CN112011490A - Pseudomonas putida strain and application thereof in degrading nicotinic acid - Google Patents
Pseudomonas putida strain and application thereof in degrading nicotinic acid Download PDFInfo
- Publication number
- CN112011490A CN112011490A CN202010970823.2A CN202010970823A CN112011490A CN 112011490 A CN112011490 A CN 112011490A CN 202010970823 A CN202010970823 A CN 202010970823A CN 112011490 A CN112011490 A CN 112011490A
- Authority
- CN
- China
- Prior art keywords
- nicotinic acid
- pseudomonas putida
- strain
- degrading
- sewage
- 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
- 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
- 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
- C12R2001/38—Pseudomonas
- C12R2001/40—Pseudomonas putida
-
- 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
- C02F3/347—Use of yeasts or fungi
-
- 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
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2305/00—Use of specific compounds during water treatment
- C02F2305/06—Nutrients for stimulating the growth of microorganisms
Abstract
The invention discloses a Pseudomonas putida strain and application thereof in degrading nicotinic acid, wherein the strain is identified as Pseudomonas putida (Pseudomonas putida), is named as N17, is currently preserved in China general microbiological culture Collection center, and has the address as follows: the microbial research institute of China academy of sciences, No. 3, Xilu No. 1, Beijing, Chaoyang, is published by letter 100101, and has a preservation date of 2020, 8, 17 days and a preservation number of CGMCC NO. 20429. The strain N-17 has a very effective effect of degrading nicotinic acid, is simple and effective for degrading nicotinic acid, is easy to operate, does not produce secondary pollution, does not influence subsequent sewage treatment, has low cost and mild reaction, and is suitable for industrial wastewater treatment.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a pseudomonas putida strain and application thereof in degradation of nicotinic acid, which are mainly applied to degradation of nicotinic acid and belong to the field of industrial microorganisms.
Background
Nicotinic acid, vitamin B3, nicotinic acid and pellagra factor. The molecular formula is as follows: c6H5NO2Molecular weight 123.11, white or off-white powder in appearance, readily soluble in alkaline solution, slightly soluble in cold water, and stable in dry state. It is one of 13 vitamins essential to human body, and is a water-soluble vitamin. Nicotinic acid is an important pyridine derivative, and the heterocyclic structure of the pyridine ring causes the nicotinic acid to be an industrial pollutant which is difficult to degrade.
In actual production, liquid phase oxidation, gas phase oxidation, nitric acid oxidation, electrooxidation, biosynthesis and the like are mainly adopted, which can industrially produce nicotinic acid, but produce industrial wastewater containing 1000-20000mg/L nicotinic acid in large amount.
There are many methods for industrial wastewater treatment: 1. physical methods such as precipitation, filtration, etc., and 2. chemical methods such as coagulation precipitation, neutralization, redox, and chemical precipitation, etc., all of which have certain disadvantages or limitations such as secondary pollution. At present, biological methods such as an activated sludge method, an adsorption regeneration method, a biofilm method and the like are most commonly and widely used, and the method has the advantages of capability of treating various kinds of waste water, small secondary pollution, low energy consumption and the like.
For conventional wastewater treatment, biological treatment processes are considered to be one of the most technically and economically viable options. However, the chemical industry that produces pyridine and pyridine derivatives operates under extreme temperature conditions and processes with many organic compounds. Acetaldehyde, ammonia and a catalyst are used to produce pyridine. Therefore, the wastewater produced by the industries is very complex, the BOD/COD value is less than 0.2, and therefore the wastewater is not very suitable for biological treatment, and the wastewater treatment difficulty of the existing process is large.
Nicotinic acid is an important pyridine derivative, and the heterocyclic structure of the pyridine ring causes the nicotinic acid to be an industrial pollutant which is difficult to degrade. And a large amount of sewage rich in nicotinic acid is generated in the industrial production process of the nicotinic acid, and the BOD/COD value of the sewage is less than 0.2, so the method is not very suitable for biological treatment, and the existing process has higher difficulty in treating the waste water.
Disclosure of Invention
The invention provides a bacterial strain N-17(Pseudomonas putida N-17) for efficiently and rapidly degrading nicotinic acid and application thereof in degrading nicotinic acid.
