CN1884480A - Method for screening allotrophic nitrobacteria - Google Patents
Method for screening allotrophic nitrobacteria Download PDFInfo
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- CN1884480A CN1884480A CN 200610010263 CN200610010263A CN1884480A CN 1884480 A CN1884480 A CN 1884480A CN 200610010263 CN200610010263 CN 200610010263 CN 200610010263 A CN200610010263 A CN 200610010263A CN 1884480 A CN1884480 A CN 1884480A
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Abstract
A sieve method of heterotrophic nitrifying bacteria belongs to sewage treatment technical sphere. The invention is to carry out heterotrophic nitrifying phenomenon from the biology angle by screening heterotrophic nitrifying bacteria and the technology is the condition precedent to carry out short-range self-synchronous nitration and denitrification. The invention is proceeded by the following steps: 1. enriched culturing of microorganism; 2. picking out single colony from beef cream protease leather solid medium and seeding them in ammonifying culture medium to culture and purify; 3. picking out single colony from ammonifying culture medium solid medium and seeding them in nitrosation culture medium to culture and purify to obtain heterotrophic nitrifying bacteria. The heterotrophic nitrifying bacteria of this invention not only can grow and breed in culture medium using ammonia nitrogen as unique nitrogen source but also can normally grow and breed in culture medium using nitrite as unique nitrogen source, which has stronger adaptive capacity for environment and has bigger development potential in actual engineering application.
Description
Technical field
The invention belongs to technical field of sewage, be specifically related to a kind of sieve method of heterotrophic nitrifying bacteria.
Background technology
Nitrate pollution is one of major reason that causes body eutrophication, so the denitrogenation processing of sewage is for keeping the water body cleaning and preventing that eutrophication all has important effect.Nitrification can be divided into two conversion processes of ammonia to nitrous acid (ammonia oxidation) and nitrous acid to nitric acid (nitrous acid oxidation), and the nitrate of formation forms nitrogen effusion water body by denitrification and finishes denitrogenation.It is generally acknowledged that two steps of nitrification are finished by two class chemoautotrophy microorganisms respectively, can the oxidation ammonia nitrogen be nitrite be Nitrosomas, can nitrite oxidation be nitrate be Nitromonas, nitrifier plays important effect in the full cycle process of nitrogen element.Traditional Nitrosomas, Nitromonas generation cycle are long, and it is extremely slow to grow, and very responsive and the application of autotrophic microorganism is restricted to the variation of environment.
For the autotrophy nitrifier, though the decomposition efficiency of the heterotrophic bacterium that has is lower since on their quantity in environment and the growth velocity often much larger than autotrophic bacteria, therefore among some environment, the contribution of heterotrophic nitrification effect can be suitable with autotrophic bacteria, even exceed.These microorganisms can carry out nitrification under the organic carbon condition, also can carry out nitrification in the organophilic clay environment, have important effect in Nitrogen Cycling.
Summary of the invention
The objective of the invention is to adopt the method for screening nitrification bacteria, realize the heterotrophic nitrification phenomenon from the biology angle, this technology is to realize the prerequisite of short distance synchronous nitration and denitrification.
The present invention carries out according to following step: one, microbial enrichment is cultivated: water sample is left standstill abandoning supernatant, getting active sludge is inoculated in the beef extract-peptone liquid nutrient medium, inoculum size is 5～10%, at 30～35 ℃, the constant temperature vibration is cultivated under the condition of 120～14rpm, when range estimation nutrient solution turbidity increases, enrichment culture liquid is inoculated in the beef extract-peptone solid medium flat board, 30 ℃ of incubators are cultivated 2～3d, picking list bacterium colony is transferred on slant tube, the bacterium colony that will the have various trait again purifying of on beef extract-peptone solid medium flat board, ruling, the dull and stereotyped incubator 2～3d that is inverted repeats above operation, and is pure until dividing;
Two, picking list bacterium colony from the beef extract-peptone solid medium, be inoculated in the ammonification medium liquid substratum, inoculum size is 5～10%, at 30～35 ℃, the constant temperature vibration is cultivated under the condition of 120～140rpm, when range estimation nutrient solution turbidity increases, enrichment culture liquid is inoculated in the ammonification substratum solid medium flat board, 30～35 ℃ of incubators are cultivated 2～3d, picking list bacterium colony is transferred on slant tube, the bacterium colony that will the have various trait again purifying of on ammonification substratum solid medium flat board, ruling, the dull and stereotyped incubator 2～3d that is inverted repeats above operation, and is pure until dividing;
Three, picking list bacterium colony from ammonification substratum solid medium, be inoculated in the nitrosification medium liquid substratum, inoculum size is 5～10%, at 30～35 ℃, the constant temperature vibration is cultivated under the condition of 120～140rpm, when range estimation nutrient solution turbidity increases, enrichment culture liquid is inoculated in the nitrosification substratum solid medium flat board, 30～35 ℃ of incubators are cultivated 2～3d, picking list bacterium colony is transferred on slant tube, the bacterium colony that will the have various trait again purifying of on nitrosification substratum solid medium flat board, ruling, the dull and stereotyped incubator 2～3d that is inverted repeats above operation, pure until dividing, obtain allotrophic nitrobacteria.
