CN103074242A - 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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CN103074242A
CN103074242A CN201110326862XA CN201110326862A CN103074242A CN 103074242 A CN103074242 A CN 103074242A CN 201110326862X A CN201110326862X A CN 201110326862XA CN 201110326862 A CN201110326862 A CN 201110326862A CN 103074242 A CN103074242 A CN 103074242A
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aerobic denitrifying
nitrogen
denitrifying bacteria
aerobic
bacterial strain
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CN103074242B (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
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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 used 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 removed the hot issue that nitrate pollution in the water has become 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 in undressed raw wastewater mainly the form with organic nitrogen and ammonia nitrogen exist; 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 that nitrate content constantly raises in the water body.The traditional biological denitrogenation comprises two processes of nitrification and denitrification, and namely ammonia nitrogen is converted into nitric nitrogen by the aerobic nitrification bacterium, and then is reduced to nitrogen by the denitrifying bacterium of anaerobism or anoxic, discharges water body.Studies show that both at home and abroad in recent years, some microorganisms also can show certain denitrifying capacity under different dissolved oxygen conditions.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.) then report less.The biological denitrificaion that appears as of aerobic denitrifying bacteria provides a brand-new approach, so that traditional biological denitrificaion approach is more perfect, also provides certain theoretical basis for denitrification process research.
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 the pH indicator in substratum according to denitrification.2. dilution method will be cultivated after the diluted sample certain multiple, then with diluting again that the pH value changes, and repetitive operation, 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 nitrite nitrogen in the water body of can under aerobic condition, effectively degrading.
In order to solve the problems of the technologies described above, the present invention is solved by following technical proposals:
The aerobic denitrifying bacteria that is used for sewage disposal, described aerobic denitrifying bacteria 16S rDNA Molecular Identification be rhodococcus erythropolis ( Rhodococcus erythropolis), called after Rhodococcus erythropolisSunda 1101, and this bacterial strain is preserved in Chinese Typical Representative culture collection center on June 23rd, 2011, 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 at first by adding restricted nitrogenous source, intermittent aeration comes the enrichment aerobic denitrifying bacteria, and then fast and effeciently screens aerobic denitrifying bacteria by add the pH indicator in substratum.
For achieving the above object, the technical scheme taked of the present invention is:
Active sludge to sewage work carries out the aeration enrichment culture, filters out aerobic bacteria, then produces the performance of alkali according to denitrifying bacteria, by add the method for brooethyl Finland indicator in substratum, effectively screens aerobic denitrifying bacteria.With the aerobic denitrification bacterium of having simulated sewage and sieving again high denitrifying capacity.
Its step is sampling successively, the aeration enrichment culture, and primary dcreening operation, multiple sieve obtains 1 strain under aerobic condition, nitric nitrogen and nitrite nitrogen in the simulated sewage of degrading simultaneously, the bacterial strain that denitrifying capacity is higher at last.
The invention provides the method for identification of strains.
Identification of strains method of the present invention mainly is the method for identifying by 16S rDNA, utilizing universal primer to amplify 16S rDNA fragment conservative in the evolutionary process checks order, then with gene library in data compare, according to the attribute of bacterial strain homology ratio-dependent bacterial strain, confirm according to systematic evolution tree more at last.
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 carried out enlarged culturing after, be inoculated in and contain the water body that nitric nitrogen and nitrite nitrogen pollute.
The present invention has significant technique effect owing to having adopted above technical scheme:
Bacterial strain provided by the invention can effectively be removed nitric nitrogen and the nitrite nitrogen in the 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 the biological denitrificaion field, and biological denitrification method is not only efficient but also cost is lower, so this bacterial strain has good prospect in being applied to the treating processes of nitrogenous effluent.
Biomaterial preservation information
The 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.
Description of drawings
Fig. 1 is the as a result figure behind the 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: will carry out the aeration enrichment culture from the active sludge that Lu Cun sewage work obtains, liquid amount 2 L/3.6 L change fresh culture every day.Leave standstill first 1h when changing fresh culture, remove the 600mL volume, and then add the 600mL fresh culture, enrichment is approximately 40 days altogether.
2. primary dcreening operation: the active sludge that enrichment is good dilutes, and is coated with at the dibromothymolsulfonphthalein substratum, then places 30 ° of C incubators to cultivate, and cultivates 2-3 days.The blue single bacterium colony that occurs on the dibromothymolsulfonphthalein substratum is classified, and then gramstaining selects the bacterium colony of each form 3-4 the separation of ruling, and places 30 ° of C incubators to cultivate, and cultivates 2-3 days.Line is separated the blue single bacterium colony that obtains classify, select characteristic bacterium colony and be saved on the inclined-plane, for multiple sieve.
