CN102559543B - Rhodococcus sp for removing nitrite nitrogen in sewage at low temperature and isolated culture method - Google Patents
Rhodococcus sp for removing nitrite nitrogen in sewage at low temperature and isolated culture method Download PDFInfo
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- CN102559543B CN102559543B CN2011104163663A CN201110416366A CN102559543B CN 102559543 B CN102559543 B CN 102559543B CN 2011104163663 A CN2011104163663 A CN 2011104163663A CN 201110416366 A CN201110416366 A CN 201110416366A CN 102559543 B CN102559543 B CN 102559543B
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- rhodococcus
- sewage
- nitrite nitrogen
- nitrate
- low temperature
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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
Abstract
The invention discloses rhodococcus sp for removing nitrite nitrogen in sewage at a low temperature and an isolated culture method. The rhodococcus sp-2 CGMCC No.5287 for removing the nitrite nitrogen in the sewage at the low temperature has the capacity of removing the nitrite nitrogen in the sewage under the low-temperature aerobic condition. Experimental results show that: although the rhodococcus sp is obviously inhibited by a low-temperature environment, the rhodococcus sp-2 CGMCC No.5287 for removing the nitrite nitrogen in the sewage has an obvious reinforcing effect of removing the nitrite nitrogen in the sewage at the low temperature, particularly at the temperature of between 8 and 12 DEG C; and compared with the sewage in which rhodococcus sp is not inoculated, the sewage in which the rhodococcus sp is inoculated has the advantage that: the removal rate of the nitrite nitrogen is improved by about 10 percent.
Description
Technical field
The present invention relates to a strain rhodococcus and purposes and isolation cultivation method.
Background technology
The reason that low temperature produces great effect to the biological treatment ability is because the reduction of temperature has influenced microbial growth and metabolism and made that change has taken place microbial population in the Sewage treatment systems.In temperature decline process, the metabolic activity of mesophilic bacteria descends gradually, just lose activity during to certain temperature, and be suitable for low temperature environment psychrophile quantity since self physiological property and the restraining effect of various ecological factors, quantitatively can't reach the status of dominant microflora, thereby cause sewage work's effluent quality in winter to descend.Therefore need badly a kind ofly can go down except the bacterial strain of sewage Central Asia nitrate nitrogen in the low-temperature aerobic condition.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of rhodococcus of removing nitrite nitrogen in the sewage is provided.
Second purpose of the present invention provides a kind of purposes of removing the rhodococcus of nitrite nitrogen in the sewage.
The 3rd purpose of the present invention provides a kind of cold condition and goes down except the isolation cultivation method of the rhodococcus of sewage Central Asia nitrate nitrogen.
Technical scheme of the present invention is summarized as follows:
Low temperature goes down except dark-2 CGMCC No.5287 of the rhodococcus of sewage Central Asia nitrate nitrogen (Rhodococcus sp.) nitre, and it has in the low-temperature aerobic condition to go down except the ability of sewage Central Asia nitrate nitrogen.
Low temperature goes down and goes down except the purposes of sewage Central Asia nitrate nitrogen in the low-temperature aerobic condition except dark-2 CGMCC No.5287 of the rhodococcus of sewage Central Asia nitrate nitrogen (Rhodococcus sp.) nitre.Preferred 8～12 ℃ of described low temperature.
