CN108486013A - Application of the adhesion sword bacterium in sewage purification - Google Patents
Application of the adhesion sword bacterium in sewage purification Download PDFInfo
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- CN108486013A CN108486013A CN201810283498.5A CN201810283498A CN108486013A CN 108486013 A CN108486013 A CN 108486013A CN 201810283498 A CN201810283498 A CN 201810283498A CN 108486013 A CN108486013 A CN 108486013A
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
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Abstract
The invention discloses application of the adhesion sword bacterium in sewage purification.The present invention experiments prove that, adhesion sword bacterium have the advantages that easily cultivate, the speed of growth soon, strong environmental adaptability, good sewage processing effect, preferable clean-up effect is all had to artificial synthesized sewage, eutrophication water and pig raising sewage etc., new strain source is provided for waste water control.
Description
Technical field
The invention belongs to technical field of sewage purification, it is more particularly related to by adhesion sword bacterium for purifying dirt
Water.
Background technology
With economic and society fast development, the mankind increasingly sharpen to the influence that aquatic ecosystem is brought, especially in recent years
It is intensive fast-developing with scale to carry out China's pig breeding industry, the excrement and urine waste of large-scale pig farm discharge is also increasing severely, excrement and urine waste
It is handled through anaerobic fermentation, most of organic matter is removed, but the indexs such as N, P and COD are still very high.The nutrients such as excess nitrogen, phosphorus
Mass flow enters water body so that algae and other zooplankters breed rapidly, and water transparency and dissolved oxygen change, and water quality is continuous
Deteriorate, to make water ecosystem and water function it is hindered and destroy.Moreover, containing Cu in the excrement and urine waste of pig farm discharge
The metallic elements such as (copper), Pb (lead) and As (arsenic), cannot such as effectively remove, will cause damages to environment.
Eutrophied water treatment and reparation have the means such as physics, chemistry and biology at present.Physical method quantities is big, consumes
Energy is high and is not thorough.It is apparent to add chemical agent short run effect, but is easy to happen water quality and rebounds and cause secondary pollution of water, meeting
Certain harmful effect is caused to the whole ecological environment of water body.Enrichment by Microorganisms nutritive water recovery technique by microorganism it
Between food chain relation establish corresponding microecosystem, accelerate the energy flow in water and substance cycle, enhancement microbiological pair
Excess nitrogen, the absorption of phosphorus, conversion and degradation circle, repair self-purification of water function with this, mitigate water body eutrophication degree in water body.
Since the input of microorganism remediation technology is less than traditional environment engineering technology, and there is environmental-friendly, non-secondary pollution, processing effect
Rate is high, processing time is short, the advantages such as easy to operate, thus becomes focus of attention.Different types of microorganisms is to each substance
Metabolism and Utilization ability difference are larger, so finding microbial resources environmental-friendly and that pollution treatment ability is strong from environment has weight
The realistic meaning wanted.
Invention content
Present invention aims at：Adhesion sword bacterium (Ensifer adhaerens sp.) is provided in artificial synthesized sewage, richness
New opplication in the purification of nutritive water, pig raising sewage etc..
The present invention will adhere sword bacterium for removing the NH in sewage4 +- N, total phosphorus (TP) and/or COD (COD).
The present invention will also adhere sword bacterium for removing Cu, Pb, As in sewage.
Compared with the existing technology, the present invention has the advantages that：
The present invention experiments prove that, adhesion sword bacterium has that easily culture, the speed of growth be fast, strong environmental adaptability, sewage
The advantages of high treating effect, all has preferable purification to sewage such as artificial synthesized sewage, eutrophication water and pig raising sewage
Effect provides new strain source for waste water control.
Description of the drawings
Fig. 1 is the nutrient agar panel bacterium colony figure of adhesion sword bacterium.
Fig. 2 is the optical microphotograph sem observation result (violet staining, oil mirror, × 100) of adhesion sword bacterium.