One of the purposes of the invention is to provide a strain capable of degrading nicotinic acid efficiently and rapidly, which is identified as Pseudomonas putida (Pseudomonas putida), is named as N17, is currently deposited in China general microbiological culture collection center, and has the address: the microbial research institute of China academy of sciences, No. 3, Xilu No. 1, Beijing, Chaoyang, is published by letter 100101, and has a preservation date of 2020, 8, 17 days and a preservation number of CGMCC NO. 20429. The strain is a mutant strain obtained by taking pseudomonas putida H-20 screened from sludge of Ribang Biotechnology Ltd as an initial strain and carrying out bacterial liquid mutagenesis screening on the initial strain. The bacterial colony of the strain is small in morphology, oval, smooth in edge, milky white and translucent.
The second purpose of the invention is to provide the bacterial liquid containing the pseudomonas putida N-17.
In some embodiments, the bacterial liquid OD600 of the invention is 1.0-2.5, preferably 1.2-1.8.
The invention also provides a sewage treatment agent containing the pseudomonas putida N-17.
The fourth purpose of the invention is to provide the application of the pseudomonas putida N-17 or the bacterial liquid containing the pseudomonas putida N-17 or the sewage treatment agent containing the pseudomonas putida N-17 in the degradation of nicotinic acid.
The fifth purpose provided by the invention is to provide the application of the pseudomonas putida N-17 or the bacterial liquid containing the pseudomonas putida N-17 or the sewage treatment agent containing the pseudomonas putida N-17 in the sewage treatment containing nicotinic acid, and in some more specific embodiments, in the sewage treatment of 3-cyanopyridine production.
In some embodiments, the application of the invention is to inoculate pseudomonas putida N-17 or a bacterial solution containing the pseudomonas putida N-17 or a sewage treatment agent containing the pseudomonas putida N-17 into sewage containing nicotinic acid; in some specific embodiments, the bacterial liquid inoculation amount of the bacterial liquid with the OD600 of 1.2-2.5 is 15-60 ml of bacterial liquid/L of sewage.
In some preferred embodiments, the application of the present invention is to introduce pseudomonas putida N-17 or a bacterial solution containing said pseudomonas putida N-17 or a sewage treatment agent containing said pseudomonas putida N-17 into sewage containing nicotinic acid, and simultaneously or sequentially add additive a, wherein additive a is a mixture of peptone and yeast powder.
In some embodiments, the additive A is prepared from peptone and yeast powder in a weight ratio of (1-5): 1, the preferable weight ratio is (2-4): 1.
in some embodiments, the additive A is added in an amount of 0.05-5 g/L of sewage, and in some preferred embodiments, the additive A is added in an amount of 0.6-5 g/L of sewage; in some more specific embodiments, the concentration is 0.8 to 3.2g/L of wastewater.
The invention has the beneficial effects that:
(1) the Pseudomonas putida N-17(Pseudomonas putida N-17) strain has good hydrochloric acid biodegradation capacity; after the strain is subjected to fermentation culture in nicotinic acid wastewater with the pH value of 6.5-8.0, the concentration (OD600) of bacterial liquid in fermentation liquor can reach 1.8-2.4, the strain has a good nicotinic acid degradation effect, can rapidly degrade high-content pyridine derivative nicotinic acid in the wastewater, can be directly used in a conventional sewage treatment system, and can achieve more than 60% of 96-hour degradation of the nicotinic acid.
(2) In the specific application process, the degradation effect of the nicotinic acid can be further improved by adding the additive, the degradation of the nicotinic acid for 48 hours can reach more than 90 percent, and the method has very important significance for sewage treatment.
(3) The Pseudomonas putida N-17(Pseudomonas putida N-17) strain disclosed by the invention metabolizes nicotinic acid, is used for synthesizing cells, partially becomes carbon dioxide, is discharged into the atmosphere, and does not generate secondary pollution.
