WO2021077452A1 - Pseudomonas balearica and application thereof in treating landfill leachate membrane concentrated solution - Google Patents

Pseudomonas balearica and application thereof in treating landfill leachate membrane concentrated solution 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/en
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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.

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Abstract

Pseudomonas balearica. The Pseudomonas balearica is named Pseudomonas balearica EBT-1, and is preserved in China Center for Type Culture Collection (CCTCC) on 17 September 2019, with a preservation number being CCTCC M 2019730. Further provided is an application of the Pseudomonas balearica in treating a landfill leachate membrane concentrated solution. High-salt-tolerant Pseudomonas balaerica EBT-1 is obtained by stepwise domestication of the landfill leachate membrane concentrated solution. The Pseudomonas balaerica EBT-1 can effectively remove the total nitrogen content of the landfill leachate membrane concentrated solution.

Description

一种巴利阿里假单胞菌及其在处理垃圾渗滤液膜浓缩液中的应用Pseudomonas balearicus and its application in processing garbage leachate membrane concentrate 技术领域Technical field
本发明涉及一种巴利阿里假单胞菌，还涉及上述巴利阿里假单胞菌在处理垃圾渗滤液膜浓缩液中的应用，属于污水处理领域。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.
背景技术Background technique
垃圾渗滤液是一种成分复杂的高浓度有机废水，具有可生化性差、氨氮浓度高、有毒有害物质含量丰富等特征。随着环境水质管控标准的日趋严格，尤其是2009年10月，《生活垃圾填埋场渗滤液处理工程技术规范(征求意见稿)》颁布，提出了深度处理应以膜处理工艺为主，进一步确定了膜工艺的标准化趋势，作为主流工艺膜法深度处理过程会产生约15％～30％的膜浓缩液。垃圾渗滤液膜浓缩液主要成份为腐殖质类物质，呈棕黑(黄)色，COD通常在1000～5000mg/L之间，总氮浓度在500～2500mg/L，并含有大量的无机离子，TDS在20000～60000mg/L之间，电导率为40000～50000us/cm，垃圾渗滤液膜浓缩液中盐度可达3％以上，废水的盐度严重影响了微生物内部渗透压，高盐度使物质吸收过程受阻，微生物生长速率缓慢，部分微生物大量死亡，严重影响该类废水的生化处理效能。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. With the increasingly stringent environmental water quality control standards, especially in October 2009, 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.
垃圾渗滤液膜浓缩液中的总氮主要来源于垃圾渗滤液生化尾水经过多级膜后的浓液。其中原水中的大部分氨氮经过硝化作用转为硝态氮，硝态氮在浓缩液总氮中的占比可达到90％以上。对于此类高盐度、高氮浓液一般作焚烧处置，但由于垃圾填埋场水量增大，废水焚烧热值低，浓液需要作废水处理，达标排放。此类废水的高盐度、高毒性，高氮，一般微生物难以处理达标，亟需适合浓缩液的生物深度脱氮方法。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. Among them, 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.
发明内容Summary of the invention
发明目的：本发明所要解决的技术问题是提供一种巴利阿里假单胞菌，该巴利阿里假单胞菌能够高效去除垃圾渗滤液膜浓缩液中的总氮含量。Objective of the invention: 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.
本发明还要解决的技术问题是提供上述巴利阿里假单胞菌在处理垃圾渗滤液膜浓缩液中的应用。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.
为解决上述技术问题，本发明所采用的技术手段为：In order to solve the above technical problems, the technical means adopted by the present invention are:
本发明的巴利阿里假单胞菌，命名为巴利阿里假单胞菌(Pseudomonas balearica)EBT-1，分类命名为巴利阿里假单胞菌(Pseudomonas balearica)，菌株号为EBT-1，其于2019年9月17日保藏于位于中国武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC M 2019730。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.
通过采集垃圾渗滤液处理厂的活性污泥，并采用高盐度的垃圾渗滤液膜浓缩液进行 逐级驯化后，获得本发明的巴利阿里假单胞菌。After collecting activated sludge from a landfill leachate treatment plant and using high-salinity landfill leachate membrane concentrate for step-by-step domestication, the Pseudomonas balearicus of the present invention is obtained.
