CN103805591A - Method for co-immobilization of nitrite bacteria-denitrifying bacteria - Google Patents

Method for co-immobilization of nitrite bacteria-denitrifying bacteria Download PDF

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Publication number
CN103805591A
CN103805591A CN201410082371.9A CN201410082371A CN103805591A CN 103805591 A CN103805591 A CN 103805591A CN 201410082371 A CN201410082371 A CN 201410082371A CN 103805591 A CN103805591 A CN 103805591A
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immobilization
bacteria
nutrient medium
nitrite
immobilized
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CN103805591B (en
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涂宝军
董玉玮
边阁
苗敬芝
徐笑笑
肖杰
朱雯雯
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Xuzhou University of Technology
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Xuzhou University of Technology
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Abstract

The invention discloses a method for co-immobilization of nitrite bacteria-denitrifying bacteria, belonging to a method for co-immobilization of cells. The method comprises the following steps: (1) embedding the denitrifying bacteria by use of sodium alginate and calcium chloride first; (2) mixing immobilized globules prepared in step (1) with the nitrite bacteria and sodium alginate, and dripping into calcium chloride for second embedding; (3) inoculating the co-immobilized nitrite bacteria-denitrifying bacteria prepared in step (2) in a culture medium containing ammonia nitrogen for culturing, wherein the co-immobilized nitrite bacteria-denitrifying bacteria can be used for degradation of the ammonia nitrogen. The method has the advantages that 1, co-immobilization culture of the nitrite bacteria and the denitrifying bacteria in the same carrier is realized for the first time; 2, the method for preparing the co-immobilized globules is simple and convenient, the operation procedures are easy to control, and the prepared co-immobilized globules are easy to store and reuse; 3, due to the co-immobilization culture of the nitrite bacteria and the denitrifying bacteria, the co-immobilized nitrite bacteria-denitrifying bacteria have higher denitrification efficiency than the traditional strain which is not immobilized or immobilized simplex strain, and is easy to be separated from the water body and recyclable.