Cold condition goes down except the isolation cultivation method of the rhodococcus of sewage Central Asia nitrate nitrogen, comprises the steps:
(1) with the sludge seeding of 10～20mL sewage disposal plant aeration tank to the Erlenmeyer flask that 250mL Nitrate bacteria nutrient solution is housed, nitrogen concentration is 50mg/L in the Sodium Nitrite in the described Nitrate bacteria nutrient solution, at 8～12 ℃, shaking speed is to tame in 120～140rpm shaking table, treat that the nitrite nitrogen degradation rate reaches at 80～90% o'clock, leave standstill, remove supernatant liquor, with the sludge seeding of Erlenmeyer flask bottom to being equipped with in second kind of Nitrate bacteria nutrient solution of 250mL, nitrite nitrogen concentration in described second kind of Nitrate bacteria nutrient solution is 150mg/L, at 8～12 ℃, in being the shaking table of 120～140rpm, rotating speed tames, treat that the nitrite nitrogen degradation rate reaches at 80～90% o'clock, namely obtains containing the Nitrate bacteria nutrient solution of low temperature resistant bacterium;
(2) get the nutrient solution that 10mL step (1) obtains, add in the 250mL Erlenmeyer flask that 90mL sterile purified water and 20～30 sterilization granulated glass spherees have been housed, vibration 10～30min makes bacterial cell fully be discharged in the supernatant liquor; Leave standstill 20～40min, get the adding of 1mL supernatant liquor and be equipped with in the test tube of 9mL sterile distilled water, obtain 10
-2Bacteria suspension, take turns doing serial dilution as stated above, accomplish 10 always
-8Till;
(3) choosing gradient is 10
-5～10
-8Bacteria suspension, get 1mL respectively and be inoculated in the Nitrate bacteria solid medium, each extent of dilution is made 3 parallel samples, the numbering; Under the condition of aerobic, preserve moisture for 10 ℃ and cultivated for 2～3 weeks;
(4) choosing colony is big in the Nitrate bacteria solid medium flat board from step (3), single bacterium colony that form is different, line separates again in Nitrate bacteria solid medium flat board, repeating plate streaking separates 3-4 time, make the homomorphosis of bacterium colony in microscope on the flat board, single, big or small similar single bacterium colony that is, called after nitre is dark-2, goes down except the rhodococcus (Rhodococcus sp.) of sewage Central Asia nitrate nitrogen through being accredited as cold condition.
Described Nitrate bacteria nutrient solution is made up of following component: NaNO
20.25g, sodium acetate 2g, MgSO
47H
2O 0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
44H
2O 0.01g, FeSO
40.01g, adding distil water is to 1000mL.
Described second kind of Nitrate bacteria nutrient solution is made up of following component: NaNO
20.74g, sodium acetate 2g, MgSO
47H
2O0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
44H
2O 0.01g, FeSO
40.01g, adding distil water is to 1000mL.
Described Nitrate bacteria solid medium is made up of following component: NaNO
20.5g, sodium acetate 2g, MgSO
47H
2O0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
44H
2O 0.01g, FeSO
40.01g, agar 20g, adding distil water be to 1000mL.
Experimental result shows: although be subjected to the obvious inhibition of low temperature environment, add a kind of dark-2 CGMCC No.5287 of rhodococcus (Rhodococcus sp.) nitre that remove nitrite nitrogen in the sewage of the present invention, at low temperatures, particularly under 8-12 ℃ condition, sewage Central Asia nitrate removal there is tangible strengthening effect, inoculate rhodococcus of the present invention and compare with the sewage of not inoculating bacterium, the nitrite nitrogen removal efficiency has improved and has been about 10%.
Description of drawings
Fig. 1 goes down except the domestication separation and Culture schema of the rhodococcus of nitrite nitrogen in the removal sewage of sewage Central Asia nitrate nitrogen for the low-temperature aerobic condition.
Fig. 2 is that dark-2 pairs of nitrite nitrogens of nitre are removed test.
Fig. 3 is dark-2 method for determining bacteria flow processs of nitre.
Embodiment
The present invention is further illustrated below by specific embodiment.
