CN103484413A - Pseudomonas putida strain and application thereof - Google Patents
Pseudomonas putida strain and application thereof Download PDFInfo
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- CN103484413A CN103484413A CN201310479966.3A CN201310479966A CN103484413A CN 103484413 A CN103484413 A CN 103484413A CN 201310479966 A CN201310479966 A CN 201310479966A CN 103484413 A CN103484413 A CN 103484413A
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Abstract
The invention aims to provide a Pseudomonas putida strain and application thereof in aquiculture and domestic sewage treatment. The Pseudomonas putida NI3 strain is collected at China Center for Type Culture Collection in Wuhan on September 29th, 2013; and the collection number is CCTCC NO:M2013457. The Pseudomonas putida NI3 strain has the advantages of aerobic denitrification property and simple nutritional requirements, has the capacity for obviously lowering nitrate nitrogen and nitrite nitrogen in the water body, and meanwhile, can effectively lower the ammonia nitrogen content in domestic sewage. The Pseudomonas putida NI3 strain can be developed into aquatic microecological preparations and sewage treating agents, and has wide application prospects.
Description
Technical field
The invention belongs to technical field of microbe application, be specifically related to a kind of pseudomonas putida strain and application thereof.
Background technology
In recent decades, China's culture fishery fast development, intensive, high-density breeding scale expanding day.But due to accumulation in aquaculture water a large amount of residual bait, ight soil and animals and plants corpse, cause water body nitrogen element content severe overweight, the eutrophication aggravation, make the breeding ecological environment be destroyed, and fish and shrimp diseases takes place frequently, and finally causes huge financial loss.Research shows, the pathogenic main contributor of aquatic animal is the severe overweight of aquaculture water Central Asia nitrate nitrogen content, and the ferrohemoglobin of nitrite in can oxidation fishes and shrimps body, make it become methemoglobin, cause it to lose the ability of transportation oxygen, finally cause fishes and shrimps death.So the nitrite nitrogen of controlling in water body becomes one of key of large-scale cultivation.
With the physics denitrogenation, compare, biological denitrificaion has its unique advantage: non-secondary pollution, denitrogenation is thorough, economic security etc.Because bio-denitrification technology has that cost is low, easy to operate, the qualified discharge reliability is strong and the advantage such as non-secondary pollution, become gradually the major way that food industrial wastewater is processed.Aerobic denitrification is that the bio-denitrification technology with unique advantage is compared in anoxic denitrification denitrogenation that propose in recent years and traditional: on the one hand, carry out denitrification under aerobic conditions, nitrification and denitrification can be carried out simultaneously in a reactor, and the equipment and manipulation cost declines to a great extent; On the other hand, the product of nitrification can avoid suppressing nitrification directly as the substrate of denitrification, the aggravation of nitrification and denitrification process.Therefore, aerobic denitrification more and more receives people's concern, also many for the screening study of aerobic denitrifying bacteria both at home and abroad, but its achievement still can not meet industrial actual demand far away.By the screening and separating aerobic denitrifying bacteria, and various growth characteristics and the Denitrification Characteristics of aerobic denitrifying bacteria are furtherd investigate, the denitrogenation processing of breeding wastewater, sanitary wastewater and industrial sewage is had to important theory value and practical significance.
Summary of the invention
The present invention is for solving the prior art problem, one strain pseudomonas putida and the application in aquaculture and sanitary sewage disposal thereof are provided, this bacterial strain has the performance of aerobic denitrification, can significantly reduce nitric nitrogen, nitrite nitrogen and ammonia-state nitrogen in water body, thereby make up the deficiencies in the prior art.
Pseudomonas putida NI3(Pseudomonas putida NI3 of the present invention) bacterial strain, be preserved in the Chinese Typical Representative culture collection administrative center of Wuhan, China Wuhan University on September 29th, 2013, deposit number is CCTCC NO:M2013457.
Pseudomonas putida NI3(Pseudomonas putida NI3 of the present invention) bacterial strain is for water body purification.
Pseudomonas putida NI3 bacterial strain of the present invention is made microbe additive with the form of bacterium powder.
