CN113862180A - Pseudomonas putida and application thereof in degrading total nitrogen in white spirit wastewater - Google Patents
Pseudomonas putida and application thereof in degrading total nitrogen in white spirit wastewater Download PDFInfo
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Abstract
The invention discloses Pseudomonas putida and application thereof in degrading total nitrogen in white spirit wastewater, belonging to the technical field of microbial degradation, wherein the Pseudomonas putida is named as Pseudomonas putida WL-2, is preserved in Guangdong province microbial strain preservation center, has the preservation date of 2021, 6 and 28 days, and has the preservation number of GDMCC NO: 61748. The pseudomonas putida has the characteristics of easy culture, strong high temperature resistance, acid and alkali resistance, excellent aerobic denitrification capability and high total nitrogen degradation rate, is aerobic denitrifying bacteria, can simultaneously carry out nitrification and denitrification under aerobic conditions, has the total nitrogen degradation rate of 60-85% in the white wine wastewater, provides an important strain for the biological denitrification process of the white wine wastewater, and has practical application value in degrading the total nitrogen in the white wine wastewater.
Description
Technical Field
The invention relates to the technical field of microbial degradation, in particular to pseudomonas putida and application thereof in degrading total nitrogen in white spirit wastewater.
Background
As the traditional brewing industry in China, the brewing of white spirit is rapidly developed in recent years, the yield of the white spirit is in an increasing state, but a large amount of white spirit wastewater is generated in the production process. The white spirit wastewater is mainly derived from distilled kettle bottom water with high organic matter concentration, fermentation blind ditch water, distillation section ground washing water, leakage water of a wine warehouse, sorghum washing water and soaking water during process operation of 'sand falling' and 'coarse sand', and the like.
At present, the liquor wastewater generally adopts a multi-stage treatment process mainly comprising anaerobic treatment and aerobic treatment so as to reach the emission standard of pollutants for fermented alcohol and liquor industrial water (GB 27631-2011). The process can effectively degrade most organic matters in the white spirit wastewater, but the nitrogen-containing compounds are still higher, so that further biological denitrification is needed. The traditional biological denitrification technology is based on two independent processes of microbial nitrification and denitrification. The first stage is carried out by nitrationUnder aerobic condition, ammonia nitrogen in water is converted into NO2-N and NO3N, carrying out denitrification by using the nitration product as a substrate. In the denitrification of the second stage, the denitrifying bacteria further convert the nitrate nitrogen and the nitrite nitrogen into nitrogen, thereby achieving the purpose of removing nitrogen ions in the water body. Along with the technical progress, heterotrophic denitrifying bacteria gradually enter the visual field of people, and can simultaneously carry out nitrification and denitrification under aerobic conditions. The aerobic denitrifying bacteria play a key role in the biological denitrification process. Therefore, the screening of the aerobic denitrifying bacteria has important significance for removing the total nitrogen in the white spirit wastewater. However, reports that pseudomonas putida degrades total nitrogen in white spirit wastewater are not seen so far.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide Pseudomonas putida (Pseudomonas putida) and application thereof in degrading total nitrogen in white spirit wastewater, wherein the strain has the characteristics of easy culture, strong high temperature resistance, acid and alkali resistance, excellent aerobic denitrification capability and high total nitrogen degradation rate.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides Pseudomonas putida (Pseudomonas putida), which is selected from activated sludge of a white spirit factory sewage treatment station, is named as Pseudomonas putida WL-2, is preserved in Guangdong province microorganism strain preservation center, has the preservation date of 2021, 6 months and 28 days, and has the preservation number of GDMCC NO: 61748.
The invention also provides application of the Pseudomonas putida (Pseudomonas putida WL-2) in degrading total nitrogen in white spirit wastewater.