该菌株的生物学性质如下：本发明巴利阿里假单胞菌(Pseudomonas balearica)EBT-1菌株来源于高盐垃圾渗滤液(盐度>3％)污水处理***中的活性污泥，分离纯化后，在LB培养基中，35℃好氧条件下生长良好，菌落呈不规则圆形，直径大小2～5mm，白色菌落，不透明，表面有由外向内的褶皱，革兰氏染色呈阴性，显微镜观察下细胞形状为杆状。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℃ 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.
本发明得到的菌株EBT-1，经16S rRNA测序，采用BLAST分析，将本发明得到的假单胞菌株EBT-1基因序列于NCBI中检索，其基因序列与巴利阿里假单胞菌(Pseudomonas balearica)相似性高达99％以上，因此鉴定得到的菌株为巴利阿里假单胞菌。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.
上述巴利阿里假单胞菌在处理垃圾渗滤液膜浓缩液中的应用。The application of the above-mentioned Pseudomonas balearicum in the treatment of the membrane concentrate of the landfill leachate.
其中，将巴利阿里假单胞菌(Pseudomonas balearica)EBT-1接种于培养基中，于30～35℃下好氧培养24～32h，培养后得到EBT-1扩培产物，然后接种于垃圾渗滤液膜浓缩液中进行处理。Among them, Pseudomonas balearica (Pseudomonas balearica) EBT-1 was inoculated into the culture medium, aerobic cultured at 30～35℃ 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.
其中，巴利阿里假单胞菌(Pseudomonas balearica)EBT-1接种于培养基中进行培养时的接种量为3％～5％。Among them, Pseudomonas balearica (Pseudomonas balearica) EBT-1 is inoculated into a medium for culture when the inoculation amount is 3% to 5%.
其中，每升培养基(水)中含有如下质量份数的组分：玉米浆(干粉)10～12.5份、酵母膏5份、磷酸氢二钾1份、氯化钠10份、无水乙酸钠2.5份以及硫酸镁0.03份。Among them, each liter of medium (water) 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.
其中，巴利阿里假单胞菌(Pseudomonas balearica)EBT-1用于降解垃圾渗滤液膜浓缩液中的含氮化合物，含氮化合物包括硝态氮、亚硝态氮和氨氮。Among them, 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.
其中，巴利阿里假单胞菌降解垃圾渗滤液膜浓缩液中的含氮化合物是在厌氧/缺氧条件下进行。Among them, Pseudomonas balearicus degrades nitrogen-containing compounds in the landfill leachate membrane concentrate under anaerobic/anoxic conditions.
其中，巴利阿里假单胞菌(Pseudomonas balearica)EBT-1在降解垃圾渗滤液膜浓缩液中含氮化合物时，按接种量0.2％～0.4％将菌液添加至垃圾渗滤液膜浓缩液厌氧/缺氧处理反应器中，反应器中废水的COD/N为4:1～6:1。Among them, when Pseudomonas balearica EBT-1 degrades nitrogenous compounds in the landfill leachate membrane concentrate, the bacterial solution is added to the landfill leachate membrane concentrate according to the inoculation amount of 0.2% to 0.4%. In the oxygen/anoxic treatment reactor, the COD/N of the wastewater in the reactor is 4:1 to 6:1.
其中，垃圾渗滤液膜浓缩液的盐度为3％以上。Among them, the salinity of the landfill leachate membrane concentrate is 3% or more.
将培养所得到的巴利阿里假单胞菌(Pseudomonas balearica)EBT-1扩大培养产物进行离心，离心后得到菌体固体。按接种量0.4％将离心固体接种至不同盐度测定配水中，在30℃，150rpm下振荡处理，跟踪不同盐度下巴利阿里假单胞菌(Pseudomonas balearica)EBT-1对含氮化合物降解速率，得到巴利阿里假单胞菌(Pseudomonas balearica)EBT-1可适应7％以下(含7％)盐度范围的废水。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℃ 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%).
其中，每升盐度测定配水中含有如下质量分数的组分：葡萄糖6.1g，KNO 3 6.07g， KH 2PO 4 1.5g，K 2HPO 4·3H 2O 6.0g，MgSO 4·7H 2O 1.0g以及根据所需盐度添加的NaCl。 Among them, 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.
