WO2017028151A1 - 一种高效苯胺降解菌制剂及其制备和应用 - Google Patents

一种高效苯胺降解菌制剂及其制备和应用 Download PDF

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WO2017028151A1
WO2017028151A1 PCT/CN2015/087210 CN2015087210W WO2017028151A1 WO 2017028151 A1 WO2017028151 A1 WO 2017028151A1 CN 2015087210 W CN2015087210 W CN 2015087210W WO 2017028151 A1 WO2017028151 A1 WO 2017028151A1
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Prior art keywords
acinetobacter
pseudomonas putida
percentage
live bacterial
composite microbial
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PCT/CN2015/087210
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English (en)
French (fr)
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曾伟明
王世民
谢慧芳
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懿科生物科技（上海）有限公司
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Priority to PCT/CN2015/087210 priority Critical patent/WO2017028151A1/zh
Priority to CN201580082434.3A priority patent/CN107922915A/zh
Publication of WO2017028151A1 publication Critical patent/WO2017028151A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • 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
    • 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
    • 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
    • C12R2001/40—Pseudomonas putida

Definitions

  • the invention relates to a microbial live bacterial preparation for treating aniline in sewage such as organic sewage, a preparation method and application thereof.
  • aniline is a widely used chemical raw material.
  • aniline substances enter the environment, seriously pollute the environment and endanger human health, and are carcinogenic.
  • certain industrial wastewaters such as printing and dyeing wastewater containing azo dyes, also produce aniline intermediates during biological treatment. Because of the toxicity of aniline compounds to living things, it has been receiving attention.
  • aniline compounds are listed as priority pollutants in the environment, and industrial drainage is strictly managed.
  • the maximum allowable emission standards are set at a concentration of 5 mg/L.
  • the drinking water standard is only 0.2mg/L.
  • aniline-contaminated water The treatment of aniline-contaminated water is difficult, and there are few domestic studies. So far, most of them have been treated by physical and chemical methods. However, these methods have high processing costs and strict technical requirements, which is difficult to promote in practical applications.
  • the biological treatment of wastewater containing aniline is an important and effective treatment method, which utilizes the action of microorganisms to decompose aniline compounds. Due to its low energy consumption and low cost, mild reaction conditions, and no secondary pollution, it has received increasing attention.
  • the aniline-degrading bacteria are essential for the complete degradation of aniline compounds.
  • Many chemical plants have applied degradable microorganisms to treat aniline compounds in industrial wastewater, and have achieved remarkable results, but they have not fully met the actual needs.
  • Chinese invention patent application 2012103644800 (inventor Zhu Wenxia, Li Liping, invention name: a microbial preparation for repairing aniline-contaminated water) through Pseudomonas aeruginosa, nitrosating bacteria, Bacillus subtilis, Alcaligenes faecalis, Saccharomyces cerevisiae
  • the compatibility between Rhodococcus, Bacillus megaterium and Aspergillus species is added to the wastewater treatment system to degrade aniline refractory compounds and promote discharge.
  • the combination and process are complicated, especially the nitrosation bacteria and Rhodococcus in the formulation are more troublesome. The cells grow slowly.
  • the invention relates to an alternative high-efficiency aniline degrading bacteria preparation, wherein the strain is easy to culture, has fast growth speed, simple assembly, simple operation, low construction investment, low operation cost, good treatment efficiency, and high-efficiency degradation of aniline compounds.
  • the high-efficiency compound microbial agent of the invention has reasonable compatibility with each strain, symbiosis and coordination, and is not antagonistic to each other, and the preparation and operation thereof are simple and convenient, and are favorable for production.
  • the present invention provides a composite microbial live bacterial preparation for efficiently degrading aniline, which comprises Acinetobacter radioresistens, Acinetobacter calcoaceticus and Pseudomonas putida. (Pseudomonas putida).
  • the number of strains per class is not less than 10% of the total number of bacteria. That is, the percentage of bacteria against Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida is not less than 10%, that is, the percentage of Acinetobacter baumannii is 10-80%, calcium acetate The percentage of Acinetobacter is 10-80%, and the percentage of Pseudomonas putida is 10-80%.
  • the ratio of bacteria to Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida is 1:1:1.
  • the A. radians is selected from one or more of ATCC Accession Nos. 43999, 499000, and 43998.
  • the Acinetobacter aceti is selected from one or more of the CGMCC Accession No. 1.6186 and the ATCC Accession Nos. 23055, 31926, 17902, BAA-347, 51432, 40897, 14987, 19638, and BAA-346. Kind.
  • Pseudomonas putida is selected from the group consisting of CGMCC deposit numbers 1.8092, 1.3301, 1.3136, 1.3124, 1.2309, 1.1836, 1.182, 1.1819, 1.1003, 1.643, and 1.593, and ATCC accession numbers 39270, 21812, 39119, 21025 One or more of 31753 and 68832.
  • the total number of microorganisms in the composite microbial live bacterial preparation of the present invention is 100 million cfu/g or more.
  • the composite microbial live bacterial preparation of the present invention comprises Acinetobacter baumannii ATCC 43999, Acinetobacter aceti CGMCC 1.6186, and Pseudomonas putida. CGMCC 1.8092.
  • the conventional microbial live bacterial preparation of the present invention uses a conventional excipient as a carrier.