Drawings
FIG. 1 is a colony morphology of Pseudomonas putida (Pseudomonas putida) N17 according to the present invention;
FIG. 2 shows the result of microscopic examination of Pseudomonas putida (Pseudomonas putida) N17 according to the present invention;
FIG. 3 is an HPLC chromatogram of a nicotinic acid standard according to the present invention;
FIG. 4 is an HPLC detection profile of initial niacin in niacin wastewater according to example 8 of the present invention;
FIG. 5 is an HPLC detection chart of nicotinic acid content at 24 hours of reaction in the experimental group 5-1 of example 8 according to the present invention;
FIG. 6 is a HPLC chromatogram for measuring the nicotinic acid content at 48 hours of reaction in the experimental group 5-1 of example 8 according to the present invention.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
In the following examples, unless otherwise specified, the media ratios were as follows:
the formula of the PYD6 solid medium is as follows: 5-30g nicotinic acid and KH2PO4 0.5-1.5g、K2HPO4 0.5-1.5g、MgSO4·7H2O 0.1-1g、NaCl 1g、(NH4)2SO41-5g of agar powder, 10-20g/L of agar powder, 1ml of trace elements and NaOH, adjusting the pH value to 7.0-7.5, and adding distilled water to 1L.
The formula of the PYD6 liquid culture medium is as follows: 5-30g nicotinic acid and KH2PO4 0.5-1.5g、K2HPO4 0.5-1.5g、MgSO4·7H2O 0.1-1g、NaCl 1g、(NH4)2SO4 1-5g, trace elements 1ml, NaOH to adjust pH to 7.0-7.5, and distilled water to 1L.
The formula of the trace elements is as follows: NaMoO4·2H2O 1.5g、MnSO4 0.13g、ZnCl2 0.23g、CuSO4·5H2O 0.05g、CoCl2 0.6g、FeCl3 0.2g、CaCl20.2g, distilled water was added to 1L.
Example 1:
obtaining sludge from a sewage treatment tank of Ribang Biotechnology Ltd, weighing 10g of the sludge sample, placing the sludge sample in a shake flask, preparing bacterial suspension with 90mL of distilled water (fully shaking with an oscillator), and preparing the bacterial suspension with the concentration of 10 by adopting a 10-fold dilution method-3、10-4、10-5、10-6The bacterial suspension of (4). Taking 0.1mL of bacterial suspension with each concentration, respectively coating the bacterial suspension on a PYD6 solid culture medium taking nicotinic acid as a unique carbon source, coating 9 plates with each concentration, respectively placing the bacterial suspensions in a constant-temperature incubator at 18 ℃, 28 ℃ and 37 ℃ for 3 times after coating, and culturing for a proper time until bacterial colonies grow out. And selecting colonies growing in the plate, carrying out plate streaking passage, separating a single colony twice or three times, and purifying the strain. The strain was identified as Pseudomonas putida (Pseudomonas putida), and was named Pseudomonas putida H-20.
Example 2: study of ultraviolet mutagenesis conditions
Inoculating activated Pseudomonas putida H-20 in LB solid culture medium into PYD6 liquid culture medium, culturing at 28 deg.C and 220rpm for 1-3 days until bacterial liquid OD600 is 0.2-0.4. Centrifuging the culture solution at 8000r/min and 4 deg.C for 10min, discarding supernatant, washing bacterial sludge with normal saline twice, suspending the bacterial sludge in normal saline, and adjusting thallus concentration to 0.01 g/mL.
16mL of the prepared bacterial liquid was placed in a dish with a diameter of 9cm, stirred on a magnetic stirrer, placed under a 15W ultraviolet lamp at a distance of 30cm, and 0.5mL of the bacterial liquid was sampled as a control. Irradiating with ultraviolet lamp preheated for 20-30min for 180s, taking 0.5mL of mutagenic bacteria liquid every 20s, diluting under red light in gradient of 10 times, spreading 0.1mL of mutagenic bacteria liquid on LB solid culture medium, wrapping with black cloth, and culturing in constant temperature incubator at 28 deg.C for 1 day.
Colonies were counted and the mortality (%) calculated as (%) (control per ml-number of bacteria per ml after treatment)/control per ml × 100%. By calculation, the fatality rate of ultraviolet lamp irradiation for 60-120s is 82-96%.
Example 3: ultraviolet mutation breeding of bacterial liquid
Inoculating activated Pseudomonas putida H-20 in LB solid culture medium into PYD6 liquid culture medium, culturing at 28 deg.C and 220rpm for 1-3 days until bacterial liquid OD600 is 0.2-0.4. Centrifuging the culture solution at 8000r/min and 4 deg.C for 10min, discarding supernatant, washing bacterial sludge with normal saline twice, suspending the bacterial sludge in normal saline, and adjusting thallus concentration to 0.01 g/mL.
