CN103275908B - Low-temperature denitrification pseudomonas fluorescens - Google Patents
Low-temperature denitrification pseudomonas fluorescens Download PDFInfo
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Abstract
The invention discloses low-temperature denitrification pseudomonas fluorescens and relates to pseudomonas fluorescens. The low-temperature denitrification pseudomonas fluorescens are pseudomonas fluorescens WSW-1001 and are collected in China General Microbiological Culture Collection Center (CGMCC), the collection number is CGMCC No.7476, the collection date is April 15th, 2013, and the collection address is No.3, No.1 Courtyard, Beichen Road, Chaoyang District, Beijing. The low-temperature denitrification pseudomonas fluorescens are heterotrophic nitrification aerobic denitrifiers, can be used for degrading NH4+-N, NO--N and NO--N (0.25 mg/L-1314.1 mg/L) with a wide concentration range, can be applied to low-nitrogen pollution source water denitrification, medium- and high-concentration wastewater denitrification and eutrophic water body treatment, and have an important meaning on low-nitrogen pollution source water denitrification, water eutrophication control and biological environment modification.
Description
Technical field
The present invention relates to a strain Pseudomonas fluorescence.
Background technology
Along with society and economic fast development, the polluted by nitrogen in China's water surrounding is on the rise.Sanitary sewage, trade effluent, breeding wastewater and farmland nitrogenous fertilizer are the main nitrogen pollution sources of water surrounding.Water body polluted by nitrogen not only causes serious environmental pollution and financial loss, even threatens human health and ecological safety.Microbial denitrogenation is considered at present most economical effective and the most promising water body and denitrogenates technology because having the advantages such as high-level efficiency, low cost, non-secondary pollution.
Nitrogen in polluted-water mainly comprises NH
4 +-N, NO
3 --N and NO
2 --N inorganic nitrogen and organonitrogen.Traditional biological denitrificaion comprises ammonification, nitrification and denitrification process, has been worked in coordination with by different oxygen, nitrification and denitrification bacterium, and the organonitrogen in sewage is ammonia nitrogen by heterotroph microbiological oxidation decomposition and inversion, is then NO by Autotrophic nitrification bacterium by mineralized nitrogen
3 --N and NO
2 --N, finally by denitrifying bacterium by NO
3 --N and NO
2 --N is converted into N
2.Recent study shows that some Heterotrophic nitrification aerobic denitrifying bacterias have synchronous nitration and denitrification function, can complete independently denitrogenation whole process.
Biological denitrificaion efficiency is limited by temperature and nitrogen concentration, and general bacterial strain is narrower to the subject range of nitrogen concentration, declines at low temperature (10 DEG C of <) denitrification ability, is not suitable for low-temperature water source denitrogenation of water.
Summary of the invention
The object of the invention is to provide a strain low-temperature denitrification Pseudomonas fluorescence.
One strain low-temperature denitrification Pseudomonas fluorescence, it is Pseudomonas fluorescens (Pseudomonasfluorescens) WSW-1001, in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC No.7476, preservation date is on April 15th, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
Pseudomonas fluorescens (Pseudomonas fluorescens) WSW-1001, the morphological specificity of bacterial strain: it is rod-short, without gemma, size is 0.2～0.4 μ m × 1.0～1.3 μ m; Bacterium colony circle, smooth, neat in edge, on NYD substratum, bacterium colony is orange-yellow, in minimal media liquid, cultivates and can produce ceroid.
Pseudomonas fluorescens (Pseudomonas fluorescens) WSW-1001, the physiological and biochemical property of bacterial strain: it is Gram-negative aerobic bacteria, oxidase positive, the catalase positive, the arginine dihydrolase positive, utilizes dextrose plus saccharose, V.P feminine gender, M.R feminine gender, liquefy gelatin; Growth temperature is 4～37 DEG C, and optimum growth temperature is 15～20 DEG C, and growth pH value is 4.0～10.0, and the most suitable growth pH value is 7.0～8.0, C/N=1～20, the suitableeest C/N=10.
