CN117187119A - Paracoccus for degrading diethylamine and triethylamine and application of Paracoccus in wastewater treatment - Google Patents
Paracoccus for degrading diethylamine and triethylamine and application of Paracoccus in wastewater treatment Download PDFInfo
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- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 title claims abstract description 240
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- 230000000593 degrading effect Effects 0.000 title claims abstract description 20
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- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 claims abstract description 42
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- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The application relates to the technical field of biological treatment of pollutants, and provides a paracoccus capable of degrading diethylamine and triethylamine simultaneously and application thereof. The application uses strain Paracoccus pantotrophus NJUST38 obtained by screening activated sludge for degrading heterocyclic compounds to carry out streak culture on culture mediums which respectively take diethylamine or triethylamine as unique carbon sources, and discovers that the strain can respectively utilize diethylamine and triethylamine as unique carbon sources for growth, and the preservation number of the strain is CCTCC NO: m2020682. The paracoccus provided by the application has high-efficiency degrading capability of ethylamine organic matters, can respectively utilize diethylamine or triethylamine as a unique carbon source and a nitrogen source for growth, synchronously realizes mineralization and degradation of the diethylamine and the triethylamine, and is suitable for removing high-concentration diethylamine and triethylamine and nondegradable organic amines in wastewater.
Description
Technical Field
The application belongs to the technical field of biological treatment of organic pollutants, and particularly relates to a paracoccus strain for degrading ethylamine organic matters and application thereof in the field of wastewater treatment.
Background
Diethylamine (DEA) and Triethylamine (TEA) belong to organic amines, and are widely used in the fields of agriculture, pharmacy, chemical industry and the like. In the organic synthesis industry, organic amine is often used for preparing polymerization inhibitors of tetrafluoroethylene, rubber vulcanization accelerators and the like, and is used as a raw material in the medicine field to participate in the synthesis of medicines such as cephalosporin, penicillin and the like, and is used as a raw material in the agriculture field to produce agricultural glyphosate and the like. Due to the large amount of organic amines, thousands of tons of diethylamine and triethylamine are directly discharged into the environment with industrial waste water and gas each year. The ethylamine is a nitrogenous organic matter, has high polarity, is easy to dissolve in water, and is easy to enter a natural water area system along with the discharge of wastewater. And the diethylamine and the triethylamine have strong ammonia odor and high corrosiveness, and the triethylamine also has the characteristics of biotoxicity, embryotoxicity, teratogenesis and the like, so the treatment of ethylamine wastewater such as the diethylamine, the triethylamine and the like becomes a difficult problem to be solved urgently.
The existing treatment method for ethylamine pollutants is mainly a physical and chemical method, and comprises a high-grade oxidation method, an incineration method, an ion exchange resin method, a distillation method and the like. The Fenton oxidation method has good degradation effect on laboratory-scale organic amine wastewater, but is limited to the fact that the industrial application cannot be realized due to the excessively high cost; the burning method burns the waste gas of diethylamine and triethylamine in the air by means of the combustion improver, which is easy to cause secondary pollution; the ion exchange resin method is often limited in practical application due to the service life of the adsorption column and excessive maintenance cost. The biological treatment method has the advantages of economy, high efficiency, small secondary pollution and the like, can realize harmless treatment, and is a proper wastewater treatment technology for treating the ethylamine wastewater.
However, due to the difficult degradation characteristics and biotoxicity of high-concentration diethylamine and triethylamine waste water, the premise of the biological treatment of ethylamine waste water is that strains with degradation functions and resistant to the biotoxicity of ethylamine waste water can be obtained. Strains with triethylamine degradation function which have been reported at present include bacillus (CN 201410018679.7), arthrobacter (CN 201010199254.2), pseudomonas (CN 201911040794.3), soxhlet bacterium (CN 201310594079.0) and the like, and pseudomonas (CN 201310083191.8) with diethylamine degradation function and the like. However, considering the complex water quality of actual ethylamine wastewater, diethylamine and triethylamine often exist in the ethylamine wastewater at the same time, the existing strain only has single treatment capacity, and the degradation effect is different, as in the Pseudomonas disclosed in patent CN201310200773.X, 32h is required for degrading 100mg/L of triethylamine, and the actual ethylamine wastewater is difficult to be qualified for high-efficiency biological treatment.
Disclosure of Invention
The application aims to provide a paracoccus capable of degrading diethylamine and triethylamine simultaneously and application thereof. The bacteria can grow by using diethylamine or triethylamine as the only carbon source and nitrogen source, and simultaneously are functional bacteria capable of efficiently degrading the diethylamine and the triethylamine.
