CN112410272A - Paracoccus for degrading N, N-dimethylformamide and application thereof in wastewater treatment - Google Patents
Paracoccus for degrading N, N-dimethylformamide and application thereof in wastewater treatment Download PDFInfo
- Publication number
- CN112410272A CN112410272A CN202011483951.0A CN202011483951A CN112410272A CN 112410272 A CN112410272 A CN 112410272A CN 202011483951 A CN202011483951 A CN 202011483951A CN 112410272 A CN112410272 A CN 112410272A
- Authority
- CN
- China
- Prior art keywords
- paracoccus
- dmf
- culture
- wastewater
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/163—Nitrates
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Environmental & Geological Engineering (AREA)
- Hydrology & Water Resources (AREA)
- Water Supply & Treatment (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention discloses a paracoccus capable of efficiently degrading DMF (dimethyl formamide) and application thereof, belonging to the field of biological treatment of organic pollutants in the environment. The invention takes activated sludge which is used for treating DMF for a long time as a bacteria source, takes an inorganic salt culture medium which takes DMF as a carbon source as a screening culture medium, separates and purifies to obtain a paracoccus which can efficiently degrade DMF and simultaneously carry out denitrification, and has a preservation number of CCTCC NO: m2020683. The paracoccus can utilize DMF as an electron donor to carry out anoxic denitrification reaction, realizes mineralization and degradation of DMF, has high-efficiency organic matter degradation capability and denitrification capability, and is suitable for removal treatment of high-concentration nitrate nitrogen and refractory organic pollutants in wastewater.
Description
Technical Field
The invention belongs to the technical field of biological treatment of organic pollutants, relates to paracoccus capable of degrading N, N-dimethylformamide, and particularly relates to paracoccus capable of performing denitrification by taking nondegradable organic pollutant N, N-dimethylformamide as an electron donor and application of the paracoccus in wastewater treatment.
Background
N, N-Dimethylformamide (DMF), an artificially synthesized organic polar solvent, is widely used in chemical, pharmaceutical, and textile industries due to its good water-miscibility and ability to dissolve various organic substances. It is reported that more than 5000 tons of DMF are discharged or transferred to the environment every year. DMF has stable structure, is not easy to hydrolyze or photolyze, is not easy to degrade by environmental microorganisms, is easy to accumulate and transfer in natural environment, and has the characteristics of biotoxicity, embryotoxicity, hepatotoxicity, teratogenesis, carcinogenesis and the like. Therefore, the development of efficient and economical wastewater treatment technology for treating industrial wastewater containing DMF has become one of the important issues in the field of environmental management at present.
At present, the treatment method of DMF-containing wastewater comprises advanced oxidation method, photocatalytic degradation method, membrane separation method, physical adsorption method and the like. Among them, the physical and chemical methods such as advanced oxidation and membrane separation are high in cost and serious in secondary pollution for treating the waste water containing DMF. The biological treatment technology has the advantages of economy, high efficiency, small secondary pollution and the like, can realize harmless treatment, and is a more appropriate wastewater treatment technology for treating the DMF-containing wastewater. However, due to the toxic and non-degradable nature of DMF, the precondition for the biological treatment of DMF contamination is to obtain strains with degradation function that are able to tolerate the biological toxicity of DMF. Among the DMF degrading strains reported so far are Paracoccus (Zhou, X., Jin, W., Sun, C., Gao, S., Chen, C., Wang, Q., Han, S., Tu, R., Latif, M.A., Wang, Q.Microbiological degradation of N, N-dimethyl for complex by sea, straight DMF-3, active slurry of Chem.Eng.J.2018,343,324-330.), Rhodococcus (Chen, X., Yang, C., Wang, W., Ge, B., Zhang, J., Liu, Y., Nan, Y, Y.Biodegradation of N, N-dimethyl for lactic recovery strain B83, G.D., S.D., Bacillus strain, S.D., Staphylococcus aureus, S.D., strain, S.D., S.A.A.D., T.A. strain, S.D., strain, S.A.A.D., strain, S.A.D.A, S.A.C., Wang., Han, S.S., S., S.A, S., T.A.S., T.A., strain, S.A. strain, S. strain, P.I. strain, S. strain, strain, n-dimethylformamidase from Alcaligenes sp.KUFA-1.J.Ferment.Bioeng.1997,84, 543-. However, the existing bacterial strains capable of degrading DMF all need to be cultured under the condition of consuming oxygen, the operation cost of processes such as power consumption aeration and the like is greatly increased in practical application, and the traditional anaerobic treatment technology has low removal efficiency and poor stability.
