WO2020097994A1 - 一株兼具异养硝化和好氧反硝化功能鞘氨醇杆菌及其应用 - Google Patents

一株兼具异养硝化和好氧反硝化功能鞘氨醇杆菌及其应用 Download PDF

Info

Publication number
WO2020097994A1
WO2020097994A1 PCT/CN2018/118165 CN2018118165W WO2020097994A1 WO 2020097994 A1 WO2020097994 A1 WO 2020097994A1 CN 2018118165 W CN2018118165 W CN 2018118165W WO 2020097994 A1 WO2020097994 A1 WO 2020097994A1
Authority
WO
WIPO (PCT)
Prior art keywords
sphingosine
microbial
sewage
sewage treatment
nitrogen
Prior art date
Application number
PCT/CN2018/118165
Other languages
English (en)
French (fr)
Inventor
邓敬轩
阮文权
黄振兴
施万胜
单晓红
Original Assignee
江南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江南大学 filed Critical 江南大学
Publication of WO2020097994A1 publication Critical patent/WO2020097994A1/zh

Links

Images

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • 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
    • C02F2101/30—Organic compounds
    • C02F2101/38—Organic compounds containing nitrogen

Definitions

  • the invention relates to a sphingosine bacillus with both heterotrophic nitrification and aerobic denitrification functions and its application, and belongs to the technical field of biological engineering and environmental engineering.
  • Ammonia nitrogen in water mainly comes from the use of farmland fertilizers, animal feces and sewage generated by aquaculture, etc. These pollutants enter the lakes and rivers through surface runoff and bring a lot of nitrate nitrogen and ammonia nitrogen, causing the deterioration of water quality and the destruction of aquatic ecological environment .
  • ammonia nitrogen is gradually transformed into nitrous nitrogen and nitrate nitrogen by autotrophic nitrification and nitrosation bacteria, and then converted into nitrogen gas by the action of denitrifying bacteria to be removed from the environment. Due to the complexity of the real environment, these two parts are relatively difficult to achieve in a real ecosystem.
  • Facultative heterotrophic nitrification and aerobic denitrification bacteria not only make it possible to synchronize the nitrification process and the denitrification process, but also the growth rate is much greater than the autotrophic bacteria, which greatly shortens the growth cycle.
  • the products of the nitrification process can directly become the substrate of the denitrification process, avoiding the inhibitory effect of the accumulation of nitrification products on the nitrification reaction, and greatly improving the biological denitrification efficiency.
  • the whole process can maintain the relative balance of acid and alkali, and the products of denitrification can supplement the environmental alkalinity, thus keeping the pH within a certain range.
  • Most facultative heterotrophic nitrification and aerobic denitrification bacteria have strong environmental adaptability and are suitable for treating large-scale polluted waters.
  • the first object of the present invention is to provide a strain of Sphingobacterium sp. (Sphingobacterium sp.), which was deposited on October 17, 2018 in the General Microbiology Center of the China Microbial Culture Collection Management Committee, with the deposit number CGMCC No. 16596, deposited The address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.
  • the second object of the present invention is to provide a microbial preparation containing sphingosine.
  • the viable bacteria content of sphingosine in the microbial preparation is ⁇ 1 ⁇ 10 9 cfu / g.
  • the microbial preparation is a biological microcapsule.
  • the preparation method of the biological microcapsules is: (1) Weighing activated carbon 10-50g / L, chitosan 5-50g / L, sodium alginate 10-50g / L Dissolve and mix well to obtain gel solution; (2) Put 50-100g solid cells into the gel solution to obtain a microbial gel solution with a cell concentration of 1 ⁇ 10 8 -1 ⁇ 10 11 cfu / ml; (3) The microbial gel solution is dropped into CaCl 2 solution with a mass concentration of 1-50 g / L at a rate of 15-25 drops / minute to obtain microbial active particles; (4) Microbial active particles are added to 5% glutaric acid Cross-linking at room temperature for 24 hours in aldehyde solution; (5) Soaking the cross-linked microbial active particles in (4) with sterile water for 24-48 hours, multiplying to obtain Sphingomonas BT1 biological microcapsules.
  • the second object of the present invention is to provide the application of the sphingosine.
  • the application includes degradation of pollutants in rivers, lakes, or sewage treatment.
  • the application is the treatment of nitrogen-containing sewage.
  • the treatment of the nitrogen-containing sewage is to put the sphingosine BT1 into the nitrogen-containing sewage at a final concentration of 10-30 mg bacterial cells / L sewage.
  • the sphingosine BT1 is put into the water body in the form of biological microcapsules.
  • the invention also claims the equipment and equipment for applying the sphingosine BT1 for sewage treatment, including but not limited to a denitrification reactor, a sewage treatment device, a constructed wetland or a denitrification tank.