  • the present invention also provides a method of preparing a composite microbial live bacterial preparation comprising preparing a mixture of Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida.
  • the number of bacteria of Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida is not less than 10% of the total number of bacteria, that is, the percentage of Acinetobacter baumannii is 10-80%, the percentage of Acinetobacter calcoaceticus is 10-80%, and the percentage of Pseudomonas putida is 10-80%.
  • the method optionally further comprises drying the mixture.
  • amplifying liquid culture is carried out separately against Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida, respectively, and then mixed in a desired ratio, mixed with the pulverized solid excipient, and dried.
  • the culture may be carried out by amplifying liquid culture against Acinetobacter baumannii, Acinetobacter calcoaceticus and Pseudomonas putida respectively before mixing, and then mixing in a desired ratio, or may be culturing the mixture after mixing. For example, it is mixed with the pulverized solid auxiliary material and cultured by solid fermentation.
  • the Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida are respectively activated by slanting, and then inoculated in a liquid medium, for example, at 26-35 ° C for 12-48 hours; Count the number of live bacteria of Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida, and the number of bacteria in Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida accounted for the total number of bacteria The percentage is not less than 10%; solid fermentation, for example, fermentation at 26-40 ° C for 24-48 hours; and drying, pulverizing, and sieving, for example drying at 40 ° C or below and / or 40-60 mesh Screened.
  • the present invention provides a method of treating sewage, such as organic sewage, comprising adding to the sewage a combined microbial live bacterial preparation of the present invention.
  • the sewage is sewage containing aniline. In one embodiment, the sewage is printing and dyeing wastewater.
  • the present invention provides a composite microbial live bacterial preparation for treating sewage, for example Application in machine sewage.
  • the sewage is aniline-containing sewage.
  • the sewage is printing and dyeing wastewater.
  • the invention also provides the use of a composite microbial live bacterial preparation for the treatment of aniline contamination.
  • aniline contamination can be, for example, an aniline sudden contamination accident.
  • the experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
  • the materials, reagents and the like used in the examples of the present invention can be obtained commercially, unless otherwise specified.
  • the strains used in the examples of the present invention were purchased from the China General Microorganisms Collection and Management Center (CGMCC), the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cultures (DSMZ).
  • live bacteria as used in the present invention means bacteria which are determined to be living bacteria by using any method known in the art for measuring live bacteria.
  • the measuring method may, for example, first dye the bacterial cells with a dye, and then count, or may dilute the solution containing the bacteria to be tested in a certain ratio, then plate the culture, and determine the live bacteria in the original solution by counting the number of colonies formed after the culture. Number (see, for example, Microbiology Experimental tutorial (2nd Edition), Zhou Deqing, Higher Education Press). Kits or devices for counting viable counts are known in the art.
  • the "living bacteria” according to the present invention is formed by using a conventional live bacteria counting method in the art, that is, a solution containing the bacteria to be tested is diluted in a certain ratio, then plated and cultured, and formed by counting culture. The number of colonies is used to determine the number of viable cells in the original solution.
  • the reference to the number of bacteria refers to the number of viable cells unless otherwise defined.
  • the term "cfu (colony forming unit)" refers to the number of colonies formed on a culture medium such as an agar plate.
  • the cfu can be determined by diluting a solution containing bacteria by a certain multiple, and then dispersing the microbial cells in a medium such as an agar plate, for example, by casting or coating, and culturing the formed colonies after culturing for a period of time. The number, and then calculate the cfu number of the original solution based on the dilution factor.
  • the dilution factor can be determined according to the experience of those skilled in the art.
  • the medium may be any medium or medium suitable for growth of the microorganism to be tested and/or for easy counting observation, such as agar or the like. Counting the number of colonies is mainly counting the number of bacterial colonies visible to the naked eye.
  • the Agrobacterium-resistant Acinetobacter, Acinetobacter aceti, and Pseudomonas putida strains contained in the composite microbial live bacterial preparation as described herein may each be changed within a range of not less than 10% of the total bacterial count.
  • the percentage of Acinetobacter baumannii is 10-80%
  • the percentage of Acinetobacter calcoaceticus is 10-80%
  • the percentage of Pseudomonas putida is 10-80%, such as Acinetobacter baumannii, acetic acid
  • the ratio of Acinetobacter baumannii and Pseudomonas putida strains may be 1:1:1. If the percentage of a certain strain is below the critical percentage of 10%, the strain will soon be eliminated.
  • the scale up culture of the bacterial solution of the present invention can be carried out by any method known in the art.
  • the present invention provides a method of preparing a composite microbial live bacterial preparation of the present invention, comprising:
  • the bevel activation refers to culturing a small amount of bacteria in a slant medium in a test tube.
  • the slant medium may be any medium suitable for culturing the bacteria.
  • One skilled in the art can determine the ramp activation time required for different bacteria, such as 12-48 hours, based on the skill in the art.
  • the temperature at which the bevel is activated may be any temperature suitable for culturing Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida, for example, 26-35 °C.
  • the formulation of the slant medium, as well as the culture conditions suitable for activating Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida, such as temperature, time, etc., are well known in the art, for example, see the Microbiology Experiment tutorial ( 2nd edition), Zhou Deqing, Higher Education Press.
  • the liquid medium may be any medium suitable for culturing Acinetobacter baumannii, Acinetobacter calcoaceticus, and Pseudomonas putida, such as LB medium, agar medium, and the like.
