Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Inorganic salt medium (MSM): 1.0g/L NH 4 NO 3 ,20g/L NaCl,0.5g/L(NH 4 ) 2 SO 4 ,0.05g/L CaCl 2 ,0.5g/L KH 2 PO 4 And 1.5g/L K 2 HPO 4 ,pH=7.0±0.2。
LB culture medium: 10.0g/L peptone, 5.0g/L NaCl,10.0g/L yeast extract, pH =7.0 ± 0.2.
The solid culture medium is prepared by adding agar into corresponding culture medium according to the proportion of 15 g/L.
Heterotrophic Nitrification Medium (HNM) (per liter of distilled water): (NH) 4 ) 2 SO 4 0.66g, 4.72g of sodium succinate 2 PO 4 0.50g,Na 2 HPO 4 0.50g,MgSO 4 ·7H 2 0.20g of O, 30.00g of NaCl, 2.00mL of trace element solution and pH =7.5.
Nitrite Denitrification Medium (NDM) (per liter of distilled water): naNO 2 0.28g, sodium succinate 3.16g 4 ·7H 2 O 0.20g,CaCl 2 0.01g,EDTA 0.07g,KH 2 PO 4 0.50g,Na 2 HPO 4 0.50g,FeSO 4 0.01g, 2.00mL of trace element solution, and pH =7.5.
Denitrification Medium (DM) (per liter of distilled water): KNO 3 1.00g, sodium succinate 4.68g 4 ·7H 2 O 0.20g,CaCl 2 0.01g,EDTA 0.07g,KH 2 PO 4 0.50g,Na 2 HPO 4 0.50g,FeSO 4 0.01g, naCl 30.00g, trace element solution 2.00mL, and pH =7.5.
Trace element solution (per liter of distilled water): EDTA.2Na 57.10g, znSO 4 ·7H 2 O 3.90g,CaCl 2 ·2H 2 O 7.00g,MnCl 2 ·4H 2 O 1.00g,FeSO 4 ·7H 2 O 5.00g,(NH4) 6 Mo 7 O 24 ·4H 2 O 1.10g,CuSO 4 ·5H 2 O 1.60g,CoCl 2 ·6H 2 O1.60g, pH6.0, and sterilizing at 121 deg.C for 30 min.
NH 4 + -N、NO 3 - -N、NO 2 - The determination and analysis methods of the N-nitrogen element are all referred to national standards: NH (NH) 4 + The determination and analysis of-N are carried out according to water quality-determination of ammonia nitrogen-Nessler reagent spectrophotometry (GB HJ 535-2009); NO 3 - Determination and analysis of N according to "determination of Water quality-nitrate Nitrogen-ultraviolet Spectrophotometry" (GB HJ/T346-2007); NO 2 - The determination and analysis of-N are based on the "determination of Water quality-nitrite Nitrogen-spectrophotometry" (GB 7493-87).
The detection method of the total phosphorus adopts an ammonium molybdate method; the detection method of Chemical Oxygen Demand (COD) adopts a chromium method; the detection method of Total Nitrogen (TN) adopts a nano reagent method.
The steps for detecting the concentration of the phthalate ester substances in the following examples are as follows:
(1) And (3) extraction: adding n-hexane with equal volume to the sample, extracting for 10min by ultrasonic oscillation (40 KHz), standing for 1 hr, collecting the upper layer organic solvent, and filtering with 0.22 μm organic filter membrane.
(2) Detecting the concentration of the phthalate ester substances by using a gas chromatograph:
a. detection conditions of a gas chromatograph:
the column was a WondaCap 5 column (GL Sciences Inc, japan.25mm. Times.30 m. Times.0.25 μm);
the detector is an Electron Capture Detector (ECD);
the sample introduction amount is 1 mu L, and the flow dividing ratio is 49:1, split-flow sample injection;
the temperature of the sample introduction chamber is 300 ℃;
the column temperature is constant at 280 ℃;
the temperature of the detector is 300 ℃;
the carrier gas is high-purity nitrogen (99.999%);
the flow rate of the carrier gas is 2mL/min;
data were obtained and analyzed by the software Labsolutions (Version 5.90, SHIMADZU, japan).
b. The standard curve establishing method comprises the following steps:
and establishing a standard curve of the relation between the concentration of the phthalate ester substance and the detection peak area by adopting an external standard method. Accurately preparing phthalate ester substance standard substances with the concentrations of 20mg/L, 40mg/L, 60mg/L, 80mg/L and 100mg/L, wherein each concentration is 3 parts, detecting the standard substance with each concentration according to the detection conditions of the gas chromatograph, thereby establishing a standard curve between the concentration of the standard substance and the peak area, and the fitting result is shown in table 1.
