CN106282069A - A kind of secondary coccus and the application in sewage purification - Google Patents
A kind of secondary coccus and the application in sewage purification Download PDFInfo
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- CN106282069A CN106282069A CN201610870685.4A CN201610870685A CN106282069A CN 106282069 A CN106282069 A CN 106282069A CN 201610870685 A CN201610870685 A CN 201610870685A CN 106282069 A CN106282069 A CN 106282069A
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- paracoccus
- fimx
- secondary coccus
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- microbial flocculant
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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
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/52—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities
- C02F1/54—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities using organic material
- C02F1/56—Macromolecular compounds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
Abstract
The invention discloses a kind of secondary coccus and the application in sewage purification, secondary coccus (Paracoccus sp.) FIMX 1, its deposit number is: CGMCC No.12826.This strain has easily cultivations, fast growth, flocculating effect is good and adaptive capacity to environment is strong feature, and its metabolite has preferable flocculating effect to sewage, for the strain that waste water control offer is new.
Description
Technical field:
The present invention relates to microbial technique and technical field of sewage, be specifically related to a kind of secondary coccus and in sewage purification
In application.
Background technology:
Microbial flocculant is the family macromolecule polymer with flocculation activity that microorganism is secreted in growth course,
Its main active has protein, polysaccharide, nucleic acid and thalline.It is to utilize biotechnology, the one prepared by fermentation
Novel, the efficient and nontoxic cheap water with bioflocculation and safety processes microbial inoculum.With tradition inorganic flocculating agent and having
Machine flocculant is compared, and it has the character of uniqueness: flocculating microbial is of a great variety, wide material sources, and growth cycle is short, easily takes
Modern biotechnology carries out industrialization, mostly the composition that flocculates is organic molecule, biodegradable, it is to avoid dirty to environment secondary
Dye, flocculating effect is relatively strong, Heat stability is good pH stable, and special heavy metal ion, Organic substance and pigment are removed specially good effect, raw
Thing flocculant not only alleviates the load of sewage disposal system, additionally it is possible to the microbial activity of enhanced water treatment biochemical stage.Therefore
Biological flocculant is the focus that water processes preparation research the most both at home and abroad, and it is sewage by partly or entirely replacing traditional flocculant
Process the new trend of development.
Summary of the invention:
It is an object of the invention to provide a kind of secondary coccus, this strain have easily cultivation, fast growth, flocculating effect good and
The feature that adaptive capacity to environment is strong, its metabolite has preferable flocculating effect to sewage, provides new bacterium for waste water control
Kind.
The present invention is achieved by the following technical programs:
Secondary coccus (Paracoccus sp.) FIMX-1 of the present invention, is preserved in China Microbiological bacterium on August 8th, 2016
Plant preservation administration committee common micro-organisms center (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China
Institute of microbiology of academy of science, postcode: 100101, its deposit number is: CGMCC No.12826.
Present invention also offers the preparation method of a kind of microbial flocculant, it is characterised in that comprise the following steps:
(1) it is seeded to pair coccus (Paracoccus sp.) FIMX-1 in fermentation medium cultivate obtain fermentation culture
Liquid, described fermentation medium every liter contains: glucose 10g, yeast extract 3g, carbamide 0.5g, peptone 0.5g, MgSO4·7H2O
0.5g, KH2PO40.2g, NaCl 0.1g, pH7.5, surplus is water；
(2) take the fermentation culture that step (1) obtains, centrifugal segregation thalline, take supernatant, add ethanol wherein and carry out
Precipitation, collects precipitation and obtains microbial flocculant.
Present invention also offers a kind of microbial flocculant prepared according to above-mentioned preparation method.
Present invention also offers microbial flocculant application in sewage purification.
Beneficial effects of the present invention is as follows: it is good that the strain that the present invention provides has easily cultivation, fast growth, flocculating effect
The feature strong with adaptive capacity to environment, its metabolite has preferable flocculating effect to sewage, provides new for waste water control
Strain.
Secondary coccus (Paracoccus sp.) FIMX-1, is preserved in Chinese microorganism strain preservation pipe on August 8th, 2016
Reason committee's common micro-organisms center (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences is micro-
Biological study institute, postcode: 100101, its deposit number is: CGMCC No.12826.
Accompanying drawing illustrates:
Fig. 1 is the whole-cell fatty acid gas chromatogram of bacterial strain of the present invention.
Detailed description of the invention:
The following is and the present invention is further illustrated rather than limitation of the present invention.