The application provides a paracoccus strain Paracoccus pantotrophus NJUST. The strain is preserved in China Center for Type Culture Collection (CCTCC) on 11 months and 6 days in 2020, and has a preservation address of university of Wuhan in Hubei province, china, and a preservation number of CCTCC NO: m2020682.
The application also provides a microbial inoculum, which contains the NJUST38 strain.
The application also provides a Paracoccus seed solution, which is prepared by activating and culturing the NJUST38 strain.
The application also provides a culture method of the NJUST38 strain, which comprises the following steps: the NJUST38 strain is inoculated in a culture medium, the pH value of the culture medium is 7.0-8.0, and the culture temperature is 30-35 ℃.
Optionally, the culture medium is LB culture medium containing diethylamine and triethylamine.
The application also provides application of the NJUST38 strain in degrading ethylamine organic matters, wherein the ethylamine organic matters comprise diethylamine and/or triethylamine.
The application also provides application of the NJUST38 strain in treatment of waste water containing diethylamine and triethylamine.
The application also provides a method for treating the ethylamine-containing organic matter wastewater, which comprises the steps of inoculating the NJUST38 strain into the wastewater to be treated; the wastewater contains diethylamine and/or triethylamine.
Optionally, in the method for treating wastewater, during inoculation, the NJUST38 strain is prepared into seed solution, and inoculated into the wastewater, and the seed solution OD 600 1.5-2.0, and the inoculation amount is 5-10% (v/v).
Optionally, the initial concentration of diethylamine or triethylamine in the wastewater is 500mg/L respectively; that is, when diethylamine is contained in the wastewater, the initial concentration of diethylamine is 500mg/L; when the wastewater contains triethylamine, the initial concentration of the triethylamine is 500mg/L.
Optionally, the pH of the wastewater is 6-7.
The Paracoccus pantotrophus NJUST provided by the application can be used for metabolism and growth by taking the diethylamine and the triethylamine as carbon sources, and simultaneously can be used for effectively degrading the diethylamine and the triethylamine. Through experiments, paracoccus pantotrophus NJUST38 is added into the simulated wastewater containing the diethylamine and the triethylamine for treatment, so that the diethylamine and the triethylamine can be synchronously and efficiently removed. The NJUST38 strain has strong adaptability to living environment and high-concentration ethylamine pollutants, and can be used for efficiently removing the ethylamine pollutants in the treatment of the ethylamine industrial wastewater, so that the economic cost is saved.
Drawings
FIG. 1 is an external view of paracoccus Paracoccus pantotrophus NJUST38 according to the present application, wherein a is a colony pattern grown on a solid medium plate and b is a scanning electron microscope image of the strain.
FIG. 2 is a graph showing the degradation effect of Paracoccus pantotrophus NJUST on diethylamine in a simulated wastewater having initial concentrations of 500mg/L for both diethylamine and triethylamine in an embodiment of the present application.
FIG. 3 is a graph showing the effect of Paracoccus pantotrophus NJUST on triethylamine degradation in simulated wastewater having initial concentrations of 500mg/L for both diethylamine and triethylamine in an embodiment of the application.
FIG. 4 is a graph showing the effect of pH of the feed water on the degradation of diethylamine when Paracoccus pantotrophus NJUST is used to degrade diethylamine and triethylamine in an embodiment of the present application.
FIG. 5 is a graph showing the effect of pH of the feed water on the degradation of triethylamine as Paracoccus pantotrophus NJUST degrades diethylamine and triethylamine in an embodiment of the present application.
Detailed Description
The technical scheme of the application is described in detail below with reference to the accompanying drawings and the specific embodiments.
The inventor screens out strain Paracoccus pantotrophus NJUST38 from activated sludge for degrading heterocyclic compounds, and performs streak culture on a culture medium which respectively takes diethylamine or triethylamine as a unique carbon source, and finds that Paracoccus pantotrophus NJUST can respectively utilize diethylamine and triethylamine as the unique carbon source for growth, and that strain Paracoccus pantotrophus NJUST38 is preserved in China Center for Type Culture Collection (CCTCC) on the 11 th month 6 of 2020, with a preservation address of university of Wuhan in China, post code 430072 and a preservation number of CCTCC NO: m2020682.
Paracoccus pantotrophus NJUST38 of the application is a first reported paracoccus capable of degrading diethylamine and triethylamine simultaneously. Compared with the reported organic amine degrading bacteria with single degradation effect, paracoccus pantotrophus NJUST38 has stronger adaptability to living environment and tolerance to high-concentration ethylamine pollutants, can simultaneously utilize diethylamine and triethylamine as carbon sources for metabolism and growth, and can effectively degrade the diethylamine and the triethylamine.