TABLE 1 Standard Curve fitting results
(3) The degradation rate of the phthalate ester is calculated by the following formula:
example 1 isolation, identification and preservation of strains
1. Isolation of the Strain
The method comprises the following steps: collecting sludge from the mangrove in intertidal zone of the seaside park of Zhanjiang city, guangdong province, packaging with a brown sample bottle, marking, and storing in a refrigerator at 4 deg.C for later use.
Step two: enriching bacteria by a unique carbon source method, specifically, taking di (2-ethyl) hexyl phthalate (DEHP) with the final concentration of 100mg/L as a unique carbon source, weighing 5g of a sample from the stored sludge, adding the sample into 50mL of MSM culture medium containing 100mg/L DEHP, and culturing for 5 days in a shaking table at 30 ℃ and 180rpm to obtain an enriched culture solution 1.
Step three: taking the enrichment culture solution 1 of the second step according to the proportion of 10% (v/v), inoculating into 10mL MSM culture medium containing 200mg/L DEHP, and placing in a shaking table to culture for 5 days at 30 ℃ and 180rpm to obtain an enrichment culture solution 2.
Step four: taking the enrichment culture solution 2 of the third step according to the proportion of 10% (v/v), inoculating into 10mL MSM culture medium containing 500mg/L DEHP, and placing in a shaking table to culture for 5 days at 30 ℃ and 180rpm to obtain an enrichment culture solution 3.
Step five: taking 3 1mL of the enrichment culture solution obtained in the step four as a reserved bacterial solution to store the strain; the remaining 9mL were treated with n-hexane at 1: the extraction ratio of 1 (v/v), the DEHP concentration is detected by using a gas chromatograph, the DEHP degradation rate is calculated, and the degradation rate is more than 95 percent.
Step six: placing the reserved bacterial liquid corresponding to the enrichment culture solution in the fifth step into a centrifuge, centrifuging for 5min at 5000rpm, washing the cell sediment by Phosphate Buffered Saline (PBS), and repeating the centrifugation and washing for three times. The cells were resuspended in 1mL of MSM liquid medium, and then, the cell suspension was spread on solid MSM medium containing 500mg/L DEHP and 0.01g/L Tween 80, cultured at 30 ℃, and the colony status was checked every day.
Step seven: selecting single colonies with hydrolytic halos on a solid MSM culture medium and good growth on the solid MSM culture medium, respectively culturing in 10mL LB enrichment culture medium for 24 hours, respectively taking 1mL culture solution, centrifuging for 5min at the rotating speed of 5000rpm, collecting thalli, washing by using PBS buffer solution, repeating the centrifugation and washing for three times, inoculating the thalli into 10mL MSM culture medium containing 100mg/L DEHP, and performing shake culture at 30 ℃ and 180rpm for 5 days;
step eight: respectively reserving 1mL of bacterial liquid, and remaining 9mL of bacterial liquid, using normal hexane to perform reaction according to the weight ratio of 1: extraction was carried out at a ratio of 1 (v/v), and the degradation rate of DEHP was again measured by gas chromatography.
Step nine: and according to the detection result of the gas chromatography in the step eight, taking the reserved bacterial liquid of the sample with the DEHP degradation rate of more than 95%, placing the reserved bacterial liquid in a centrifuge, centrifuging for 5min at 5000rpm, washing the cell sediment by Phosphate Buffered Saline (PBS), and repeating the centrifugation and washing for three times. The cells were resuspended in 1mL of MSM liquid medium, and then, the cell suspension was spread on solid MSM medium containing 500mg/L DEHP and 0.01g/L Tween 80, cultured at 30 ℃ and checked for colony status every day.
Step ten: and (3) firstly repeating the steps seven, eight and nine to purify the strains twice, and then repeating the steps seven and eight to purify the strains once, wherein from the results obtained in the step eight, the DEHP degradation rates of one strain in the three purification plates are all 100%, the degradation capability is stable, the bacterial colony is single, and the strain is named as the strain RL-GZ01.
