WO2021077452A1 - Pseudomonas balearica et son application dans le traitement d'une solution concentrée de membrane de lixiviat de décharge - Google Patents

Pseudomonas balearica et son application dans le traitement d'une solution concentrée de membrane de lixiviat de décharge Download PDF

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WO2021077452A1
WO2021077452A1 PCT/CN2019/114170 CN2019114170W WO2021077452A1 WO 2021077452 A1 WO2021077452 A1 WO 2021077452A1 CN 2019114170 W CN2019114170 W CN 2019114170W WO 2021077452 A1 WO2021077452 A1 WO 2021077452A1
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pseudomonas
landfill leachate
balearica
ebt
pseudomonas balearica
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PCT/CN2019/114170
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Chinese (zh)
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黄开龙
张徐祥
叶林
任洪强
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南京大学
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Priority to JP2021544869A priority Critical patent/JP7062322B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas
    • 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
    • C02F3/02—Aerobic processes
    • C02F3/12—Activated sludge processes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • 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
    • C02F2103/06—Contaminated groundwater or leachate
    • 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

Definitions

  • the present invention relates to a kind of Pseudomonas balearicum, and also relates to the application of the above-mentioned Pseudomonas balearicum in the treatment of garbage leachate membrane concentrates, and belongs to the field of sewage treatment.
  • Landfill leachate is a kind of high-concentration organic wastewater with complex composition, which has the characteristics of poor biodegradability, high ammonia nitrogen concentration, and rich content of toxic and harmful substances.
  • the "Technical Specifications for Landfill Leachate Treatment Engineering (Draft for Comment)" was promulgated, and it was proposed that the advanced treatment should be based on the membrane treatment process.
  • the standardization trend of membrane technology is determined. As the mainstream technology, the advanced treatment process of membrane method will produce about 15%-30% of membrane concentrate.
  • the main components of the landfill leachate membrane concentrate are humic substances, which are brown-black (yellow).
  • the COD is usually between 1000 and 5000 mg/L, the total nitrogen concentration is between 500 and 2500 mg/L, and contains a large amount of inorganic ions, TDS Between 20,000 ⁇ 60,000mg/L, the conductivity is 40,000 ⁇ 50,000us/cm, and the salinity in the landfill leachate membrane concentrate can reach more than 3%.
  • the salinity of wastewater seriously affects the internal osmotic pressure of microorganisms, and high salinity makes substances The absorption process is blocked, the growth rate of microorganisms is slow, and some microorganisms die in large numbers, which seriously affects the biochemical treatment efficiency of this type of wastewater.
  • the total nitrogen in the membrane concentrate of the landfill leachate mainly comes from the concentrated liquid of the biochemical tail water of the landfill leachate after passing through the multi-stage membrane.
  • most of the ammonia nitrogen in the raw water is converted into nitrate nitrogen through nitrification, and the proportion of nitrate nitrogen in the total nitrogen of the concentrated liquid can reach more than 90%.
  • Such high-salinity, high-nitrogen concentrated liquids are generally treated by incineration, but due to the increase in the amount of water in the landfill, the waste water incineration has a low calorific value, and the concentrated liquid needs to be treated as wastewater to meet the discharge standards.
  • This kind of wastewater has high salinity, high toxicity, and high nitrogen, and it is difficult for general microorganisms to treat it up to the standard. There is an urgent need for a biological deep denitrification method suitable for concentrated liquid.
  • the technical problem to be solved by the present invention is to provide a Pseudomonas balearicum which can efficiently remove the total nitrogen content in the membrane concentrate of the landfill leachate.
  • the technical problem to be solved by the present invention is to provide the application of the above-mentioned Pseudomonas balearicum in the treatment of landfill leachate membrane concentrate.
  • the technical means adopted by the present invention are:
  • the Pseudomonas balearica of the present invention is named Pseudomonas balearica (Pseudomonas balearica) EBT-1, the classification is named Pseudomonas balearica (Pseudomonas balearica), and the strain number is EBT-1, It was deposited at the Chinese Type Culture Collection at Wuhan University, China on September 17, 2019, and the deposit number is CCTCC M 2019730.
  • the Pseudomonas balearicus of the present invention is obtained.
  • the biological properties of the strain are as follows:
  • the Pseudomonas balearica EBT-1 strain of the present invention is derived from the activated sludge in the high-salt landfill leachate (salinity> 3%) sewage treatment system, and is separated and purified Later, in LB medium, 35°C aerobic conditions grow well, the colonies are irregularly round, 2 ⁇ 5mm in diameter, white colonies, opaque, with folds from the outside to the inside, and Gram staining is negative.
  • the cell shape is rod-shaped under microscope observation.
  • the strain EBT-1 obtained in the present invention was sequenced by 16S rRNA and BLAST analysis was used to retrieve the gene sequence of the Pseudomonas strain EBT-1 obtained in the present invention in NCBI, and its gene sequence was similar to that of Pseudomonas balearicus (Pseudomonas balearica) has a similarity of more than 99%, so the identified strain is Pseudomonas balearica.
