CN113862180B - Pseudomonas putida and application thereof in degradation of total nitrogen in white spirit wastewater - Google Patents

Pseudomonas putida and application thereof in degradation of total nitrogen in white spirit wastewater Download PDF

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Publication number
CN113862180B
CN113862180B CN202111083694.6A CN202111083694A CN113862180B CN 113862180 B CN113862180 B CN 113862180B CN 202111083694 A CN202111083694 A CN 202111083694A CN 113862180 B CN113862180 B CN 113862180B
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pseudomonas putida
total nitrogen
white spirit
wastewater
liquid
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CN113862180A (en
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方春玉
吕枫
安明哲
赵长青
寇治刚
赵兴秀
苏建
张富勇
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Sichuan University of Science and Engineering
Wuliangye Yibin Co Ltd
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Sichuan University of Science and Engineering
Wuliangye Yibin Co Ltd
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    • 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
    • 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
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/32—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters
    • C02F2103/325—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters from processes relating to the production of wine products
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

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 Pseudomonas putida WL-2 and is preserved in the Guangdong province microorganism strain preservation center, the preservation date is 2021, 6 months and 28 days, and the preservation number is 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 denitrification bacteria, can simultaneously perform nitrification and denitrification under aerobic conditions, has the total nitrogen degradation rate of 60-85% on white wine wastewater, provides an important strain for the biological denitrification process of white wine wastewater, and has practical application value on degrading total nitrogen in white wine wastewater.

Description

Pseudomonas putida and application thereof in degradation of total nitrogen in white spirit wastewater
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
White spirit brewing is used as the traditional Chinese brewing industry, and is rapidly developed in recent years, the white spirit yield is in an ascending state, but a large amount of white spirit wastewater is generated in the production process. The white spirit waste water mainly comes from distilled pot bottom water containing high organic matter concentration, fermentation blind drain water, ground flushing water of a distillation section, water leakage of a wine depot, sorghum flushing water and soaking water during the process operation of 'sand setting' and 'coarse sand', and the like, and the waste water has high COD (chemical oxygen demand), SS (suspended solids) value (suspended solids in water), acidic pH value and complex components, and has high concentration of organic matters, nitrogen and phosphorus, high chromaticity and high treatment difficulty, so that extremely high environmental pressure and economic pressure are brought to white spirit enterprises.
At present, the white spirit wastewater generally adopts a multi-stage treatment process mainly comprising anaerobic and aerobic to reach the emission standard of industrial water pollutants of fermented alcohol and white spirit (GB 27631-2011). The process can effectively degrade most of organic matters in the white spirit wastewater, but the nitrogen-containing compounds are still higher, so that further biological denitrification is needed. The conventional biological denitrification technology is based on two independent processes of microbial nitrification and denitrification. The first stage converts ammonia nitrogen in water into NO under the aerobic condition by nitration 2 -N and NO 3 And N, performing denitrification by taking the nitrified product as a substrate. In the denitrification of the second stage, the denitrifying bacteria further convert nitrate nitrogen and nitrite nitrogen into nitrogen, so that the aim of removing nitrogen ions in the water body is fulfilled. With the technical progress, the iso-aerobic denitrifying bacteria gradually enter the field of vision of people, and the bacteria can simultaneously perform nitrification and denitrification under the aerobic condition. 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, no report of degrading total nitrogen in white spirit wastewater by pseudomonas putida is known 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, and 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 above purpose, the invention adopts the following technical scheme:
the invention provides pseudomonas putida (Pseudomonas putida), which is screened from activated sludge of a sewage treatment station of a wine plant, is named Pseudomonas putida WL-2, is preserved in the microorganism strain preservation center of Guangdong province, has a preservation date of 2021, 6 and 28 days, and has a 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 total nitrogen in white spirit wastewater comprises the following steps:
step (1): preparing pseudomonas putida (Pseudomonas putida WL-2) activated bacterial liquid;
step (2): and (3) putting the activated pseudomonas putida (Pseudomonas putida WL-2) bacterial liquid obtained in the step (1) into white spirit wastewater for degradation.
Further, the concentration of the Pseudomonas putida (Pseudomonas putida WL-2) activated bacterial liquid in the step (1) is 2×10 7 ～10×10 7 CFU/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 step (2) are: the degradation temperature is 25-30 ℃, the rotation speed is 150-210 r/min, and the degradation time is 24-48 h.