In a specific embodiment, the NJUST38 strain can be prepared to form a microbial inoculum; the microbial inoculum can be used for degrading diethylamine and triethylamine.
In one embodiment, the NJUST38 strain can be subjected to activation culture to obtain paracoccus seed solution.
The application also provides a culture method of the NJUST38 strain, which comprises the steps of inoculating the NJUST38 strain into a culture medium, wherein the pH of the culture medium is 7.0-8.0, and the culture temperature is 30-35 ℃. Preferably, the medium may be an LB medium containing diethylamine and triethylamine.
The application also provides application of the NJUST38 strain in degrading ethylamine organic matters, namely application in degrading diethylamine and/or triethylamine. The applications include degradation of diethylamine and triethylamine in different environmental media. Such as diethylamine and triethylamine in the treatment of wastewater, in waste gas leaches, in landfill leachate, in soil leachate and so on in different media.
The application also provides application of the NJUST38 strain in treating ethylamine-containing organic matter wastewater. Namely, a method for treating waste water containing diethylamine and/or triethylamine is provided, namely, the strain NJUST38 is inoculated into waste water to be treated for biochemical treatment. The biochemical treatment comprises biological treatment processes of waste water such as AO, A2O, SBR, MBR and the like based on an activated sludge process.
Specifically, when the strain is applied to wastewater treatment, in a specific implementation mode, the NJUST38 strain is subjected to activation culture to prepare seed liquid, and the seed liquid is inoculated into wastewater; the seed liquid OD 600 1.5-2.0, and the inoculation amount is 5-10% (v/v).
Preferably, the initial concentration of diethylamine or triethylamine in the wastewater is 500mg/L. Here, the concentration of diethylamine or triethylamine is 500mg/L, respectively. In practice, the initial concentration of diethylamine or triethylamine in the wastewater may also be below 500mg/L, or slightly above 500mg/L, for example 300-600 mg/L, or the total concentration of both may be about 1 g/L.
Preferably, the wastewater pH may be 6 to 7. The pH may also be from 6.5 to 7.5, more preferably the pH is 7.0. The temperature of the wastewater is normal temperature.
The medium formulation used in the examples below is as follows.
The composition of the LB medium was as follows: 10g L -1 Tryptone, 5g L -1 Yeast extract, 10g L -1 Sodium chloride.
The composition of the inorganic salt medium is as follows: 1.53g L -1 NaHPO 4 ·12H 2 O，0.38g L -1 KH 2 PO 4 ，0.1gL - 1 MgSO 4 ·7H 2 O，0.05g L -1 CaCl 2 ，10mL·L -1 The amounts of the microelement solution SL-4, diethylamine and triethylamine are added according to the experimental requirements.
Microelement SL-4: 0.5. 0.5g L -1 EDTA，0.2g L -1 FeSO 4 ·7H 2 O, trace element SL-6 is 100mL L -1 。
Microelement SL-6: 0.01g L -1 ZnSO 4 ·7H 2 O，0.03g L -1 MnCl 2 ·4H 2 O，0.3g L -1 H 3 BO 4 ，0.2g L -1 CoCl 2 ·6H 2 O，0.01g L -1 CuCl 2 ·2H 2 O，0.02g L -1 NiCl 2 ·6H 2 O，0.03g L -1 Na 2 MoO 4 ·2H 2 O。
Adding 20g L on the basis of inorganic salt culture medium -1 After sterilizing for 20 minutes at 121 ℃ in a high-pressure steam sterilizing pot, pouring the agar into a sterile culture dish, and cooling to room temperature to obtain an inorganic salt agar solid culture medium plate.
Example 1
Screening, separating and identifying Paracoccus pantotrophus NJUST.
(1) Screening and isolation of strains
Collecting the mixture for degrading the heterocyclic compound (containing nitrogen methyl pyrrolidone)After sludge acclimation for 30 days by using wastewater containing heterocyclic compounds, 5g of the activated sludge is sampled, and the activated sludge is added into 100mL of sterile physiological saline (0.85% sodium chloride solution) and is stirred uniformly and then stands for two hours. Adding 1mL supernatant into inorganic salt liquid culture medium sterilized at 121deg.C for 20min, concentrating and culturing for three days in 180 rpm shaking table, concentrating for three times, and gradient diluting the culture solution with sterile physiological saline (0.85% sodium chloride solution) to 10 -4 -10 -10 Multiple times. Preparing inorganic salt agar solid culture medium plates, taking 20 mu L of diluted culture solution, respectively coating the diluted culture solution on the inorganic salt agar solid culture medium plates, and placing the plates in a biochemical incubator for culturing for three days at the constant temperature of 30-35 ℃. Single bacterial colony with obvious difference on the culture dish is selected, purified culture is carried out by adopting a plate streak separation method, and after five times of continuous purification, single bacterial strain is obtained and inclined surface preservation is carried out.
