/v), and culturing at 30 deg.C under anaerobic condition in dark place for 5 days to obtain seed solution.
Sodium benzoate is used as an electron donor, ferric citrate is used as an electron acceptor, and the reduction capability of anaerobic benzoic acid degradation coupled iron of the strain PLL0 is evaluated. Inoculating fresh logarithmic phase sodium benzoate degrading strain PLL0 seed liquid into benzoic acid-ferric citrate culture medium according to the inoculation amount of 10% (v/v), wherein three strains are arranged in parallel; placing in a constant temperature incubator at 30 ℃ for dark culture, sampling once every 7 days, and determining samples obtained at different times.
And detecting the degradation condition of sodium benzoate by using a High Performance Liquid Chromatograph (HPLC), and monitoring the reduction condition of ferric citrate by using a phenazine method.
HPLC method for detecting sodium benzoate: the concentration of benzoate was measured by an HPLC system equipped with a Wondasil C18 reverse phase column (250 mM. times.4.6 mM, pore diameter 5 μm; Shimadzu, Japan) with a UV detector wavelength of 271nm, a mobile phase of 50mM, pH 4.5 of sodium acetate: acetonitrile (volume ratio 80%: 20%), and a flow rate of 1.2 mL/min. The benzoic acid label is shown in FIG. 4.
The iron Fe (II) and Fe (III) concentrations were measured as follows:
(1) preparing a feloxazine solution:
1) dissolving 11.96g of HEPES (molecular weight 238.3) in 800mL of ultrapure water;
2) adding 1.0g of felazine, fully and uniformly mixing, and adjusting the pH to 7.0;
3) the volume is adjusted to 1L by ultrapure water to obtain the feloxazine solution, and the feloxazine solution is stored in a refrigerator at 4 ℃ in a dark place.
(2) Fe (ii) standard curve configuration:
1) ferrous ammonium sulfate was formulated to the following concentrations: 0.1 mM, 5mM, 10mM, 20mM, 40mM, 80 mM;
2) diluting each concentration by 50 times with 0.5M dilute hydrochloric acid, and digesting for 15min to obtain digestion solution;
3) sucking 0.05mL of digestion solution, adding into 2.45mL of phenazine solution, mixing uniformly, and developing;
4) measuring the light absorption value by using an enzyme-labeling instrument at the wavelength of 562 nm;
5) fe (ii) standard curve at 50-fold dilution, as shown in fig. 5;
(3) determination of Fe (II) in the sample:
1) quickly adding 0.1mL of sample into 4.9mL of 0.5M hydrochloric acid, uniformly mixing, and digesting for 15min to obtain a digestion solution;
2) 0.05mL of digestion solution and 2.45mL of phenanthroline solution are sucked and mixed uniformly, and color development is carried out;
3) measuring the light absorption value at the wavelength of 562 nm;
4) the fe (ii) standard curve (fig. 5) was substituted and the fe (ii) concentration in the sample was calculated.
(4) Determination of total iron:
1) adding 0.2mL of 6.25M hydroxylamine hydrochloride into a test tube containing 4.7mL of 0.5M hydrochloric acid, and uniformly mixing to obtain a mixed solution of hydrochloric acid and hydroxylamine hydrochloride;
2) quickly sucking 0.1mL of sample, adding the sample into the mixed solution of hydrochloric acid and hydroxylamine hydrochloride, uniformly mixing, and then placing the mixture in a dark place for digestion for 2 hours to obtain a digestion solution; the ferrihydrite needs to be digested for 12 hours;
3) 0.05mL of digestion solution and 2.45mL of phenanthroline solution are sucked and mixed uniformly, and color development is carried out;
4) if any precipitate exists, the mixed solution needs to be filtered;
5) measuring the absorbance at 562nm wavelength;
6) the calculation was carried out with the Fe (II) standard curve (FIG. 5).
As shown in FIG. 6, 1.85mM sodium benzoate was degraded to 0.5mM in 60 days, the degradation rate was 73%, the average daily oxidation rate of 45. mu.M sodium benzoate, and the Fe (III) reduction rate was 70%, and the average daily reduction rate was 0.66 mM.