Embodiment 1: secondary coccus (Paracoccus sp.) separation of FIMX-1, purification and qualification
1, sample collecting: bacteria samples picks up from Foochow eutrophication river bottom mud, uses self-control sediment sampler to take
Bed mud puts into sterile bag, is placed in 4 DEG C of Refrigerator stores.
2, strain separating
(1) primary dcreening operation: weigh 5g bed mud under aseptic condition, is placed in filling in conical flask without cingula with magnetic stir bar, adds
Entering mass fraction is 0.8% physiological saline solution 100mL, and vibrate on magnetic stirring apparatus 30min, breaks up zoogloea, makes bacterium
Suspension.By sterilized pipette, extract 5mL, it is separately added in the screening culture medium containing beef extract-peptone equipped with 100mL,
28 DEG C, enrichment culture 3-4d of vibrating on the shaking table of 120r/min.Drawing 0.5mL culture fluid, it is 0.8% aseptic for adding mass fraction
In normal saline, obtain 10-1, 10-2, 10-3, 10-4, 10-5With 10-6The dilute solution of gradient, the dilution drawing variable concentrations is molten
The each 0.2mL of liquid coats on nutrient agar flat board, cultivates 2～3d for 28 DEG C.Select single bacterium colony.Repeated 3 coatings,
Choose kind, consistent to colony characteristics, the bacterium colony person of appearance without exception, it is believed that be Economical Purification.Single colony inoculation is to nutrient agar slopes
Cultivate in culture medium, preserve.The a small amount of thalline of picking from nutrient agar slant medium, is seeded to equipped with 100mL fermentation culture
In the 500mL triangular flask of base, 28 DEG C, shaken cultivation 3d on the shaking table of 120r/min.2mL fermentation liquid of respectively asking for is equipped with 50mL
Kaolin clay suspension (4g L-1) color comparison tube in.Add 2mL CaCl2Solution (mass fraction is 1%), mix homogeneously, quiet
Put 10min, range estimation can make suspension flocculate into oarse-grained be flocculation primary dcreening operation active bacterial strain.
Described screening culture medium: Carnis Bovis seu Bubali cream 5g L-1, peptone 10g L-1, sodium chloride 5g L-1, pH 7.2,121 DEG C
Autoclaving 15min.
Described nutrient agar: peptone 10g, Carnis Bovis seu Bubali cream 3g, sodium chloride 5g, agar 15～20g, distilled water
1000mL, 121 DEG C of autoclaving 15min.
Described fermentation medium: glucose 10g L-1, yeast extract 3g L-1, carbamide 0.5g L-1, peptone
0.5g·L-1, MgSO4·7H2O 0.5g·L-1, KH2PO4 0.2g·L-1, NaCl 0.1g L-1, pH7.5, solvent is water, will
Mentioned component is by after its content mix homogeneously, and 121 DEG C of autoclaving 15min are standby.
(2) multiple sieve: the flocculation primary dcreening operation active bacterial strain obtained by step (1) primary dcreening operation is inoculated in equipped with 50mL fermentation medium
In triangular flask, 28 DEG C, shaken cultivation 3-4d on the shaking table of 120r/min.Absorption 1mL fermentation liquid joins 50mL concentration and is
0.1g·L-1Aqueous suspension ofkaolin in.First high degree of agitation 2min, then low rate mixing 5min, finally stand 10min, take supernatant
Liquid, with its absorbance at 550nm of spectrophotometric determination, with the distilled water without flocculant the most as a control group,
It is all provided with three Duplicate Samples, calculates flocculating rate.The flocculation activity of bacterial strain uses flocculating rate to represent, chooses the bacterial strain that flocculating rate is the highest
(flocculating rate 90%), is FIMX-1 by this Strain Designation.
Flocculating rate=(N0-N)/N0 × 100%
Wherein, N0 is comparison supernatant absorbance at 550nm, and N is testing sample supernatant extinction at 550nm
Degree.
