CN104277996A - Microbacterium keratanolyticum, and culture method and application thereof - Google Patents

Microbacterium keratanolyticum, and culture method and application thereof Download PDF

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Publication number
CN104277996A
CN104277996A CN201410348045.8A CN201410348045A CN104277996A CN 104277996 A CN104277996 A CN 104277996A CN 201410348045 A CN201410348045 A CN 201410348045A CN 104277996 A CN104277996 A CN 104277996A
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bacterial strain
microbacterium
phosphorus
keratin
strain
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CN104277996B (en
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蒋冬花
谢祥聪
胡优
李晓倩
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Zhejiang Normal University CJNU
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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
    • 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
    • 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
    • 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

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  • Water Supply & Treatment (AREA)
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  • Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
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Abstract

The invention relates to a Microbacterium keratanolyticum Mk-6 strain. The Microbacterium keratanolyticum Mk-6 strain has high environmental adaptability, can quickly grow in a low-nutrient synthetic sewage liquid culture medium with the phosphorus concentration of 14.9 mg/L (the water body can be eutrophicated when the concentration reaches 0.2 mg/L), and can remove the phosphorus; and the Microbacterium keratanolyticum Mk-6 strain has the advantages of high and stable phosphorus removal efficiency and no secondary pollution, and the phosphorus removal efficiency after culture for 20 hours can reach 60-70%. Therefore, the Mk-6 strain can be used for phosphorus-containing sewage treatment, is an important microbial resource in the water body biological phosphorus removal technique, and has favorable application prospects.