16mL of the prepared bacterial liquid was placed in a dish with a diameter of 9cm, stirred on a magnetic stirrer, placed under a 15W ultraviolet lamp at a distance of 30cm, and 0.5mL of the bacterial liquid was sampled as a control. Irradiating with ultraviolet lamp preheated for 20-30min for 70-120s, taking 0.5mL of mutagenic bacteria liquid every 10s, diluting under red light for 10 times, spreading 0.1mL of mutagenic bacteria liquid on PYD6 solid culture medium, wrapping with black cloth, and culturing in 28 deg.C incubator for 2-3 days.
And selecting single bacterial colonies which grow rapidly on the flat plate after mutagenesis and have large bacterial colonies, carrying out streak culture on a PYD6 solid culture medium at 28 ℃, then selecting trace bacteria, transferring the trace bacteria into a PYD6 liquid culture medium, carrying out culture at 28 ℃, carrying out 220rpm, and measuring the OD600 of the bacterial liquid every 24 hours. Carrying out subculture on the strain which rapidly grows in a PYD6 liquid culture medium with nicotinic acid as a unique carbon source for 4-5 times, and then carrying out the next round of ultraviolet mutagenesis on the dominant strain with stable genetic characters.
Example 4: research on nitrosoguanidine mutagenesis conditions
The dominant pseudomonas putida mutant strain after multiple rounds of ultraviolet mutagenesis was activated in PYD6 solid medium. Selecting a single clone, inoculating the single clone into a PYD6 liquid culture medium, culturing for 1d at 28 ℃ and 220rpm until the bacterial liquid OD600 is between 0.2 and 0.4. Centrifuging the culture solution at 8000r/min and 4 deg.C for 10min, discarding supernatant, washing bacterial sludge with normal saline twice, suspending the bacterial sludge in normal saline, and adjusting thallus concentration to 0.01 g/mL.
Taking 3 equal parts of bacterial liquid, adding a certain amount of nitrosoguanidine solution to ensure that the final concentration of the nitrosoguanidine in the mixed liquid is 0.05mg/mL, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL and 0.4mg/mL respectively, and carrying out water bath at the temperature of 28 ℃ for 30 min. Then centrifuging for 10min at 12000r/min and 4 ℃, discarding the supernatant, washing the thalli for 3-5 times by using sterile normal saline, terminating the mutagenesis reaction, after that, respectively coating 0.1mL of the thalli on a PYD6 solid culture medium plate after 10-fold gradient dilution, and culturing for 2 days in a constant temperature incubator at 28 ℃.
Colonies were counted and the mortality (%) calculated as (%) (control per ml-treated bacteria per ml)/control 100% bacteria per ml. By calculation, the lethality was 90% at a final concentration of 0.1mg/mL of nitrosoguanidine.
Example 5: mutagenesis of nitrosoguanidine
The mutagenized strain of example 4 was activated in PYD6 solid medium. Selecting a single clone, inoculating the single clone into a PYD6 liquid culture medium, culturing for 1d at 28 ℃ and 220rpm until the bacterial liquid OD600 is 0.2-0.4. Centrifuging the culture solution at 8000r/min and 4 deg.C for 10min, discarding supernatant, washing bacterial sludge with normal saline twice, suspending the bacterial sludge in normal saline, and adjusting thallus concentration to 0.01 g/mL.
Taking 3 equal parts of bacterial liquid, adding a certain amount of nitrosoguanidine solution to ensure that the final concentration of the nitrosoguanidine in the mixed liquid is 0.1mg/mL, and carrying out water bath for 30min at the temperature of 28 ℃. Then centrifuging for 10min at 12000r/min and 4 ℃, discarding the supernatant, washing the thalli for 3-5 times by using sterile normal saline, terminating the mutagenesis reaction, after that, respectively coating 0.1mL of the thalli on a PYD6 solid culture medium plate after 10-fold gradient dilution, and culturing for 2 days in a constant temperature incubator at 28 ℃.
