CN104911133A - Pseudomonas aeruginosa and application - Google Patents
Pseudomonas aeruginosa and application Download PDFInfo
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- CN104911133A CN104911133A CN201510362940.XA CN201510362940A CN104911133A CN 104911133 A CN104911133 A CN 104911133A CN 201510362940 A CN201510362940 A CN 201510362940A CN 104911133 A CN104911133 A CN 104911133A
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- denitrification
- pseudomonas aeruginosa
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Classifications
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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
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- 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
- C12R2001/385—Pseudomonas aeruginosa
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/34—Chemical or biological purification of waste gases
- B01D53/46—Removing components of defined structure
- B01D53/54—Nitrogen compounds
- B01D53/56—Nitrogen oxides
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/34—Chemical or biological purification of waste gases
- B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
- B01D53/84—Biological processes
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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
- C02F3/02—Aerobic processes
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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
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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
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
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- 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
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- Engineering & Computer Science (AREA)
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- Life Sciences & Earth Sciences (AREA)
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- Biodiversity & Conservation Biology (AREA)
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- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
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- Tropical Medicine & Parasitology (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a pseudomonas aeruginosa and an application. The pseudomonas aeruginosa CP1 is collected by China Centre for Type Culture Collection, wherein the collection number is CCTCC NO: 2015197, and the collection date is April 6, 2015. The strain is used for a denitrification treatment for nitrogen-containing wastewater or used for an aerobic denitrification denitration treatment for NOx smoke or waste gas. In case of no impressed current, the optimal culture condition of the strain comprises a temperature of 30-40 DEG C and C/N of not less than 12. The strain is still capable of maintaining an efficient nitrate degradation process under an aerobic condition. However, the strain is capable of achieving a removal rate of 99.01% or above for nitrate nitrogen with an initial concentration of 250mg/L within 16 hours in the case that the C/N is reduced to 4-6 under the current simulation of 10mA, and hardly generates nitrate accumulation.
Description
Technical field
The invention belongs to environmental engineering, biological field, Pseudomonas aeruginosa (pseudomonas aeruginosa) the CP1 bacterial strain and removing in high temperature environments in waste water relating to the electroactive aerobic denitrification capability of a strain tool removes the application of nitrate radical in water and nitrite anions.
Background technology
China is large agricultural country, the annual agricultural wastes enormous amount produced.And for a long time, China has all been placed on city the emphasis of Pollution abatement, but ignore account for national total area up to 90% vast rural area.The waste of a large amount of farm crop is arbitrarily burnt or is naturally rotted, add air pollution index and have influence on traffic and air transportation cause, agricultural chemicals, form garbage and dejection etc. are residual enters soil, the inflow stream such as nitrate, nitrite causing being formed, lake permeating the ground, cause the azotate pollution of surface water and groundwater.Have influence on the yield and quality of agricultural-food on the one hand, also restrict Economic development on the other hand, also serious harm is to HUMAN HEALTH.Not only serious destruction is caused to environment, also result in a large amount of wastings of resources simultaneously.
In the research of numerous treatment of nitric acid nitrogen waste water, biological eliminating method as a kind of green high-efficient treatment technology and be more and more taken seriously, biologic treating technique has the advantages such as light to influence degree, non-secondary pollution, technique are simple, processing power is strong, mode is flexible, become denitrogenation of waste water technology most widely used in recent years, the efficient biological denitrification method of exploiting economy and technology have become one of field of water pollution control focus.
Chinese scholars has carried out research extensively and profoundly in aerobic denitrification field, proves that aerobic denitrification exists clear superiority than conventional denitrification technology.The main body of aerobic denitrification application is aerobic denitrification flora, in recent years, constantly has aerobic denitrifying bacteria to be separated, and is present in paracoccus, Rhodopseudomonas, Alcaligenes, bacillus etc. more than 50 and belongs to.Traditionally, when process is containing high-nitrogen waste water, carbon source deficiency is a sewage treatment area difficult problem always, needs to consume a large amount of additional organic carbon sources to meet denitrification process, and along with more strict to the emission control of nitrogen phosphorus in sewage, carbon resource shortage problem is more outstanding.So how effectively to reduce carbon source cost and reduce Nitrous Oxide N while efficient process nitrogenous effluent
2the discharge of O is study hotspot problem always.Analyze in conjunction with above, if electrochemistry can be utilized in conjunction with aerobic denitrifying method, make aerobic microbiological can carry out transfer transport with negative electrode directly or indirectly.This means not need to add a large amount of organic carbon sources, in environmental improvement, not only can prevent excessive organism from entering water outlet, also likely make biosolids output reduce.