Disclosure of Invention
1. Problems to be solved
Aiming at the problems that the prior process for treating wastewater containing a pollutant DMF by a biological method needs to be carried out under an oxygen consumption condition and the operation cost is high, the invention provides a Paracoccus (Paracoccus) strain for efficiently degrading the DMF, the strain can utilize the DMF as an electron donor and nitrate nitrogen as an electron acceptor to carry out denitrification reaction, and the degradation of the DMF is realized while anoxic denitrification is carried out.
2. Technical scheme
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention takes activated sludge which is used for treating DMF wastewater for a long time as a bacterial source, takes a culture medium which takes DMF as a carbon source as a screening culture medium, separates and purifies to obtain a Paracoccus which utilizes DMF as an electron donor for denitrification and is named as Paracoccus sp.NJUST48, and the Paracoccus is preserved in China Center for Type Culture Collection (CCTCC) at 11 and 06 days in 2020, the preservation address is Wuhan university in Wuhan City, the preservation number is CCTCC NO: m2020683.
The paracoccus 16S rRNA gene sequence is shown in SEQ ID NO:1 (Table 1).
The invention also provides a screening method of the Paracoccus sp.NJUST48, which takes the activated sludge which is used for treating the DMF wastewater for a long time as a bacterial source to separate, purify and identify the bacterial strain.
Preferably, the screening comprises the steps of:
(1) sampling, namely sampling from activated sludge which is used for treating DMF wastewater for a long time, adding the activated sludge into sterile physiological saline, uniformly stirring and standing.
Further, 10mL of the sample was taken out, added to 100mL of sterile physiological saline, and allowed to stand for 2 hours.
(2) And (3) enriching, taking the supernatant in the step (1), adding the supernatant into an inorganic salt liquid culture medium, and carrying out enrichment culture and repeating.
Further, taking 1mL of the supernatant in the step (1), adding the supernatant into 50mL of inorganic salt liquid culture medium, and carrying out enrichment culture in a constant-temperature shaking incubator for three days (28-32 ℃, 180 revolutions per minute) for three times.
Preferably, the composition of the mineral salts medium is: NaHPO4·12H2O(1.53g L-1)，KH2PO4(0.38g L-1)，MgSO4·7H2O(0.1g L-1)，CaCl2(0.05g L-1) Microelement solution SL-4(10mL L)-1)。
Microelement SL-4: EDTA (0.5g L)-1)，FeSO4·7H2O(0.2g L-1) Trace element SL-6(100mL L)-1)。
Microelement SL-6: ZnSO4·7H2O(0.01g L-1)，MnCl2·4H2O(0.03g L-1)，H3BO4(0.3g L-1)，CoCl2·6H2O(0.2g L-1)，CuCl2·2H2O(0.01g L-1)，NiCl2·6H2O(0.02g L-1)，Na2MoO4·2H2O(0.03g L-1)。
(3) And (3) diluting, and taking the culture solution in the step (2) to perform gradient dilution by using sterile physiological saline.
Further, dilute to 10-4-10-10The dilution factor can also be adjusted according to the actual situation.
(4) Coating and culturing a solid culture medium, purifying and culturing to prepare an inorganic salt agar solid culture medium plate, respectively coating the diluted culture solution on the inorganic salt agar solid culture medium plate, and culturing in a biochemical incubator; and selecting single colonies with obvious differences on the culture dish, and performing purification culture.
Further, 20. mu.L of the suspension was applied under the following culture conditions: 28-32 ℃ for 3 days.
Further, the purification culture is carried out by adopting a plate streaking separation method, and the purification culture is repeated as many times as required. Further, five successive purifications were carried out to obtain a single strain.
(5) And (4) liquid culture and preservation, namely culturing the single colony purified and cultured in the step (4) in an anaerobic inorganic salt liquid culture medium containing DMF and nitrate nitrogen, monitoring the concentration change of the DMF and the nitrate nitrogen in the culture process, and selecting a strain capable of most effectively removing the DMF and the nitrate nitrogen for preservation.
Preferably, the inorganic salt liquid culture medium is selected, DMF and nitrate nitrogen are added, and pure helium is aerated to remove dissolved oxygen.
Preferably, the culture is carried out in a constant-temperature shaking incubator under the following culture conditions: 180 revolutions per minute and 28 ℃ to 32 ℃.
(7) And identifying the preserved strain by one or more of morphological, physiological and biochemical tests or molecular biological identification.
The invention also provides a culture method of the Paracoccus, which is to inoculate Paracoccus sp.NJUST48 in an LB culture medium, wherein the pH value of the culture medium is 6.5-7.5, and the culture temperature is 28-32 ℃.
