CN103923867B - Mixed bacterial microbial preparation and in process containing the application in nitric nitrogen waste water - Google Patents
Mixed bacterial microbial preparation and in process containing the application in nitric nitrogen waste water Download PDFInfo
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Abstract
The invention discloses a kind of mixed bacterial microbial preparation, the bacteria suspension be made up of series bacillus, Pseudomonas stutzeri, pseudomonas pseudoalcaligenes and Pseudomonas oleovorans, wherein, class bacillus viable count is 1.69 × 10
10more than CFU/ml, Pseudomonas stutzeri viable count is 2.35 × 10
10more than CFU/ml, pseudomonas pseudoalcaligenes viable count is 2.18 × 10
10more than CFU/ml, Pseudomonas oleovorans viable count is 2.26 × 10
10more than CFU/ml.Described mixed bacterial microbial preparation may be used for process containing nitric nitrogen waste water, concrete application method is: adjustment is containing pH value to 7.0 ~ 7.2 of nitric nitrogen waste water, then by mixed bacterial microbial preparation with 1% ~ 10% inoculum size be inoculated into containing in nitric nitrogen waste water, 30 ~ 35 DEG C of quiescent culture, cultivate denitrification effect after 4 days obvious, complete denitrogenation can be realized.
Description
Technical field
The present invention relates to a kind of mixed bacterial microbial preparation, and in process containing the application in nitric nitrogen waste water, belong to environmental technology field.
Background technology
At present, along with the development of national economy, the pollution of China's Water is more and more serious, body eutrophication outstanding problem, and water scarcity, water surrounding worsen oneself increasingly through becoming the important factor of restriction China Economic development.The underground water in the most of area of China is being subjected to nitrate-N pollution in varying degrees.Nitrate excessive in water body is very big to the Health hazard of human body, and in human body, nitric nitrogen can be reduced into nitrite under microbial process, causes methemoglobinemia and baby's cyanosis.NO3-N and NO2-N can change into nitrosamine and nitrous acid amides under certain condition in addition, and they are materials that are highly carcinogenic, that cause change, teratogenesis, therefore water body control and administer extremely urgent.Drinking Water in China standard specifies, nitrate nitrogen content must lower than 10mg/L.The method of current process nitrate nitrogen in water body has multiple, is mainly divided into physico-chemical processes and biological denitrification method.Biological denitrification method, compared with physico-chemical processes, has the feature of low energy, efficient, non-secondary pollution.Under anti-nitration reaction occurs in anoxic or anaerobic condition, by heterotrophic bacterium with NO
2 -and NO
3 -as electron acceptor(EA), be reduced into gaseous substance and discharged, thus nitrogen cycle is carried out smoothly, thus reduced because NO3-N and NO2-N accumulates the poisonous infringement caused biology, water quality protection is had great significance.
Research shows, denitrifying microorganism is extensively present in soil, lamination rock and water ecological environment, but distribution unfixing on taxonomy.Up to the present, in 50 150 kinds belonged to, the microorganism of denitrifying capacity detected, comprise bacterium, actinomycetes, Archimycetes and fungi, and the scope of denitrifying microorganism is also in continuous expansion.Wherein Rhodopseudomonas (Pseudomonas), neisseria (Neisseria) and Bacillus (Bacillus) are comparatively common denitrifying bacteriums, and its denitrification process all occurs under anaerobism and half anaerobic condition.Rhodopseudomonas contains the whole enzyme systems in denitrification, can by NO
3 -be reduced into N
2.Bacillus can utilize NO
3 -or NO
2 -as electron acceptor(EA).
Had at present research from waste water isolation identification denitrifying microorganism as Achromobacter, Alcaligenes, Aquaspirillum, Azoarcus, Bacillus, Brachymonas, Paracoccus, Pseudomonas, Rhodocyclus, Thauera etc., successfully can carry out denitrification under the condition of mixed culture.
Summary of the invention
For above-mentioned prior art, in conjunction with the denitrification bacterial strain that China's Water Pollution Problem and laboratory, contriver place are separated in earlier stage from wetland plant rhizosphere soil and active sludge, the invention provides a kind of mixed bacterial microbial preparation, and in process containing the application in nitric nitrogen waste water, and concrete application method.