Description

A kind of method of co-immobilization nitrite bacteria-denitrifying bacterium
Technical field
The present invention relates to a kind of method of co-immobilization cell, be specifically related to a kind of method of co-immobilization nitrite bacteria-denitrifying bacterium.
Background technology
In recent years, many large-lot producer, factory discharge does not in large quantities have treated industrial sewage, such as coking chemical waste water, coal pressure gasification waste water, nitrogenous fertilizer waste water etc., cause the sharply increase of nitrogenous compound, thereby cause serious polluted by nitrogen phenomenon, cause the series of environmental problems such as eutrophication and water ecosystem disorder of water body, as affect fish and some the hydrobiological oxygen transmission in water, cause their blood to reduce in conjunction with oxygen ability, cause fish and some hydrobiont death, when serious, even can destroy the eubiosis.Therefore, the solution of polluted by nitrogen problem is very urgent.
The application of co-immobilization cell technology in nitrite bacteria and denitrifying bacterium at present still awaits further research.
Summary of the invention
The object of the invention is to provide a kind of method of co-immobilization nitrite bacteria-denitrifying bacterium, by the denitrogenation of co-immobilization cell, compared with traditional denitride technology, there is feature quick, efficient, that can reuse.
The object of the present invention is achieved like this: take the bacterial classification of nitrite bacteria, denitrifying bacterium liquid culture as co-immobilization object, adopt immobilized cell technology, first denitrifying bacterium is carried out to embedding with sodium alginate and calcium chloride, immobilized spherule after embedding is mixed with nitrite bacteria and sodium alginate again, splash into and in calcium chloride, carry out embedding for the second time, 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 of selecting is the Nitrosomonas of nitrite bacteria and denitrifying bacterium two large classes nitrosomonas sp.with aerobic pseudomonas pseudomonassp., after ordinary method activation, adopt the liquid nutrient medium that contains ammonia nitrogen to cultivate nitrite bacteria, adopt the liquid nutrient medium that contains nitric nitrogen to cultivate denitrifying bacterium;
(2) embedding for the first time: be chosen at the good denitrifying bacterium of growing way in liquid nutrient medium, precipitation, abandons supernatant liquor, adds sodium alginate to mix, and final concentration is at 40-45g/L.Draw mixed solution and splash into the CaCl of 19-21g/L with No. 5 syringes 2in solution, become bead, 4 ℃ of fixing 6h of refrigerator, deionized water rinsing 2-3 time for bead;
(3) embedding for the second time: be chosen at the good nitrite bacteria of growing way in liquid nutrient medium, precipitation, abandon supernatant liquor, add sodium alginate to mix, final concentration, at 40-45g/L, adds the immobilized spherule in step (2), uses the glass pipette of diameter 0.5cm-0.7cm after mixing, absorption is mixed with the immobilized spherule of bacterium liquid, splashes into the CaCl of 19-21g/L 2in solution, become bead, 4 ℃ of fixing 4-6h of refrigerator, deionized water rinsing 2-3 time for bead;
(4) preparation simulated sewage liquid nutrient medium: in simulated sewage liquid nutrient medium, ammonia-nitrogen content is 200-250mg/L, add calcium carbonate as carbon source, in 250mL shaking flask, add 100mL-160mL simulated sewage liquid nutrient medium: be 2/mL liquid nutrient medium according to inoculum size, add co-immobilization bead, 28 ℃-30 ℃, 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 ℃-28 ℃, 130-160r/min shaking table is cultivated 24h-48h and carried out the anti-nitration reaction stage;
(6) according to the content of State Environmental Protection Administration's " water and effluent monitoring analytical procedure " mensuration nitrite nitrogen, ammonia nitrogen, nitrate nitrogen.
Beneficial effect, owing to having adopted such scheme: immobilized cell technology has huge superiority and development potentiality in field of waste water treatment.Interaction by means of two or more microorganism in same environment realizes.Some bacterial classifications in mixed bacterium can provide important degrading enzyme like this, and other bacterial classification can provide tensio-active agent or somatomedin, and their physiological property can be supplemented mutually.Different cells are fixed in identical carrier simultaneously and form 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 functionalitys in same system.Because the product of nitrite bacteria can be used as the substrate of denitrifying bacterium, nitrosification and two stages of denitrification can complete in same reactor, realize short distance nitration-denitrification, improve the efficiency of biological denitrificaion.Concrete following advantage:
(1) the present invention has realized 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 of making is easy to preserve and recycling;
(3) cultivate by nitrite bacteria, denitrifying bacterium co-immobilization, have than tradition not immobilized bacteria or immobilization single culture nitric efficiency high, the advantage such as easily separate, can reuse with water body.