Preferably, the LB medium further contains DMF and sodium nitrate.
Preferably, the molar ratio of DMF to sodium nitrate is (0.8-1.5): 1. Further, the molar ratio of DMF to sodium nitrate was 0.8: 1.
Preferably, the concentration of DMF is 11.7-15.3 mM, and the concentration of sodium nitrate is 15.6-19.6 mM.
The invention also provides application of the paracoccus in treatment of DMF-containing wastewater.
Preferably, the wastewater contains nitrate nitrogen, or nitrate nitrogen is added to the wastewater.
Preferably, the temperature of the wastewater treatment is 28-32 ℃, and/or the pH of the wastewater treatment is 6.5-7.5.
Preferably, the paracoccus bacteria are prepared into seed liquid and added into the wastewater. Further, the OD600 of the paracoccus seed liquid is 1.5-2.0, and the inoculation amount is 5-10%.
Preferably, the paracoccus is prepared into a microbial inoculum and added into the wastewater.
The invention also provides a bacterial agent for degrading DMF, which contains the screened Paracoccus sp.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) the Paracoccus sp.NJUST48 provided by the invention has stronger adaptability and tolerance to the biological environment, can treat DMF in wastewater under an anoxic condition, can reduce an oxygen-consuming aeration section when being applied to the process treatment of industrial wastewater compared with the oxygen-consuming condition, and saves the economic cost.
(2) According to Paracoccus sp.NJUST48, DMF is used as an electron donor, nitrate nitrogen is used as an electron acceptor for metabolism and growth, and meanwhile, the Paracoccus sp.NJUST48 has high-efficiency DMF degradation capability and denitrification capability, can be successfully applied to biochemical treatment of wastewater containing both nitrate nitrogen and DMF, and realizes high-efficiency removal of the nitrate nitrogen and the DMF in the wastewater. As shown in fig. 2, the efficiency of the treatment was higher than that under the conventional anaerobic condition.
(3) The invention utilizes the characteristic that the industrial wastewater usually contains nitrate with higher concentration to obtain the high-efficiency bacterial strain which takes DMF as an electron donor and nitrate nitrogen as an electron acceptor, realizes the degradation of DMF while denitrifying by anoxia, and has important significance for the low-cost and harmless treatment of the industrial wastewater containing DMF and nitrate nitrogen.
Drawings
Fig. 1 is a scanning electron microscope image of Paracoccus sp.
FIG. 2 is a graph showing the effect of Paracoccus sp.NJUST48 on the degradation of DMF in a liquid medium at an initial concentration of DMF of 13.7. + -. 0.9 mM.
FIG. 3 is a graph showing the denitrification effect of Paracoccus sp.NJUST48 on nitrate nitrogen in a liquid medium having an initial concentration of nitrate nitrogen of 17.6. + -. 1.9 mM.
FIG. 4 is a graph showing the effect of Paracoccus sp.NJUST48 on DMF degradation and simultaneous denitrification under different influent pH conditions.
Detailed Description
The invention is further described with reference to specific examples.
It should be noted that the terms "upper", "lower", "left", "right" and "middle" used in the present specification are for the sake of clarity, and are not intended to limit the scope of the present invention, and changes or adjustments of the relative relationship thereof may be made without substantial technical changes.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; as used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
As used herein, the term "about" is used to provide the flexibility and inaccuracy associated with a given term, measure or value. The degree of flexibility for a particular variable can be readily determined by one skilled in the art.
As used herein, at least one of the terms "is intended to be synonymous with one or more of. For example, "at least one of A, B and C" explicitly includes a only, B only, C only, and combinations thereof, respectively.
Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limit values of 1 to about 4.5, but also include individual numbers (such as 2, 3, 4) and sub-ranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed to include all of the aforementioned values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or feature being described.
Any steps recited in any method or process claims may be executed in any order and are not limited to the order presented in the claims.
Example 1
Screening, separating and identifying paracoccus capable of degrading DMF.
(1) Screening and isolation of strains
10mL of activated sludge, which had been used for a long period of time for the treatment of DMF wastewater, was sampled, added to 100mL of sterile physiological saline (0.85% sodium chloride solution), stirred well, and allowed to stand for two hours. Adding 1mL of supernatant into 50mL of inorganic salt liquid culture medium, carrying out enrichment culture in a constant-temperature shaking incubator for three days (28-32 ℃, 180 r/min), after three times of enrichment, taking culture solution, and carrying out gradient dilution to 10 degrees by using sterile normal saline (0.85% sodium chloride solution)-4-10-10And (4) doubling.
