JP7062322B2 - Use in the treatment of membrane concentrates of Pseudomonas balealica and its debris leachate - Google Patents

Description

CCTCC CCTCC M 2019730

The present invention belongs to the field of sewage treatment with respect to Pseudomonas balealica and further to use in the treatment of a membrane concentrate of the above-mentioned Pseudomonas balealica waste leachate.

The waste leachate is a high-concentration organic wastewater having a complicated component, has poor biodegradability, has a high concentration of ammonia nitrogen, and has a high content of toxic and harmful substances. As the environmental water quality management standards become more and more strict, especially when the "Technical Specifications for Leachate Treatment Work for Household Waste Landfills (Open Draft)" was promulgated in October 2009, advanced treatment will be mainly based on the membrane treatment process. It is suggested that this should be done, and the standardization tendency of the membrane process is further determined, resulting in about 15% to 30% membrane concentrate in the advanced treatment by the membrane method, which is the mainstream process. The membrane concentrate of garbage leachate is mainly composed of rotaceous substances, has a brown-black (yellow) color, has a COD of usually 1000 to 5000 mg / L, and has a total nitrogen concentration of 500 to 2500 mg / L, and is a large amount. It contains inorganic ions, has a TDS of 20,000 to 60,000 mg / L, a conductivity of 40,000 to 50,000 us / cm, and the salt content of the leachate in the wastewater landfill can reach 3% or more, and the salt content of wastewater. Has a great effect on the osmotic pressure inside the microorganism, the absorption of substances is hindered by high salt content, the growth rate of the microorganism is slowed down, some microorganisms die in large quantities, and the biochemical treatment effect of such wastewater Is seriously damaged.

The total nitrogen in the membrane concentrate of the dust leachate is mainly derived from the concentrate in which the biochemically treated wastewater of the dust leachate has passed through the multi-stage membrane. Most of the ammonia nitrogen in the raw water is converted to nitrate nitrogen by nitrification, and the ratio of nitrate nitrogen in the total nitrogen of the concentrate can reach 90% or more. Incinerator treatment is generally used for such high-salt and high-nitrogen concentrates, but due to the high water content of the landfill disposal site, the amount of heat generated by incineration of wastewater is low, and the concentrate should be treated with wastewater. It is necessary to meet the standard and discharge. Since such wastewater has high salt content, high toxicity, and high nitrogen content, it cannot meet the standard when treated with general microorganisms, and an advanced biological denitrification method suitable for a concentrate is expected.

An object of the present invention: The technical problem to be solved by the present invention is to provide Pseudomonas balealica capable of effectively removing the total nitrogen content in the membrane concentrate of the dust leachate.

The technical problem to be solved by the present invention is to provide the use in the treatment of the membrane concentrate of the above-mentioned Pseudomonas balealica dust leachate.

The technical means adopted by the present invention in order to solve the above technical problems are as follows.

The Pseudomonas balealica of the present invention was named Pseudomonas balearica EBT-1, classified as Pseudomonas balealica, and the strain number was EBT-1 in September 2019. An international deposit based on the Pseudomonas pseudomonas was made to the China Typical Culture Deposit Center of Wuhan University in China on the same day, and the deposit number is CCTCC M 2009730.

Activated sludge from a waste leachate treatment plant is collected and gradually acclimatized to a membrane concentrate of high-salt waste leachate to obtain Pseudomonas balealica of the present invention.

The biological properties of the strain are as follows. The Pseudomonas balearica EBT-1 strain of the present invention is derived from activated sludge in a high-salt waste leachate (salt> 3%) sewage treatment system, and after separation and purification, it is separated and purified in an aerobic condition at 35 ° C. in an LB medium. The colonies are irregularly rounded, 2-5 mm in diameter, white and opaque, with wrinkles from the outside to the inside on the surface, and are negative when stained with Gram. When observed under a microscope, the shape of the cells is rod-shaped.

The strain EBT-1 obtained in the present invention was sequenced with 16S rRNA, subjected to BLAST analysis, and the Pseudomonas strain EBT-1 gene sequence obtained in the present invention was detected in NCBI. The similarity with (Pseudomonas baleria) is as high as 99% or more, and therefore, the obtained strain is identified as Pseudomonas balealica.

Use in the treatment of the membrane concentrate of the above-mentioned Pseudomonas balealica waste leachate.

Pseudomonas balearica EBT-1 was inoculated into the medium and aerobic cultured at 30-35 ° C. for 24-32 hours to obtain the EBT-1 expanded culture product, and then a membrane concentrate of dust leachate. Inoculate and process.

