CN104277996B - Solve keratan microbacterium and its cultural method and application - Google Patents
Solve keratan microbacterium and its cultural method and application Download PDFInfo
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- CN104277996B CN104277996B CN201410348045.8A CN201410348045A CN104277996B CN 104277996 B CN104277996 B CN 104277996B CN 201410348045 A CN201410348045 A CN 201410348045A CN 104277996 B CN104277996 B CN 104277996B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/205—Bacterial isolates
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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
- 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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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/105—Phosphorus compounds
Abstract
The present invention is one plant of solution keratan microbacterium（Microbacterium keratanolyticum）6 bacterial strains of Mk, strong environmental adaptability, can be 14.9 mg/L in low nutrition, phosphorus concentration（Body eutrophication will be caused by reaching 0.2 mg/L）Artificial wastewater fluid nutrient medium in fast-growth, and by phosphorus ligands；Dephosphorization efficiency is high and stablizes, and 20 h phosphorus efficiency of culture are up to 60% ~ 70%, non-secondary pollution.Therefore, 6 bacterial strains of Mk can be applied to phosphorous sewage disposal, is microbial resources important on aqueous bio dephosphorization process, has a good application prospect.
Description
Technical field
The invention belongs to field of environment microorganism, and in particular to one plant of solution keratan microbacterium（Microbacterium keratanolyticum）Separation, culture and its application in sewage dephosphorization of Mk-6 bacterial strains.
Background technology
Phosphorus is one of indispensable element of biological growth, but phosphorus content will cause water body rich more than 0.2 mg/L in water body
Nutrient laden, causes water quality to reduce, and the harmful substance increase in water body, drinking water sources reduce, the health of people and animals is impacted,
The balance of the ecosystem is destroyed, causes economic loss.China Environmental State Bulletin in 2013 is shown, Song Hua River, Haihe River, too
The waters such as lake, Chaohu, Dian Chi, it is exceeded because of indexs such as total phosphorus, for many years in eutrophic state.Therefore, control industry
Waste water, sanitary sewage phosphorus content have become one of great environmental problem for needing to solve in a hurry.
At present, it is water body dephosphorized to be broadly divided into biological and chemical dephosphorization.Chemical dephosphorization has easy to operate, efficient dephosphorization, steady
The advantages that determining；But the method is of high cost, easily causes secondary pollution.Biological phosphate-eliminating is using the phosphorus in polyP bacteria enrichment water body, is passed through
The mode dephosphorization of remaining phosphorus containing sludge is excluded, has the advantages that energy saving, sludge output is few, cost is low, non-secondary pollution, is extensive
The main sewage dephosphorization method of sewage treatment plant.
PolyP bacteria is a kind of microorganism with " excess suction phosphorus " feature, can be unnecessary phosphorus with the shape of polyphosphate
Formula is stored in vivo, is substantially reduced the phosphorus content in external environment, is the main executive of aqueous bio dephosphorization, is removed in biology
Play a decisive role in phosphorus.
Laboratory uses low nutrition artificial wastewater fluid nutrient medium（Phosphorus content is 14.9 mg/L）It is water body dephosphorized to detect bacterial strain
Ability, polyP bacteria of the screening with efficient dephosphorization ability.Artificial wastewater fluid nutrient medium phosphorus concentration is to cause body eutrophication most
Low-phosphorous concentration（0.2 mg/L）74.5 times, screened by low nutrition superelevation phosphorus concentration artificial wastewater fluid nutrient medium poly-
Phosphorus bacterium, can well adapt to natural water environment, and the more efficient phosphorus removed in natural water.
The present invention is sieved to the polyP bacteria solution keratan microbacterium of plant height effect biological phosphate-eliminating characteristic from natural habitat
（Microbacterium keratanolyticum）Mk-6 bacterial strains, it is intended to which abundant polyP bacteria resource, expands polyP bacteria strain
Storehouse, strain is provided for biological removal of phosphorus in wastewater.
The content of the invention
In order to solve above-mentioned technical problem, of the invention first purpose is to provide one plant of solution keratan microbacterium
（Microbacterium keratanolyticum）Mk-6 bacterial strains, the bacterial strain dephosphorization efficiency is high and stablizes.The second of the present invention
A purpose is to provide the cultural method of above-mentioned bacterial strain.Second object of the present invention is to provide the application of above-mentioned bacterial strain.
