CN103805591B - A kind of method of co-immobilization nitrite bacteria-denitrifying bacterium - Google Patents
A kind of method of co-immobilization nitrite bacteria-denitrifying bacterium Download PDFInfo
- Publication number
- CN103805591B CN103805591B CN201410082371.9A CN201410082371A CN103805591B CN 103805591 B CN103805591 B CN 103805591B CN 201410082371 A CN201410082371 A CN 201410082371A CN 103805591 B CN103805591 B CN 103805591B
- Authority
- CN
- China
- Prior art keywords
- immobilization
- denitrifying bacterium
- nutrient medium
- nitrite bacteria
- liquid nutrient
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
A method for co-immobilization nitrite bacteria-denitrifying bacterium, belongs to the method for co_immobilized cell.The method comprising the steps of: (1) first embeds denitrifying bacterium with sodium alginate and calcium chloride; (2) immobilized spherule obtained in step (1) is mixed with nitrite bacteria and sodium alginate again, in instillation calcium chloride, carry out second time embedding; (3) co-immobilization nitrosification-denitrifying bacterium obtained in step (2) is inoculated in the substratum containing ammonia nitrogen and cultivates, can be used for the degraded of ammonia nitrogen.Advantage: 1, achieve nitrite bacteria, the denitrifying bacterium co-immobilization in identical carrier first and cultivate; 2, prepare co-immobilization bead method easy, schedule of operation is easy to control, and the co-immobilization bead made is easy to preserve and recycling; 3, cultivated by nitrite bacteria, denitrifying bacterium co-immobilization, have than the non-immobilized bacteria of tradition or immobilization single culture nitric efficiency is high, be easily separated with water body, can reuse.
Description
Technical field
The present invention relates to a kind of method of co_immobilized cell, be specifically related to the method for a kind of co-immobilization nitrite bacteria-denitrifying bacterium.
Background technology
In recent years, many large-lot producer, factory discharge in large quantities does not have treated industrial sewage, such as coking chemical waste water, coal pressure gasification wastewater, nitrogenous fertilizer waste water etc., cause the sharply increase of nitrogenous compound, thus cause serious polluted by nitrogen phenomenon, the series of environmental problems such as cause the eutrophication of water body and water ecosystem is disorderly, as affected fish in water and some hydrobiological oxygen transmission, their blood is caused to reduce in conjunction with oxygen ability, cause fish and the death of some hydrobionts, time serious, even can destroy the eubiosis.Therefore, the solution of polluted by nitrogen problem is very urgent.
The application of current co_immobilized cell technology in nitrite bacteria and denitrifying bacterium still awaits further research.
Summary of the invention
The object of the invention is the method that a kind of co-immobilization nitrite bacteria-denitrifying bacterium will be provided, by co_immobilized cell denitrogenation, compared with traditional denitride technology, there is feature that is quick, efficient, that can reuse.
The object of the present invention is achieved like this: with the bacterial classification of nitrite bacteria, denitrifying bacterium liquid culture for co-immobilization object, adopt immobilized cell technology, first denitrifying bacterium is embedded with sodium alginate and calcium chloride, immobilized spherule after embedding is mixed with nitrite bacteria and sodium alginate again, second time embedding is carried out in instillation calcium chloride, form co-immobilization nitrosification-denitrifying bacterium, can be used for the degraded of ammonia nitrogen.