When Pseudomonas balearica EBT-1 is inoculated into a medium and cultured, the inoculation amount is 3% to 5%.

1 L of medium (water) contains 10 to 12.5 parts by mass of corn steep liquor (dry powder), 5 parts by mass of yeast extract, 1 part by mass of dipotassium hydrogen phosphate, 10 parts by mass of sodium chloride, and 2 parts by mass of anhydrous sodium acetate. Includes 5.5 parts by mass and 0.03 parts by mass of magnesium sulfate.

Pseudomonas balearica EBT-1 is used for the decomposition of nitrogen-containing compounds in the membrane concentrate of waste leachate, the nitrogen-containing compounds containing nitrate nitrogen, nitrite nitrogen, and ammonia nitrogen.

Degradation of nitrogen-containing compounds in the membrane concentrate of waste leachate by Pseudomonas balealica is carried out under anaerobic / hypoxic conditions.

When Pseudomonas balearica EBT decomposes nitrogen-containing compounds in the membrane concentrate of the leachate, the inoculum is 0.2% to 0.4%, and the bacterial solution is the membrane concentrate of the leachate. The COD / N of the waste water added to the anaerobic / low oxygen treatment reactor is 4: 1 to 6: 1.

The salt content of the membrane concentrate of the dust leachate is 3% or more.

The Pseudomonas barealica EBT-1 expanded culture product obtained by culturing is centrifuged to obtain a bacterial cell solid. Pseudomonas balealica EBT- As a result of tracking the decomposition rate of No. 1, Pseudomonas balearica EBT-1 can be applied to wastewater in the range of 7% or less (7% content) salt.

Salinity measurement Per 1 L of water supply, in terms of mass fraction, glucose 6.1 g, KNO ₃ 6.07 g, KH ₂ PO ₄ 1.5 g, K ₂ HPO _4.3 H ₂ O 6.0 g, _Л4.7 H ₂ O 1. NaCl added according to 0 g and a desired salt content is added as a component.

The Pseudomonas barealica EBT-1 expanded culture product obtained by culturing is centrifuged to obtain a bacterial cell solid. Centrifugated solids were inoculated into a low oxygen bottle containing a membrane concentrate of leachate at an inoculation amount of 0.4% and shaken at 30 ° C. and 150 rpm. Pseudomonas against the membrane concentrate of leachate -The total nitrogen removal rate of Pseudomonas balearica EBT-1 is as high as 99%.

The effective volume of the anaerobic / low oxygen treatment reactor of the membrane concentrate of the dust leachate is 2 L, the original sludge in the reactor has a concentration of 60,000 to 80,000 mg / L, and the hydraulic residence time is 12 hours. The ratio of carbon to nitrogen was set to 4: 1 by externally adding a carbon source, and the Pseudomonas balearica EBT-1 expanded culture solution obtained by culturing was trashed at an inoculation amount of 0.2%. When added to the anaerobic / low oxygen treatment reactor of the membrane concentrate of the leachate, the sludge activity in the reactor is effectively improved, the total nitrogen removal rate is effectively increased from 30% to 98.89%, and The quality of the discharged water is stable and reaches the standard. It is shown that by adding Pseudomonas balearica EBT-1 of the present invention to an existing sludge system, the denitrification efficiency of the sludge system can be effectively improved.

Beneficial Effects In the present invention, a membrane concentrate of leachate is gradually acclimatized to obtain a highly salt-tolerant Pseudomonas balealica EBT-1, which is a Pseudomonas balealica. , Efficient biological denitrification of dust leachate against membrane concentrate can be achieved, the denitrification rate of existing activated sludge can be effectively improved, the denitrification effect of sludge can be enhanced, and the quality of discharged water can be improved. Is stable.

FIG. 3 is a salt-adaptive denitrification effect diagram of EBT-1 in Example 3. It is a high denitrification efficiency diagram with respect to the membrane concentrate of the dust leachate of EBT-1 in Example 4. It is a structural schematic diagram of the anaerobic / hypoxic treatment reactor of the membrane concentrate of the dust leachate. It is a denitrification strengthening efficiency diagram with respect to the membrane concentrate reactor of the dust leachate of EBT-1 in Example 5.

Hereinafter, the technical proposal of the present invention will be further described with reference to the drawings and specific examples.

Example 1: Isolation and Identification of Strain Strain EBT-1 is an aerobic denitrifying bacterium having denitrifying activity, screened from activated sludge of a waste leachate sewage treatment system in a garbage incineration plant.