Further, the application of the Pseudomonas putida (Pseudomonas putida WL-2) in degrading the total nitrogen in the white spirit wastewater comprises the following steps:
step (1): preparing activated bacterium liquid of Pseudomonas putida (Pseudomonas putida WL-2);
step (2): and (2) putting the activated Pseudomonas putida (Pseudomonas putida WL-2) bacterial liquid obtained in the step (1) into the white spirit wastewater for degradation.
Further, the concentration of the activated bacterial liquid of Pseudomonas putida (Pseudomonas putida WL-2) in the step (1) is 2X 107～10×107CFU/mL。
Further, the total nitrogen concentration in the white spirit wastewater in the step (2) is 4500-5000 mg/L.
Further, the degradation conditions in the step (2) are as follows: the degradation temperature is 25-30 ℃, the rotating speed is 150-210 r/min, and the degradation time is 24-48 h.
Further, the inoculum size of the Pseudomonas putida (Pseudomonas putida WL-2) is 1-5 wt%.
Further, the preparation method of the Pseudomonas putida (Pseudomonas putida WL-2) activated bacterium solution comprises the following steps:
1) inoculating a Pseudomonas putida (Pseudomonas putida WL-2) strain into a seed liquid culture medium, and carrying out shake cultivation at the temperature of 25-30 ℃ and at the speed of 150-210 r/min for 12-24 h to obtain a seed liquid;
2) placing the enriched liquid culture medium in a conical flask, sterilizing and cooling, then inoculating 4-8 wt% of the seed liquid obtained in the step 1) on an aseptic operation table, and then placing the seed liquid at 25-30 ℃ and 150-210 r/min for shake cultivation for 1-2 days to obtain the Pseudomonas putida (Pseudomonas putida WL-2) activated bacterial liquid.
Further, the formula of the seed liquid culture medium in the step 1) comprises the following components in parts by weight: 3-7 parts of yeast extract, 8-12 parts of peptone, 0.1-0.4 part of monopotassium phosphate, 0.1-0.4 part of dipotassium phosphate, 0.1-0.4 part of magnesium sulfate and 800-1100 parts of distilled water.
Further, the enriched liquid in the step 2) comprises the following components in parts by weight: 14-18 parts of glucose, 4-8 parts of peptone, 0.5-1.5 parts of magnesium sulfate, 1-4 parts of monopotassium phosphate and 800-1100 parts of distilled water.
Further, the pH value of the enriched liquid in the step 2) is 6.5-7.5.
Further, the culture medium is sterilized for 20-30 min at 120-125 ℃.
In summary, the invention has the following advantages:
1. the liquor wastewater is acidic (the pH value is about 5.95-6.85), a common nitrifying strain cannot grow in a large amount, and the Pseudomonas putida (Pseudomonas putida WL-2) provided by the invention has strong environmental adaptability and is easy to culture.
2. The white spirit wastewater belongs to high-concentration organic wastewater, the total nitrogen content is high, and the Pseudomonas putida (Pseudomonas putida WL-2) provided by the invention has higher capability of degrading the total nitrogen. The strain is subjected to subculture for many times, and the strain is still stably inherited and has stable degradation capability; during the passage, no obvious variant strain appears.
3. The Pseudomonas putida (Pseudomonas putida WL-2) is aerobic denitrifying bacteria, can simultaneously carry out nitrification and denitrification under aerobic conditions, has a total nitrogen degradation rate of 60-85% in the white spirit wastewater, provides an important strain for the biological denitrification process of the white spirit wastewater, and has practical application value in degrading the total nitrogen in the white spirit wastewater.