The method of screening nitrification bacteria of the present invention realizes the heterotrophic nitrification phenomenon from the biology angle, allotrophic nitrobacteria has the generation cycle weak point with respect to the autotrophy nitrobacteria, growth and breeding speed is fast, to the insensitive advantage of the variation of environment, the allotrophic nitrobacteria that is screened only can utilize ammonia nitrogen as carrying out growth and breeding on the substratum of unique nitrogenous source, and can nitrite as the substratum of only nitrogen source in normal growth breeding, environment there is stronger adaptive faculty, bigger development potentiality is arranged in practical engineering application.
Embodiment
Embodiment one: present embodiment is screened allotrophic nitrobacteria according to following steps:
One, be formulated as follows substratum:
Beef-protein medium: extractum carnis 1g, NaCl 1g, peptone 3g, sodium acetate 1g, K
2HPO
40.5g, MnSO
44H
2O 0.01g, FeSO
40.01g, H
2O 1000mL, pH=7.0～7.2.
Ammonification substratum: NH
4Cl 0.382g, sodium acetate 2g, MgSO
47H
2O 0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
44H
2O 0.01g, FeSO
40.01g, H
2O 1000mL.
Nitrosification substratum: 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, H
2O 1000mL.
Two, screening allotrophic nitrobacteria:
1, microbial enrichment is cultivated: water sample is left standstill abandoning supernatant, getting active sludge is inoculated in the beef extract-peptone liquid nutrient medium, inoculum size is 5～10%, the constant temperature vibration is cultivated under 30～35 ℃, the condition of 120～140rpm, when seeing obvious growth arranged (range estimation nutrient solution turbidity increases), then enrichment culture liquid is inoculated in the beef extract-peptone solid medium flat board, 30 ℃ of incubators are cultivated 2～3d.Picking list bacterium colony is transferred on slant tube, the bacterium colony that will the have various trait again purifying of ruling on beef extract-peptone solid medium flat board, the dull and stereotyped incubator 2～3d that is inverted.Repeat above operation, pure until dividing.
2, picking list bacterium colony from the beef extract-peptone solid medium is inoculated in the ammonification medium liquid substratum.Inoculum size is 5～10%, the constant temperature vibration is cultivated under 30～35 ℃, the condition of 120～140rpm, when seeing obvious growth is arranged (range estimation nutrient solution turbidity increases), then enrichment culture liquid is inoculated in the ammonification substratum solid medium flat board, 30～35 ℃ of incubators are cultivated 2～3d.Picking list bacterium colony is transferred on slant tube, the bacterium colony that will the have various trait again purifying of ruling on ammonification substratum solid medium flat board, the dull and stereotyped incubator 2～3d that is inverted.Repeat above operation, pure until dividing.
3, from " ammonification substratum solid medium " picking list bacterium colony, be inoculated in the nitrosification medium liquid substratum.Inoculum size is 5～10%, the constant temperature vibration is cultivated under 30～35 ℃, the condition of 120～140rpm, when seeing obvious growth is arranged (range estimation nutrient solution turbidity increases), then enrichment culture liquid is inoculated in the nitrosification substratum solid medium flat board, 30～35 ℃ of incubators are cultivated 2～3d.Picking list bacterium colony is transferred on slant tube, the bacterium colony that will the have various trait again purifying of ruling on nitrosification substratum solid medium flat board, the dull and stereotyped incubator 2～3d that is inverted.Repeat above operation, pure until dividing.