Separation screening method: Waste leachate (high salt content) Sewage treatment Active sludge is taken out from the biochemical system, and 5 g of active sludge is inoculated into a DM inorganic salt medium (salt content in the medium> 3%) having a nitrate nitrogen concentration of 100 mg / L. Incubate for 1 day in a shaker at a temperature of 35 ° C. and a rotation speed of 150 rpm to obtain the first bacterial solution, and inoculate the first bacterial solution at a dose of 5% (body integration rate) to a nitrate nitrogen concentration of 200 mg / L. Inoculate a DM inorganic salt medium (salt content in the medium> 3%), incubate for 1 day in a shaker at a temperature of 35 ° C. and a rotation speed of 150 rpm to obtain a second bacterial solution, and inoculate at a dose of 5%. The two bacterial solutions were inoculated into a DM inorganic salt medium having a nitrate nitrogen concentration of 300 mg / L and cultured for one day in a shaker at a temperature of 35 ° C. and a rotation speed of 150 rpm to obtain a tertiary bacterial solution to obtain a tertiary bacterial solution. Is absorbed, applied to a DM inorganic salt solid medium (salt content in the medium> 3%), allowed to stand at 35 ° C. and cultured, a strain having a fast growth rate is selected, and a strain having a fast growth rate is bromotimol blue. Streak in a DM inorganic salt solid medium (salt content in the medium> 3%) containing Further streaked and purified in a medium (salt content in the medium> 3%) to select a single colony, which colony is Pseudomonas balealica of the present invention, the strain number is EBT-1, and the strain EBT. -1 can realize efficient biodenitration of the high-salt dust leachate against the membrane concentrate.

The characteristics of the colony of the strain EBT-1 are as follows. After separation and purification, it grows well in LB medium under aerobic conditions at 35 ° C., the colonies are irregularly round, 2-5 mm in diameter, white and opaque, and the surface. There are wrinkles from the outside to the inside, and when Gram stain is performed, it shows a negative result, and when observed under a microscope, the cell shape is rod-shaped.

The EBT-1 bacterium 16S rRNA full sequence obtained by PCR amplification is as follows (SEQ ID NO. 1).

gcttgcggcagactacacatgcagtcgagcggcagcgggtccttcgggatgccggcgagcggcggacgggtgagtaatgcctaggaatctgcctggtagtgggggataactcggggaaactcgagctaataccgcatacgtcctacgggagaaagcgggggatcttcggacctcgcgctaccagatgagcctaggtcggattagctagttggtgaggtaaaggctcaccaaggcgacgatccgtagctggtctgagaggatgatcagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattggacaatgggcgaaagcctgatccagccatgccgcgtgtgtgaagaaggtcttcggattgtaaagcactttaagttgggaggaagggcagtaagctaatatcttgctgttttgacgttaccgacagaataagcaccggctaacttcgtgccagcagccgcggtaatacgaagggtgcaagcgttaatcggaattactgggcgtaaagcgcgcgtaggtggtttgataagttggatgtgaaagccccgggctcaacctgggaattgcatccaaaactgtctgactagagtatggcagagggtggtggaatttcctgtgtagcggtgaaatgcgtagatataggaaggaacaccagtggcgaaggcgaccatctgggctaatactgacactgaggtgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgtcgactagccgttgggatccttgagatcttagtggcgcagctaacgcattaagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggccttgacatgcagagaactttccagagatggattggtgccttcgggaactctgac acaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgtaacgagcgcaacccttgtccttagttaccagcacgttaaggtgggcactctaaggagactgccggtgacaaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacggcctgggctacacacgtgctacaatggtcggtacaaagggttgccaagccgcgaggtggagctaatcccataaaaccgatcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcggaatcgctagtaatcgtgaatcagaatgtcacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggtagtgggttgctccagaagtaagcgaagtctaaccttcggggggacggtaccacggagatactg

As a result of comparison, the gene sequence has a high similarity of 99% or more to Pseudomonas balealica, and therefore, the obtained strain is identified as Pseudomonas balealica.

Example 2: Expanded culture of Pseudomonas barealica EBT-1 of the present invention The medium used for the expanded culture contains 10 g of corn steep liquor (dry powder), 5 g of yeast extract, and sodium chloride per 1 L of water. It contains 10 g, anhydrous sodium acetate 2.5 g, dipotassium hydrogen phosphate 1 g and magnesium sulfate 0.03 g, and the initial pH of the medium is 6.5.

Medium production process: 10 g of corn steep liquor (dry powder), 5 g of yeast extract, 10 g of sodium chloride, 2.5 g of anhydrous sodium acetate, 1 g of dipotassium hydrogen phosphate and 0.03 g of magnesium sulfate are dissolved in 1000 g of water and alkaline. The pH was adjusted to 6.5 and sterilized at 121 ° C. for 20 minutes to obtain a medium.