Pseudomonas fluorescens (Pseudomonas fluorescens) WSW-1001, the molecular biology identification result of bacterial strain: analyze by 16S rDNA sequence alignment, the most approaching with the 16S rDNA sequence of Pseudomonas fluorescence, similarity is 99%; In conjunction with morphological features, growth characteristics, physio-biochemical characteristics determine that bacterial strain WSW-1001 is Pseudomonas fluorescens (Pseudomonas fluorescens).
The present invention's one strain low-temperature denitrification Pseudomonas fluorescence, it is Pseudomonas fluorescens (Pseudomonasfluorescens) WSW-1001, in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC No.7476, preservation date is on April 15th, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
Low-temperature denitrification Pseudomonas fluorescence in the present invention can be under 8 DEG C of conditions the NH of the low middle and high concentration in efficient degradation water
4 +-N, NO
3 --N and NO
2-N, the NH of 24h to low middle and high concentration
4 +-N (0.25-4.53mg/L, 48.34-365.32mg/L, 522.13-1314.24mg/L) degradation rate is 78.4-72.3%, 71.4%-50.7%, 23.2%-12.4%, to the NO of low middle and high concentration
3 -the degradation rate of-N (0.24-5.08mg/L, 50.41-352.35mg/L, 513.44-1201.8mg/L) is 83.3%-81.2%, 81.8%-66.9%, 40.5%-16.4%; To the NO of low middle and high concentration
2 -the degradation rate of-N (0.27-5.24mg/L, 49.83-308.92mg/L, 518.67-1084.45mg/L) is 84.4%-83.2%, 80.2%-76.5%, 34.5%-18.9%.
Low-temperature denitrification Pseudomonas fluorescence in the present invention is Heterotrophic nitrification aerobic denitrifying bacteria, can under 8 DEG C of conditions, synchronously carry out nitrated and aerobic anti-nitration reaction, can independently efficiently complete denitrogenation whole process.Initial ammonia nitrogen is 48.34mg/L, 8 DEG C, 24h can be that nitrogen is got rid of by 47.8% mineralized nitrogen, residual ammonia nitrogen 18.81 ± 0.15mg/L, there is a small amount of nitric nitrogen (5.04 ± 0.12mg/L), nitrite nitrogen (0.002 ± 0.001mg/L) and the N of trace
2o (0.003 ± 0.0013mg/L) produces, and approximately 20.9% nitrogen becomes cellular material.
Low-temperature denitrification Pseudomonas fluorescence in the present invention, the NH of the extensive concentration range of can degrading
4 +-N, NO
3 --N and NO
2 --N (0.25mg/L-1314.1mg/L), can be applicable to the source water denitrogenation of low polluted by nitrogen, denitrogenation of waste water and the eutrophication water processing of senior middle school's concentration, to low temperature (10 DEG C of <) source water denitrogenation, body eutrophication is administered and ecotope is repaired significant.
Brief description of the drawings
Fig. 1 is the colonial morphology figure of low-temperature denitrification Pseudomonas fluorescence on NYD substratum in embodiment one;
Fig. 2 is the phyletic evolution tree graph of low-temperature denitrification Pseudomonas fluorescence in embodiment one.
Embodiment
Embodiment one: present embodiment one strain low-temperature denitrification Pseudomonas fluorescence, it is Pseudomonas fluorescens (Pseudomonas fluorescens) WSW-1001, in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC No.7476, preservation date is on April 15th, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
A strain low-temperature denitrification Pseudomonas fluorescence in present embodiment, its separation screening from Heilongjiang Province's Songhuajiang River Water in March obtains, screening process is as follows: get 100mL river and put into 500mL triangular flask, add 50mg ammonium sulfate, 300mg sodium-acetate and 5mL trace element solution, 8 DEG C, 160r/min enrichment culture 5 days, getting 1mL enrichment culture liquid adds in the triangular flask that contains 50mL basic culture solution, 3 Duplicate Samples, 8 DEG C, 160r/min concussion is cultivated 3 days, by nutrient solution doubling dilution to 1000 times, getting 100 μ L nutrient solutions is coated on the solid plate containing minimum medium, flat board is placed in 8 DEG C of incubators and is cultivated until grow single bacterium colony, to after single bacterium colony line purifying, carry out the test of low temperature ammonia nitrogen degradation, can obtain low temperature ammonia nitrogen degradation bacterial strain, wherein every 1L basic culture solution is made up of the distilled water of 0.5g ammonium sulfate, 5g sodium-acetate, 50mL trace element solution and surplus, 121 DEG C of sterilizing 20min, described trace element solution (g/L): phosphorus 5g acid hydrogen dipotassium, 2.5g magnesium sulfate, 2.5g sodium-chlor, 0.05g ferric sulfate, 0.05g manganous sulfate and 1L distilled water water, minimum medium is added with agar and is formed by basic culture solution.