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 the bacterial classification in Chinese Academy of Sciences's outsourcing, after the order-checking of Shanghai Sheng Gong biotech firm is identified, be the Nitrosomonas of the large class of nitrite bacteria nitrosomonas sp.aerobic pseudomonas with the large class of denitrifying bacterium pseudomonassp.: existing by the laboratory preservation of Xuzhou Engineering Institute food microbiology.
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 3mix dissolving, 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 3mix dissolving, pH 7.5, NH 3-N concentration is 206 mg/L.
Embodiment 1: take the bacterial classification of nitrite bacteria, denitrifying bacterium liquid culture as co-immobilization object, adopt immobilized cell technology, first denitrifying bacterium is carried out to embedding with sodium alginate and calcium chloride, immobilized spherule after embedding is mixed with nitrite bacteria and sodium alginate again, splash into and in calcium chloride, carry out embedding for the second time, 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 of selecting is the Nitrosomonas of nitrite bacteria and denitrifying bacterium two large classes nitrosomonas sp.with aerobic pseudomonas pseudomonassp., after ordinary method activation, adopt the liquid nutrient medium that contains ammonia nitrogen to cultivate nitrite bacteria, adopt the liquid nutrient medium that contains nitric nitrogen to cultivate denitrifying bacterium;
(2) embedding for the first time: be chosen at the good denitrifying bacterium of growing way in liquid nutrient medium, precipitation, abandons supernatant liquor, adds sodium alginate to mix, and final concentration is at 40-45g/L.Draw mixed solution and splash into the CaCl of 19-21g/L with No. 5 syringes 2in solution, become bead, 4 ℃ of fixing 6h of refrigerator, deionized water rinsing 2-3 time for bead;
(3) embedding for the second time: be chosen at the good nitrite bacteria of growing way in liquid nutrient medium, precipitation, abandon supernatant liquor, add sodium alginate to mix, final concentration, at 40-45g/L, adds the immobilized spherule in step (2), uses the glass pipette of diameter 0.5cm-0.7cm after mixing, absorption is mixed with the immobilized spherule of bacterium liquid, splashes into the CaCl of 19-21g/L 2in solution, become bead, 4 ℃ of fixing 4-6h of refrigerator, deionized water rinsing 2-3 time for bead;
(4) preparation simulated sewage liquid nutrient medium: in simulated sewage liquid nutrient medium, ammonia-nitrogen content is 200-250mg/L, add calcium carbonate as carbon source, in 250mL shaking flask, add 100mL-160mL simulated sewage liquid nutrient medium: be 2/mL liquid nutrient medium according to inoculum size, add co-immobilization bead, 28 ℃-30 ℃, 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 ℃-28 ℃, 130-160r/min shaking table is cultivated 24h-48h and carried out the anti-nitration reaction stage;
(6) according to the content of State Environmental Protection Administration's " water and effluent monitoring analytical procedure " mensuration nitrite nitrogen, ammonia nitrogen, nitrate nitrogen.
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 to mix, sodium alginate final concentration is 40-45g/L, draws mixed solution and splash into the CaCl of 21g/L with No. 5 syringes 2in solution, become bead, the about 0.3cm of particle diameter, 4 ℃ of fixing 4h of refrigerator.Deionized water rinsing 2-3 time for bead.Be chosen at the good denitrifying bacterium of growing way in liquid nutrient medium, precipitation, abandon supernatant liquor, according to 20 nitrite bacteria beads that fixed of every gram of thalline access, mix, according to denitrifying bacterium: sodium alginate=1:1(w/v) ratio add sodium alginate to mix, sodium alginate final concentration is 40-45g/L, draw bead and mixed solution with the Glass tubing of diameter 0.5cm, splash into the CaCl of 21g/L 2in solution, become co-immobilization bead, 4 ℃ of fixing 4h of refrigerator.
2, denitrification process
Be 2-3/mL co-immobilization bead according to inoculum size, join in the 250mL shaking flask that fills 140mL simulated sewage liquid nutrient medium, 8.0,30 ℃ of pH, 110-140r/min shaking table is cultivated.Every 4-6h measures ammonia nitrogen, nitrite nitrogen, nitrate nitrogen content, in the time that nitrite nitrogen content is higher, adds 5g/L Trisodium Citrate, continues to measure ammonia nitrogen, nitrite nitrogen, nitrate nitrogen content.Result is as follows.
When the anti-54h of nitrosification, 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; While adding after Trisodium Citrate 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, reuse technique
In the time that nitrite nitrogen content is reduced to lower value by maximum value, old simulated sewage liquid nutrient medium is outwelled, rejoined 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.Change after culture medium culturing for the 4th time, co-immobilization bead quantity has reduced 3/4, therefore reusable 3-4 time of co-immobilization bead.
4, cryopreservation technique
The co-immobilization bead preparing is placed on after 4 ℃ of Refrigerator store 7d, adds simulated sewage liquid nutrient medium to cultivate, and result shows, after cryopreservation, noticeable change does not occur for the denitrogenation of co-immobilization bead and nitrite nitrogen removal capacity.
Above result shows: preparation method is simple for co-immobilization nitrosification-denitrifying bacterium, and nitric efficiency is high, reusable, is easy to preserve.