In order to realize first above-mentioned purpose, present invention employs following technical solution：
Solve keratan microbacterium（Microbacterium keratanolyticum）Mk-6 bacterial strains, the bacterial strain preservation are in place
In China typical culture collection center（CCTCC）, preservation date is on 06 24th, 2014, and deposit number is：CCTCC NO:
M 2014279, preservation address：Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University collection.
Water body example of the bacterial strain screening according to the present invention from Jinhua, Zhejiang Province city suburbs pond.Using gradient dilution method
Pure bacterial strain is obtained, it is micro- for solution keratan according to morphological feature, physiological and biochemical property, 16S rDNA gene order identification of M k-6 bacterial strains
Bacillus（Microbacterium keratanolyticum）.
In order to realize second above-mentioned purpose, present invention employs following technical solution：
The cultural method of above-mentioned solution keratan microbacterium Mk-6 bacterial strains, this method comprise the following steps：Go bail for be stored in-
The 20 μ L of solution keratan microbacterium Mk-6 bacterial strains of 20 DEG C of glycerine are coated on the tablet of beef extract-peptone solid medium,
Single bacterium is chosen after 28 DEG C of 24 h of culture and falls within the mixing of 0.3 mL sterile waters, takes 100 μ L to be coated on beef extract-peptone solid culture
On the tablet of base, take 2 holes to be connected in 15 mL beef extract-peptone fluid nutrient mediums with card punch, be placed in 28 DEG C, 170 r/min
24 h are cultivated in shaking table and are used as seed liquor.
In order to realize second above-mentioned purpose, present invention employs following technical solution：
Above-mentioned solution keratan microbacterium Mk-6 bacterial strains are used for dephosphorization.The biology for being preferred for municipal sewage plant removes
Phosphorus.
The present invention is one plant of solution keratan microbacterium（Microbacterium keratanolyticum）Mk-6 bacterial strains, ring
Border is adaptable, can be 14.9 mg/L in low nutrition, phosphorus concentration（Body eutrophication will be caused by reaching 0.2 mg/L）
Artificial wastewater fluid nutrient medium in fast-growth, and by phosphorus ligands；Dephosphorization efficiency is high and stablizes, and 20 h phosphorus efficiency of culture are reachable
60% ~ 70%, non-secondary pollution.Therefore, Mk-6 bacterial strains can be applied to phosphorous sewage disposal, be weight on aqueous bio dephosphorization process
The microbial resources wanted, have a good application prospect.
Brief description of the drawings
The colony characteristics of Fig. 1 Mk-6 bacterial strains.
The thalli morphology of Fig. 2 Mk-6 bacterial strains（×1000）.
Fig. 3 Mk-6 bacterial strain 16S rDNA gene orders.
The Microbacterium phylogenetic tree that Fig. 4 is established based on 16s rDNA gene orders.
Growth curve of Fig. 5 Mk-6 bacterial strains in low nutrition high phosphorus artificial wastewater nutrient solution.
Influence of Fig. 6 liquid amounts to Mk-6 bacterial strain dephosphorizing rates.
Embodiment
The separation screening of the efficient polyP bacteria solution keratan microbacterium Mk-6 bacterial strains of embodiment 1 and identification
（One）Culture medium
Beef extract-peptone solid medium：3 g/L of beef extract, 10 5 g/L of g/L, NaCl of peptone, 20 g/ of agar
L, pH 7.0 ~ 7.2（Fluid nutrient medium is not added with agar）, separation, purifying for bacterium.
