CN103923867A - Mixed bacterial colony microbial preparation and application thereof in treatment of wastewater containing nitrate nitrogen - Google Patents
Mixed bacterial colony microbial preparation and application thereof in treatment of wastewater containing nitrate nitrogen Download PDFInfo
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Abstract
The invention discloses a mixed bacterial colony microbial preparation which is a bacterial suspension consisting of paenibacillus, pseudomonas stutzeri, pseudomonas pseudoalcaligenes and pseudomonas oleovoran, wherein the viable count of paenibacillus is more than 1.69*10<10>CFU/Ml, the viable count of pseudomonas stutzeri is more than 2.35*10<10>CFU/Ml, the viable count of pseudomonas pseudoalcaligenes is more than 2.18*10<10>CFU/Ml, and the viable count of pseudomonas oleovoran is more than 2.26*10<10>CFU/Ml. The mixed bacterial colony microbial preparation can be used for treating wastewater containing nitrate nitrogen, and the specific application method comprises the steps of regulating the pH value of the wastewater containing nitrate nitrogen to 7.0-7.2, then inoculating the wastewater containing nitrate nitrogen with the mixed bacterial colony microbial preparation according to the inoculation amount of 1-10%, and performing static culture at 30-35 DEG C. The denitrification effect can be obvious after 4 days culture and complete denitrification can be realized.
Description
Technical field
The present invention relates to a kind of mixed bacterial microbial preparation, and processing 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 areas 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.Nitrate and nitrite can change into nitrosamine and nitrous acid amides under certain condition in addition, and they are materials highly carcinogenic, that cause change, teratogenesis, therefore the control of water body and administer extremely urgent.Drinking Water in China standard regulation, nitrate nitrogen content must be lower than 10mg/L.The current method of processing 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.Anti-nitration reaction occurs under anoxic or anaerobic condition, by heterotrophic bacterium with NO
2 -and NO
3 -as electron acceptor(EA), be reduced into gaseous substance and discharged, thereby nitrogen cycle is carried out smoothly, thereby reduced poisonous infringement biology being caused due to nitrate and nitrite accumulation, water quality protection is had great significance.
Research shows, denitrifying microorganism is extensively present in soil, lamination rock and water ecological environment, but on taxonomy, does not have fixing distribution.Up to the present, the microorganism of denitrifying capacity in 150 kinds of 50 genus, 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 enzymes system in denitrification, can be 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. can successfully carry out denitrification under the condition of mixed culture.
Summary of the invention
For above-mentioned prior art, the denitrification bacterial strain separating from wetland plant rhizosphere soil and active sludge early stage in conjunction with China's water pollution situation and laboratory, contriver place, the invention provides a kind of mixed bacterial microbial preparation, and contain the application in nitric nitrogen waste water in processing, and concrete application method.
The present invention is achieved by the following technical solutions:
A kind of mixed bacterial microbial preparation, the bacteria suspension being formed by 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 CCTCC NO:M2011120, this bacterium classification called after: Paenibacillus sp.XP1, preservation date is on April 10th, 2011, depositary institution is: Chinese Typical Representative 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 CCTCC NO:M2011431, this bacterium classification called after Pseudomonas stutzeri XP2, preservation date is on November 27th, 2011, depositary institution is: Chinese Typical Representative 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 CCTCC NO:M2012225, this bacterium classification called after Pseudomonas pseudoalcaligenes CL-1, preservation date is on June 13rd, 2012, depositary institution is: Chinese Typical Representative 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 CCTCC NO:M2012226, this bacterium classification called after Pseudomonas oleovorans CL-3, preservation date is on June 13rd, 2012, depositary institution is: Chinese Typical Representative 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:
With plate streaking activation series bacillus 1～3 day; After activation, picking list colony inoculation in seed culture fluid, 30～35 DEG C, 150～180r/min, 18～24h is cultivated in shaking table concussion, obtains seed liquor; Get seed liquor and be inoculated in enrichment culture liquid (inoculum size is 15～20%, percent by volume), 30～35 DEG C, 150～180r/min, 18～24h is cultivated in shaking table concussion, 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 with sterilized water, obtains series bacillus bacteria suspension; Same method (step, parameter, substratum are all identical) prepares Pseudomonas stutzeri bacteria suspension, pseudomonas pseudoalcaligenes bacteria suspension and Pseudomonas oleovorans bacteria suspension; Four kinds of bacteria suspension equal volume amounts are mixed, mix, obtain mixed bacterial microbial preparation.
When described plate streaking activation, substratum used is LB substratum, is existing conventional medium in prior art.
Described seed culture fluid is LB nutrient solution, is existing conventional medium in prior art.