  • Pseudomonas balearica (Pseudomonas balearica) EBT-1 was inoculated into the culture medium, aerobic cultured at 30 ⁇ 35°C for 24 ⁇ 32h, after the culture, the expanded culture product of EBT-1 was obtained, and then it was inoculated into garbage The percolate membrane concentrate is processed.
  • Pseudomonas balearica (Pseudomonas balearica) EBT-1 is inoculated into a medium for culture when the inoculation amount is 3% to 5%.
  • each liter of medium contains the following components by mass: 10 to 12.5 parts of corn steep liquor (dry powder), 5 parts of yeast extract, 1 part of dipotassium hydrogen phosphate, 10 parts of sodium chloride, and anhydrous acetic acid 2.5 parts of sodium and 0.03 parts of magnesium sulfate.
  • Pseudomonas balearica (Pseudomonas balearica) EBT-1 is used to degrade nitrogen-containing compounds in the landfill leachate membrane concentrate.
  • the nitrogen-containing compounds include nitrate nitrogen, nitrite nitrogen and ammonia nitrogen.
  • Pseudomonas balearicus degrades nitrogen-containing compounds in the landfill leachate membrane concentrate under anaerobic/anoxic conditions.
  • the bacterial solution is added to the landfill leachate membrane concentrate according to the inoculation amount of 0.2% to 0.4%.
  • the COD/N of the wastewater in the reactor is 4:1 to 6:1.
  • the salinity of the landfill leachate membrane concentrate is 3% or more.
  • the expanded culture product of Pseudomonas balearica (Pseudomonas balearica) EBT-1 obtained by the culture is centrifuged, and the bacterial solid is obtained after centrifugation.
  • the centrifugal solid was inoculated into the water for determination of different salinity according to the inoculation amount of 0.4%, and shaken at 30°C and 150rpm to track the degradation of nitrogen-containing compounds by Pseudomonas balearica EBT-1 under different salinities Rate, obtained Pseudomonas balearica (Pseudomonas balearica) EBT-1 can adapt to wastewater with salinity below 7% (including 7%).
  • each liter of salinity determination water contains the following mass fraction components: glucose 6.1g, KNO 3 6.07g, KH 2 PO 4 1.5g, K 2 HPO 4 ⁇ 3H 2 O 6.0g, MgSO 4 ⁇ 7H 2 O 1.0g and NaCl added according to the required salinity.
  • the expanded culture product of Pseudomonas balearica (Pseudomonas balearica) EBT-1 obtained by the culture is centrifuged, and the bacterial solid is obtained after centrifugation.
  • the centrifugal solid was inoculated into an anoxic bottle containing a landfill leachate membrane concentrate according to the inoculation amount of 0.4%, and then shaken at 30°C and 150 rpm.
  • Pseudomonas balearica EBT-1 was effective on landfill infiltration.
  • the total nitrogen removal rate of the filtrate membrane concentrate is as high as 99%.
  • the effective volume of the anaerobic/anoxic treatment reactor of the landfill leachate membrane concentrate is 2L
  • the original sludge concentration in the reactor is 60000 ⁇ 80,000mg/L
  • the hydraulic retention time is 12h
  • the carbon source is added to make the carbon to nitrogen ratio 4:4: 1.
  • the expanded culture broth of Pseudomonas balearica (Pseudomonas balearica) EBT-1 is added to the anaerobic/anoxic treatment reactor of the landfill leachate membrane concentrate according to the inoculation amount of 0.2%.
  • Can significantly improve the sludge activity in the reactor the total nitrogen removal rate is effectively increased from 30% to 98.89%
  • the effluent quality is stable and up to standard.
  • the present invention obtains high-salt-tolerant Pseudomonas balearica EBT-1 after stepwise domestication of the garbage percolate membrane concentrate.
  • the Pseudomonas balearica ) EBT-1 can achieve high-efficiency biological denitrification of the landfill leachate membrane concentrate; in addition, it can effectively increase the denitrification rate of the existing activated sludge, enhance the sludge denitrification effect, and stabilize the effluent quality.
  • Figure 1 is a graph showing the adaptive denitrification effect of EBT-1 on salinity in Example 3;
  • Figure 2 is a graph showing the efficiency of EBT-1 for deep denitrification of landfill leachate membrane concentrate in Example 4;
  • Figure 3 is a schematic diagram of the structure of the anaerobic/anoxic treatment reactor for the membrane concentrate of landfill leachate;
  • Fig. 4 is an efficiency diagram of the enhanced denitrification of the landfill leachate membrane concentrate reactor by EBT-1 in Example 5.
  • the strain EBT-1 is an aerobic denitrifying bacteria with denitrification activity screened from the activated sludge of the waste incineration leachate sewage treatment system.
  • the separation and screening method is: remove the activated sludge from the waste leachate (high salt) sewage treatment biochemical system; inoculate 5 g of activated sludge into a DM inorganic salt medium with a nitrate nitrogen concentration of 100 mg/L (salinity in the medium) >3%), incubate on a shaker with a temperature of 35°C and a rotating speed of 150 rpm for 1 day to obtain the first bacterial solution; inoculate the first bacterial solution to a nitrate nitrogen concentration of 200 mg at an inoculum of 5% (volume fraction) /L of DM inorganic salt medium (salinity in the medium>3%), cultured on a shaker with a temperature of 35°C and a rotating speed of 150rpm for 1 day to obtain the second bacterial solution; The bacterial solution was inoculated into a DM inorganic salt medium with a nitrate nitrogen concentration of 300 mg/L, and cultured on a shaker at
  • strain EBT-1 The colony characteristics of strain EBT-1 are as follows: After isolation and purification, it grows well in LB medium under 35°C aerobic conditions. The colonies are irregularly round, with a diameter of 2 ⁇ 5mm, white colonies, opaque, and outwardly directed. The inner folds were negative by Gram stain, and the cell shape was rod-shaped under microscope observation.
  • Example 2 Expanded culture of Pseudomonas balearica EBT-1 of the present invention:
  • the medium used for the expansion culture includes the following quality components: 10g corn steep liquor (dry powder) per liter of water, 5g yeast extract, 10g sodium chloride, 2.5g anhydrous sodium acetate, 1g dipotassium hydrogen phosphate and 0.03g magnesium sulfate ,
  • the initial pH of the medium is 6.5.
  • the production process of the culture medium is as follows: dissolve 10g corn steep liquor (dry powder), 5g yeast extract, 10g sodium chloride, 2.5g anhydrous sodium acetate, 1g dipotassium hydrogen phosphate and 0.03g magnesium sulfate in 1000g water, adjust it with alkali The pH was 6.5, and sterilized at 121°C for 20 minutes to obtain a culture medium.
  • Pseudomonas balearica (Pseudomonas balearica) EBT-1 stock solution was inoculated into the culture medium according to the inoculation amount of 3% to 5% (volume fraction), and cultured at 30 to 35°C for 28 to 32 hours.
  • the bacteria solution obtained by the expansion culture is counted by the dilution plate method, and the target effective bacteria amount is 5 ⁇ 10 8 to 1 ⁇ 10 9 CFU/mL.
  • Example 3 The present invention Pseudomonas balearica (Pseudomonas balearica) EBT-1 salinity adaptability investigation
  • the Pseudomonas balearica EBT-1 expansion medium obtained by cultivation in Example 2 was centrifuged at 6000 rpm for 5 minutes, and the supernatant was removed to obtain activated bacterial solids.
  • Each liter of salinity determination water contains the following mass fraction components: glucose 6.1g, KNO 3 6.07g, KH 2 PO 4 1.5g, K 2 HPO 4 ⁇ 3H 2 O 6.0g, MgSO 4 ⁇ 7H 2 O 1.0g , NaCl is adjusted according to the required salinity.
  • the centrifugal cell solids were inoculated into the salinity determination water according to the inoculation amount of 0.4%, and were shaken at 30°C and 150 rpm in a 250 mL blue cap bottle.
  • the nitrogen removal rate of Pseudomonas balearica (Pseudomonas balearica) EBT-1 decreases with the increase of salinity. Below 5% salinity, the total nitrogen removal rate can reach more than 90%; At 7% salinity, the total nitrogen removal rate is above 50%.
  • the patent Pseudomonas balearica EBT-1 is obtained from the sludge in the biochemical system of high-salt landfill leachate. It has certain salt tolerance characteristics and is obtained through high-efficiency denitrification and domestication screening. Strong salt tolerance and denitrification performance.
  • Example 4 Analysis of the effect of Pseudomonas balearica EBT-1 of the present invention on biological denitrification of landfill leachate membrane concentrate.
  • the Pseudomonas balearica (Pseudomonas balearica) EBT-1 expansion broth obtained in Example 2 was transferred to a new centrifuge tube, centrifuged at 6000 rpm for 5 minutes, and the supernatant was removed to obtain the activated Bacterial solids, take a clean blue cap bottle, add 200mL landfill leachate membrane concentrate (salinity 3.5%) to the blue cap bottle, and then add anhydrous sodium acetate as the carbon source to make the carbon-nitrogen ratio in the waste liquid 4:1, finally add Pseudomonas balearica EBT-1 according to the inoculum amount 0.4%, and shake at 30°C and 150rpm.
  • Implementation case 5 Effect analysis of Pseudomonas balearica EBT-1 of the present invention on enhanced biological denitrification of landfill leachate membrane concentrate
  • the structure of the anaerobic/anoxic reactor for the membrane concentrate of landfill leachate is shown in Figure 3.
  • the mixed liquid of the landfill leachate membrane concentrate and the external carbon source enters from the bottom of the reactor, passes through the anaerobic/anoxic reaction of the activated sludge, and flows out from the upper part of the reactor.
  • the effective volume of the reactor is 2L
  • the hydraulic retention time is 12h
  • the sludge concentration is 60000mg/L ⁇ 80000mg/L
  • the carbon source carbon to nitrogen ratio is 4:1
  • the water is continuously fed in and out
  • the nitrogen compounds in the water are detected every day to calculate the denitrification efficiency.
  • Figure 4 shows the results of stable effluent denitrification efficiency before and after the addition of bacteria in the landfill leachate membrane concentrate anaerobic/anoxic reactor.
  • Figure 4 Phase I the total nitrogen concentration of the influent of the anaerobic/anoxic reactor of the landfill leachate membrane concentrate is 547.48 mg/L.
  • the denitrification activated sludge has been exposed to Due to the impact of the amount of carbon source, the community structure changes, resulting in poor sludge denitrification activity, and the total nitrogen removal rate of the effluent fluctuates between 30% and 50%; Pseudomonas balearica EBT-1 is added After the bacterial agent and the reactor sludge are fully mixed (Figure 4 Phase II), the total nitrogen removal rate of the effluent is effectively improved. As the adaptation period increases, the total nitrogen removal rate increases significantly, and the final total nitrogen removal rate of the effluent can reach 98.89 %; Then increase the total nitrogen concentration of the influent to 1000mg/L ( Figure 4 Phase III), and the total nitrogen removal rate of the system is still stable above 90%.
  • Pseudomonas balearica (Pseudomonas balearica) EBT-1 bacterial agent has high-efficiency denitrification performance. After adding the denitrification activated sludge system, the community structure of denitrification and denitrification bacteria in the system is enriched, and the sludge is greatly improved. The denitrification performance. In addition, the bacterial agent can better adapt to the landfill leachate membrane concentrate, grow and reproduce in the system, and is not easy to be lost, thereby ensuring the stability of the effluent water quality.
  • the Pseudomonas balearica EBT-1 of the present invention has high removal efficiency of total nitrogen in the landfill leachate membrane concentrate, and can effectively solve the problem of deep denitrification in the landfill leachate membrane concentrate.
  • Pseudomonas balearica EBT-1 of the present invention is added to an existing sludge system, the denitrification efficiency of the sludge system can be effectively increased.