Obtained bacterial strain is carried out to form, cultural characters and physio-biochemical characteristics qualification, and concrete steps are as follows: the thalline that low temperature is cultivated 48 hours carries out under 1000 power microscopes, observing thalline size and thalline color after gramstaining; To after bacterium liquid doubling dilution, be coated on minimum medium and NYD culture medium flat plate, observe colonial morphology and color; Liquid bacteria liquid is cultivated to 4-7 days, observe and have or not pigment formation; Bacterial strain is carried out to oxydase, catalase, arginine dihydrolase, dextrose plus saccharose fermentation, V.P, M.R and gelatin liquification test.
Result: this bacterial strain is rod-short, without gemma, size is 0.2～0.4 μ m × 1.0～1.3 μ m; Bacterium colony circle, smooth, neat in edge (as Fig. 1), on NYD substratum, bacterium colony is orange-yellow, in minimal media liquid, cultivates and can produce ceroid.This bacterial strain is Gram-negative aerobic bacteria, oxidase positive, and the catalase positive, the arginine dihydrolase positive, utilizes dextrose plus saccharose, V.P feminine gender, M.R feminine gender, liquefy gelatin; Growth temperature is 4～37 DEG C, and optimum growth temperature is 15～20 DEG C.
Obtained bacterial strain is carried out to 16S rDNA sequential analysis: get the centrifugal collection thalline of 1mL bacterium liquid, add 100 μ L sterilized waters, after suspension, boil 10min, centrifugal rear collection supernatant liquor obtains total DNA, carry out pcr amplification with bacterial 16 S rDNA universal primer, PCR product is carried out to agargel electrophoresis, reclaiming size checks order at the unique DNA band at 1500bp place, result obtains 1400bp DNA sequence dna, in GenBank, carry out homologous sequence comparison, with Mega4.0 software building phylogenetic tree (as Fig. 2), the 16S rDNA sequence homology 99% of result and Pseudomonas fluorescence, combining form, cultural characters and physio-biochemical characteristics, be accredited as Pseudomonas fluorescens (Pseudomonas fluorescens), therefore the bacterial strain that obtained is a novel bacterial, be committed to the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC No.7476, preservation date is on April 15th, 2013.
The Heterotrophic nitrification of low-temperature denitrification Pseudomonas fluorescence and aerobic denitrification:
The utilization of bacterial strain to different carbon sources: taking ammonium sulfate as only nitrogen source, change carbon source, carbon source is respectively citric acid, trisodium citrate, glucose, sucrose, sodium-acetate, sodium carbonate and glycerine.Ammonium sulfate content in nutrient solution is 0.05%, and carbon source content is 0.5%, trace element 5%, pH7.2.50mL nutrient solution is contained in 250mL triangular flask, and 8 DEG C, 160r/min concussion is cultivated 2 days, and 3 of each processing are parallel, measure bacterium turbidity (OD
600).The results are shown in Table 1, bacterial strain can finely utilize 7 kinds of carbon sources, Dichlorodiphenyl Acetate salt utilize effect best.
The impact of the different carbon sources of table 1 on strain growth
The utilization of bacterial strain to different nitrogen sources: taking sodium-acetate as sole carbon source, change nitrogenous source, nitrogenous source is respectively ammonium chloride, ammonium sulfate, lemon acid amide, azanol, saltpetre, Sodium Nitrite.Sodium acetate content in nutrient solution is 0.5%, and nitrogenous source content is 0.05%, trace element 5%, pH7.2.50ml nutrient solution is contained in 250ml triangular flask, and 8 DEG C, 160r/min concussion is cultivated 2 days, and 3 of each processing are parallel, measure bacterium turbidity (OD
600).The results are shown in Table 2, bacterial strain can finely utilize 6 kinds of nitrogenous sources.