Further, the inoculation amount of the pseudomonas putida (Pseudomonas putida WL-2) is 1 to 5 weight percent.
Further, the preparation method of the pseudomonas putida (Pseudomonas putida WL-2) activated bacterial liquid comprises the following steps:
1) Inoculating Pseudomonas putida (Pseudomonas putida WL-2) strain into a seed liquid culture medium, and culturing for 12-24 hours at 25-30 ℃ in a shaking table at 150-210 r/min to obtain seed liquid;
2) Placing the enriched liquid culture medium in a conical flask, sterilizing and cooling, inoculating 4-8wt% of the seed liquid obtained in the step 1) on a sterile operation table, and then placing the seed liquid in a shaking table for shake cultivation at 25-30 ℃ for 150-210 r/min for 1-2 days to obtain pseudomonas putida (Pseudomonas putida WL-2) activated bacteria liquid.
Further, the formula of the seed liquid culture medium in the step 1) comprises the following components in parts by weight: 3 to 7 parts of yeast extract, 8 to 12 parts of peptone, 0.1 to 0.4 part of monopotassium phosphate, 0.1 to 0.4 part of dipotassium phosphate, 0.1 to 0.4 part of magnesium sulfate and 800 to 1100 parts of distilled water.
Further, the enriched liquid in step 2) comprises the following components in parts by weight: 14 to 18 parts of glucose, 4 to 8 parts of peptone, 0.5 to 1.5 parts of magnesium sulfate, 1 to 4 parts of monopotassium phosphate and 800 to 1100 parts of distilled water.
Further, the pH of the enriched liquid in step 2) is between 6.5 and 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 white spirit wastewater is acidic (pH is about 5.95-6.85), a large amount of common nitrifying strains cannot grow, 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, and has high total nitrogen content, and pseudomonas putida (Pseudomonas putida WL-2) provided by the invention has higher total nitrogen degradation capability. The strain is subjected to multiple subculture, and the strain is still stably inherited and has stable degradation capability; during passage no obvious variant strains appeared.
3. The pseudomonas putida (Pseudomonas putida WL-2) is aerobic denitrifying bacteria, can perform nitrification and denitrification simultaneously under aerobic conditions, has a total nitrogen degradation rate of 60-85% on white wine wastewater, provides an important strain for the biological denitrification process of white wine wastewater, and has practical application value on degrading total nitrogen in white wine wastewater.
Drawings
FIG. 1 is a colony morphology of Pseudomonas putida (Pseudomonas putida WL-2) of the present invention on LB plate medium;
FIG. 2 is a graph showing the result of gram staining of Pseudomonas putida (Pseudomonas putida WL-2) according to the present invention;
FIG. 3 is a scanning electron microscope image of Pseudomonas putida (Pseudomonas putida WL-2) of the present invention;
FIG. 4 is a diagram of the sequence evolution tree of Pseudomonas putida (Pseudomonas putida WL-2) of the present invention;
FIG. 5 is a graph showing the results of the growth and denitrification test of Pseudomonas putida (Pseudomonas putida WL-2) in the present invention;
FIGS. 6-7 are graphs showing the results of acid and alkali resistance and high temperature resistance tests of Pseudomonas putida (Pseudomonas putida WL-2) of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the particular embodiments described herein are illustrative only and are not intended to limit the invention, i.e., the embodiments described are merely some, but not all, of the embodiments of the invention.
Thus, the following detailed description of the embodiments of the invention, as provided, 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 made by a person skilled in the art without making any inventive effort, are intended to be within the scope of the present invention.