Limit phosphorus（Rich phosphorus）Solid medium：Take 30 mL deionized water dissolvings, 8.372 g 3- N-morpholinyls, 0.717g
N- tri-（Methylol）Methylglycine, adjusts pH to 7.4, in the following order solubilization liquid with the KOH of 10 mol/L：0.01 mL
1.830% FeSO4Solution（Now match somebody with somebody）、5 mL 1.9 mol/L NH4Cl、1 mL 0.276 mol/L K2SO4、0.025 mL
0.02 mol/L CaCl2·2H2O、0.42 mL 1.25 mol/L MgCl2·6 H2O、20 mL 2.5 mol/L NaCl、
0.02 mL micro-mixed liquors（Ammonium heptamolybdate 3 × 10-6 mol/L、H3BO3 4×10-4 mol/L、CoCl2 3×10-5
mol/L、CuSO4 10-5 mol/L、MnCl2 8×10-5 mol/L、ZnSO4 10-5 mol/L）, 10 mL, 1% glucose, 0.4
1.686% vitamin B1s of mL, 250 μ L, 6.928% K2HPO4（Rich phosphorus adds 5 mL）、50 mg para-totuidine are blue, add water to be settled to
500 mL, mix and solid training are made with sterilization treatment after filtration sterilization dissolved with 20 g agar and not solidified 500 mL solution
Support base, the screening for polyP bacteria.
（Two）Method
Using gradient dilution partition method.1 mL of water sampling, adds 9 mL sterile waters to dilute, and carries out 10-1~10-3Gradient dilution,
Take 10-2、10-3100 μ L of dilution are coated on beef extract-peptone solid medium tablet, 28 DEG C of 24 h of culture.Picking is thin
Bacterium single bacterium colony, is separated, is purified；Bacterial strain is further purified and uses method of scoring, the pure bacterial strain of gained, which is put in -20 DEG C of refrigerators, to be preserved,
It is spare.
The pure bacterial strain of bacterium of preservation is inoculated with respectively on limit phosphorus and rich phosphorus solid medium, with Lv Shi methylene blues decoration method, Soviet Union
Red black decoration method, blue hickie method and Ammonium Molybdate Spectrophotometric Method for Determination bacterium bacterial strain are to the absorbability of phosphorus, through primary dcreening operation and secondary screening
It is the bacterium bacterial strain that Mk-6 efficiently inhales phosphorus to obtain one plant of numbering afterwards（Attached drawing 2）.
Morphology, Physiology and biochemistry and the identification of 16S r DNA sequence dnas are carried out to Mk-6 bacterial strains.
（Three）As a result
The morphological feature of Mk-6 bacterial strains：Bacterium colony circle, yellow, edge are complete together, swell, is smooth, sticky, moisten, is translucent
（Attached drawing 1）；Thalline is in short and small rod-shaped, and Gram's staining is negative, amphitrichous, no gemma（Attached drawing 2）.
The physiological and biochemical property of Mk-6 bacterial strains：Mk-6 bacterial strains Starch Hydrolysis, indoles, hydrogen sulfide and the examination of L-arginine decarboxylase
Test and be positive, it is impossible to which using citrate, do not liquefy gelatin, and VP, methyl red, glucose fermentation, nitrate reduction test are equal
For feminine gender（Table 1）；Strain growth temperature range is 15 DEG C ~ 40 DEG C, and optimum growth temperature is 25 DEG C ~ 28 DEG C, suitable growth
PH is 7.0 ~ 9.0.
Mk-6 bacterial strain 16S rDNA gene orders（Attached drawing 3）：16S rDNA gene sequencing analysis results show, its base sequence
Row total length is 1422 bp, carries out homology analysis with online Blast softwares, chooses the strain construction system of 19 plants of Microbacteriums
System development tree（Attached drawing 4）.Shown by Fig. 4, Mk-6 bacterial strains and solution keratan microbacterium（Microbacterium keratanolyticum）In the same branch of chadogram, two bacterial strain homologys are up to 98%.With reference to strain morphology feature, life
Biochemical character is managed, identifies the bacterial strain for solution keratan microbacterium（Microbacterium keratanolyticum）.
The physiological and biochemical test result of 1 Mk-6 bacterial strains of table
| Pilot project | As a result | Pilot project | As a result |
| Indoles | + | Methyl red | - |
| Hydrogen sulfide | + | Gelatin liquefaction | - |
| Starch Hydrolysis | + | Glucose fermentation | - |
| L-arginine decarboxylase | + | Nitrate reduction | - |
| VP | - | Citric acid utilizes | - |
Note："+" is the positive, and "-" is feminine gender.
2 Mk-6 bacterial strains of embodiment growth curve in low nutrition high phosphorus artificial wastewater nutrient solution measures
（One）Bacterial strain：Mk-6 bacterial strains.