Drawings
FIG. 1 is a colony morphology of Pseudomonas putida (Pseudomonas putida WL-2) on LB plate medium in the present invention;
FIG. 2 is a graph showing the results of gram-staining Pseudomonas putida (Pseudomonas putida WL-2) in the present invention;
FIG. 3 is a scanning electron micrograph of Pseudomonas putida (Pseudomonas putida WL-2) according to the present invention;
FIG. 4 is a sequence evolutionary tree diagram of Pseudomonas putida (Pseudomonas putida WL-2) according to the present invention;
FIG. 5 is a graph showing the results of growth and denitrification tests of Pseudomonas putida (Pseudomonas putida WL-2) according to the present invention;
FIGS. 6 to 7 are graphs showing the results of acid and alkali resistance and high temperature resistance tests of Pseudomonas putida (Pseudomonas putida WL-2) according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Thus, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 isolation, purification and screening of Pseudomonas putida (Pseudomonas putida WL-2)
The separation, purification and screening of Pseudomonas putida (Pseudomonas putida WL-2) comprises the following steps:
(1) sampling in a liquor factory sewage treatment station, selecting activated sludge, and filling into a sterile sampling bag;
(2) weighing 10g of the activated sludge obtained in the step (1) and placing the activated sludge into a 250mL conical flask filled with 90mL of DM basal medium for domestication culture;
(3) taking the supernatant of the mixed solution in the step (2) to be sequentially diluted into 10 times by a 10-fold dilution method-5、10-6、10-7、10-7、10-8、10-9A double dilution solution;
(4) respectively transferring 0.1mL of each diluent obtained in the step (3) by using a sterile liquid transfer gun, coating the diluent on a BTB solid culture medium filled with 20mL of BTB solid culture medium, and uniformly coating the bacterial liquid on the surface of the culture medium by using a coating rod;
(5) placing the culture medium obtained in the step (4) in a constant temperature incubator at 30 ℃ for 2 d;
(6) selecting bacteria with blue single colony in the culture medium, and respectively selecting single colony with different forms to separate and purify in a BTB solid culture medium;
(7) culturing in 30 deg.C constant temperature incubator for 2d, and repeating the steps until each bacterium is pure colony; when the cultured strain is purified, storing the strain in a refrigerator at-20 ℃ for later use;
(8) inoculating the strains obtained by primary screening into a seed liquid culture medium, and performing shake cultivation at 30 ℃ and 160r/min for 24h to obtain seed liquid; the formula of the seed liquid culture medium is as follows: 5g of yeast extract, 10g of peptone, 0.25g of dihydrogen phosphate, 0.3g of dipotassium hydrogen phosphate, 0.3g of magnesium sulfate and 1L of deionized water;
(9) taking 100mL of DM liquid culture medium to be based on a 250mL conical flask, sterilizing and cooling, inoculating 10 wt% of seed liquid on a sterile operating platform, and then placing the seed liquid on a shaking table at 30 ℃ and 160r/min for 2 days; centrifuging at the normal temperature of 5000r/min for 10min after culture, measuring the total nitrogen index of the supernatant, and selecting a strain with higher total nitrogen degradation rate; wherein the strain with the number of WL-2 is named as Pseudomonas putida (Pseudomonas putida WL-2), and the total nitrogen degradation rate is 64.18%.
The formula of the DM basic culture medium is 1.5g of monopotassium phosphate, 0.01g of magnesium sulfate, 7.9g of disodium hydrogen phosphate, 5.66g of sodium citrate and 0.84g of sodium nitrate, 1L of deionized water is added, and the pH value is adjusted to 7; the BTB solid culture medium is a DM basic culture medium, 0.0006g of bromothymol blue, 15g of agar and 1L of deionized water, and the pH is adjusted to 7.0; the culture medium is sterilized at 121 deg.C for 20 min.
Example 2 identification of Pseudomonas putida (Pseudomonas putida WL-2)
Identification of Pseudomonas putida (Pseudomonas putida WL-2) comprising the following steps:
(1) observation of strain morphology and culture characteristics
Inoculating the strain with the best total nitrogen degradation obtained by rescreening in example 1 on a beef extract peptone (NA) solid medium, culturing at 37 ℃ for 18h, and observing the morphological characteristics of colonies, wherein the colonies are light yellow, round and flat, wet in surface, smooth, neat in edge and opaque, and are gram-negative bacteria as shown in figures 1 and 2; meanwhile, a scanning electron micrograph thereof is shown in fig. 3.
