CN113502237B - Enterobacter reuteri for degrading ammonia nitrogen in white spirit wastewater and application thereof - Google Patents

Enterobacter reuteri for degrading ammonia nitrogen in white spirit wastewater and application thereof Download PDF

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CN113502237B
CN113502237B CN202110499544.7A CN202110499544A CN113502237B CN 113502237 B CN113502237 B CN 113502237B CN 202110499544 A CN202110499544 A CN 202110499544A CN 113502237 B CN113502237 B CN 113502237B
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enterobacter
ludwigii
enterobacter ludwigii
ammonia nitrogen
reuteri
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赵长青
赵阳
曾钰
冉光雨
吕枫
方春玉
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Sichuan University of Science and Engineering
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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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • 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
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    • 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
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    • 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

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Abstract

The invention relates to the field of microorganisms, in particular to enterobacter ledebuae, a screening method and application. The activated sludge screened by Enterobacter reuteri ludwigii ZY-10 in a white spirit factory sewage treatment station has been preserved in Guangdong province microbial strain preservation center in 2021, 4 and 29 days, the preservation number is GDMCC No.61633, and the preservation address is No. 59 building 5 of Michelia Tomentosa No. 100 of Guangzhou city, Guangdong province. The strain is easy to culture, and the ammonia nitrogen degradation rate is high. Ammonia nitrogen in the white spirit wastewater is treated by the strain, and the ammonia nitrogen degradation reaches 60-85%. The invention provides an important strain for the nitrification process in the biological denitrification of the white spirit wastewater, lays a foundation for later construction of a composite strain for treating the white spirit wastewater, and has higher application value.

Description

Enterobacter reuteri for degrading ammonia nitrogen in white spirit wastewater and application thereof
Technical Field
The invention belongs to the technical field of microbial degradation, and particularly relates to enterobacter reuteri for degrading ammonia nitrogen in white spirit wastewater and application thereof.
Background
The Chinese liquor industry is rapidly developed, the economic yield is rapidly increased, but a large amount of liquor wastewater is generated in the production process. The liquor wastewater mainly comprises distillation pot bottom water, ancient air washing pool water, kiln ground water, soaking drainage water and the like, and belongs to typical high-concentration organic wastewater. The main pollutants comprise starch, sugar, alcohol, lipid, cellulose and the like, so the treatment difficulty is high, if the white spirit wastewater is directly discharged without being treated, the water eutrophication is caused, and the surrounding environment is also polluted.
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 anaerobic process mainly comprises an Upflow Anaerobic Sludge Blanket (UASB), an anaerobic biological filter (AF), an anaerobic Expanded Granular Sludge Blanket (EGSB), an internal circulation anaerobic reactor (IC) and the like, and can effectively degrade most organic matters in the white spirit wastewater, but the nitrogen-containing compounds are still higher, so that the further biological denitrification is needed. Biological denitrification means that nitrogen-containing compounds are firstly converted into ammonia nitrogen under the action of ammoniated bacteria, then converted into nitrate or nitrite under the action of nitrobacteria, and finally converted into gaseous nitrogen, NO or N under the action of denitrifying bacteria 2 And O. Nitrifying bacteria play a key role in the biological denitrification process. Therefore, the screening of the nitrifying bacteria has important significance for removing the ammonia nitrogen in the white wine wastewater.
Disclosure of Invention
The invention aims to provide the Enterobacter reuteri for degrading ammonia nitrogen in the white spirit wastewater, the strain is easy to culture, can be better used for degrading ammonia nitrogen in the white spirit wastewater, has higher degradation capability, can degrade the ammonia nitrogen by 60-85 percent, and provides an important strain for the nitrification process in the biological denitrification of the white spirit wastewater.
