CN106047765A - Pseudomonas strain MHGC424 for degrading marine organic matters and application of pseudomonas strain MHGC424 - Google Patents
Pseudomonas strain MHGC424 for degrading marine organic matters and application of pseudomonas strain MHGC424 Download PDFInfo
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- 230000000593 degrading effect Effects 0.000 title abstract 3
- 239000013535 sea water Substances 0.000 claims abstract description 34
- 241000589774 Pseudomonas sp. Species 0.000 claims abstract description 20
- 238000004321 preservation Methods 0.000 claims abstract description 10
- 230000001580 bacterial effect Effects 0.000 claims description 26
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- 241000894006 Bacteria Species 0.000 description 31
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- 238000006731 degradation reaction Methods 0.000 description 22
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 9
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- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
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- 239000008346 aqueous phase Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- ZYWFEOZQIUMEGL-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol;phenol Chemical compound ClC(Cl)Cl.CC(C)CCO.OC1=CC=CC=C1 ZYWFEOZQIUMEGL-UHFFFAOYSA-N 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N1/205—Bacterial isolates
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- 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
- C12R2001/38—Pseudomonas
-
- 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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Abstract
The invention discloses a pseudomonas strain MHGC424 for degrading marine organic matters. A preservation name of the pseudomonas strain MHGC424 is pseudomonas MHGC424 (Pseudomonas sp. MHGC424), the pseudomonas strain MHGC424 is preserved in the China Center for Type Culture Collection in the Wuhan University in Wuhan in China on 28 August, 2009, and a preservation number of the pseudomonas strain MHGC424 is CCTCC NO:M209188. The pseudomonas strain MHGC424 has the advantages that the pseudomonas strain MHGC424 is capable of efficiently degrading the organic matters, the organic matters in seawater polluted by the organic matters can be quickly and effectively removed by the pseudomonas strain MHGC424, and the pseudomonas strain MHGC424 is free of poisoning and pathogenic effects on marine aquaculture organisms; the pseudomonas strain MHGC424 for removing the high-concentration organic matters in marine aquaculture environments is safe, is environmentally friendly and is high in efficiency.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of pseudomonad strain for marine organisms degraded
MHGC424 and the application in processing Organic Pollution sea water thereof.
Background technology
In recent years, while seawater cage culture industry develops rapidly, cultivation " self-pollution " problem shows especially out day by day
Coming, the organic pollutant that wherein breeding process produces usually can cause that the water body of seawater cage culture is smelly, depositional environment is disliked
Changing, cause oxygen in water to reduce, cultured fishes are dead, and water ecology balance is destroyed.
Seawater cage culture system, as a kind of high density, the artificial cultivation ecosystem of high bait throwing in, affects breeding environment
The underlying cause is in breeding process the organic depositions such as a large amount of residual bait of generation, feces and dead animals and plants remains.
Current physics and chemistry go removal organic polluter method all to have certain limitation.Wherein to there is efficiency low for physical method
Under, the shortcoming that cost is high；Although chemical method instant effect, but often cause secondary pollution.For reaching quick to organic pollution, high
Effect is removed, and the present invention intends concentration and separation purification from sea water culture environment deposit and obtains a strain microorganism, and use it for right
In the improvement of Organic Pollution.
Summary of the invention
First purpose of the present invention is to provide a kind of pseudomonad strain for marine organisms degraded
MHGC424, this bacterial strain has efficient organic matter degradation ability, can fast and effeciently remove the Organic substance in briny environment.
Second object of the present invention is to provide above-mentioned pseudomonad strain MHGC424 for marine organisms degraded
Application in processing organic pollutant in seawater.
Being experimentally verified that, sea-farming biology is not poisoned pathogenic by pseudomonad strain MHGC424 of the present invention
Effect.
First purpose of the present invention is achieved by the following technical solution: a kind of for marine organisms degraded
Pseudomonad strain MHGC424, the preservation of this bacterial strain is entitled: pseudomonas MHGC424 (Pseudomonas
Sp.MHGC424), depositary institution is: China typical culture collection center, and preservation address is: Wuhan, China Wuhan University, protects
Hide the date be: on August 28th, 2009, deposit number is: CCTCC NO:M209188.