Pseudomonas putida NI3 bacterial strain of the present invention has the performance of aerobic denitrification, can degradable concentration in its 8h the nitrite nitrogen that is 5-10mg/L, can effectively reduce the content of nitric nitrogen and nitrite nitrogen in breeding wastewater; This bacterial strain also has obvious Degradation to the ammonia-state nitrogen in sanitary sewage, and its degradation rate of 7 days can reach 64.95%.Therefore, this bacterial strain is expected to be developed as aquatic products probiotics and sewage-treating agent, and effect is remarkable.
Embodiment
Below to pseudomonas putida NI3(Pseudomonas putida NI3 of the present invention) bacterial strain and effect thereof, application be described in detail.
Separation screening and the evaluation of embodiment 1 bacterial strain
1, the component of substratum used in the present invention is as follows:
Primary dcreening operation BTB substratum: extractum carnis 3.0g; Peptone 10.0g; Sucrose 15.0g; Sodium-chlor 20.0g; Teepol1.0g; Bromothymol blue 0.00006g; Water 1000ml.
Nutrient broth medium: peptone 10g; Beef powder 3g; Sodium-chlor 5.0g; Glucose 1g; Water 1000ml.
Screening culture medium: NaNO
20.1g(about 20mg/L NO
2-N); Glucose 5.0g; K
2hPO
41.0g; NaCl2.0g; MgSO
4.7H
2o0.5g; Water 1000ml.
2, screening method
Adopt the gradient dilution method by after the mud sample dilution, get 0.1mL and evenly coat the BTB media surface, insert constant incubator, after cultivating 2～3d under 30 ℃, with the transfering loop picking, make substratum on every side single bacterium colony of blue haloing occur, carry out separation and purification and be the primary dcreening operation bacterial strain.Altogether picking the single bacterium colonies of 13 strains, called after NK1, Y1-13, BX, BY, N1, N4, NI1, NI2, NI3, WU1, WU2, WU3, XAO1 respectively.Inoculation primary dcreening operation bacterial strain is to nutrient broth medium, carry out liquid culture 24h under 30 ℃, 205r/min condition after, through centrifugal resuspended, inoculum size with 1% is inoculated in screening culture medium, carry out the shaking flask processing, sampling and measuring experimental system Central Asia nitrate after 24h under 30 ℃, 205r/min condition.Wherein, nitrite nitrogen NO
2 –n content is by diazonium-azo spectrophotometry, and biomass is measured by coating method, and measurement result is as shown in table 1.
Table 1: NO in each strain culturing 24h secondary fermentation liquid
2 -n content
| ? | Initial NO 2-N value (mg/L) | 24hNO 2-N value (mg/L) | Degradation rate |
| NK1 | 22.4 | 21.4 | 4.5% |
| Y1-13 | 22.0 | 22.0 | 0% |
| N1 | 22.2 | 21.6 | 2.7% |
| N4 | 22.4 | 7.8 | 65.2% |
| BX | 22.6 | 15.8 | 30.1% |
| BY | 22.1 | 21.3 | 3.6% |
| NI1 | 22.4 | 0 | 100% |
| NI2 | 22.8 | 22.7 | 0.4% |
| NI3 | 22.4 | 5.2 | 76.8% |
| WU1 | 22.6 | 0 | 100% |
| WU2 | 22.2 | 21.8 | 1.8% |
| WU3 | 22.0 | 22.0 | 0% |
| XAO1 | 22.1 | 15.7 | 29.0% |
| Blank | 22.2 | 22.2 | 0% |
As can be seen from Table 1, the NI3 bacterial strain has very strong degradation capability to nitrite nitrogen, and degradation rate is 76.8%.
3, identify
1) colony morphology characteristic: the NI3 bacterial strain is cultivated on nutrient agar to 18-24 hour, its colony morphology characteristic is as follows:
The NI3 bacterium colony is rendered as canescence, opaque, surface wettability, non-wrinkled, diameter 1-2mm, and its cell of microscopy is rod-short.
2) utilize test kit to extract the genomic dna of NI3 bacterial strain, utilize round pcr its 16S rRNA sequence that increases.The 16S rRNA sequence similarity degree of pseudomonas putida of finding NI3 bacterial strain and announcement by the BLAST compare of analysis is the highest, consistent with the biochemical identification result.By NI3 bacterial strain called after pseudomonas putida NI3(Pseudomonas putida NI3).