3. sieve again: the bacterial strain that primary dcreening operation is preserved accesses thalline enlarged culturing base, 30 ℃, 200 rpm, cultivate 24h, then access respectively 100mL in 1% ratio and detect substratum, cultivate 24 h, 30 ℃, 200 rpm measure the variation of inoculation front and back nitric nitrogen and nitrite nitrogen.The bacterial strain that selection nitric nitrogen degradation rate reaches more than 40% and the nitrite nitrogen degradation rate reaches more than 25% carries out the denitrification capability test, obtains relatively preferably bacterial strain of a strain performance.
4. detection method:
4.1 the mensuration of nitric nitrogen: ultraviolet spectrophotometry, referring to the environment protection industry standard HJ/T 346-2007 of the People's Republic of China (PRC).
4.2 the mensuration of nitrite nitrogen: N-(1-naphthyl)-quadrol hydrochloric acid spectrophotometry is referring to GB GB 7493-87.
4.3 the mensuration of dry cell weight (DCW): get bacterial culture fluid, centrifugal 5min under the condition of 8000 rpm, then abandoning supernatant is dried to the 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 the 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 evaluation of aerobic denitrifying bacteria
1. extraction bacterial genomes
With purpose bacterial strain access LB substratum, after the cultivation overnight, get the centrifugal thalline that gets of 1mL, then extract bacterial genomes with the 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 the PCR reaction, then carry out agarose gel electrophoresis (1%) checking, uv analyzer checks electrophoresis result, and the PCR clip size is 1500 bp approximately.
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 ℃ of 5 min of denaturation; 94 ℃ of 35 sec of sex change; 55 ℃ of 45sec anneal; Extend 72 ℃ 2 minutes; Goto step2 circulates 30 times; 72 ℃ of 10 min of extension is 1 time eventually; 12 ℃ of for ever.3. PCR product purification
Utilize PCR product purification test kit to carry out glue and reclaim, then will reclaim fragment and send to the living worker's order-checking in Shanghai.
4. sequence alignment and submission
The gained fragment that will check order is compared at ncbi database, through comparison find this bacterial strain with Rhodococcus erythropolisHomology up to 97%, determine that tentatively this bacterial strain is Rhodococcus erythropolis
5. bacterial strain homology analysis
After comparison, select several representational fragments in the acquired results and carry out the systematic evolution tree analysis, further determine the title of bacterial strain.Show according to the result of Fig. 2, on 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.
The detection substratum (/L): KH 2PO 41g; MgSO 47H 2O 1g; Soduxin 2.8g; NaNO 30.6 or NaNO 20.50.
2. processing condition:
Temperature: 30 ℃; Shaking speed: 150rpm; Liquid amount: 100/250mL adds without stream.
3. the application of aerobic denitrifying bacteria
Denitrifying bacteria picking one articulating on the inclined-plane is entered the enlarged culturing base, at 30 ℃, cultivate 24h under the condition of 150rpm, 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 ℃, 150rpm cultivates after 24 hours centrifugal 5min under the condition of 8000rpm, detect nitric nitrogen and nitrite nitrogen content in the supernatant liquor, the nitrogen of Units of Account thalline is removed ability.
Table 1 bacterial strain is to the removal ability of nitric nitrogen
Figure 181088DEST_PATH_IMAGE002
Annotate: dry cell weight (DCW) is 1.0 g/L
Table 2 bacterial strain is to the removal ability of nitrite nitrogen
Figure 160545DEST_PATH_IMAGE004
Annotate: dry cell weight (DCW) is 1.0 g/L
Embodiment 4
The application of aerobic denitrifying bacteria in actual sewage is processed
The denitrification bacterial classification added in certain SBR of municipal wastewater treatment plant technique (sequencing batch reactor) aeration tank by 0.1% throwing bacterium amount (dry cell weight (DCW) is 20 g/L) carry out denitrogenation processing.Influent quality is: ammonia nitrogen 64 mgL -1, chemical oxygen demand (COD) (COD) 475 mgL -1The control group ammonia nitrogen removal frank that does not add denitrifying bacteria is 78%, and chemical oxygen demand (COD) (COD) clearance is 70%; Add that ammonia nitrogen removal frank is 94% behind the denitrifying bacteria, chemical oxygen demand (COD) (COD) clearance is 95%.Compare with control group, denitrifying bacteria can obviously improve the removal efficient of ammonia nitrogen.
In a word, the above only is preferred embodiment of the present invention, and all equalizations of doing according to the present patent application claim 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〉strain is used for the aerobic denitrifying bacteria of sewage disposal
<210>?1
<211>1210
<212>DNA
＜213〉rhodococcus erythropolis ( Rhodococcus erythropolis）AAAAAGGGACCCAGCATTAACACATGCAAGTCGAGCGGTAAGGCCTTTCGGGGTACACGAGCGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACTTCAGGTTGCATGACTTGGGGTGGAAAGATTTATCGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGACGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAATTACTGGGCGTAAAGAGTTCGTAGGCGGTTTGTCGCGTCGTTTGTGAAAACCAGCAGCTCAACTGCTGGCTTGCAGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAACGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTCCTTCCACGGAATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATATACCGGCAAGCTGCAGAGATGTGGACCCCCTTGTGGTCGGTATACAGGTGGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGTTAAGTCCCGCAACGAGCGCACCCCTATCTATGTGCCAGCACGTTATGTGCTACTCGTAAGAGACTGCCGGGGTCCACCTCGGAGGTAGTGGACGACGTCCAAGTCATCATGCCCTTATGTCCAGGCTTCACAATGCCTAACATTGCCAGTTACAGAGGGCCTTGCCGAGAAACCGTTTAT
 