（Two）Culture medium
Beef extract-peptone solid medium：Formula is the same as embodiment 1
Artificial wastewater fluid nutrient medium：0.925 g/L of sodium acetate, 0.1 g/L of peptone, yeast extract 0.01 g/L, NaCl
0.05 g/L、KH2PO4 0.0655 g/L、MgCl2·6H2O 0.1412 g/L、CaCl20.025 g/L, pH 7.0 ~ 7.2.
（Three）Method
Go bail for and be stored in -20 DEG C of 20 μ L of glycerol stock and be coated on the tablet of beef extract-peptone solid medium, 28 DEG C
Cultivate and single bacterium is chosen after 24 h fall within the mixing of 0.3 mL sterile waters, take 100 μ L to be coated on the flat of beef extract-peptone solid medium
On plate, card punch is used（0.5 cm of aperture）Take 2 holes to be connected in 15 mL beef extract-peptone fluid nutrient mediums, be placed in 28 DEG C, 170
24 h are cultivated in r/min shaking tables（OD600≈1.5）As seed liquor；100 mL artificial wastewaters are equipped with 8% inoculum concentration access again
In 250 mL conical flasks of fluid nutrient medium, 28 DEG C are placed in, is cultivated in 170 r/min shaking tables, OD is measured every 2 h600Value, is painted
The growth curve of Mk-6 bacterial strains processed.
（Four）As a result
After Mk-6 bacterial strains are activated, in low nutrition high phosphorus（Phosphorus content is 14.9 mg/L）In artificial wastewater fluid nutrient medium
Culture, growth is very fast, and it is longer to stablize growth period（Attached drawing 5）, can be the strain that biological removal of phosphorus in wastewater provides.Attached drawing 5 can be seen that：
The h of 0 h of Mk-6 strain culturings ~ 8 is lag phase, and the h of 8 h ~ 20 is exponential phase, and the h of 20 h ~ 36 is stablizes growth period, the 36th h
Into decline phase.
Influence of 3 liquid amount of embodiment to Mk-6 bacterial strain biological phosphor-removing effects
（One）Bacterial strain：Mk-6 bacterial strains.
（Two）Culture medium
Beef extract-peptone solid medium：Formula is the same as embodiment 1；
Artificial wastewater fluid nutrient medium：Formula is the same as embodiment 2.
（Three）Method
Go bail for and be stored in -20 DEG C of 20 μ L of glycerol stock and be coated on the tablet of beef extract-peptone solid medium, 28 DEG C
Cultivate and single bacterium is chosen after 24 h fall within the mixing of 0.3 mL sterile waters, take 100 μ L to be coated on the flat of beef extract-peptone solid medium
On plate, card punch is used（0.5 cm of aperture）Take 2 holes to be connected in 15 mL beef extract-peptone fluid nutrient mediums, be placed in 28 DEG C, 170
24 h are cultivated in r/min shaking tables（OD600≈1.5）As seed liquor；It is dirty equipped with different amounts of synthesis with 8% inoculum concentration access again
Water liquid culture medium（Liquid amount is respectively 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL）In 250 mL triangular flasks,
In 28 DEG C of temperature, 170 r/min of shaking table speed, starting pH cultivates 20 h under conditions of being 7.2.30 mL bacteria suspensions are taken in 50
In the centrifuge tube of mL, 5 min are centrifuged with 12 000 r/min, take supernatant to survey phosphorus concentration by the assay method of total phosphorus, total phosphorus
Measure uses ammonium molybdate spectrophotometric method, and dephosphorizing rate is calculated according to control.
（Four）As a result
Different liquid amounts have a certain impact AJ-3 bacterial strain phosphor-removing effects, when liquid amount is to fill 40 in 250 mL triangular flasks
ML, 28 DEG C of temperature, 170 r/min of shaking table speed, artificial wastewater fluid nutrient medium（PH is 7.2）20 h of lower culture, dephosphorizing rate can
Up to 67.43%；The suitable liquid amount of Mk-6 bacterial strains is 40 mL（Attached drawing 6）.