2. Morphological characteristics of Strain RL-GZ01
The morphological characteristics of the strain RL-GZ01 were observed under an electron microscope, and the results are shown in FIG. 1, and it can be seen that the strain has a rod-shaped body, no flagella and no spores.
The colony morphology of the strain RL-GZ01 is shown in FIG. 2, and the colony is orange and round, has smooth edges and has a protrusion on the surface.
3. Physiological and biochemical characteristics of strain RL-GZ01
RL-GZ01 is a gram-positive strain; the hydrogen sulfide reaction is black, and the reaction is positive; obvious blue appears in the denitrification color development plate, and the plate is positive; a great amount of bubbles are generated in the catalase test, and the catalase test also shows positive results; urease test also showed positive; the fermentation test of V-P, methyl red, indole, gelatin and glucose has no corresponding positive reaction phenomenon, and the result is negative. In an antibiotic resistance test, sterile circular filter paper sheets respectively dripped with Amp, kan and tetracycline are placed in the middle of an LB culture medium which is fully coated with a strain RL-GZ01 by a coating plate method, the flat plate is placed in a constant temperature incubator at 30 ℃ for culturing for several days, the flat plate is observed, and a circle of non-growing bacteria surround the circular filter paper sheets, which indicates that the strains have no resistance to Amp, kan and tetracycline. The physiological and biochemical identification of the strain RL-GZ01 is shown in Table 2.
TABLE 2 physiological and biochemical identification results of strain RL-GZ01
4. 16S rRNA gene identification of strain RL-GZ01
Inoculating the strain RL-GZ01 into an LB culture medium, culturing for 12h at 30 ℃ and 180rpm, taking 1mL of Bacterial liquid, centrifugally collecting thalli, extracting Bacterial genome DNA by using a Bacterial genome extraction Kit (TaKaRaMiniBEST Bacterial Genomic DNA extraction Kit Ver.3.0), detecting the obtained gene DNA by using 0.8% agarose gel electrophoresis, determining the concentration of the Genomic DNA by using a Micro spectrophotometer (Micro Drop, BIO-DL, china), and storing the obtained Genomic DNA at-20 ℃ for later use.
A pair of universal primers was designed for amplification of 16S rDNA sequences: (27F.
PCR amplification was carried out using Premix TaqTM DNA polymerase (Takara, japan) using the extracted genomic DNA of strain RL-GZ01 as a template, the PCR product was detected by 1% Agarose Gel electrophoresis, purified with a DNA Purification recovery Kit (TaKaRaMiniBEST Agarose Gel DNA extraction Kit Ver.4.0), ligated to a pMD19-T vector, transformed into E.coli DH 5. Alpha. Competent cells, spread on an LB solid medium plate containing 0.1% (v/v) ampicillin, cultured at 37 ℃ for 12 hours, white colonies were picked up into a liquid LB medium, cultured with shaking at 37 ℃ and 180rpm for 12 hours, extracted with a Plasmid extraction Kit (TaKaRaMiniBEST Plasmid Purification Kit Ver.4.0), and sent to HakkenProducer Biolabs for sequencing. The sequencing results were analyzed by Blast alignment on NCBI website (http:// www. NCBI. Nlm. Nih. Gov /), and phylogenetic trees were constructed using MEGA software (version: 7.0), and the results are shown in FIG. 3.
The strain RL-GZ01 is identified as Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) by integrating the thallus morphology, physiological and biochemical characteristics and 16S rRNA gene sequence, and is named as Rhodococcus pyridinivorans RL-GZ01 strain.
5. Strain preservation
Rhodococcus pyridinivorans RL-GZ01 strain is preserved in Guangdong province culture collection center in 2022 at 20.4 months, with the preservation number being GDMCC No:62401, the storage address is: building No. 59, building No. 5 of the prefecture midroad No. 100 yard in Guangzhou city.
EXAMPLE 2 degradation of phthalate esters by Strain RL-GZ01
1. Experimental methods
(1) Preparing a seed solution: selecting Rhodococcus pyridinivorans RL-GZ01 single colony from LB plate, and performing shaking culture in LB liquid culture medium at 30 ℃ until OD is reached 600 =1.5, obtain seed liquid.