(2) Measurement of physiological and biochemical Properties
The physiological and biochemical characteristics are measured according to the handbook of identifying common bacteria systems, and comprise gram staining, spore staining, catalase reaction, nitrate reduction reaction and hydrogen sulfide (H)2S) test, citrate test, starch hydrolysis test, sugar oxidation fermentation test and the like.
The results of the physiological and biochemical tests are shown in the attached table 1, and the nitrate reduction test is positive, the V-P test is negative, the methyl red test is negative, the catalase test is positive, the sugar fermentation test is positive, and the citrate test is positive.
TABLE 1 physiological and biochemical identification results
Note: in the table, "+" is positive and "-" is negative.
(3) Strain molecular biology identification and construction of phylogenetic tree thereof
And identifying by 16S rRNA to obtain a nucleotide sequence with a sequence SEQ ID No: 1 (sequence table). Through NBCI comparison, phylogenetic trees are drawn (as shown in figure 4), and the strain provided by the invention has 100 percent of homology with the pseudomonas putida, and is the pseudomonas putida of the same type and different strains.
According to morphological observation, physiological and biochemical experiments and 16S rRNA identification, the strain WL-2 is Pseudomonas putida and is named as Pseudomonas putida WL-2.
Example 3
A method for culturing Pseudomonas putida WL-2, comprising the following steps:
(1) 100mL of seed liquid culture medium was prepared in a 250mL Erlenmeyer flask and sterilized at 121 ℃ for 20 min. The formula of the seed liquid culture medium comprises 5g of yeast extract, 10g of peptone, 0.25g of dihydrogen phosphate, 0.3g of dipotassium hydrogen phosphate, 0.3g of magnesium sulfate and 1L of deionized water;
(2) inoculating the Pseudomonas putida Putida WL-2 strain into the seed liquid culture medium obtained in the step (1) on an aseptic operating platform;
(3) placing the seed liquid culture medium obtained in the step (2) at 30 ℃ for shake cultivation for 24 hours at 160r/min to obtain seed liquid;
(4) preparing 100mL of enrichment liquid culture medium based on a 250mL conical flask, and sterilizing for 20min at 121 ℃; wherein the formula of the enrichment liquid culture medium is 10g of glucose, 5g of peptone, 1g of magnesium sulfate, 2g of monopotassium phosphate and 1L of deionized water, and the pH value is adjusted to 7;
(5) inoculating 6 wt% of seed liquid into the enriched liquid culture medium on an aseptic operation table;
(6) shaking-culturing at 30 deg.C and 160r/min for 2 days, and measuring OD once every 2h with visible spectrophotometer600nmObtaining the activated bacterium liquid of Pseudomonas putida WL-2 by the value and the total nitrogen value;
(7) sequentially diluting the Pseudomonas putida WL-2 activated bacteria liquid obtained in the step (6) into 10 times by a 10-fold dilution method-2、10-3、10-4、10-5、10-6、10-7Diluting bacterial liquid by times;
(8) respectively transferring 0.1mL of each diluted bacterial liquid by using a sterile liquid transfer gun and coating the diluted bacterial liquid in an enrichment solid culture medium containing 15 mL; wherein the formula of the enrichment solid medium is 10g of glucose, 5g of peptone, 1g of magnesium sulfate, 2g of monopotassium phosphate, 1L of deionized water and 15g of agar, and the pH value is adjusted to 7;
(9) culturing in 30 deg.C incubator for 1d, recording colony number, and calculating Pseudomonas putida WL-2 activated bacteria solution concentration.
According to the plate count, the viable bacteria concentration of the activated bacterium liquid WL-2 of the Pseudomonas putida is 1.2X 107The CFU/mL, growth curve and denitrification curve are shown in FIG. 5.