The screening and separating qualification process of pseudomonad strain MHGC424 for marine organisms degraded in the present invention
For: take the deposit of seawater cage culture place, after laboratory utilizes Selective agar medium to cultivate, isolated and purified bacterial strain, according to
The bacterial strain power to bait organic matter degradation ability, finishing screen selects a strain the bacterium of stronger degradation capability to bait Organic substance
Strain, numbered MHGC424, then according to colony morphology characteristic, cultural characteristic and the physiological and biochemical property of bacterial strain, and
16SrDNA gene order is through being analyzed discovery with the gene order that logged in Genbank, and bacterial strain MHGC424 is through mirror
It is set to Rhodopseudomonas, named pseudomonas MHGC424 (Pseudomonas sp.MHGC424).
Second object of the present invention is achieved by the following technical solution: above-mentioned for marine organisms degraded
The application in processing organic pollutant in seawater of pseudomonad strain MHGC424.
Detailed process in processing Organic Pollution sea water is: above-mentioned isolated and purified pseudomonas of learning from else's experience
(Pseudomonas sp.) mono-bacterium colony of MHGC424, cultivates 40-48h in activation amplification culture base, by sea water and bacterium solution volume
Ratio is 40-60:1, is inoculated in pseudomonas MHGC424 (Pseudomonas sp.MHGC424) containing the organic sea water of bait
In, 25-28 DEG C of constant-temperature shaking culture 3-5d.
The composition of above-mentioned activation amplification culture base is preferably: peptone 5.0g, yeast extract 1.0g, high ferric phosphate 0.1g, old
Sea water 1000mL, pH 7.6-8.2,121 DEG C of sterilizing 20min.
The present invention compared with prior art has the advantage that the present invention screens from the deposit of marine culture area
The new strains arrived, has stronger degradation capability, to bait Organic substance in 1-5d to organic pollution especially bait Organic substance
COD clearance reach 23.6～40.9%.
Accompanying drawing explanation
Fig. 1 is the PCR primer sepharose electrophoresis figure of bacterial strain MHGC424 in embodiment 2；
Fig. 2 is the electron microscopic picture of bacterial strain MHGC424 in embodiment 3；
Fig. 3 is the bod in the high concentration bait Organic substance sea water adding bacterial strain MHGC424 in embodiment 5
(BOD) variation diagram；
Fig. 4 is the chemical oxygen consumption (COC) in the high concentration bait Organic substance sea water adding bacterial strain MHGC424 in embodiment 5
(COD) variation diagram.
Detailed description of the invention
The present invention will be further described with embodiment below in conjunction with the accompanying drawings, but limits the present invention the most in any form.
Embodiment 1
Screening for pseudomonad strain MHGC424 of marine organisms degraded
One, material prepares
1, bacterium source and experiment wastewater
(1) deposit in the Dapeng'ao Cove Capacity Limit in Marine Net Cage Fish Farm district of Shenzhen that bacterium source has more than 20 year to cultivate history.
(2) experiment wastewater (containing high concentration bait Organic substance sea water): trash fish gruel 5g, Chen Haishui 1000mL, pH 7.6～
8.2,121 DEG C of sterilizing 20min.
2, culture medium
(1) Selective agar medium: trash fish gruel 20g, Chen Haishui 1000mL, pH 7.6～8.2,121 DEG C of sterilizing 20min.
(2) separation and slant medium: peptone 5g, yeast extract 1g, high ferric phosphate 0.1g, agar powder 20g, Chen Haishui
1000mL, pH 7.6～8.2.
(3) activation amplification culture base: peptone 5g, yeast extract 1g, high ferric phosphate 0.1g, Chen Haishui 1000mL, pH 7.6
～8.2,121 DEG C of sterilizing 20min.
3, experimental instruments
U.S.'s HACH BOD Trak analyzer；
Sea, Guangdong profit electromagnetic type air compressor ACO-009E；
Suzhou safe and sound superclean bench SW-CJ-1BU；
Upper Nereid grand biochemical cultivation case SHP-150；
Europe times, Suzhou shaken cultivation case OBS-2F；
Germany's HYDRO-BIOS-KIEL bottom sampler；
Japan HIRAYAMA autoclave HVN-85；
Hitachi's H-7650 transmission electron microscope；
PC818 type PCR instrument；
Electrophresis apparatus and electrophoresis tank；
Heating plate and titrator；
Gel ultraviolet visualizer etc..
