CN103074242B - Aerobic denitrifying bacterium for sewage treatment and application thereof - Google Patents

Aerobic denitrifying bacterium for sewage treatment and application thereof Download PDF

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CN103074242B
CN103074242B CN201110326862.XA CN201110326862A CN103074242B CN 103074242 B CN103074242 B CN 103074242B CN 201110326862 A CN201110326862 A CN 201110326862A CN 103074242 B CN103074242 B CN 103074242B
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denitrifying bacteria
nitrogen
aerobic denitrifying
application
aerobic
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CN103074242A (en
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郑展望
杨瑾
王斌
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Zhe Jiang Shuangliang Sunda Environment Protection Co ltd
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ZHEJIANG SHANGDA ENVIRONMENTAL PROTECTION CO Ltd
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

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  • Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to the fields of biotechnology, environmental microorganisms and water treatment and discloses an aerobic denitrifying bacterium for sewage treatment and application thereof. The strain of the bacterium provided by the invention is screened from activated sludge in a sewage treatment plant and can effectively remove nitrate nitrogen and nitrite nitrogen in sewage, with removal capability of nitrate nitrogen and nitrite nitrogen respectively being 3.01 mgN/(g.L.H) and 1.80 mgN/(g.L.H). A denitrogenation method by using the bacterial strain belongs to the field of biological denitrogenation, and the biological denitrogenation method has the advantages of high efficiency and low cost; and the bacterial strain has a good prospect in treatment of nitrogen-containing waste water.