Preparing an inorganic salt agar solid medium plate, respectively coating 20 mu L of the diluted culture solution on the inorganic salt agar solid medium plate, and placing the plate in a biochemical incubator for culturing for three days at the temperature of between 28 and 32 ℃. And selecting single colonies with obvious differences on a culture dish, performing purification culture by adopting a plate streaking separation method, and continuously purifying for five times to obtain a single strain.
Preparing 100mL of inorganic salt liquid culture medium containing DMF and nitrate nitrogen, filling the inorganic salt liquid culture medium into a 120mL serum bottle, aerating the inorganic salt liquid culture medium by using pure helium to remove dissolved oxygen, sterilizing the inorganic salt liquid culture medium at the high temperature of 121 ℃, inoculating, separating and purifying the obtained pure strain, culturing the pure strain in a constant-temperature shaking incubator at the temperature of 28-32 ℃ at 180 revolutions per minute, and monitoring the concentration change of the DMF and the nitrate nitrogen in the culture process. Selecting a strain capable of effectively removing DMF and nitrate nitrogen in the culture medium, named NJUST48, and performing slant storage and low-temperature storage at-80 ℃.
The inorganic salt culture medium comprises the following components: NaHPO4·12H2O(1.53g L-1)，KH2PO4(0.38g L-1)，MgSO4·7H2O(0.1g L-1)，CaCl2(0.05g L-1) Microelement solution SL-4(10mL L)-1)。
Microelement SL-4: EDTA (0.5g L)-1)，FeSO4·7H2O(0.2g L-1) Trace element SL-6(100mL L)-1)。
Microelement SL-6: ZnSO4·7H2O(0.01g L-1)，MnCl2·4H2O(0.03g L-1)，H3BO4(0.3g L-1)，CoCl2·6H2O(0.2g L-1)，CuCl2·2H2O(0.01g L-1)，NiCl2·6H2O(0.02g L-1)，Na2MoO4·2H2O(0.03g L-1)。
The amount of DMF and sodium nitrate is added according to the experimental requirements.
Adding 20g L on the basis of liquid culture medium-1The agar is sterilized by autoclaving at 121 ℃ for 20 minutes in a sterilizer, and then poured into a sterile culture dish to be cooled to room temperature, so as to obtain an inorganic salt agar solid culture medium plate.
(2) Identification of strains
Performing morphological, physiological and biochemical tests and molecular biological identification on the strain NJUST48 obtained in the step (1), determining the 16S rRNA gene sequence of the strain, performing homology comparison on the 16S rRNA gene sequence of the strain and gene sequences in a GenBank database, analyzing the result, and determining the genus of the strain on the molecular biological level.
TABLE 1 Paracoccus 16S rRNA Gene sequences
Morphological characteristics: NJUST48 colonies are yellowish, smooth and opaque in surface, neat in edge, glossy, and diffusively turbid in liquid medium. The bacterial strain has short rod-shaped cells, more secretion on the cell surface, cell size of 0.37-0.56 mu m multiplied by 0.93-1.48 mu m, and FIG. 1 is a scanning electron microscope image of the bacterium NJUST 48.
Physiological and biochemical characteristics: gram negative bacteria, nitrate reductase positive.
③ identifying in molecular biology: taking nuclear DNA of the NJUST48 strain as a template, carrying out PCR amplification by using a universal primer for bacterial amplification, and determining the gene sequence of the strain NJUST48, wherein the gene sequence is shown as SEQ ID NO. 1. The 16S rRNA gene sequence of the strain is submitted to a GenBank database for homology comparison, and the result shows that the sequence similarity of NJUST48 and Paracoccus sp.Y3B-1(HM018693.1) reaches more than 99%. Based on morphological, physiological and biochemical tests and molecular biological analysis of NJUST48, NJUST48 was identified as Paracoccus sp.
Example 2
The application of the strain Paracoccus sp.NJUST48 in the treatment of waste water containing DMF and nitrate nitrogen.
Inoculating Paracoccus sp.NJUST48 into LB liquid culture medium containing 6.8mM of DMF, adjusting the pH to 6.5-7.5, carrying out shake culture at the temperature of 28-32 ℃ for 180 r/min, enriching the strain of NJUST48, and after the strain enters a logarithmic growth phase (about 48 hours), centrifuging the obtained bacterial liquid for 10 min (4 ℃, 6000 r/min) by using an ultra-low temperature centrifuge to obtain deposited bacteria. And (3) resuspending the strain in a sterilized inorganic salt liquid culture medium, centrifuging, repeatedly washing for three times, resuspending the strain in the sterilized inorganic salt liquid culture medium, and detecting that OD600 is 1.5-2.0 to obtain a seed solution.