Claims (1)

1. the method for a co-immobilization nitrite bacteria-denitrifying bacterium, it is characterized in that: take the bacterial classification of nitrite bacteria, denitrifying bacterium liquid culture as co-immobilization object, adopt immobilized cell technology, first denitrifying bacterium is carried out to embedding with sodium alginate and calcium chloride, immobilized spherule after embedding is mixed with nitrite bacteria and sodium alginate again, splash in calcium chloride and carry out embedding for the second time, 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 of selecting is the Nitrosomonas of nitrite bacteria and denitrifying bacterium two large classes nitrosomonas sp.with aerobic pseudomonas pseudomonassp., after ordinary method activation, adopt the liquid nutrient medium that contains ammonia nitrogen to cultivate nitrite bacteria, adopt the liquid nutrient medium that contains nitric nitrogen to cultivate denitrifying bacterium;
(2) embedding for the first time: be chosen at the good denitrifying bacterium of growing way in liquid nutrient medium, precipitation, abandons supernatant liquor, adds sodium alginate to mix, and final concentration is at 40-45g/L; Draw mixed solution and splash into the CaCl of 19-21g/L with No. 5 syringes 2in solution, become bead, 4 ℃ of fixing 6h of refrigerator, deionized water rinsing 2-3 time for bead;
(3) embedding for the second time: be chosen at the good nitrite bacteria of growing way in liquid nutrient medium, precipitation, abandon supernatant liquor, add sodium alginate to mix, final concentration, at 40-45g/L, adds the immobilized spherule in step (2), uses the glass pipette of diameter 0.5cm-0.7cm after mixing, absorption is mixed with the immobilized spherule of bacterium liquid, splashes into the CaCl of 19-21g/L 2in solution, become bead, 4 ℃ of fixing 4-6h of refrigerator, deionized water rinsing 2-3 time for bead;
(4) preparation simulated sewage liquid nutrient medium: in simulated sewage liquid nutrient medium, ammonia-nitrogen content is 200-250mg/L, add calcium carbonate as carbon source, in 250mL shaking flask, add 100mL-160mL simulated sewage liquid nutrient medium: be 2/mL liquid nutrient medium according to inoculum size, add co-immobilization bead, 28 ℃-30 ℃, 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 ℃-28 ℃, 130-160r/min shaking table is cultivated 24h-48h and carried out the anti-nitration reaction stage;
(6) according to the content of State Environmental Protection Administration's " water and effluent monitoring analytical procedure " mensuration nitrite nitrogen, ammonia nitrogen, nitrate nitrogen.
CN201410082371.9A 2014-03-07 2014-03-07 A kind of method of co-immobilization nitrite bacteria-denitrifying bacterium Expired - Fee Related CN103805591B (en)

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CN108342380A (en) * 2017-01-24 2018-07-31 苏州百源基因技术有限公司 A kind of common immobilization method and the application of archaeal dna polymerase and reverse transcriptase
CN109234265A (en) * 2018-10-10 2019-01-18 中南民族大学 A kind of microcarrier and preparation method thereof improving nitrobacteria culture density
CN110092482A (en) * 2019-03-22 2019-08-06 山东大学 A method of realizing that synchronous PND-DNRA handles carbon containing, nitrogen waste water based on immobilized spherule
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

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108342380A (en) * 2017-01-24 2018-07-31 苏州百源基因技术有限公司 A kind of common immobilization method and the application of archaeal dna polymerase and reverse transcriptase
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
CN109234265A (en) * 2018-10-10 2019-01-18 中南民族大学 A kind of microcarrier and preparation method thereof improving nitrobacteria culture density
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
CN110092482A (en) * 2019-03-22 2019-08-06 山东大学 A method of realizing that synchronous PND-DNRA handles carbon containing, nitrogen waste water based on immobilized spherule
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

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