CN103911332A - Method for culturing nitrogen and phosphorus removal composite bacteria of black and odorous water body - Google Patents
Method for culturing nitrogen and phosphorus removal composite bacteria of black and odorous water body Download PDFInfo
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Abstract
The invention discloses a method for culturing nitrogen and phosphorus removal composite bacteria of a black and odorous water body. The method comprises the following four steps: (1) collecting a strain sample, (2) carrying out multiplication culturing, (3) screening and acclimating the strain, and (4) purifying the strain. According to the method disclosed by the invention, an indigenous microorganism with a nitrogen and phosphorus removal effect is firstly screened from activated sludge and black and odorous bottom mud of a municipal sewage plant, sewage of the black and odorous water body is used as a main carbon source and energy, other nutrient elements necessary for strain growth are added, and composite bacteria with a nitrogen and phosphorus removal effect are obtained from the local environment by acclimation. The strain cultured by the method disclosed by the invention has the advantages of fast proliferation and obvious nitrogen and phosphorus removal effect on the black and odorous water body, and the like, the composite bacteria obtained by the method disclosed by the invention are used for treating the black and odorous water body, so as to avoid secondary pollution due to use of a chemical water purifier, the safety of the strain does not need to be worried, and the treatment efficiency is much higher than that of cultivation of aquatic plants.
Description
Technical field
The present invention relates to microbial strains culture technique field, be specifically related to a kind of method of cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria.
Background technology.
At the city inland river of China, sealing water body, small watershed all exists serious body eutrophication to pollute, black smelly, peculiar smell, even blue-green algae red tide, is causing huge infringement aspect water body safety and HUMAN HEALTH.The nutritive substance such as nitrogen, phosphorus surplus is to cause the basic reason of the black smelly eutrophication of water body, as N concentration >0.2mg/L in water body, just has the possibility that eutrophication occurs when P concentration >0.02mg/L.Not comprehensive due to the planning of China urban pipe network, the impact that causes many city inland rivers to be all subject to sanitary sewage discharge becomes sewage ditch, loses self-purification capacity.And sanitary sewage is the maximum emission source of the nutritive substance such as nitrogen, phosphorus, the nitrogen phosphorus that therefore how to solve black and odorous water is restoration of the ecosystem city inland river, sealing water body, the key problem of small watershed.The more methods such as chemical water purification agent, additional bacterial classification and planting aquatic plants that add that use at present.Add chemical water purification agent and can reduce fast the phosphorus content in water body, but produce inorganic sediment, cause bed mud state deteriorating, produce secondary pollution, and nitrogen is not acted on; Additional bacterial classification, can increase the quantity of microorganisms of nitrogen and phosphors removal in water body, but has the adaptation of external bacterial classification to water body and the risk of alien species long-term safety, and cost is higher, and the expense of the additional bacterial classification of every square meter water body is in 1-2 unit; Plantation has the waterplant of separating the poly-phosphorus of nitrogen, can absorb the nitrogen phosphorus in water body, but growth cycle is longer, and needs harvesting in time, and labour cost is higher, acts on limited.
In view of the above problems, be necessary to cultivate one and solve the problems referred to above for black and odorous water denitrogenation dephosphorizing composite bacteria method of cultivation.
Summary of the invention
The object of the present invention is to provide one for black and odorous water denitrogenation dephosphorizing composite bacteria method of cultivation, to effectively solving the problem of nitrogen phosphorus surplus in black and odorous water.
Technical scheme of the present invention is:
For a method of cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria, comprise the following steps:
(1) bacterial classification sample collecting: gather respectively the active sludge of municipal wastewater treatment plant and the bed mud of black and odorous water, put into thermostat container stand-by, temperature is 28 ℃～35 ℃;
(2) amplification cultivation: under aerobic condition, add respectively liquid nutrient medium to carry out the steady cultivation of perseverance in two kinds of mud samples, temperature is 28 ℃～35 ℃, and incubation time is 2～3 days;
(3) screening domesticated strain: two kinds of mud mixed liquids are mixed and added in screening domestication substratum according to 1～3:1, and aerobic vibration at 28 ℃～35 ℃ temperature, 2～3 days is an acclimation period, needs 5-8 acclimation period; After each acclimation period finishes, get 5-10mL nutrient solution and be inoculated in fresh screening domestication substratum;
(4) purifying bacterial classification: the bacterial classification having screened is inoculated in activation enrichment medium, 28 ℃～35 ℃ temperature, cultivates 2-3 days, use supercentrifuge centrifugal treating, then with phosphate buffered saline buffer cleaning, the more centrifugal bacterial classification suspension of making.
