CN102199561A - Aerobic denitrifying bacterium treating nitrite as nitrogen source and screening method thereof - Google Patents
Aerobic denitrifying bacterium treating nitrite as nitrogen source and screening method thereof Download PDFInfo
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- CN102199561A CN102199561A CN2011100676345A CN201110067634A CN102199561A CN 102199561 A CN102199561 A CN 102199561A CN 2011100676345 A CN2011100676345 A CN 2011100676345A CN 201110067634 A CN201110067634 A CN 201110067634A CN 102199561 A CN102199561 A CN 102199561A
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Abstract
The invention relates to a denitrifying bacterium and a screening method thereof, and concretely relates to an aerobic denitrifying bacterium treating nitrite as a nitrogen source and the screening method thereof. The aerobic denitrifying bacterium treating nitrite as the nitrogen source is Pseudomonas sp. yy7, and belongs to Pseudomonas, a preservation number of the bacterium is CCTCC No: M 2011157, and a preservation date of the bacterium is 2011, 01, 20. The screening method comprises the following steps: 1, preparing an enriched nutrient solution; 2, preparing single colonies on a solid selective medium and enlarging cultivation; and 3, inoculating a bacteria liquid after cultivation enlargement into a liquid selective medium, screening, selecting the bacterium liquid with a NO2<->-N removal efficiency of higher than 80%, separating, and purifying. So the method is completed. According to the invention, the yy7 grows and metabolizes with treating NO2<->-N as the only nitrogen source, and provides provenances for biological intensification short-cut simultaneous nitrification and denitrification technologies.
Description
Technical field
The present invention relates to a strain denitrifying bacterium and a screening method thereof.
Background technology
Denitrification process can only carry out under anaerobism and anoxia condition in traditional biological denitrificaion theory, and complete denitrification denitrogenation process is nitrate (NO
3 --N) → nitrite (NO
2 --N) → nitrogen protoxide (NO) → nitrous oxide (N
2O) → nitrogen (N
2), it is generally acknowledged that the existence of oxygen can stop NO in this course
3 --N and NO
2 --N is reduced as final electron acceptor(EA), but along with being found of aerobic denitrifying bacteria Thiosphaera pantotropha, Robertson has confirmed first under aerobic condition, NO
3 --N and NO
2 --N can be used as electron acceptor(EA) equally and is reduced to N by Thiosphaera pantotropha
2, this phenomenon is proved in the metabolic process research to many aerobic denitrification bacterial strains subsequently.
The acquisition of aerobic denitrifying bacteria provides new approach for realizing synchronous nitration and denitrification (SND), the biological reinforcing technology of use aerobic denitrifying bacteria has been proved can realize the SND denitrogenation in a reactor, with by two sections combinations of aerobic nitrification and anaerobic denitrifying being realized the technology of SND denitrogenation compares, its technical superiority is: (1) carries out nitrated-anti-nitration reaction in same reactor, can significantly reduce floor space and construction fund, shorten the processing cycle; (2) use aerobic denitrifying bacteria, can reduce the chemical substance that adds regulation system pH in the treating processes, reduce cost; (3) in treating processes, the easier control of aerobic denitrifying bacteria.Yet for realizing short-cut nitrification and denitrification technology, the aerobic denitrification bacterial strain that adds also needs to possess effective degraded NO
2 --N ability.Therefore, increase economic efficiency, just need isolate efficiently with NO in order to save the denitrogenation cost greatly
2 --N is the aerobic denitrification bacterial strain of metabolism substrate.
Summary of the invention
The invention provides aerobic denitrifying bacteria and screening method thereof that a strain is nitrogenous source with the nitrite; Purpose is to provide provenance for biological reinforced short distance synchronous nitration-denitrification process.
With the nitrite is the aerobic denitrifying bacteria of nitrogenous source, it is false pseudomonas bacillus (Pseudomonas sp.) yy7, belong to Rhodopseudomonas (Pseudomonas), in China's typical culture collection center preservation, deposit number is CCTCC No:M 2011157, and preservation date is on 01 20th, 2011; It is the Gram-negative aerobic bacteria, is tyrothricin, and length is 1.9 μ m～2.2 μ m, and wide is 0.5 μ m～0.6 μ m, no gemma and flagellum; On beef-protein medium, form opaque oyster white bacterium colony, bacterium colony protuberance and surface and edge-smoothing.