Example 6 screening and identification of strains capable of efficiently degrading nicotinic acid
After mutagenesis, single colonies which grow rapidly on the plate and are large in colony size in example 5 are selected and streaked on a PYD6 solid culture medium at 28 ℃, then micro-thalli are selected and transferred into a PYD6 liquid culture medium, the culture is carried out at 28 ℃ and 220rpm, and the OD600 of bacterial liquid is measured every 24 hours. Carrying out 3-5 times of subculture on a strain which rapidly grows in a PYD6 liquid culture medium with nicotinic acid as a unique carbon source, and then carrying out seed preservation on a dominant strain with stable genetic character to obtain a strain capable of efficiently and rapidly degrading nicotinic acid. The obtained strain was subjected to 16S rDNA amplification and sequence analysis, and bacterial DNA was extracted using QIAamp kit following the kit procedures strictly, and 16S rDNA of the bacterium was amplified by PCR reaction. PCR reaction (50. mu.L): 50ng of template DNA, 20pmol/L of Primer F (5 '-AGAGTTTGATCMTGGCTCAG-3'), 20pmol/L of Primer R (5 '-TACGGYTACCTTGTTACGACT-3'), 2.5U of Taq DNA polymerase, and 50. mu.L of ultrapure water. And (3) PCR reaction conditions: 5min at 94 ℃; 94 ℃ 30sec, 57 ℃ 45sec, 72 ℃ 1min (32 cycles); 5min at 72 ℃. Separating the PCR product by 2% agarose electrophoresis, after a band of about 1500bp is confirmed, sending the purified PCR product to a Shanghai bioengineering company for sequencing, processing a sequencing result by SerialCloner software, wherein a 16S rDNA sequence is shown as SEQ ID NO:1, performing Blast comparison analysis on the 16S rDNA sequence and a related 16S rDNA sequence in a GeneBank database, and identifying the bacterium as Pseudomonas putida (Pseudomonas putida) according to the result, wherein the bacterium is named as N-17. After 48h, the concentration of the bacterial liquid (OD600) is 1.5, and the color of the bacterial liquid is light yellow, which is different from black brown before mutagenesis, and the bacterial liquid is small in shape, oval, smooth in edge, milky white and semitransparent (as shown in figures 1 and 2). Submitting China general microbiological culture collection management center collection on 17.8.2020, addresses: the microbial research institute of China academy of sciences, No. 3, Xilu No. 1, Beijing, Chaoyang, is published by letter 100101, and has a preservation date of 2020, 8, 17 days and a preservation number of CGMCC NO. 20429.
Example 7
EXAMPLE 6 comparison of the screened Pseudomonas putida N-17(Pseudomonas putida N-17) with the original strain H-20 of example 1 in the degradation effect of nicotinic acid
The nicotinic acid wastewater is taken from the production wastewater of 3-cyanopyridine of Biotechnology Limited, Anhui, and contains nicotinic acid (7000-30000ppm), 3-cyanopyridine (100-5000mg/L), 3-methylpyridine (500-4000mg/L), pyridine (100-2000mg/L), ammonia nitrogen (500-3000mg/L), total nitrogen (500-5000mg/L) and pH (6.5-8.0).
The method specifically comprises the following steps:
1. seed culture:
single colonies of Pseudomonas putida N-17(Pseudomonas putida N-17) and Pseudomonas putida H-20(Pseudomonas putida H-20) were inoculated into PYD6 broth, activated, and cultured at 28 ℃ for 2-3 days to OD600 to 1.2-1.8, respectively.
2. Fermentation culture:
inoculating the seed solution obtained in the step 1) into 50ml of nicotinic acid wastewater according to the inoculation amount of 2%, and culturing at 28 ℃ and 220 rpm.
3. Determination of nicotinic acid degradation Effect
The reaction mixture was incubated at 28 ℃ and 220rpm, and the nicotinic acid concentration was measured by sampling every 24 hours (HPLC method).
TABLE 1 comparison of Pseudomonas putida H-20 and Pseudomonas putida N-17 Niacin degrading Capacity
As can be seen from Table 1, Pseudomonas putida N-17(Pseudomonas putida N-17) has a good nicotinic acid degradation effect.