Example 4
Acid and alkali resistance and high temperature resistance of the Pseudomonas putida WL-2 are measured. The method comprises the following steps:
(1) preparing 100mL of enrichment liquid culture medium based on a 250mL conical flask, adjusting the pH value to 1, 2, 3, 7 and 11, 12 and 13 respectively, and sterilizing at 121 ℃ for 20 min; wherein the formula of the enrichment liquid culture medium comprises 10g of glucose, 5g of peptone, 1g of magnesium sulfate, 2g of monopotassium phosphate and 1L of deionized water;
(2) respectively inoculating 6 wt% of seed liquid into the enrichment liquid culture medium;
(3) culturing at 30 deg.C at 160r/min for 24 hr, passing through OD600nmCalculating the survival rate according to the absorbance under the value;
(4) preparing 100mL of enrichment liquid culture medium based on a 250mL conical flask, sterilizing at 121 ℃ for 20min, and respectively inoculating 6 wt% of seed liquid; wherein the formula of the enrichment liquid culture medium comprises 10g of glucose, 5g of peptone, 1g of magnesium sulfate, 2g of monopotassium phosphate and 1L of deionized water;
(5) heating in water bath at 30 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C and 100 deg.C for 2 hr respectively;
(6) culturing at 30 deg.C at 160r/min for 24 hr, passing through OD600nmAbsorbance at value, survival rate was calculated.
According to the results, as shown in FIGS. 6 and 7, in this example, Pseudomonas putida WL-2 survived at a pH of 3 to 11 and a temperature of 80 ℃. Therefore, the Pseudomonas putida WL-2 has good tolerance to acid and alkali and high temperature.
Example 5
The Pseudomonas putida WL-2 degrades the total nitrogen in the white spirit wastewater. The method comprises the following steps:
(1) sampling at a sewage treatment station of a liquor factory, and filling liquor wastewater at the inlet of an A/O reaction tank into a sterile plastic barrel;
(2) taking 100mL of white spirit wastewater, adjusting the pH to 6.5, placing the wastewater into a 250mL conical flask, and sterilizing the wastewater for 20min at 121 ℃;
(3) inoculating 2 wt% Pseudomonas putida WL-2 activated bacteria solution (viable bacteria concentration is 5.2X 10) into the conical flask obtained in the step (2) on a sterile operating platform7CFU/mL)；
(4) Placing the Erlenmeyer flask obtained in the step (3) and inoculated with Pseudomonas putida WL-2 activated bacterium liquid in a shaking table at the temperature of 27.5 ℃ and at the speed of 145r/min for culturing for 24 hours to serve as an experimental group;
(5) replacing the activated bacterium liquid of the Pseudomonas putida WL-2 in the steps (1) to (4) with sterile normal saline, and using the rest conditions consistent with the experimental groups obtained in the steps (1) to (4) as blank groups;
(6) after the culture is finished, the total nitrogen concentration of the experimental group and the blank group is measured, and the total nitrogen degradation rate is calculated.
The specific method for measuring the total nitrogen index comprises the following steps: and (3) carrying out digestion by an alkaline potassium persulfate method and carrying out ultraviolet spectrophotometry. Taking two clean air digestion colorimetric tubes, adding 4mL of distilled water into one tube, and adding ten times of sewage sample liquid into the other tube in equivalent dilution; respectively adding 2mL of total nitrogen reagent I, screwing a pipe cover, shaking uniformly, putting the pipe cover into a digestion instrument for digestion at 120 ℃ for 30min, cooling, and then respectively adding 1mL of total nitrogen reagent I; and taking two clean clearance digestion tubes, adding 5mL of total nitrogen reagent III, taking 1mL of digestion solution of the blank sample and the sewage sample respectively, screwing, shaking uniformly, standing for 10min for color development, and measuring the total nitrogen concentration on a multi-parameter water quality analyzer. And (4) calculating the total nitrogen degradation rate of the white spirit wastewater.
The results of the measurements showed that the degradation rate of Pseudomonas putida WL-2 in this example was 71.45% of the total nitrogen in the white spirit wastewater.