Be the aerobic denitrifying bacteria of nitrogenous source with the nitrite among the present invention, it is false pseudomonas bacillus (Pseudomonas sp.) yy7, it utilizes glucose, the catalase positive, oxidase negative, methyl red test feminine gender, the acetyl methyl carbinol positive, the H2S test is positive, starch test is positive, the gelatin test is positive, produce indoles, growth pH value is 7～8, and growth temperature is 10～30 ℃.
Be the aerobic denitrifying bacteria of nitrogenous source with the nitrite among the present invention, it is false pseudomonas bacillus (Pseudomonas sp.) yy7, useful length is about the nucleotide sequence (the sequence accession number among the GenBank is FJ440553) of 580bp on its 16S rRNA gene, this sequence is put into GenBank compares, result's demonstration is the highest with the similarity of the 16S rRNA sequence of pseudomonas sp., reaches 94%.In conjunction with ne ar feature, growth conditions, Physiology and biochemistry qualification result, determine that false pseudomonas bacillus (Pseudomonas sp.) yy7 is the novel bacterial of Rhodopseudomonas.
The present invention is the aerobic denitrifying bacteria of nitrogenous source with the nitrite, it is false pseudomonas bacillus (Pseudomonas sp.) yy7, belong to Rhodopseudomonas (Pseudomonas), in China's typical culture collection center preservation, deposit number is CCTCC No:M 2011157, and preservation date is on 01 20th, 2011.
The screening method that with the nitrite is the aerobic denitrifying bacteria of nitrogenous source is realized according to the following steps: one, get Harbin Sewage Plant A
2The aerobic section mud 5mL of/O technology is inoculated in the LB liquid nutrient medium of 60mL, temperature be under 25 ℃, the aerobic condition of 120r/min behind the constant-temperature shaking culture 24h mud enrichment culture liquid; Two, adopt coubling dilution that mud enrichment culture liquid is carried out gradient dilution, and be applied on the solid selective medium, be put in the thermostat container and under 25 ℃ of conditions, cultivate 120h, obtain single bacterium colony, being seeded to then in the LB liquid nutrient medium of 15mL, is constant temperature vibration enlarged culturing 48h under 25 ℃, the aerobic condition of 120r/min in temperature; Three, get enlarged culturing after bacterium liquid 1mL be inoculated in the 20mL fitting of fluids substratum, under temperature is 25 ℃, the aerobic condition of 150r/min, cultivate 36h, then with NO in the fitting of fluids substratum
2 -The removal efficient of-N is screened as index, chooses NO
2 --N removes efficient and is higher than 80% bacterium liquid, behind separation, purifying, obtains the bacterial strain that a strain has the aerobic denitrification function, and promptly finishing with the nitrite is the screening of the aerobic denitrifying bacteria of nitrogenous source;
Wherein in the step 2 the every L of solid selective medium by the NaNO of 0.1g
2, 0.5g KH
2PO
4, 0.05g FeCl
26H
2The CaCl of O, 0.02g
2, 0.5g MgSO
47H
2The liquid microelement of the sodium acetate of O, 0.3g, the agar of 2g, 1mL and the water of surplus are formed, and the pH value is 7.2～7.5,115 ℃ of autoclaving 25min, and the every L of described liquid microelement is by the FeSO of 3g
47H
2The H of O, 0.01g
3BO
3, 0.01g Na
2MoO
42H
2The MnSO of O, 0.02g
4H
2The CuSO of O, 0.01g
45H
2The ZnSO of O, 0.01g
4, the EDTA of 0.5g and surplus water form;
The every L of fitting of fluids substratum is by the NaNO of 0.1g in the step 3
2, 0.5g KH
2PO
4, 0.05g FeCl
26H
2The CaCl of O, 0.02g
2, 0.5g MgSO
47H
2The water of the sodium acetate of O, 0.3g, the liquid microelement of 1mL and surplus is formed, and the pH value is 7.2～7.5,115 ℃ of autoclaving 25min, and the every L of described liquid microelement is by the FeSO of 3g
47H
2The H of O, 0.01g
3BO
3, 0.01g Na
2MoO
42H
2The MnSO of O, 0.02g
4H
2The CuSO of O, 0.01g
45H
2The ZnSO of O, 0.01g
4, the EDTA of 0.5g and surplus water form.
Aerobic denitrification bacterial strain provided by the invention (false pseudomonas bacillus (Pseudomonas sp.) yy7) is with NO
2 --N is that only nitrogen source carries out growth metabolism, for biological reinforced short distance synchronous nitration-denitrification process provides provenance, helps the application of short distance synchronous nitration-denitrification process, has saved the denitrogenation cost and has increased economic efficiency, and has very big promotional value.False pseudomonas bacillus among the present invention (Pseudomonas sp.) yy7 is to COD, NO
2 --N clearance has reached 70.7% and 97.3% respectively.