Description

Separate keratin microbacterium and cultural method thereof and application
Technical field
The invention belongs to field of environment microorganism ,be specifically related to a strain solution keratin microbacterium ( microbacterium keratanolyticum) separation of Mk-6 bacterial strain, cultivation and the application in sewage dephosphorization thereof.
Background technology
Phosphorus is one of essential element of biological growth, but in water body, phosphorus content will cause body eutrophication more than 0.2 mg/L, causes water quality to reduce, objectionable impurities in water body increases, and drinking water sources reduces, and impacts the health of people and animals, destroy the balance of the ecosystem, cause financial loss.With regard to China Environmental State Bulletin display in 2013, the waters such as Song Hua River, Haihe River, Taihu Lake, Chaohu, Dian Chi, exceeding standard all because of indexs such as total phosphorus, was in eutrophic state for many years.Therefore, control trade effluent, sanitary sewage phosphorus content have become one of great environmental problem of eager needs solution.
At present, water body dephosphorizedly biological and chemical dephosphorization is mainly divided into.Chemical dephosphorization has simple to operate, efficient dephosphorization, the advantage such as stable; But this method cost is high, easily causes secondary pollution.Biological phosphate-eliminating utilizes the phosphorus in polyP bacteria enrichment water body, by getting rid of the mode dephosphorization of residue phosphorus containing sludge, has the advantages such as energy-conservation, sludge output is few, cost is low, non-secondary pollution, is the main sewage dephosphorization method of extensive sewage work.
PolyP bacteria is the microorganism that a class has " excess suction phosphorus " feature, can unnecessary phosphorus with the form of polyphosphoric acid salt storage in vivo, phosphorus content in external environment is significantly reduced, is the main executive of aqueous bio dephosphorization, plays a decisive role in biological phosphate-eliminating.
Laboratory uses low nutrition artificial wastewater liquid nutrient medium (phosphorus content is 14.9 mg/L) to detect the water body dephosphorized ability of bacterial strain, and screening has the polyP bacteria of efficient dephosphorization ability.Artificial wastewater liquid nutrient medium phosphorus concentration is 74.5 times that cause the minimum phosphorus concentration of body eutrophication (0.2 mg/L), the polyP bacteria obtained is screened by low nutrition superelevation phosphorus concentration artificial wastewater liquid nutrient medium, can reform of nature water body environment preferably, and remove the phosphorus in natural water more efficiently.
The present invention be sieved to from natural habitat a plant height effect biological phosphate-eliminating characteristic polyP bacteria solution keratin microbacterium ( microbacterium keratanolyticum) Mk-6 bacterial strain, be intended to the polyP bacteria resource enriched, expand polyP bacteria strain library, for biological removal of phosphorus in wastewater provides bacterial classification.
Summary of the invention
In order to solve above-mentioned technical problem, first object of the present invention be to provide a strain solution keratin microbacterium ( microbacterium keratanolyticum) Mk-6 bacterial strain, this bacterial strain dephosphorization efficiency by using is high and stable.Second object of the present invention 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 object, present invention employs following technical scheme:
Solution keratin microbacterium ( microbacterium keratanolyticum) Mk-6 bacterial strain, this bacterial strain is deposited in and is 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: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center.
Bacterial strain screening involved in the present invention is from the water body example in pond, suburbs, Jinhua, Zhejiang Province city.Adopt gradient dilution method to obtain pure bacterial strain, according to morphological specificity, physiological and biochemical property, 16S rDNA gene order identification of M k-6 bacterial strain for separate keratin microbacterium ( microbacterium keratanolyticum).
In order to realize second above-mentioned object, present invention employs following technical scheme:
The cultural method of above-mentioned solution keratin microbacterium Mk-6 bacterial strain, the method comprise the following steps: to go bail for solution keratin microbacterium Mk-6 bacterial strain 20 μ L of the glycerine being stored in-20 DEG C coats on the flat board of beef extract-peptone solid medium, choose single bacterium colony after 28 DEG C of cultivation 24 h to mix in 0.3 mL sterilized water, getting 100 μ L coats on the flat board of beef extract-peptone solid medium, getting 2 holes with punch tool receives in 15 mL beef extract-peptone liquid nutrient mediums, be placed in 28 DEG C, cultivate 24 h in 170 r/min shaking tables and be used as seed liquor.
In order to realize second above-mentioned object, present invention employs following technical scheme:
Above-mentioned solution keratin microbacterium Mk-6 bacterial strain is used for dephosphorization.Be preferred for the biological phosphate-eliminating of municipal sewage plant.
The present invention be a strain solution keratin microbacterium ( microbacterium keratanolyticum) Mk-6 bacterial strain, adaptive capacity to environment is strong, can be that 14.9 mg/L(reach 0.2 mg/L and will cause body eutrophication at low nutrition, phosphorus concentration) artificial wastewater liquid nutrient medium in grow fast, and by phosphorus ligands; Dephosphorization efficiency by using is high and stable, cultivates 20 h phosphorus efficiency and can reach 60% ~ 70%, non-secondary pollution.Therefore, Mk-6 bacterial strain can be applicable to phosphorous sewage disposal, is Microbial resources important on aqueous bio dephosphorization process, has a good application prospect.
Accompanying drawing explanation
The colony characteristics of Fig. 1 Mk-6 bacterial strain.
The thalli morphology (× 1000) of Fig. 2 Mk-6 bacterial strain.
Fig. 3 Mk-6 bacterial strain 16S rDNA gene order.
The Microbacterium phylogenetic tree that Fig. 4 sets up based on 16s rDNA gene order.
The growth curve of Fig. 5 Mk-6 bacterial strain in low nutrition high phosphorus artificial wastewater nutrient solution.
Fig. 6 liquid amount is on the impact of Mk-6 bacterial strain dephosphorizing rate.
Embodiment
the separation screening of the efficient polyP bacteria solution keratin microbacterium Mk-6 bacterial strain of embodiment 1 and qualification
(1) substratum
Beef extract-peptone solid medium: extractum carnis 3 g/L, peptone 10 g/L, NaCl 5 g/L, agar 20 g/L, pH 7.0 ~ 7.2 (liquid nutrient medium does not add agar), for separation, the purifying of bacterium.