  • the medium for Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida may be the same or different, and the medium may be the same as or different from the slant medium.
  • the liquid culture temperature may be any temperature suitable for culturing Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida, for example, 26-35 ° C, such as 30 ° C.
  • the culture time may be any desired time suitable for the proliferation of Acinetobacter baumannii, Acinetobacter calcoaceticus, and Pseudomonas putida, for example, 12-48 hours, such as 24 hours.
  • the required ratio is such that the percentage of each type of strain in the total number of bacteria is not less than 10% (ie, the percentage of Acinetobacter baumannii is 10-80%, and the percentage of Acinetobacter aceti is 10-80). %, the percentage of Pseudomonas putida is 10-80%), for example, the required ratio can be So 1:1:1.
  • the solid fermentation can also use any suitable fermentation medium.
  • the solid fermentation medium consists of 73% rice bran, 10% bran, 7% soy flour, 8% milk powder, 1% trace element, and the other difference is water.
  • Both the fermentation temperature and the fermentation time may be any temperature and time suitable for culturing the bacteria, for example, a temperature of 26-40 ° C, such as 28 ° C, for a period of 24-48 hours, such as 48 hours.
  • the drying and pulverization may use any suitable temperature and other conditions known in the art, such as a drying temperature of 40 ° C or less.
  • the sieving may use any mesh known in the art suitable for screening for the desired bacteria, for example 40-60 mesh, such as 40, 50 or 60 mesh.
  • liquid culture and solid fermentation are carried out according to the routine operation in the art, wherein the liquid medium and the solid fermentation substrate adopt the formulas commonly used in the art, for example, see “Microbiology Experiment Course (2nd Edition)", Zhou Deqing, Higher Education Publishing Society.
  • the above method for expanding culture and preparing a solid preparation is not unique, and those skilled in the art can select a suitable medium and expand the culture method according to common sense, so that the number of viable bacteria reaches 100 million cfu/g or more, and microbial solids are prepared according to conventional methods. Preparation of the method of preparation.
  • the composite microbial agent of the invention can form a dominant bacterial species, namely Acinetobacter baumannii, Acinetobacter calcoaceticus and Pseudomonas putida to be formulated into a high-efficiency microbial preparation, and is added to the wastewater treatment system in a certain amount. Accelerate the degradation of pollutants by microorganisms to improve the biological treatment efficiency of the system and ensure the stable operation of the system. It contains a variety of microorganisms with excellent degradation ability for refractory pollutants. Each strain has reasonable compatibility, symbiosis coordination, non-antagonism, high activity, large biomass, rapid reproduction, and is added to the wastewater treatment system.
  • the aniline-contaminated water has good degradation effect and can achieve high COD removal rate at the same time. It is suitable for industrial sewage treatment, which can improve the treatment water volume and water quality, reduce operating costs and promote emission standards.
  • the culture conditions of each microorganism are as follows: the culture temperature is 30 ° C, and the culture time is 48 hours; for the formulation of each medium, see “Microbiology Experiment Course (2nd Edition)", Zhou Deqing, Higher Education Press.
  • the ATCC has a storage number of 43999 for A. radians and CGMCC with a storage number of 1.6186, and the Acinetobacter calcoaceticum is activated by slanting, and then inoculated in a liquid medium, and cultured at 30 ° C for 24 hours to realize the seed liquid. Amplify the liquid culture.
  • Liquid medium formula: glucose 3%, pancreatic fistula 1.5%, beef extract 3%, peptone 2%, Tween 80 1%, sodium acetate 0.5%, ammonium citrate 0.2%, magnesium sulfate 0.058%, manganese sulfate 0.25%, pH 6.5.
  • Liquid medium formulation glucose 3%, peptone 2%, yeast extract 0.2%, sodium acetate 0.5%, pH 7.0.
  • Viable count Dilute the solution containing the bacteria to be tested, then plate culture, and count the number of viable cells in the original solution by counting the number of colonies formed after the culture (see, for example, "Microbiology Experiment Course (2nd Edition)", Zhou Deqing , Higher Education Press). The number of bacteria against Acinetobacter baumannii, Acinetobacter aceti, and Pseudomonas putida was measured, and the number of each strain was mixed in a ratio of 1:1:1. Only in this step, the percentage of each strain is not less than 10%, so as to ensure that the percentage of each strain in the subsequent solid sample is not less than 10%.
  • the simulated wastewater containing aniline 10 mg/L was used as a test object.
  • the simulated wastewater was a concentration of glucose and aniline solution with COD as control index, and added Ca, Mg, Fe, Co, Trace elements such as Ni and Zn (1 mL of trace element solution is added per liter of wastewater, and the specific components of the trace element solution are shown in Table 1).
  • the chemical oxygen demand (COD) is 350-360 mg/L
  • the temperature is 30 ° C
  • the mixed liquid suspension solid concentration (MLSS) is 5.7 g/L.
  • SBR sequential batch activated sludge process
  • the simulated azo dye wastewater prepared by using Acid Orange II is a 353 mg/L acid orange II and a certain concentration (with COD as a control index) starch solution, and the COD is controlled to be about 2000 mg/L. .