The allocation ratio of the liquid nutrient medium described in step (2) is: in 1000mL substratum, add K
2hPO
40.0-1.5g, MgSO
47H
2o1-3g, extractum carnis 1-3g, (NH
4)
2sO
41-2g, NaH
2pO43H
2o1-2g, urea 0.2-0.3g, FeSO
47H
2o0.002-0.003g, MnSO
47H
2o0.003-0.004g, ZnSO
47H
2o0.003-0.004g, CoSO
47H
2o0.001-0.002g, glucose 15-16g, adjusting pH value is 7.0, and autoclaving.
The allocation ratio that substratum is tamed in the described screening of step (3) is: the sewage 50-100g, the K that in 1000mL substratum, add black and odorous water
2hPO
41.5-2.5g, NaH
2pO
43H
2o1-2g, MgSO
47H
2o3-4g,, NH
4cl2-3g, vitamins B
10.1-0.2g, adjusting pH value is 7.0, and autoclaving.
The allocation ratio of the activation enrichment medium described in step (4) is: in 1000mL substratum, add NH
4cl2-3g, extractum carnis 5-6g, glucose 15-16g, agar 15-16g, adjusting pH value is 7.0, and autoclaving.
Through identifying, gained bacterial classification comprises following four kinds of microorganisms: bacillus cereus, Paracoccus denitrificans, Pseudomonas stutzeri, subtilis.
First the present invention filters out the indigenous microorganism with denitrogenation dephosphorizing in the active sludge of municipal wastewater factory and black and odorous water bed mud, with the sewage of black and odorous water as main carbon source and energy, add again the required nutritive element of other growth, obtain from the composite bacteria with denitrogenation dephosphorizing in native environment by domestication.It is fast that the bacterial classification that the present invention turns out has propagation, to the feature such as the Nitrogen/Phosphorus Removal of black and odorous water is obvious, the composite bacteria processing black and odorous water that use the present invention obtains had both avoided using the secondary pollution of chemical water purification agent, also do not need to worry the security of bacterial classification, processing efficiency is also far above planting aquatic plants.
Accompanying drawing explanation
Fig. 1 is the schema for black and odorous water denitrogenation dephosphorizing composite bacteria method of cultivation of the present invention.
Embodiment
Embodiment:
Schema for black and odorous water denitrogenation dephosphorizing composite bacteria method of cultivation of the present invention refers to Fig. 1, and concrete implementation step is as follows:
(1) bacterial classification sample collecting: gather the active sludge of Nanjing one municipal wastewater treatment plant and the each 1000mL of bed mud in Yi Heichou inland river, city with sterile sampling bottle respectively, put into thermostat container stand-by, temperature is 28 ℃～35 ℃.
(2) amplification cultivation: add liquid nutrient medium according to the ratio of 10g/L respectively in two kinds of mud samples, and carry out aeration constant temperature culture, temperature is 28 ℃～35 ℃, and incubation time is 2-3 days.Wherein the composition of liquid nutrient medium is: in 1000mL substratum, add K
2hPO
41.5g, MgSO
47H
2o1g, extractum carnis 1g, (NH
4)
2sO
41g, NaH
2pO
43H
2o1g, urea 0.2g, FeSO
47H
2o0.002g, MnSO
47H
2o0.003g, ZnSO
47H
2o0.003g, CoSO
47H
2o0.001g, glucose 15g, adjusting pH value is 7.0, and autoclaving.