EXAMPLE 1 isolation, purification and screening of Pseudomonas putida (Pseudomonas putida WL-2)
Isolation, purification and screening of pseudomonas putida (Pseudomonas putida WL-2) comprising the steps of:
(1) Sampling at a sewage treatment station of a white spirit factory, selecting activated sludge, and filling the activated sludge into a sterile sampling bag;
(2) Weighing 10g of activated sludge obtained in the step (1), placing the activated sludge in a 250mL conical flask filled with 90mL of DM basic culture medium, and performing domestication culture;
(3) Taking the supernatant of the mixed solution in the step (2), and sequentially diluting the supernatant into 10 according to a 10-fold dilution method -5 、10 -6 、10 -7 、10 -7 、10 -8 、10 -9 A multiple of the diluted solution;
(4) Respectively transferring 0.1mL of each diluent obtained in the step (3) by using a sterile pipetting gun, coating the diluent in a solid culture medium filled with 20mL of BTB, and uniformly coating 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 2d of culture;
(6) Selecting the bacteria which are blue single colonies in the culture medium, and respectively picking the single colonies with different forms into a BTB solid culture medium for separation and purification;
(7) Culturing in a constant temperature incubator at 30deg.C for 2d, and repeating the steps until each strain is pure colony; after the cultured bacteria are purified, the strain is preserved in a refrigerator at the temperature of minus 20 ℃ for standby;
(8) Inoculating the strain obtained by primary screening into a seed solution culture medium, and performing shake culture at 30 ℃ and 160r/min for 24 hours to obtain seed solution; 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 based on a 250mL conical flask, sterilizing, cooling, inoculating 10wt% seed liquid on a sterile operation table, and then placing the mixture in a shaking table at 30 ℃ for 2 days; centrifuging at normal temperature for 10min at 5000r/min after culturing, measuring total nitrogen index of supernatant, and selecting strain with high total nitrogen degradation rate; among them, the strain numbered WL-2, designated Pseudomonas putida (Pseudomonas putida WL-2), had a total nitrogen degradation rate of 64.18%.
The DM basal medium formula comprises 1.5g of monopotassium phosphate, 0.01g of magnesium sulfate, 7.9g of disodium hydrogen phosphate, 5.66g of sodium citrate, 0.84g of sodium nitrate and 1L of deionized water, and the pH value is adjusted to 7; the formula of the BTB solid medium is DM basal medium, bromothymol blue 0.0006g, agar 15g, deionized water 1L, and the pH is adjusted to 7.0; the culture medium is sterilized at 121deg.C for 20min.
EXAMPLE 2 identification of Pseudomonas putida (Pseudomonas putida WL-2)
Identification of pseudomonas putida (Pseudomonas putida WL-2), comprising the steps of:
(1) Observation of strain morphology and culture characteristics
Inoculating the strain with the best total nitrogen degradation obtained in the re-screening of the example 1 on a beef extract peptone (NA) solid culture medium, culturing for 18 hours at 37 ℃, and observing the morphological characteristics of a bacterial colony, wherein the bacterial colony is light yellow, round, flat, moist, smooth, neat in edge and opaque and is a gram-negative bacterium as shown in fig. 1 and 2; meanwhile, the scanning electron microscope diagram is shown in fig. 3.
(2) Determination of physiological and biochemical characteristics
The physiological and biochemical characteristics are determined by referring to the manual for identifying common bacterial systems, including gram staining, spore staining, contact enzyme reaction, nitrate reduction reaction and hydrogen sulfide (H) 2 S) test, citrate test, starch hydrolysis test, sugar oxidation fermentation measurement, etc.
The physiological and biochemical test results are shown in the attached table 1, the nitrate reduction is positive, the V-P test is negative, the methyl red test is negative, the contact enzyme test is positive, the sugar fermentation test is positive, and the citrate test is positive.
TABLE 1 physiological and Biochemical identification results
Note that: in the table, "+" is positive and "-" is negative.
(3) Molecular biological identification of strain and construction of phylogenetic tree thereof
The nucleotide sequence is obtained through 16S rRNA identification, and the sequence SEQ ID No:1 (sequence table). Through NBCI comparison, a phylogenetic tree is drawn (shown as figure 4), and the homology of the strain provided by the invention and pseudomonas putida is 100%, and the strain is pseudomonas putida of the same type and different strains.
Based on morphological observation, physiological and biochemical experiments and 16S rRNA identification, the WL-2 strain was Pseudomonas putida Pseudomonas putida, and designated Pseudomonas putida Pseudomonas putida WL-2.