  • Adding NH 4 Cl and KH 2 PO 4 to adjust CODcr: N:P 200:5:1; adding 60mg/L NaHCO 3 and trace elements such as Ca, Mg, Fe, Co, Ni, Zn (per liter of wastewater Add 1mL of trace element solution, the specific composition of trace element solution is shown in Table 1).
  • the domestication of anaerobic sludge is started. Starting from the initial acid orange II concentration of 10 mg/L, the water is changed every day, and the dye concentration is gradually increased. After 1 month of domestication, the dye concentration reaches 353 mg/L. Use anaerobic sludge.
  • the domesticated anaerobic sludge was inoculated at 40 °C for 48 h, and the pH was adjusted to neutral.
  • the COD was 940-1005 mg/L
  • the ammonia nitrogen content was 14-17 mg/L
  • the aniline content was 11.2-11.9 mg. /L.
  • An SBR reactor was used, in which 1 ppm of the live bacterial preparation of Example 1 was added in the same manner as in Example 2, first, the bacterial agent was activated, and the activated bacterial liquid was taken and added in an amount of 1 mL/L of wastewater.
  • the test results are shown in Table 3.
  • the dyeing wastewater 1 was finally discharged from the printing and dyeing factory as the test object. After anaerobic hydrolysis for 48 hours in the same manner as in Example 3, the pH was adjusted to neutral, the COD content was 440-474 mg/L, and the aniline content was 1-1.2 mg. /L.
  • An SBR reactor was used, in which 1 ppm of the live bacterial preparation of Example 1 was added in the same manner as in Example 2, first, the bacterial agent was activated, and the activated bacterial liquid was taken and added in an amount of 1 mL/L of wastewater. Temperature 30 ° C, MLSS 5.5g / L, 150rpm rotating bed, aerobic reaction for 24h. The test results are shown in Table 4.
  • the combined effect of the combined microbial live bacterial preparation and the activated sludge of the present invention is better than the conventional activated sludge method, and the COD removal rate can be increased.
  • the printing and dyeing wastewater 2 finally discharged from the printing and dyeing factory was used as the test object, and the pH was adjusted to neutral after anaerobic hydrolysis for 48 hours in the same manner as in Example 3.
  • the COD content was 449-465 mg/L, and the aniline content was 6.0-6.7. Mg/L.
  • An SBR reactor was used, in which 1 ppm of the live bacterial preparation of Example 1 was added in the same manner as in Example 2, first, the bacterial agent was activated, and the activated bacterial liquid was taken and added in an amount of 1 mL/L of wastewater.
  • the test results are shown in Table 5.