Pseudomonas balearica EBT-1 bacterial mother broth was inoculated into the medium at an inoculation amount of 3% to 5% (volume fraction) and cultured at 30 to 35 ° C. for 28 to 32 hours.

After the expanded culture using the medium of the present invention, the expanded bacterial solution was counted by the dilution plate method, and the target effective bacterial amount was 5 × 10 ⁸ to 1 × 10 ⁹ CFU / mL.

Example 3: Examination of salt adaptability of Pseudomonas balealica of the present invention The Pseudomonas balealica EBT-1 expanded culture solution cultured in Example 2 was centrifuged at 6000 rpm for 5 min. The supernatant was discarded to obtain an activated cell solid. Four salt gradients of 0%, 3%, 7% and 10% were set. Salinity measurement Per 1 L of water supply, in terms of mass fraction, glucose 6.1 g, KNO ₃ 6.07 g, KH ₂ PO ₄ 1.5 g, K ₂ HPO _4.3 H ₂ O 6.0 g, י _4.7 H ₂ O 1. 0 g, NaCl (adjusted according to the desired salt content) is contained as an ingredient. Centrifugal cells are inoculated into salt-measured water supply at an inoculation amount of 0.4%, and shaken at 30 ° C. and 150 rpm in a 250 mL bottle with a blue lid.

Changes in total nitrogen in different salt condition reactions were detected and the results are shown in FIG. As shown in FIG. 1, the denitrification rate of Pseudomonas barealica EBT-1 decreases with increasing salt content, and at 5% salt content or less, the total nitrogen removal rate reaches 90% or more, 7 At% salt content, the total nitrogen removal rate is 50% or more. The Pseudomonas barealica EBT-1 of the present patent is obtained from sludge in the biochemical system of high-salt waste leachate, which itself has certain salt resistance and is efficient denitrification. It is obtained by acclimatization and screening, and has high salt resistance and denitrification performance.

Example 4: Analysis of the biological denitrification effect of the Pseudomonas balealica EBT-1 dust exudate of the present invention on the membrane concentrate of the Pseudomonas balealica EBT-1 expanded culture solution cultured in Example 2. Transfer the bacterial solution from Pseudomonas to a new centrifuge tube, centrifuge at 6000 rpm for 5 minutes, discard the supernatant, obtain activated bacterial cell solids, prepare a clean bottle with a blue lid, and put dust in the bottle with a blue lid. Add 200 mL of leachate membrane concentrate (3.5% salt), then add anhydrous sodium acetate as a carbon source to a 4: 1 ratio of carbon to nitrogen in the waste liquid, and finally Pseudomonas barealica. ) EBT-1 was added at an inoculum of 0.4% and shaken at 30 ° C. and 150 rpm.

Changes in total nitrogen in the membrane concentrate of the dust leachate during the reaction were detected, and the results are shown in FIG. As shown in FIG. 2, after 72 hours of treatment, the total nitrogen concentration in the membrane concentrate of the dust leachate decreased from the initial concentration of 1110.74 mg / L to 13.37 mg / L, and as can be seen from this, the Pseudomonas of the present invention. -Pseudomonas balearica EBT-1 has a high total nitrogen removal rate of 99% with respect to the membrane concentrate of the dust leachate. Pseudomonas balearica EBT-1 is a facultative anaerobic bacterium, has aerobic denitrification properties, and has a high denitrification function even in a hypoxic environment.

Example 5: Analysis of the effect of enhancing biological denitrification on the membrane concentrate of Pseudomonas barealica EBT-1 of the present invention The structure of the anaerobic / hypoxic reactor of the membrane concentrate of the dust leachate It is shown in FIG. The membrane concentrate of the dust leachate and the external carbon source mixture enter from the bottom of the reactor, undergo an anaerobic / hypoxic reaction with activated sludge, and then flow out from the top of the reactor. The reactor has an effective volume of 2 L, a hydraulic residence time of 12 hours, a sludge concentration of 60,000 mg / L to 80,000 mg / L, and a carbon-to-nitrogen ratio of an external carbon source of 4: 1 for water supply and discharge. Pseudomonas balealica cultured in Example 2 after continuous operation, daily detection of the index of nitrogen-containing compound in the discharged water, detection of denitrification efficiency, and stable water quality of the discharged water from the reactor. (Pseudomonas barealica) EBT-1 expanded culture solution The bacterial solution is charged into the reactor sludge at an inoculation amount of 0.2%, and the denitrification efficiency of the water stably discharged before and after the addition of the bacterial agent is followed.