Claims (5)

1. a strain is used for the aerobic denitrifying bacteria of sewage disposal, it is characterized in that: this bacterial strain through 16S rDNA Molecular Identification be rhodococcus erythropolis ( Rhodococcus erythropolis), culture presevation numbering: CCTCC M 2011211, preservation time: on June 23rd, 2011, preservation place: Chinese Typical Representative culture collection center.
2. the aerobic denitrifying bacteria for sewage disposal according to claim 1 is characterized in that: described aerobic denitrifying bacteria is applied to nitric nitrogen and the nitrite nitrogen in the water body of pollution degradation.
3. the aerobic denitrifying bacteria for sewage disposal according to claim 2 is characterized in that: the ratio input in 1% after the aerobic denitrifying bacteria enlarged culturing is contained in the water body of nitric nitrogen.
4. the aerobic denitrifying bacteria for sewage disposal according to claim 2 is characterized in that: the ratio input in 1% after the aerobic denitrifying bacteria enlarged culturing is contained in the water body of nitrite nitrogen.
5. according to claim 3 or 4 described aerobic denitrifying bacterias for sewage disposal, it is characterized in that: described enlarged culturing condition is 30 ℃, cultivates 24h under the 200 rpm conditions.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468621A (en) * 2013-09-29 2013-12-25 青岛蔚蓝生物集团有限公司 Compound microorganism preparation capable of purifying water
CN104560858A (en) * 2015-01-26 2015-04-29 鹭滨环保科技（上海）有限公司 Rhodococcus erythropolis, construction method thereof and application in sewage treatment
CN104611279A (en) * 2015-03-02 2015-05-13 中蓝连海设计研究院 Rhodococcus erythropolis LH-N13 as well as microbial agent and use thereof
CN109182192A (en) * 2018-09-26 2019-01-11 北京化工大学 One plant of aerobic denitrifying bacteria HY3-2 and its application in sewage water denitrification
CN113005062A (en) * 2021-03-24 2021-06-22 中国科学院成都生物研究所 Facultative ammonia oxidizing bacteria and application thereof

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US6337204B1 (en) * 1999-06-17 2002-01-08 Institut Francais Du Petrole Biological culture containing Rhodococcus erythropolis erythropolis and/or Rhodococcus rhodnii and process for desulfurization of petroleum fraction
CN101492220A (en) * 2008-01-22 2009-07-29 范兆科 Method for advanced treatment of industrial wastewater with biological technique and membrane technique

Non-Patent Citations (1)

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吴晟雯，等: "白腐菌和红平红球菌共固定化处理增塑剂废水", 《 化 工 环 保》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468621A (en) * 2013-09-29 2013-12-25 青岛蔚蓝生物集团有限公司 Compound microorganism preparation capable of purifying water
CN104560858A (en) * 2015-01-26 2015-04-29 鹭滨环保科技（上海）有限公司 Rhodococcus erythropolis, construction method thereof and application in sewage treatment
CN104611279A (en) * 2015-03-02 2015-05-13 中蓝连海设计研究院 Rhodococcus erythropolis LH-N13 as well as microbial agent and use thereof
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
CN109182192A (en) * 2018-09-26 2019-01-11 北京化工大学 One plant of aerobic denitrifying bacteria HY3-2 and its application in sewage water denitrification
CN113005062A (en) * 2021-03-24 2021-06-22 中国科学院成都生物研究所 Facultative ammonia oxidizing bacteria and application thereof

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