  • the combined treatment of the combined microbial live bacteria preparation and the activated sludge of the invention is better than the traditional activated sludge method, and more aniline and COD can be removed in a shorter time. After a long period of treatment, the aniline can be completely degraded and the COD removal rate is higher than that of the activated sludge process.

Abstract

本发明提供了一种高效降解废水中苯胺的复合微生物活菌制剂及其制备方法和应用。该复合微生物活菌制剂含有抗辐射不动杆菌(Acinetobacter radioresistens)、醋酸钙不动杆菌(Acinetobacter calcoaceticus)、恶臭假单胞菌(Pseudomonas putida)。本发明还提供了处理污水的方法，包括向所述污水中加入所述复合微生物活菌制剂。

Description

一种高效苯胺降解菌制剂及其制备和应用 技术领域
本发明涉及处理污水例如有机污水中苯胺的微生物活菌制剂及其制备方法和应用。
背景技术
随着城市人口的日益膨胀和工农业的不断发展，水环境污染事故发生频繁，严重的危害了人、畜的健康乃至危及生命。工业污水成分更加复杂，苯胺是广泛应用的化工原料，由此进入环境中的苯胺类物质越来越多，严重污染环境和危害人体健康，并具有致癌性。同时，某些工业废水，如含偶氮类染料的印染废水在生物处理过程中也会产生苯胺类中间产物。由于苯胺类化合物对生物的毒性，所以一直受到人们关注。我国把苯胺类化合物列入环境中优先控制的重点污染物，并对工业排水进行严格管理，制定了最高容许排放标准，其浓度为5mg/L。而饮用水标准仅为0.2mg/L。
苯胺类化合物污染的水体治理较为困难，国内研究较少，至今为止，绝大多数采用物理、化学方法加以处理，但这些方法处理费用偏高，技术要求较为严格，在实际应用上难以推广。
生物处理含苯胺类物质废水是比较重要而有效的一类处理方法，它是利用微生物的作用，使苯胺类化合物得到分解。由于其能耗和成本低、反应条件温和、无二次污染等优点，日益得到重视。
苯胺类化合物降解菌对苯胺类化合物的彻底降解是十分必需的。许多化工厂已经应用降解性微生物处理工业废水中的苯胺类化合物，并且已经取得了显著的效果，但是还不能完全满足实际的需要。例如中国发明专利申请2012103644800(发明人朱文霞,李丽萍，发明名称：一种修复苯胺类污染水体的微生物制剂)通过铜绿假单孢菌、亚硝化细菌、枯草芽孢杆菌、粪产碱杆菌、酿酒酵母、红球菌、巨大芽孢杆菌、黑曲霉菌种之间配伍，投加在废水处理系统中，降解苯胺类难降解化合物，促进达标排放。但其组配和工序较为复杂，特别是其配方中的亚硝化细菌、红球菌培养较麻烦， 菌体生长缓慢。
本发明涉及一种替代性的高效苯胺降解菌制剂，其中的菌株易于培养，生长速度快，组配简单、操作简便、建设投资少、运行成本低、处理效率好，对苯胺类化合物具有高效降解能力，并能同时达到较高的COD去除率，兼具显著的经济和环境效益，具有非常好的应用前景。
发明内容
本发明的高效复合微生物菌剂，各菌种之间合理配伍，共生协调，互不拮抗，其制备及操作简便易行，利于生产。
在一个方面，本发明提供了一种高效降解苯胺的复合微生物活菌制剂，其特征在于其含有抗辐射不动杆菌(Acinetobacter radioresistens)、醋酸钙不动杆菌(Acinetobacter calcoaceticus)和恶臭假单胞菌(Pseudomonas putida)。
在一个实施方案中，每一类菌株数量占总菌数的百分比不低于10％。即抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的菌数占总菌数的百分比均不低于10％，即抗辐射不动杆菌的百分比为10-80％，醋酸钙不动杆菌的百分比为10-80％，恶臭假单胞菌的百分比为10-80％，。
在一个实施方案中，抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的菌数比例是1:1:1。
在一个实施方案中，抗辐射不动杆菌选自ATCC保藏号为43999、49000和43998中的一种或多种。
在一个实施方案中，醋酸钙不动杆菌选自CGMCC保藏号为1.6186以及ATCC保藏号为23055、31926、17902、BAA-347、51432、40897、14987、19638和BAA-346中的一种或多种。
在一个实施方案中，恶臭假单胞菌选自CGMCC保藏号为1.8092、1.3301、1.3136、1.3124、1.2309、1.1836、1.1820、1.1819、1.1003、1.643和1.593以及ATCC保藏号为39270、21812、39119、21025、31753和68832中的一种或多种。
在一个实施方案中，本发明所述复合微生物活菌制剂中微生物的总菌数为1亿cfu/克或以上。
在一个实施方案中，本发明所述复合微生物活菌制剂包含抗辐射不动杆菌ATCC 43999、醋酸钙不动杆菌CGMCC 1.6186和恶臭假单胞菌 CGMCC 1.8092。
在一个实施方案中，本发明所述复合微生物活菌制剂中以常规辅料作为载体。
在另一个方面，本发明还提供了复合微生物活菌制剂的制备方法，包括制备抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的混合物。
在一个实施方案中，所述抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的菌数占总菌数的百分比均不低于10％，即抗辐射不动杆菌的百分比为10-80％，醋酸钙不动杆菌的百分比为10-80％，恶臭假单胞菌的百分比为10-80％。
在一个实施方案中，所述方法任选还包括将所述混合物干燥。
在一个实施方案中，包括分别对抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌进行放大液体培养，然后按所需比例混合，与粉碎过筛后的固体辅料混合，干燥。