Specific implementation mode
In order to make the purpose of the present invention, technical solution and advantageous effects be more clear, with reference to embodiments, to this
Invention is further elaborated.It should be understood that embodiment described in this specification is just for the sake of this hair of explanation
It is bright, be not intended to limit the present invention, parameter, ratio of embodiment etc. can adaptation to local conditions make a choice and substance had no to result
It influences.
Separation, purifying and the identification of the adhesion sword bacterium of embodiment 1
1. sample collection：Bacteria samples acquire the pond water from Foochow eutrophication.Water sample sample is placed in 4 DEG C of refrigerators and protects
It deposits.
2. strain separating：
2.1 primary dcreening operation：Under aseptic condition, 10ml water sample samples are drawn, are added equipped with 200mL denitrification enriched mediums
In 500mL triangular flasks, in 25~28 DEG C, 120r/min 3~5d of shaken cultivation, first generation enrichment culture object is obtained.By the first generation
Enrichment culture object subculture, method are same as above, shaken cultivation, but bacterial load will be reduced by generation.Or so 10 generation of subculture,
Final enrichment culture object is obtained, at this point, microscopy observation is with the presence of a large amount of bacteriums.0.1mL culture solutions are drawn, 0.8% nothing is added
In bacterium physiological saline, 10 are obtained-1, 10-2, 10-3, 10-4, 10-5With 10-6The dilute solution of gradient draws the dilution of various concentration
Each 0.2mL of solution is coated on bromthymol blue (BTB) selective medium, 25~28 DEG C of 2~3d of culture.From these dilution ladders
1 most suitable gradient is picked out in degree, it is desirable that bacterium colony distribution is clear uniformly (clump count is at 20-200) on tablet, from tablet
Upper picking blue or surrounding have the single bacterium colony of blue halos.Kind is chosen in repeated 3 coatings, until colony characteristics are consistent, bacterium without exception
Drop out existing person, it is believed that be Economical Purification.Single bacterium colony is inoculated on nutrient agar slant medium and cultivates.It is chosen from slant medium
A small amount of thalline is taken, is seeded in the test tube equipped with the sterile denitrification enriched mediums of 10mL, with the closed bottleneck of rubber plug, sets incubator
In 25~28 DEG C of constant temperature incubations.Sample 1mL at regular intervals, be separately added into 2 clean tubes, respectively be added dropwise diphenylamines and
Green Si Shi reagent 0.2mL contain quantitative change according to what the color change of solution understood nitrate nitrogen and nitrite nitrogen in culture medium
Change.Changes of contents according to nitrate nitrogen and nitrite nitrogen qualitatively judges denitrification rate, choose several plants of denitrification rates compared with
High bacterial strain.By bacterial strain dibbling in limit phosphorus and the excessive glucose-MOPS culture mediums of phosphorus, 25~28 DEG C of 2~3d of culture.Observation is blue
The growing state of hickie.Picking all generates the bacterial strain of locus coeruleus simultaneously on two kinds of culture mediums.
Denitrification enriched medium：KNO32.0g, MgSO4·7H20 0.2g, K2HPO40.5g, sodium potassium tartrate tetrahydrate 20g,
CaCl20.02g, H2O 1000mL, pH 7.2.
Bromthymol blue (BTB) selective medium：Agar 20g, KNO31g, KH2PO41g, FeCl2·6H2O 0.5g,
CaCl2·7H2O 0.2g, MgSO4·7H2O 1g, sodium succinate 8.5g, BTB (1% is dissolved in alcohol) 1mL；Distilled water 1000mL,
PH7.0-7.3 is adjusted with 1mol/LNaOH.
Nutrient agar (NA)：Peptone 10g, beef extract 3g, sodium chloride 5g, 15~20g of agar, distilled water
1000mL, 121 DEG C of high pressure sterilization 15min.