The present invention is achieved by the following technical solutions:
A kind of mixed bacterial microbial preparation, the bacteria suspension be made up of series bacillus, Pseudomonas stutzeri, pseudomonas pseudoalcaligenes and Pseudomonas oleovorans, wherein, class bacillus viable count is 1.69 × 10
10more than CFU/ml, Pseudomonas stutzeri viable count is 2.35 × 10
10more than CFU/ml, pseudomonas pseudoalcaligenes viable count is 2.18 × 10
10more than CFU/ml, Pseudomonas oleovorans viable count is 2.26 × 10
10more than CFU/ml.
Further, described series bacillus is selected from the bacterial classification that deposit number is CCTCCNO:M2011120, this bacterium classification called after: Paenibacillussp.XP1, preservation date is on April 10th, 2011, depositary institution is: China typical culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.Be documented in another Chinese patent of applicant, the patent No. 201110121394.2, publication number 102220265A.
Further, described Pseudomonas stutzeri is selected from the bacterial classification that deposit number is CCTCCNO:M2011431, this bacterium classification called after PseudomonasstutzeriXP2, preservation date is on November 27th, 2011, depositary institution is: China typical culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.Be documented in another Chinese patent application of applicant, number of patent application 201210201671.5, publication number 102899263A.
Further, described pseudomonas pseudoalcaligenes is selected from the bacterial classification that deposit number is CCTCCNO:M2012225, this bacterium classification called after PseudomonaspseudoalcaligenesCL-1, preservation date is on June 13rd, 2012, depositary institution is: China typical culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.Be documented in another Chinese patent application of applicant, number of patent application 201310032730.5, publication number 103114062A.
Further, described Pseudomonas oleovorans is selected from the bacterial classification that deposit number is CCTCCNO:M2012226, this bacterium classification called after PseudomonasoleovoransCL-3, preservation date is on June 13rd, 2012, depositary institution is: China typical culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.Be documented in another Chinese patent of applicant, the patent No. 201210269891.1, publication number 102776145A.
The preparation method of described mixed bacterial microbial preparation is as follows:
Series bacillus 1 ~ 3 day is activated with plate streaking; After activation, picking list colony inoculation in seed culture fluid, 30 ~ 35 DEG C, 150 ~ 180r/min, shaking table concussion cultivation 18 ~ 24h, obtains seed liquor; Getting seed liquor is inoculated in enrichment culture liquid (inoculum size is 15 ~ 20%, percent by volume), 30 ~ 35 DEG C, 150 ~ 180r/min, and shaking table concussion cultivation 18 ~ 24h, obtains enrichment culture bacterium liquid; Enrichment culture bacterium liquid centrifugal 15 ~ 20min under 5000 ~ 8000r/min, goes supernatant liquor to obtain concentrated somatic cells, is diluted to the bacteria suspension of 1.5g wet thallus/L, obtains series bacillus bacteria suspension with sterilized water; Same method (step, parameter, substratum are all identical) prepares Pseudomonas stutzeri bacteria suspension, pseudomonas pseudoalcaligenes bacteria suspension and Pseudomonas oleovorans bacteria suspension; By four kinds of bacteria suspension equal volume amounts mixing, mixing, obtains mixed bacterial microbial preparation.
During described plate streaking activation, substratum used is LB substratum, is conventional medium existing in prior art.
Described seed culture fluid is LB nutrient solution, is conventional medium existing in prior art.
Consisting of (unit of the percentage ratio of following each component is g/ml) of described enrichment culture liquid: Seignette salt 2%; KNO
30.2%; K
2hPO
40.05%; MgSO
47H
2o0.02%; Surplus is water; PH value 7.0 ~ 7.2.During preparation, by each component mixing mixing, adjust ph to 7.0 ~ 7.2 (sodium hydroxide solution or hydrochloric acid regulate), 110 DEG C of autoclaving 20min, to obtain final product.
Described mixed bacterial microbial preparation, may be used for process containing nitric nitrogen waste water, concrete application method is: adjustment is containing pH value to 7.0 ~ 7.2 (with sodium hydroxide solution or hydrochloric acid adjustment) of nitric nitrogen waste water, then mixed bacterial microbial preparation of the present invention is inoculated into containing in nitric nitrogen waste water with the inoculum size of 1% ~ 10% (percent by volume), 30 ~ 35 DEG C of quiescent culture.