  • the equipment is more specific, and may be a high-efficiency denitrification reactor, an integrated sewage treatment device, a sewage treatment plant denitrification tank, and the like.
  • the invention also claims to protect the biofilm used in the sewage treatment process.
  • the biofilm is prepared from at least one of kitchen waste, plant straw or shells, and supports the sphingosine.
  • the present invention provides a strain of sphingosine, which has the characteristics of heterotrophic nitrification and aerobic denitrification, and can be applied to the field of nitrogen-containing sewage treatment and river treatment.
  • the removal rate of ammonia nitrogen is greater than 90% and The environment has no pollution, and the effects of rapid biological nitrogen removal and restoration of the self-purification ability of the river are of great significance in the field of environmental water treatment.
  • a strain of Sphingobacterium (Sphingobacterium sp.) was deposited on October 17, 2018 in the General Microbiology Center of the China Microbial Culture Collection Management Committee.
  • the deposit number is CGMCC No. 16596.
  • the deposit address is Beichen West Road, Chaoyang District, Beijing No. 3, No. 1 Institute of Microbiology, Chinese Academy of Sciences.
  • Figure 1 shows the growth and degradation curves of sphingosine BT1
  • Figure 2 is a graph showing the degradation of sphingosine BT1 on nitrate degradation
  • Figure 3 is a graph showing the degradation of sphingosine BT1 on nitrous nitrogen degradation
  • Figure 4 is a gel electrophoresis diagram of amoA gene verification; among them, Marker: DL2000 (100bp ⁇ 2000bp); 1: Gene fragment: amoA;
  • Fig. 5 is a gel electrophoresis diagram of nirK gene verification; among them, Marker: DL2000 (100bp-2000bp); 1: gene fragment is nirS; 2: gene fragment is nirK.
  • Vickers salt solution (g / L): K 2 HPO 4 5.0, MgSO 4 .7H 2 O 2.5, NaCl 2.5, MnSO 4 .4H 2 O 0.05, FeSO 4 .7H 2 O 0.05.
  • Aerobic denitrification medium nitrate medium: KNO 3 2g, sodium citrate 5g, K 2 HPO 4 1g, KH 2 PO 4 1g, MgSO 4 ⁇ 7H 2 O 0.2g, trace element solution 2mL, supplemented with distilled water to 1L, pH7.2-7.5
  • Nitrite medium NaNO 2 2g, sodium citrate 5g, K 2 HPO 4 1g, KH 2 PO 4 1g, MgSO 4 ⁇ 7H 2 O 0.2g, trace element solution 2mL, supplemented with distilled water to 1L, pH7.2- 7.5
  • Trace element solution EDTA 50.0g, ZnSO 4 2.2g, CaCl 2 ⁇ 2H 2 O 5.5g, MnCl 2 ⁇ 4H 2 O 5.06g, FeSO 4 ⁇ 7H 2 O 5.0g, (NH 4 ) 6Mo 7 O 2 ⁇ 4H 2 O 1.1g, CuSO 4 ⁇ 5H 2 O 1.57g, CoCl 2 ⁇ 6H 2 O 1.61g, make up distilled water to 1L, pH 6.0
  • Activated sludge of a certain period of stable operation of landfill leachate aerobic fermentation tank is used as separation sludge.
  • the pure sphingosine BT1 strain was isolated by dilution mixed plate method.
  • the genome of the strain Sphingomonas BT1 was extracted and subjected to 16S rDNA sequencing. The similarity of the sequencing results with known sequences in the Genbank database was compared. The results showed that the strain Sphingomobacterium BT1 had the highest sequence homology with Sphingobacterium and was identified as Sphingomobacterium.
  • the growth curve of the strain can be obtained from the 1OD 600 index in Table 1.
  • the strain only enters the exponential phase after a lag period of 8h. Within 24h, the OD value reaches 0.0149 from 1.349, and then enters the stable phase after 24h. Compared with the common nitrifying bacteria growth cycle, it shortens by 24-48 hours.
  • the ammonia nitrogen decreased from 90.21mg / L to 4.49mg / L, the ammonia nitrogen degradation rate reached 95.01%, the total nitrogen decreased from 93.77mg / L to 5.11mg / L, and the degradation rate of total nitrogen was 94.65%.
  • Sphingomonas BT1 Compared with common nitrifying bacteria or anaerobic denitrifying bacteria, Sphingomonas BT1 has both heterotrophic nitrification and aerobic denitrification functions. The nitrification process and the denitrification process can be carried out simultaneously, and the nitrification products can be directly used as Substrate. It can be seen from Table 1 that Sphingomonas BT1 has a short growth cycle, reaching a plateau within 24 hours, and its growth rate is much greater than that of autotrophic nitrifying bacteria; it has strong environmental adaptability and strong tolerance to high concentrations of ammonia nitrogen.
  • the sphingosine BT1 starts to grow at the 46th hour and reaches a peak at 80 hours.
  • the nitrate nitrogen decreased from 240.42mg / L to 133.32mg / L
  • the degradation rate of nitrate nitrogen was 44.5%
  • the nitrite accumulated from 0 to 40.59mg / L, indicating that Sphingomonas BT1 has a negative effect on nitrate
  • the removal rate of nitrogen is good, and it has a strong denitrification function.
  • the nitrite culture medium was used to screen and characterize the denitrification performance of Sphingomonas BT1. On the basis of the nitrate medium, potassium nitrate was replaced with sodium nitrite.