Therefore, screening and separating has efficient aerobic denitrifying bacteria and just can overcome the above problems to a certain extent the research process nitrogenous effluent of the mechanism of its aerobic denitrification under the condition of extra electric field catalysis.
Summary of the invention
The object of the present invention is to provide a kind of bacterial strain and the electrochemistry aerobic denitrification purposes thereof can under mesophilic condition with high-efficiency aerobic denitrification capability.
Object of the present invention is achieved through the following technical solutions:
The bacterial strain that the present invention has high-efficiency aerobic denitrification capability is Pseudomonas aeruginosa (Pseudomonasaeruginosa) CP1, and this bacterial strain has following characteristics:
(1) colony characteristics: bacterium colony is flat, light brown, smooth surface is opaque, and edge is irregular, shaft-like, Gram-negative, under anaerobic can grow, belong to amphimicrobe;
(2) glucose oxidase fermenting experiment is shown as non-fermented type, and catalase, oxydase, gelatine liquefication experiment, nitrate reduction experiment, the experiment of D-Glucose fermenting experiment, fructose fermenting experiment, pyocyanin, arginine dihydrolase experiment, Citrate trianion utilize and be all positive; Hydrogen sulfide is tested, V-P identifies, lysine decarboxylase is tested, ONPG measures, Starch Hydrolysis experiment is feminine gender, can carry out denitrification, can with ammonia nitrogen, nitric nitrogen or nitrite nitrogen for nitrogenous source grows;
(3) the bacterial genomes DNA extraction kit adopting different expressions gram Bioisystech Co., Ltd in Beijing to produce is carried out extraction to the genomic dna of this bacterium and can be obtained, the 16S rDNA gene sequence characteristic of this bacterial strain CP1: the fragment length that its 16S rDNA has sequence is 1387bp, the sequence BLAST software recorded is compared with the 16SrDNA sequence of known bacterium in GenBank database, finds out the known bacterial strain the highest with CP1 similarity.Adopt NJ method phylogenetic tree construction in MEGA software, to determine the evolutionary degree of bacterial strain CP1.
The result of more than testing shows, the homology of bacterial strain CP1 and Pseudomonas aeruginosa is 99%, and wherein the most similar to type strain Pseudomonas aeruginosa strain N002, homology reaches 99%.Application MEGA software calculates evolutionary distance drafting 16S rDNA phylogenetic tree by Neighbor-Joining method and analyzes.Simultaneously in conjunction with the result of Physiology and biochemistry qualification, determine that this bacterial strain is most possibly Pseudomonas aeruginosa, called after Pseudomonas aeruginosa strain CP1.
Described Pseudomonas aeruginosa CP1, is suitable for growing in temperature 30 ~ 40 DEG C of environment, especially, not to grow and 41 DEG C of growths at 4 DEG C.
Described Pseudomonas aeruginosa is used for the denitrogenation processing of nitrogenous effluent, also may be used for the aerobic denitrification denitration process containing NOx flue gas or waste gas.
Described denitrogenation and denitration process are all carry out under the stimulation of impressed current.
Described strength of current is 5 ~ 15mA.
Described strength of current is 10mA.
In described denitrogenation and denitration process used medium, carbon source is Soduxin, Trisodium Citrate or glucose.
The C/N ratio of described substratum is 4 ~ 6.
Described denitrogenation and denitration process are all carried out under 30 ~ 40 DEG C of temperature condition.
Pseudomonas aeruginosa (Pseudomonas aeruginosa) CP1 that the present invention relates to is comparatively strong to the tolerance of oxygen, has nowadays grasped its some physiological-biochemical characteristics.Under the environment of research high oxygen concentration in denitrating flue gas process, carry out aerobic denitrification preliminary experiment to this bacterium to show, under 30-40 DEG C of temperature, aerobic condition, this bacterium effectively can remove the nitrate nitrogen in nutrient solution, the accumulation of nitrite nitrogen is not detected in reaction process, and showing the optimum pH value of its denitrification in the denitrification process NOx process in early stage is weakly alkaline, insensitive to 0 ~ 11.2mg/L dissolved oxygen concentration.By adopting high-density aerobic denitrifying bacteria cultural method, developing and utilizing impressed current to improve biomass to strengthen the treatment process of aerobic denitrification.After PB experiment and response surface optimization have been carried out to the aerobic denitrification reaction conditions under different strength of current stimulation, be that under the condition of 10mA, biomass and Denitrification rate all reach maximum value at impressed current.