Cold condition goes down except the isolation cultivation method of the rhodococcus of sewage Central Asia nitrate nitrogen, comprises the steps:
(1) with the sludge seeding of 15mL Tianjin discipline village sewage disposal plant aeration tank to the Erlenmeyer flask that 250mL Nitrate bacteria nutrient solution is housed, nitrogen concentration is 50mg/L in the Sodium Nitrite in the described Nitrate bacteria nutrient solution, at 10 ℃, shaking speed is to tame in the 130rpm shaking table, treat that the nitrite nitrogen degradation rate reaches at 90% o'clock, leave standstill, remove supernatant liquor, with the sludge seeding of Erlenmeyer flask bottom to being equipped with in second kind of Nitrate bacteria nutrient solution of 250mL, nitrite nitrogen concentration in described second kind of Nitrate bacteria nutrient solution is 150mg/L, at 10 ℃, in being the shaking table of 130rpm, rotating speed tames, treat that the nitrite nitrogen degradation rate reaches at 90% o'clock, namely obtain containing the Nitrate bacteria nutrient solution of low temperature resistant bacterium;
(2) get the nutrient solution that 10mL step (1) obtains, add in the 250mL Erlenmeyer flask that 90mL sterile purified water and 30 sterilization granulated glass spherees have been housed, vibration 20min makes bacterial cell fully be discharged in the supernatant liquor; Leave standstill 30min, get the adding of 1mL supernatant liquor and be equipped with in the test tube of 9mL sterile distilled water, obtain 10
-2Bacteria suspension, take turns doing serial dilution as stated above, accomplish 10 always
-8Till;
(3) choosing gradient is 10
-5～10
-8Bacteria suspension, get 1mL respectively and be inoculated in the Nitrate bacteria solid medium, each extent of dilution is made 3 parallel samples, the numbering; Under the condition of aerobic, preserve moisture for 10 ℃ and cultivated for 3 weeks;
(4) choosing colony is big in the Nitrate bacteria solid medium flat board from step (3), single bacterium colony that form is different, line separates again in Nitrate bacteria solid medium flat board, repeating plate streaking separates 3 times, make the homomorphosis of bacterium colony in microscope on the flat board, single, big or small similar single bacterium colony that is, called after nitre is dark-2, goes down except the rhodococcus (Rhodococcus sp.) of sewage Central Asia nitrate nitrogen through being accredited as cold condition.
The Nitrate bacteria nutrient solution that adopts among each embodiment is made up of following component: NaNO
20.25g, sodium acetate 2g, MgSO
47H
2O 0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
44H
2O 0.01g, FeSO
40.01g, adding distil water is to 1000mL.
Second kind of Nitrate bacteria nutrient solution is made up of following component: NaNO
20.74g, sodium acetate 2g, MgSO
47H
2O 0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
44H
2O 0.01g, FeSO
40.01g, adding distil water is to 1000mL.
The Nitrate bacteria solid medium is made up of following component: NaNO
20.5g, sodium acetate 2g, MgSO
47H
2O 0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
44H
2O 0.01g, FeSO
40.01g, agar 20g, adding distil water be to 1000mL.
Rhodococcus of the present invention (Rhodococcus sp.) has following Microbiological Characteristics:
1. morphological characteristic
Examine under a microscope, for spherical, diameter is 1.5 μ m; Form the oyster white bacterium colony at the Nitrate bacteria solid medium.
2. cultivate and learn characteristic
Under 10 ℃, when cultivating in the Nitrate bacteria solid medium, Rhodococcus sp. bacterial strain of the present invention has following characteristic.
Smooth surface is moistening, rat, and the neat in edge rule, individual rounded, thalline is creamy white.
3. physiological property
Aerobic condition goes down except the gram positive bacterium of sewage Central Asia nitrate nitrogen, and the catalase positive, methyl red test feminine gender, hydrolyzed starch, liquefy gelatin, edwardsiella hoshinae, growth pH value are 6～8,5～30 ℃ of growth temperatures.
| Gram | Catalase | Methyl red | The starch hydrolysis | The gelatin test | Indole test | p H | Temperature |
| G + | + | - | + | + | - | 6～8 | 5～30 |
The 16s Sequence Identification of bacterium of the present invention
16SrDNA is bacterium class prokaryotic organism very conservative sequences in very long evolutionary process, in the taxonomy of modern microorganism, playing the part of important role, as the most frequently used phyletic evolution tagged molecule of bacterial flora structural analysis, it is more efficient and convenient to carry out structural analysis of microbial community with 16S rDNA.
Carrying out the Bacteria Identification flow process with the 16s sequence is undertaken by Fig. 3.
Beef-protein medium among Fig. 3 is formed: extractum carnis 3g, and peptone 10g, sodium-chlor 5g, adding distil water are to 1000mL, and pH 7.0～7.2.
The LB substratum: tryptone 10g, yeast extract 5g, NaCl 10g, adding distil water is to 1000mL.