Preparing 100mL of inorganic salt liquid culture medium containing heterocyclic compounds, filling the inorganic salt liquid culture medium into a 120mL serum bottle, aerating with pure helium gas to remove dissolved oxygen, sterilizing at the high temperature of 121 ℃ for 20 minutes, inoculating the pure strain obtained by separation and purification, culturing in a constant-temperature shaking incubator at 180 rpm and at the temperature of 30-35 ℃, and monitoring the concentration change of the heterocyclic compounds. Selecting strain capable of effectively removing heterocyclic compound in culture medium, named NJUST38, and performing inclined plane preservation and low temperature preservation at-80deg.C.
(2) Identification of strains
Carrying out morphological, physiological and biochemical tests and molecular biological identification on the strain NJUST38, carrying out gene sequencing through 16S rRNA, submitting a nucleotide sequence obtained by sequencing to a GenBank database, and carrying out BLAST comparison and analysis to determine the genus of the strain.
(1) Morphological features: colonies of NJUST38 were round, yellowish in color, moist on surface, opaque, and clean-edged (FIG. 1 a). According to the scanning electron microscope image (figure 1 b) of the strain, the cells of the strain NJUST38 are observed to be spherical, substances such as polysaccharide, protein and the like are easy to secrete on the surfaces of the cells in the growth process, and the cell diameter range is 0.48-0.69 mu m.
(2) Physiological and biochemical characteristics: the strain NJUST38 is a gram-negative bacterium, and both the oxidase reaction and the nitrate reduction reaction are positive.
(3) Molecular biology identification: the nuclear gene of the NJUST38 strain is used as a template, the nuclear gene is amplified by using a Polymerase Chain Reaction (PCR) technology, the obtained nucleotide sequence is delivered to a GenBank database for BLAST similarity and homology comparison, and clustering analysis is carried out by adopting software MEGA5.1 comparison results, so that the result shows that the strain NJUST38 has sequence homology of up to 100 percent with the strain Paracoccus pantotrophus strain B-3 (KT 715779.1) and the strain Paracoccus pantotrophus strain JCM6892 (AB 598745.1).
Based on the morphological, physiological and biochemical tests and molecular biological analysis of NJUST38, it was comprehensively confirmed that strain NJUST38 belongs to Paracoccus Paracoccus pantotrophus, which was designated Paracoccus pantotrophus NJUST.
Example 2
The strain Paracoccus pantotrophus NJUST has the property of degrading diethylamine and triethylamine.
Strain Paracoccus pantotrophus NJUST is inoculated to LB liquid medium containing 500mg/L of diethylamine and triethylamine, 180 rpm shaking culture is carried out at 30-35 ℃, strain enrichment of Paracoccus pantotrophus NJUST is carried out, after the strain enters the logarithmic phase (about 48 h), the obtained bacterial liquid is centrifuged by an ultra-low temperature centrifuge for 5 minutes (4 ℃ and 7000 rpm), and deposited bacterial cells are obtained. Re-suspending with sterilized inorganic salt liquid culture medium, centrifuging, repeatedly washing for three times, and suspending the strain in sterile inorganic salt liquid culture medium to obtain seed solution (controlling OD) 600 About 1.5 to 2.0).
100mL of inorganic salt liquid culture medium containing diethylamine and triethylamine with initial concentrations of 500mg/L respectively was added to a 250mL Erlenmeyer flask as simulated wastewater to construct a biodegradation system. Subsequently, the conical flask was sealed with six layers of sterile gauze, autoclaved at 121 ℃ for 20 minutes and allowed to stand to room temperature. Inoculating the seed liquid into a conical flask, performing shake culture at the inoculum size of 5%, performing shake culture at the temperature of 30-35 ℃ and the rotation speed of 180 r/min, periodically sampling, monitoring concentration changes of diethylamine and triethylamine in wastewater (the concentration of diethylamine and triethylamine is measured by a gas chromatograph (Agilent 7890B), the gas chromatograph is under the conditions that the column temperature is 50 ℃ for 3min, the temperature is increased to 180 ℃ for 10min, the temperature rising rate is 10 ℃/min, the temperature of a sample injector is 200 ℃, the temperature of a detector is 320 ℃, the carrier gas is high-purity nitrogen, the volume flow is 4mL/min, the split ratio is 5:1), and setting an experimental group without adding the strain Paracoccus pantotrophus NJUST38 as a non-biological control system, wherein the rest of the operations are the same as the experimental system.