  • Synthesizing the effect of sphingosine BT1 to degrade nitrate and nitrite it can be concluded that when sphingosine BT1 uses nitrate or nitrite as a nitrogen source, there will be a 25-40 hour incubation period The number growth period is 12-22 hours, and after 30 hours it begins to enter the stable and declining period. Sphingomonas BT1 can simply use nitrate or nitrite as a nitrogen source to achieve the effect of denitrification, thus proving that Sphingomonas BT1 has a strong denitrification function.
  • amoA1F (5’-GGGGTTTCTACTGGTGGT-3 ’);
  • amoA2R (5’-CCCCTCKGSAAAGCCTTCTTC-3 ’)
  • the PCR product is a 491 bp band, which has been sequenced to be consistent with the sequence of the amoA gene. From this, it can be confirmed that the amoA gene exists in Sphingomonas BT1 and has nitrification function.
  • Nir gene is divided into two types, namely nirK and nirS.
  • the nirK gene encodes Cu-type nitrite reductase, and another nirS gene encodes cytochrome reductase. It should be noted that these two coding genes will not exist in the same strain at the same time.
  • PCR was used to verify the presence of nirK and nirS genes in Sphingomonas BT1. The primer sequences are as follows:
  • nirS-R3cd (5’-GASTTCGGRTGSGTCTTGA)
  • nirK-F1aCu (5’-ATCATGGTSCTGCCGCG)
  • nirK-R3Cu (5’-GCCTCGATCAGRTTGTGGTT)
  • nirS gene is not present in the sphingosine BT1 strain, and the nirK gene is present. This indicates that the sphingosine Bacillus BT1 has Cu-type nitrite reductase, and the genotype is consistent with its aerobic denitrification function.
  • the DN reactor is a high-efficiency denitrification reactor with a water distribution system and a three-phase separator.
  • the core is built, and the height-to-diameter ratio is set to> 2.
  • the controllable parameter is HRT. When the HRT is set to 48h, the reactor operating parameters are as follows:
  • the above parameters show that Sphingomonas BT1 can effectively remove ammonia nitrogen in practical applications.
  • the ammonia nitrogen content decreased from 2000mg / L to 35mg / L, and the removal rate reached 40.9mg / L * h.
  • Total nitrogen (TN) content decreased from 2500mg / L to 200mg / L within 48h
  • COD decreased from 8000mg / L to 700mg / L.
  • the degradation rates of total nitrogen and COD reached 92% and 91.25%, respectively. DO is always maintained at less than 1mg / L, which indicates that the sphingosine BT1 has low requirements for dissolved oxygen concentration and strong adaptability, and is particularly suitable for the treatment of eutrophic and high ammonia nitrogen water bodies.
  • the sphingosine BT1 bacterial solution was added to the front of the aerobic tank at a concentration of 20 ppm.
  • the ammonia nitrogen content was reduced from 20mg / L to 5mg / L, the ammonia nitrogen removal rate reached 75%, and the daily sewage treatment capacity could reach 55000-60000m 3 , indicating that Sphingomonas BT1 can be used to treat large-scale sewage. Effectively reduce the concentration of ammonia nitrogen in nitrogen-containing sewage.
  • the sphingosine BT1 strain is combined with a biological carrier to prepare an immobilized microorganism that is suitable for river water and can be slowly released.
  • the steps for preparing immobilized strains are:
  • Fermentation culture of strains pure culture of sphingosine BT1 in a mechanically stirred fermenter by liquid fermentation, the medium is heterotrophic nitrification liquid medium. After performing pure culture at 30 ° C. for 12-48 hours, a high-concentration pure sphingosine BT1 pure culture liquid is obtained, and the viable count of the liquid is 1 ⁇ 10 8 -1 ⁇ 10 11 cfu / ml.
  • Preparation of solid cells After the fermentation of the strain is completed, the fermentation broth is removed by centrifugation to obtain solid cells.
  • Preparation and immobilization of biological carriers (1) Weigh and dissolve and mix evenly 10-50 g / L of activated carbon, 5-50 g / L of chitosan, and 10-50 g / L of sodium alginate to obtain a gel solution. (2) 50-100 g of solid cells are inserted into the gel solution to obtain a microbial gel solution with a cell concentration of 1 ⁇ 10 8 -1 ⁇ 10 11 cfu / ml. (3) The microbial gel solution is dropped into a CaCl 2 solution with a mass concentration of 1-50 g / L at a rate of 15-25 drops / minute to obtain microbial active particles.
  • the amount of sphingosine Bt1 microcapsules released in 30 days was more than 85%, the bacterial survival rate was more than 5%, and the number of effective viable bacteria reached 10 9 cfu / g after verification.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Inorganic Chemistry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Hydrology & Water Resources (AREA)
  • Environmental & Geological Engineering (AREA)
  • Water Supply & Treatment (AREA)
  • Molecular Biology (AREA)
  • Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一株兼具异养硝化和好氧反硝化功能鞘氨醇杆菌及其应用，属于生物工程、环境工程技术领域。所述鞘氨醇杆菌已于2018年10月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.16596，该菌株具有异养硝化、好氧反硝化特性，能够应用于含氮污水处理、河道治理领域，在对氨氮的去除率大于90％并对环境无污染。