将培养所得到的巴利阿里假单胞菌(Pseudomonas balearica)EBT-1扩大培养产物进行离心，离心后得到菌体固体。按接种量0.4％将离心固体接种至装有垃圾渗滤液膜浓缩液的缺氧瓶中，在30℃，150rpm下振荡处理，巴利阿里假单胞菌(Pseudomonas balearica)EBT-1对垃圾渗滤液膜浓缩液的总氮去除率高达99％。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%.
其中垃圾渗滤液膜浓缩液厌氧/缺氧处理反应器有效体积为2L，反应器中原始污泥浓度为60000～80000mg/L，水力停留时间为12h，外加碳源使碳氮比为4:1，将培养所得到的巴利阿里假单胞菌(Pseudomonas balearica)EBT-1扩大培养菌液按接种量0.2％将菌液添加至垃圾渗滤液膜浓缩液厌氧/缺氧处理反应器中，可显著提高反应器中污泥活力，总氮去除率由30％有效提升至98.89％，且出水水质稳定达标。从而说明将本发明巴利阿里假单胞菌(Pseudomonas balearica)EBT-1添加入已有的污泥体系后，能够有效增大污泥体系的脱氮效率。Among them, 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, and 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%, and the effluent quality is stable and up to standard. This shows that the addition of Pseudomonas balearica EBT-1 of the present invention into the existing sludge system can effectively increase the denitrification efficiency of the sludge system.
有益效果：本发明通过对垃圾渗滤液膜浓缩液的逐级驯化后，获得耐高盐的巴利阿里假单胞菌(Pseudomonas balearica)EBT-1，该巴利阿里假单胞菌(Pseudomonas balearica)EBT-1能够实现对垃圾渗滤液膜浓缩液的高效生物脱氮；另外，可以有效提高已有活性污泥的脱氮速率，增强污泥脱氮效果，出水水质稳定。Beneficial effects: 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.
附图说明Description of the drawings
图1为实施例3中EBT-1对盐度适应性脱氮效果图；Figure 1 is a graph showing the adaptive denitrification effect of EBT-1 on salinity in Example 3;
图2为实施例4中EBT-1对垃圾渗滤液膜浓缩液深度脱氮的效率图；Figure 2 is a graph showing the efficiency of EBT-1 for deep denitrification of landfill leachate membrane concentrate in Example 4;
图3为垃圾渗滤液膜浓缩液厌氧/缺氧处理反应器结构示意图；Figure 3 is a schematic diagram of the structure of the anaerobic/anoxic treatment reactor for the membrane concentrate of landfill leachate;
图4为实施例5中EBT-1对垃圾渗滤液膜浓缩液反应器强化脱氮的效率图。Fig. 4 is an efficiency diagram of the enhanced denitrification of the landfill leachate membrane concentrate reactor by EBT-1 in Example 5.
具体实施方式Detailed ways
以下结合附图和具体实施例对本发明的技术方案做进一步说明。The technical solution of the present invention will be further described below in conjunction with the drawings and specific embodiments.
实施例1：菌株的分离与鉴定Example 1: Isolation and identification of strains
菌株EBT-1是从垃圾焚烧厂垃圾渗滤液污水处理***的活性污泥中筛选出的具有反硝化活力的好氧反硝化细菌。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.