When there is impressed current, the demand of carbon source obviously declines, and C/N is only 4.0 and can realizes complete denitrification and there is the accumulation of nitrite hardly.And initial nitrogen concentration, pH and temperature of reaction there is no too large remarkably influenced in PB test.Therefore, the reaction process of whole extra electric field strengthening aerobic denitrification can bear change in reaction conditions to a certain degree, ensure that CP1 can as the dominant bacteria in treatment system, even if occur that processing load and temperature fluctuation also can keep the nitrate subtractive process of more efficient.Best strength of current is 10mA, and best C/N ratio is 4 ~ 5.
Compared with prior art, tool of the present invention has the following advantages:
When there is not impressed current, this bacterial strain optimal culture conditions is 30 ~ 40 DEG C, C/N >=12.This bacterial strain feature is insensitive to the oxygen of 0 ~ 11.2mg/L concentration, shows that it is under aerobic condition, still can maintain efficient nitrate degradation process.Under the galvanism of 10mA, this bacterial strain can reach more than 99.01% to nitrate nitrogen (in the N content) clearance that starting point concentration is 250mg/L when reducing to 4 ~ 6 by C/N in 16 hours, and there is nitrite accumulation hardly.Show that this bacterial classification is when carbon source is comparatively not enough, denitrification can be completed efficiently with extra electric field is collaborative.
Pseudomonas aeruginosa of the present invention (pseudomonas aeruginosa) CP1, by China typical culture collection center preservation (being called for short CCTCC), its deposit number is CCTCC NO:M 2015197, preservation date is on April 6th, 2015, and preservation address is Wuhan, China university.
Accompanying drawing explanation
Fig. 1 is the phylogeny tree graph of Pseudomonas aeruginosa CP1;
Fig. 2 is the denitrification capability figure of bacterium Pseudomonas aeruginosa CP1 under different carbon source;
Fig. 3 is the denitrification capability figure of bacterium Pseudomonas aeruginosa CP1 under differing temps;
Fig. 4 is the denitrification capability figure of bacterium Pseudomonas aeruginosa CP1 under different strength of current;
Fig. 5 is that no current stimulates different C/N than the denitrification capability figure of lower bacterium Pseudomonas aeruginosa CP1;
Fig. 6 be under constant current different C/N than the denitrification capability figure of lower bacterium Pseudomonas aeruginosa CP1;
Fig. 7 is the denitrification capability figure cultivating bacterium Pseudomonas aeruginosa CP1 under various inlet NO concentration;
Fig. 8 is denitrating flue gas biofiltration device schematic diagram, flue gas 1, gas regulator 2, temperature controlling instruments 3, bio-trickling filter 4, microorganisms reproduction device 5, pump 6, venting port 7.
Embodiment
Embodiment 1: the separation of bacterial classification, Isolation and characterization
This research has biomembranous filler to the triangular flask that the aseptic denitrification nutrient solution of 90mL is housed from taking out a block length in the reactor of above-mentioned biological dripping and filtering system, in an oscillator the microbial film on filler being shaken to coming off, continuing microbial film to shake up to smash after taking out filler.Microbial film after smashing again extracts and adds in new aseptic denitrification liquid nutrient medium, and places it in 30 DEG C, in the gas bath constant-temperature table shaking culture of 160r/min.After 24h, the inoculum size being 10% with volume ratio accesses in the fresh aseptic denitrification nutrient solution of 100mL.Repeat this process and find bacterium liquid well-grown afterwards in three days, and denitrification percent reaches more than 80%.Bacterium liquid is got 1ml in the test tube that 9ml sterilized water is housed, fully obtain 10 after mixing
-1the bacteria suspension of gradient, prepares 10 by that analogy
-2~ 10
-8the bacteria suspension of different gradient, simultaneously according to the bacterial concentration in stoste, chooses 10
-4, 10
-5, 10
-6the bacteria suspension 0.1ml of gradient is applied in the denitrification solid medium developed the color with dibromothymolsulfonphthalein (BTB) prepared, after being inverted into 30 DEG C of fixed temperature and humidity incubator 48h, long in visible flat board have single bacterium colony, due to the effect of BTB developer, the periphery of bacterial colonies that denitrification occurs will produce blue halos, repeat to select also plate streaking and obtain pure bacterium colony three times.Single bacterium colony of the different shape after above-mentioned separation obtains by the multiple sieve stage is inoculated in respectively and is equipped with in the aseptic denitrification substratum of 50ml, shakes cultivation 24 hours under above-mentioned the same terms, gets the bacterium liquid after cultivation and measures OD
600nitric nitrogen in value and nutrient solution and the concentration of nitrite nitrogen, investigate its upgrowth situation in 24h and nitric nitrogen removal effect, finally filter out a strain dominant bacteria, be named as Pseudomonas aeruginosa CP1.