The impact of table 2 different nitrogen sources on strain growth
The degradation capability of bacterial strain to different concns nitrogenous source: taking sodium-acetate as sole carbon source, nitrogenous source is used respectively ammonium sulfate, saltpetre and Sodium Nitrite, concentration is divided 3 sections, 0.2-6.0.mg/L, 50-400mg/L, 500-1000mg/L.50mL nutrient solution is contained in 250mL triangular flask, 8 DEG C, 160r/min concussion is cultivated 1 day, 3 of each processing are parallel, measure residual nitrogen content and (adopt nessler reagent light-intensity method according to GB7480-87 ammonia nitrogen, nitrite adopts N-Nai Ji-quadrol light-intensity method, and nitrate adopts Pbenoldisulfonic Acid light-intensity method).The results are shown in Table 3, the NH of the extensive concentration range of bacterial strain degradable
4 -n, NO
3 -n and NO
2 -n, to the degradation capability of high density nitrogenous source (> 500mg/L) lower than low middle concentration.
The degradation effect of table 3 bacterial strain to different concns nitrogenous source
| Starting point concentration mg/L | Final concentration mg/L | Nitrogen degradation rate % | |
| NH 4 --N | 0.25±0.12 | 0.054±0.15 | 78.4 |
| 4.53±0.15 | 1.257±0.18 | 72.3 | |
| 48.34±0.21 | 18.81±0.23 | 71.4 | |
| 165.43±0.14 | 62.19±0.12 | 62.4 | |
| 365.32±0.17 | 180.09±0.13 | 50.7 | |
| 522.13±0.19 | 400.97±0.16 | 23.2 | |
| 1314.24±0.16 | 1151.16±0.12 | 12.4 | |
| N0 3 --N | 0.24±0.12 | 0.04±0.14 | 83.3 |
| 5.08±0.17 | 0.95±0.14 | 81.2 | |
| 50.41±013 | 9.17±0.12 | 81.8 | |
| 97.83±0.16 | 19.95±0.20 | 79.6 | |
| 352.35±0.21 | 116.63±0.18 | 66.9 | |
| 513.44±0.23 | 305.50±0.19 | 40.5 | |
| 1201.80±0.14 | 1004.71±0.19 | 16.4 | |
| NO 2 --N | 0.27±0.25 | 0.05±0.21 | 84.4 |
| 5.24±0.22 | 0.88±0.19 | 83.2 | |
| 49.83±0.20 | 9.86±0.23 | 80.2 |
| 124.53±0.14 | 27.65±0.17 | 77.8 | |
| 308.92±0.21 | 72.6±0.24 | 76.5 | |
| 518.67±0.18 | 339.73±0.23 | 34.5 | |
| 1084.45±0.26 | 879.49±0.20 | 18.9 |
Claims (1)
1. a strain low-temperature denitrification Pseudomonas fluorescence, it is characterized in that it is Pseudomonas fluorescens (Pseudomonas fluorescens) WSW-1001, in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC No.7476, preservation date is on April 15th, 2013, and preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute.
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| CN104164389B (en) * | 2014-07-09 | 2016-09-07 | 青岛蔚蓝天成生物科技有限公司 | One fluorescent pseudomonads and the application in aquaculture thereof |
| CN109179650A (en) * | 2018-09-21 | 2019-01-11 | 西安吉利电子化工有限公司 | A kind of high-performance bio compounded carbons and preparation method thereof |
| CN112625942B (en) * | 2020-12-01 | 2022-06-14 | 华南理工大学 | Aerobic denitrifying bacterium and application thereof |
| CN113151077B (en) * | 2021-04-14 | 2023-03-21 | 广西大学 | Pseudomonas aeruginosa and screening method and application thereof |
| CN114058554B (en) * | 2021-12-10 | 2022-09-13 | 青岛蔚蓝赛德生物科技有限公司 | Composting pseudomonas strain and application thereof |
| CN114836355B (en) * | 2022-05-30 | 2024-04-02 | 沈阳风景园林股份有限公司 | Pseudomonas DT04 and application thereof in sewage denitrification |
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| CN102061276A (en) * | 2010-11-15 | 2011-05-18 | 北京大学 | Pseudomonas sp. strain for biological denitrification under low temperature and application thereof |
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