分离筛选方法为：从垃圾渗滤液(高盐)污水处理生化***中取出活性污泥；将5g活性污泥接种至硝态氮浓度为100mg/L的DM无机盐培养基(培养基中盐度>3％)中，在温度35℃、转速为150rpm的摇床上培养1天，得到第一菌液；按5％(体积分数)的接种量将第一菌液接种至硝态氮浓度为200mg/L的DM无机盐培养基(培养基中盐度>3％)中，在温度35℃、转速为150rpm的摇床上培养1天，得到第二菌液；按5％的接种量将第二菌液接种至硝态氮浓度为300mg/L的DM无机盐培养基中，在温度 35℃、转速为150rpm的摇床上培养1天，得到第三菌液；吸取第三菌液，涂布于DM无机盐固体培养基(培养基中盐度>3％)中，于35℃下静置培养，挑选出生长速率较快的菌株；将生长速率较快的菌株于含有溴百里酚蓝的DM无机盐固体培养基(培养基中盐度>3％)上划线，于35℃下静置培养，得到平板变蓝的菌株；将筛选得到的菌株在DM无机盐固体培养基(培养基中盐度>3％)上进一步划线纯化，至挑选得到单菌落，该菌落即为本发明巴利阿里假单胞菌，菌株号为EBT-1，该菌株EBT-1能实现对高盐度的垃圾渗滤液膜浓缩液的高效生物脱氮。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 a temperature of 35°C and a rotating speed of 150 rpm for 1 day to obtain a third bacterial solution; aspirate the third bacterial solution and spread it on In the DM inorganic salt solid medium (salinity in the medium>3%), stand still culture at 35℃, and select the strains with faster growth rate; The DM inorganic salt solid medium (salinity in the medium>3%) was streaked and cultured at 35°C to obtain the strains that turned blue; the strains were screened in the DM inorganic salt solid medium (medium (Medium salinity>3%) is further streaked and purified until a single colony is selected. This colony is the Pseudomonas balearicus of the present invention, and the strain number is EBT-1. This strain EBT-1 can achieve high salt content. High-efficiency biological denitrification of solid landfill leachate membrane concentrate.
菌株EBT-1的菌落特征如下：分离纯化后，在LB培养基中，35℃好氧条件下生长良好，菌落呈不规则圆形，直径大小2～5mm，白色菌落，不透明，表面有由外向内的褶皱，革兰氏染色呈阴性，显微镜观察下细胞形状为杆状。The colony characteristics of strain EBT-1 are as follows: After isolation and purification, it grows well in LB medium under 35℃ 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.
通过PCR扩增，得到的EBT-1菌16S rRNA全序列如下(SEQ ID NO.1)：Through PCR amplification, the full sequence of the 16S rRNA of EBT-1 strain obtained is as follows (SEQ ID NO.1):
Figure PCTCN2019114170-appb-000001
Figure PCTCN2019114170-appb-000001
经比对，其基因序列与巴利阿里假单胞菌(Pseudomonas balearica)相似性高达99％以上，因此鉴定得到的菌株为巴利阿里假单胞菌。After comparison, its gene sequence is as high as 99% similar to Pseudomonas balearica, so the identified strain is Pseudomonas balearica.
实施例2：本发明巴利阿里假单胞菌(Pseudomonas balearica)EBT-1的扩大培养：Example 2: Expanded culture of Pseudomonas balearica EBT-1 of the present invention:
扩大培养采用的培养基包括如下质量的组分：每升水中含玉米浆(干粉)10g、酵母膏5g、氯化钠10g、无水乙酸钠2.5g、磷酸氢二钾1g以及硫酸镁0.03g，培养基的初始pH为6.5。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.
培养基的制作工艺为：将玉米浆(干粉)10g、酵母膏5g、氯化钠10g、无水乙酸钠2.5g、磷酸氢二钾1g以及硫酸镁0.03g溶解于1000g水中，用碱调整其pH为6.5，于121℃下灭菌20min，得到培养基。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.
将巴利阿里假单胞菌(Pseudomonas balearica)EBT-1菌母液按接种量3％～5％(体积分数)接种于培养基中，于30～35℃下培养28～32h。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.
采用本发明培养基扩培后，将扩大培养得到的菌液用稀释平板法计数，得到目标有效菌量为5×10 8～1×10 9CFU/mL。 After the culture medium of the present invention is used for expansion, 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.
实施例3：本发明巴利阿里假单胞菌(Pseudomonas balearica)EBT-1盐度适应性考察Example 3: The present invention Pseudomonas balearica (Pseudomonas balearica) EBT-1 salinity adaptability investigation
将实施例2中培养得到的巴利阿里假单胞菌(Pseudomonas balearica)EBT-1扩培液，于6000rpm下离心5min，去掉上清液，得到活化后的菌体固体。设定0％，3％，7％,，10％四个盐度梯度。每升盐度测定配水中含有如下质量分数的组分：葡萄糖6.1g，KNO 3 6.07g，KH 2PO 4 1.5g，K 2HPO 4·3H 2O 6.0g，MgSO 4·7H 2O 1.0g，NaCl根据所需盐度调整。将离心菌体固体按接种量0.4％接种于盐度测定配水中，于250mL蓝盖瓶中30℃下，150rpm振荡处理。 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. Set four salinity gradients of 0%, 3%, 7%, and 10%. 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.