Example 3
The culture method of pseudomonas putida Pseudomonas putida WL-2 comprises the following steps:
(1) 100mL seed solution culture was prepared based on 250mL Erlenmeyer flasks and sterilized at 121℃for 20min. 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 pseudomonas putida Pseudomonas putida WL-2 strain to the seed liquid culture medium obtained in the step (1) on a sterile operating table;
(3) Placing the seed liquid culture medium obtained in the step (2) at 30 ℃ and 160r/min for shake cultivation for 24 hours to obtain seed liquid;
(4) Preparing 100mL enrichment liquid culture based on a 250mL conical flask, and sterilizing at 121 ℃ for 20min; 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, and the pH value is adjusted to 7;
(5) On a sterile operation table, 6wt% of seed liquid is inoculated into the enrichment liquid culture medium;
(6) Shaking at 30deg.C for 2 days at 160r/min, and measuring OD every 2 hr with visible spectrophotometer 600nm The value and the total nitrogen value are obtained to obtain pseudomonas putida Pseudomonas putida WL-2 activated bacteria liquid;
(7) Sequentially diluting Pseudomonas putida WL-2 activated bacteria liquid obtained in the step (6) into 10 according to a 10-fold dilution method -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 Multiple diluted bacteria liquid;
(8) Respectively transferring 0.1mL of each diluted bacterial liquid by using a sterile pipetting gun, and coating the diluted bacterial liquids in a solid enrichment culture medium filled with 15 mL; wherein the formula of the enriched solid culture medium comprises 10g of glucose, 5g of peptone, 1g of magnesium sulfate, 2g of monopotassium phosphate, 1L of deionized water, 15g of agar and regulating the pH to 7;
(9) Culturing in a constant temperature incubator at 30deg.C for 1d, recording colony number, and calculating the concentration of Pseudomonas putida Pseudomonas putida WL-2 activated bacteria liquid.
According to the plate count, the living bacteria concentration of the pseudomonas putida Pseudomonas putida WL-2 activated bacteria liquid is 1.2X10 7 CFU/mL, growth curves and denitrification curves are shown in FIG. 5.
Example 4
Acid and alkali resistance and high temperature resistance of pseudomonas putida Pseudomonas putida WL-2 are measured. The method comprises the following steps:
(1) Preparing 100mL enrichment liquid culture, adjusting the pH value of the enrichment liquid culture to 1, 2, 3, 7, 11, 12 and 13 respectively based on a 250mL conical flask, and sterilizing at 121 ℃ for 20min; 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;
(2) Respectively inoculating 6wt% seed solution into the enriched liquid culture medium;
(3) Culturing at 30deg.C for 24 hr at 160r/min, and passing through OD 600nm Absorbance at the value, calculating survival;
(4) Preparing 100mL enrichment liquid culture based on a 250mL conical flask, sterilizing at 121 ℃ for 20min, and respectively inoculating 6wt% seed liquid; 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;
(5) Heating in water bath at 30deg.C, 50deg.C, 60deg.C, 70deg.C, 80deg.C, 90deg.C and 100deg.C for 2 hr;
(6) Culturing at 30deg.C for 24 hr at 160r/min, and passing through OD 600nm The absorbance at the value was used to calculate the survival rate.
According to the results, as shown in FIGS. 6 and 7, pseudomonas putida Pseudomonas putida WL-2 in this example survived at a pH of 3 to 11 and a temperature of 80 ℃. Pseudomonas putida Pseudomonas putida WL-2 is therefore well-resistant to acids and bases and high temperatures.
Example 5
Pseudomonas putida Pseudomonas putida WL-2 degrades the total nitrogen in the white wine wastewater. The method comprises the following steps:
(1) Sampling at a sewage treatment station of a white spirit factory, taking white spirit wastewater at the inlet of an A/O reaction tank, and filling the white spirit wastewater into a sterile plastic bucket;
(2) Taking 100mL of white spirit wastewater, wherein the total nitrogen in the wastewater is 4613mg/L, adjusting the pH to 6.5, placing the white spirit wastewater into a 250mL conical flask, and sterilizing the white spirit wastewater at 121 ℃ for 20min;
(3) On a sterile operating table, 2wt% of Pseudomonas putida Pseudomonas putida WL-2 activated bacteria solution (active bacteria concentration is 5.2X10) is introduced into the conical flask obtained in the step (2) 7 CFU/mL)；
(4) Placing the conical flask which is accessed with the pseudomonas putida Pseudomonas putida WL-2 activated bacterial liquid obtained in the step (3) into a shaking table at the temperature of 27.5 ℃ and the speed of 145r/min for culturing for 24 hours, and taking the conical flask as an experimental group;
(5) The Pseudomonas putida Pseudomonas putida WL-2 activated bacteria liquid in the step (1) -step (4) is replaced by sterile physiological saline, and the rest conditions are consistent with the experimental group obtained in the step (1) -step (4) to be used as a blank group;
(6) After the end of the incubation, the total nitrogen concentration of the experimental group and the blank group was measured, and the total nitrogen degradation rate was calculated.