Description of drawings
Fig. 1 is the atom mechanics microscopic examination figure of false pseudomonas bacillus in the embodiment one (Pseudomonas sp.) yy7; Fig. 2 is the phylogenetic tree spectrogram that the 16S rRNA gene order of (Pseudomonas sp.) yy7 of false pseudomonas bacillus in the embodiment two and close bacterial strain makes up.
Embodiment
Embodiment one: present embodiment is the aerobic denitrifying bacteria of nitrogenous source with the nitrite, it is false pseudomonas bacillus (Pseudomonas sp.) yy7, belong to Rhodopseudomonas (Pseudomonas), in China's typical culture collection center preservation, deposit number is CCTCC No:M 2011157, and preservation date is on 01 20th, 2011; It is the Gram-negative aerobic bacteria, is tyrothricin, and length is 1.9 μ m～2.2 μ m, and wide is 0.5 μ m～0.6 μ m, no gemma and flagellum; On beef-protein medium, form opaque oyster white bacterium colony, bacterium colony protuberance and surface and edge-smoothing.
False pseudomonas bacillus in the present embodiment (Pseudomonas sp.) yy7 (see figure 1), according to " uncle Jie Shi bacteriology identification handbook " it is carried out conventional Physiology and biochemistry and identify that experimental result is: utilize glucose, the catalase positive, oxidase negative, methyl red test feminine gender, the acetyl methyl carbinol positive, H
2The S test is positive, starch test is positive, the gelatin test is positive, produce indoles.
The growth pH value of false pseudomonas bacillus in the present embodiment (Pseudomonas sp.) yy7 is 7～8, and growth temperature is 10～30 ℃.
Embodiment two: present embodiment is that the screening method of the aerobic denitrifying bacteria of nitrogenous source is realized according to the following steps with the nitrite: one, get Harbin Sewage Plant A
2The aerobic section mud 5mL of/O technology is inoculated in the LB liquid nutrient medium of 60mL, temperature be under 25 ℃, the aerobic condition of 120r/min behind the constant-temperature shaking culture 24h mud enrichment culture liquid; Two, adopt coubling dilution that mud enrichment culture liquid is carried out gradient dilution, and be applied on the solid selective medium, be put in the thermostat container and under 25 ℃ of conditions, cultivate 120h, obtain single bacterium colony, being seeded to then in the LB liquid nutrient medium of 15mL, is constant temperature vibration enlarged culturing 48h under 25 ℃, the aerobic condition of 120r/min in temperature; Three, get enlarged culturing after bacterium liquid 1mL be inoculated in the 20mL fitting of fluids substratum, under temperature is 25 ℃, the aerobic condition of 150r/min, cultivate 36h, then with NO in the fitting of fluids substratum
2 -The removal efficient of-N is screened as index, chooses NO
2 --N removes efficient and is higher than 80% bacterium liquid, behind separation, purifying, obtains the bacterial strain that a strain has the aerobic denitrification function, and promptly finishing with the nitrite is the screening of the aerobic denitrifying bacteria of nitrogenous source;
Wherein in the step 2 the every L of solid selective medium by the NaNO of 0.1g
2, 0.5g KH
2PO
4, 0.05g FeCl
26H
2The CaCl of O, 0.02g
2, 0.5g MgSO
47H
2The liquid microelement of the sodium acetate of O, 0.3g, the agar of 2g, 1mL and the water of surplus are formed, and the pH value is 7.2～7.5,115 ℃ of autoclaving 25min, and the every L of described liquid microelement is by the FeSO of 3g
47H
2The H of O, 0.01g
3BO
3, 0.01g Na
2MoO
42H
2The MnSO of O, 0.02g
4H
2The CuSO of O, 0.01g
45H
2The ZnSO of O, 0.01g
4, the EDTA of 0.5g and surplus water form;
The every L of fitting of fluids substratum is by the NaNO of 0.1g in the step 3
2, 0.5g KH
2PO
4, 0.05g FeCl
26H
2The CaCl of O, 0.02g
2, 0.5g MgSO
47H
2The water of the sodium acetate of O, 0.3g, the liquid microelement of 1mL and surplus is formed, and the pH value is 7.2～7.5,115 ℃ of autoclaving 25min, and the every L of described liquid microelement is by the FeSO of 3g
47H
2The H of O, 0.01g
3BO
3, 0.01g Na
2MoO
42H
2The MnSO of O, 0.02g
4H
2The CuSO of O, 0.01g
45H
2The ZnSO of O, 0.01g
4, the EDTA of 0.5g and surplus water form.