(3) screening domesticated strain: two kinds of mud mixed liquids are got to 5mL altogether according to 1:1 ratio and mix, add 1000mL screening domestication substratum, aerobic vibration at 28 ℃～35 ℃ temperature, 2 days is an acclimation period, carries out altogether 6 acclimation periods.After each acclimation period finishes, get 5mL nutrient solution and be inoculated in fresh 1000mL screening domestication substratum.Wherein screening domestication medium component is: the sewage 100g, the K that in 1000mL substratum, add black and odorous water
2hPO
41.5g, NaH
2pO
43H
2o1.0g, MgSO
47H
2o3.0g,, NH
4cl2g, vitamins B
10.1g, adjusting pH value is 7.0, and autoclaving.
(4) purifying bacterial classification: the bacterial classification 5mL having screened is inoculated in 1000mL activation enrichment medium, 28 ℃～35 ℃ temperature, cultivate 2 days, with supercentrifuge (8000r/min) centrifugal treating 10min, clean with phosphate buffered saline buffer again, more centrifugal 10min makes bacterial classification suspension.Wherein activating enrichment culture based component is to add NH in 1000mL substratum
4cl2g, extractum carnis 5g,, glucose 15g, agar 15g, adjusting pH value is 7.0, and autoclaving.
(5) strain identification: the extraction providing with MIDI lipid acid identification systems, the bacterium colony that authentication method purification Identification goes out.The bacterial classification identifying comprises following four kinds of microorganisms, and number consecutively is: A bacillus cereus B Paracoccus denitrificans C Pseudomonas stutzeri D subtilis.
A bacillus cereus falls for oyster white, and irregularly shaped, gramstaining is positive; B Paracoccus denitrificans, milk yellow, the individual ovalize of bacterial strain, gramstaining is negative; C Pseudomonas stutzeri falls for yellow, straight or micro-curved bacilliform, and gramstaining is negative; D bacillus subtilis bacterium colony is micro-yellow, oval to column, Gram-positive.The ratio that four kinds of bacterium survive in substratum is: A bacillus cereus 38%, B Paracoccus denitrificans 35%, C Pseudomonas stutzeri 18%, D subtilis 9%.By the composite bacteria suspension obtaining, at 28-35 ℃ of temperature, add liquid nutrient medium, cultivate 2 days, aerobic treatment is taken from the sewage in black smelly river, experiment discovery, according to 1% dosage, the aerobic treatment of 8-16 hour can efficiently be removed the suspended substance (SS) in sewage, CODcr, ammonia nitrogen and total phosphorus (TP).As table 1, by the processing of 16 hours, can reach the requirement of surface water four class standards.
Table 1
Above-described embodiment is interpreted as being only not used in and limiting the scope of the invention for the present invention is described.After having read the content of the present invention's record, those skilled in the art can make various changes or modifications the present invention, and these equivalences change and modification falls into the scope of the claims in the present invention equally.
Claims (4)
1. for a method of cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria, it is characterized in that, comprise the following steps:
(1) bacterial classification sample collecting: gather respectively the active sludge of municipal wastewater treatment plant and the bed mud of black and odorous water, put into thermostat container stand-by, temperature is 28 ℃～35 ℃;
(2) amplification cultivation: under aerobic condition, add respectively liquid nutrient medium to carry out the steady cultivation of perseverance in two kinds of mud samples, temperature is 28 ℃～35 ℃, and incubation time is 2～3 days;
(3) screening domesticated strain: two kinds of mud mixed liquids are mixed and added in screening domestication substratum according to 1～3:1, and aerobic vibration at 28 ℃～35 ℃ temperature, 2～3 days is an acclimation period, needs 5-8 acclimation period; After each acclimation period finishes, get 5-10mL nutrient solution and be inoculated in fresh screening domestication substratum;
(4) purifying bacterial classification: the bacterial classification having screened is inoculated in activation enrichment medium, 28 ℃～35 ℃ temperature, cultivates 2-3 days, centrifugal treating, then clean then the centrifugal bacterial classification suspension made from phosphate buffered saline buffer.