Example 8: experiment for rapidly degrading nicotinic acid
1. Seed culture:
a single colony of Pseudomonas putida N-17(Pseudomonas putida N-17) selected in example 6 was inoculated into PYD6 medium and activated, and cultured at 28 ℃ for 2 to 3 days to OD600 of 1.2 to 1.8.
2. Fermentation culture:
inoculating the seed solution obtained in the step 1 into 50ml of nicotinic acid wastewater according to the inoculation amount of 2%, and adding an additive A (the weight ratio of peptone to yeast powder is 3: 2) into the nicotinic acid wastewater to improve the degradation effect of the nicotinic acid. The additive A addition amounts are respectively adjusted as follows: 0g, 0.005g, 0.01g, 0.02g, 0.04g, 0.08g, 0.16 g. The reaction mixture was incubated at 28 ℃ and 220rpm, and the nicotinic acid concentration was measured by sampling every 24 hours (HPLC method).
3. Results
As shown in Table 2 and FIGS. 3 to 6, the nicotinic acid degradation rate reached 99.5% or more and the effect of degrading nicotinic acid with Pseudomonas putida (Pseudomonas putida N-17) was optimized when 0.04g or more of additive A was used as the amount of the reaction mixture.
TABLE 250 ml PYD6 Medium with additive A for increasing the Niacin degradation
In addition, experiments show that the Pseudomonas putida N-17(Pseudomonas putida N-17) strain disclosed by the invention metabolizes nicotinic acid for synthesizing cells, partially becomes carbon dioxide, is discharged into the atmosphere and does not generate secondary pollution.
Sequence listing
<110> Anhui Ribang Biotech Co., Ltd
<120> Pseudomonas putida strain and application thereof in degradation of nicotinic acid
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1536
<212> DNA
<213> Pseudomonas putida
<400> 1
aactgaagag tttgatcatg gctcagattg aacgctggcg gcaggcctaa cacatgcaag 60
tcgagcggat gacgggagct tgctccttga ttcagcggcg gacgggtgag taatgcctag 120
gaatctgcct ggtagtgggg gacaacgttt cgaaaggaac gctaataccg catacgtcct 180
acgggagaaa gcaggggacc ttcgggcctt gcgctatcag atgagcctag gtcggattag 240
ctagttggtg gggtaatggc tcaccaaggc gacgatccgt aactggtctg agaggatgat 300
cagtcacact ggaactgaga cacggtccag actcctacgg gaggcagcag tggggaatat 360
tggacaatgg gcgaaagcct gatccagcca tgccgcgtgt gtgaagaagg tcttcggatt 420
gtaaagcact ttaagttggg aggaagggca gtaagttaat accttgctgt tttgacgtta 480
ccgacagaat aagcaccggc taactctgtg ccagcagccg cggtaataca gagggtgcaa 540
gcgttaatcg gaattactgg gcgtaaagcg cgcgtaggtg gtttgttaag ttggatgtga 600
aagccccggg ctcaacctgg gaactgcatc caaaactggc aagctagagt acggtagagg 660
gtggtggaat ttcctgtgta gcggtgaaat gcgtagatat aggaaggaac accagtggcg 720
aaggcgacca cctggactga tactgacact gaggtgcgaa agcgtgggga gcaaacagga 780
ttagataccc tggtagtcca cgccgtaaac gatgtcaact agccgttgga atccttgaga 840
ttttagtggc gcagctaacg cattaagttg accgcctggg gagtacggcc gcaaggttaa 900
aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960
aacgcgaaga accttaccag gccttgacat gcagagaact ttccagagat ggattggtgc 1020
cttcgggaac tctgacacag gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg 1080
ggttaagtcc cgtaacgagc gcaacccttg tccttagtta ccagcacgtt atggtgggca 1140
ctctaaggag actgccggtg acaaaccgga ggaaggtggg gatgacgtca agtcatcatg 1200
gcccttacgg cctgggctac acacgtgcta caatggtcgg tacagagggt tgccaagccg 1260
cgaggtggag ctaatctcac aaaaccgatc gtagtccgga tcgcagtctg caactcgact 1320
gcgtgaagtc ggaatcgcta gtaatcgcga atcagaatgt cgcggtgaat acgttcccgg 1380
gccttgtaca caccgcccgt cacaccatgg gagtgggttg caccagaagt agctagtcta 1440
accttcggga ggacggttac cacggtgtga ttcatgactg gggtgaagtc gtaacaaggt 1500
agccgtaggg gaacctgcgg ctggatcacc tcctta 1536
Claims (10)
1. A bacterial strain N-17 for degrading nicotinic acid has a preservation number of CGMCC NO. 20429.
2. A bacterial liquid comprising the strain according to claim 1.
3. A wastewater treatment agent comprising pseudomonas putida N-17 as set forth in claim 1.
4. The use of the strain N-17 of claim 1, the bacterial liquid of claim 2 or the sewage treatment agent of claim 3 in the degradation of nicotinic acid.