Description

一株兼具异养硝化和好氧反硝化功能鞘氨醇杆菌及其应用 技术领域
本发明涉及一株兼具异养硝化和好氧反硝化功能鞘氨醇杆菌及其应用，属于生物工程、环境工程技术领域。
背景技术
河流、湖泊中氨氮含量超标常常导致水体富营养化，水中溶氧减少，这已成为我国水体污染的主要原因之一。水体中氨氮主要来源于农田化肥的使用，养殖业产生的动物粪便污水等等，这些污染物质通过地表径流进入湖泊河流带入大量的硝氮和氨氮，造成水体质量恶化和水生态环境结构的破坏。
传统的生物脱氮理论中，氨氮经自养硝化、亚硝化细菌逐步转化成亚硝态氮、硝态氮，再经反硝化菌作用转化成氮气从环境中去除。由于现实环境复杂，这两个部分在真实的生态系统中，是比较难实现的。
兼性异养硝化和好氧反硝化菌不仅仅能使硝化过程和反硝化成过程同步进行成为可能，而且生长速率远大于自养菌，大大缩短了生长周期，。硝化过程的产物可直接成为反硝化过程的底物，避免了硝化产物积累对硝化反应的抑制作用，大大提高生物脱氮效率。整个过程能保持酸碱相对平衡，反硝化作用的产物能够补充环境碱度，从而是使pH保持在一定范围之内。大部分兼性异养硝化和好氧反硝化菌环境适应性强，适合治理大面积污染水域。
目前，研究者对于兼性异养硝化和好氧反硝化菌的研究还很少，且仅有的一些研究也多是聚焦在其降解机理。因此，对兼性异养硝化和好氧反硝化菌的生理生化特性及脱氮效能进行深入研究是必要的，具有重要的理论和实际应用价值。
发明内容
本发明的第一个目的是提供一株鞘氨醇杆菌(Sphingobacterium sp.)，于2018年10月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.16596，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
本发明的第二个目的是提供含有鞘氨醇杆菌的微生物制剂。
在本发明的一种实施方式中，所述微生物制剂中鞘氨醇杆菌的活菌含量≥1×10 9cfu/g。
在本发明的一种实施方式中，所述微生物制剂为生物微胶囊。
在本发明的一种实施方式中，所述生物微胶囊的制备方式为：(1)将活性炭10-50g/L、壳聚糖5-50g/L、海藻酸钠10-50g/L称量溶解并混合均匀，得到凝胶液；(2)将50-100g固态 菌体接入凝胶液中，得到菌体浓度为1×10 8-1×10 11cfu/ml的微生物凝胶液；(3)将微生物凝胶液按照15-25滴/分钟的速度滴入质量浓度为1-50g/L的CaCl 2溶液中，得到微生物活性颗粒；(4)将微生物活性颗粒于5％戊二醛溶液中在室温下交联24小时；(5)将(4)中交联后的微生物活性颗粒用无菌水浸泡24-48小时后，增殖得到鞘氨醇杆菌BT1生物微胶囊。
本发明的第二个目的是提供所述鞘氨醇杆菌的应用。
在本发明的一种实施方式中，所述应用包括降解河道、湖泊污染物或污水处理等。
在本发明的一种实施方式中，所述应用是对含氮污水进行处理。
在本发明的一种实施方式中，所述对含氮污水进行处理是将所述鞘氨醇杆菌BT1按终浓度10～30mg菌体/L污水投放至含氮污水中。
在本发明的一种实施方式中，所述鞘氨醇杆菌BT1以生物微胶囊的形式投放至水体中。
本发明还要求保护应用所述鞘氨醇杆菌BT1进行污水处理的装置设备，包括但不限于脱氮反应器、污水处理装置、人工湿地或反硝化池。
在本发明的一种实施方式中，所述设备更具体的，可以是高效脱氮反应器、一体化污水处理装置、污水处理厂反硝化池等。
本发明还要求保护用于污水处理过程的生物膜，所述生物膜以餐厨垃圾、植物秸秆或贝壳中的至少一种制备生物膜载体，负载所述的鞘氨醇杆菌。
有益效果：本发明提供了一株鞘氨醇杆菌，该菌株具有异养硝化、好氧反硝化特性，能够应用于含氮污水处理、河道治理领域，在对氨氮的去除率大于90％并对环境无污染，达到快速生物脱氮、恢复河道自净能力的效果，在环境水处理领域具有重大意义。
生物材料保藏
一株鞘氨醇杆菌(Sphingobacterium sp.)，于2018年10月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.16596，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
附图说明
图1为鞘氨醇杆菌BT1生长及氨氮降解曲线；
图2为鞘氨醇杆菌BT1对硝氮降解变化曲线图；
图3为鞘氨醇杆菌BT1对亚硝氮降解变化曲线图；
图4为amoA基因验证的凝胶电泳图；其中，Marker：DL2000(100bp～2000bp)；1：基因片段：amoA；
图5为nirK基因验证的凝胶电泳图；其中，Marker：DL2000(100bp～2000bp)；1：基因 片段为nirS；2：基因片段为nirK。
具体实施方式
异养硝化培养基(g/L)：(NH 4) 2SO 40.47,C 4H 4Na 2O 45.62，维氏盐溶液50ml，C/N＝10，pH＝7.0，琼脂2％。