2. the method for cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria according to claim 1, is characterized in that, the allocation ratio of the described liquid nutrient medium of step (2) is: in 1000mL substratum, add K
2hPO
40.0-1.5g, MgSO
47H
2o1-3g, extractum carnis 1-3g, (NH
4)
2sO
41-2g, NaH
2pO43H
2o1-2g, urea 0.2-0.3g, FeSO
47H
2o0.002-0.003g, MnSO
47H
2o0.003-0.004g, ZnSO
47H
2o0.003-0.004g, CoSO
47H
2o0.001-0.002g, glucose 15-16g, adjusting pH value is 7.0, and autoclaving.
3. the method for cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria according to claim 1, is characterized in that, the allocation ratio that substratum is tamed in the described screening of step (3) is: the sewage 50-100g, the K that in 1000mL substratum, add black and odorous water
2hPO
41.5-2.5g, NaH
2pO
43H
2o1-2g, MgSO
47H
2o3-4g,, NH
4cl2-3g, vitamins B
10.1-0.2g, adjusting pH value is 7.0, and autoclaving.
4. the method for cultivation for black and odorous water denitrogenation dephosphorizing composite bacteria according to claim 1, is characterized in that, the allocation ratio of the described activation enrichment medium of step (4) is: in 1000mL substratum, add NH
4cl2-3g, extractum carnis 5-6g,, glucose 15-16g, agar 15-16g, adjusting pH value is 7.0, and autoclaving.
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Cited By (16)
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| CN104877916A (en) * | 2015-06-01 | 2015-09-02 | 云南省烟草农业科学研究院 | Paracoccus limosus, bacterium thereof, method for preparing bacterium and application thereof |
| CN106148246A (en) * | 2016-08-30 | 2016-11-23 | 蓝德环保科技集团股份有限公司 | Purify compounding microbial inoculum of black and odorous water and preparation method thereof |
| CN106337028A (en) * | 2015-07-17 | 2017-01-18 | 上海海洋大学 | Cleaning agent, and preparation method and application thereof |
| CN106396133A (en) * | 2016-12-01 | 2017-02-15 | 东莞理工学院 | Method for treating black and odorous water by using ethanol making straw waste residue immobilized phosphorus-accumulating bacterium |
| CN106676022A (en) * | 2015-11-06 | 2017-05-17 | 丹阳市尚德生物科技有限公司 | Method for preparing microbe bacteria liquid for treating black-odor riverway |
| CN106745804A (en) * | 2016-12-16 | 2017-05-31 | 北京桑德环境工程有限公司 | A kind of microorganism High-speed clarification agent for black and odorous water and preparation method thereof |
| CN108178327A (en) * | 2017-12-26 | 2018-06-19 | 中冶京诚工程技术有限公司 | For the microbe to screen enrichment method of black and odorous water river regulation and its application |
| CN109052624A (en) * | 2018-08-08 | 2018-12-21 | 中国科学院水生生物研究所 | A kind of method and device of movable purifying sewage and black and odorous water |
| CN109231711A (en) * | 2018-11-06 | 2019-01-18 | 江苏澳洋生态园林股份有限公司 | A kind of black-odor riverway sewage disposal system and method |
| CN109576189A (en) * | 2018-12-29 | 2019-04-05 | 合肥市东方美捷分子材料技术有限公司 | A kind of black and odorous water composite bacteria agent and its preparation method and application |
| CN109576201A (en) * | 2019-01-31 | 2019-04-05 | 上海同瑞环保科技有限公司 | A method of culture indigenous microorganism efficiently removes water body ammonia nitrogen |
| CN109678257A (en) * | 2019-03-01 | 2019-04-26 | 北京高能时代环境技术股份有限公司 | A method of being suitable for the functional flora construction method and application flora improvement black and odorous water of black and odorous water improvement |
| CN110484458A (en) * | 2018-08-28 | 2019-11-22 | 华中科技大学 | It is a kind of to handle acclimation and screening method of the low C/N than the aerobic denitrification polyP bacteria of sewage |
| CN111268857A (en) * | 2020-01-21 | 2020-06-12 | 武汉工程大学 | Method for removing high-concentration ammonia nitrogen in residual ammonium salt leachate of rare earth leaching site by chemical-biological method |