The pure strain obtained after above-mentioned separation and purification is carried out Physiology and biochemistry qualification and 16S rDNA checks order.PCR primer is a pair universal primer 16SF:5 '-AGAGTTTGATCATGGCTCAG-3 ' (upstream primer) and 16SR:5 '-GGTACCTTGTTACGACTT-3 ' (downstream primer).Pcr amplification reaction condition is: denaturation 5min at 95 DEG C, 94 DEG C of sex change 0.5min, 56 DEG C of annealing 0.5min, 72 DEG C of amplification 2min, and sex change is to amplification through 30 circulations, and last 72 DEG C extend 10min.The product obtained carries out 16S rDNA order-checking (the handsome Bioisystech Co., Ltd in Shanghai), and the 16SrDNA length of the bacterial strain CP1 obtained is 1387bp, and sequencing result is shown in sequence table.Sequencing result is carried out tetraploid rice by blast program in GeoBank database, reaches 99% with Pseudomonas aeruginosa homology, called after CP1, adopt NJ method phylogenetic tree construction in MEGA software, see Fig. 1.
After this electroactive domestication is carried out to this bacterium, to screen and carry out enlarged culturing in the CP1 preserved access LB substratum, culture condition is 35 DEG C, 160rpm, access being equipped with in the triangular flask of denitrification liquid nutrient medium of sterilizing according to the inoculum size of 10% (volume ratio) afterwards, culture condition is the same.With this simultaneously, in this triangular flask, (anode is stainless steel to electrode insertion rod, negative electrode is graphite carbon rod), joining power also keeps received current constant for 5mA cultivates 12 hours, by above-mentioned condition, CP1 is accessed in another triangular flask again afterwards, constant current is upgraded to 10mA and cultivates 12 hours, be upgraded to 20mA to constant current by that analogy, so three times repeatedly, and measure the OD of bacterial classification
600value and denitrification efficiency, investigate its denitrification removal efficiency outside under electrical field stimulation and growth performance.Finally can think that this bacterial strain CP1 has electrochemical activity and can well grow under the condition having external electric field to stimulate and carry out aerobic denitrification.
Embodiment 2: Pseudomonas aeruginosa Pseudomonas aeruginosa CP1 denitrification capability detects
1, the denitrification capability of Pseudomonas aeruginosa CP1 under different carbon source
Nitrogenous source (KNO in maintenance denitrification substratum
3) constant when, investigate bacterial strain for the utilization power of this several carbon source of Soduxin, Trisodium Citrate, glucose, sodium acetate and sucrose.Concrete operation step is as follows:
By 1mL bacterial suspension inoculation in 99mL denitrification substratum, following (g/L): the KNO of denitrification medium component
31; Na
2hPO
47.9; KH
2pO
41.5; MgSO
47H
2o 0.1; Trace element solution 2mL/L; PH 7.0 ~ 7.5.Trace element solution composition (g/L): EDTA 50.0; ZnSO
42.2; CaCl
25.5; MnCl
24H
2o 5.06; FeSO
47H
2o 5.0; (NH
4)
6mo
7o
24H
2o 1.1; CuSO
45H
2o 1.57; CoCl
26H
2o 1.61.Add above 5 kinds of carbon sources (concentration is respectively 9.4g/L, 6.79g/L, 4.16g/L, 5.68g/L, 3.95g/L) respectively, make C/N be 12, in 30 DEG C, shaking culture under 160rpm condition.