In order to achieve the above purpose, the specific technical scheme of the invention is as follows:
enterobacter ludwigiiEnterobacter ludwigii ZY-10, the activated sludge screened from the sewage treatment station of the liquor factory is preserved in Guangdong province microorganism strain preservation center in 29 th 4 th 2021, the preservation number is GDMCC No.61633, the preservation address is No. 59 building 5 of Michelia Sunryi 100 of Guangdong province, Guangzhou City, the preservation address is postal code: 510070.
the second purpose of the invention is to provide an Enterobacter reuteri strainEnterobacter ludwigii The ZY-10 is applied to degrading the white spirit wastewater.
Further, it is toFor the Enterobacter reuteriEnterobacter ludwigii The ZY-10 is applied to degrading ammonia nitrogen in white spirit wastewater.
Preferably, the Enterobacter reuteri bacteriumEnterobacter ludwigii The application method of ZY-10 in degrading ammonia nitrogen in the white spirit wastewater comprises the following steps:
1) preparation of Enterobacter reuteriEnterobacter ludwigii ZY-10 activated bacterium liquid;
2) activated Enterobacter reuteriEnterobacter ludwigii Adding ZY-10 bacterial liquid into the white spirit wastewater;
3) enterobacter ludwigiiEnterobacter ludwigii ZY-10 degrades ammonia nitrogen in the white wine wastewater.
Preferably, Enterobacter reuteriEnterobacter ludwigii The concentration of ZY-10 activated bacterium liquid is 4-20 x 10 5 CFU/mL。
Preferably, the ammonia nitrogen concentration in the white spirit wastewater is 100-400 mg/L; enterobacter ludwigiiEnterobacter ludwigii The inoculation amount of ZY-10 is 4-10%.
Preferably, the degradation temperature in the step 3) is 30-40 ℃, the rotating speed is 120-360 r/min, and the degradation time is 24-72 h.
Preferably, Enterobacter reuteriEnterobacter ludwigii The culture method of ZY-10 comprises the following steps:
1) enterobacter reuteriEnterobacter ludwigii Inoculating the ZY-10 strain into a seed solution culture medium, and performing shake culture at 35-39 ℃ at 150-210 r/min for 24-48 h to obtain a seed solution;
2) taking a certain amount of nitrobacteria enriched liquid to culture on the basis of a conical flask, sterilizing and cooling, inoculating 4-8% of seed liquid on an aseptic operation table, and then placing the seed liquid at 35-39 ℃ for 2-4 days in a shaking table at 150-210 r/min to obtain the enterobacter reuteriEnterobacter ludwigii ZY-10 activated bacterial liquid.
Preferably, the seed liquid culture medium formula comprises the following components in parts by weight: 4-8 parts of yeast extract, 8-12 parts of peptone, 8-12 parts of sodium chloride and 800-1100 parts of distilled water.
Preferably, the nitrifying bacteria enriched liquid comprises the following components in parts by weight: 8-10 parts of trisodium citrate, 0.5-1.5 parts of dipotassium phosphate, 2-6 parts of ammonium sulfate, 0.4-0.8 part of magnesium sulfate heptahydrate, 0.4-0.8 part of sodium chloride, 0.4-0.8 part of ferrous sulfate heptahydrate and 800-1100 parts of distilled water, and the pH value is adjusted to 6.5-7.5.
Preferably, the culture medium is sterilized at 121 ℃ for 20 min.
Compared with the prior art, the positive effects of the invention are as follows:
firstly, the white spirit wastewater is slightly acidic, and a common nitrifying strain cannot grow in large quantity, so that the enterobacter lewinii provided by the inventionEnterobacter ludwigii ZY-10 has strong tolerance to acid environment and is easy to culture.
(II) the white spirit wastewater belongs to high-concentration organic wastewater, the ammonia nitrogen content of the white spirit wastewater is higher, and the Enterobacter ludwigii provided by the inventionEnterobacter ludwigii ZY-10 has higher capability of degrading ammonia nitrogen. The strain is subjected to subculture for many times, and the strain is still stably inherited and has stable degradation capability; no obvious variant strains appear in the process of passage at present.