The specific method for measuring the total nitrogen index comprises the following steps: alkaline potassium persulfate method is used for digestion of ultraviolet spectrophotometry. Taking two clean empty digestion colorimetric tubes, wherein one tube is added with 4mL of distilled water, and the other tube is added with sewage sample liquid which is diluted ten times in equivalent; adding 2mL of total nitrogen reagent I respectively, tightening a pipe cover, shaking uniformly, putting the pipe cover into a digestion instrument for digestion for 30min at 120 ℃, cooling, and then adding the total nitrogen reagent 1mL respectively; and (3) taking two clean empty digestion pipes, adding 5mL of total nitrogen reagent III, respectively taking 1mL of blank sample and sewage sample digestion solution, uniformly twisting, standing for 10min for color development, and measuring the total nitrogen concentration on a multi-parameter water quality analyzer. And calculating the total nitrogen degradation rate of the white spirit wastewater.
The measurement result shows that the total nitrogen degradation rate of pseudomonas putida Pseudomonas putida WL-2 in the example on the white wine wastewater is 71.45%.
Example 6
Pseudomonas putida Pseudomonas putida WL-2 degrades the total nitrogen in the white wine wastewater. The method comprises the following steps:
(1) Sampling at a sewage treatment station of a white spirit factory, taking white spirit wastewater at the inlet of an A/O reaction tank, and filling the white spirit wastewater into a sterile plastic bucket.
(2) Taking 100mL of white spirit wastewater, wherein the total nitrogen in the wastewater is 4613mg/L, adjusting the pH to 7.5, placing the white spirit wastewater into a 250mL conical flask, and sterilizing the white spirit wastewater at 121 ℃ for 20min;
(3) On a sterile operating table, 2% Pseudomonas putida Pseudomonas putida WL-2 activated bacteria solution (active bacteria concentration of 2.1X10) was introduced into a conical flask 7 CFU/mL)；
(4) Placing the conical flask which is accessed with the pseudomonas putida Pseudomonas putida WL-2 activated bacterial liquid obtained in the step (3) into a shaking table at the temperature of 27.5 ℃ and at the speed of 175r/min for culturing for 24 hours;
(5) The Pseudomonas putida Pseudomonas putida WL-2 activated bacteria liquid in the step (1) -step (4) is replaced by sterile physiological saline, and the rest conditions are consistent with the experimental group obtained in the step (1) -step (4) to be used as a blank group;
(6) After the end of the incubation, the total nitrogen concentration of the experimental group and the blank group was measured, and the total nitrogen degradation rate was calculated.
The measurement result shows that the total nitrogen degradation rate of pseudomonas putida Pseudomonas putida WL-2 in the white wine wastewater is 66.27%.
Example 7
Pseudomonas putida Pseudomonas putida WL-2 degrades the total nitrogen in the white wine wastewater. The method comprises the following steps:
(1) Sampling at a sewage treatment station of a white spirit factory, taking white spirit wastewater at the inlet of an A/O reaction tank, and filling the white spirit wastewater into a sterile plastic bucket.
(2) Taking 100mL of white spirit wastewater, wherein total nitrogen in the wastewater is 4613mg/L, adjusting pH to 7.0, placing the white spirit wastewater into a 250mL conical flask, and sterilizing at 121 ℃ for 20min.
(3) On a sterile operating table, 2wt% Pseudomonas putida Pseudomonas putida WL-2 activated bacteria solution (active bacteria concentration 8.2X10) was inoculated into a conical flask 7 CFU/mL)；
(4) Placing the conical flask which is accessed with the pseudomonas putida Pseudomonas putida WL-2 activated bacterial liquid obtained in the step (3) in a shaking table at 30 ℃ for culturing for 24 hours at 175 r/min;
(5) The Pseudomonas putida Pseudomonas putida WL-2 activated bacteria liquid in the step (1) -step (4) is replaced by sterile physiological saline, and the rest conditions are consistent with the experimental group obtained in the step (1) -step (4) to be used as a blank group;
(6) After the end of the incubation, the total nitrogen concentration of the experimental group and the blank group was measured, and the total nitrogen degradation rate was calculated.
The measurement result shows that the total nitrogen degradation rate of pseudomonas putida Pseudomonas putida WL-2 in the white wine wastewater is 76.61%.