Pseudomonas putida NI3(Pseudomonas putida NI3) bacterial strain, be preserved in the Chinese Typical Representative culture collection administrative center of Wuhan, China Wuhan University on September 29th, 2013, deposit number is CCTCC NO:M2013457.
The degradation effect of embodiment 2NI3 bacterial strain to nitrite nitrogen
1, substratum:
Activation medium: nutrient broth;
Screening culture medium: NaNO
20.1g(about 20mg/L NO
2-N); Glucose 5.0g; K
2hPO
41.0g; NaCl2.0g; MgSO
4.7H
2o0.5g; Water 1000ml.
Detect substratum:
High density nitrite nitrogen substratum: NaNO
20.05g(about 10mg/L NO
2-N); Glucose 5.0g; K
2hPO
41.0g; NaCl2.0g; MgSO
4.7H
2o0.5g; Water 1000ml.
Lower concentration nitrite nitrogen substratum: NaNO
20.025g(about 5mg/L NO
2-N); Glucose 5.0g; K
2hPO
41.0g; NaCl2.0g; MgSO
4.7H
2o0.5g; Water 1000ml.
2, experimental technique:
NI3 bacterial strain list colony inoculation on the picking flat board, to the test tube that the 8ml nutrient broth is housed, is placed in 30 ℃, 205r/min shaking table and is activated; 5ml bacterium liquid is transferred in the aseptic centrifuge tube of 10ml, the centrifugal 10min of 4000r/min, and clean twice with physiological saline, obtain bacteria suspension; Get 1ml bacterial suspension inoculation (inoculum size approximately 1%) to the 250ml triangular flask that about 100ml screening culture medium is housed, three of each treatment group are parallel, are placed in 30 ℃, 205r/min shaking table and cultivate; Detect bacterium amount and NO in 4h, 8h, 16h and 24h sampling respectively
2--N concentration; Get about 3ml nutrient solution with disposable syringe, wherein the 1.5ml nutrient solution is transferred in centrifuge tube, by nutrient agar plate meter total number of bacterial colony; 0.22 μ m membrane filtration for all the other 1.5ml nutrient solutions, obtain sterile liquid and be placed in centrifuge tube, uses wherein nitrite nitrogen NO of diazonium-azo spectrphotometric method for measuring
2--N concentration.
Table 2:NI3 bacterial strain in 24h in to the degraded situation of nitrite nitrogen
From the results shown in Table 2, the nitrite nitrogen that NI3 bacterial strain of the present invention can degradable concentration in 8h is 5-10mg/L, have the degradation efficiency of outstanding nitrite nitrogen.
The treatment effect of embodiment 3NI3 bacterial strain to aquiculture waste water
The NI3 bacterial strain of the present invention's screening is activated, uses the nutrient broth enlarged culturing, and through liquid state fermentation, centrifugal collection bacterium mud, add starch carrier, dries the bacterium powder of making 10,000,000,000/g.Breeding wastewater is taken from loach pool breeding wastewater, its nitrate nitrogen content is 45.62 ± 1.15mg/L after measured, nitrite nitrogen is 0.58 ± 0.03mg/L, and experiment container is plastic tank, and the experiment water body is 10L, experimental group is added the bacterium powder of above three kinds of bacterium according to mass ratio 50ppm, control group does not add the bacterium powder, experimental group and control group all arrange 3 parallel, the uninterrupted aeration of experimental session, each container aeration rate is adjusted to unanimously, and experimental temperature is 28 ℃ of constant temperature.Measure the nitric nitrogen NO in each experimental group and control group water body when 3d
3 –n and nitrite nitrogen NO
2 –the content of N, the results are shown in Table 3.
Table 3: the content of nitric nitrogen and nitrite nitrogen in each treatment group of breeding wastewater
| Group | NO 3 –N（mg/L） | NO 2 –N（mg/L） |
| The 24h control group | 22.8 | 1.2 |
| The NI3 bacterial strain | 20.4 | 0.8 |
| The 48h control group | 10.3 | 0.9 |
| The NI3 bacterial strain | 2.7 | 0.2 |
From the results shown in Table 3, NI3 bacterial strain provided by the invention can effectively reduce the content of nitric nitrogen and nitrite nitrogen in breeding wastewater.