5. Use of the strain N-17 according to claim 1, the bacterial liquid according to claim 2 or the sewage treatment agent according to claim 3 in the treatment of sewage containing nicotinic acid.
6. The use of claim 5, wherein the wastewater containing nicotinic acid is wastewater from 3-cyanopyridine production.
7. The use according to claim 5, wherein the bacterial liquid of the strain N-17 of claim 1 or the bacterial liquid of claim 2 or the sewage treatment agent of claim 3 is inoculated into sewage containing nicotinic acid; preferably, the bacterial liquid inoculation amount of the OD600 of 1.2-2.5 is 15-60 ml bacterial liquid/L sewage.
8. The use according to claim 7, wherein additive A is added to the sewage containing nicotinic acid, wherein additive A is a mixture of peptone and yeast powder.
9. The application of claim 8, wherein the additive A is prepared from peptone and yeast powder in a weight ratio of (1-5): 1; the preferable weight ratio is (2-4): 1.
10. the application of the composition according to claim 8, wherein the additive A is added in an amount of 0.05-5 g/L sewage; the preferable addition amount is 0.6-5 g/L sewage; more preferably, the amount of the wastewater is 0.8-3.2 g/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010970823.2A CN112011490B (en) | 2020-09-16 | 2020-09-16 | Pseudomonas putida strain and application thereof in degrading nicotinic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010970823.2A CN112011490B (en) | 2020-09-16 | 2020-09-16 | Pseudomonas putida strain and application thereof in degrading nicotinic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112011490A true CN112011490A (en) | 2020-12-01 |
| CN112011490B CN112011490B (en) | 2022-07-19 |
Family
ID=73522039
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010970823.2A Active CN112011490B (en) | 2020-09-16 | 2020-09-16 | Pseudomonas putida strain and application thereof in degrading nicotinic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112011490B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877264A (en) * | 2021-04-12 | 2021-06-01 | 江南大学 | Pseudomonas putida mutant strain and application thereof in biological preparation of 6-hydroxynicotinic acid |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005106006A2 (en) * | 2004-05-05 | 2005-11-10 | Jubilant Organosys Limited | Biotransformation of nicotinic acid to 6-hydroxynicotinic acid |
| CN102337233A (en) * | 2011-05-06 | 2012-02-01 | 江南大学 | Pseudomonas putida and method for producing nicotinic acid or isonicotinic acid through converting Pseudomonas putida |
| WO2015191582A1 (en) * | 2014-06-09 | 2015-12-17 | BiOWiSH Technologies, Inc. | Microbial compositions for hydrocarbon remediation and methods of use thereof |
| CN106148448A (en) * | 2016-08-28 | 2016-11-23 | 山东昆达生物科技有限公司 | A kind of method utilizing pseudomonas putida to prepare nicotinic acid |
-
2020
- 2020-09-16 CN CN202010970823.2A patent/CN112011490B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005106006A2 (en) * | 2004-05-05 | 2005-11-10 | Jubilant Organosys Limited | Biotransformation of nicotinic acid to 6-hydroxynicotinic acid |
| CN102337233A (en) * | 2011-05-06 | 2012-02-01 | 江南大学 | Pseudomonas putida and method for producing nicotinic acid or isonicotinic acid through converting Pseudomonas putida |
| WO2015191582A1 (en) * | 2014-06-09 | 2015-12-17 | BiOWiSH Technologies, Inc. | Microbial compositions for hydrocarbon remediation and methods of use thereof |
| CN106148448A (en) * | 2016-08-28 | 2016-11-23 | 山东昆达生物科技有限公司 | A kind of method utilizing pseudomonas putida to prepare nicotinic acid |
Non-Patent Citations (4)
| Title |
|---|
| JOSÉ I. JIMÉNEZ 等: "Deciphering the genetic determinants for aerobic nicotinic acid degradation: the nic cluster from Pseudomonas putida KT2440", 《PROC NATL ACAD SCI U S A.》 * |