| CN111268807A (en) * | 2020-01-21 | 2020-06-12 | 武汉工程大学 | Method for removing ammonia nitrogen in residual ammonium salt leachate of rare earth leaching site by using indigenous denitrification microbial flora |
| CN112520854A (en) * | 2020-12-30 | 2021-03-19 | 魏一明 | Method for treating sewage by using biological membrane |
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| CN104877916A (en) * | 2015-06-01 | 2015-09-02 | 云南省烟草农业科学研究院 | Paracoccus limosus, bacterium thereof, method for preparing bacterium and application thereof |
| CN106337028A (en) * | 2015-07-17 | 2017-01-18 | 上海海洋大学 | Cleaning agent, and preparation method and application thereof |
| CN106676022A (en) * | 2015-11-06 | 2017-05-17 | 丹阳市尚德生物科技有限公司 | Method for preparing microbe bacteria liquid for treating black-odor riverway |
| CN106148246A (en) * | 2016-08-30 | 2016-11-23 | 蓝德环保科技集团股份有限公司 | Purify compounding microbial inoculum of black and odorous water and preparation method thereof |
| CN106148246B (en) * | 2016-08-30 | 2019-11-12 | 蓝德环保科技集团股份有限公司 | Purify the compounding microbial inoculum and preparation method thereof of black and odorous water |
| CN106396133B (en) * | 2016-12-01 | 2019-03-26 | 东莞理工学院 | A method of black and odorous water is handled with ethyl alcohol stalk waste residue immobilization polyP bacteria processed |
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| CN106745804B (en) * | 2016-12-16 | 2020-12-04 | 北京桑德环境工程有限公司 | Microbial quick clarifying agent for black and odorous water body and preparation method thereof |
| CN108178327A (en) * | 2017-12-26 | 2018-06-19 | 中冶京诚工程技术有限公司 | For the microbe to screen enrichment method of black and odorous water river regulation and its application |
| CN109052624A (en) * | 2018-08-08 | 2018-12-21 | 中国科学院水生生物研究所 | A kind of method and device of movable purifying sewage and black and odorous water |
| CN109052624B (en) * | 2018-08-08 | 2021-08-03 | 中国科学院水生生物研究所 | Mobile method and device for purifying sewage and black and odorous water |
| CN110484458A (en) * | 2018-08-28 | 2019-11-22 | 华中科技大学 | It is a kind of to handle acclimation and screening method of the low C/N than the aerobic denitrification polyP bacteria of sewage |
| CN109231711A (en) * | 2018-11-06 | 2019-01-18 | 江苏澳洋生态园林股份有限公司 | A kind of black-odor riverway sewage disposal system and method |
| CN109576189A (en) * | 2018-12-29 | 2019-04-05 | 合肥市东方美捷分子材料技术有限公司 | A kind of black and odorous water composite bacteria agent and its preparation method and application |
| CN109576189B (en) * | 2018-12-29 | 2022-04-19 | 合肥市东方美捷分子材料技术有限公司 | Composite microbial inoculum for black and odorous water body and preparation method and application thereof |
| CN109576201A (en) * | 2019-01-31 | 2019-04-05 | 上海同瑞环保科技有限公司 | A method of culture indigenous microorganism efficiently removes water body ammonia nitrogen |
| CN109678257A (en) * | 2019-03-01 | 2019-04-26 | 北京高能时代环境技术股份有限公司 | A method of being suitable for the functional flora construction method and application flora improvement black and odorous water of black and odorous water improvement |
| CN111268857A (en) * | 2020-01-21 | 2020-06-12 | 武汉工程大学 | Method for removing high-concentration ammonia nitrogen in residual ammonium salt leachate of rare earth leaching site by chemical-biological method |
| CN111268807A (en) * | 2020-01-21 | 2020-06-12 | 武汉工程大学 | Method for removing ammonia nitrogen in residual ammonium salt leachate of rare earth leaching site by using indigenous denitrification microbial flora |
| CN112520854A (en) * | 2020-12-30 | 2021-03-19 | 魏一明 | Method for treating sewage by using biological membrane |
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