维氏盐溶液(g/L)：K 2HPO 45.0，MgSO 4·7H 2O 2.5，NaCl 2.5，MnSO 4·4H 2O 0.05，FeSO 4·7H 2O 0.05。
好氧反硝化培养基：硝酸盐培养基：KNO 3 2g，柠檬酸钠5g，K 2HPO 4 1g，KH 2PO 4 1g，MgSO 4·7H 2O 0.2g，微量元素溶液2mL，补充蒸馏水至1L，pH7.2-7.5
亚硝酸盐培养基：NaNO 2 2g，柠檬酸钠5g，K 2HPO 4 1g，KH 2PO 4 1g，MgSO 4·7H 2O 0.2g，微量元素溶液2mL，补充蒸馏水至1L，pH7.2-7.5
微量元素溶液：EDTA 50.0g，ZnSO 4 2.2g，CaCl 2·2H 2O 5.5g，MnCl 2·4H 2O 5.06g，FeSO 4·7H 2O 5.0g，(NH 4)6Mo 7O 2·4H 2O 1.1g，CuSO 4·5H 2O 1.57g，CoCl 2·6H 2O 1.61g，补蒸馏水至1L，pH 6.0
实施例1
垃圾渗滤液好氧发酵罐运行稳定的某周期的活性污泥作为分离用泥。在异养硝化培养基上，采用稀释混合平板法进行分离获得纯种鞘氨醇杆菌BT1菌株。提取菌株鞘氨醇杆菌BT1的基因组，进行16S rDNA测序。将测序结果与Genbank数据库中的已知序列进行相似度对比，结果显示，菌株鞘氨醇杆菌BT1与鞘氨醇杆菌属(Sphingobacterium)序列同源性最高，鉴定为鞘氨醇杆菌。
实施例2鞘氨醇杆菌(Sphingobacterium)鞘氨醇杆菌BT1氨氮降解能力测定
将鞘氨醇杆菌(Sphingobacterium)鞘氨醇杆菌BT1活化富集培养后，移取1ml悬浊液至含有100ml不含琼脂的异养硝化培养基中，在30℃，160rpm/min条件下培养48小时。每隔8小时取培养液。取部分培养液在600nm处测定鞘氨醇杆菌BT1的生长曲线，然后剩余培养液经0.22μm微孔滤膜过滤，检测滤液氨氮、亚硝氮、硝氮和COD等指标。结果如表1及图1所示。
表1鞘氨醇杆菌BT1对氨氮降解速率的变化
Figure PCTCN2018118165-appb-000001
Figure PCTCN2018118165-appb-000002
由表1OD 600指标可以得到菌株的生长曲线。菌株只需要经过8h的迟缓期就可进入指数期，在24h内，OD值从0.01达到了1.349，24h后进入稳定期，与常见的硝化细菌生长周期相比，缩短了24-48小时。
在40h内，氨氮从90.21mg/L降至4.49mg/L，氨氮降解率达到95.01％，总氮从93.77mg/L降至5.11mg/L，总氮的降解率为94.65％。
需要注意的是，48小时内，亚硝氮含量从0mg/L积累至0.3mg/L，硝氮从0.6mg/L降低至0.05mg/L，表明鞘氨醇杆菌BT1具有积累亚硝氮的和降解硝氮的能力，由此我们推测鞘氨醇杆菌BT1可能具有好氧反硝化的能力。
与常见的硝化细菌或者厌氧反硝化细菌相比，鞘氨醇杆菌BT1兼具异养硝化和好氧反硝化功能，硝化过程和反硝化过程可同时进行，硝化的产物可直接作为反硝化的底物。从表1可以看出，鞘氨醇杆菌BT1生长周期短，24小时内即可达到平台期，生长速率远大于自养硝化菌；环境适应力强，对高浓度氨氮的耐受力强。
实施例3鞘氨醇杆菌(Sphingobacterium)BT1好氧反硝化能力测定
(1)采用硝酸盐培养基对鞘氨醇杆菌BT1的反硝化性能进行筛选和表征。
将鞘氨醇杆菌(Sphingobacterium)BT1活化富集培养后，移取1ml悬浊液至含有100ml不含琼脂的硝酸盐培养基或亚硝酸盐培养基中，在30℃，160rpm/min条件下培养，每隔8小时取培养液分别测定细菌浓度(OD 600)、硝氮、亚硝氮。
表2鞘氨醇杆菌BT1对硝氮降解变化
Figure PCTCN2018118165-appb-000003
从表2及图2可知，鞘氨醇杆菌BT1在第46小时开始生长，在80小时达到峰值。在100小时内，硝态氮从240.42mg/L下降至133.32mg/L，硝态氮降解率为44.5％，亚硝酸盐从0累积至40.59mg/L，表明鞘氨醇杆菌BT1对硝态氮的去除率较好，具有较强的反硝化功能。
(2)采用亚硝酸盐培养基对鞘氨醇杆菌BT1的反硝化性能进行筛选和表征。在硝酸盐培养基的基础上，将其中的硝酸钾替换为亚硝酸钠。
表3鞘氨醇杆菌BT1对亚硝氮降解变化
Figure PCTCN2018118165-appb-000004
从表3和图3中可以看出，菌种在接种后第27小时开始生长，在68小时达到顶峰。在100小时内，亚硝氮含量从439.86mg/L降至375.97mg/L，亚硝酸盐含量下降了14.5％，硝态氮从80.27mg/L降至53.13mg/L，含量下降了33.8％。