Concrete steps are as follows:
(1) actication of culture, liquid culture: the bacterial classification selected is the Nitrosomonas of the large class of nitrite bacteria and denitrifying bacterium two
nitrosomonas sp.with aerobic pseudomonas
pseudomonassp., after ordinary method activation, adopt the liquid nutrient medium containing ammonia nitrogen to cultivate nitrite bacteria, adopt the liquid nutrient medium containing nitric nitrogen to cultivate denitrifying bacterium;
(2) first time embedding: be chosen at the good denitrifying bacterium of growing way in liquid nutrient medium, precipitation, abandons supernatant liquor, add sodium alginate mixing, final concentration is at 40-45g/L.The CaCl of mixed solution instillation 19-21g/L is drawn with No. 5 syringes
2in solution, become bead, 6h fixed by 4 DEG C of refrigerators, bead deionized water rinsing 2-3 time;
(3) second time embedding: be chosen at the good nitrite bacteria of growing way in liquid nutrient medium, precipitation, abandon supernatant liquor, add sodium alginate mixing, final concentration, at 40-45g/L, adds the immobilized spherule in step (2), with the glass pipette of diameter 0.5cm-0.7cm after mixing, draw the immobilized spherule being mixed with bacterium liquid, the CaCl of instillation 19-21g/L
2in solution, become bead, 4-6h fixed by 4 DEG C of refrigerators, bead deionized water rinsing 2-3 time;
(4) simulated sewage liquid nutrient medium is prepared: in simulated sewage liquid nutrient medium, ammonia-nitrogen content is 200-250mg/L, add calcium carbonate as carbon source, 100mL-160mL simulated sewage liquid nutrient medium is added: be 2/mL liquid nutrient medium according to inoculum size in 250mL shaking flask, add co-immobilization bead, 28 DEG C-30 DEG C, 110-140r/min shaking table is cultivated 48h-60h and is carried out the nitrosation reaction stage;
(5) add organic carbon source: add 4-6g/L Trisodium Citrate as organic carbon source, 26 DEG C-28 DEG C, 130-160r/min shaking table is cultivated 24h-48h and is carried out the anti-nitration reaction stage;
(6) content of nitrite nitrogen, ammonia nitrogen, nitrate nitrogen is measured according to State Environmental Protection Administration's " water and waste water method for monitoring and analyzing ".
Beneficial effect, owing to have employed such scheme: immobilized cell technology has huge superiority and development potentiality in field of waste water treatment.The interaction of microorganism in same environment by means of two or more realizes.Some bacterial classifications in such mixed bacterium can provide important degrading enzyme, and other bacterial classification can provide tensio-active agent or somatomedin, and their physiological property can be supplemented mutually.Different cell is fixed in identical carrier simultaneously and forms co-immobilization system, combine the advantage of mixed fermentation and immobilization technology.System stability, can act synergistically the cell and crganelle of several difference in functionality in same system.Product due to nitrite bacteria can be used as the substrate of denitrifying bacterium, and nitrosification and denitrification two benches can complete in same reactor, realize short-cut nitrification-denitrification, improves the efficiency of biological denitrificaion.Concrete following advantage:
(1) the present invention achieves the co-immobilization cultivation in identical carrier of nitrite bacteria, denitrifying bacterium first;
(2) prepare co-immobilization bead method easy, schedule of operation is easy to control, and the co-immobilization bead made is easy to preserve and recycling;
(3) cultivated by nitrite bacteria, denitrifying bacterium co-immobilization, have than the non-immobilized bacteria of tradition or immobilization single culture nitric efficiency is high, be easily separated with water body, the advantage such as can to reuse.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail:
1, bacterial classification: bacterial classification is nitrite bacteria and denitrifying bacterium.
Nitrite bacteria (
nitrosomonas sp.), denitrifying bacterium (
pseudomonassp.) be bacterial classification in Chinese Academy of Sciences's outsourcing, after the order-checking qualification of Shanghai Sheng Gong biotech firm, be the Nitrosomonas of the large class of nitrite bacteria
nitrosomonas sp.with the aerobic pseudomonas of the large class of denitrifying bacterium
pseudomonassp.: existing by Xuzhou Engineering Institute food microbiology Laboratories Accession.