The sample getting 0h and 24h detects nitrate radical, nitrite anions, OD
600content, result as shown in Figure 2, there are some difference in the impact of different carbon source on bacterial strain CP1 denitrification effect, bacterial strain with Soduxin and Trisodium Citrate for carbon source time, bacterial strain CP1 denitrification effect is best, denitrification percent reaches 95.82% and 92.31% respectively, there is not the accumulation of nitrite simultaneously; Next is using glucose as carbon source, denitrification percent is 87.34%, have decline slightly relative to first two carbon source, this may be because the organic acid that glucose needs to be hydrolyzed to molecular weight could be utilized by Institute of Micro-biology, so effect is not as Soduxin and Trisodium Citrate.
2, under differing temps, the denitrification capability of bacterium Pseudomonas aeruginosa CP1 compares;
The identical denitrification liquid nutrient medium of same 1% volume ratio bacterium liquid inoculation is had to be put in the constant-temperature shaking incubator of 20 DEG C, 25 DEG C, 30 DEG C, 37 DEG C, 40 DEG C and 45 DEG C respectively, after all cultivating 24h with the rotating speed of 160r/min, survey its thalli growth absorbance (OD
600), NO
3 -and NO
2 -content, to investigate at different temperatures, the upgrowth situation of bacterial strain CP1 and denitrification effect.Wherein, denitrification liquid nutrient medium take Soduxin as carbon source, and other components are consistent with above experiment.If no special instructions, following examples are all sole carbon source with Soduxin, and concrete usage quantity is adjusted with C/N (element mass ratio).
As shown in Figure 3, when temperature is 20 DEG C, the growth of bacterial strain CP1 is suppressed experimental result, and denitrification percent is only 21.79%, and the accumulation rate of nitrite is higher, reaches 20.88%.When temperature is increased to 25 DEG C, although OD
600had rising slightly with denitrification percent, but the accumulation rate of nitrite also adds thereupon, illustrate under cryogenic, bacterium can not utilize extraneous nutritive substance normally, makes its growth velocity and nitric efficiency all lower; Along with the continuation of temperature raises, thalli growth is good, and denitrification percent also raises gradually, when temperature is elevated to 30 DEG C, and the OD of bacterial strain
600value reaches the highest, and denitrification percent is also elevated to 97.54%, and substantially without the accumulation of nitrite.When temperature is elevated to 40 DEG C always, thalline OD
600just some reduces slightly for value and denitrification percent, and denitrification percent still can remain on about 95%, illustrates that bacterial strain CP1 is at 30 ~ 40 DEG C of equal energy well-growns, and has good denitrification effect; Therefore in subsequent experimental, the temperature of reaction is decided to be 35 DEG C.When temperature is 45 DEG C, the OD of bacterium liquid
600value declines slightly to some extent, but its denitrification percent declines clearly, reduce to 8.5%, major cause is that accumulation rate of nitrite nitrogen is very high, not consumed by thalline further after in water, nitric nitrogen major part changes into nitrite, may be due under the high temperature conditions, although the activity of the growth of bacterium and nitrate reductase just declines slightly, but the nitrite reductase in bacterial strain body is subject to obvious suppression, the nitrite in water can not be converted further.
3, the denitrification capability of Pseudomonas aeruginosa CP1 under different strength of current incentive condition
Inoculum size by 1% is by bacterium liquid inoculation denitrification substratum, and in denitrification substratum, component is constant, the strength of current in regulating direct supply to make to react is respectively 5,10,15,20mA, temperature is 35 DEG C, 160rpm shaking culture.This experiment can show in strength of current situation, the denitrification of bacterial strain CP1, to check the domestication result of early stage to this bacterial strain.
The sample getting 0h and 24h detects nitrate radical, nitrite anions content, and as shown in Figure 4, the denitrification percent of bacterial classification all can reach more than 95% to result.Best strength of current is 10mA, and denitrification percent can reach 99%.Illustrate that this bacterial strain has good chemical property, this makes this bacterial classification can have Application Areas widely in Practical Project.
4, different C/N (C, N element mass ratio) is than the electricity of lower Pseudomonas aeruginosa CP1
Stream catalytic reduction performance
Investigate when additional constant current I=10mA, C/N is than when being respectively 2,4,6,8, the denitrification capability of Pseudomonas aeruginosa CP1, and the difference of the denitrification capability performance of contrast different C/N ratio (1,3,6,9,12,15) when not having extra electric field.