3, the qualification of strain:
Taking FIMX-1 strain culturing that multiple sieve obtains to bacterium solution 10mL of exponential phase, 8000r/min is centrifuged 15min,
Incline supernatant, collects thalline.CTAB/NaCl method is used to extract phage gene group DNA.Step is as follows: add in thalline
13.5mL TE solution (pH 8.0), suspends, and adds 3mL 10% (mass fraction) sodium lauryl sulphate (SDS), 150 μ L
100mg/mL lysozyme and 150 μ L 100mg/mL E.C. 3.4.21.64s, mixing, 37 DEG C of water-bath 1h, add 2.5mL 5mol/L NaCl and
2mL CTAB/NaCl solution, is mixed, 65 DEG C of water-bath 10min, with isopyknic phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol
Each extracting 3 times, isopropanol precipitating DNA, DNA are dissolved in 50 μ L TE, and-20 DEG C save backup.Utilize 1% agarose gel electrophoresis
The genomic DNA integrity of detection extracting.Use the universal primer 27F (5'-of 16S rRNA gene
AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') to extract DNA carry out PCR
Amplification.DNA product after amplification is delivered to Shanghai biological engineering company limited check order.With BLAST software with NCBI's
The 16S rRNA sequence that GenBank data base includes compares.16S rRNA (its nucleotide sequence of FIMX-1 bacterial strain
As shown in SEQ ID NO.1) and Paracoccus solventivorans strain TLB-6 and Paracoccus
The similarity of the 16S rRNA gene order of solventivorans strain TLA-29 is 98%, with type strain
Paracoccus solventivorans strain ATCC 700252 and Paracoccus solventivorans
Strain DSM 6637T similarity is 97%.Thus determine that bacterial strain FIMX-1 belongs to paracoccus (Paracoccus sp.),
Named secondary coccus (Paracoccus sp.) FIMX-1.
Microbial DNA base composition has species specificity, and (G+C) value of DNA is the most stable in cell, can be as antibacterial
Identifying important Heredity index, and it is not affected by other factors beyond cell age and Mutagen, (G+C) content analysis becomes
For a kind of important method of microbial genetics classification, relation and set up new point between discriminating bacteria kind and between kind
During class unit significant.Use the G+C of high effective liquid chromatography for measuring pair coccus (Paracoccus sp.) FIMX-1
Molar fraction, the G+C molar fraction of secondary coccus (Paracoccus sp.) FIMX-1 is 63.7% (mol%).Paracoccus bacterium
Strain Paracoccus solventivorans strain TLB-6, Paracoccus solventivorans strain
TLA-29、Paracoccus solventivorans strain DSM 6637T、Paracoccus sp.kocurii、
G+C mole point of Paracoccus sp.G1212, Paracoccus sediminis sp.nov and Paracoccus sp.WB1
Number be respectively 65.8% (mol%), 66.0% (mol%), 68.5% (mol%), 71.0% (mol%), 62.0%
(mol%), 62.2% (mol%) and 68.8% (mol%).Secondary coccus (Paracoccus sp.) FIMX-1 with it has been reported that
All there is certain difference in the G+C molar fraction of main paracoccus bacterial strain.
Fatty acid (fatty acid, FA), as the fundamental component of cell, often exists with esterified state, and component is the most steady
Determining, the antibacterial of different genera, the composition of its fatty acid and content show difference in various degree, the kind of fatty acid and quantity
Closely related with the feature of antibacterial, heredity, composition and the content of bacterial fatty acid contains abundant taxonomic information, can make
One of biomarker for Bacteria Identification.If fatty acid chromatograph main peak exists significant difference, then point out different species or genus
Antibacterial, if fatty acid chromatograph main peak is similar, only exists the difference of peak height, then points out identical antibacterial kind, different bacterial strains.By dividing
Analysis result can draw, the fatty acid species of secondary coccus (Paracoccus sp.) FIMX-1 and content and most of paracoccus
The composition of the fatty acid of strain is consistent.The gas chromatogram of the whole-cell fatty acid of secondary coccus (Paracoccus sp.) FIMX-1
Analysis result is shown in Table 1 and Fig. 1, and MIDI data storehouse comparison result is as shown in table 1.In TSBA6 (6.10) data base, secondary coccus
(Paracoccus sp.) FIMX-1's to Paracoccus-solventivorans and Paracoccus-alkenifer is similar
Degree is respectively 0.537 and 0.208.