(2) Degradation experiment of phthalate ester substances:
taking OD 600 200 mu L of Rhodococcus pyridinovorus RL-GZ01 strain seed liquid is centrifuged for 4min at 5000rpm, bacterial precipitates are collected, the bacterial precipitates are fully suspended by 200 mu L of PBS, the operations of centrifugation and suspension are repeated for 3 times, and the bacterial precipitates are sequentially added into 10mL of MSM culture medium which takes one of di (2-ethylhexyl) phthalate (DEHP), dicyclohexyl phthalate (DCHP), di-n-octyl phthalate (DNOP), butyl Benzyl Phthalate (BBP), di-n-butyl phthalate (DBP), diethyl phthalate (DEP) and dimethyl phthalate (DMP) as a unique carbon source, the final concentration of the phthalate substances is 100mg/L, and then the culture is carried out for 60h under the conditions of constant temperature shaking at 30 ℃.
A control group (MSM medium with PAEs as the sole carbon source) was set without addition of bacteria, and 3 replicates were set for all experiments.
And detecting the concentration of the PAEs every 12h, and drawing a PAEs concentration histogram.
2. Results of the experiment
The degradation of different PAEs by the Rhodococcus pyridinivorans RL-GZ01 strain is shown in FIG. 4A, the concentration of the PAEs in a control group is shown in FIG. 4B, and the Rhodococcus pyridinivorans RL-GZ01 strain can completely degrade 100mg/L of di-n-butyl phthalate (DBP), butyl Benzyl Phthalate (BBP), diethyl phthalate (DEP) and dimethyl phthalate (DMP) when cultured for 12 h; when cultured for 24 hours, the Rhodococcus pyridinivorans RL-GZ01 strain can thoroughly degrade 100mg/L of di (2-ethylhexyl) phthalate (DEHP), dicyclohexyl phthalate (DCHP) and di-n-octyl phthalate (DNOP).
The results show that Rhodococcus pyridinivorans RL-GZ01 strain has the ability to completely degrade di (2-ethylhexyl) phthalate, dicyclohexyl phthalate, di-n-octyl phthalate, butyl benzyl phthalate, di-n-butyl phthalate, diethyl phthalate and dimethyl phthalate.
EXAMPLE 3 Denitrification Performance of Strain RL-GZ01
1. Experimental method
(1) Preparing a seed solution: picking single colony from LB plate to LB liquid culture medium, shaking culture at 30 deg.C until OD 600 =1.5, obtain seed liquid.
(2) And (3) denitrification experiment:
taking OD 600 200 mu L of Rhodococcus pyridinivorans RL-GZ01 strain seed liquid of =1.5, centrifuging for 4min under the condition of 5000rpm, collecting thalli, fully suspending with 1mL of HNM culture medium, NDM culture medium and DM culture medium respectively, repeating the operations of centrifuging and suspending for 3 times, supplementing 200mL of corresponding culture medium respectively, and culturing for 72h in a constant temperature shaking table at 30 ℃ and in the dark.
Three control groups (HNM medium, NDM medium, DM medium, respectively) were set without addition of bacteria, and 3 replicates were set for all experiments.
NH in each culture medium was detected every 12h 4 + -N、NO 2 - -N、NO 3 - -concentration of N and plotting a nitrogen content histogram.
2. Results of the experiment
The clearance of Rhodococcus pyridinivorans RL-GZ01 on different nitrogen sources is shown in FIG. 5, and it can be seen that the Rhodococcus pyridinivorans RL-GZ01 strain on NH after being cultured for 72 hours 4 + -N、NO 2 - -N、NO 3 - The denitrification rates of-N reach 93.6%, 77.0% and 77.3% respectively, and the Rhodococcus pyridinivorans RL-GZ01 strain is treated with NH respectively 4 + -N、NO 2 - -N、NO 3 - the-N shows good denitrification capability in the culture medium with the only nitrogen source, which indicates that the Rhodococcus pyridinivorus RL-GZ01 strain has good nitrification and denitrification capability, can effectively remove nitrogen in different forms, and can be used for repairing the environment polluted by nitrogen.
EXAMPLE 4 simultaneous denitrification and DEHP degradation Performance of Strain RL-GZ01
1. Experimental methods
(1) Preparing a seed solution: picking single colony from LB plate to LB liquid culture medium, shaking culture at 30 deg.C until OD 600 =1.5, obtain seed liquid.