Description

One strain is for aerobic denitrifying bacteria and the application thereof of sewage disposal
Technical field
The present invention relates to biotechnology, environmental microorganism and water treatment field, related in particular to a kind of aerobic denitrifying bacteria for sewage disposal and application thereof.
Background technology
Day by day serious along with body eutrophication, removing nitrate pollution in water has become a hot issue in current water prevention and cure of pollution field.Nitrogen in sewage mainly with molecular nitrogen, organonitrogen, ammonia nitrogen, nitric nitrogen, nitrite nitrogen, thiocyanide and prussiate form exist, and mainly exist with the form of organic nitrogen and ammonia nitrogen in undressed raw wastewater; Main remaining ammonia nitrogen and nitric nitrogen after secondary biochemical treatment.For a long time, removing of ammonia nitrogen in sewage mainly emphasized in denitrogenation, nitric nitrogen do not processed, and causes nitrate content in water body constantly to raise.Traditional biological denitrogenation comprises two processes of nitrification and denitrification, and ammonia nitrogen is converted into nitric nitrogen by aerobic nitrification bacterium, and then is reduced to nitrogen by the denitrifying bacterium of anaerobism or anoxic, discharges water body.Domestic and international research in recent years shows, some microorganisms, under different dissolved oxygen conditions, also can show certain denitrifying capacity.Such as what reported thiosphaera pantotropha, Alcaligenes faecalis, Pseudomonas aeruginosa, Thaurea mechernichensis, Acinetobacter calcoaceticus, Providencia rettgeri, Pseudomonas stutzeriwith bacillusstrains etc. all can carry out denitrification under aerobic condition, and for Rhod ( rhodococcussp.) report less.The biological denitrificaion that appears as of aerobic denitrifying bacteria provides a brand-new approach, makes traditional biological denitrificaion approach more perfect, also for denitrification process research provides certain theoretical basis.
The screening method of aerobic denitrifying bacteria is a lot, mainly comprises: 1. enrichment culture method: utilize selective medium to screen, add restricted Carbon and nitrogen sources in substratum, or be that the process that alkali increases is added pH indicator in substratum according to denitrification.2. dilution method, will cultivate after diluted sample certain multiple, and what then pH value is changed dilutes again, repetitive operation, and not dominant aerobic denitrifying bacteria was separated originally.3. intermittent aeration method, aerobic denitrifying bacteria can utilize oxygen molecule and nitric nitrogen simultaneously, and frequent transitions aerobic, anoxic is conducive to its status that gets the mastery in competition.
Summary of the invention
The invention provides a strain aerobic denitrifying bacteria, nitric nitrogen and the nitrite nitrogen that can under aerobic condition, effectively degrade in water body.
In order to solve the problems of the technologies described above, the present invention is solved by following technical proposals:
For the aerobic denitrifying bacteria of sewage disposal, described aerobic denitrifying bacteria 16S rDNA Molecular Identification be rhodococcus erythropolis ( rhodococcus erythropolis), called after rhodococcus erythropolissunda 1101, this bacterial strain, on June 23rd, 2011, is preserved in Chinese Typical Representative culture collection center, and culture presevation is numbered CCTCC M 2011211.
The invention provides a kind of method of screening aerobic denitrifying bacteria.
Bacterial screening of the present invention adopts multiple screening method to combine, and first, by adding restricted nitrogenous source, intermittent aeration carrys out enrichment aerobic denitrifying bacteria, and then fast and effeciently screens aerobic denitrifying bacteria by add pH indicator in substratum.
For achieving the above object, the technical scheme that the present invention takes is:
The active sludge of sewage work is carried out to aeration enrichment culture, filter out aerobic bacteria, then produce the performance of alkali according to denitrifying bacteria, by add the method for brooethyl Finland indicator in substratum, effectively screen aerobic denitrifying bacteria.With the aerobic denitrification bacterium of having simulated sewage and sieving again high denitrifying capacity.
Its step is sampling successively, aeration enrichment culture, and primary dcreening operation, multiple sieve finally obtains 1 strain under aerobic condition, nitric nitrogen and the nitrite nitrogen that can degrade in simulated sewage, the bacterial strain that denitrifying capacity is higher simultaneously.
The invention provides the method for identification of strains.
Identification of strains method of the present invention is mainly the method for identifying by 16S rDNA, utilizing universal primer to amplify 16S rDNA fragment conservative in evolutionary process checks order, then with gene library in data compare, according to the attribute of bacterial strain homology ratio-dependent bacterial strain, finally confirm according to systematic evolution tree again.
Aerobic denitrifying bacteria of the present invention is applied to nitric nitrogen and the nitrite nitrogen in the water body of pollution degradation.Be specially thalline is carried out after enlarged culturing, be inoculated in the water body containing nitric nitrogen and nitrite nitrogen pollution.
The present invention, owing to having adopted above technical scheme, has significant technique effect:
Bacterial strain provided by the invention can effectively be removed nitric nitrogen and the nitrite nitrogen in sewage, and the ability of removing nitric nitrogen and removal nitrite nitrogen is respectively 3.01 mgN/ (gLh) and 1.80 mgN/ (gLh).The denitrogenation method of this bacterial strain belongs to biological denitrificaion field, and biological denitrification method is not only efficient but also cost is lower, and therefore this bacterial strain has good prospect in the treating processes that is applied to nitrogenous effluent.
biomaterial preservation information
Biomaterial title: rhodococcus erythropolis ( rhodococcus erythropolis);
Depositary institution: Chinese Typical Representative culture collection center;
Preservation date: on June 23rd, 2011;
Deposit number: CCTCC M 2011211.
Brief description of the drawings
Fig. 1 is the result figure after 16S rDNA pcr amplification of the present invention.
Fig. 2 is the sibship figure of bacterial strain of the present invention and other bacterial strain.
Embodiment
The present invention is described in further detail with embodiment below in conjunction with accompanying drawing 1 to accompanying drawing 2:
embodiment 1
The screening of aerobic denitrifying bacteria
1. enrichment culture: the active sludge that sewage work of Jiang Conglu village obtains carries out aeration enrichment culture, liquid amount 2 L/3.6 L, change fresh culture every day.While changing fresh culture, first leave standstill 1h, remove 600mL volume, and then add 600mL fresh culture, altogether enrichment approximately 40 days.