Example 6
The Pseudomonas putida WL-2 degrades the total nitrogen in the white spirit wastewater. The method comprises the following steps:
(1) sampling at a sewage treatment station of a liquor factory, and filling liquor wastewater at an inlet of an A/O reaction tank into a sterile plastic barrel.
(2) Taking 100mL of white spirit wastewater, adjusting the pH to 7.5, placing the wastewater with total nitrogen of 4613mg/L into a 250mL conical flask, and sterilizing the wastewater for 20min at 121 ℃;
(3) inoculating 2% Pseudomonas putida WL-2 activated bacteria solution (viable bacteria concentration is 2.1X 10) into the conical flask on a sterile operating platform7CFU/mL)；
(4) Placing the Erlenmeyer flask obtained in the step (3) and inoculated with Pseudomonas putida WL-2 activated bacterium liquid in a shaking table at the temperature of 27.5 ℃ and at the speed of 175r/min for culturing for 24 hours;
(5) replacing the activated bacterium liquid of the Pseudomonas putida WL-2 in the steps (1) to (4) with sterile normal saline, and using the rest conditions consistent with the experimental groups obtained in the steps (1) to (4) as blank groups;
(6) after the culture is finished, the total nitrogen concentration of the experimental group and the blank group is measured, and the total nitrogen degradation rate is calculated.
The determination result shows that the degradation rate of Pseudomonas putida WL-2 on the total nitrogen in the white spirit wastewater is 66.27%.
Example 7
The Pseudomonas putida WL-2 degrades the total nitrogen in the white spirit wastewater. The method comprises the following steps:
(1) sampling at a sewage treatment station of a liquor factory, and filling liquor wastewater at an inlet of an A/O reaction tank into a sterile plastic barrel.
(2) Taking 100mL of white spirit wastewater, adjusting the pH to 7.0 when the total nitrogen in the wastewater is 4613mg/L, filling the wastewater into a 250mL conical flask, and sterilizing the wastewater for 20min at 121 ℃.
(3) 2 wt% Pseudomonas putida WL-2 activated bacteria solution (viable bacteria concentration 8.2X 10) was inoculated into the conical flask on a sterile operating table7CFU/mL)；
(4) Placing the Erlenmeyer flask obtained in the step (3) and inoculated with Pseudomonas putida WL-2 activated bacterium liquid in a shaking table at 30 ℃ and 175r/min for culturing for 24 h;
(5) replacing the activated bacterium liquid of the Pseudomonas putida WL-2 in the steps (1) to (4) with sterile normal saline, and using the rest conditions consistent with the experimental groups obtained in the steps (1) to (4) as blank groups;
(6) after the culture is finished, the total nitrogen concentration of the experimental group and the blank group is measured, and the total nitrogen degradation rate is calculated.
The determination result shows that the degradation rate of Pseudomonas putida WL-2 on the total nitrogen in the white spirit wastewater is 76.61%.
Example 8
The Pseudomonas putida WL-2 degrades the total nitrogen in the white spirit wastewater. The method comprises the following steps:
(1) sampling at a sewage treatment station of a liquor factory, and filling liquor wastewater at the inlet of an A/O reaction tank into a sterile plastic barrel;
(2) taking 100mL of white spirit wastewater, adjusting the pH to 7.0, placing the wastewater with the total nitrogen of 4613mg/L into a 250mL conical flask, and sterilizing the wastewater for 20min at 121 ℃;
(3) inoculating 2% Pseudomonas putida WL-2 activated bacteria solution (viable bacteria concentration is 1.0X 10) into the conical flask on a sterile operating platform8CFU/mL)；
(4) Placing the Erlenmeyer flask obtained in the step (3) and inoculated with Pseudomonas putida WL-2 activated bacterium liquid in a shaking table at the temperature of 27.5 ℃ and at the speed of 175r/min for culturing for 24 hours;
(5) replacing the activated bacterium liquid of the Pseudomonas putida WL-2 in the steps (1) to (4) with sterile normal saline, and using the rest conditions consistent with the experimental groups obtained in the steps (1) to (4) as blank groups;
(6) after the culture is finished, the total nitrogen concentration of the experimental group and the blank group is measured, and the total nitrogen degradation rate is calculated.