在一个实施方案中，还包括在混合之前分别培养例如在液体培养基中培养抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌，和/或在混合之后培养所述混合物。所述培养可以是在混合之前分别对抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌进行放大液体培养，然后按所需比例混合，也可以是在混合之后对所述混合物进行培养，例如与粉碎过筛后的固体辅料混合，经固体发酵培养。
在一个实施方案中，将抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌分别经斜面活化后，接种于液体培养基中，例如在26-35℃培养12-48小时；分别计数抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的活菌数并混合，其中抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的菌数占总菌数的百分比均不低于10％；固体发酵，例如在26-40℃发酵24-48小时；及烘干，粉碎，并且过筛，例如在40℃或以下烘干和/或经40-60目过筛。
在另一个方面，本发明提供了一种处理污水例如有机污水的方法，包括向所述污水中加入本发明所述的复合微生物活菌制剂。
在一个实施方案中，所述污水是含有苯胺的污水。在一个实施方案中，所述污水是印染废水。
在另一个方面，本发明提供了复合微生物活菌制剂在处理污水例如有 机污水中的应用。
在一个实施方案中，所述污水是含苯胺的污水。
在一个实施方案中，所述污水是印染废水。
在另一个方面，本发明还提供了复合微生物活菌制剂在处理苯胺污染中的应用。所述苯胺污染可以是例如苯胺突发污染事故。
本发明实施例中所用的实验方法如无特殊说明，均为常规方法。本发明实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。本发明实施例中所用的菌种均从中国普通微生物菌种保藏管理中心(CGMCC)、美国典型培养物保藏中心(ATCC)或德国微生物菌种保藏中心(DSMZ)购得。
本发明所述的“活菌”是指通过使用本领域已知的任何测定活菌的方法测定为活菌的细菌。所述测定方法例如可以先用染料染色细菌细胞、然后计数，或者可以将包含待测细菌的溶液按一定比例稀释，然后铺板培养，通过计数培养后形成的菌落数来确定原溶液中的活菌数(参见例如《微生物学实验教程(第2版)》，周德庆，高等教育出版社)。本领域已知计数活菌数的试剂盒或设备。在本发明的一个实施方案中，本发明所述的“活菌”是通过使用本领域常规活菌计数方法，即将包含待测细菌的溶液按一定比例稀释，然后铺板培养，通过计数培养后形成的菌落数来确定原溶液中的活菌数。
在本文中，除非特别定义，提及菌数时均是指活菌数。
如本文所述，术语“cfu(菌落形成单位)”是指在培养介质例如琼脂平板上形成的菌落数。例如，cfu可以如下测定：将含有细菌的溶液稀释一定倍数，然后将稀释液例如通过浇注或涂布方式让微生物单细胞一一分散在介质例如琼脂平板上，培养一段时间后，计数形成的菌落数，再根据稀释倍数计算原溶液的cfu数。稀释倍数可以根据本领域技术人员的经验确定。所述介质可以是适于待测微生物生长和/或便于计数观察的任何介质或培养基，例如琼脂等。计数菌落数主要是对肉眼可见的细菌菌落数进行计数。
如本文所述的复合微生物活菌制剂所包含的抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌菌株各自可以在不低于总菌数10％的范围内变 动，即抗辐射不动杆菌的百分比为10-80％，醋酸钙不动杆菌的百分比为10-80％，恶臭假单胞菌的百分比为10-80％，例如抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌菌株的比例可以是1：1：1。若某类菌株的百分比低于10％的临界百分比，该类菌株将很快被淘汰。
本发明菌液的放大培养可以采用本领域已知的任何方法进行。例如，本发明提供了一种制备本发明复合微生物活菌制剂的方法，包括：
-将抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌分别经斜面活化后，接种于液体培养基中培养；
-活菌计数，分别计数抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的活菌数，按照所需比例将菌液进行混合；
-固体发酵；
-烘干，粉碎并过筛。
所述斜面活化是指将少量细菌挑于试管中的斜面培养基中进行培养。所述斜面培养基可以是适于培养所述细菌的任何培养基。本领域技术人员根据本领域技术知识可以确定不同细菌所需的斜面活化时间，例如12-48小时。斜面活化的温度可以是适于培养抗辐射不动杆菌、醋酸钙不动杆菌以及恶臭假单胞菌的任何温度，例如26-35℃。
斜面培养基的配方，以及适于活化抗辐射不动杆菌、醋酸钙不动杆菌以及恶臭假单胞菌的培养条件例如温度、时间等均为本领域所熟知，例如参见《微生物学实验教程(第2版)》，周德庆，高等教育出版社。
所述液体培养基可以是适于培养抗辐射不动杆菌、醋酸钙不动杆菌以及恶臭假单胞菌的任何培养基，例如LB培养基、琼脂培养基等。用于抗辐射不动杆菌、醋酸钙不动杆菌以及恶臭假单胞菌的培养基可以相同或不同，该培养基与斜面培养基也可以相同或不同。
液体培养温度可以是适于培养抗辐射不动杆菌、醋酸钙不动杆菌以及恶臭假单胞菌的任何温度，例如26-35℃，如30℃。培养时间可以是适于抗辐射不动杆菌、醋酸钙不动杆菌以及恶臭假单胞菌增殖的任意所需时间，例如12-48小时，如24小时。
所述所需比例是可以保证每一类菌株数量占总菌数的百分比不低于10％(即抗辐射不动杆菌的百分比为10-80％，醋酸钙不动杆菌的百分比为10-80％，恶臭假单胞菌的百分比为10-80％)的任意比例，例如所需比例可 以是1:1:1。
所述固体发酵也可以使用任何合适的发酵培养基。在一个实施方案中，所述固体发酵培养基组成为米糠73％，麸皮10％，大豆粉7％，奶粉8％，微量元素1％，其它差额部分为水。发酵温度以及发酵时间均可以是适于培养所述细菌的任何温度和时间，例如温度为26-40℃，如28℃，时间为24-48小时，如48小时。
所述烘干和粉碎可以使用本领域已知的任何合适温度和其他条件，例如烘干温度为40℃或以下。所述过筛可以使用本领域已知的任何适于筛选所需细菌的筛目，例如40-60目，如40、50或60目。