综合鞘氨醇杆菌BT1降解硝酸盐和亚硝酸盐的效果，可以得出，鞘氨醇杆菌BT1以硝酸盐或亚硝酸盐为氮源时，会有一个25-40小时的潜伏期，菌株的对数生长期为12-22小时，30小时后开始进入稳定期和衰亡期。鞘氨醇杆菌BT1可以单纯利用硝酸盐或者亚硝酸盐作为氮源，达到脱氮的效果，从而证明鞘氨醇杆菌BT1具有较强的反硝化功能。
实施例4鞘氨醇杆菌(Sphingobacterium)鞘氨醇杆菌BT1好氧反硝化能力测定
(1)采用PCR验证鞘氨醇杆菌BT1中是否存在硝化功能基因amoA。引物序列如下：
amoA1F(5’-GGGGTTTCTACTGGTGGT-3’)；
amoA2R(5’-CCCCTCKGSAAAGCCTTCTTC-3’)
反应条件：95℃3min；94℃1min，54.5℃45s，72℃1min，35个循环；72℃，10min；PCR产物使用0.8％琼脂糖凝胶电泳检测，结果如图4所示：
PCR产物为491bp大小的条带，经测序，与amoA基因序列一致，由此可以确认鞘氨醇杆菌BT1中存在amoA基因，具有硝化功能。
(2)反硝化功能基因Nir：Nir基因分为两种，分别是nirK和nirS。nirK基因编码Cu型亚硝酸盐还原酶，另一种nirS基因编码细胞色素还原酶。需要注意的是，这两种编码基因不会同时存在于同一株菌中。采用PCR验证鞘氨醇杆菌BT1中是否存在nirK和nirS基因。引物序列如下：
nirS-cd3aF(5’-GTSAACGTSAAGGARACSGG)
nirS-R3cd(5’-GASTTCGGRTGSGTCTTGA)
nirK-F1aCu(5’-ATCATGGTSCTGCCGCG)
nirK-R3Cu(5’-GCCTCGATCAGRTTGTGGTT)
反应条件：95℃10min；95℃30s，56℃30s，72℃30s，35个循环；72℃，10min；PCR 产物使用0.8％琼脂糖凝胶电泳检测，结果如图5所示。
从图中可以看出，菌株鞘氨醇杆菌BT1中不存在nirS基因，存在nirK基因。即表明鞘氨醇杆菌BT1中存在Cu型亚硝酸盐还原酶，基因型与其表现出来的好氧反硝化功能吻合。
实施例5
鞘氨醇杆菌BT1在异养硝化培养基上培养24小时后，按照1％的体积比例投入DN反应器中，DN反应器为高效脱氮反应器，内有布水系统和三相分离器等核心构建，并设定高径比＞2，可控参数为HRT，当HRT设定为48h时，反应器运行参数如下：
Figure PCTCN2018118165-appb-000005
以上参数表明，鞘氨醇杆菌BT1在实际应用中能够高效的去除氨氮，，在48小时内，氨氮含量从2000mg/L降低至35mg/L，去除速率达到了40.9mg/L*h，同时，总氮(TN)含量在48h内从2500mg/L降低至200mg/L，COD从8000mg/L降低至700mg/L，总氮和COD的降解率分别达到了92％和91.25％。DO始终保持在小于1mg/L，这表明鞘氨醇杆菌BT1对溶氧的浓度要求低，适应性强，特别适用于治理富营养化、高氨氮水体。
实施例6菌株鞘氨醇杆菌BT1在含氮污水处理方面的应用
在使用鞘氨醇杆菌BT1对南京某污水处理厂含氮污水处理实验中，按照20ppm的浓度在好氧池前端流加鞘氨醇杆菌BT1菌液。48小时内，氨氮含量从20mg/L降低至5mg/L，氨氮去除率达到了75％，日处理污水量可达到55000-60000m 3，表明鞘氨醇杆菌BT1用于处理大规模污水时，可以有效的减少含氮污水中的氨氮浓度。
实施例7菌株鞘氨醇杆菌BT1在微生物固定化方面的应用
将鞘氨醇杆菌BT1菌株与生物载体结合，制备成一种适用于河道水体并能缓慢释放的固定化微生物。
制备固定化菌株的步骤为：
菌株的发酵培养；采用液体发酵的方式在机械搅拌式发酵罐中进行鞘氨醇杆菌BT1的纯培养，培养基为异养硝化液体培养基。在30℃下进行纯培养达到12-48小时后，得到高浓度 的鞘氨醇杆菌BT1纯培养菌液，菌液活菌计数在1×10 8-1×10 11cfu/ml。
固态菌体的制备：菌株发酵结束后，离心去除发酵液，得到固态菌体。
生物载体的制备及固定化：(1)将活性炭10-50g/L、壳聚糖5-50g/L、海藻酸钠10-50g/L称量溶解并混合均匀，得到凝胶液。(2)将50-100g固态菌体接入凝胶液中，得到菌体浓度为1×10 8-1×10 11cfu/ml的微生物凝胶液。(3)将微生物凝胶液按照15-25滴/分钟的速度滴入质量浓度为1-50g/L的CaCl 2溶液中，得到微生物活性颗粒。(4)将微生物活性颗粒于5％戊二醛溶液中以1:50在室温下交联24小时。(5)将(4)中交联后的微生物活性颗粒用无菌水浸泡24-48小时后，增殖得到鞘氨醇杆菌BT1生物微胶囊。
获得的鞘氨醇杆菌BT1生物微胶囊在30天内的释放量达85％以上，菌存活率大于5％，有效活菌数经过验证后达到10 9cfu/g。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2018118165-appb-000006
Figure PCTCN2018118165-appb-000007