(III) Enterobacter ludwigii of the present inventionEnterobacter ludwigii The ZY-10 has the degradation rate of ammonia nitrogen in the white spirit wastewater reaching 60-85%, provides an important strain for the nitrification process in the biological denitrification of the white spirit wastewater, and has great practical significance for degrading ammonia nitrogen in the white spirit wastewater.
Detailed Description
Enterobacter ludwigiiEnterobacter ludwigii ZY-10, which was deposited at 29.4.2021 in Guangdong province center for microbial cultures, with the deposit number GDMCC No.61633, with the deposit address of Mieli Zhonglu 100, Midlao 5, Guangdong province institute for microbiology, and with the deposit address of postal code: 510070.
preferably, the enterobacter reuteri is applied to degradation of white spirit wastewater.
Preferably, the enterobacter reuteri is applied to degrading ammonia nitrogen in white spirit wastewater.
Preferably, the application of the Enterobacter ludwigii in degrading ammonia nitrogen in white spirit wastewater comprises the following steps
1) Preparation of Lushi sausageBacillusEnterobacter ludwigii ZY-10 activated bacterium liquid; enterobacter ludwigiiEnterobacter ludwigii The concentration of ZY-10 activated bacterium liquid is 4-20 x 10 5 CFU/mL。
2) Activated Enterobacter reuteriEnterobacter ludwigii Adding ZY-10 bacterial liquid into the white spirit wastewater;
3) enterobacter ludwigiiEnterobacter ludwigii ZY-10 degrades ammonia nitrogen in the white wine wastewater. The concentration of ammonia nitrogen in the white spirit wastewater is 100-400 mg/L; enterobacter ludwigiiEnterobacter ludwigii The inoculation amount of ZY-10 is 4-10%. The degradation temperature in the step 3) is 30-40 ℃, the rotating speed is 120-360 r/min, and the degradation time is 24-72 h.
Preferably, the Enterobacter reuteri bacteriumEnterobacter ludwigii A method for culturing ZY-10, which comprises the following steps:
1) enterobacter reuteriEnterobacter ludwigii The ZY-10 strain is inoculated in a seed liquid culture medium and is subjected to shake cultivation at the temperature of 35-39 ℃ and at the speed of 150-210 r/min for 24-48 h to obtain seed liquid;
2) taking a certain amount of nitrobacteria enriched liquid to culture on the basis of a conical flask, sterilizing and cooling, inoculating 4-8% of seed liquid on an aseptic operation table, and then placing the seed liquid at 35-39 ℃ and performing shake culture at 150-210 r/min for 2-4 days to obtain the enterobacter reuteriEnterobacter ludwigii ZY-10 activated bacterial liquid.
Preferably, the seed liquid culture medium formula comprises the following components in parts by weight: 4-8 parts of yeast extract, 8-12 parts of peptone, 8-12 parts of sodium chloride and 800-1100 parts of distilled water.
Preferably, the nitrifying bacteria enriched liquid comprises the following components in parts by weight: 8-10 parts of trisodium citrate, 0.5-1.5 parts of dipotassium phosphate, 2-6 parts of ammonium sulfate, 0.4-0.8 part of magnesium sulfate heptahydrate, 0.4-0.8 part of sodium chloride, 0.4-0.8 part of ferrous sulfate heptahydrate and 800-1100 parts of distilled water, and the pH value is adjusted to 6.5-7.5.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of being modified in various respects
Various modifications and adaptations can be made without departing from the spirit of the invention. It is to be noted that the features in the following embodiments and examples may be combined with each other without conflict.
The raw materials and reagents referred to in this application are all commercially available:
example 1
Enterobacter ludwigiiEnterobacter ludwigii The screening of ZY-10 comprises the following steps:
(1) sampling in a liquor factory sewage treatment station, selecting activated sludge, and filling in a sterile sampling bag.
(2) 2g of activated sludge is weighed into a 250mL conical flask filled with 90mL of sterile physiological saline and shaken up.
(3) Sequentially diluting the supernatant of the mixed solution to 10 times by 10 times dilution method -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 Double dilution of the solution.