2, substratum
Nitrite bacteria liquid nutrient medium: 4g/L (NH
4)
2sO
4, 1g/L K
2hPO
4, 0.5g/L MgSO
4, 2g/L NaCl, 0.4g/L FeSO
4, 5g/L CaCO
3mixing is dissolved, pH 8.0-8.2;
Denitrifying bacterium liquid culture: 2g/LKNO
3, 0.2g/LMgSO
4, 1g/LK
2hPO
4, 1g/LKH
2pO
4, 5g/L Trisodium Citrate, pH 7.2-7.6;
Simulated sewage liquid nutrient medium: 0.65g/L NH
4cl, 5g/L Trisodium Citrate (adding afterwards), 0.2g/L K
2hPO
4, 0.1g/L MgSO
4, 0.08g/L FeSO
4, 2g/L CaCO
3mixing is dissolved, pH 7.5, NH
3-N concentration is 206 mg/L.
Embodiment 1: with the bacterial classification of nitrite bacteria, denitrifying bacterium liquid culture for co-immobilization object, adopt immobilized cell technology, first denitrifying bacterium is embedded with sodium alginate and calcium chloride, immobilized spherule after embedding is mixed with nitrite bacteria and sodium alginate again, second time embedding is carried out in instillation calcium chloride, form co-immobilization nitrosification-denitrifying bacterium, can be used for the degraded of ammonia nitrogen.
Concrete steps are as follows:
(1) actication of culture, liquid culture: the bacterial classification selected is the Nitrosomonas of the large class of nitrite bacteria and denitrifying bacterium two
nitrosomonas sp.with aerobic pseudomonas
pseudomonassp., after ordinary method activation, adopt the liquid nutrient medium containing ammonia nitrogen to cultivate nitrite bacteria, adopt the liquid nutrient medium containing nitric nitrogen to cultivate denitrifying bacterium;
(2) first time embedding: be chosen at the good denitrifying bacterium of growing way in liquid nutrient medium, precipitation, abandons supernatant liquor, add sodium alginate mixing, final concentration is at 40-45g/L.The CaCl of mixed solution instillation 19-21g/L is drawn with No. 5 syringes
2in solution, become bead, 6h fixed by 4 DEG C of refrigerators, bead deionized water rinsing 2-3 time;
(3) second time embedding: be chosen at the good nitrite bacteria of growing way in liquid nutrient medium, precipitation, abandon supernatant liquor, add sodium alginate mixing, final concentration, at 40-45g/L, adds the immobilized spherule in step (2), with the glass pipette of diameter 0.5cm-0.7cm after mixing, draw the immobilized spherule being mixed with bacterium liquid, the CaCl of instillation 19-21g/L
2in solution, become bead, 4-6h fixed by 4 DEG C of refrigerators, bead deionized water rinsing 2-3 time;
(4) simulated sewage liquid nutrient medium is prepared: in simulated sewage liquid nutrient medium, ammonia-nitrogen content is 200-250mg/L, add calcium carbonate as carbon source, 100mL-160mL simulated sewage liquid nutrient medium is added: be 2/mL liquid nutrient medium according to inoculum size in 250mL shaking flask, add co-immobilization bead, 28 DEG C-30 DEG C, 110-140r/min shaking table is cultivated 48h-60h and is carried out the nitrosation reaction stage;
(5) add organic carbon source: add 4-6g/L Trisodium Citrate as organic carbon source, 26 DEG C-28 DEG C, 130-160r/min shaking table is cultivated 24h-48h and is carried out the anti-nitration reaction stage;
(6) content of nitrite nitrogen, ammonia nitrogen, nitrate nitrogen is measured according to State Environmental Protection Administration's " water and waste water method for monitoring and analyzing ".