Two, the separation of bacterial strain and screening
1, the enrichment of bacterial strain
Take the deposit at the 5cm of below Dapeng'ao Cove Capacity Limit in Marine Net Cage Fish Farm deposit top layer and put into sampler bag, then put
Enter in 0 DEG C of ice chest and take back laboratory.Take 250g deposit to be added in the enrichment bottle of 2L, add 750mL and go out Selective agar medium, and add
Entering appropriate cellucotton, at ambient temperature continuous aeration enrichment culture 2 months, sterilizing sea water is irregularly added in centre according to situation
And Selective agar medium.
2, organic matter degradation antibacterial is isolated and purified
Select and directly rinse the cellucotton that antibacterial adhesion amount is bigger, obtaining being enriched with bacterium solution, suitably dilute, be spread evenly across
On isolation medium flat board, cultivate 3d, after bacterium colony grows, single bacterium colony that picking comes in every shape for 28 DEG C, put down at isolation medium
Carry out line on plate to separate, until obtaining pure single bacterium colony.By in isolated and purified microbionation to slant medium and in ice
The preservation of 4 DEG C of case.
3, the screening of organic matter degradation antibacterial
By weighing bacterial strain Utilization ability organic to bait, with organic bio-degradable by BOD and COD value
Property (BOD5/COD0) represent that in the 5d time, antibacterial utilizes the organic degradation capability of bait.
Take the isolated and purified bacterial strain obtained 25-28 DEG C of cultivation 40-48h in activation amplification culture base, by sea water and bacterium solution
Volume ratio 40-60:1, is inoculated into containing in high concentration (5%, weight/mass percentage composition) the organic sea water of bait, takes and do not inoculate bacterium solution
Bait Organic substance sea water as blank, 25-28 DEG C of temperature oscillation is cultivated, and response time 1-5d monitors bait Organic substance
BOD and COD of sea water.
The bacterial strain of a numbered MHGC424 of strain, i.e. marine organisms degradation bacteria strains MHGC424 is obtained through screening.
Embodiment 2
The qualification of marine organisms degradation bacteria strains MHGC424
1, the qualification to marine organisms degradation bacteria strains MHGC424
Marine organisms degradation bacteria strains MHGC424 has been carried out qualification and the mirror of 16S rDNA molecule of Physiology and biochemistry
Fixed, from molecular level, and combine Gern morphology feature and Analysis of The Physiological And Biochemical Properties determines the kind of bacterial strain.
16S rDNA sequence analysis is essentially according to following steps:
(1) extraction of antibacterial core DNA:
1) choose single colony inoculation overnight incubation in amplification culture base with autoclaved toothpick, take 1.5mL bacterium solution in
In Eppendorf pipe, 8000rpm room temperature is centrifuged 5min, thoroughly removes supernatant.
2) add STE (Sodium-Tris-EDTA) buffer 1.5mL washed once, be centrifuged and abandon supernatant, add 0.6mLTE
(Tris-EDTA) slow solution, resuspended antibacterial.
3) lysozyme of 30 μ L 10mg/mL, 37 DEG C of water-bath 45min are added.
4) SDS (dodecyl sodium sulfate) of 65 μ L 10% is added, the E.C. 3.4.21.64 3 μ L of 20mg/mL, 50 DEG C of water-bath 2h, extremely
Solution becomes clear.
5) equal-volume phenol chloroform is added three times, till can't see protein.
6) in supernatant, add the NaAc (pH5.2) of the 3M of 1/10 volume.
7) add isopyknic isopropanol, precipitate DNA.It is wound around with Glass rod and chooses DNA filament, then use volumn concentration
Be 70% washing with alcohol once, naturally dry, and DNA be dissolved in 100 μ L TE solution.
8) adding the Rnase (RNase) of final concentration of 50 μ g/mL, 37 DEG C, 30min, removal RNA ,-20 DEG C save backup.