Preferably, quiescent culture 8 ~ 96 hours, most preferred, quiescent culture 96 hours (4 days), denitrification effect is obvious.
The present inventor is through research and practice discovery repeatedly, the mixed bacterial microbial preparation utilizing series bacillus (Paenibacillussp.), Pseudomonas stutzeri (Pseudomonasstutzeri), pseudomonas pseudoalcaligenes (Pseudomonaspseudoalcaligenes) and Pseudomonas oleovorans (Pseudomonasoleovorans) to form processes containing nitric nitrogen waste water, can realize the efficient removal of nitrogen.Achievement of the present invention can be applicable to the biological denitrification process in Industrial Wastewater Treatment, simultaneously to the NO of various environment
3 -/ NO
2 -the eutrophication pollution control of reparation and water body is also with a wide range of applications.
Accompanying drawing explanation
Fig. 1: different ratios mixed bacterial is to the nitric nitrogen removal effect figure of waste water.
Fig. 2: mixed bacterial is inoculated into the apparent figure containing nitric nitrogen waste water.
Fig. 3: mixed bacterial changes containing the growth curve in the experiment of nitric nitrogen waste water in process.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.Embodiment for illustration of the present invention, instead of limits the present invention.
Embodiment 1 prepares mixed bacterial microbial preparation
Preparation method is as follows:
Series bacillus 2 days are activated with plate streaking; After activation, picking list colony inoculation in 50ml seed culture fluid, 30 ~ 35 DEG C, 180r/min, shaking table concussion cultivate 24h, obtain seed liquor; Getting seed liquor is inoculated in enrichment culture liquid (inoculum size is 20%, percent by volume), 30 ~ 35 DEG C, 180r/min, and 24h is cultivated in shaking table concussion, obtains enrichment culture bacterium liquid; Enrichment culture bacterium liquid centrifugal 20min under 7000r/min, goes supernatant liquor to obtain concentrated somatic cells, is diluted to the bacteria suspension of 1.5g wet thallus/L, obtains series bacillus bacteria suspension with sterilized water;
Same method (step, parameter, substratum are all identical) prepares Pseudomonas stutzeri bacteria suspension, pseudomonas pseudoalcaligenes bacteria suspension and Pseudomonas oleovorans bacteria suspension; By four kinds of bacteria suspension equal volume amounts mixing, mixing, obtain mixed bacterial microbial preparation, after testing, in mixed bacterial microbial preparation, class bacillus viable count is 1.69 × 10
10cFU/ml, Pseudomonas stutzeri viable count is 2.35 × 10
10cFU/ml, pseudomonas pseudoalcaligenes viable count is 2.18 × 10
10cFU/ml, Pseudomonas oleovorans viable count is 2.26 × 10
10cFU/ml.
Described series bacillus, deposit number is CCTCCNO:M2011120, and Classification And Nomenclature is: Paenibacillussp.XP1, preservation date is on April 10th, 2011, depositary institution is: China typical culture collection center, and preservation address is: China. Wuhan. and Wuhan University, postcode 430072.
Described Pseudomonas stutzeri, deposit number is CCTCCNO:M2011431, Classification And Nomenclature is PseudomonasstutzeriXP2, preservation date is on November 27th, 2011, depositary institution is: China typical culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.
Described pseudomonas pseudoalcaligenes, deposit number is CCTCCNO:M2012225, Classification And Nomenclature is PseudomonaspseudoalcaligenesCL-1, preservation date is on June 13rd, 2012, depositary institution is: China typical culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.
Described Pseudomonas oleovorans, deposit number is CCTCCNO:M2012226, Classification And Nomenclature is PseudomonasoleovoransCL-3, preservation date is on June 13rd, 2012, depositary institution is: China typical culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.
During described plate streaking activation, substratum used is LB substratum, is conventional medium existing in prior art.
Described seed culture fluid is LB nutrient solution, is conventional medium existing in prior art.