Limit phosphorus and the excessive glucose-MOPS culture mediums of phosphorus：10 × MOPS mixtures (8.372g MOPS+ of 100ml
0.717g tricine+30ml deionized waters, 10mol L- 1KOH adjust pH to 7.4, total volume to 44ml, 0.01%1ml
The FeSO of brand-new4Solution, in the following order solubilization liquid：5ml 1.9mol L- 1NH4Cl, 1ml 0.276mol L- 1K4SO4,
0.025ml 0.02mol L- 1CaCl2·2H2O, 0.21ml 2.5mol L- 1MgCl2·6H2O, 10ml 5mol L- 1NaCl,
0.02ml micro-mixed liquors, 38.7ml deionized waters, glucose 0.1g), take 50ml to be placed in the triangular flask of two 500ml
In, 0.0087g K are added into a triangular flask2HPO4, become limit phosphorus culture medium；0.1732g is added into another bottle
K2HPO4, become the excessive culture medium of phosphorus.Thiamine solution and deionized water is added into two kinds of culture mediums, uses respectively thin
After the filtering of bacterium filter, it is sub-packed in the triangular flask of sterilized 150ml.
Micro-mixed liquor：MnCl2·4H2O 12.98g, CuSO4·5H2O 1.83g, ZnSO4·7H2O 3.20g,
FeSO40.05g, constant volume to 1L.
NH4 +- N is measured using reagent colorimetric method；TP uses potassium persulfate oxidation-Ammonium Molybdate Spectrophotometric Method for Determination；
COD is measured using dichromate titration.
2.2 secondary screening：It is sky not add microbial inoculum in the denitrogenation dephosphorizing function bacterium access artificial wastewater that primary dcreening operation is obtained respectively
White control group sets up three parallel groups, 25~28 DEG C, 120rpm shake cultures.NH in water body is measured by sampling in 96h4 +- N, TP and
The content of COD.The wherein one plant bacterial strain best to wastewater treatment efficiency is chosen, is NY003 by the Strain Designation.NY003 bacterial strains
Treated sewage 96h, NH4 +- N removal rates are that 88%, TP removal rates are 78%, and COD degradation rate is 79%.
The preparation of artificial synthesized sewage, composition (mg/L)：Glucose 360, yeast extract 80.0, KH2PO414.0,
MgSO4·7H2O 24.0, NH4Cl 60.0, CaCl218.0 NaHCO324.0 MnSO4·7H2O 6.0, FeCl30.3,
NaNO230.0 KNO330.0, it is dissolved in 1L distilled water, adjusts pH to 7.0.It is sub-packed in 500mL triangular flasks.121 DEG C of high pressures
Sterilize 15min.
3. the identification of strain：The bacterium solution 5mL, 8000r/min for taking culture to exponential phase centrifuge 15min, and incline supernatant
Liquid collects thalline.Using CTAB/NaCl methods extraction phage gene group DNA.Steps are as follows：1.35mLTE solution is added in thalline
(pH 8.0) suspends, 10% lauryl sodium sulfate of 0.3mL (SDS) and 150 μ L 100mg/ml lysozymes and 150 μ L is added
100mg/mL Proteinase Ks, mixing, 60 DEG C of water-bath 1h add 0.25mL 5mol/LNaCl and 0.2mL CTAB/NaCl solution, 65 DEG C
Water-bath 10min is respectively extracted 3 times with isometric phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol, 10000r/min centrifugations
0.6 times of volume isopropanol is added in 10min, Aspirate supernatant, is positioned over -20 DEG C and precipitates DNA, and DNA is dissolved in 50 μ L TE, and -20
It DEG C saves backup.Utilize the genomic DNA integrality of 1% agarose gel electrophoresis detection extracting.Using 16S rRNA genes
Universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') are to carrying
The DNA taken carries out PCR amplification.Supreme marine growth Engineering Co., Ltd is sent to be sequenced the DNA product after amplification.Use BLAST
The 16S rRNA sequences that the GenBank databases of software and NCBI are included are compared.NY003 and adhesion sword bacterium
The 16S rRNA genes of Ensifer adhaerens strain WJB133 and Ensifer adhaerens strain WJB36
The similitude of sequence is 99%.Physiology and biochemistry and the Molecular Identification of comprehensive bacterial strain are as a result, determine that bacterial strain NY003 is adhesion sword
Bacterium, 16S rRNA sequences such as SEQ ID NO:Shown in 1.