检测不同盐度条件反应过程中总氮变化，结果见图1。如图1所示，巴利阿里假单胞菌(Pseudomonas balearica)EBT-1脱氮速率随着盐度的增加而降低，在5％盐度以下，总氮脱除率可以达到90％以上；在7％盐度下，总氮去除率在50％以上。本专利巴利阿里假单胞菌(Pseudomonas balearica)EBT-1是由高盐垃圾渗滤液生化***中污泥得到，其本身具备一定的耐盐特性，经过高效脱氮驯化筛选得到，自身有较强的耐盐、脱氮性能。The changes in total nitrogen during the reaction under different salinity conditions were detected, and the results are shown in Figure 1. As shown in Figure 1, 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.
实施例4：本发明巴利阿里假单胞菌(Pseudomonas balearica)EBT-1对垃圾渗滤液膜浓缩液生物脱氮的效果分析。Example 4: Analysis of the effect of Pseudomonas balearica EBT-1 of the present invention on biological denitrification of landfill leachate membrane concentrate.
将实施例2中培养得到的巴利阿里假单胞菌(Pseudomonas balearica)EBT-1扩培液菌液转移到新的离心管中，于6000rpm下离心5min，去掉上清液，得到活化后的菌体固体，取干净的蓝盖瓶，往蓝盖瓶中加入200mL垃圾渗滤液膜浓缩液(盐度为3.5％)，再加入无水乙酸钠为碳源，使废液中碳氮比为4:1，最后按接种量0.4％加入巴利阿里假单胞菌(Pseudomonas balearica)EBT-1，于30℃下，150rpm振荡处理。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℃ and 150rpm.
检测反应过程中垃圾渗滤液膜浓缩液中总氮的变化，结果见图2。如图2所示，经过72h处理，垃圾渗滤液膜浓缩液中的总氮浓度由初始浓度为1110.74mg/L降至13.37mg/L，由此可知，说明本发明巴利阿里假单胞菌(Pseudomonas balearica)EBT-1对垃圾渗滤液膜浓缩液的总氮去除率高达99％。巴利阿里假单胞菌(Pseudomonas balearica) EBT-1为兼性厌氧菌，同时具有好氧反硝化特性，在缺氧环境中仍然能够有较高的反硝化功能。The change of total nitrogen in the membrane concentrate of the landfill leachate during the reaction was detected, and the result is shown in Figure 2. As shown in Figure 2, after 72 hours of treatment, the total nitrogen concentration in the landfill leachate membrane concentrate dropped from the initial concentration of 1110.74 mg/L to 13.37 mg/L. This shows that the Pseudomonas balearicus of the present invention (Pseudomonas balearica) The total nitrogen removal rate of EBT-1 on the landfill leachate membrane concentrate is as high as 99%. Pseudomonas balearica (Pseudomonas balearica) EBT-1 is a facultative anaerobe with aerobic denitrification properties and can still have a high denitrification function in anoxic environment.
实施案例5：本发明巴利阿里假单胞菌(Pseudomonas balearica)EBT-1对垃圾渗滤液膜浓缩液生物脱氮强化的效果分析Implementation case 5: Effect analysis of Pseudomonas balearica EBT-1 of the present invention on enhanced biological denitrification of landfill leachate membrane concentrate
垃圾渗滤液膜浓缩液厌氧/缺氧反应器结构如图3所示。垃圾渗滤液膜浓缩液及外加碳源混合液由反应器底部进入，通过活性污泥厌氧/缺氧反应后，由反应器上部流出。反应器有效容积为2L，水力停留时间12h，污泥浓度60000mg/L～80000mg/L，外加碳源碳氮比4:1，连续进出水，每天检测出水含氮化合物指标，计算脱氮效率，待反应器出水水质稳定后，按接种量0.2％投加实施例2中培养得到的巴利阿里假单胞菌(Pseudomonas balearica)EBT-1扩培液菌液至反应器污泥中，跟踪投加菌剂前后稳定出水脱氮效率。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, and the nitrogen compounds in the water are detected every day to calculate the denitrification efficiency. After the effluent quality of the reactor is stable, add the Pseudomonas balearica EBT-1 culture solution cultured in Example 2 to the reactor sludge according to the inoculum amount 0.2%, and follow-up Before and after adding bacteria, the effluent denitrification efficiency is stabilized.