Adding 100mL of inorganic salt liquid culture medium with the initial concentration of DMF being 13.7 +/-0.9 mM and the initial concentration of sodium nitrate being 17.6 +/-1.9 mM into a 120mL serum bottle to serve as simulated wastewater, and continuously introducing high-purity helium to remove oxygen dissolved in the serum bottle and the wastewater so as to construct an anoxic denitrification system. Subsequently, the serum bottle was sealed with a butyl rubber stopper and an aluminum cap, autoclaved at 121 ℃ for 20 minutes, and allowed to stand to room temperature. Inoculating the seed solution into a serum bottle, wherein the inoculation amount is 5-10%, performing shake culture at 28-32 ℃ under the condition of 180 r/min, and monitoring the concentration change of DMF (dimethyl formamide) and nitrate nitrogen in wastewater. And (3) setting an experimental group without adding sodium nitrate as an anaerobic control system, and carrying out the rest operations in the anoxic denitrification system. An experimental group which is not inoculated with NJUST48 is set as an abiotic control system, and the rest operation is the same as that of an anoxic denitrification system.
As shown in FIG. 2, 13.7. + -. 0.9mM DMF was completely degraded within 22 hours in the anoxic denitrification system containing sodium nitrate. As shown in FIG. 3, in the anoxic denitrification system containing sodium nitrate, complete denitrification was achieved within 22 hours with 17.6. + -. 1.9mM of nitrate nitrogen. However, the removal of DMF at the same concentration was less than 30% in the anaerobic control system without the addition of the electron acceptor sodium nitrate (fig. 2). Furthermore, the concentrations of nitrate nitrogen and nitrogen methyl pyrrolidone did not change significantly in the non-biological control system not inoculated with NJUST48 (figure 2). This example illustrates that the separated Paracoccus sp.njustt 48 can be successfully applied to biochemical treatment of wastewater containing both nitrate nitrogen and DMF, and efficient removal of nitrate nitrogen and DMF in the wastewater is achieved.
Example 3
The application of the strain Paracoccus sp.NJUST48 in the treatment of wastewater containing DMF and nitrate nitrogen under different pH conditions.
Preparing 100mL of inorganic salt liquid culture medium containing DMF with initial concentration of 13.5 +/-1.8 mM, nitrate nitrogen with initial concentration of 17.6 +/-2.0 mM and initial pH of 5.0, 6.0, 7.0, 8.0 and 9.0 respectively as simulated wastewater, adding the simulated wastewater into a 120mL serum bottle, and continuously introducing high-purity helium gas to remove oxygen dissolved in the serum bottle and the wastewater so as to construct an anoxic denitrification system. Subsequently, the serum bottle was sealed with a butyl rubber stopper and an aluminum cap, autoclaved at 121 ℃ for 20 minutes, and allowed to stand to room temperature. Inoculating the seed solution into a serum bottle, wherein the inoculation amount is 5-10%, performing shake culture at 28-32 ℃ under the condition of 180 r/min, and monitoring the concentration change of DMF (dimethyl formamide) and nitrate nitrogen in wastewater.
Experimental results as shown in fig. 4, removal of DMF and nitrate nitrogen was achieved by each of Paracoccus sp.njustt 48 at initial pH of 6.0 and 7.0, and DMF and nitrate nitrogen were completely removed at pH of 7.0; under the conditions that the initial pH is 5.0 and 8.0, the rate of removing DMF and nitrate nitrogen by Paracoccus sp.NJUST48 is greatly reduced; under the condition of pH 9.0, the removal rate of DMF and nitrate nitrogen is as low as below 8%.
This example illustrates that in an anoxic denitrification system, the pH range suitable for DMF degradation and denitrification by the strain Paracoccus sp.njustt 48 is from weak acidity to neutral, and the removal efficiency is highest in a neutral environment, and the alkaline environment can significantly reduce the removal capacity of DMF and nitrate nitrogen.