Limit phosphorus (rich phosphorus) solid medium: get 30 mL deionized water dissolving 8.372 g 3-morpholine propanesulfonic acid, 0.717g N-tri-(methylol) methylglycine, regulate pH to 7.4 with the KOH of 10 mol/L, add solution in the following order: 0.01 mL 1.830% FeSO 4solution (now joining), 5 mL 1.9 mol/L NH 4cl, 1 mL 0.276 mol/L K 2sO 4, 0.025 mL 0.02 mol/L CaCl 22H 2o, 0.42 mL 1.25 mol/L MgCl 26 H 2o, 20 mL 2.5 mol/L NaCl, 0.02 mL trace element mixed solution (Ammonium Heptamolybdate 3 × 10 -6mol/L, H 3bO 34 × 10 -4mol/L, CoCl 23 × 10 -5mol/L, CuSO 410 -5mol/L, MnCl 28 × 10 -5mol/L, ZnSO 410 -5mol/L), 10 mL 1% glucose, 0.4 mL 1.686% VITMAIN B1,250 μ L 6.928% K 2hPO 4(rich phosphorus adds 5 mL) ,50 mg para-totuidine are blue, add water and are settled to 500 mL, after filtration sterilization with sterilising treatment be dissolved with 20 g agar and not solidified 500 mL solution mix and make solid medium, for the screening of polyP bacteria.
(2) method
Adopt gradient dilution partition method.Water sampling 1 mL, adds 9 mL sterilized water dilutions, carries out 10 -1~ 10 -3gradient dilution, gets 10 -2, 10 -3diluent 100 μ L coats on beef extract-peptone solid medium flat board, cultivates 24 h for 28 DEG C.The single bacterium colony of picking bacterium, carries out being separated, purifying; Be further purified bacterial strain and adopt method of scoring, the pure bacterial strain of gained is put in-20 DEG C of refrigerators and is preserved, for subsequent use.
Inoculate the pure bacterial strain of bacterium of preservation respectively on limit phosphorus and rich phosphorus solid medium, with Lv Shi methylene blue staining, sudan black staining, blue hickie method and Ammonium Molybdate Spectrophotometric Method for Determination bacterial isolates to the receptivity of phosphorus, after primary dcreening operation and multiple sieve, obtain a strain be numbered the bacterial isolates (accompanying drawing 2) that Mk-6 efficiently inhales phosphorus.
The qualification of morphology, Physiology and biochemistry and 16S r DNA sequence dna is carried out to Mk-6 bacterial strain.
(3) result
The morphological specificity of Mk-6 bacterial strain: bacterium colony is circular, yellow, complete neat, the protuberance in edge, smooth, thickness, moistening, translucent (accompanying drawing 1); Thalline is short and small shaft-like, and gramstaining is negative, amphitrichous, without gemma (accompanying drawing 2).
The physiological and biochemical property of Mk-6 bacterial strain: the test of Mk-6 bacterial strain Starch Hydrolysis, indoles, hydrogen sulfide and L-arginine decarboxylase is all positive, Citrate trianion can not be utilized, not liquefy gelatin, VP, methyl red, glucose fermentation, nitrate reduction test are 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, and suitable growth pH is 7.0 ~ 9.0.
Mk-6 bacterial strain 16S rDNA gene order (accompanying drawing 3): 16S rDNA gene sequencing analytical results shows, its base sequence total length is 1422 bp, use online Blast software to carry out homology analysis, choose the strain construction phylogenetic tree (accompanying drawing 4) of 19 strain Microbacteriums.Shown by Fig. 4, Mk-6 bacterial strain with separate keratin microbacterium ( microbacterium keratanolyticum) on same of evolutionary tree, two bacterial strain homologys are up to 98%.In conjunction with strain morphology feature, physiological and biochemical property, identify this bacterial strain for separate keratin microbacterium ( microbacterium keratanolyticum).
The physiological and biochemical test result of table 1 Mk-6 bacterial strain
Test subject Result Test subject Result
Indoles + Methyl red -
Hydrogen sulfide + Gelatine liquefication -
Starch Hydrolysis + Glucose fermentation -
L-arginine decarboxylase + Nitrate reduction -
VP - Citric acid utilizes -
Note: "+" is positive, "-" is negative.
embodiment 2 Mk-6 bacterial strain growth curve in low nutrition high phosphorus artificial wastewater nutrient solution measures
(1) bacterial strain:mk-6 bacterial strain.
(2) substratum
Beef extract-peptone solid medium: same embodiment 1 of filling a prescription
Artificial wastewater liquid nutrient medium: sodium acetate 0.925 g/L, peptone 0.1 g/L, yeast extract paste 0.01 g/L, NaCl 0.05 g/L, KH 2pO 40.0655 g/L, MgCl 26H 2o 0.1412 g/L, CaCl 20.025 g/L, pH 7.0 ~ 7.2.
(3) method
The glycerol stock 20 μ L being stored in-20 DEG C that goes bail for coats on the flat board of beef extract-peptone solid medium, choose single bacterium colony after 28 DEG C of cultivation 24 h to mix in 0.3 mL sterilized water, getting 100 μ L coats on the flat board of beef extract-peptone solid medium, getting 2 holes with punch tool (aperture 0.5 cm) receives in 15 mL beef extract-peptone liquid nutrient mediums, be placed in 28 DEG C, in 170 r/min shaking tables, cultivate 24 h(OD 600≈ 1.5) as seed liquor; Be equipped with in 250 mL Erlenmeyer flasks of 100 mL artificial wastewater liquid nutrient mediums with the inoculum size access of 8% again, be placed in 28 DEG C, cultivate in 170 r/min shaking tables, measure OD every 2 h 600value, draws the growth curve of Mk-6 bacterial strain.
(4) result
After Mk-6 bacterial strain is activated, cultivate in low nutrition high phosphorus (phosphorus content is 14.9 mg/L) artificial wastewater liquid nutrient medium, growth is very fast, the stable growth phase longer (accompanying drawing 5), can be the bacterial classification that biological removal of phosphorus in wastewater provides.Accompanying drawing 5 can be found out: Mk-6 strain culturing 0 h ~ 8 h is lag phase, and 8 h ~ 20 h are logarithmic phase, and 20 h ~ 36 h are the stable growth phase, and the 36th h enters decline phase.
embodiment 3 liquid amount is on the impact of Mk-6 bacterial strain biological phosphor-removing effect
(1) bacterial strain:mk-6 bacterial strain.
(2) substratum
Beef extract-peptone solid medium: same embodiment 1 of filling a prescription;
Artificial wastewater liquid nutrient medium: same embodiment 2 of filling a prescription.
(3) method
The glycerol stock 20 μ L being stored in-20 DEG C that goes bail for coats on the flat board of beef extract-peptone solid medium, choose single bacterium colony after 28 DEG C of cultivation 24 h to mix in 0.3 mL sterilized water, getting 100 μ L coats on the flat board of beef extract-peptone solid medium, getting 2 holes with punch tool (aperture 0.5 cm) receives in 15 mL beef extract-peptone liquid nutrient mediums, be placed in 28 DEG C, in 170 r/min shaking tables, cultivate 24 h(OD 600≈ 1.5) as seed liquor; Be equipped with in artificial wastewater liquid nutrient medium (liquid amount is respectively 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL) the 250 mL triangular flasks of different amount with the inoculum size access of 8% again, temperature 28 DEG C, shaking table speed 170 r/min, initial pH cultivate 20 h under the condition of 7.2.Get 30 mL bacteria suspensions in the centrifuge tube of 50 mL, with centrifugal 5 min of 12 000 r/min, get supernatant liquor and survey phosphorus concentration by the measuring method of total phosphorus, total phosphorus yield adopts ammonium molybdate spectrophotometric method, calculates dephosphorizing rate according to contrast.
(4) result
Different liquid amount has a certain impact to AJ-3 bacterial strain phosphor-removing effect, when liquid amount is fill 40 mL in 250 mL triangular flasks, temperature 28 DEG C, shaking table speed 170 r/min, cultivate 20 h under artificial wastewater liquid nutrient medium (pH is 7.2), dephosphorizing rate can reach 67.43%; It is 40 mL (accompanying drawings 6) that Mk-6 bacterial strain is suitable for liquid amount.
　
<110> Zhejiang Normal University
<120> separates keratin microbacterium and cultural method thereof and application
<160>
 