垃圾渗滤液膜浓缩液厌氧/缺氧反应器投加菌剂前后稳定出水脱氮效率结果见图4。如图4所示，投加菌剂前(图4第I阶段)，垃圾渗滤液膜浓缩液厌氧/缺氧反应器进水总氮浓度为547.48mg/L，反硝化活性污泥由于受过量碳源冲击，群落结构发生变化，而导致污泥反硝化活力差，出水总氮去除率在30％～50％之间波动；投加巴利阿里假单胞菌(Pseudomonas balearica)EBT-1菌剂与反应器污泥充分混合后(图4第II阶段)，其出水总氮去除率有效提高，随着适应周期的增加，总氮去除率显著增加，最终出水总氮去除率可以达到98.89％；随后提高进水总氮浓度至1000mg/L(图4第III阶段)，***总氮去除率仍然稳定在90％以上。巴利阿里假单胞菌(Pseudomonas balearica)EBT-1菌剂具备高效反硝化性能，在加入反硝化活性污泥体系后，丰富了该体系中反硝化脱氮菌群落结构，大大提高了污泥的反硝化性能。并且该菌剂能够较好的适应垃圾渗滤液膜浓缩液，在体系中生长繁殖，不易流失，进而保证出水水质稳定。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. As shown in Figure 4, before the addition of bacterial agents (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.
本发明巴利阿里假单胞菌(Pseudomonas balearica)EBT-1对垃圾渗滤液膜浓缩液中总氮的去除效率高，能够有效解决垃圾渗滤液膜浓缩液中深度脱氮这一难题。并且将本发明巴利阿里假单胞菌(Pseudomonas balearica)EBT-1添加到已有的污泥体系后，能够有效增大污泥体系的脱氮效率。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. In addition, after 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.
Figure PCTCN2019114170-appb-000002
Figure PCTCN2019114170-appb-000002
Figure PCTCN2019114170-appb-000003
Figure PCTCN2019114170-appb-000003

Claims (10)

  1. 一种巴利阿里假单胞菌，其特征在于：所述巴利阿里假单胞菌命名为巴利阿里假单胞菌(Pseudomonas balearica)EBT-1，其于2019年9月17日保藏于中国典型培养物保藏中心，保藏编号为CCTCC M 2019730。A kind of Pseudomonas balearica, characterized in that: the Pseudomonas balearica is named Pseudomonas balearica (Pseudomonas balearica) EBT-1, which was deposited on September 17, 2019 China Type Culture Collection, the deposit number is CCTCC M 2019730.
  2. 权利要求1所述的巴利阿里假单胞菌在处理垃圾渗滤液膜浓缩液中的应用。The use of the Pseudomonas balearicus described in claim 1 in the treatment of landfill leachate membrane concentrates.
  3. 根据权利要求2所述的巴利阿里假单胞菌在处理垃圾渗滤液膜浓缩液中的应用，其特征在于：将巴利阿里假单胞菌(Pseudomonas balearica)EBT-1接种于培养基中，于30～35℃下好氧培养24～32h，培养后得到EBT-1扩培产物，然后接种于垃圾渗滤液膜浓缩液中进行处理。The application of Pseudomonas balearica in the treatment of membrane concentrates of landfill leachate according to claim 2, characterized in that: Pseudomonas balearica (Pseudomonas balearica) EBT-1 is inoculated into the culture medium , Aerobic culture at 30～35℃ for 24～32h, after culture, EBT-1 expanded culture product is obtained, and then it is inoculated into landfill leachate membrane concentrate for treatment.