In conjunction with bacterial strain 16S rDNA sequence alignment result, the G+C molar fraction of DNA and the gas chromatogram of whole-cell fatty acid
Analysis result, comprehensively determines that bacterial strain FIMX-1 belongs to paracoccus (Paracoccus sp.), is paracoccus (Paracoccus
Sp.) a novel species, this pair coccus (Paracoccus sp.) FIMX-1 is preserved in China Microbiological bacterium on August 8th, 2016
Plant preservation administration committee common micro-organisms center (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China
Institute of microbiology of academy of science, postcode: 100101, its deposit number is: CGMCC No.12826
The parameter list of the gas chromatograph results of the whole-cell fatty acid of secondary coccus (Paracoccus sp.) FIMX-1 of table 1
Embodiment 2: prepared by microbial flocculant
Described microbial flocculant is using the metabolite of described secondary coccus (Paracoccus sp.) FIMX-1 as mainly
Active component, its preparation comprises the following steps:
(1) slant culture: embodiment 1 is sieved secondary coccus (Paracoccus sp.) FIMX-1 obtained again and is seeded to nutrition
In agar (LB) culture medium, cultivate 2～3d in 28 DEG C of incubators and obtain slant medium FIMX-1 thalline；Described Nutrient agar is trained
Support based formulas with embodiment 1.
(2) fermentation culture: provoke the FIMX-1 thalline on the slant medium that a small amount of step (1) obtains with inoculating loop and access
In fermentation medium, in 28 DEG C, shaken cultivation 2～3d obtains fermentation culture, described fermentation medium on the shaking table of 120r/min
Formula with embodiment 1.
(3) preparation of microbial flocculant: take the fermentation culture that step (2) obtains, 4000～5000rpm/min from
The heart, removes thalline, takes supernatant, adds the pre-cooled ethanol of 1.5 times of volumes, and centrifugal collecting precipitation obtains target product, is micro-
Biological flocculant.
Embodiment 3: described secondary coccus (Paracoccus sp.) FIMX-1 application in sewage purification
(1) purification to inland river sewage
Inland river sewage removes large particulate matter through 2 layers of filtered through gauze in advance.The microbial flocculant of Example 2 preparation
(metabolite of described secondary coccus (Paracoccus sp.) FIMX-1 is as active component) 0.15g, joins 50mL inland river
In sewage (pH is 6.5), it is simultaneously introduced the CaCl that 0.2mL mass fraction is 1%2Solution.First high degree of agitation 2min, then stir at a slow speed
Mix 5min, finally stand 10min, take supernatant, with its absorbance at 550nm of spectrophotometric determination, simultaneously not add
Add the distilled water of flocculant as a control group, be all provided with three Duplicate Samples, calculate flocculating rate.Described microbial flocculant is to inland river
The flocculating rate of sewage is up to 75% (flocculating rate of matched group is 19%), and secondary coccus (Paracoccus sp.) FIMX-1 is to inland river
Sewage has preferable flocculating and purifying effect.
(2) anerobic sowage of raising pigs is gone out water purification
The microbial flocculant of Example 2 preparation (produce by the metabolism of described secondary coccus (Paracoccus sp.) FIMX-1
Thing is as main active) 0.3g, join 50mL dilute 2 times raise pigs in anerobic sowage water outlet in (pH is 8.1), simultaneously
Adding 0.2mL mass fraction is the CaCl of 1%2Solution.First high degree of agitation 2min, then low rate mixing 5min, finally stand
10min, takes supernatant, with its absorbance at 550nm of spectrophotometric determination, simultaneously with the distilled water without flocculant
As a control group, it is all provided with three Duplicate Samples, calculates flocculating rate.The described microbial flocculant wadding to anerobic sowage water outlet of raising pigs
Solidifying rate, up to 51% (flocculating rate of matched group is 12%), has preferable flocculating and purifying effect to anerobic sowage water outlet of raising pigs.
Claims (5)
1. secondary coccus (Paracoccus sp.) FIMX-1, its deposit number is: CGMCC No.12826.
2. secondary coccus (Paracoccus sp.) FIMX-1 application in sewage purification described in claim 1.
3. the preparation method of a microbial flocculant, it is characterised in that comprise the following steps:
(1) it is seeded in fermentation medium cultivate by secondary coccus (Paracoccus sp.) FIMX-1 described in claim 1
To fermentation culture, described fermentation medium every liter contains: glucose 10g, yeast extract 3g, carbamide 0.5g, peptone 0.5g,
MgSO4·7H2O 0.5g, KH2PO40.2g, NaCl 0.1g, pH7.5, surplus is water；
(2) take the fermentation culture that step (1) obtains, centrifugal segregation thalline, take supernatant, add ethanol wherein and sink
Form sediment, collect precipitation and obtain microbial flocculant.
4. the microbial flocculant prepared according to the preparation method described in claim 3.
5. the application in sewage purification of the microbial flocculant described in claim 4.
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