(4) Respectively transferring 0.1mL of each diluent by using a sterile liquid transfer gun, coating the diluent on a solid culture medium containing 30mL of nitrobacteria enrichment, and uniformly coating the bacterial liquid on the surface of the culture medium by using a coating rod; the inoculation amount is too large, the surface of the coated culture medium is not easy to dry, and the contamination is easy to cause when the culture medium is inverted.
(5) Placing in a constant temperature incubator at 37 ℃ for 2 d.
(6) Selecting bacteria in the culture medium which are single colonies, respectively selecting the single colonies with different forms, and separating and purifying the single colonies in the nitrobacteria enrichment solid culture medium.
(7) The cells were incubated at 37 ℃ for 2 days and the procedure was repeated until each strain was a pure colony. When the cultured strain is purified, the strain is stored in a freezer at-20 ℃ for further use. The formula of the nitrobacteria enrichment solid culture medium is as follows: 10g of trisodium citrate, 2g of dipotassium phosphate, 4g of ammonium sulfate, 0.4g of magnesium sulfate heptahydrate, 0.4g of sodium chloride, 0.4g of ferrous sulfate heptahydrate, 20g of agar and 1L of distilled water, and the pH value is adjusted to 7. The culture medium is sterilized at 121 deg.C for 20 min.
(8) Inoculating the strains obtained by primary screening into a seed liquid culture medium, and performing shake cultivation at 37 ℃ and 180r/min for 24h to obtain the seed liquid. The formula of the seed liquid culture medium is as follows: 5g of yeast extract, 10g of peptone, 10g of sodium chloride and 1L of distilled water.
(9) Taking 100mL of nitrobacteria enriched liquid to culture in a 250mL conical flask, sterilizing and cooling, inoculating 10% seed liquid on a sterile operating platform, and then placing the seed liquid at 37 ℃ and culturing in a shaking table at 180r/min 2
And (5) day. Centrifuging at the normal temperature of 4000r/min for 10min after culture, measuring the ammonia nitrogen index of supernatant, and selecting a strain with higher ammonia nitrogen degradation rate. Wherein the bacterial strain numbered ZY-10 has the ammonia nitrogen degradation rate of 67.56 percent.
The formula of the nitrifying bacteria enriched liquid culture medium is as follows: 8g of trisodium citrate, 1g of dipotassium phosphate, 2g of ammonium sulfate, 0.4g of magnesium sulfate heptahydrate, 0.6g of sodium chloride, 0.5g of ferrous sulfate heptahydrate and 1L of distilled water, and the pH value is adjusted to 6.5. The culture medium is sterilized at 121 deg.C for 20 min.
(10) And (3) identification: and selecting the ZY-10 strain for identification. And identifying by 16sRNA to obtain a nucleotide sequence of SEQ ID No: 1 (sequence table). Through NBCI comparison, the homology of the Enterobacter ludwigii strain provided by the invention and a standard strain is 99.72 percent, and the Enterobacter ludwigii strain is an Enterobacter ludwigii strain of different strains of the same species.
(11) Naming: the ZY-10 strain was identified as Enterobacter reuteri according to 16 sRNA: (Enterobacter ludwigii) Is named as Enterobacter ludwigiaeEnterobacter ludwigii ZY-10。
(12) And (4) preservation: the Enterobacter reuteri strain is preparedEnterobacter ludwigii ZY-10 was deposited in the culture Collection of microorganisms of Guangdong province at 29.4.2021 under the accession number GDMCC No.61633, and the deposit address is No. 59, 5 th of Michelia Tokyo 100, Guangzhou, Guangdong province.
Example 2
Enterobacter ludwigiiEnterobacter ludwigii The ZY-10 culturing process includes 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 4g of yeast extract, 8g of peptone, 9g of sodium chloride and 1L of distilled water.
(2) On a sterile operating platform, the Enterobacter reuteri is putEnterobacter ludwigii ZY-10 strain is inoculated in seed liquid culture medium.