(2) DEHP degradation experiment and denitrification experiment:
taking OD 600 200 mu L of Rhodococcus pyridinivorans RL-GZ01 strain seed liquid with the concentration of 1.5 is centrifuged for 4min at 5000rpm to collect thalli, then 200 mu L of HNM culture medium, NDM culture medium and DM culture medium are respectively used for full suspension, the operations of centrifugation and suspension are repeated for 3 times, 10mL of corresponding culture medium containing 100mg/L DEHP is respectively supplemented, and the shake cultivation is carried out for 60 hours at 30 ℃, 180rpm and under the dark condition.
Three control groups (10 mL of HNM medium, NDM medium, DM medium containing 100mg/L DEHP, respectively) were set without addition of bacteria, and 3 replicates were set for all experiments.
The degradation of DEHP and NH in each medium were measured every 12 hours 4 + -N、NO 2 - -N、NO 3 - N concentration and plotting a nitrogen content histogram and a DEHP degradation graph.
2. Results of the experiment
Denitrification of different nitrogen sources by Rhodococcus pyridinivorans RL-GZ01 strain in medium with different nitrogen sources containing 100mg/L DEHP is shown in FIG. 6, and degradation of DEHP by Rhodococcus pyridinivorans RL-GZ01 strain in medium with different nitrogen sources containing 100mg/L DEHP is shown in FIG. 7.
As can be seen, rhodococcus pyridinivorans RL-GZ01 strain 72h was cultured in a medium supplemented with DEHP 3 - The denitrogenation rate of-N is more than 60%, NH 4 + -N、NO 2 - The denitrification rates of-N and 100mg/L DEHP respectively reach 95.1% and 100%, and the degradation rate of 100mg/L DEHP reaches 100%, so that the Rhodococcus pyridinivorans RL-GZ01 strain has the capacity of degrading DEHP and denitrifying simultaneously.
In addition, compared to the HNM medium or NDM medium of example 3 containing no DEHP, the medium was adjusted to NH 4 + -N、NO 2 - The denitrification rate of-N is improved because the addition of DEHP increases the carbon source in the culture medium, changes the carbon-nitrogen ratio in the original culture medium, and influences the denitrification condition of the strain.
Example 5 Strain RL-GZ01 against different concentrations of NH 4 + Scavenging Properties of-N and DEHP
1. Experimental methods
(1) Preparing a seed solution: picking single colony from LB plate to LB liquid culture medium, shaking culture at 30 deg.C until OD 600 And =1.5, obtaining a seed liquid.
(2) DEHP degradation experiment and denitrogenation experiment:
preparation of different NH 4 + HNM medium at N and DEHP concentrations: first of all, NH is prepared 4 + HNM medium with final N concentrations of 20mg/L, 60mg/L, 200mg/L and 500mg/L, 3 parallel samples are arranged at each concentration, DEHP is added into each parallel sample respectively, so that the final DEHP concentrations are 5mg/L, 20mg/L and 50mg/L, and 12 groups of medium are finally obtained.
Taking OD 600 200 μ L of Rhodococcus pyridinivorans RL-GZ01 strain seed liquid, centrifuging at 5000rpm for 4min to collect bacterial precipitate, suspending with 200 μ L of the above 12 groups of HNM medium, repeating centrifuging and suspending for 3 times, and repeatingThe cells were each supplemented with 10mL of the corresponding medium and shake-cultured at 30 ℃ and 180rpm in the dark for 60 hours. The specific test design is shown in table 3:
TABLE 3
The control group without added bacteria of the above 12 tests was set up, and 3 replicates were set up for all tests.
The DEHP degradation and NH were measured every 12 hours 4 + N concentration, plotting DEHP degradation curves and histograms of nitrogen content.
2. Results of the experiment
Rhodococcus pyridinovorus RL-GZ01 strain NH at 20mg/L, 60mg/L and 200mg/L 4 + The degradation curves of DEHP at different concentrations (5 mg/L, 20mg/L, 50 mg/L) at N concentration are shown in FIG. 8, in which only 3 curves of the experimental group, i.e. different NH values, are visible 4 + At the N concentration, the DEHP degradation curves at the same concentration are overlapped, which shows that when NH is generated 4 + When the concentration of N is 20mg/L, 60mg/L and 200mg/L, the degradation capability of the strain RL-GZ01 on DEHP is not influenced by NH 4 + Influence of the N concentration, the concentration of DEHP all dropping to 0 after 24h of incubation; in addition, only three curves of the control group are shown in the figure, because the detection finds that the concentrations of DEHP in the experiment process are quite close to each other for the control group with the same initial concentration of DEHP, so that when data are processed, the DEHP degradation results of the control group with the same initial concentration of DEHP are plotted into one curve by means of averaging, and the data are conveniently read from the figure.