Adhesion sword bacterium was preserved in Fujian Microorganism Inst. on March 13rd, 2018, and preserving number is：FIM-
20180313003。
The preparation of the adhesion sword bacterium of embodiment 2
(1) inclined-plane culture：
1 gained adhesion sword bacterium of embodiment is seeded on nutrient agar, in 28 DEG C of 2~3d of incubator culture.Battalion
Agar medium formula is supported with embodiment 1.
(2) fermented and cultured：
With oese the adhesion sword thalline on a small amount of slant medium is provoked to access in fermentation medium, in 25~28 DEG C,
3~5d of shaken cultivation on the shaking table of 120r/min.
Fermentative medium formula (g/L)：Tryptone 10.0, yeast powder 5.0, sodium chloride 10.0, surplus are water, and pH is certainly
So, after mixing by above-mentioned composition, it sterilizes spare.
Cleaning test of the adhesion sword bacterium of embodiment 3 to eutrophication water
Eutrophic water is derived from eutrophication pond waters.Adhesion sword bacterium 5~10mL of bacterium solution prepared by Example 2,
8000r/min centrifuges 5min, abandons supernatant and obtains thalline.The culture medium on thalline is washed away with 0.8% physiological saline, uses sterile water
Dissolving, centrifugation, is repeated 3 times to obtain wet thallus.It measures 3L eutrophic waters to be added in process container, adds 0.05% (v/v's)
It adheres sword bacterium thalline, NH in treatment fluid4 +The initial value of-N, TP and COD is respectively：18.3mg/L, 1.5mg/L and 87mg/L.With
It is blank control group not add microbial inoculum, sets up three parallel groups.Intermittent aerating mode is used at room temperature, that is, is aerated 1h, is stopped
Expose 1h.NH in water body is measured by sampling after 96h4 +The content of-N, TP and COD.NH in blank control group treatment fluid at this time4 +-N、TP
Value with COD is respectively：13.4mg/L, 1.0mg/L and 56.6mg/L.And add the NH in the treatment fluid of microbial inoculum4 +- N, TP and
The value of COD is respectively：1.6mg/L, 0.2mg/L and 12.2mg/L.
The result shows that compared with not adding bacterium, NH in the treatment fluid of Amur microbacterium is added4 +- N removal rates are 91%, TP
Removal rate is 87%, and COD degradation rate is 86%.The sword bacterium NY003 that adheres is notable to the clean-up effect of eutrophic water.
Cleaning test of the adhesion sword bacterium of embodiment 4 to wastewater from pig farm anaerobic effluent
Adhesion sword bacterium bacterium solution 5~10mL, 8000r/min centrifugation 5min prepared by Example 2, abandons supernatant and obtains bacterium
Body.The culture medium on thalline is washed away with 0.8% physiological saline, with sterile water dissolution, centrifugation is repeated 3 times to obtain wet thallus.Measure 3L
Wastewater from pig farm anaerobic effluent treatment fluid (50mL anaerobic effluent+150mL tap water), is added in process container, wastewater from pig farm is detested
Add the thalline of 0.1% (v/v) in oxygen water outlet treatment fluid, thalline inoculum concentration is 0.1% (V/V), NH in treatment fluid4 +-N、TP、
The initial value of COD, Cu, Pb and As is respectively：339.7mg/L, 5.6mg/L, 513mg/L, 0.58mg/L, 0.03mg/L and
0.02mg/L.Not add microbial inoculum as blank control group, three parallel groups are set up.Intermittent aerating mode is used at room temperature,
It is aerated 1h, stops exposing 1h.NH in water body is measured by sampling after 96h4 +The content of-N, TP, COD, Cu, Pb and As.At blank control group
Manage the NH in liquid4 +The value of-N, TP and COD is respectively：221mg/L, 3.9mg/L, 418.3mg/L, 0.58mg/L, 0.03mg/L and
0.02mg/L.Add the NH in the treatment fluid of microbial inoculum4 +- N, TP, COD, Cu, Pb and As value be respectively：44.16mg/L、
1.06mg/L, 112.86mg/L, 0.28mg/L, 0.02mg/L and 0.01mg/L.