(3) Placing in a shaking table at 37 deg.C and 150r/min for 24h to obtain seed solution.
(4) 100mL of nitrobacteria enriched liquid culture medium is prepared in a 250mL conical flask, and sterilization is carried out for 20min at 121 ℃. The nitrobacteria enrichment liquid medium comprises 8g of trisodium citrate, 1g of dipotassium phosphate, 2g of ammonium sulfate, 0.4g of magnesium sulfate heptahydrate, 0.6g of sodium chloride, 0.5g of ferrous sulfate heptahydrate and 1L of distilled water, and the pH value is adjusted to 6.5.
(5) Inoculating 6% seed liquid into the nitrobacteria enriched liquid culture medium on an aseptic operation table.
(6) Culturing in shaking bed at 37 deg.C and 150r/min for 2 days to obtain Enterobacter reuteriEnterobacter ludwigii ZY-10 activated bacterial liquid.
(7) Enterobacter reuteriEnterobacter ludwigii Sequentially diluting ZY-10 activated bacteria liquid to 10 times by a 10-fold dilution method -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 Diluted bacterial liquid.
(8) 0.1mL of each diluted bacterial solution was transferred by a sterile pipette and spread on 30mL of solid medium containing nitrobacteria. The nitrobacteria enrichment solid medium comprises 8g of trisodium citrate, 1g of dipotassium phosphate, 2g of ammonium sulfate, 0.4g of magnesium sulfate heptahydrate, 0.6g of sodium chloride, 0.5g of ferrous sulfate heptahydrate, 1L of distilled water and 20g of agar powder, and the pH value is adjusted to 6.5.
(9) Culturing in 37 deg.C constant temperature incubator for 2d, recording colony number, and calculating Enterobacter reuteriEnterobacter ludwigii ZY-10 activated bacteria liquid concentration.
According to plate count, Enterobacter ludwigiiEnterobacter ludwigii The viable bacteria concentration of ZY-10 activated bacteria liquid is 7.2 multiplied by 10 6 CFU/mL。
Example 3
Enterobacter ludwigiiEnterobacter ludwigii ZY-10 degrades ammonia nitrogen in the white wine wastewater. The method comprises the following steps:
(1) sampling in a sewage treatment station of a liquor factory, taking liquor wastewater at the inlet of the secondary treatment pool, and filling into a sterile plastic barrel.
(2) Diluting ammonia nitrogen in the white spirit wastewater to 150mg/L, wherein the pH is 4.3, putting 100mL into a 250mL conical flask, and sterilizing at 121 ℃ for 20 min.
(3) Inoculating 8% Enterobacter reuteri into the conical flask on a sterile operating platformEnterobacter ludwigii ZY-10 activated bacterium liquid (viable bacteria concentration is 7.0X 10) 5 CFU/mL）。
(4) Culturing in a shaker at 37 deg.C and 150r/min for 48 h.
(5) Blank group Enterobacter ledeburinusEnterobacter ludwigii ZY-10 activated bacteria liquid is replaced by sterile normal saline, and other conditions are consistent with those of the experimental group.
(6) And after the culture is finished, measuring the ammonia nitrogen concentration of the experimental group and the blank group, and calculating the ammonia nitrogen degradation rate.
The specific method for measuring the ammonia nitrogen index comprises the following steps: and (4) transferring the white spirit wastewater after the culture into a centrifugal tube, centrifuging at the normal temperature of 4000r/min for 10min, taking supernatant, and diluting by 20 times by using distilled water. Taking 5mL of diluent to a sample tube, adding 3 drops of ammonia nitrogen reagent I, screwing a tube cover, and turning over the tube evenly. And opening the tube cover, adding 3 drops of ammonia nitrogen reagent II, screwing the tube cover, and turning upside down uniformly. Standing for 10min, and measuring the ammonia nitrogen concentration on a multi-parameter water quality analyzer. And calculating the ammonia nitrogen degradation rate of the white spirit wastewater.