By through seed culture medium enlarged culturing bacterium liquid with 1% volume ratio be inoculated in the liquid nutrient medium of above-mentioned different C/N ratio, shaking culture under 160rpm rotating speed, 30 DEG C of temperature condition.Denitrification medium component is described above, adds Soduxin as sole carbon source.Result as shown in Figure 5,6, the nitrate radical clearance of bacterial strain under the condition not having electrical field stimulation is the highest needs to reach about 12, but under the effect that there is 10mA cathodic current, C/N declines to a great extent to 4 ~ 6 can complete removing and there is not the accumulation of nitrite of nitrate.Therefore, this bacterium has very good electroactive, can be good at adapting to the environment of extra electric field and aerobic denitrification is greatly promoted by electric current, compared with the reaction conditions stimulated with no current, impressed current can make this bacterial strain greatly reduce carbon source cost in denitrification process, and this makes this bacterium more extensive in industrial denitration application.
5, in culturing process under no current incentive condition different rotating speeds Pseudomonas aeruginosa
The denitrification capability of CP1 compares
Inoculum size by 1% is by bacterium liquid inoculation denitrification substratum, in denitrification substratum, component is constant, regulate shaking speed to be respectively 0,40,80,120,160,180,200rpm, the process that corresponding respectively anoxic and oxygen concn progressively rise, temperature is 30 DEG C, shaking culture.This experiment can show in different oxygen concentrations situation, the denitrification of CP1.
The sample getting 0h and 24h detects nitrate radical, nitrite anions content, and result is as shown in table 1, and when nitrate initial in nutrient solution is about 140mg/L, the denitrification percent of bacterium liquid all can reach about 95%.Illustrate that this bacterial strain has good oxytolerant performance, this makes this bacterial classification can have Application Areas widely in Practical Project.
Table 1 different rotating speeds is on the impact of bacterial strain CP1 denitrification effect
Embodiment 3: the no current denitrating flue gas experiment of Pseudomonas aeruginosa Pseudomonas aeruginosa CP1
The experiment of NO is removed under utilizing bio-trickling filter to carry out hot environment.Biological filter system is as Fig. 8.In biological filtering tower combined working biofilm phase liquid storage tank, nutrition composition is with the denitrification substratum of embodiment 2, by microbionation biofilm in bio-trickling filter, investigates the effect that this system removes NOx in flue gas under long-time running situation.First by after enlarged culturing bacterial strain CP1 access circulation fluid reservoir in.What biofilm process adopted is the mode that interval soaks filler, specifically comprise two kinds of modes: by circulation fluid by pump delivery to the top of tower, get back in reservoir after whole filter tower again, in reservoir, put into aeration head to strengthen the dissolved oxygen of circulation fluid simultaneously, utilize circulation fluid to carry out biofilm with the contact of filler in flow process; Circulation fluid is poured in bio-trickling filter and also all fillers are soaked, aeration head is installed in the bottom of tower aeration is carried out to circulation fluid.The periodic conversion of two kinds of biofilm modes within one day, drips filter to soak for one day.The circulation fluid all more renewed every day, and the bacterium liquid newly cultivated is accessed with 10% ratio, thalline quantity also can be avoided too much to form biological flock so simultaneously, and non-cohesive on filling surface.After the biofilm of 20 days, filling surface there is the biofilm formation of one deck brown color, and under the condition of sterile liquid access in circulation fluid, NO
3 -removal efficiency, close to 100%, illustrates biofilm success, starts to pass into flue gas.After starting to pass into industrial smoke, in circulation fluid, no longer access bacterial classification, and add NO wherein
3 -concentration reduce gradually, make the change of microorganism to nitrogenous source kind have an adaptive process.The flow of flue gas is 1L/min, and the corresponding empty bed residence time is 1.5min, and oxygen concn (volume ratio) is 8%, and in air inlet, NO concentration is by 100ppm (133.9mg/m
3) start to raise gradually, circulation fluid is changed once to maintain microbial growth and activity for every two days, and its content is except nitrogenous source changes, and all the other substances content are constant.
Research object using the clearance of NO as system removal effect.Bio-trickling filter has run one month continuously under various inlet NO concentration, and its operation conditions as shown in Figure 7.