Claims (19)

  1. 一株鞘氨醇杆菌(Sphingobacterium sp.)，于2018年10月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.16596，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
  2. 含有权利要求1所述鞘氨醇杆菌的微生物制剂。
  3. 根据权利要求2所述的微生物制剂，其特征在于，鞘氨醇杆菌的活菌含量≥1×10 9CFU/g。
  4. 根据权利要求2所述的微生物制剂，其特征在于，鞘氨醇杆菌的活菌含量≥1×10 9CFU/mL。
  5. 根据权利要求2或3所述的微生物制剂，其特征在于，所述微生物制剂为生物微胶囊。
  6. 根据权利要求4所述的微生物制剂，其特征在于，所述生物微胶囊包含有壳体以及壳体内部芯材；壳体材料包括但不限于海藻酸钠凝胶；壳体内部芯材包括但不限于氯化钙溶液，所述壳体内部芯材中分散有1×10 8～1×10 11CFU/mL所述鞘氨醇杆菌的细胞。
  7. 根据权利要求6所述的微生物制剂，其特征在于，所述生物微胶囊的制备方式为：(1)将活性炭10-50g/L、壳聚糖5-50g/L、海藻酸钠10-50g/L混合，得到凝胶液；(2)将菌体与凝胶液混合，得到菌体浓度为1×10 8-1×10 11cfu/ml的微生物凝胶液；(3)将微生物凝胶液滴入质量浓度为1-50g/L的CaCl 2溶液中，得到微生物活性颗粒；(4)将微生物活性颗粒于5％戊二醛溶液中在室温下交联；(5)将步骤(4)交联后的微生物活性颗粒用无菌水浸泡，增殖得到鞘氨醇杆菌BT1生物微胶囊。
  8. 权利要求1所述的鞘氨醇杆菌在化工、环境领域降解河道、湖泊污染物或污水处理方面的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述污水是含氮污水。
  10. 根据权利要求8或9所述的应用，其特征在于，所述污水包括生活污水、工业废水、垃圾渗滤液。
  11. 一种污水处理方法，其特征在于，将权利要求1所述的鞘氨醇杆菌按终浓度10～30mg菌体/L污水投放至污水中。
  12. 根据权利要求8所述的方法，其特征在于，所述鞘氨醇杆菌以生物微胶囊的形式投放至水体中。
  13. 应用权利要求1所述菌株进行污水处理的装置设备。
  14. 根据权利要求13所述的设备，其特征在于，所述设备包括脱氮反应器、污水处理装置、人工湿地或反硝化池。
  15. 根据权利要求14所述的设备，其特征在于，所述设备为高效脱氮反应器。
  16. 根据权利要求14所述的设备，其特征在于，所述设备为一体化污水处理装置。
  17. 根据权利要求14所述的设备，其特征在于，所述设备为污水处理厂反硝化池。
  18. 用于污水处理过程的生物膜，其特征在于，含有权利要求1所述的鞘氨醇杆菌。
  19. 根据权利要求18所述的生物膜，其特征在于，以餐厨垃圾、植物秸秆或贝壳中的至少一种制备生物膜载体。
PCT/CN2018/118165 2018-11-13 2018-11-29 一株兼具异养硝化和好氧反硝化功能鞘氨醇杆菌及其应用 WO2020097994A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811346777.8A CN109161513B (zh) 2018-11-13 2018-11-13 一株鞘氨醇杆菌及其应用
CN201811346777.8 2018-11-13

Publications (1)

Publication Number Publication Date
WO2020097994A1 true WO2020097994A1 (zh) 2020-05-22

Family

ID=64877134

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/118165 WO2020097994A1 (zh) 2018-11-13 2018-11-29 一株兼具异养硝化和好氧反硝化功能鞘氨醇杆菌及其应用

Country Status (2)

Country Link
CN (1) CN109161513B (zh)
WO (1) WO2020097994A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115806916A (zh) * 2022-12-21 2023-03-17 中国科学院沈阳应用生态研究所 一株耐低温异养硝化-好氧反硝化争论贪噬菌及其应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109825450A (zh) * 2019-01-17 2019-05-31 广东博沃特生物科技有限公司 一株耐高氨氮异养硝化细菌及其应用
CN114058552B (zh) * 2021-12-06 2023-07-25 楚雄云泉酱园有限责任公司 一株用于豆瓣酱发酵的食鞘氨醇杆菌