(2) the PCR amplification of 16S rDNA gene
P1 (forward primer): 27F (5 '-AGA GTT TGA TCC TGG CTC AG-3 ')
P2 (downstream primer): 1492R (5 '-AAG TCG TAA CAA GGT AAC C-3 ')
In the reaction volume of 50 μ L, add 1 μ L template DNA (0.1ug), 0.5 μ LP1 and P2 (final concentration of 0.5 μM), 1
μ L dNTP (every kind of NTP0.2mM), 0.5 μ LTaq polymerase (2U) and 5 μ L10 × PCR buffer.PCR amplification condition is: 94 DEG C
Denaturation 5min, at 94 DEG C of degeneration 30s, 61-65 DEG C of annealing 30s, 72 DEG C extend 1min, circulate 30 times；Last 72 DEG C extend eventually
10min。
(3) recovery of PCR primer
After PCR primer is carried out electrophoresis with 1% agarose gel (mass percent), cut under uviol lamp containing being intended to reclaim
The gel of fragment, puts into and adds 2 times of volume TE in 1.5mL centrifuge tube, adds equal-volume water-saturated phenol extracting one after 65 DEG C of water-bath 10min
Secondary centrifugal after take upper strata aqueous phase again with phenol-chloroform-isoamyl alcohol extraction once, collection is reset and added the 10mol/L acetic acid of 0.1 times of volume
Ammonium and 2 times of volume dehydrated alcohol precipitations, centrifugal, wash precipitation once with the ethanol that volumn concentration is 70%, be dissolved in after air-drying
Appropriate sterilizing distilled water.
(4) partial sequence of 16S rDNA measures and analyzes
P-f:CACGACGTTGTAAAACGACAGTTTGATCCTGGCTC；
P-r:GGATAACAATTTCACACAGGAAGGAGGTGATCCAGCC.
Adding 1 μ L template DNA (＜ 0.1 μ g), PCR expands 30 circulations (94 DEG C of 1min, 55 DEG C of 30s, 72 DEG C of 2min).Adopt
Reaction is terminated with dye terminator in ABI PRISM sequencing kit.Then, survey at Applied Biosystem373A DNA
Check order on sequence instrument.The 16S rDNA sequence recorded uses BLAST software and GenBank data base's comparative analysis, finally from molecule
The kind of this bacterium is determined in level.
2,16S rDNA sequencing
The present invention uses the method to 16S rDNA Sequencing and Characterization that antibacterial carries out the qualification of molecular level.With carefully
The core DNA of bacterium is template, and the universal primer expanded with the PCR of 16S rDNA gene, as primer, carries out PCR amplification, obtains length
Amplified band for 1498bp detects with 1% agarose gel electrophoresis, as shown in Figure 1.After PCR primer is purified, measure its total order
Row.
Embodiment 3
Marine organisms degradation bacteria strains MHGC424 colonial morphology, physiology and biochemical character
Marine organisms degradation bacteria strains MHGC424 colonial morphology, physiology and biochemical character are shown in Table 1；Cellular morphology is shown in Fig. 2.
The colony morphology characteristic of table 1 marine organisms degradation bacteria strains MHGC424 and main physiological and biochemical property
Remarks: "+": the bacterial strain of more than 90% is positive, "-": the bacterial strain of more than 90% is negative.
Embodiment 4
The determination of marine organisms degradation bacteria strains MHGC424
By in the 16S rDNA gene order of the marine organisms a length of 1498bp of degradation bacteria strains MHGC424 and GenBank
Listed gene order compares, marine organisms degradation bacteria strains MHGC424 and Rhodopseudomonas
The homology of (Pseudomonas sp.) bacterial strain is up to more than 96.3%.
The Physiology and biochemistry of comprehensive bacterial strain and Molecular Identification result, it may be determined that marine organisms degradation bacteria strains MHGC424 is false
Zygosaccharomyces (Pseudomonas sp.), the named pseudomonas of bacterial strain MHGC424 (Pseudomonas sp.) MHGC424
(i.e. for pseudomonad strain MHGC424 of marine organisms degraded).Pseudomonas (Pseudomonas sp.) MHGC424
Being preserved in China typical culture collection center (being called for short CCTCC) on August 28th, 2009, preservation is entitled: pseudomonas
MHGC424 (Pseudomonas sp.MHGC424), deposit number is: CCTCC NO:M209188, and preservation address is: China is military
Chinese Wuhan University.
The present invention separates, the marine organisms degradation bacteria strains MHGC424 of screening, has stronger organic matter degradation ability.
This has just widened people to Rhodopseudomonas (Pseudomonas sp.) in the applied research thinking of its function aspects, and for going
Except the Organic substance in sea water polluted by organic matter provides useful bacterium source and technology, and sea-farming biology is not poisoned
Pathogenic effects, has safe and environment-friendly and efficient actual application value.