500mg/L NH of Rhodococcus pyridinivorans RL-GZ01 strain 4 + The degradation curves for DEHP at different concentrations (5 mg/L, 20mg/L, 50 mg/L) at N concentration are shown in FIG. 9, and it can be seen that the concentration of DEHP at 5mg/L, 20mg/L drops to 0 after 24h of incubation; after 36h of incubation, the DEHP concentration, at a concentration of 50mg/L, was reduced to 0. Indicating that the Rhodococcus pyridinivorans RL-GZ01 strain has NH of 500mg/L 4 + The complete degradability towards DEHP is maintained at N concentration.
The results in FIGS. 8 and 9 show that the NH concentration is different 4 + The Rhodococcus pyridinivorus RL-GZ01 strain has complete degradation capability on DEHP with the concentration of 5mg/L, 20mg/L and 50mg/L under the condition of N concentration, but NH 4 + The concentration of-N influences the rate of DEHP degradation, particularly at NH 4 + The concentration of-N reaches 500mg/L and above, with NH 4 + The degradation rate of DEHP also slows down with increasing N concentration.
Rhodococcus pyridinovorus RL-GZ01 strain under the condition of different concentrations (5 mg/L, 20mg/L and 50 mg/L) of DEHP to NH with the concentration of 20mg/L 4 + The denitrification of-N is shown in FIG. 10, which shows that NH concentration of 20mg/L is present in the presence of DEHP 4 + The concentration of N reaches a stable value after 24 hours, and approaches 0; indicating NH at 20mg/L 4 + N Environment, addition of DEHP at different concentrations degrades NH against Rhodococcus pyridinivorans RL-GZ01 strain 4 + The performance of-N has no significant effect.
Rhodococcus pyridinovorus RL-GZ01 strain under the condition of different concentrations (5 mg/L, 20mg/L and 50 mg/L) of DEHP to NH with the concentration of 60mg/L 4 + The denitrification of-N is shown in FIG. 11, which shows that the concentration of NH is 60mg/L under the condition that the DEHP concentration is 5mg/L and 20mg/L 4 + -the concentration after N24 hours is substantially close to 0; at a DEHP concentration of 50mg/L, a concentration of 60mg/L NH 4 + The concentration after 24 hours of-N is about 15mg/L, and after 36 hours, the concentration is close to 0.
Rhodococcus pyridinivorans RL-GZ01 strain under the condition of different concentrations (5 mg/L, 20mg/L and 50 mg/L) of DEHP to NH with the concentration of 200mg/L 4 + The denitrification of-N is shown in FIG. 12, which shows that after 72 hours of incubation under conditions of different concentrations of DEHP, the concentration of NH was 200mg/L 4 + The concentration of-N is reduced to about 20 mg/L.
Rhodococcus pyridinivorans RL-GZ01 strain under the condition of different concentrations (5 mg/L, 20mg/L and 50 mg/L) of DEHP to NH with the concentration of 500mg/L 4 + The denitrification of-N is shown in FIG. 13, which shows that the DEHP was present at different concentrationsThen, after 72 hours of culture, NH was added at a concentration of 500mg/L 4 + The concentration of N is reduced to about 65 mg/L.
The results in FIGS. 10, 11, 12, and 13 show that Rhodococcus pyridinivorans RL-GZ01 strain at concentrations of 20mg/L, 60mg/L, 200mg/L, and 500mg/L NH under different concentrations of (5 mg/L, 20mg/L, and 50 mg/L) DEHP 4 + N has excellent scavenging ability, but the concentration of DEHP affects the denitrification efficiency, particularly in NH 4 + At lower N concentrations (20 mg/L), the addition of DEHP at different concentrations had little effect on denitrification; when NH is present 4 + When the concentration of N is higher (60 mg/L), the denitrification rate is reduced along with the DEHP concentration reaching 50 mg/L; but when NH 4 + When the concentration of-N is very high (200 mg/L and 500 mg/L), the denitrification condition can not obviously change due to the change of DEHP concentration. This is because the change in the carbon-nitrogen ratio affects the denitrification efficiency.