The determination result shows that the degradation rate of Pseudomonas putida WL-2 on the total nitrogen in the white spirit wastewater is 78.95%.
The foregoing is merely exemplary and illustrative of the present invention and it is within the purview of one skilled in the art to modify or supplement the embodiments described or to substitute similar ones without the exercise of inventive faculty, and still fall within the scope of the claims.
Sequence listing
<110> Sichuan university of light chemical industry
<120> pseudomonas putida and application thereof in degrading total nitrogen in white spirit wastewater
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cgacagaata agcaccggct aactctgtgc cagcagccgc ggtaatacag agggtgcaag 480
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tagataccct ggtagtccac gccgtaaacg atgtcaacta gccgttggaa tccttgagat 780
tttagtggcg cagctaacgc attaagttga ccgcctgggg agtacggccg caaggttaaa 840
actcaaatga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca 900
acgcgaagaa ccttaccagg ccttgacatg cagagaactt tccagagatg gattggtgcc 960
ttcgggaact ctgacacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg 1020
gttaagtccc gtaacgagcg caacccttgt ccttagttac cagcacgtta tggtgggcac 1080
tctaaggaga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa gtcatcatgg 1140
cccttacggc ctgggctaca cacgtgctac aatggtcggt acagagggtt gccaagccgc 1200
gaggtggagc taatctcaca aaaccgatcg tagtccggat cgcagtctgc aactcgactg 1260
cgtgaagtcg gaatcgctag taatcgcgaa tcagaatgtc gcggtgaata cgttcccggg 1320
ccttgtacac accgcccgtc acaccatggg agtgggttgc accagaagta gctagtctaa 1380
ccttcggg 1388
Claims (8)
1. Pseudomonas putida (Pseudomonas putida), which is named as Pseudomonas putida WL-2 and is preserved in Guangdong province microorganism culture collection center with the preservation date of 2021, 6 months and 28 days and the preservation number is GDMCC NO: 61748.
2. Use of Pseudomonas putida WL-2 according to claim 2 for degrading total nitrogen in liquor wastewater.
3. The use according to claim 2, comprising the steps of:
step (1): preparing Pseudomonas putida WL-2 activated bacterium liquid;
step (2): and (2) putting the activated Pseudomonas putida bacterial liquid WL-2 obtained in the step (1) into the white spirit wastewater for degradation.
4. The use according to claim 3, wherein the concentration of the Pseudomonas putida WL-2 activated bacteria solution in step (1) is 2 x 107～10×107CFU/mL。
5. The application of claim 3, wherein the total nitrogen concentration in the white spirit wastewater in the step (2) is 4500-5000 mg/L.
6. The use of claim 3, wherein the conditions for degradation in step (2) are: the degradation temperature is 25-30 ℃, the rotating speed is 150-210 r/min, and the degradation time is 24-48 h.
7. The use according to claim 3, wherein the Pseudomonas putida WL-2 is inoculated in an amount of 1 to 5 wt.%.
8. The use of claim 3, wherein the preparation method of the Pseudomonas putida WL-2 activated bacterium solution comprises the following steps:
1) inoculating the Pseudomonas putida WL-2 strain into a seed liquid culture medium, and performing shake cultivation at the temperature of 25-30 ℃ and at the speed of 150-210 r/min for 12-24 h to obtain a seed liquid;
2) placing the enriched liquid culture medium in a conical flask, sterilizing and cooling, inoculating 4-8 wt% of the seed liquid obtained in the step 1) on an aseptic operation table, and then placing the seed liquid in a shaker at 25-30 ℃ and 150-210 r/min for 1-2 days to obtain the pseudomonas putida activated bacterial liquid.
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