Example 8
Pseudomonas putida Pseudomonas putida WL-2 degrades the total nitrogen in the white wine wastewater. The method comprises the following steps:
(1) Sampling at a sewage treatment station of a white spirit factory, taking white spirit wastewater at the inlet of an A/O reaction tank, and filling the white spirit wastewater into a sterile plastic bucket;
(2) Taking 100mL of white spirit wastewater, wherein the total nitrogen in the wastewater is 4613mg/L, adjusting the pH to 7.0, placing the white spirit wastewater into a 250mL conical flask, and sterilizing the white spirit wastewater at 121 ℃ for 20min;
(3) On a sterile operating table, 2% Pseudomonas putida Pseudomonas putida WL-2 activated bacteria solution (active bacteria concentration 1.0X10) was introduced into a conical flask 8 CFU/mL)；
(4) Placing the conical flask which is accessed with the pseudomonas putida Pseudomonas putida WL-2 activated bacterial liquid obtained in the step (3) in a shaking table at the temperature of 27.5 ℃ and at the speed of 175r/min for culturing for 24 hours;
(5) The Pseudomonas putida Pseudomonas putida WL-2 activated bacteria liquid in the step (1) -step (4) is replaced by sterile physiological saline, and the rest conditions are consistent with the experimental group obtained in the step (1) -step (4) to be used as a blank group;
(6) After the end of the incubation, the total nitrogen concentration of the experimental group and the blank group was measured, and the total nitrogen degradation rate was calculated.
The measurement result shows that the total nitrogen degradation rate of pseudomonas putida Pseudomonas putida WL-2 in the white wine wastewater is 78.95%.
The foregoing is merely illustrative and explanatory of the invention as it is claimed, as modifications and additions may be made to, or similar to, the particular embodiments described, without the benefit of the inventors' inventive effort, and as alternatives to those of skill in the art, which remain within the scope of this patent.
Sequence listing
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taaagcactt taagttggga ggaagggcag taagttaata ccttgctgtt ttgacgttac 420
cgacagaata agcaccggct aactctgtgc cagcagccgc ggtaatacag agggtgcaag 480
cgttaatcgg aattactggg cgtaaagcgc gcgtaggtgg tttgttaagt tggatgtgaa 540
agccccgggc tcaacctggg aactgcatcc aaaactggca agctagagta cggtagaggg 600
tggtggaatt tcctgtgtag cggtgaaatg cgtagatata ggaaggaaca ccagtggcga 660
aggcgaccac ctggactgat actgacactg aggtgcgaaa gcgtggggag caaacaggat 720
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 (7)

1. Pseudomonas putidaPseudomonas putida) The application of the pseudomonas putida in degrading the total nitrogen in the white spirit wastewater is characterized in that the pseudomonas putida is named asPseudomonas putidaWL-2, deposited in the Guangdong province microorganism strain collection center, the date of deposition is 2021, 6 and 28 days, and the deposition number is GDMCC NO 61748; degradation conditions of pseudomonas putida for degrading total nitrogen in white spirit wastewater are as follows: the pH value is 3-11, and the temperature is 30-70 ℃.
2. The use according to claim 1, comprising the steps of:
step (1): preparation of Pseudomonas putidaPseudomonas putidaActivating bacterial liquid by WL-2;
step (2): the activated pseudomonas putida obtained in the step (1) is treatedPseudomonas putidaAnd (3) adding the WL-2 bacterial liquid into the white spirit wastewater for degradation.
3. The use according to claim 2, wherein the pseudomonas putida in step (1)Pseudomonas putidaThe concentration of WL-2 activated bacteria liquid is 2×10 7 ~10×10 7 CFU/mL。
4. The use according to claim 2, wherein the total nitrogen concentration in the white spirit wastewater in the step (2) is 4500-5000 mg/L.
5. The use according to claim 2, wherein the degradation conditions in step (2) are: the degradation temperature is 25-30 ℃, the rotation speed is 150-210 r/min, and the degradation time is 24-48 h.
6. The use according to claim 2, wherein the pseudomonas putida isPseudomonas putidaThe inoculation amount of WL-2 added into the white spirit wastewater is 1-5 wt%.
7. The use according to claim 2, wherein the preparation method of the pseudomonas putida Pseudomonas putida WL-2 activated bacterial liquid comprises the following steps:
1) Inoculating pseudomonas putida Pseudomonas putida WL-2 strain into a seed liquid culture medium, and performing shake culture at 25-30 ℃ for 12-24 hours at 150-210 r/min to obtain seed liquid;
2) Placing the enriched liquid culture medium in a conical flask, sterilizing and cooling, inoculating 4-8wt% of the seed liquid obtained in the step 1) on a sterile operation table, and then placing the seed liquid in a shaking table at 25-30 ℃ for culturing for 1-2 days at 150-210 r/min to obtain the pseudomonas putida activated bacteria liquid.
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