| JOSÉ IGNACIO JIMÉNEZ 等: "A finely tuned regulatory circuit of the nicotinic acid degradation pathway in Pseudomonas putida", 《ENVIRON MICROBIOL.》 * |
| LI AIWEN 等: "Characterization and Genome Analysis of a Nicotine and Nicotinic Acid-Degrading Strain Pseudomonas putida JQ581 Isolated from Marine", 《MAR DRUGS.》 * |
| XIAO YUJIE 等: "FinR Regulates Expression of nicC and nicX Operons, Involved in Nicotinic Acid Degradation in Pseudomonas putida KT2440", 《APPL ENVIRON MICROBIOL.》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877264A (en) * | 2021-04-12 | 2021-06-01 | 江南大学 | Pseudomonas putida mutant strain and application thereof in biological preparation of 6-hydroxynicotinic acid |
| CN112877264B (en) * | 2021-04-12 | 2022-04-05 | 江南大学 | Pseudomonas putida mutant strain and application thereof in biological preparation of 6-hydroxynicotinic acid |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112011490B (en) | 2022-07-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109576187B (en) | Cyanide degradation strain and method for degrading cyanide by using same | |
| CN110643548B (en) | Microbacterium flavum for degrading aniline and application thereof | |
| CN110655198B (en) | Method for treating nitrogen-containing wastewater by using heterotrophic nitrification-aerobic denitrification paracoccus strain | |
| CN110791444B (en) | Pseudomonas stutzeri, composite microbial inoculum prepared from pseudomonas stutzeri and application of composite microbial inoculum | |
| CN109456913B (en) | Pseudomonas aeruginosa and method for treating organic wastewater by applying pseudomonas aeruginosa | |
| CN108060101B (en) | Ocean Dietzia W02-3a and application thereof in denitrification | |
| CN110656057A (en) | Heterotrophic nitrification-aerobic denitrification paracoccus strain, seed liquid, preparation method and application thereof | |
| CN112011490B (en) | Pseudomonas putida strain and application thereof in degrading nicotinic acid | |
| CN109055259B (en) | Pseudomonas XD-3, application thereof and microbial flocculant | |
| CN107164280B (en) | Vomitoxin degrading bacterium and application thereof | |
| CN113684154B (en) | Pseudomonas Kunmingensis strain and application thereof in environment-friendly water treatment | |
| CN109468251A (en) | The method of one plant of thiocarbamide degradation bacteria strains and the application bacterial strain processing waste water containing thiocarbamide | |
| CN112300961B (en) | Quinoline degrading strain and application thereof in quinoline-containing wastewater | |
| CN115960745A (en) | Salt-tolerant epichlorohydrin degrading bacterium and application thereof in treatment of epichlorohydrin in high-salinity water | |
| CN115651855A (en) | Pseudomonas stutzeri and its product and use | |
| CN109666613B (en) | Facultative autotrophic rhizobium with nitrate reduction ferrous oxidation function and application thereof | |
| CN113755400A (en) | Bacillus cereus for degrading aureomycin and application thereof | |
| CN111763633B (en) | Salt-tolerant denitrifying bacterium and application thereof | |
| CN115449492B (en) | Salt-tolerant bacillus capable of efficiently degrading N, N-dimethylformamide and application thereof | |
| CN114480221B (en) | Equilibrium brevifolium and application thereof in formaldehyde degradation | |
| CN111235053B (en) | Bacillus amyloliquefaciens Z1112 and application thereof | |
| CN114874943B (en) | Efficient denitrification compound preparation and application thereof | |
| CN114058554B (en) | Composting pseudomonas strain and application thereof | |
| CN110468077B (en) | Microbacterium Bn-88, microbial agent and application thereof in degrading petroleum hydrocarbon | |
| CN112501080B (en) | Application of sphingosine bacillus clarfungi in preparation of microbial agent for removing COD (chemical oxygen demand) in petroleum wastewater |
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 |