The every 1000mL of LB liquid nutrient medium is soaked by the yeast of the Tryptones of 10g, 5g and powder, 10gNaCl forms with water surplus in the present embodiment step 1 and two, and the pH value is 6.0～8.0, and the LB liquid nutrient medium is using behind sterilization 15～30min down at 121 ℃.
To being that the aerobic denitrifying bacteria of nitrogenous source carries out conventional Physiology and biochemistry and identifies with the nitrite, adopt the CTAB method to carry out the extraction of bacteria total DNA according to " uncle Jie Shi bacteriology identification handbook ".Gained bacterial genomes DNA is carried out pcr amplification with bacterial 16 S rRNA universal primer, the PCR product is reclaimed in rubber tapping behind the electrophoresis, change E.coli DH5 α over to after product is connected to the pMD18 carrier, obtain positive colony, adopt the order-checking of ABI3730 sequenator by ammonia benzyl resistance and the screening of blue hickie; The sequence and the existing sequence among the GenBank that record are carried out the Blast comparison, and by the NJ method 16S rRNA gene order of bacterial strain is classified and the Phylogenetic Analysis (see figure 2) with MEGA4.1 software; Present embodiment screening obtains is that the most close kind of aerobic denitrifying bacteria of nitrogenous source is Rhodopseudomonas (Pseudomonas) with the nitrite, homology reaches 94%, in conjunction with ne ar feature, growth conditions, Physiology and biochemistry qualification result, find that belonging to other bacterial classification with this has any different, be defined as the novel bacterial of Rhodopseudomonas, false pseudomonas bacillus (Pseudomonas sp.) yy7 of called after.
The present embodiment screening nitrite that is able to is the aerobic denitrifying bacteria of nitrogenous source, and it is the growth metabolism analysis of false pseudomonas bacillus (Pseudomonas sp.) yy7:
The right cylinder device of employing synthetic glass preparation (Φ 10cm * 20cm); 20mL bacterium liquid is inoculated in the 300mL artificial culture liquid, and the control aeration rate is 0.06L/min, and stirring velocity is 180r/min; The every L of artificial culture liquid is by the NaNO of 0.1g
2, 0.5g KH
2PO
4, 0.05g FeCl
26H
2The CaCl of O, 0.02g
2, 0.5g MgSO
47H
2The water of the sodium acetate of O, 1g, the liquid microelement of 1mL and surplus is formed, and the pH value is 7.2～7.5,115 ℃ of autoclaving 25min.
Growth metabolism characteristic: when initial COD is 1200mg/L, NO
2 --N is 20mg/L, and when keeping the constant aerating amount and being 0.06L/min, bacterial strain yy7 can normally carry out growth metabolism, and has shown tangible aerobic denitrification feature; Each parameter of its metabolic process changed detects, the result be bacterial strain yy7 in its normal growth metabolic process, can utilize COD, O simultaneously
2And NO
2 --N, wherein COD is as electron donor, O
2And NO
2 --N is as electron acceptor(EA), and this meets forefathers aerobic denitrification bacterial strain pathways metabolism is explained; Accordingly, COD, NO
2 --N clearance has reached 70.7% and 97.3% respectively.
Claims (2)
1. a strain is the aerobic denitrifying bacteria of nitrogenous source with the nitrite, it is characterized in that it is false pseudomonas bacillus (Pseudomonas sp.) yy7, belong to Rhodopseudomonas (Pseudomonas), in China's typical culture collection center preservation, deposit number is CCTCC No:M 2011157, and preservation date is on 01 20th, 2011.