Consisting of (unit of the percentage ratio of following each component is g/ml) of described enrichment culture liquid: Seignette salt 2%; KNO
30.2%; K
2hPO
40.05%; MgSO
47H
2o0.02%; Surplus is water; PH value 7.0.During preparation, by each component mixing mixing, adjust ph to 7.0,110 DEG C of autoclaving 20min, to obtain final product.
The process of embodiment 2 mixed bacterial microbial preparation is containing the experiment of nitric nitrogen waste water
Experiment is carried out in 250mL triangular flask, every bottle of 200mL is containing nitric nitrogen waste water (nitrate is up to 136mg/L), totally three bottles, respectively to mixed bacterial microbial preparation (percent by volume prepared by the embodiment 1 adding 1%, 5%, 10% in three bottles, the amount of the mixed bacterial microbial preparation namely added in three bottles is respectively 2ml, 10ml, 20ml), quiescent culture at 30 ~ 35 DEG C, and get waste water by corresponding time (every 8 hours) respectively, suction filtration under the filter membrane of 0.22 μm, measure nitrate nitrogen content in filtrate, calculate denitrification percent.
Result as shown in Figure 1, shows in figure, adds the triangular flask of the mixed bacterial microbial preparation of 1%, the removal of nitrate nitrogen when 8h, NO
3 --N clearance reaches 60.3%.During 96h, NO
3 --N clearance reaches 98.2%.
Add the triangular flask of the mixed bacterial microbial preparation of 5%, the removal of nitrate nitrogen mainly concentrates on first 8 hours, during 8h, NO
3 --N clearance reaches 83%.During 16h, NO
3 --N clearance reaches 93.4%.During 96h, NO
3 --N clearance reaches 100%, realizes complete denitrogenation.
Add the triangular flask of the mixed bacterial microbial preparation of 10%, the removal of nitrate nitrogen mainly concentrates on first 8 hours, during 8h, NO
3 --N clearance reaches 82.7%.During 96h, NO
3 --N clearance reaches 93.7%.
Described consisting of (unit of the percentage ratio of following each component is g/ml) containing nitric nitrogen waste water: sodium acetate 0.2%; KNO
30.1%; K
2hPO
40.04%; MgSO
47H
2o0.06%; Surplus is water; PH value 7.0.During preparation, by each component mixing mixing, adjust ph to 7.0,110 DEG C of autoclaving 20min, to obtain final product.
Embodiment 3 mixed bacterial changes containing the growth curve in the experiment of nitric nitrogen waste water in process
Experiment is carried out in 250mL triangular flask, every bottle of 200mL is containing nitric nitrogen waste water (preparation method is with embodiment 2), the mixed bacterial microbial preparation adding embodiment 1 preparation (establishes three triangular flasks, the amount of the mixed bacterial microbial preparation added is respectively 1%, 5%, 10%, that is: 2ml, 10ml, 20ml), quiescent culture at 30 ~ 35 DEG C, the OD of timing Spectrophotometric Assays waste water
600(result as shown in Figure 3).After mixed bacterial is inoculated into waste water, along with the carrying out of time, bacterium amount reproduction, Bacterial Denitrification at One Time effect is remarkable, occurs a large amount of bubble in waste water, and gradually muddy (see Fig. 2).After flora is inoculated into waste water, the growth of its biomass is mainly at front 24h, OD
600increase gradually.After 24h, bacterium reduces gradually, and each concentration mixed bacterial all presents downtrending, and this may be due to microorganism amount reproduction, and carbon source limited in competition waste water, microbial growth metabolism is suppressed.
Claims (6)
1. a mixed bacterial microbial preparation, is characterized in that: the bacteria suspension be made up of series bacillus, Pseudomonas stutzeri, pseudomonas pseudoalcaligenes and Pseudomonas oleovorans, and wherein, class bacillus viable count is 1.69 × 10
10more than CFU/ml, Pseudomonas stutzeri viable count is 2.35 × 10
10more than CFU/ml, pseudomonas pseudoalcaligenes viable count is 2.18 × 10
10more than CFU/ml, Pseudomonas oleovorans viable count is 2.26 × 10
10more than CFU/ml, described series bacillus is selected from the bacterial classification that deposit number is CCTCCNO:M2011120, this bacterium classification called after: Paenibacillussp.XP1, preservation date is on April 10th, 2011, depositary institution is: China typical culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072;
Described Pseudomonas stutzeri is selected from the bacterial classification that deposit number is CCTCCNO:M2011431, this bacterium classification called after PseudomonasstutzeriXP2, preservation date is on November 27th, 2011, depositary institution is: China typical culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072;
Described pseudomonas pseudoalcaligenes is selected from the bacterial classification that deposit number is CCTCCNO:M2012225, this bacterium classification called after PseudomonaspseudoalcaligenesCL-1, preservation date is on June 13rd, 2012, depositary institution is: China typical culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072;
Described Pseudomonas oleovorans is selected from the bacterial classification that deposit number is CCTCCNO:M2012226, this bacterium classification called after PseudomonasoleovoransCL-3, preservation date is on June 13rd, 2012, depositary institution is: China typical culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.