Sequence listing
<110> Nanjing university of science and technology
Nanjing Institute of environmental science, Ministry of ecology and environment
<120> Paracoccus for degrading N, N-dimethylformamide and application thereof in wastewater treatment
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1214
<212> DNA
<213> Paracoccus (Paracoccus)
<400> 1
gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct gttccgcgat 60
tactagcgat tccaacttca tggggtcgag ttgcagaccc caatccgaac tgagatggct 120
tttggggatt aacccactgt caccaccatt gtagcacgtg tgtagcccaa cccgtaaggg 180
ccatgaggac ttgacgtcat ccacaccttc ctccgactta tcatcggcag ttcttccaga 240
gtgcccaacc aaatgatggc aactggaagt gtgggttgcg ctcgttgccg gacttaaccg 300
aacatctcac gacacgagct gacgacagcc atgcagcacc tgtccacagg tctcttacga 360
gaagacccga tctctcgggc tgtcctgcga tgtcaagggt tggtaaggtt ctgcgcgttg 420
cttcgaatta aaccacatgc tccaccgctt gtgcgggccc ccgtcaattc ctttgagttt 480
taatcttgcg accgtactcc ccaggcggaa tgcttaatcc gttaggtgtg tcaccgaaca 540
gcatgctgcc cgacgactgg cattcatcgt ttacggcgtg gactaccagg gtatctaatc 600
ctgtttgctc cccacgcttt cgcacctcag cgtcagtatc gagccagtga gccgccttcg 660
ccactggtgt tcctccgaat atctacgaat ttcacctcta cactcggaat tccactcacc 720
tctctcgaac tccagaccaa tagttttgaa ggcagttccg aggttgagcc ccgggatttc 780
acccccaact ttctggtccg cctacgtgcg ctttacgccc agtaattccg aacaacgcta 840
gccccctccg tattaccgcg gctgctggca cggagttagc cggggcttct tctgctggta 900
ccgtcattat cttcccagct gaaagagctt tacaacccta aggccttcat cactcacgcg 960
gcatggctag atcagggttg cccccattgt ctaagattcc ccactgctgc ctcccgtagg 1020
agtctgggcc gtgtctcagt cccagtgtgg ctgatcatcc tctcaaacca gctatggatc 1080
gtaggcttgg taggccatta ccccaccaac tacctaatcc aacgcgggcc gatccttctc 1140
cgataaatct ttcccccaag gggcgtatac ggtattactc ccagtttccc agggctattc 1200
cgtagagaag ggca 1214
Claims (12)
1. A Paracoccus is named Paracoccus sp.NJUST48, and the preservation number is CCTCC NO: m2020683.
2. The paracoccus bacterium of claim 1, wherein the paracoccus 16S rRNA gene sequence is shown as SEQ ID NO. 1.
3. The method for screening Paracoccus according to claim 1 or 2, comprising the steps of,
(1) sampling, namely sampling from activated sludge which is used for treating DMF wastewater for a long time, adding the activated sludge into sterile physiological saline, uniformly stirring, and standing;
(2) enriching, namely adding the supernatant in the step (1) into an inorganic salt liquid culture medium, and carrying out enrichment culture in a constant-temperature shaking incubator and repeating;
(3) diluting, namely taking the culture solution in the step (2) to perform gradient dilution by using sterile physiological saline;
(4) solid coating and purification culture: preparing an inorganic salt agar solid culture medium plate, and respectively coating the diluted culture solution on the inorganic salt agar solid culture medium plate for culture; selecting single colonies with obvious differences on a culture dish, and performing purification culture;
(5) liquid culture and preservation: and (4) culturing the single colony purified and cultured in the step (4) in an anaerobic inorganic salt liquid culture medium containing DMF and nitrate nitrogen, monitoring the concentration change of the DMF and the nitrate nitrogen in the culture process, and selecting the strain with reduced concentration for storage.
4. The method for culturing Paracoccus according to claim 1 or 2, wherein Paracoccus is inoculated into LB medium, pH of the medium is 6.5 to 7.5, and the culture temperature is 28 ℃ to 32 ℃.
5. The method for culturing Paracoccus according to claim 4, wherein the LB medium further contains DMF and sodium nitrate.
6. The method for culturing Paracoccus according to claim 5, wherein the molar ratio of DMF to sodium nitrate is (0.8-1.5): 1, and/or the concentration of DMF is 11.7-15.3 mM and the concentration of sodium nitrate is 15.6-19.6 mM.
7. Use of paracoccus as claimed in claim 1 or 2 in the treatment of DMF containing wastewater.
8. A use of Paracoccus according to claim 7, wherein the wastewater further contains nitrate nitrogen.
9. A use of Paracoccus according to claim 7 or 8, wherein said Paracoccus is formulated as a seed solution to be added to wastewater; or the paracoccus is prepared into a microbial inoculum and added into the wastewater.
10. The use of Paracoccus as claimed in claim 9 in the treatment of DMF containing wastewater, wherein the OD600 of the prepared seed solution is 1.5-2.0, and the inoculation amount is 5-10%.