2. screening is the method for the aerobic denitrifying bacteria of nitrogenous source with the nitrite, it is characterized in that with the nitrite being that the screening method of the aerobic denitrifying bacteria of nitrogenous source is realized according to the following steps: one, get Harbin Sewage Plant A
2The aerobic section mud 5mL of/O technology is inoculated in the LB liquid nutrient medium of 60mL, temperature be under 25 ℃, the aerobic condition of 120r/min behind the constant-temperature shaking culture 24h mud enrichment culture liquid; Two, adopt coubling dilution that mud enrichment culture liquid is carried out gradient dilution, and be applied on the solid selective medium, be put in the thermostat container and under 25 ℃ of conditions, cultivate 120h, obtain single bacterium colony, being seeded to then in the LB liquid nutrient medium of 15mL, is constant temperature vibration enlarged culturing 48h under 25 ℃, the aerobic condition of 120r/min in temperature; Three, get enlarged culturing after bacterium liquid 1mL be inoculated in the 20mL fitting of fluids substratum, under temperature is 25 ℃, the aerobic condition of 150r/min, cultivate 36h, then with NO in the fitting of fluids substratum
2 -The removal efficient of-N is screened as index, chooses NO
2 --N removes efficient and is higher than 80% bacterium liquid, behind separation, purifying, obtains the bacterial strain that a strain has the aerobic denitrification function, and promptly finishing with the nitrite is the screening of the aerobic denitrifying bacteria of nitrogenous source;
Wherein in the step 2 the every L of solid selective medium by the NaNO of 0.1g
2, 0.5g KH
2PO
4, 0.05g FeCl
26H
2The CaCl of O, 0.02g
2, 0.5g MgSO
47H
2The liquid microelement of the sodium acetate of O, 0.3g, the agar of 2g, 1mL and the water of surplus are formed, and the pH value is 7.2～7.5,115 ℃ of autoclaving 25min, and the every L of described liquid microelement is by the FeSO of 3g
47H
2The H of O, 0.01g
3BO
3, 0.01g Na
2MoO
42H
2The MnSO of O, 0.02g
4H
2The CuSO of O, 0.01g
45H
2The ZnSO of O, 0.01g
4, the EDTA of 0.5g and surplus water form;
The every L of fitting of fluids substratum is by the NaNO of 0.1g in the step 3
2, 0.5g KH
2PO
4, 0.05g FeCl
26H
2The CaCl of O, 0.02g
2, 0.5g MgSO
47H
2The water of the sodium acetate of O, 0.3g, the liquid microelement of 1mL and surplus is formed, and the pH value is 7.2～7.5,115 ℃ of autoclaving 25min, and the every L of described liquid microelement is by the FeSO of 3g
47H
2The H of O, 0.01g
3BO
3, 0.01g Na
2MoO
42H
2The MnSO of O, 0.02g
4H
2The CuSO of O, 0.01g
45H
2The ZnSO of O, 0.01g
4, the EDTA of 0.5g and surplus water form.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525730A (en) * | 2013-10-14 | 2014-01-22 | 青岛蔚蓝生物集团有限公司 | Pseudomonas otitidis strain and application thereof |
| CN104531595A (en) * | 2015-01-20 | 2015-04-22 | 重庆大学 | Method for rapidly screening and separating heterotrophic nitrification-aerobic denitrification bacterium |
| 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 |
| CN107794235A (en) * | 2017-09-11 | 2018-03-13 | 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 |
| CN111647539A (en) * | 2020-07-06 | 2020-09-11 | 重庆工商大学 | Method for strengthening film forming capability of aerobic denitrifying bacteria |
| CN115838665A (en) * | 2022-11-15 | 2023-03-24 | 中国科学院城市环境研究所 | Enterobacter strain TCD1-1 T And uses thereof |
| CN115838665B (en) * | 2022-11-15 | 2024-05-31 | 中国科学院城市环境研究所 | Enterobacter strain TCD1-1TAnd applications thereof |
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2011
- 2011-03-21 CN CN2011100676345A patent/CN102199561A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525730A (en) * | 2013-10-14 | 2014-01-22 | 青岛蔚蓝生物集团有限公司 | Pseudomonas otitidis strain and application thereof |
| CN103525730B (en) * | 2013-10-14 | 2015-12-02 | 青岛蔚蓝生物集团有限公司 | A kind of otitis pseudomonas strains and application thereof |
| CN104531595A (en) * | 2015-01-20 | 2015-04-22 | 重庆大学 | Method for rapidly screening and separating heterotrophic nitrification-aerobic denitrification bacterium |
| 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 |
| CN107794235A (en) * | 2017-09-11 | 2018-03-13 | 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 |
| CN107794235B (en) * | 2017-09-11 | 2019-09-24 | 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 |
| CN111647539A (en) * | 2020-07-06 | 2020-09-11 | 重庆工商大学 | Method for strengthening film forming capability of aerobic denitrifying bacteria |
| CN115838665A (en) * | 2022-11-15 | 2023-03-24 | 中国科学院城市环境研究所 | Enterobacter strain TCD1-1 T And uses thereof |
| CN115838665B (en) * | 2022-11-15 | 2024-05-31 | 中国科学院城市环境研究所 | Enterobacter strain TCD1-1TAnd applications thereof |
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Application publication date: 20110928 |