Embodiment 2: co-immobilization nitrosification-denitrifying bacterium
1, co-immobilization technique
Be chosen at the good nitrite bacteria of growing way in liquid nutrient medium, precipitation, abandon supernatant liquor, according to nitrite bacteria: sodium alginate=1:1(w/v) ratio get a certain amount of thalline, add sodium alginate mixing, sodium alginate final concentration is 40-45g/L, draws with No. 5 syringes the CaCl that mixed solution is added dropwise to 21g/L
2in solution, become bead, particle diameter is about 0.3cm, and 4h fixed by 4 DEG C of refrigerators.Bead deionized water rinsing 2-3 time.Be chosen at the good denitrifying bacterium of growing way in liquid nutrient medium, precipitation, abandon supernatant liquor, 20 nitrite bacteria beads fixed are accessed according to every gram of thalline, mixing, according to denitrifying bacterium: sodium alginate=1:1(w/v) ratio add sodium alginate mixing, sodium alginate final concentration is 40-45g/L, the Glass tubing being about 0.5cm with diameter draws bead and mixed solution, is added dropwise to the CaCl of 21g/L
2in solution, become co-immobilization bead, 4h fixed by 4 DEG C of refrigerators.
2, denitrification process
Be 2-3/mL co-immobilization bead according to inoculum size, join in the 250mL shaking flask filling 140mL simulated sewage liquid nutrient medium, pH 8.0,30 DEG C, 110-140r/min shaking table is cultivated.Every 4-6h measures ammonia nitrogen, nitrite nitrogen, nitrate nitrogen content, when nitrite nitrogen content is higher, adds 5g/L Trisodium Citrate, continues to measure ammonia nitrogen, nitrite nitrogen, nitrate nitrogen content.Result is as follows.
During nitrosification anti-54h, ammonia-nitrogen content has been down to 7.85 mg/L, and when ammonia nitrogen removal frank is 96.19%, 48h, nitrite nitrogen content reaches 180.55 mg/L; After adding Trisodium Citrate, during 78h, nitrite nitrogen has been down to 8.25 mg/L, and clearance is 95.43%.
Result shows, co-immobilization bead nitrogen removal performance is good, and speed of response is very fast.
3, technique is reused
When nitrite nitrogen content is reduced to lower value by maximum value, old simulated sewage liquid nutrient medium is outwelled, rejoin new simulated sewage liquid nutrient medium, again cultivate and measure ammonia nitrogen, nitrite nitrogen, nitrate nitrogen content.Result is as follows.
Increase along with reusing number of times, the color of co-immobilization bead starts bleach, and the small ball's diameter increases gradually and breaks, and quantity reduces gradually.After 4th time replaced medium is cultivated, co-immobilization bead quantity decreases 3/4, therefore reusable 3-4 time of co-immobilization bead.
4, cryopreservation technique
After the co-immobilization bead prepared is placed on 4 DEG C of Refrigerator store 7d, add simulated sewage liquid nutrient medium and cultivate, result shows, after cryopreservation, noticeable change does not occur for the denitrogenation of co-immobilization bead and nitrite nitrogen removal capacity.
Above result shows: co-immobilization nitrosification-denitrifying bacterium preparation method is simple, and nitric efficiency is high, reusable, is easy to preserve.