FIG. 4 shows the results of the denitrification efficiency of the water that is stably discharged before and after the bacterial agent is put into the anaerobic / hypoxic reactor of the membrane concentrate of the dust leachate. As shown in FIG. 4, the total nitrogen concentration of the anaerobic / hypoxic reactor of the membrane concentrate of the dust exudate was 547.48 mg / L before the addition of the bacterial agent (stage I in FIG. 4). As the denitrifying active sludge is shocked by an excessive carbon source, the structure of the community changes, which results in poor denitrifying activity of the sludge and a total nitrogen removal rate of wastewater between 30% and 50%. When the Pseudomonas barealica EBT-1 fungus and the sludge of the reactor are sufficiently mixed (Phase II in FIG. 4), the total nitrogen removal rate of the discharged water is effective. With the increase in the adaptation cycle, the total nitrogen removal rate was clearly improved, the total nitrogen removal rate of the final discharged water reached 98.89%, and then the total nitrogen concentration of the feed water was 1000 mg / It was improved to L (stage III in FIG. 4), and even in this way, the total nitrogen removal rate of the system was stabilized to 90% or more. Pseudomonas barealica EBT-1 fungus has high efficiency denitrification performance, and when added to a denitrifying activated sludge system, enriches the structure of the denitrifying denitrifying community in the system and sludge. Greatly improves the denitrification performance of. The fungal agent can be effectively applied to the membrane concentrate of the leachate, grows and propagates in the system, and is less likely to be washed away, thereby ensuring the stability of the water quality of the discharged water.

The Pseudomonas barealica EBT-1 of the present invention has a high efficiency of removing total nitrogen from the membrane concentrate of the dust leachate, and can effectively solve the difficult problem of high denitrification of the membrane concentrate of the dust leachate. When the Pseudomonas barealica EBT-1 of the present invention is added to an existing sludge system, the denitrification efficiency of the sludge system can be effectively improved.

Claims (10)

Pseudomonas Balearica,
Pseudomonas balealica, named Pseudomonas balealica, was deposited at the China Typical Culture Depositary Center on September 17, 2019, and has a deposit number of CCTCC M 2009730. Use in the treatment of the membrane concentrate of the Pseudomonas balealica dust leachate according to claim 1. Pseudomonas balearica EBT-1 was inoculated into the medium and aerobic cultured at 30-35 ° C. for 24-32 hours to obtain the EBT-1 expanded culture product, and then a membrane concentrate of trash leachate. The use in the treatment of the membrane concentrate of the Pseudomonas balealica dust leachate according to claim 2, wherein the treatment is carried out by inoculating the Pseudomonas balea. The film of Pseudomonas balealica dust exudate according to claim 3, wherein the inoculation amount of Pseudomonas balearica EBT-1 when inoculated into a medium and cultured is 3% to 5%. Use in the processing of concentrates. As components, 10 parts by mass of corn steep liquor, 5 parts by mass of yeast extract, 1 part by mass of dipotassium hydrogen phosphate, 10 parts by mass of sodium chloride, 2.5 parts by mass of anhydrous sodium acetate, and 0.03 parts of magnesium sulfate per 1 L of medium. Use in the treatment of a membrane concentrate of the dust leachate of Pseudomonas balealica according to claim 3, wherein the mass is contained. The use in the treatment of the membrane concentrate of the Pseudomonas balealica dust leachate according to claim 3, wherein the initial pH of the medium is 6.5 to 7.5. Pseudomonas balearica EBT-1 is used to degrade nitrogen-containing compounds in the membrane concentrate of leachate, which nitrogen-containing compounds include nitrate nitrogen, nitrite nitrogen and ammonia nitrogen. Use in the treatment of a membrane concentrate of the Pseudomonas balealica dust leachate according to claim 2. The membrane concentrate of Pseudomonas balealica according to claim 7, wherein the decomposition of the nitrogen-containing compound in the membrane concentrate of the dust leachate by Pseudomonas balealica is carried out under anaerobic / low oxygen conditions. Use in processing. When Pseudomonas balealica EBT-1 decomposes nitrogen-containing compounds in the membrane concentrate of leachate, the inoculum is 0.2% to 0.4%, and the bacterial solution is the membrane concentrate of the leachate. The Pseudomonas balealica dust leachate according to claim 7, wherein the COD / N of the waste water in the reactor is 4: 1 to 6: 1 when added to the anaerobic / low oxygen treatment reactor. Use in the treatment of membrane concentrates. The use in the treatment of the membrane concentrate of Pseudomonas balealica according to claim 7, wherein the membrane concentrate of the dust leachate has a salt content of 3% or more.

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