The 16s sequence that the present invention records is as follows:
ACGCTGGCGGCGTGCTTAACGCACTGCAAGTCCCAGCGGTGAGGCCTTTCGGGGTACACGAGCGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCTCCTATCGCATGGTGGGTGGTGGAAAGATTTATCGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGACGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAATTACTGGGCGTAAAGAGTTCGTAGGCGGTTTGTCGCGTCGTTTGTGAAAACCAGCAGCTCAACTGCTGGCTTGCAGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAACGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTCCTTCCACGGAATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATATACCGGAAAGCTGCAGAGATGTGGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTATGTTGCCAGCACGTTATGGTGGGGACTCGTAAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGCGAGACCGTGAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCTTAACCCCGAGTGGGAAccording to the microbiological characteristics; named strain of the invention nitrate deep -2; classified named Rhodococcus equi (Rhodococcus sp.) Was September 22, 2011 Deposit of Microorganisms deposited at the China General Microbiological Centre Management Committee; Accession No. as CGMCC No.5287; preservation address: Beichen West Road, Chaoyang District Court No. 1 on the 3rd; Institute of Microbiology and has survived.
Embodiment 2
The degradation effect of dark-2 pairs of nitrite nitrogens of nitre
In the beaker of 1L, add 500mL Nitrate bacteria nutrient solution, initial nitrite nitrogen concentration is adjusted into 50mg/L, add nitre of the present invention dark-2, connecing three rings nitre of the present invention dark-2 from the Nitrate bacteria solid plate goes into the above-mentioned Nitrate bacteria nutrient solution, keeping pH is 7.5, measures the degraded situation of 72 hours dark-2 pairs of nitrite nitrogens of nitre, the results are shown in Figure 2.
As seen from Figure 2, the degraded through 72 hours, nitrite nitrogen concentration is 6mg/L in the solution, and nitrite nitrogen is removed substantially, and clearance is 88.0%, proves that dark-2 pairs of nitrite nitrogens of nitre among the present invention have good removal ability.
Embodiment 3
Cold condition goes down except the isolation cultivation method of the rhodococcus of sewage Central Asia nitrate nitrogen, comprises the steps:
(1) with the sludge seeding of 10mL Tianjin discipline village sewage disposal plant aeration tank to the Erlenmeyer flask that 250mL Nitrate bacteria nutrient solution is housed, nitrogen concentration is 50mg/L in the Sodium Nitrite in the described Nitrate bacteria nutrient solution, at 8 ℃, shaking speed is to tame in the 140rpm shaking table, treat that the nitrite nitrogen degradation rate reaches at 80% o'clock, leave standstill, remove supernatant liquor, with the sludge seeding of Erlenmeyer flask bottom to being equipped with in second kind of Nitrate bacteria nutrient solution of 250mL, nitrite nitrogen concentration in described second kind of Nitrate bacteria nutrient solution is 150mg/L, at 8 ℃, in being the shaking table of 140rpm, rotating speed tames, treat that the nitrite nitrogen degradation rate reaches at 80% o'clock, namely obtain containing the Nitrate bacteria nutrient solution of low temperature resistant bacterium;
(2) get the nutrient solution that 10mL step (1) obtains, add in the 250mL Erlenmeyer flask that 90mL sterile purified water and 20 sterilization granulated glass spherees have been housed, vibration 30min makes bacterial cell fully be discharged in the supernatant liquor; Leave standstill 20min, get the adding of 1mL supernatant liquor and be equipped with in the test tube of 9mL sterile distilled water, obtain 10
-2Bacteria suspension, take turns doing serial dilution as stated above, accomplish 10 always
-8Till;
(3) choosing gradient is 10
-5～10
-8Bacteria suspension, get 1mL respectively and be inoculated in the Nitrate bacteria solid medium, each extent of dilution is made 3 parallel samples, the numbering; Under the condition of aerobic, preserve moisture for 10 ℃ and cultivated for 2 weeks;
(4) choosing colony is big in the Nitrate bacteria solid medium flat board from step (3), single bacterium colony that form is different, line separates again in Nitrate bacteria solid medium flat board, repeating plate streaking separates 4 times, make the homomorphosis of bacterium colony in microscope on the flat board, single, big or small similar single bacterium colony that is, called after nitre is dark-2, goes down except the rhodococcus (Rhodococcus sp.) of sewage Central Asia nitrate nitrogen through being accredited as cold condition.