  4. 根据权利要求3所述的巴利阿里假单胞菌在处理垃圾渗滤液膜浓缩液中的应用，其特征在于：巴利阿里假单胞菌(Pseudomonas balearica)EBT-1接种于培养基中进行培养时的接种量为3％～5％。The application of Pseudomonas balearica in the treatment of membrane concentrates of landfill leachate according to claim 3, characterized in that: Pseudomonas balearica (Pseudomonas balearica) EBT-1 is inoculated in the culture medium. The amount of inoculation during culture is 3% to 5%.
  5. 根据权利要求3所述的巴利阿里假单胞菌在处理垃圾渗滤液膜浓缩液中的应用，其特征在于：每升培养基中含有如下质量份数的组分：玉米浆10份、酵母膏5份、磷酸氢二钾1份、氯化钠10份、无水乙酸钠2.5份以及硫酸镁0.03份。The application of Pseudomonas balearicus in the treatment of landfill leachate membrane concentrates according to claim 3, characterized in that: each liter of culture medium contains the following components by mass: 10 parts of corn steep liquor, yeast 5 parts of paste, 1 part of dipotassium hydrogen phosphate, 10 parts of sodium chloride, 2.5 parts of anhydrous sodium acetate and 0.03 parts of magnesium sulfate.
  6. 根据权利要求3所述的巴利阿里假单胞菌在处理垃圾渗滤液膜浓缩液中的应用，其特征在于：所述培养基的初始pH为6.5～7.5。The application of Pseudomonas balearicus in the treatment of membrane concentrates of landfill leachate according to claim 3, characterized in that the initial pH of the culture medium is 6.5-7.5.
  7. 根据权利要求2所述的巴利阿里假单胞菌在处理垃圾渗滤液膜浓缩液中的应用，其特征在于：巴利阿里假单胞菌(Pseudomonas balearica)EBT-1用于降解垃圾渗滤液膜浓缩液中的含氮化合物，含氮化合物包括硝态氮、亚硝态氮和氨氮。The application of Pseudomonas balearica in the treatment of landfill leachate membrane concentrate according to claim 2, characterized in that: Pseudomonas balearica (Pseudomonas balearica) EBT-1 is used to degrade landfill leachate Nitrogen-containing compounds in the membrane concentrate include nitrate nitrogen, nitrite nitrogen and ammonia nitrogen.
  8. 根据权利要求7所述的巴利阿里假单胞菌在处理垃圾渗滤液膜浓缩液中的应用，其特征在于：巴利阿里假单胞菌降解垃圾渗滤液膜浓缩液中的含氮化合物是在厌氧/缺氧条件下进行。The application of Pseudomonas balearicus in the treatment of landfill leachate membrane concentrates according to claim 7, characterized in that: Pseudomonas baleari degrades nitrogen-containing compounds in the landfill leachate membrane concentrates Performed under anaerobic/anoxic conditions.
  9. 根据权利要求7所述的巴利阿里假单胞菌在处理垃圾渗滤液膜浓缩液中的应用，其特征在于：巴利阿里假单胞菌(Pseudomonas balearica)EBT-1在降解垃圾渗滤液膜浓缩液中含氮化合物时，按接种量0.2％～0.4％将菌液添加至垃圾渗滤液膜浓缩液厌氧/缺氧处理反应器中，反应器中废水的COD/N为4∶1～6∶1。The application of Pseudomonas balearica in the treatment of landfill leachate membrane concentrates according to claim 7, characterized in that: Pseudomonas balearica (Pseudomonas balearica) EBT-1 is used to degrade landfill leachate membranes When nitrogen compounds are contained in the concentrate, add the bacterial solution to the landfill leachate membrane concentrate anaerobic/anoxic treatment reactor according to the inoculation amount 0.2%～0.4%. The COD/N of the wastewater in the reactor is 4:1～ 6:1.