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101580309A (zh) * 2005-04-21 2009-11-18 揖斐电株式会社 含有机物废液的处理方法
JP2010158649A (ja) * 2009-01-09 2010-07-22 Sun Chan Yan 廃水の処理方法
KR20160041361A (ko) * 2014-10-07 2016-04-18 주식회사 오이코스 윤활유분해 미생물을 이용한 토양정화방법 및 토양정화제

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2458119C1 (ru) * 2010-12-29 2012-08-10 Государственное научное учреждение Татарский научно-исследовательский институт агрохимии и почвоведения Российской академии сельскохозяйственных наук Штамм sphingobacterium multivorum, используемый для получения бактериального удобрения под томаты и огурцы
CN105779344A (zh) * 2016-03-29 2016-07-20 江苏蓝星化工环保股份有限公司 一种复合微生物菌剂
CN105886437B (zh) * 2016-05-11 2020-03-03 西安紫瑞生物科技有限公司 用于治理水体污染的组合物或复合菌剂
CN106635904B (zh) * 2016-12-27 2019-05-07 曲阜师范大学 一种具有降解苯胺能力的鞘氨醇杆菌及其应用
CN107857422A (zh) * 2017-08-15 2018-03-30 广东高龙环保科技有限公司 一种规模化畜禽养殖污水处理方法
CN107502580A (zh) * 2017-09-30 2017-12-22 重庆融极环保工程有限公司 一种用于处理畜禽养殖废水的功能菌剂及其制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101580309A (zh) * 2005-04-21 2009-11-18 揖斐电株式会社 含有机物废液的处理方法
JP2010158649A (ja) * 2009-01-09 2010-07-22 Sun Chan Yan 廃水の処理方法
KR20160041361A (ko) * 2014-10-07 2016-04-18 주식회사 오이코스 윤활유분해 미생물을 이용한 토양정화방법 및 토양정화제

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XIANG, HONGWEI: "Study on the Treatment of Low Carbon Source Wastewater and its Microbial Community Dynamics in Small Towns by Bioaugmentation and Solid Carbon Sources", CHINESE MASTER S THESES FULL-TEXT DATABASE, ENGINEERING SCIENCE & TECHNOLOGY 1, vol. 1, no. 06, 15 June 2018 (2018-06-15), pages 14; 42 - 43 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115806916A (zh) * 2022-12-21 2023-03-17 中国科学院沈阳应用生态研究所 一株耐低温异养硝化-好氧反硝化争论贪噬菌及其应用

Also Published As

Publication number Publication date
CN109161513B (zh) 2020-09-04
CN109161513A (zh) 2019-01-08

Similar Documents

Publication Publication Date Title
CN107201325B (zh) 假单胞菌菌株及其培养方法和应用
Jiang et al. Rapid formation of aniline-degrading aerobic granular sludge and investigation of its microbial community succession
WO2020097994A1 (zh) 一株兼具异养硝化和好氧反硝化功能鞘氨醇杆菌及其应用
WO2024113537A1 (zh) 一种异养硝化-好氧反硝化复合菌剂及其制备方法和应用
CN114890555B (zh) 一种用于治理农村黑臭水体的固体微生物制剂及其制备方法与应用
CN112142199A (zh) 一种提高一体式部分反硝化-厌氧氨氧化耦合脱氮性能的装置及方法
CN106676038B (zh) 一种脱氨氮的复合微生物菌剂及其应用
Rong et al. Towards advanced mariculture wastewater treatment by bacterial-algal symbiosis system with different bacteria and algae inoculation ratios
CN110699285A (zh) 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
CN115386520B (zh) 一株嗜吡啶红球菌rl-gz01菌株及其应用
CN107473404B (zh) 一种自成型块状碳载体固定微生物的净水剂及其制备方法
CN104611279B (zh) 一种红城红球菌lh‑n13及其微生物菌剂与用途
Xu et al. The contributions and mechanisms of iron-microbes-biochar in constructed wetlands for nitrate removal from low carbon/nitrogen ratio wastewater
Zeng et al. Improving a compact biofilm reactor to realize efficient nitrogen removal performance: step-feed, intermittent aeration, and immobilization technique
CN113151063B (zh) 弗氏柠檬酸杆菌as11及其在污水处理中的应用
CN108410754B (zh) 一种高盐重金属难降解有机废水处理及抗菌除臭的高效jm菌技术
CN102978145B (zh) 一株兼具异养硝化-好氧反硝化与除磷功能的喹啉降解菌qg6及其应用
CN116395861A (zh) 活性自维持液体反硝化含菌材料及其制法与应用
CN106754453B (zh) 一种功能强化脱氮微生物的富集培养方法
CN110407338B (zh) 一株低温脱氮除磷菌及其应用
CN110468066B (zh) 一种好氧反硝化菌株及其应用
CN109652330B (zh) 香矛醇假单胞菌wxp-4及其在脱氮中的应用
CN110818180A (zh) 一种微生物制剂消解黑臭河道的方法
CN113979548B (zh) 一种节杆菌ty2用于水处理的用途和用于水处理的菌剂
Hassan et al. The removal performance of nitrates in the novel 3D-BERS with GAC and diversity of immobilized microbial communities treating nitrate-polluted water: Effects of pH and COD/NO 3--N ratio

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18939994

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18939994

Country of ref document: EP

Kind code of ref document: A1