The consisting of of described enrichment culture liquid (unit of the percentage ratio of following each component is g/ml): Seignette salt 2%; KNO
30.2%; K
2hPO
40.05%; MgSO
47H
2o0.02%; Surplus is water; PH value 7.0～7.2.When preparation, each component is mixed, regulate pH value 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, can be for the treatment of containing nitric nitrogen waste water, concrete application method is: adjust pH value to 7.0～7.2 (with sodium hydroxide solution or hydrochloric acid adjustment) containing 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 leave standstill cultivation.
Preferably, leave standstill and cultivate 8～96 hours, most preferred, leave standstill and cultivate 96 hours (4 days), denitrification effect is obvious.
The present inventor is through research and practice discovery repeatedly, utilize the mixed bacterial microbial preparation of series bacillus (Paenibacillus sp.), Pseudomonas stutzeri (Pseudomonas stutzeri), pseudomonas pseudoalcaligenes (Pseudomonas pseudoalcaligenes) and Pseudomonas oleovorans (Pseudomonas oleovorans) composition to process 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 all-environment NO
3 -/ NO
2 -the eutrophication pollution control of reparation and water body is also with a wide range of applications.
Brief description of the drawings
Fig. 1: the nitric nitrogen removal effect figure of different ratios mixed bacterial to waste water.
Fig. 2: mixed bacterial is inoculated into the apparent figure containing nitric nitrogen waste water.
Fig. 3: mixed bacterial changes at the growth curve of processing containing in the experiment of nitric nitrogen waste water.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.Embodiment is used for illustrating the present invention, instead of limits the present invention.
Embodiment 1 prepares mixed bacterial microbial preparation
Preparation method is as follows:
With plate streaking activation series bacillus 2 days; After activation, picking list colony inoculation in 50ml seed culture fluid, 30～35 DEG C, 180r/min, 24h is cultivated in shaking table concussion, obtains seed liquor; Get seed liquor and be inoculated in enrichment culture liquid (inoculum size is 20%, percent by volume), 30～35 DEG C, 180r/min, 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 with sterilized water, obtains series bacillus bacteria suspension;
Same method (step, parameter, substratum are all identical) prepares Pseudomonas stutzeri bacteria suspension, pseudomonas pseudoalcaligenes bacteria suspension and Pseudomonas oleovorans bacteria suspension; Four kinds of bacteria suspension equal volume amounts are mixed, mix, 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 CCTCC NO:M2011120, Classification And Nomenclature is: Paenibacillus sp.XP1, preservation date is on April 10th, 2011, depositary institution is: Chinese Typical Representative culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.
Described Pseudomonas stutzeri, deposit number is CCTCC NO:M2011431, Classification And Nomenclature is Pseudomonas stutzeri XP2, preservation date is on November 27th, 2011, depositary institution is: Chinese Typical Representative culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.
Described pseudomonas pseudoalcaligenes, deposit number is CCTCC NO:M2012225, Classification And Nomenclature is Pseudomonas pseudoalcaligenes CL-1, preservation date is on June 13rd, 2012, depositary institution is: Chinese Typical Representative culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.
Described Pseudomonas oleovorans, deposit number is CCTCC NO:M2012226, Classification And Nomenclature is Pseudomonas oleovorans CL-3, preservation date is on June 13rd, 2012, depositary institution is: Chinese Typical Representative culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.
When described plate streaking activation, substratum used is LB substratum, is existing conventional medium in prior art.
Described seed culture fluid is LB nutrient solution, is existing conventional medium in prior art.
The consisting of of described enrichment culture liquid (unit of the percentage ratio of following each component is g/ml): Seignette salt 2%; KNO
30.2%; K
2hPO
40.05%; MgSO
47H
2o0.02%; Surplus is water; PH value 7.0.When preparation, each component is mixed, regulate pH value to 7.0,110 DEG C of autoclaving 20min, to obtain final product.
Embodiment 2 mixed bacterial microbial preparations are processed 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, mixed bacterial microbial preparation (the percent by volume of preparing to the embodiment 1 that adds 1%, 5%, 10% in three bottles respectively, the amount of the mixed bacterial microbial preparation adding in three bottles is respectively 2ml, 10ml, 20ml), at 30～35 DEG C, leave standstill and cultivate, and get waste water by the corresponding time (every 8 hours) respectively, suction filtration under the filter membrane of 0.22 μ m, measure nitrate nitrogen content in filtrate, calculate denitrification percent.
Result as shown in Figure 1, shows in figure, adds the triangular flask of 1% mixed bacterial microbial preparation, the removal of nitrate nitrogen in the time of 8h, NO
3 --N clearance reaches 60.3%.When 96h, NO
3 --N clearance reaches 98.2%.