<110>Zhejiang Normal University
<120>Solve keratan microbacterium and its cultural method and application
<160>
<210> 1
<211> 1422
<212> rDNA
<213>Solve keratan microbacterium
<400> 1
1 GCAGGAGCTT GCTCTTGTGG ATCAGTGGCG AACGGGTGAG TAACACGTGA GCAACCTGCC
61 CCGGACTCTG GGATAAGCGC TGGAAACGGC GTCTAATACT GGATACGAGT AGCGACCGCA
121 TGGTCAGCTA TTGGAAAGAA CTTCGGTCTG GGATGGGCTC GCGGCCTATC AGCTTGTTGG
181 TGAGGTAATG GCTCACCAAG GCGTCGACGG GTAGCCGGCC TGAGAGGGTG ACCGGCCACA
241 CTGGGACTGA GACACGGCCC AGACTCCTAC GGGAGGCAGC AGTGGGGAAT ATTGCACAAT
301 GGGCGCAAGC CTGATGCAGC AACGCCGCGT GAGGGATGAC GGCCTTCGGG TTGTAAACCT
361 CTTTTAGTAG GGAAGAAGCG AAAGTGACGG TACCTGCAGA AAAAGCGCCG GCTAACTACG
421 TGCCAGCAGC CGCGGTAATA CTTATGGCGC AAGCGTTATC CGGAATTATT GGGCGTAAAG
481 AGCTCGTAGG CGGTTTGTCG CGTCTGCTGT GAAATCTGGG GGCTCAACCC CCAGCCTGCA
541 GTGGGTACGG GCAGACTAGA GTGCGGTAGG GGAGATTGGA ATTCCTGGTG TAGCGGTGGA
601 ATGCGCAGAT ATCAGGAGGA ACACCGATGG CGAAGGCAGA TCTCTGGGCC GTAACTGACG
661 CTGAGGAGCG AAAGGGTGGG GAGCAAACAG GCTTAGATAC CCTGGTAGTC CACCCCGTAA
721 ACGTTGGGAA CTAGTTGTGG GGTCCATTCC ACGGATTCCG TGACGCAGCT AACGCATTAA
781 GTTCCCCGCC TGGGGAGTAC GGCCGCAAGG CTAAAACTCA AAGGAATTGA CGGGGACCCG
841 CACAAGCGGC GGAGCATGCG GATTAATTCG ATGCAACGCG AAGAACCTTA CCAAGGCTTG
901 ACATATACGA GAACGGGCCA GAAATGGTCA ACTCTTTGGA CACTCGTAAA CAGGTGGTGC
961 ATGGTTGTCG TCAGCTCGTG TCGTGAGATG TTGGGTTAAG TCCCGCAACG AGCGCAACCC
1021 TCGTTCTATG TTGCCAGCAC GTAATGGTGG GAACTCATGG GATACTGCCG GGGTCAACTC
1081 GGAGGAAGGT GGGGATGACG TCAAATCATC ATGCCCCTTA TGTCTTGGGC TTCACGCATG
1141 CTACAATGGC CGGTACAAAG GGCTGCAATA CCGCGAGGTG GAGCGAATCC CAAAAAGCCG
1201 GTCCCAGTTC GGATTGAGGT CTGCAACTCG ACCTCATGAA GTCGGAGTCG CTAGTAATCG
1261 CAGATCAGCA ACGCTGCGGT GAATACGTTC CCGGGTCTTG TACACACCGC CCGTCAAGTC
1321 ATGAAAGTCG GTAACACCTG AAGCCGGTGG CCTAACCCTT GTGGAAGGGG AAGCCGTCGG
1381 AAAGGTGGGA TCGGTAAATT AGGACTAAGT CGTAACATGG AG
Claims (2)
1. solve keratan microbacterium（Microbacterium keratanolyticum）Mk-6 bacterial strains are used for the application of dephosphorization, institute
The Mk-6 bacterial strains stated are deposited in positioned at China typical culture collection center（CCTCC）, preservation date is on 06 24th, 2014,
Deposit number is：CCTCC NO:M 2014279, preservation address：In the preservation of Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University
The heart.
2. solve keratan microbacterium（Microbacterium keratanolyticum）Mk-6 bacterial strains are used for municipal sewage treatment
The application of the biological phosphate-eliminating of factory, the Mk-6 bacterial strains are deposited in positioned at China typical culture collection center（CCTCC）, preservation
Date is on 06 24th, 2014, and deposit number is：CCTCC NO:M 2014279, preservation address：Wuhan City, Hubei Province Wuchang
Luo Jia Shan Wuhan University collection.
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