2. primary dcreening operation: the active sludge that enrichment is good dilutes is coated with on dibromothymolsulfonphthalein substratum, is then placed in 30 ° of C incubators and cultivates, and cultivates 2-3 days.The blueness list bacterium colony occurring on dibromothymolsulfonphthalein substratum is classified, and gramstaining, then selects the bacterium colony of each form 3-4 the separation of ruling, and is placed in 30 ° of C incubators and cultivates, and cultivates 2-3 days.Line is separated to the blueness list bacterium colony obtaining and classify, select characteristic bacterium colony and be saved on inclined-plane, for multiple sieve.
3. multiple sieve: the bacterial strain access thalline enlarged culturing base that primary dcreening operation is preserved, 30 DEG C, 200 rpm, cultivate 24h, then access respectively 100mL in 1% ratio and detect substratum, cultivate 24 h, 30 DEG C, 200 rpm, measure the variation of inoculation front and back nitric nitrogen and nitrite nitrogen.Select the bacterial strain that nitric nitrogen degradation rate reaches more than 40% and nitrite nitrogen degradation rate reaches more than 25% to carry out denitrification capability test, obtain strain performance bacterial strain relatively preferably.
4. detection method:
The mensuration of 4.1 nitric nitrogens: ultraviolet spectrophotometry, referring to the environment protection industry standard HJ/T 346-2007 of the People's Republic of China (PRC).
The mensuration of 4.2 nitrite nitrogens: N-(1-naphthyl)-quadrol hydrochloric acid spectrophotometry, referring to GB GB 7493-87.
The mensuration of 4.3 dry cell weights (DCW): get bacterial culture fluid, centrifugal 5min under the condition of 8000 rpm, abandoning supernatant is then dried to thalline constant weight in the thermostatic drying chamber of 105 ° of C.
5. relevant substratum
Enrichment medium (g/L): (NH 4) 2sO 40.5; Soduxin 2.17; Vickers salts solution 50ml; PH 7.2.
Vickers salts solution (g/L): K 2hPO 45.0; MgSO 47H 2o 2.5; NaCl 2.5; FeSO 47H 2o 0.05; MnSO 44H 2o 0.05.
Primary dcreening operation substratum (g/L): agar 20; KNO 31; KH 2pO 41; FeSO 47H 2o 0.5; CaCl 20.2; MgSO 47H 2o 1; Soduxin 8.5; Dibromothymolsulfonphthalein (get 0.1g dibromothymolsulfonphthalein and be dissolved in 10mL alcohol) 1mL, pH 7.0～7.3.
Thalline enlarged culturing base (g/L): KNO 31; KH 2pO 41; FeSO 47H 2o 0.05; CaCl 20.02; MgSO 47H 2o 1; Soduxin 6H 2o 8.5.
Detect substratum (g/L): KH 2pO 41; MgSO 47H 2o 1; Soduxin 2.8; NaNO 30.6 or NaNO 20.50.
embodiment 2
The qualification of aerobic denitrifying bacteria
1. extract bacterial genomes
By object bacterial strain access LB substratum, after cultivation overnight, get the centrifugal thalline that obtains of 1mL, then extract bacterial genomes with genome test kit, agarose gel electrophoresis (1%) checking, uv analyzer checks electrophoresis result.
2. pcr amplification 16S rDNA fragment
Utilize universal primer (8f:5 '-AGAGTTTGATCCTGGCTCAG-3', 1492r:5'-GGTTACCTTGTTACGACTT-3) carry out PCR reaction, then carry out agarose gel electrophoresis (1%) checking, uv analyzer checks electrophoresis result, PCR clip size approximately 1500 bp.
PCR reaction system (Takara LA Taq 50 μ L): 10 × Buffer, 5 μ L; DNTP 8 μ L; Primer1 1 μ L; Premier2 1 μ L; DNA profiling 1 μ L; Enzyme 0.5 μ L; ddH 2o 33.5 μ L.
PCR condition: 94 DEG C of 5 min of denaturation; 94 DEG C of 35 sec of sex change; 55 DEG C of 45sec anneal; Extend 72 DEG C 2 minutes; Goto step2, circulates 30 times; 72 DEG C of 10 min of extension 1 time eventually; 12 DEG C of for ever.3. PCR product purification
Utilize PCR product purification test kit to carry out glue recovery, then recovery fragment is sent to the raw work order-checking in Shanghai.
4. sequence alignment and submission
Order-checking gained fragment is compared at ncbi database, through comparison find this bacterial strain with rhodococcus erythropolishomology up to 97%, tentatively determine this bacterial strain be rhodococcus erythropolis.
5. bacterial strain homology analysis
After comparison, in acquired results, select several representational fragments and carry out systematic evolution tree analysis, further determine the title of bacterial strain.Show according to the result of Fig. 2, in sibship, Sunda 1101 with rhodococcus erythropolismore approaching.
embodiment 3
Aerobic denitrifying bacteria treatment of simulated sewage
1. relevant substratum:
Thalline enlarged culturing base (/L): KNO 31g; KH 2pO 41g; FeSO 47H 2o 0.05g; CaCl 20.02g; MgSO 47H 2o 1g; Soduxin 8.5g.
Detect substratum (/L): KH 2pO 41g; MgSO 47H 2o 1g; Soduxin 2.8g; NaNO 30.6 or NaNO 20.50.
2. processing condition:
Temperature: 30 DEG C; Shaking speed: 150rpm; Liquid amount: 100/250mL adds without stream.
3. the application of aerobic denitrifying bacteria
Denitrifying bacteria picking one articulating on inclined-plane is entered to enlarged culturing base, at 30 DEG C, under the condition of 150rpm, cultivate 24h, get a part of stereometry dry cell weight, rest part accesses respectively in the simulated sewage that is rich in nitric nitrogen and nitrite nitrogen in 1% ratio, 30 DEG C, 150rpm, cultivates after 24 hours centrifugal 5min under the condition of 8000rpm, detect nitric nitrogen and nitrite nitrogen content in supernatant liquor, the nitrogen of Units of Account thalline is removed ability.
The removal ability of table 1 bacterial strain to nitric nitrogen
Note: dry cell weight (DCW) is 1.0 g/L
The removal ability of table 2 bacterial strain to nitrite nitrogen
Note: dry cell weight (DCW) is 1.0 g/L
embodiment 4
The application of aerobic denitrifying bacteria in actual sewage is processed
Denitrification bacterial classification is added in certain SBR of municipal wastewater treatment plant technique (sequencing batch reactor) aeration tank and carries out denitrogenation processing by 0.1% throwing bacterium amount (dry cell weight (DCW) is 20 g/L).Influent quality is: ammonia nitrogen 64 mgL -1, chemical oxygen demand (COD) (COD) 475 mgL -1.The control group ammonia nitrogen removal frank that does not add denitrifying bacteria is 78%, and chemical oxygen demand (COD) (COD) clearance is 70%; Adding ammonia nitrogen removal frank after denitrifying bacteria is 94%, and chemical oxygen demand (COD) (COD) clearance is 95%.Compared with control group, denitrifying bacteria can obviously improve the removal efficiency of ammonia nitrogen.
In a word, the foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to the covering scope of patent of the present invention.
<110> Zhejiang Prov Sunda Environmental Protection Co., Ltd
<120> mono-strain is for the aerobic denitrifying bacteria of sewage disposal
<210>?1
<211>1210
<212>DNA
<213> rhodococcus erythropolis ( rhodococcus erythropolis）AAAAAGGGACCCAGCATTAACACATGCAAGTCGAGCGGTAAGGCCTTTCGGGGTACACGAGCGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACTTCAGGTTGCATGACTTGGGGTGGAAAGATTTATCGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGACGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAATTACTGGGCGTAAAGAGTTCGTAGGCGGTTTGTCGCGTCGTTTGTGAAAACCAGCAGCTCAACTGCTGGCTTGCAGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAACGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTCCTTCCACGGAATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATATACCGGCAAGCTGCAGAGATGTGGACCCCCTTGTGGTCGGTATACAGGTGGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGTTAAGTCCCGCAACGAGCGCACCCCTATCTATGTGCCAGCACGTTATGTGCTACTCGTAAGAGACTGCCGGGGTCCACCTCGGAGGTAGTGGACGACGTCCAAGTCATCATGCCCTTATGTCCAGGCTTCACAATGCCTAACATTGCCAGTTACAGAGGGCCTTGCCGAGAAACCGTTTAT
 