The triangular flask that adds 5% mixed bacterial microbial preparation, the removal of nitrate nitrogen mainly concentrates on first 8 hours, when 8h, NO
3 --N clearance reaches 83%.When 16h, NO
3 --N clearance reaches 93.4%.When 96h, NO
3 --N clearance reaches 100%, realizes complete denitrogenation.
The triangular flask that adds 10% mixed bacterial microbial preparation, the removal of nitrate nitrogen mainly concentrates on first 8 hours, when 8h, NO
3 --N clearance reaches 82.7%.When 96h, NO
3 --N clearance reaches 93.7%.
Described consist 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.When preparation, each component is mixed, regulate pH value to 7.0,110 DEG C of autoclaving 20min, to obtain final product.
Embodiment 3 mixed bacterials change at the growth curve of processing containing in 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 (preparation method is with embodiment 2), add mixed bacterial microbial preparation prepared by embodiment 1 (to establish three triangular flasks, the amount of the mixed bacterial microbial preparation adding is respectively 1%, 5%, 10%, that is: 2ml, 10ml, 20ml), at 30～35 DEG C, leave standstill and cultivate, the OD of timing Spectrophotometric Assays waste water
600(result as shown in Figure 3).Mixed bacterial is inoculated into after waste water, along with the carrying out of time, and bacterium amount reproduction, Bacterial Denitrification at One Time effect is remarkable, occurs a large amount of bubbles in waste water, and muddy (seeing Fig. 2) gradually.Flora is inoculated into after waste water, and 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, limited carbon source in competition waste water, and microbial growth metabolism is suppressed.
Claims (7)
1. a mixed bacterial microbial preparation, is characterized in that: the bacteria suspension being 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.
2. mixed bacterial microbial preparation according to claim 1, is characterized in that:
Described series bacillus is selected from the bacterial classification that deposit number is CCTCC NO:M2011120, this bacterium classification called after: Paenibacillus sp.XP1, preservation date is on April 10th, 2011, depositary institution is: Chinese Typical Representative 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 CCTCC NO:M2011431, this bacterium classification called after Pseudomonas stutzeri XP2, preservation date is on November 27th, 2011, depositary institution is: Chinese Typical Representative 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 CCTCC NO:M2012225, this bacterium classification called after Pseudomonas pseudoalcaligenes CL-1, preservation date is on June 13rd, 2012, depositary institution is: Chinese Typical Representative 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 CCTCC NO:M2012226, this bacterium classification called after Pseudomonas oleovorans CL-3, preservation date is on June 13rd, 2012, depositary institution is: Chinese Typical Representative culture collection center, preservation address is: China. Wuhan. and Wuhan University, postcode 430072.
3. the preparation method of the mixed bacterial microbial preparation described in claim 1 or 2, is characterized in that: with plate streaking activation series bacillus 1～3 day; After activation, picking list colony inoculation in seed culture fluid, 30～35 DEG C, 150～180r/min, 18～24h is cultivated in shaking table concussion, obtains seed liquor; Get seed liquor and be inoculated in enrichment culture liquid, 30～35 DEG C, 150～180r/min, 18～24h is cultivated in shaking table concussion, 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 with sterilized water, obtains series bacillus bacteria suspension; Same method prepares Pseudomonas stutzeri bacteria suspension, pseudomonas pseudoalcaligenes bacteria suspension and Pseudomonas oleovorans bacteria suspension; Four kinds of bacteria suspension equal volume amounts are mixed, mix, obtain mixed bacterial microbial preparation.
4. preparation method according to claim 3, 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.When preparation, each component is mixed, regulate pH value to 7.0～7.2,110 DEG C of autoclaving 20min, to obtain final product.
5. the mixed bacterial microbial preparation described in claim 1 or 2 contains the application in nitric nitrogen waste water in processing.
6. application according to claim 5, it is characterized in that: concrete application method is: adjust pH value to 7.0～7.2 containing nitric nitrogen waste water, then mixed bacterial microbial preparation is inoculated into containing in nitric nitrogen waste water with 1%～10% inoculum size, 30～35 DEG C leave standstill cultivation.
7. application according to claim 6, is characterized in that: leave standstill and cultivate 8～96 hours.
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| 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 |
| CN110396479A (en) * | 2019-08-02 | 2019-11-01 | 自然资源部第三海洋研究所 | One acidproof flora and its application for handling Fenton acidic effluent |
| 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 |
| CN112553100A (en) * | 2019-09-26 | 2021-03-26 | 有研工程技术研究院有限公司 | Composite microbial agent and method for soil fertility improvement and ecological restoration of heavy metal-containing field by using same |
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