The results of the assay showed that Enterobacter reuteriEnterobacter ludwigii The degradation rate of ZY-10 to ammonia nitrogen in the white wine wastewater is 65.78 percent.
Example 4
Enterobacter ludwigiiEnterobacter ludwigii ZY-10 degrades ammonia nitrogen in the white wine wastewater. The method comprises the following steps:
(1) sampling in a sewage treatment station of a liquor factory, taking liquor wastewater at the inlet of the secondary treatment pool, and filling into a sterile plastic barrel.
(2) Diluting ammonia nitrogen in the white spirit wastewater to 250mg/L, wherein the pH is 4.1, taking 100mL, filling into a 250mL conical flask, and sterilizing at 121 ℃ for 20 min.
（3）Inoculating Enterobacter leydis 6% into the Erlenmeyer flask on a sterile operating tableEnterobacter ludwigii ZY-10 activated bacterium liquid (viable bacteria concentration is 1.1X 10) 6 CFU/mL）。
(4) Culturing in a shaker at 36 deg.C and 160r/min for 48 h.
(5) Blank group Enterobacter ledeburinusEnterobacter ludwigii ZY-10 activated bacteria liquid is replaced by sterile normal saline, and other conditions are the same as those of the experimental group.
(6) And after the culture is finished, measuring the ammonia nitrogen concentration of the experimental group and the blank group, and calculating the ammonia nitrogen degradation rate.
The results of the assay showed that Enterobacter reuteriEnterobacter ludwigii The degradation rate of ZY-10 to ammonia nitrogen in the white wine wastewater is 72.38 percent.
Example 5
Enterobacter ludwigiiEnterobacter ludwigii ZY-10 degrades ammonia nitrogen in the white wine wastewater. The method comprises the following steps:
(1) sampling in a sewage treatment station of a liquor factory, taking liquor wastewater at the inlet of the secondary treatment pool, and filling into a sterile plastic barrel.
(2) Diluting ammonia nitrogen in the white spirit wastewater to 300mg/L, wherein the pH is 3.9, taking 100mL, filling into a 250mL conical flask, and sterilizing at 121 ℃ for 20 min.
(3) Inoculating 8% Enterobacter reuteri into the conical flask on a sterile operating platformEnterobacter ludwigii ZY-10 activated bacterium liquid (viable bacteria concentration is 7.8 multiplied by 10) 5 CFU/mL）。
(4) Culturing in a shaker at 32 deg.C and 200r/min for 54 h.
(5) Blank group Enterobacter ledeburinusEnterobacter ludwigii ZY-10 activated bacteria liquid is replaced by sterile normal saline, and other conditions are the same as those of the experimental group.
(6) And after the culture is finished, measuring the ammonia nitrogen concentration of the experimental group and the blank group, and calculating the ammonia nitrogen degradation rate.
The results of the assay showed that Enterobacter reuteriEnterobacter ludwigii The degradation rate of ZY-10 to ammonia nitrogen in the white wine wastewater is 68.79%.
Example 6
Enterobacter ludwigiiEnterobacter ludwigii ZY-10 degrades ammonia nitrogen in the white wine wastewater. The method comprises the following steps:
(1) sampling in a sewage treatment station of a liquor factory, taking liquor wastewater at the inlet of the secondary treatment tank, and filling into a sterile plastic barrel.
(2) Diluting ammonia nitrogen in the white spirit wastewater to 400mg/L, wherein the pH is 3.7, putting 100mL into a 250mL conical flask, and sterilizing at 121 ℃ for 20 min.
(3) Inoculating 10% Enterobacter reuteri into the conical flask on a sterile operating platformEnterobacter ludwigii ZY-10 activated bacterium liquid (viable bacteria concentration is 1.5 multiplied by 10) 6 CFU/mL）。
(4) Culturing in a shaking table at 35 deg.C and 180r/min for 72 h.
(5) Blank group Enterobacter ledeburinusEnterobacter ludwigii ZY-10 activated bacteria liquid is replaced by sterile normal saline, and other conditions are consistent with those of the experimental group.