Embodiment 4
Cold condition goes down except the isolation cultivation method of the rhodococcus of sewage Central Asia nitrate nitrogen, comprises the steps:
(1) with the sludge seeding of 20mL Tianjin discipline village sewage disposal plant aeration tank to the Erlenmeyer flask that 250mL Nitrate bacteria nutrient solution is housed, nitrogen concentration is 50mg/L in the Sodium Nitrite in the described Nitrate bacteria nutrient solution, at 12 ℃, shaking speed is to tame in the 120rpm shaking table, treat that the nitrite nitrogen degradation rate reaches at 90% o'clock, leave standstill, remove supernatant liquor, with the sludge seeding of Erlenmeyer flask bottom to being equipped with in second kind of Nitrate bacteria nutrient solution of 250mL, nitrite nitrogen concentration in described second kind of Nitrate bacteria nutrient solution is 150mg/L, at 12 ℃, in being the shaking table of 120rpm, rotating speed tames, treat that the nitrite nitrogen degradation rate reaches at 90% o'clock, namely obtain containing the Nitrate bacteria nutrient solution of low temperature resistant bacterium;
(2) get the nutrient solution that 10mL step (1) obtains, add in the 250mL Erlenmeyer flask that 90mL sterile purified water and 30 sterilization granulated glass spherees have been housed, vibration 10min makes bacterial cell fully be discharged in the supernatant liquor; Leave standstill 40min, get the adding of 1mL supernatant liquor and be equipped with in the test tube of 9mL sterile distilled water, obtain 10
-2Bacteria suspension, take turns doing serial dilution as stated above, accomplish 10 always
-8Till;
(3) choosing gradient is 10
-5～10
-8Bacteria suspension, get 1mL respectively and be inoculated in the Nitrate bacteria solid medium, each extent of dilution is made 3 parallel samples, the numbering; Under the condition of aerobic, preserve moisture for 10 ℃ and cultivated for 3 weeks;
(4) choosing colony is big in the Nitrate bacteria solid medium flat board from step (3), single bacterium colony that form is different, line separates again in Nitrate bacteria solid medium flat board, repeating plate streaking separates 3 times, make the homomorphosis of bacterium colony in microscope on the flat board, single, big or small similar single bacterium colony that is, called after nitre is dark-2, goes down except the rhodococcus (Rhodococcus sp.) of sewage Central Asia nitrate chlorine through being accredited as cold condition.
Experiment showed, that embodiment 3 and embodiment 4 separation and Culture remove the effect that the rhodococcus (Rhodococcus sp.) of nitrite nitrogen in the sewage also has good degrading nitrite nitrogen, clearance is respectively 87.0% and 87.3%.
Claims (2)
- A cold condition go down except rhodococcus (Rhodococcus sp.) nitre of sewage Central Asia nitrate nitrogen dark-2CGMCC No.5287, it has in the low-temperature aerobic condition to go down except the ability of sewage Central Asia nitrate nitrogen, described low temperature is 8-12 ℃.
- A kind of cold condition of claim 1 go down except rhodococcus (Rhodococcus sp.) nitre of sewage Central Asia nitrate nitrogen dark-2CGMCC No.5287 goes down except the purposes of sewage Central Asia nitrate nitrogen in the low-temperature aerobic condition, described low temperature is 8-12 ℃.
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Citations (1)
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| CN101875909A (en) * | 2010-01-26 | 2010-11-03 | 中国科学院成都生物研究所 | Heterotrophic nitrification aerobic denitrifying bacteria and culture method and application thereof |
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| CN101875909A (en) * | 2010-01-26 | 2010-11-03 | 中国科学院成都生物研究所 | Heterotrophic nitrification aerobic denitrifying bacteria and culture method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| 大菱鲆工厂化养殖水质净化菌的筛选与系统发育分析;张艳等;《海洋水产研究》;20070815(第04期);第21-25页 * |
| 好氧反硝化菌的分离鉴定及特性研究;张光亚等;《微生物学杂志》;20051130(第06期);第23-26页 * |
| 张光亚等.好氧反硝化菌的分离鉴定及特性研究.《微生物学杂志》.2005,(第06期), |
| 张艳等.大菱鲆工厂化养殖水质净化菌的筛选与系统发育分析.《海洋水产研究》.2007,(第04期), |
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