Embodiment two: present embodiment realizes by following steps:
1, microbial enrichment is cultivated: water sample is left standstill abandoning supernatant, getting active sludge is inoculated in the beef extract-peptone liquid nutrient medium, inoculum size is 5%, 30 ℃, 120rpm constant temperature vibration cultivation, when seeing obvious growth arranged (range estimation nutrient solution turbidity increases), then enrichment culture liquid is inoculated in the beef extract-peptone solid medium flat board, 30 ℃ of incubators are cultivated 2～3d.Picking list bacterium colony is transferred on slant tube, the bacterium colony that will the have various trait again purifying of ruling on beef extract-peptone solid medium flat board, the dull and stereotyped incubator 2～3d that is inverted.Repeat above operation, pure until dividing.
2, from beef extract-peptone solid medium picking list bacterium colony, be inoculated in the ammonification medium liquid substratum.Inoculum size is that 5%, 30 ℃, 120rpm constant temperature vibration are cultivated, and seeing has obvious growth when (range estimation nutrient solution turbidity increases), then enrichment culture liquid is inoculated in the ammonification substratum solid medium flat board, and 30 ℃ of incubators are cultivated 2～3d.Picking list bacterium colony is transferred on slant tube, the bacterium colony that will the have various trait again purifying of ruling on ammonification substratum solid medium flat board, the dull and stereotyped incubator 2～3d that is inverted.Repeat above operation, pure until dividing.
3, from ammonification substratum solid medium picking list bacterium colony, be inoculated in the nitrosification medium liquid substratum.Inoculum size is that 5%, 30 ℃, 120rpm constant temperature vibration are cultivated, and seeing has obvious growth when (range estimation nutrient solution turbidity increases), then enrichment culture liquid is inoculated in the nitrosification substratum solid medium flat board, and 30 ℃ of incubators are cultivated 2～3d.Picking list bacterium colony is transferred on slant tube, the bacterium colony that will the have various trait again purifying of ruling on nitrosification substratum solid medium flat board, the dull and stereotyped incubator 2～3d that is inverted.Repeat above operation, pure until dividing.
4, with the microbial inoculant that is separated in ammonification medium liquid substratum and nitrosification medium liquid substratum, adopt ordinary method to measure its nitrated performance.
Claims (4)
1, a kind of sieve method of heterotrophic nitrifying bacteria is characterized in that described screening method is:
One, microbial enrichment is cultivated: water sample is left standstill abandoning supernatant, getting active sludge is inoculated in the beef extract-peptone liquid nutrient medium, inoculum size is 5～10%, at 30～35 ℃, the constant temperature vibration is cultivated under the condition of 120～140rpm, when range estimation nutrient solution turbidity increases, enrichment culture liquid is inoculated in the beef extract-peptone solid medium flat board, 30 ℃ of incubators are cultivated 2～3d, picking list bacterium colony is transferred on slant tube, the bacterium colony that will the have various trait again purifying of on beef extract-peptone solid medium flat board, ruling, the dull and stereotyped incubator 2～3d that is inverted repeats above operation, and is pure until dividing;
Two, picking list bacterium colony from the beef extract-peptone solid medium, be inoculated in the ammonification medium liquid substratum, inoculum size is 5～10%, at 30～35 ℃, the constant temperature vibration is cultivated under the condition of 120～140rpm, when range estimation nutrient solution turbidity increases, enrichment culture liquid is inoculated in the ammonification substratum solid medium flat board, 30～35 ℃ of incubators are cultivated 2～3d, picking list bacterium colony is transferred on slant tube, the bacterium colony that will the have various trait again purifying of on ammonification substratum solid medium flat board, ruling, the dull and stereotyped incubator 2～3d that is inverted repeats above operation, and is pure until dividing;
Three, picking list bacterium colony from ammonification substratum solid medium, be inoculated in the nitrosification medium liquid substratum, inoculum size is 5～10%, at 30～35 ℃, the constant temperature vibration is cultivated under the condition of 120～140rpm, when range estimation nutrient solution turbidity increases, enrichment culture liquid is inoculated in the nitrosification substratum solid medium flat board, 30～35 ℃ of incubators are cultivated 2～3d, picking list bacterium colony is transferred on slant tube, the bacterium colony that will the have various trait again purifying of on nitrosification substratum solid medium flat board, ruling, the dull and stereotyped incubator 2～3d that is inverted repeats above operation, pure until dividing, obtain allotrophic nitrobacteria.