(6) And after the culture is finished, measuring the ammonia nitrogen concentration of the experimental group and the blank group, and calculating the ammonia nitrogen degradation rate.
The results of the assay showed that Enterobacter reuteriEnterobacter ludwigii The degradation rate of ZY-10 to ammonia nitrogen in the white wine wastewater is 83.65%.
While the foregoing shows and describes the fundamental principles and principal features of the invention, together with the advantages thereof, the foregoing embodiments and description are illustrative only of the principles of the invention, and various changes and modifications can be made therein without departing from the spirit and scope of the invention, which will fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
SEQUENCE LISTING
<110> Sichuan university of light chemical industry
<120> Enterobacter ludwigii for degrading ammonia nitrogen in white spirit wastewater and application
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1433
<212>　16S rRNA
<213> Enterobacter ludwigii
<400> 1
1 CATGCAAGTC GAACGGTAGC ACAGAGAGCT TGCTCTCGGG TGACGAGTGG CGGACGGGTG
61 AGTAATGTCT GGGAAACTGC CTGATGGAGG GGGATAACTA CTGGAAACGG TAGCTAATAC
121 CGCATAACGT CGCAAGACCA AAGAGGGGGA CCTTCGGGCC TCTTGCCATC AGATGTGCCC
181 AGATGGGATT AGCTAGTAGG TGGGGTAATG GCTCACCTAG GCGACGATCC CTAGCTGGTC
241 TGAGAGGATG ACCAGCCACA CTGGAACTGA GACACGGTCC AGACTCCTAC GGGAGGCAGC
301 AGTGGGGAAT ATTGCACAAT GGGCGCAAGC CTGATGCAGC CATGCCGCGT GTATGAAGAA
361 GGCCTTCGGG TTGTAAAGTA CTTTCAGCGG GGAGGAAGGT GTTGTGGTTA ATAACCGCAG
421 CAATTGACGT TACCCGCAGA AGAAGCACCG GCTAACTCCG TGCCAGCAGC CGCGGTAATA
481 CGGAGGGTGC AAGCGTTAAT CGGAATTACT GGGCGTAAAG CGCACGCAGG CGGTCTGTCA
541 AGTCGGATGT GAAATCCCCG GGCTCAACCT GGGAACTGCA TTCGAAACTG GCAGGCTAGA
601 GTCTTGTAGA GGGGGGTAGA GTTCCAGGTG TAGCGGTGAA ATGCGTAGAG ATCTGGAGGA
661 ATACCGGTGG CGAAGGCGGC CCCCTGGACA AAGACTGACG CTCAGGTGCG AAAGCGTGGG
721 GAGCAAACAG GATTAGATAC CCTGGTAGTC CACGCCGTAA ACGATGTCGA CTTGGAGGTT
781 GTGCCCTTGA GGCGTGGCTT CCGGAGCTAA CGCGTTAAGT CGACCGCCTG GGGAGTACGG
841 CCGCAAGGTT AAAACTCAAA TGAATTGACG GGGGCCCGCA CAAGCGGTGG AGCATGTGGT
901 TTAATTCGAT GCAACGCGAA GAACCTTACC TACTCTTGAC ATCCAGAGAA CTTAGCAGAG
961 ATGGTTTGGT GCCTTCGGGA ACTCTGAGAC AGGTGCTGCA TGGCTGTCGT CAGCTCGTGT
1021 TGTGAAATGT TGGGTTAAGT CCCGCAACGA GCGCAACCCT TATCCTTTGT TGCCAGCGGT
1081 CCGGCCGGGA ACTCAAAGGA GACTGCCAGT GATAAACTGG AGGAAGGTGG GGATGACGTC
1141 AAGTCATCAT GGCCCTTACG AGTAGGGCTA CACACGTGCT ACAATGGCGC ATACAAAGAG
1201 AAGCGACCTC GCGAGAGCAA GCGGACCTCA TAAAGTGCGT CGTAGTCCGG ATTGGAGTCT
1261 GCAACTCGAC TCCATGAAGT CGGAATCGCT AGTAATCGTA GATCAGAATG CTACGGTGAA
1321 TACGTTCCCG GGCCTTGTAC ACACCGCCCG TCACACCATG GGAGTGGGTT GCAAAAGAAG
1381 TAGGTAGCTT AACCTTCGGG AGGGCGCTAC CAC 1413

Claims (10)

1. Enterobacter ludwigiiEnterobacter ludwigii ZY-10, which was deposited at 29.4.2021 in the microbial culture collection center of Guangdong province with the collection number GDMCC No.61633, and the collection address of No. 59, 5 th of Ju Dazhou 100 Mc. Ming Lizhou, Guangzhou, Guangdong province.