The experimental results are shown in FIGS. 2 and 3, in the biodegradation system containing 500mg/L of diethylamine and triethylamine, 500mg/L of diethylamine and triethylamine were completely degraded within 18 hours and 24 hours, respectively. In the non-biological control system without Paracoccus pantotrophus NJUST inoculated, both diethylamine and triethylamine had slight volatilities. The embodiment shows that Paracoccus pantotrophus NJUST38 obtained by separation can be successfully applied to biochemical treatment of waste water containing diethylamine and triethylamine at the same time, and realizes synchronous and efficient removal of the diethylamine and the triethylamine in the waste water.
Example 3
The strain Paracoccus pantotrophus NJUST is applied to the treatment of the waste water containing diethylamine and triethylamine under different water inlet pH conditions (5.0-9.0).
100mL of inorganic salt liquid culture mediums with initial concentrations of diethylamine and triethylamine of 500mg/L and initial pH values of 5.0, 6.0, 7.0, 8.0 and 9.0 are respectively prepared as simulated wastewater, and added into a 250mL conical flask to construct a biodegradation system. Subsequently, the conical flask mouth was sealed with six layers of sterile gauze, autoclaved at 121 ℃ for 20min, and allowed to stand to room temperature. Inoculating the seed liquid into a conical flask, wherein the inoculum size is 5% -10%, shake culturing at 30-35 ℃ and 180 rpm, periodically sampling, and monitoring the concentration change of diethylamine and triethylamine in the simulated wastewater.
As shown in fig. 4 and 5, paracoccus pantotrophus NJUST can remove diethylamine and triethylamine at initial pH of 6.0 and 7.0, and can completely remove diethylamine and triethylamine at pH of 7.0; the removal rate of diethylamine and triethylamine by Paracoccus pantotrophus NJUST38 was greatly reduced at initial pH of 5.0 and 8.0; and the removal rate of diethylamine and triethylamine is as low as 10% or less at an initial pH of 9.0.
This example demonstrates that in a biodegradation system, the appropriate pH range for the strain Paracoccus pantotrophus NJUST to degrade diethylamine and triethylamine is weak to neutral, and the efficiency of Paracoccus pantotrophus NJUST to remove diethylamine and triethylamine is highest under neutral conditions, and the weak alkaline environment reduces the removal capacity of Paracoccus pantotrophus NJUST38 for diethylamine and triethylamine, and the alkaline environment greatly reduces the removal capacity of strain Paracoccus pantotrophus NJUST38 for diethylamine and triethylamine.
The application has been described in detail in connection with the specific embodiments and exemplary examples thereof, but such description is not to be construed as limiting the application. It will be understood by those skilled in the art that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present application and its embodiments without departing from the spirit and scope of the present application, and these fall within the scope of the present application.
Claims (10)
1. Paracoccus Paracoccus pantotrophus NJUST strain with a preservation number of CCTCC NO: m2020682.
2. A microbial agent comprising the strain NJUST38 as claimed in claim 1.
3. A Paracoccus seed solution, characterized in that the Paracoccus seed solution is prepared by activating and culturing the strain NJUST38 according to claim 1.
4. The method for culturing an NJUST38 strain according to claim 1, wherein the NJUST38 strain is inoculated into a medium having a pH of 7.0 to 8.0 and a culture temperature of 30℃to 35 ℃.
5. The method for culturing an NJUST38 strain according to claim 4, wherein the medium is LB medium containing diethylamine and triethylamine.
6. The use of the strain NJUST38 according to claim 1 for degrading organic compounds of the ethylamine class, characterized in that the organic compounds of the ethylamine class comprise diethylamine and/or triethylamine.
7. A method for treating wastewater containing ethylamine organic matters, which is characterized in that the NJUST38 strain of claim 1 is inoculated into the wastewater to be treated; the wastewater contains diethylamine and/or triethylamine.
8. The method according to claim 7, wherein the NJUST38 strain is prepared into seed solution, inoculated into waste water, and the seed solution OD 600 1.5-2.0, and the inoculation amount is 5-10% (v/v).
9. The method according to claim 7, wherein when diethylamine is contained in the wastewater, the initial concentration of diethylamine is 500mg/L; when the wastewater contains triethylamine, the initial concentration of the triethylamine is 500mg/L.
10. The method of claim 7, wherein the wastewater has a pH of 6 to 7.
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