  10. 根据权利要求7所述的巴利阿里假单胞菌在处理垃圾渗滤液膜浓缩液中的应用，其特征在于：垃圾渗滤液膜浓缩液的盐度为3％以上。The application of Pseudomonas balearicum in the treatment of landfill leachate membrane concentrates according to claim 7, wherein the salinity of the landfill leachate membrane concentrates is more than 3%.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114836355A (en) * 2022-05-30 2022-08-02 沈阳风景园林股份有限公司 Pseudomonas DT04 and application thereof in sewage denitrification
CN118165898A (en) * 2024-05-14 2024-06-11 黄河三角洲京博化工研究院有限公司 Pseudomonas strain Ba Li Ali and application thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113003726B (en) * 2021-03-04 2022-07-01 重庆理工大学 Treatment method of reverse osmosis membrane filtration concentrated solution of landfill leachate
CN113684159B (en) * 2021-10-27 2022-02-11 佛山市玉凰生态环境科技有限公司 Palyra barbadensis and application thereof in denitrification and dephosphorization of river water body

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6019900A (en) * 1998-08-03 2000-02-01 The Regents Of The University Of California Single stage denitrification anaerobic digestion
CN102703349A (en) * 2012-05-29 2012-10-03 北京大学 Small brevibacterium strain capable of carrying out biological denitrification under high-salt condition and application of small brevibacterium strain to wastewater treatment
CN103270133A (en) * 2010-11-01 2013-08-28 纳幕尔杜邦公司 Methods, strains, and compositions useful for microbially enhanced oil recovery: pseudomonas stutzeri
CN103373767A (en) * 2012-04-29 2013-10-30 中国石油化工股份有限公司 Method for biologically denitrifying high-salinity sewage generated in production process of catalysts
CN103773723A (en) * 2014-01-17 2014-05-07 南京曜动节能环保科技有限公司 Salt-resistant pseudomonas stutzeri having low-temperature biological denitrification function
CN108546657A (en) * 2018-04-09 2018-09-18 深圳市长隆科技有限公司 A kind of Bali Ali pseudomonad FX-1 and its application
CN109486699A (en) * 2018-08-21 2019-03-19 四川大学 One plant of salt tolerant aerobic denitrifying bacteria and application
CN110054302A (en) * 2019-04-01 2019-07-26 黄山市益天士生物科技有限公司 Inhibit the processing material and preparation method thereof of algal grown

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102703350B (en) * 2012-05-29 2013-05-22 北京大学 Application of salt-tolerant nitrogen and phosphorus removing bacillus altitudinis to wastewater treatment
CN108546656B (en) * 2018-04-08 2021-03-09 哈尔滨工业大学（威海） Salt-tolerant alteromonas and application thereof
CN110182961A (en) * 2019-04-01 2019-08-30 黄山市益天士生物科技有限公司 For adjusting the regulator and preparation method thereof of artificial breeding pond water quality

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6019900A (en) * 1998-08-03 2000-02-01 The Regents Of The University Of California Single stage denitrification anaerobic digestion
CN103270133A (en) * 2010-11-01 2013-08-28 纳幕尔杜邦公司 Methods, strains, and compositions useful for microbially enhanced oil recovery: pseudomonas stutzeri
CN103373767A (en) * 2012-04-29 2013-10-30 中国石油化工股份有限公司 Method for biologically denitrifying high-salinity sewage generated in production process of catalysts
CN102703349A (en) * 2012-05-29 2012-10-03 北京大学 Small brevibacterium strain capable of carrying out biological denitrification under high-salt condition and application of small brevibacterium strain to wastewater treatment
CN103773723A (en) * 2014-01-17 2014-05-07 南京曜动节能环保科技有限公司 Salt-resistant pseudomonas stutzeri having low-temperature biological denitrification function
CN108546657A (en) * 2018-04-09 2018-09-18 深圳市长隆科技有限公司 A kind of Bali Ali pseudomonad FX-1 and its application
CN109486699A (en) * 2018-08-21 2019-03-19 四川大学 One plant of salt tolerant aerobic denitrifying bacteria and application
CN110054302A (en) * 2019-04-01 2019-07-26 黄山市益天士生物科技有限公司 Inhibit the processing material and preparation method thereof of algal grown

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114836355A (en) * 2022-05-30 2022-08-02 沈阳风景园林股份有限公司 Pseudomonas DT04 and application thereof in sewage denitrification
CN114836355B (en) * 2022-05-30 2024-04-02 沈阳风景园林股份有限公司 Pseudomonas DT04 and application thereof in sewage denitrification
CN118165898A (en) * 2024-05-14 2024-06-11 黄河三角洲京博化工研究院有限公司 Pseudomonas strain Ba Li Ali and application thereof

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