2. Enterobacter ludwigii according to claim 1Enterobacter ludwigii Application of ZY-10 in degrading white spirit wastewater.
3. Enterobacter ludwigii according to claim 1Enterobacter ludwigii The ZY-10 is applied to degrading ammonia nitrogen in the white spirit wastewater.
4. Enterobacter reuteri according to claim 3Enterobacter ludwigii The application of ZY-10 in degrading ammonia nitrogen in the white spirit wastewater is characterized by comprising the following steps:
1) preparation of Enterobacter reuteriEnterobacter ludwigii ZY-10 activated bacterium liquid;
2) activated Enterobacter reuteriEnterobacter ludwigii Adding ZY-10 bacterial liquid into the white spirit wastewater;
3) enterobacter ludwigiiEnterobacter ludwigii ZY-10 degrades ammonia nitrogen in the white wine wastewater.
5. The use of claim 4, wherein: enterobacter ludwigiiEnterobacter ludwigii The concentration of ZY-10 activated bacterium liquid is 4-20 x 10 5 CFU/mL。
6. The use of claim 4, wherein: the concentration of ammonia nitrogen in the white spirit wastewater is 100-400 mg/L; enterobacter ludwigiiEnterobacter ludwigii The inoculation amount of ZY-10 is 4-10%.
7. The use of claim 4, wherein: the degradation temperature in the step 3) is 30-40 ℃, the rotating speed is 120-360 r/min, and the degradation time is 24-72 h.
8. Enterobacter ludwigii according to claim 1Enterobacter ludwigii The ZY-10 culture method is characterized by comprising the following steps:
1) enterobacter reuteriEnterobacter ludwigii The ZY-10 strain is inoculated in a seed liquid culture medium and is subjected to shake cultivation at the temperature of 35-39 ℃ and at the speed of 150-210 r/min for 24-48 h to obtain seed liquid;
2) taking a certain amount of nitrobacteria enriched liquid for culture based on a conical flask, sterilizing and cooling, inoculating 4-8% of seed liquid on an aseptic operation table, and then placing the seed liquid on a shaker at 35-39 ℃ and 150-210 r/min for 2-4 days to obtain the enterobacter reuteriEnterobacter ludwigii ZY-10 activated bacterial liquid.
9. Enterobacter reuteri according to claim 8Enterobacter ludwigii The ZY-10 culture method is characterized in that the formula of the seed liquid culture medium comprises the following components in parts by weight: 4-8 parts of yeast extract, 8-12 parts of peptone, 8-12 parts of sodium chloride and 800-1100 parts of distilled water.
10. Enterobacter reuteri according to claim 8Enterobacter ludwigii The ZY-10 culture method is characterized in that the nitrobacteria enriched liquid comprises the following components in parts by weight: 8-10 parts of trisodium citrate, 0.5-1.5 parts of dipotassium phosphate, 2-6 parts of ammonium sulfate, 0.4-0.8 part of magnesium sulfate heptahydrate, 0.4-0.8 part of sodium chloride, 0.4-0.8 part of ferrous sulfate heptahydrate and 800-1100 parts of distilled water, and the pH value is adjusted to 6.5-7.5.
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