Example 6 Performance of the strain RL-GZ01 in urban wastewater for degrading PAEs, removing nitrogen and removing phosphorus simultaneously
1. Experimental method
(1) Preparing a seed solution: picking single colony from LB plate to LB liquid culture medium, shaking culture at 30 deg.C until OD 600 =1.5, obtain seed liquid.
(2) Co-degradation experiments:
the urban wastewater is sampled from a Zhanjiang Xixia mountain wastewater factory, the initial pH is 7.13, the total phosphorus concentration is 10.89mg/L, the total nitrogen concentration is 74.51mg/L, the ammonia nitrogen concentration is 70.91mg/L, the DEHP concentration is 0.13mg/L, and the COD is 622.4mg/L. Before the experiment, the concentration of DEHP in the urban wastewater is adjusted to 5mg/L in a mode of manually adding DEHP, and the rest is unchanged to obtain experimental urban wastewater, and the experimental urban wastewater is placed in a refrigerator at 4 ℃ for later use.
Taking OD 600 200 mu L of Rhodococcus pyridinivorans RL-GZ01 strain seed liquid of 1.5, centrifuging for 4min at 5000rpm to collect thalli, fully suspending 200 mu L of the experimental urban wastewater, repeating the operations of centrifuging and suspending for 3 times, adding 10mL of experimental urban wastewater, and performing shake culture at 30 ℃, 180rpm and in the dark for 84 hours. And setting a control group (experimental urban wastewater) without bacteria.
The DEHP concentration is detected every 6h, and the COD, TP and NH are detected every 12h 4 + -N、NO 3 - -N、NO 2 - N, TN, DEHP concentrations, each detection was performed in 3 replicates.
2. Results of the experiment
The change of COD value of the urban wastewater is shown in FIG. 14, and it can be seen that the COD value of the urban wastewater is reduced from 622.4mg/L to 101.0mg/L by Rhodococcus pyridinivorans RL-GZ01 strain after being cultured for 84 hours, and the COD value of the control group has no obvious change within 84 hours.
The degradation condition of the Rhodococcus pyridinivorans RL-GZ01 strain on TP of the experimental urban wastewater is shown in FIG. 15, and it can be seen that the Rhodococcus pyridinivorans RL-GZ01 strain can reduce the total phosphorus concentration of the experimental urban wastewater from 10.89mg/L to 2.37mg/L after being cultured for 84 hours, and the phosphorus removal rate reaches 78.23%.
Rhodococcus pyridinivorans RL-GZ01 strain for testing TN and NH of urban wastewater 4 + -N、NO 3 - -N、NO 2 - The degradation condition of the-N is shown in figure 16, and therefore, after the Rhodococcus pyridinivorans RL-GZ01 strain is cultured for 84 hours, the ammonia nitrogen concentration of the experimental urban wastewater can be reduced from 70.91mg/L to 2.2mg/L, and the removal rate reaches 96.9%; after being cultured for 84 hours, the Rhodococcus pyridinivorans RL-GZ01 strain can reduce the total nitrogen concentration of the experimental urban wastewater from 74.51mg/L to 3.4mg/L, and the removal rate reaches 95.4%. Meanwhile, the change condition of nitrate nitrogen and nitrite nitrogen in the experimental urban wastewater is the same as that of a control group, and no obvious accumulation occurs. The Rhodococcus pyridinivorans RL-GZ01 strain is shown to have the capability of synchronous nitrification and denitrification.
The degradation condition of the Rhodococcus pyridinivorans RL-GZ01 strain on DEHP in the experimental urban wastewater is shown in FIG. 17, and therefore the Rhodococcus pyridinivorans RL-GZ01 strain can completely degrade 5mg/L DEHP in the experimental urban wastewater.
The results show that the Rhodococcus pyridinivorans RL-GZ01 strain has good adaptability in urban wastewater, has the capabilities of degrading PAEs, removing nitrogen and removing phosphorus simultaneously, and can effectively repair the complex environment polluted by phthalate substances and/or nitrogen and phosphorus.
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.