Claims (4)

1. a strain is for the application of the aerobic denitrifying bacteria of sewage disposal, it is characterized in that: this bacterial strain is rhodococcus erythropolis (Rhodococcus erythropolis) through 16S rDNA Molecular Identification, culture presevation numbering: CCTCC M 2011211, the preservation time: on June 23rd, 2011, preservation place: Chinese Typical Representative culture collection center; Described aerobic denitrifying bacteria is applied to nitric nitrogen and the nitrite nitrogen in the water body of pollution degradation.
2. according to the application of the aerobic denitrifying bacteria for sewage disposal described in claim 1, it is characterized in that: the ratio in 1% after aerobic denitrifying bacteria enlarged culturing is dropped into containing in the water body of nitric nitrogen.
3. according to the application of the aerobic denitrifying bacteria for sewage disposal described in claim 2, it is characterized in that: the ratio in 1% after aerobic denitrifying bacteria enlarged culturing is dropped into containing in the water body of nitrite nitrogen.
4. according to the application of the aerobic denitrifying bacteria for sewage disposal described in claim 2 or 3, it is characterized in that: described enlarged culturing condition is 30 DEG C, under 200 rpm conditions, cultivate 24h.
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CN103468621B (en) * 2013-09-29 2020-04-14 青岛蔚蓝生物集团有限公司 Compound microbial preparation for purifying water body
CN104560858B (en) * 2015-01-26 2018-04-13 鹭滨环保科技（上海）股份有限公司 A kind of red city Rhodococcus sp and its construction method and the application in sewage disposal
CN104611279B (en) * 2015-03-02 2017-06-09 中蓝连海设计研究院 A kind of red city Rhodococcus sp LH N13 and its microbial bacterial agent and purposes
CN109182192B (en) * 2018-09-26 2021-02-19 北京化工大学 Aerobic denitrifying bacterium HY3-2 and application thereof in sewage denitrification
CN113005062B (en) * 2021-03-24 2022-05-13 中国科学院成都生物研究所 Facultative ammonia oxidizing bacteria and application thereof

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