Embodiment 5
The application of marine organisms degradation bacteria strains MHGC424
Application example one:
Rhodopseudomonas (Pseudomonas sp.) the mono-bacterium colony of MHGC424 of separated purification in Example 1, in work
Change and amplification culture base is cultivated 40-48h, be 40-60:1 by sea water and bacterium solution volume ratio, be inoculated into containing the organic sea water of bait
In, if blank, stirrer stir culture, cultivation temperature 25-28 DEG C, the BOD of monitoring bait Organic substance sea water is (biochemical continuously
Oxygen consumption), react 2-7d.
From figure 3, it can be seen that the bait Organic substance of inoculation Rhodopseudomonas (Pseudomonas sp.) MHGC424 bacterial strain
BOD in sea water is gradually increased in time, and 4-7d remains stable.Rhodopseudomonas (Pseudomonas sp.) MHGC424 bacterial strain
At 4-7d, Organic substance had stronger degradation capability.
Application example two:
Rhodopseudomonas (Pseudomonas sp.) the mono-bacterium colony of MHGC424 of separated purification in Example 1, in work
Change and amplification culture base is cultivated 40-48h, be 40-60:1 by sea water and bacterium solution volume ratio, be inoculated into containing the organic sea water of bait
In, if blank, stirrer stir culture, cultivation temperature 25-28 DEG C, the COD in monitoring bait Organic substance sea water (changes continuously
Learn oxygen consumption), react 1-5d.
From fig. 4, it can be seen that the bait Organic substance of inoculation Rhodopseudomonas (Pseudomonas sp.) MHGC424 bacterial strain
COD in sea water gradually decreases in time, until 4d reduces to minimum, the COD clearance in sea water is 40.9%.Pseudomonas
Belong to (Pseudomonas sp.) MHGC424 bacterial strain and can reduce the COD in bait Organic substance sea water quickly and effectively.
Application example three:
Pseudomonas (Pseudomonas sp.) the mono-bacterium colony of MHGC424 of separated purification in Example 1, in activation
Amplification culture base is cultivated 40-48h, is 40-60:1 by sea water and bacterium solution volume ratio, is inoculated into the glass equipped with 2L filtering sea
In circle cylinder, and with the filtering sea that is not added with bacterium solution for comparison, put 40 urosomes respectively in a suitable place to breed and be about the Penaeus monodon (Penaeus of 1cm
Monodon) shrimp Seedling and 20 urosomes are about black porgy (Sparus macrocephalus) prelarva of 3cm, each point of 3 parallel laboratory tests
Group, water temperature 25-28 DEG C, after 96h, statistical analysis shrimp Seedling survival rate 75%～85.5%, larval survival rate 75%～
85.5%, survival rate between experimental group and matched group without significant difference.Visible, pseudomonas (Pseudomonas sp.)
Sea-farming biology is not poisoned pathogenic effects by MHGC424.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (2)
1., for a pseudomonad strain MHGC424 of marine organisms degraded, it is characterized in that: the preservation title of this bacterial strain
For: pseudomonas MHGC424 (Pseudomonas sp.MHGC424), depositary institution is: China typical culture collection center,
Preservation address is: Wuhan, China Wuhan University, and preservation date is: on August 28th, 2009, deposit number is: CCTCC NO:
M209188。
2. pseudomonad strain MHGC424 for marine organisms degraded described in claim 1 is organic in processing sea water
Application in pollutant.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876617A (en) * | 2012-10-18 | 2013-01-16 | 浙江大学 | Pseudomonas and purpose of pseudomonas |
| CN103756933A (en) * | 2013-12-31 | 2014-04-30 | 江南大学 | Pseudomonas strain capable of degrading PVA (polyvinyl alcohol) |
| CN105039234A (en) * | 2015-07-10 | 2015-11-11 | 中国海洋大学 | Microbial complex flora for eliminating sulfide in sediments |
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2016
- 2016-07-26 CN CN201610596713.8A patent/CN106047765A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876617A (en) * | 2012-10-18 | 2013-01-16 | 浙江大学 | Pseudomonas and purpose of pseudomonas |
| CN103756933A (en) * | 2013-12-31 | 2014-04-30 | 江南大学 | Pseudomonas strain capable of degrading PVA (polyvinyl alcohol) |
| CN105039234A (en) * | 2015-07-10 | 2015-11-11 | 中国海洋大学 | Microbial complex flora for eliminating sulfide in sediments |
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| 莫照兰等: "虾池有机污染物降解细菌的筛选", 《水产学报》 * |
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