2. the preparation method of mixed bacterial microbial preparation according to claim 1, is characterized in that: activate series bacillus 1 ~ 3 day with plate streaking; After activation, picking list colony inoculation in seed culture fluid, 30 ~ 35 DEG C, 150 ~ 180r/min, shaking table concussion cultivation 18 ~ 24h, obtains seed liquor; Getting seed liquor is inoculated in enrichment culture liquid, 30 ~ 35 DEG C, 150 ~ 180r/min, and shaking table concussion cultivation 18 ~ 24h, obtains enrichment culture bacterium liquid; Enrichment culture bacterium liquid centrifugal 15 ~ 20min under 5000 ~ 8000r/min, goes supernatant liquor to obtain concentrated somatic cells, is diluted to the bacteria suspension of 1.5g wet thallus/L, obtains series bacillus bacteria suspension with sterilized water; Same method prepares Pseudomonas stutzeri bacteria suspension, pseudomonas pseudoalcaligenes bacteria suspension and Pseudomonas oleovorans bacteria suspension; By four kinds of bacteria suspension equal volume amounts mixing, mixing, obtains mixed bacterial microbial preparation.
3. preparation method according to claim 2, is characterized in that:
Described seed culture fluid is LB nutrient solution;
Consisting of of described enrichment culture liquid: Seignette salt 2%; KNO
30.2%; K
2hPO
40.05%; MgSO
47H
2o0.02%; Surplus is water; PH value 7.0 ~ 7.2; During preparation, by each component mixing mixing, adjust ph to 7.0 ~ 7.2,110 DEG C of autoclaving 20min, to obtain final product.
4. mixed bacterial microbial preparation according to claim 1 contains the application in nitric nitrogen waste water in process.
5. application according to claim 4, it is characterized in that: embody rule method is: adjustment is containing pH value to 7.0 ~ 7.2 of nitric nitrogen waste water, then by mixed bacterial microbial preparation with 1% ~ 10% inoculum size be inoculated into containing in nitric nitrogen waste water, 30 ~ 35 DEG C of quiescent culture.
6. application according to claim 5, is characterized in that: quiescent culture 8 ~ 96 hours.
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| CN104889153B (en) * | 2015-06-24 | 2017-03-29 | 中国科学院新疆生态与地理研究所 | A kind of method of bacterial immobilisation Mercury in Soil under anaerobic condition |
| CN106676021A (en) * | 2015-11-06 | 2017-05-17 | 丹阳市尚德生物科技有限公司 | Microbial preparation for river course water quality restoration, and preparation method thereof |
| CN109205795A (en) * | 2017-12-21 | 2019-01-15 | 向勇 | A kind of compound nitrifier and its production method |
| CN109609406A (en) * | 2018-12-27 | 2019-04-12 | 广州市金龙峰环保设备工程股份有限公司 | A kind of slow-release microbial bacterial agent and preparation method thereof administered for black and odorous water |
| CN109467405A (en) * | 2019-01-08 | 2019-03-15 | 浙江工业大学 | A kind of paper mill sludge brick and preparation method |
| CN110396479B (en) * | 2019-08-02 | 2021-03-09 | 自然资源部第三海洋研究所 | Acid-resistant flora and application thereof in treatment of Fenton acidic effluent |
| CN112553100B (en) * | 2019-09-26 | 2023-01-06 | 有研资源环境技术研究院(北京)有限公司 | Composite microbial agent and method for soil fertility improvement and ecological restoration of heavy metal-containing field by using same |
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