Abstract

Pseudomonas balearica. Le Pseudomonas balearica est désigné par Pseudomonas balearica EBT-1, et est conservé auprès du China Center for Type Culture Collection (CCTCC), en date du 17 septembre 2019, avec un numéro de conservation CCTCC M 2019730. L'invention concerne en outre une application du Pseudomonas balearica dans le traitement d'une solution concentrée de membrane de lixiviat de décharge. Le Pseudomonas balaerica EBT-1 à tolérance de sel élevée est obtenu par une fermentation par étapes de la solution concentrée de membrane de lixiviat de décharge. Le Pseudomonas balaerica EBT-1 peut éliminer efficacement la teneur totale en azote de la solution concentrée de membrane de lixiviat de décharge.
PCT/CN2019/114170 2019-10-24 2019-10-30 Pseudomonas balearica et son application dans le traitement d'une solution concentrée de membrane de lixiviat de décharge WO2021077452A1 (fr)

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CN113003726B (zh) * 2021-03-04 2022-07-01 重庆理工大学 垃圾渗滤液反渗透膜滤浓缩液的处理方法
CN113684159B (zh) * 2021-10-27 2022-02-11 佛山市玉凰生态环境科技有限公司 一株巴利阿里假单胞菌及其在河道水体脱氮除磷中的应用

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CN114836355A (zh) * 2022-05-30 2022-08-02 沈阳风景园林股份有限公司 一种假单胞菌dt04及其在污水脱氮的应用
CN114836355B (zh) * 2022-05-30 2024-04-02 沈阳风景园林股份有限公司 一种假单胞菌dt04及其在污水脱氮的应用

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