2, sieve method of heterotrophic nitrifying bacteria according to claim 1 is characterized in that the composed as follows of described beef-protein medium: extractum carnis 1g, NaCl 1g, peptone 3g, sodium acetate 1g, K
2HPO
40.5g, MnSO
44H
2O 0.01g, FeSO
40.01g, H
2O 1000mL, pH=7.0～7.2.
3, sieve method of heterotrophic nitrifying bacteria according to claim 1 is characterized in that the composed as follows of described ammonification substratum: NH
4Cl 0.382g, sodium acetate 2g, MgSO47H
2O 0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
44H
2O 0.01g, FeSO
40.01g, H
2O 1000mL.
4, sieve method of heterotrophic nitrifying bacteria according to claim 1 is characterized in that the composed as follows of described nitrosification substratum: 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, H
2O 1000mL.
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|---|---|---|---|
| CN 200610010263 CN1884480A (en) | 2006-07-07 | 2006-07-07 | Method for screening allotrophic nitrobacteria |
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|---|---|---|---|
| CN 200610010263 CN1884480A (en) | 2006-07-07 | 2006-07-07 | Method for screening allotrophic nitrobacteria |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952767A (en) * | 2012-05-17 | 2013-03-06 | 哈尔滨工业大学宜兴环保研究院 | Pseudomonas psychrophila HA-4 low-temperature sulfamethoxazole degrading bacteria as well as screening method and application thereof |
| CN103194381A (en) * | 2013-04-03 | 2013-07-10 | 天津市兴源环境技术工程有限公司 | Screening and identification kit of denitrifying bacteria and screening and identification method |
| CN104046575A (en) * | 2013-03-13 | 2014-09-17 | 都江堰惠农生物技术有限责任公司 | Method for separating denitrifying bacteria |
| CN104450568A (en) * | 2014-12-01 | 2015-03-25 | 镇江拜因诺生物科技有限公司 | Preparation method of nitrobacteria inoculation liquid |
| CN104531595A (en) * | 2015-01-20 | 2015-04-22 | 重庆大学 | Method for rapidly screening and separating heterotrophic nitrification-aerobic denitrification bacterium |
| CN106554928A (en) * | 2015-09-30 | 2017-04-05 | 中国石油化工股份有限公司 | A kind of Enrichment culture method of allotrophic nitrobacteria |
-
2006
- 2006-07-07 CN CN 200610010263 patent/CN1884480A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952767A (en) * | 2012-05-17 | 2013-03-06 | 哈尔滨工业大学宜兴环保研究院 | Pseudomonas psychrophila HA-4 low-temperature sulfamethoxazole degrading bacteria as well as screening method and application thereof |
| CN104046575A (en) * | 2013-03-13 | 2014-09-17 | 都江堰惠农生物技术有限责任公司 | Method for separating denitrifying bacteria |
| CN103194381A (en) * | 2013-04-03 | 2013-07-10 | 天津市兴源环境技术工程有限公司 | Screening and identification kit of denitrifying bacteria and screening and identification method |
| CN104450568A (en) * | 2014-12-01 | 2015-03-25 | 镇江拜因诺生物科技有限公司 | Preparation method of nitrobacteria inoculation liquid |
| CN104531595A (en) * | 2015-01-20 | 2015-04-22 | 重庆大学 | Method for rapidly screening and separating heterotrophic nitrification-aerobic denitrification bacterium |
| CN106554928A (en) * | 2015-09-30 | 2017-04-05 | 中国石油化工股份有限公司 | A kind of Enrichment culture method of allotrophic nitrobacteria |
| CN106554928B (en) * | 2015-09-30 | 2020-06-09 | 中国石油化工股份有限公司 | Enrichment culture method of heterotrophic nitrifying bacteria |
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Open date: 20061227 |