本文中，液体培养和固体发酵的操作均按本领域常规操作，其中液体培养基和固体发酵基质采用本领域常用配方，例如参见《微生物学实验教程(第2版)》，周德庆，高等教育出版社。
上述菌种扩大培养及制备固体制剂的方法不是唯一的，本领域技术人员可以根据常识选择合适的培养基及扩大培养方法，使活菌数达到1亿cfu/克以上，以及按照常规制备微生物固体制剂的方法制备。
本发明的复合菌剂将能形成优势菌群的菌种，即抗辐射不动杆菌、醋酸钙不动杆菌以及恶臭假单胞菌配制成高效微生物制剂，按一定量投加到废水处理系统中，加速微生物对污染物的降解，以提高系统的生物处理效率，保证系统稳定运行。其含有多种对难降解污染物有优良降解能力的微生物，各菌种之间合理配伍，共生协调，互不拮抗，活性高，生物量大，繁殖快，投加在废水处理系统中，对苯胺类污染水体有良好的降解效果，并能同时达到较高的COD去除率，适于工业污水处理，可提高处理水量和处理水质，降低运行费用，促进达标排放。
具体实施方式
本发明通过下述实施例进一步阐明，但任何实施例或其组合不应当理解为对本发明的范围或实施方式的限制。在不偏离本发明的精神和范围的前提下，本领域技术人员可以对本发明的技术方案进行任何修改或改变，这种修改和改变也包含在本发明的范围内。
下述实施例中所用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
除非特别指出，各微生物培养条件如下：培养温度为30℃，培养时间为48小时；各培养基配方参见《微生物学实验教程(第2版)》，周德庆，高等教育出版社。
实施例1
复合微生物活菌制剂的制备
(1)分别进行各类菌株的放大液体培养：
①将ATCC的保藏号为43999的抗辐射不动杆菌和CGMCC的保藏号为1.6186的醋酸钙不动杆菌分别经斜面活化后，接种于液体培养基中，30℃培养24小时，实现种子液的放大液体培养。液体培养基配方：葡萄糖3％，胰胨1.5％，牛肉膏3％，蛋白胨2％，吐温80 1％，乙酸钠0.5％，柠檬酸铵0.2％，硫酸镁0.058％，硫酸锰0.25％，pH 6.5。
②将CGMCC的保藏号为1.8092的恶臭假单胞菌经斜面活化后，接种于液体培养基中，28℃培养24小时，实现种子液的放大液体培养。液体培养基配方：葡萄糖3％，蛋白胨2％，酵母膏0.2％，乙酸钠0.5％，pH 7.0。
活菌计数：将包含待测细菌的溶液稀释，然后铺板培养，通过计数培养后形成的菌落数来确定原溶液中的活菌数(参见例如《微生物学实验教程(第2版)》，周德庆，高等教育出版社)。测定抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的菌数，每一类菌株数量按照1:1:1等比例进行混合。只有该步骤达到每株菌的百分比不低于10％，才能保证后续固体样品中每株菌的百分比不低于10％。
(2)固体发酵罐中加入发酵培养基(按以下质量百分比：米糠73％，麸皮10％，大豆粉7％，奶粉8％，微量元素1％，其它差额部分为水)，121℃灭菌30分钟，待罐温降至40℃以下，在发酵罐旋转的情况下喷入如上获得的混合菌液，28℃固体发酵48小时，实现固体扩大培养。40℃烘干，粉碎，经40目过筛，备用。活菌计数，总菌数达到1亿cfu/克以上，包装装袋。
实施例2
以含苯胺10mg/L的模拟废水作为测试对象，所述模拟废水是一定浓度(以COD为控制指标)的葡萄糖和苯胺溶液，同时添加Ca、Mg、Fe、Co、 Ni、Zn等微量元素(每升废水中投加1mL的微量元素溶液，微量元素溶液具体成分见表1)。
表1
微量元素 添加药品 浓度(mg/L) 微量元素 添加药品 浓度(mg/L)
Ca CaCl2·2H2O 330 Co CoCl2·6H2O 240
Mg MgSO4 500 B H3BO4 14
Cu CuSO4·5H2O 250 Mn MnCl2·4H2O 990
Ni NiCl2·6H2O 190 Mo NH4MoO4·4H2O 9
Fe FeCl3·6H2O 500 Zn ZnSO4·7H2O 250
添加60mg/L NaHCO3以保证反应器内具有足够缓冲能力。其化学需氧量(COD)为350-360mg/L，温度30℃，混合液悬浮固体浓度(MLSS)5.7g/L。采用序批式活性污泥法(SBR)反应器，其中添加实施例1中制得的活菌制剂1ppm，添加方式为：首先将上述活菌制剂活化，活化在200mL模拟废水中添加4g菌剂，在30℃条件下，曝气12h～16h。取活化后的菌液，按1mL/L废水的用量加入。150rpm转速摇床，好氧反应24h，并于4h和24h分别取样测试。测试结果见表2。
表2
Figure PCTCN2015087210-appb-000001
可见，对于含有苯胺的模拟废水，本发明的复合微生物活菌制剂与活性污泥组合的处理效果明显好于传统的活性污泥法，可在较短时间内去除较多的苯胺和COD；经过较长时间处理，可完全降解苯胺，并达到较高的COD去除率。
实施例3
以酸性橙II配制的模拟偶氮染料废水作为测试对象，所述模拟偶氮染料废水是353mg/L的酸性橙Ⅱ和一定浓度(以COD为控制指标)淀粉溶液，控制COD约为2000mg/L。同时加入NH4Cl和KH2PO4调节CODcr：N：P＝200：5：1；加入60mg/L NaHCO3和Ca、Mg、Fe、Co、Ni、Zn等微量元素(每升废水中投加1mL的微量元素溶液，微量元素溶液具体成分见表1)。
首先进行厌氧污泥的驯化，从起始酸性橙II浓度为10mg/L开始，每天换水，逐渐增加染料浓度，驯化1个月后，染料浓度达到353mg/L，此时污泥作为接种用厌氧污泥。
接种驯化后的厌氧污泥，于40℃厌氧水解48h后，调节pH值至中性，其COD为940-1005mg/L，氨氮含量为14-17mg/L，苯胺含量为11.2-11.9mg/L。采用SBR反应器，其中添加实施例1中的活菌制剂1ppm，添加方式为：如实施例2所述，首先将菌剂活化，取活化后的菌液，按1mL/L废水的用量加入。温度30℃，MLSS5.75g/L，150rpm转速摇床，好氧反应24h，并于4h和24h分别取样测试。测试结果见表3。
表3
Figure PCTCN2015087210-appb-000002
可见，对于模拟偶氮染料废水，本发明的复合微生物活菌制剂与活性污泥组合的处理效果明显好于传统活性污泥法，可在较短时间内去除较多的苯胺、COD；经过较长时间处理，可完全降解苯胺，达到较高的COD和氨氮去除率。