The metabolic pathway for degrading benzoic acid under the anaerobic condition of the sodium benzoate-degrading strain PLL0 is predicted as shown in FIG. 7, and from FIG. 7, 8 enzymes exist in the sodium benzoate-degrading strain PLL0, and 14 genes are probably involved in the anaerobic degradation of benzoic acid.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> river-south university
<120> one strain for degrading sodium benzoate and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1459
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> 16S rRNA gene sequence of Strain PLL0
<400> 1
agtggcggac gggtgagtaa cgcgtgggta acctacccat aagaccggga caacccctgg 60
aaacgggggc taataccgga tgagcttagc ttgaggcatc tcaagctaag gaaaggcggc 120
ctctgaagat gctgccgttt atggatggac ccgcgtctga ttagctagtt ggtggggtag 180
aggcctacca aagcaacgat cagtagccgg cctgagaggg tgaacggcca cactgggact 240
gagacacggc ccagactcct acgggaggca gcagtgggga atcttccgca atggacgaaa 300
gtctgacgga gcaacgccgc gtgtatgacg aaggccttcg ggttgtaaag tactgtcttt 360
ggggacgaac ggtatctatg taaataatgc agatacatga cggtacccaa ggaggaagcc 420
ccggctaact acgtgccagc agccgcggta atacgtaggg ggcgagcgtt gtccggaatc 480
attgggcgta aagggcgcgt aggcgggtca ttaagtctgg tgtgaaaggc aggggctcaa 540
cccctggaag gcaccggaaa ctggtgatct tgaggacagg agaggaaagc ggaattccac 600
gtgtagcggt gaaatgcgta gatatgtgga ggaacaccag tggcgaaggc ggctttctgg 660
actgtaactg acgctgaggc gcgaaagcgt ggggagcaaa caggattaga taccctggta 720
gtccacgccg taaacgatga gtgctaggtg tagagggtat cgaccccttc tgtgccgcag 780
ttaacacaat aagcactccg cctggggagt acggccgcaa ggttgaaact caaaggaatt 840
gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgacgcaacg cgaagaacct 900
taccaaggct tgacatcctc agaaccctgt ggaaacacgg gggtgccctt cggggaactg 960
agagacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1020
caacgagcgc aacccctatt tttagttgct aacgggtaaa gctgagcact ctagagagac 1080
tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgtct 1140
tgggctacac acgtgctaca atggccggta cagacggaag cgaagccgtg aggcggagca 1200
aatccgagaa agccggtctc agttcggatt gttctctgca actcgagagc atgaagtcgg 1260
aatcgctagt aatcgcaggt cagcacactg cggtgaatac gttcccgggc cttgtacaca 1320
ccgcccgtca caccacgaaa gtctgcaaca cccgaagccg gtgagctaac catctggagg 1380
cagccgtcga aggtggggta gatgattggg gtgaagtcgt aacaaggtag ccgtatcgga 1440
aggtgcggct ggatcacct 1459
Claims (4)
1. A sodium benzoate degrading strain is characterized by being named as (Desulobacterium ferrireducens) PLL0, being preserved in China general microbiological culture Collection center (CGMCC) of China institute of microbiology, institute No. 3 of China academy of sciences, West Lu No. 1, North Chen, south China, in Beijing city, 13 days at 2021, and the preservation number is CGMCC No. 22520.
2. A microbial agent comprising the sodium benzoate-degrading strain of claim 1.
3. The application of the sodium benzoate degrading strain of claim 1 or the microbial agent containing the sodium benzoate degrading strain of claim 2, wherein the sodium benzoate degrading strain in the sodium benzoate degrading strain or the microbial agent containing the sodium benzoate degrading strain can be coupled with Fe (II) reduced and oxidized by benzoate under anaerobic conditions for environmental remediation.
4. Use according to claim 3, characterized in that the environmental remediation is soil remediation.
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