The result shows that NH in the treatment fluid of addition microbial inoculum group4 +- N removal rates are that 87%, TP removal rates are 81%, COD degradation
Rate is 78%, and the removal rate to Cu, Pb and As is respectively 51%, 33% and 50%.Sword bacterium NY003 adhere to wastewater from pig farm anaerobism
The clean-up effect of water outlet is notable.
Cleaning test of the adhesion sword bacterium of embodiment 5 to artificial synthesized sewage
Adhesion sword bacterium bacterium solution 5~10mL, 8000r/min centrifugation 5min prepared by Example 2, abandons supernatant and obtains bacterium
Body.The culture medium on thalline is washed away with 0.8% physiological saline, with sterile water dissolution, centrifugation is repeated 3 times to obtain wet thallus.Measure 3L
Artificial wastewater is added in process container, adds the thalline of 0.05% (v/v), NH in treatment fluid4 +The initial value of-N, TP and COD
Respectively：25.67mg/L, 7.13mg/L and 168mg/L.Not add microbial inoculum as blank control group, three parallel groups are set up.
Intermittent aerating mode is used at room temperature, that is, is aerated 1h, stops exposing 1h.NH in water body is measured by sampling after 96h4 +- N, TP and COD's contains
Amount.NH in blank control group treatment fluid4 +The value of-N, TP and COD is respectively：9.53mg/L, 2.39mg/L and 74.3mg/L.Add
Add the NH in the treatment fluid of microbial inoculum4 +The value of-N, TP and COD is respectively：4.36mg/L, 1.78mg/L and 36.96mg/L.
The result shows that NH in the treatment fluid of addition microbial inoculum group4 +- N removal rates are that 83%, TP removal rates are 75%, COD degradation
Rate is 78%.The sword bacterium NY003 that adheres is notable to the clean-up effect of wastewater from pig farm anaerobic effluent.
The preparation of artificial synthesized sewage, composition (mg/L)：Glucose 360, yeast extract 80.0, KH2PO414.0,
MgSO4·7H2O 24.0, NH4Cl 60.0, CaCl218.0 NaHCO324.0 MnSO4·7H2O 6.0, FeCl30.3,
NaNO230.0 KNO330.0, it is dissolved in 1L distilled water, adjusts pH to 7.0.It is sub-packed in 500mL triangular flasks.121 DEG C of high pressures
Sterilize 15min.
The announcement and guidance of book according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula carries out change and modification appropriate.Therefore, the invention is not limited in specific implementation modes disclosed and described above, to this
Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.In addition, although this specification
In used some specific terms, these terms are merely for convenience of description, does not limit the present invention in any way.
Sequence table
<110>Fujian Microorganism Inst.
<120>Application of the adhesion sword bacterium in sewage purification
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1000
<212> DNA/RNA
<213>Adhere sword bacterium 16S rRNA (Ensifer adhaerens sp.)