11. Use of paracoccus bacterium according to claim 10 in the treatment of DMF-containing wastewater, wherein: when the wastewater is treated, the temperature is 28-32 ℃, and/or the pH is 6.5-7.5.
12. A DMF degrading bacterial preparation comprising the Paracoccus bacterium according to claim 1 or 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011483951.0A CN112410272B (en) | 2020-12-16 | 2020-12-16 | Paracoccus for degrading N, N-dimethylformamide and application thereof in wastewater treatment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011483951.0A CN112410272B (en) | 2020-12-16 | 2020-12-16 | Paracoccus for degrading N, N-dimethylformamide and application thereof in wastewater treatment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112410272A true CN112410272A (en) | 2021-02-26 |
| CN112410272B CN112410272B (en) | 2022-04-29 |
Family
ID=74775201
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011483951.0A Active CN112410272B (en) | 2020-12-16 | 2020-12-16 | Paracoccus for degrading N, N-dimethylformamide and application thereof in wastewater treatment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112410272B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113104992A (en) * | 2021-04-06 | 2021-07-13 | 长沙理工大学 | Culture method of anaerobic inoculation mud for treating N, N-dimethylformamide wastewater |
| CN113980851A (en) * | 2021-11-08 | 2022-01-28 | 浙江工业大学 | Paracoccus YBH-X with dimethylacetamide degradation capability and application thereof |
| CN114045238A (en) * | 2021-11-08 | 2022-02-15 | 浙江树人学院（浙江树人大学） | Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide and application thereof |
| CN114456974A (en) * | 2022-01-14 | 2022-05-10 | 浙江台州秀川科技有限公司 | Paracoccus thiooxidans capable of efficiently degrading DMF (dimethyl formamide) and application thereof in treatment of waste water containing DMF |
| CN114933990A (en) * | 2022-05-31 | 2022-08-23 | 南京理工大学 | N-methylpyrrolidone degradation synchronous denitrifying bacteria and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106986462A (en) * | 2017-02-28 | 2017-07-28 | 广东省食品工业研究所有限公司 | One kind is using N, the method for N dimethylformamides in microbial degradation Sucralose waste water |
| CN110656071A (en) * | 2019-11-11 | 2020-01-07 | 黄河三角洲京博化工研究院有限公司 | Paracoccus huilkii for efficiently degrading DMF (dimethyl formamide) and application thereof |
-
2020
- 2020-12-16 CN CN202011483951.0A patent/CN112410272B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106986462A (en) * | 2017-02-28 | 2017-07-28 | 广东省食品工业研究所有限公司 | One kind is using N, the method for N dimethylformamides in microbial degradation Sucralose waste water |
| CN110656071A (en) * | 2019-11-11 | 2020-01-07 | 黄河三角洲京博化工研究院有限公司 | Paracoccus huilkii for efficiently degrading DMF (dimethyl formamide) and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| S. SANJEEVKUMAR ET AL.: "Paracoccus denitrificans SD1 mediated augmentation with indigenous mixed cultures for enhanced removal of N,N-dimethylformamide from industrial effluents", 《BIOCHEMICAL ENGINEERING JOURNAL》 * |
| WANG ET AL.: "Nitrate stimulation of N-Methylpyrrolidone biodegradation by Paracoccus pantotrophus: Metabolite mechanism and Genomic characterization", 《BIORESOURCE TECHNOLOGY》 * |
| ZHOU ET AL.: "Microbial degradation of N,N-dimethylformamide by Paracoccus sp. strain DMF-3 from activated sludge", 《CHEMICAL ENGINEERING JOURNAL》 * |
| 卢伟强等: "脱氮副球菌DYTN-1 高效去除污水中的总氮", 《食品与发酵工业》 * |
| 陈建华等: "一株N，N-二甲基甲酰胺高效降解菌的筛选鉴定及其生物降解特性", 《净水技术》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113104992A (en) * | 2021-04-06 | 2021-07-13 | 长沙理工大学 | Culture method of anaerobic inoculation mud for treating N, N-dimethylformamide wastewater |
| CN113980851A (en) * | 2021-11-08 | 2022-01-28 | 浙江工业大学 | Paracoccus YBH-X with dimethylacetamide degradation capability and application thereof |
| CN114045238A (en) * | 2021-11-08 | 2022-02-15 | 浙江树人学院（浙江树人大学） | Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide and application thereof |
| CN113980851B (en) * | 2021-11-08 | 2023-06-02 | 浙江工业大学 | Paracoccus YBH-X with dimethylacetamide degradation capability and application thereof |
| CN114045238B (en) * | 2021-11-08 | 2023-09-29 | 浙江树人学院（浙江树人大学） | Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide and application thereof |