<210>?1
<211>?1422
<212>?rDNA
<213> separates keratin 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 (4)

1. separate keratin microbacterium ( microbacterium keratanolyticum) Mk-6 bacterial strain, this bacterial strain is deposited in and is 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: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center.
2. the cultural method of solution keratin microbacterium Mk-6 bacterial strain according to claim 1, it is characterized in that the method comprise the following steps: to go bail for solution keratin microbacterium Mk-6 bacterial strain 20 μ L of the glycerine being stored in-20 DEG C coats on the flat board of beef extract-peptone solid medium, choose single bacterium colony after 28 DEG C of cultivation 24 h to mix in 0.3 mL sterilized water, getting 100 μ L coats on the flat board of beef extract-peptone solid medium, getting 2 holes with punch tool receives in 15 mL beef extract-peptone liquid nutrient mediums, be placed in 28 DEG C, cultivate 24 h in 170 r/min shaking tables and be used as seed liquor.
3. solution keratin microbacterium Mk-6 bacterial strain according to claim 1 is used for dephosphorization.
4. solution keratin microbacterium Mk-6 bacterial strain according to claim 1 is used for the biological phosphate-eliminating of municipal sewage plant.
CN201410348045.8A 2014-07-21 2014-07-21 Solve keratan microbacterium and its cultural method and application Active CN104277996B (en)

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CN113481131A (en) * 2021-08-17 2021-10-08 河南工业大学 Composite phosphorus-accumulating microbial inoculum and preparation method and application thereof

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111004742A (en) * 2019-12-16 2020-04-14 浙江工业大学 Microbacterium ZY with dichloromethane degradation performance and application thereof
CN113481131A (en) * 2021-08-17 2021-10-08 河南工业大学 Composite phosphorus-accumulating microbial inoculum and preparation method and application thereof
CN113481131B (en) * 2021-08-17 2024-03-26 河南工业大学 Composite phosphorus accumulating bacterial agent and preparation method and application thereof

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