When ventilating at first, in circulation fluid, add 0.5g/L nitrogenous source KNO
3, reduce gradually every day subsequently, from the 9th day, no longer add KNO
3.As seen from the figure, at the first day of system cloud gray model, air inlet NO concentration is 100ppm, and clearance only has 46.5%, the adaptation changed denitrification approach along with microbial film, and the clearance of NO also improves thereupon, is increased to 62.1% at the 5th day NO clearance.Subsequently, the inlet gas concentration of NO increased about 100ppm, NO clearance and also continues along with rising every five days.When air inlet NO concentration is increased to about 500ppm, clearance maintains about 90%, reaches 91.3% time the highest.The 25th day time, the clearance of NO is down to 81.7% from 90.9% suddenly, and this may be that clearance returns to 90.5% again gradually subsequently because NO load increases suddenly.Long-time running situation illustrates that this bio-trickling filter has good removal effect to NO under aerobic conditions.
This embodiment proves under aerobic concentration conditions, and strain Pseudomonas aeruginosa CP1 has efficient aerobic denitrification capability, can NO in effective elimination flue gas.
Embodiment 4: Pseudomonas aeruginosa Pseudomonas aeruginosa CP1 combined with electrochemical means are used for mariculture denitrogenation of waste water
This bacterium is used for the denitrogenation processing of certain marine site breeding wastewater aerobic section.The correlation water index entering aerobic section is that water quality contains nitrite nitrogen 1.27mg/L, and ammonia nitrogen 44.83mg/L, water temperature is at about 30 DEG C.Treating processes is, unloading phase by 1% volume ratio CP1 bacterium liquid is added in aerobic reaction pond.With 24 hours for the cycle, stimulate (10mA) 12h recession removing DC source at enforcement constant current, circulation repeatedly.
After stabilization, ammonia nitrogen and the nitrite concentration of water outlet is measured.In the effect of constant current, nitrite clearance is basically stable at about 95%, aqueous concentration is lower than 0.079mg/L, ammonia nitrogen aqueous concentration is 0.884mg/L, and not apply electrical field stimulation as a control group, its water outlet nitrite concentration is 0.283mg/L, ammonia nitrogen aqueous concentration is 4.96mg/L, can find out, the effect of electric field makes bacterial strain CP1 treatment stage low concentration ammonia nitrogen and the denitrification rate in nitrite stage are obviously greater than and do not apply electrical field stimulation.Therefore, the interpolation of this bacterium and the applying of extra electric field significantly improve denitrification ability, can be good at the water quality adapting to the low C/N ratio of breeding wastewater.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a Pseudomonas aeruginosa, it is characterized in that, described Pseudomonas aeruginosa (pseudomonasaeruginosa) CP1, by China typical culture collection center preservation, its deposit number is CCTCC NO:M 2015197, and preservation date is on April 6th, 2015.
2. the application of Pseudomonas aeruginosa described in claim 1, is characterized in that, this Pseudomonas aeruginosa is used for the denitrogenation processing of nitrogenous effluent, or for the aerobic denitrification denitration process containing NOx flue gas or waste gas.
3. application according to claim 2, is characterized in that, described denitrogenation and denitration process are all carry out under the stimulation of impressed current.
4. application according to claim 3, is characterized in that, described strength of current is 5 ~ 15mA.
5. application according to claim 4, is characterized in that, described strength of current is 10mA.
6. application according to claim 5, is characterized in that, described denitrogenation and denitration process are all carried out under 30 ~ 40 DEG C of temperature condition.
7. the application according to Claims 2 or 3 or 4 or 5 or 6, is characterized in that, the carbon source in described denitrogenation and denitration process used medium is Soduxin, Trisodium Citrate or glucose.
8. application according to claim 7, is characterized in that, the C/N ratio of described substratum is 4 ~ 6.
9. application according to claim 8, is characterized in that, described medium component is as follows: carbon source is 2.00 ~ 8.735, KNO
31; Na
2hPO
47.9; KH
2pO
41.5; MgSO
47H
2o 0.1, unit g/L; Trace element solution 2mL/L; PH 7.0 ~ 7.5.
10. application according to claim 9, is characterized in that, described trace element solution composition is as follows: EDTA50.0; ZnSO
42.2; CaCl
25.5; MnCl
24H
2o 5.06; FeSO
47H
2o 5.0; (NH
4)
6mo
7o
24H
2o 1.1; CuSO
45H
2o 1.57; CoCl
26H
2o 1.61, unit g/L.
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