Claims (1)
1. the method for co-immobilization nitrite bacteria-denitrifying bacterium, it is characterized in that: with the bacterial classification of nitrite bacteria, denitrifying bacterium liquid culture for co-immobilization object, adopt immobilized cell technology, first denitrifying bacterium is embedded with sodium alginate and calcium chloride, immobilized spherule after embedding is mixed with nitrite bacteria and sodium alginate again, carry out second time embedding in instillation calcium chloride, form co-immobilization nitrosification-denitrifying bacterium, can be used for the degraded of ammonia nitrogen;
Concrete steps are as follows:
(1) actication of culture, liquid culture: the bacterial classification selected is the Nitrosomonas of the large class of nitrite bacteria and denitrifying bacterium two
nitrosomonas sp.with aerobic pseudomonas
pseudomonassp., after ordinary method activation, adopt the liquid nutrient medium containing ammonia nitrogen to cultivate nitrite bacteria, adopt the liquid nutrient medium containing nitric nitrogen to cultivate denitrifying bacterium;
(2) first time embedding: be chosen at the good denitrifying bacterium of growing way in liquid nutrient medium, precipitation, abandons supernatant liquor, add sodium alginate mixing, final concentration is at 40-45g/L; The CaCl of mixed solution instillation 19-21g/L is drawn with No. 5 syringes
2in solution, become bead, 6h fixed by 4 DEG C of refrigerators, bead deionized water rinsing 2-3 time;
(3) second time embedding: be chosen at the good nitrite bacteria of growing way in liquid nutrient medium, precipitation, abandon supernatant liquor, add sodium alginate mixing, final concentration, at 40-45g/L, adds the immobilized spherule in step (2), with the glass pipette of diameter 0.5cm-0.7cm after mixing, draw the immobilized spherule being mixed with bacterium liquid, the CaCl of instillation 19-21g/L
2in solution, become bead, 4-6h fixed by 4 DEG C of refrigerators, bead deionized water rinsing 2-3 time;
(4) simulated sewage liquid nutrient medium is prepared: in simulated sewage liquid nutrient medium, ammonia-nitrogen content is 200-250mg/L, add calcium carbonate as carbon source, 100mL-160mL simulated sewage liquid nutrient medium is added: be 2/mL liquid nutrient medium according to inoculum size in 250mL shaking flask, add co-immobilization bead, 28 DEG C-30 DEG C, 110-140r/min shaking table is cultivated 48h-60h and is carried out the nitrosation reaction stage;
(5) add organic carbon source: add 4-6g/L Trisodium Citrate as organic carbon source, 26 DEG C-28 DEG C, 130-160r/min shaking table is cultivated 24h-48h and is carried out the anti-nitration reaction stage;
(6) content of nitrite nitrogen, ammonia nitrogen, nitrate nitrogen is measured according to State Environmental Protection Administration's " water and waste water method for monitoring and analyzing ".
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410082371.9A CN103805591B (en) | 2014-03-07 | 2014-03-07 | A kind of method of co-immobilization nitrite bacteria-denitrifying bacterium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410082371.9A CN103805591B (en) | 2014-03-07 | 2014-03-07 | A kind of method of co-immobilization nitrite bacteria-denitrifying bacterium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103805591A CN103805591A (en) | 2014-05-21 |
| CN103805591B true CN103805591B (en) | 2015-10-28 |
Family
ID=50702978
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410082371.9A Expired - Fee Related CN103805591B (en) | 2014-03-07 | 2014-03-07 | A kind of method of co-immobilization nitrite bacteria-denitrifying bacterium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103805591B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108342380B (en) * | 2017-01-24 | 2021-04-27 | 苏州百源基因技术有限公司 | Co-immobilization method and application of DNA polymerase and reverse transcriptase |
| CN109234265B (en) * | 2018-10-10 | 2022-04-01 | 中南民族大学 | Microcarrier for improving culture density of nitrobacteria and preparation method thereof |
| CN110092482B (en) * | 2019-03-22 | 2021-08-10 | 山东大学 | Method for realizing synchronous PND-DNRA (pneumatic digital-to-analogue conversion) treatment of carbon-nitrogen-containing wastewater based on immobilized beads |
| CN111454935A (en) * | 2020-04-25 | 2020-07-28 | 北京博泰至淳生物科技有限公司 | Immobilized enzyme for sewage denitrification and preparation method and application thereof |