<400> 1
tgcaatgcgg cagcttacca tgcagtcgag cgccccgcaa ggggagcggc agacgggtga 60
gtaacgcgtg ggaatctacc cttttctacg gaataacgca gggaaacttg tgctaatacc 120
gtatacgccc ttcgggggaa agatttatcg ggaaaggatg agcccgcgtt ggattagcta 180
gttggtgggg taaaggccta ccaaggcgac gatccatagc tggtctgaga ggatgatcag 240
ccacattggg actgagacac ggcccaaact cctacgggag gcagcagtgg ggaatattgg 300
acaatgggcg caagcctgat ccagccatgc cgcgtgagtg atgaaggccc tagggttgta 360
aagctctttc accggtgaag ataatgacgg taaccggaga agaagccccg gctaacttcg 420
tgccagcagc cgcggtaata cgaagggggc tagcgttgtt cggaattact gggcgtaaag 480
cgcacgtagg cggacattta agtcaggggt gaaatcccgg ggctcaaccc cggaactgcc 540
tttgatactg ggtgtctaga gtatggaaga ggtgagtgga attccgagtg tagaggtgaa 600
attcgtagat attcggagga acaccagtgg cgaaggcggc tcactggtcc attactgacg 660
ctgaggtgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgccgtaa 720
acgatgaatg ttagccgtcg ggcagtttac tgttcggtgg cgcagctaac gcattaaaca 780
ttccgcctgg ggagtacggt cgcaagatta aaactcaaag gaattgacgg gggcccgcac 840
aagcggtgga gcatgtgggt ttaattcgaa gcaacgcgca gaaccttacc agcccttgac 900
atcccgatcg cggattacgg agacgttttc cttcagttcg gctggatcgg agacaggtgc 960
tgcatgctgt cgtcagctcg tgtcgtgaga tgtgggtaag 1000
Claims (4)
1. adhere NH of the sword bacterium in removing sewage4 +Application in-N.
2. application of the sword bacterium in the total phosphorus in removing sewage of adhering.
3. the application in COD of the sword bacterium in sewage of degrading of adhering.
4. application of the sword bacterium in Cu, Pb, As in removing sewage of adhering.
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| CN201810283498.5A CN108486013A (en) | 2018-04-02 | 2018-04-02 | Application of the adhesion sword bacterium in sewage purification |
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| CN201810283498.5A CN108486013A (en) | 2018-04-02 | 2018-04-02 | Application of the adhesion sword bacterium in sewage purification |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114042748A (en) * | 2021-08-30 | 2022-02-15 | 齐齐哈尔大学 | Application of toxophila agglomerans JB19 in remediation of heavy metal contaminated soil |
| CN114292797A (en) * | 2022-03-08 | 2022-04-08 | 佛山市玉凰生态环境科技有限公司 | Physarum viscosum and application of microbial flocculant thereof in sewage treatment |
| CN114317634A (en) * | 2021-12-29 | 2022-04-12 | 苏州科宁多元醇有限公司 | Efficient preparation method and application of extracellular polysaccharide of sword-like adhesion bacterium |
| CN116904349A (en) * | 2023-06-15 | 2023-10-20 | 中国科学院上海高等研究院 | Adhesive sword bacteria with aerobic denitrification capability and application thereof |
-
2018
- 2018-04-02 CN CN201810283498.5A patent/CN108486013A/en not_active Withdrawn
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114042748A (en) * | 2021-08-30 | 2022-02-15 | 齐齐哈尔大学 | Application of toxophila agglomerans JB19 in remediation of heavy metal contaminated soil |
| CN114317634A (en) * | 2021-12-29 | 2022-04-12 | 苏州科宁多元醇有限公司 | Efficient preparation method and application of extracellular polysaccharide of sword-like adhesion bacterium |
| CN114292797A (en) * | 2022-03-08 | 2022-04-08 | 佛山市玉凰生态环境科技有限公司 | Physarum viscosum and application of microbial flocculant thereof in sewage treatment |
| CN114292797B (en) * | 2022-03-08 | 2022-05-24 | 佛山市玉凰生态环境科技有限公司 | Physarum viscosum and application of microbial flocculant thereof in sewage treatment |
| CN116904349A (en) * | 2023-06-15 | 2023-10-20 | 中国科学院上海高等研究院 | Adhesive sword bacteria with aerobic denitrification capability and application thereof |
| CN116904349B (en) * | 2023-06-15 | 2024-03-22 | 中国科学院上海高等研究院 | Adhesive sword bacteria with aerobic denitrification capability and application thereof |
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Application publication date: 20180904 |