| CN114456974A (en) * | 2022-01-14 | 2022-05-10 | 浙江台州秀川科技有限公司 | Paracoccus thiooxidans capable of efficiently degrading DMF (dimethyl formamide) and application thereof in treatment of waste water containing DMF |
| CN114456974B (en) * | 2022-01-14 | 2024-04-09 | 浙江台州秀川科技有限公司 | Paracoccus thiooxidans capable of efficiently degrading DMF and application thereof in DMF-containing wastewater treatment |
| CN114933990A (en) * | 2022-05-31 | 2022-08-23 | 南京理工大学 | N-methylpyrrolidone degradation synchronous denitrifying bacteria and application thereof |
| CN114933990B (en) * | 2022-05-31 | 2023-09-05 | 南京理工大学 | N-methylpyrrolidone degradation synchronous denitrifying bacterium and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112410272B (en) | 2022-04-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112410272B (en) | Paracoccus for degrading N, N-dimethylformamide and application thereof in wastewater treatment | |
| CN109913387B (en) | Enterobacter for degrading N-methylpyrrolidone and application of enterobacter in wastewater treatment | |
| CN112574916B (en) | N-methylpyrrolidone degrading bacterium and application thereof in wastewater treatment | |
| CN114908016A (en) | Pseudomonas denitrificans and application thereof in field of sewage and wastewater purification | |
| CN113462622B (en) | Pseudomonas for efficiently degrading various aromatic pollutants and application thereof | |
| CN114933990B (en) | N-methylpyrrolidone degradation synchronous denitrifying bacterium and application thereof | |
| CN114806921B (en) | Denitrifying bacteria with N-methylpyrrolidone as electron donor and application thereof | |
| CN110229766B (en) | Microbacterium oxydans and application thereof in degradation of organic pollutants | |
| CN112961790A (en) | Heterotrophic nitrifying bacteria resistant to high-salt environment and application thereof | |
| CN111909885A (en) | Salt-tolerant COD-reducing strain, culture method and application | |
| CN109486725B (en) | Bacterial strain capable of degrading benzene series and petroleum hydrocarbon and application thereof | |
| CN115386520B (en) | Rhodococcus pyridine-philic RL-GZ01 strain and application thereof | |
| CN107760636B (en) | Denitrifying strain taking low-quality carbon source phenol as electron donor and application thereof | |
| CN109052624B (en) | Mobile method and device for purifying sewage and black and odorous water | |
| CN101993838A (en) | Delftia tsuruhatensis strain H1 with chloroaniline degradation capacity and application thereof | |
| CN115305218A (en) | Plateau bacillus SX-3 and application thereof in degrading industrial sewage | |
| CN110938567B (en) | Bacillus subtilis, microbial agent and application thereof | |
| CN110982728B (en) | Bacterial strain for degrading chlorobenzene volatile organic compounds and culture method and application thereof | |
| Parvanova-Mancheva et al. | Biodegradation potential of Pseudomonas putida to phenol compared to Xanthobacter autotrophicus GJ10 and Pseudomonas denitrificans strains | |
| CN115975891A (en) | Ochrobactrum capable of degrading N-methylpyrrolidone and application of Ochrobactrum capable of degrading N-methylpyrrolidone in wastewater treatment | |
| CN114940957B (en) | Paracoccus ubitus with facultative denitrification synchronous denitrification and dephosphorization performances | |
| CN114958659B (en) | Paracoccus variabilis with aerobic nitrification, denitrification and dephosphorization performances | |
| CN113800652B (en) | Salt-tolerant aerobic denitrifying bacterium and application of coupling activated carbon thereof in strengthening water body pollution treatment | |
| CN116004474B (en) | Ochrobactrum intermedium, microbial inoculum, and method and device for treating chemical wastewater by using same | |
| CN114231439B (en) | Microbial preparation for degrading oil in high-salinity water |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20210226 Assignee: JIANGSU YIYU ENVIRONMENTAL PROTECTION TECHNOLOGY CO.,LTD. Assignor: NANJING University OF SCIENCE AND TECHNOLOGY|NANJING INSTITUTE OF ENVIRONMENTAL SCIENCES, MEE Contract record no.: X2023980030539 Denomination of invention: Paracoccus degrading N, N-dimethylformamide and its application in wastewater treatment Granted publication date: 20220429 License type: Exclusive License Record date: 20230111 |
|
| EE01 | Entry into force of recordation of patent licensing contract |