Embodiment 4 sanitary sewage water treatment effects
By the activation of NI3 bacterial strain, enlarged culturing, through liquid state fermentation, centrifugal collection bacterium mud, add starch carrier, dries the bacterium powder of making 10,000,000,000/g.Sanitary wastewater is taken from waterways, Licang District Li Cun food market, Qingdao City, get its supernatant liquor after staticly settling, measuring its ammonia nitrogen content is 206.54mg/L, and experiment container is plastic tank, and the experiment water body is 10L, experimental group is added few NI3 bacterium powder according to mass ratio 10ppm, control group does not add the bacterium powder, experimental group and control group all arrange 3 parallel, the uninterrupted aeration of experimental session, each container aeration rate is adjusted to unanimously, and experimental temperature is 28 ℃ of constant temperature.The content of ammonia-state nitrogen measure respectively each experimental group and control group water body when the 4th day and the 7th day in
Table 4: the clearance of ammonia-state nitrogen in each treatment group of sanitary sewage
From the results shown in Table 4, NI3 bacterial strain provided by the invention has obvious Degradation to the ammonia-state nitrogen in sanitary sewage, and its degradation rate of 7 days can reach 64.95%.
In sum, the pseudomonas putida NI3 bacterial strain that the present invention screens can effectively degrade nitric nitrogen and nitrite nitrogen in water body, also have significant degradation effect for the ammonia-state nitrogen in sanitary sewage.And this bacterium is aerobic type bacterial strain, less demanding to living environment, therefore can be widely used in the processing of breeding wastewater or sanitary sewage.
Claims (5)
1. a pseudomonas putida NI3(Pseudomonas putida NI3) bacterial strain, its deposit number is CCTCC NO:M2013457.
2. the pseudomonas putida NI3(Pseudomonas putida NI3 claimed in claim 1) application of bacterial strain in water body purification.
3. application as claimed in claim 2, is characterized in that described water body is aquaculture system or sanitary sewage.
4. the goods for water body purification, is characterized in that, described goods are for pseudomonas putida NI3(Pseudomonas putida NI3 claimed in claim 1) microbial preparation for preparing of bacterial strain.
5. goods as claimed in claim 4, is characterized in that described microbial preparation is the bacterium powder.
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| CN104232543A (en) * | 2014-09-17 | 2014-12-24 | 常州大学 | Method for removing nitrate nitrogen and total phosphorus in printing and dyeing wastewater by denitrifying poly-phosphorus accumulating organisms (B8) |
| CN105543152A (en) * | 2016-02-24 | 2016-05-04 | 长治学院 | Pseudomonas putida and application thereof to promotion of growth of cercidiphyllum japonicum |
| WO2016179390A1 (en) * | 2015-05-05 | 2016-11-10 | BiOWiSH Technologies, Inc. | Microbial compositions and methods for denitrification at high dissolved oxygen levels |
| CN106967640A (en) * | 2017-04-11 | 2017-07-21 | 上海莱泰生物科技有限公司 | A kind of preparation method of deodorizing microorganism microbial inoculum |
| CN107686820A (en) * | 2017-09-11 | 2018-02-13 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of aerobic denitrifying bacteria and its application in water body denitrification |
| US10252928B2 (en) | 2014-10-31 | 2019-04-09 | BiOWiSH Technologies, Inc. | Method for reducing cyanuric acid in recreational water systems |
| US10336636B2 (en) | 2015-11-02 | 2019-07-02 | BiOWiSH Technologies, Inc. | Methods for reducing evaporative loss from swimming pools |
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| US10988728B2 (en) | 2013-05-20 | 2021-04-27 | BiOWiSH Technologies, Inc. | Microbial-based waste water treatment compositions and methods of use thereof |
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| US10252928B2 (en) | 2014-10-31 | 2019-04-09 | BiOWiSH Technologies, Inc. | Method for reducing cyanuric acid in recreational water systems |
| WO2016179390A1 (en) * | 2015-05-05 | 2016-11-10 | BiOWiSH Technologies, Inc. | Microbial compositions and methods for denitrification at high dissolved oxygen levels |
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