实施例4
以得自印染厂最终排出印染废水1作为测试对象，以同实施例3的方式厌氧水解48h后，调节pH值到中性，COD含量为440-474mg/L，苯胺含量为1-1.2mg/L。采用SBR反应器，其中添加实施例1中的活菌制剂1ppm，添加方式为：如实施例2所述，首先将菌剂活化，取活化后的菌液，按1mL/L废水的用量加入。温度30℃，MLSS 5.5g/L，150rpm转速摇床，好氧反应24h。测试结果见表4。
表4
Figure PCTCN2015087210-appb-000003
可见，对于实际经厌氧水解后的印染废水，本发明的复合微生物活菌制剂与活性污泥组合的处理效果好于传统活性污泥法，可增加COD的去除率。
实施例5
以得自印染厂最终排出的印染废水2作为测试对象，以同实施例3的方式厌氧水解48h后，调节pH值到中性，COD含量为449-465mg/L，苯胺含量为6.0-6.7mg/L。采用SBR反应器，其中添加实施例1中的活菌制剂1ppm，添加方式为：如实施例2所述，首先将菌剂活化，取活化后的菌液，按1mL/L废水的用量加入。温度30℃，MLSS 5.5g/L，150rpm转速摇床，好氧反应24h，并于4h和24h分别取样测试。测试结果见表5。
表5
Figure PCTCN2015087210-appb-000004
可见，对于实际经厌氧水解后的印染废水，本发明的复合微生物活菌制剂与活性污泥组合的处理效果好于传统活性污泥法，可在较短时间内去除较多的苯胺和COD；经过较长时间处理，可完全降解苯胺，并达到比活性污泥法较高的COD去除率。

Claims (11)

  1. 一种降解苯胺的复合微生物活菌制剂，其特征在于其含有抗辐射不动杆菌(Acinetobacter radioresistens)、醋酸钙不动杆菌(Acinetobacter calcoaceticus)和恶臭假单胞菌(Pseudomonas putida)。
  2. 权利要求1所述的复合微生物活菌制剂，其特征在于抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的菌数占总菌数的百分比均不低于10％，即抗辐射不动杆菌的百分比为10-80％，醋酸钙不动杆菌的百分比为10-80％，恶臭假单胞菌的百分比为10-80％，例如所述抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的菌数比例是1:1:1。
  3. 权利要求1-2任一项所述的复合微生物活菌制剂，其特征在于
    (i)所述抗辐射不动杆菌选自ATCC保藏号为43999、49000和43998中的一种或多种；或者
    (ii)所述醋酸钙不动杆菌选自CGMCC保藏号为1.6186以及ATCC保藏号为23055、31926、17902、BAA-347、51432、40897、14987、19638和BAA-346中的一种或多种；或者
    (iii)所述恶臭假单胞菌选自CGMCC保藏号为1.8092、1.3301、1.3136、1.3124、1.2309、1.1836、1.1820、1.1819、1.1003、1.643和1.593以及ATCC保藏号为39270、21812、39119、21025、31753、68832中的一种或多种。
  4. 权利要求1-3任一项所述的复合微生物活菌制剂，其特征在于所述复合微生物活菌制剂中微生物的总菌数为1亿cfu/克或以上。
  5. 权利要求1-4任一项所述的复合微生物活菌制剂，其特征在于所述复合微生物活菌制剂包含抗辐射不动杆菌ATCC 43999、醋酸钙不动杆菌CGMCC 1.6186和恶臭假单胞菌CGMCC 1.8092。
  6. 一种制备权利要求1-5任一项所述的复合微生物活菌制剂的方法，包括：
    制备抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的混合物，优 选抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的菌数占总菌数的百分比均不低于10％，即抗辐射不动杆菌的百分比为10-80％，醋酸钙不动杆菌的百分比为10-80％，恶臭假单胞菌的百分比为10-80％，优选抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的菌数比例是1:1:1，
    任选还包括将所述混合物干燥，
    任选还包括在混合之前分别培养例如在液体培养基中培养抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌，和/或在混合之后培养所述混合物。
  7. 权利要求6所述的方法，包括以下步骤：
    (a)将抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌分别经斜面活化后，接种于液体培养基中培养，例如在26-35℃培养12-48小时；
    (b)分别计数抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的菌数并混合，其中抗辐射不动杆菌、醋酸钙不动杆菌和恶臭假单胞菌的菌数占总菌数的百分比均不低于10％；
    (c)固体发酵，例如在26-40℃发酵24-48小时；及
    (d)烘干，粉碎，并且过筛，例如在40℃或以下烘干和/或经40-60目过筛。
  8. 一种处理污水例如有机污水的方法，包括向所述污水中加入权利要求1-5任一项的复合微生物活菌制剂。
  9. 权利要求1-5任一项所述的复合微生物活菌制剂在处理污水例如有机污水中的应用。
  10. 权利要求8的方法或权利要求9的应用，其中所述污水是含苯胺的污水或印染废水。
  11. 权利要求1-5任一项所述的复合微生物活菌制剂在处理苯胺污染中的应用。
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CN112960778A (zh) * 2021-02-23 2021-06-15 吴奇桐 一种降解多环类有机物的组合物及其制备方法和应用
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