| CN111500567A (en) * | 2020-04-28 | 2020-08-07 | 深圳文科园林股份有限公司 | Preparation method of co-immobilized pellet for synchronous nitrification and denitrification and co-immobilized pellet |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1699220A (en) * | 2005-05-23 | 2005-11-23 | 南京工业大学 | Method and apparatus for immobilizing cells to treat wastewater |
-
2014
- 2014-03-07 CN CN201410082371.9A patent/CN103805591B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1699220A (en) * | 2005-05-23 | 2005-11-23 | 南京工业大学 | Method and apparatus for immobilizing cells to treat wastewater |
Non-Patent Citations (4)
| Title |
|---|
| Characterization of nitrifying and denitrifying bacteria coimmobilized in PVA and kinetics model of biological nitrogen removal by coimmobilized cells;Guo-min Cao et al.;《Enzyme and Microbial Technology》;20021231;第30卷;49-55 * |
| The nature of interaction between immobilized nitrification and denitrification bacteria;E. Kotlar et al.;《Journal of Biotechnology》;19961231;第51卷;251-258 * |
| 亚硝化-反硝化固定化细胞捷径生物脱氮的研究;廖雪义 蓝 荣;《襄樊学院学报》;20080831;第29卷(第8期);35-39 * |
| 共固定化技术研究进展;薄涛 侯红萍;《酿酒科技》;20101231(第193期);77-79 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103805591A (en) | 2014-05-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mujtaba et al. | Removal of nutrients and COD from wastewater using symbiotic co-culture of bacterium Pseudomonas putida and immobilized microalga Chlorella vulgaris | |
| CN103805591B (en) | A kind of method of co-immobilization nitrite bacteria-denitrifying bacterium | |
| CN103695412B (en) | A kind of imbedded microbe microbial inoculum purifying for water body in-situ and preparation method thereof | |
| CN108467118B (en) | Method for removing nitrogen and phosphorus in aquaculture wastewater by using immobilized algae bacteria | |
| CN109956563B (en) | Preparation method and application of efficient aerobic denitrification phosphorus-accumulating bacteria immobilized pellet | |
| CN102392011A (en) | Preparation method and application of immobilized bacteria for improving denitrification efficiency of constructed wetland | |
| Wang et al. | Treatment of rich ammonia nitrogen wastewater with polyvinyl alcohol immobilized nitrifier biofortified constructed wetlands | |
| CN104862257A (en) | Enrichment culture method of indigenous nitrogen transforming microorganism and application of same to treatment of water ammonia-nitrogen pollution | |
| Manonmani et al. | Research advances and challenges in anammox immobilization for autotrophic nitrogen removal | |
| CN106479935B (en) | A kind of preparation method of use in waste water treatment denitrification denitrogenation microbial inoculum | |
| CN104232546A (en) | Construction method and application of immobilized biological bacterium agent for micro-polluted water source | |
| Xiao et al. | Removal of ammonium-N from ammonium-rich sewage using an immobilized Bacillus subtilis AYC bioreactor system | |
| CN106635923A (en) | Method for preparing high-density salt-tolerant denitrifying bacterium agent applicable to wastewater treatment | |
| CN103255123B (en) | Method for mycelium pellet to form mixed mycelium pellet by adsorbing photosynthetic bacteria | |
| CN113151247A (en) | Dunaliella salina-halophilic bacteria immobilized microsphere and preparation method and application thereof | |
| CN105753160A (en) | Preparation method of floating bed composite substrate for efficient nitrogen and phosphorus removal | |
| CN107630012A (en) | A kind of embedding method of aerobic denitrifying bacteria | |
| CN114908002A (en) | Biological nano-selenium reinforced composite strain and application thereof | |
| KR20140074451A (en) | Microbial agent for improving water quality of aquarium | |
| Petrovič et al. | Effect of Chlorella sorokiniana on the biological denitrification of drinking water | |
| CN112322570A (en) | Preparation method and application of humus reducing bacteria growth promoter | |
| CN106701632A (en) | Strain of pseudomonas poae and application thereof to sewage treatment | |
| CN101983938B (en) | Preparation method of bagasse-based biofilm | |
| CN110699313A (en) | Screening and culturing method of specific microbial flora for degrading industrial wastewater | |
| CN114031189B (en) | Method for treating sewage with low carbon-nitrogen ratio |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151028 Termination date: 20170307 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |