CN110272850A - New category bacterial strain with molten algae ability and its to the application of phaeocystis globosa - Google Patents
New category bacterial strain with molten algae ability and its to the application of phaeocystis globosa Download PDFInfo
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Abstract
The invention discloses a kind of new category bacterial strain with molten algae ability, deposit number is CCTCC NO:M 2019404, and classification naming is Mesobaculum littorinae M0103.The bacterial strain screening is simple, is easy culture, and inventor has also set up corresponding cultural method.Studies have shown that M0103 bacterial strain algicidal effect is good, fermentation liquid has Beibu Bay advantage algae phaeocystis globosa to be inhibited and killing effect well, for 24 hours under treatment conditions molten algae rate up to 90% or more.Since M0103 bacterial strain comes from Beibu Bay snail, thus also have the characteristics that ecological security is good, not will cause secondary pollution in use process.The discovery of the bacterial strain enriches having a good application prospect in terms of controlling phaeocystis globosa red tide using microbial resources except algae effect stability, efficient, environmental-friendly advantage for China.
Description
Technical field
The invention belongs to technical field of environmental microorganism more particularly to a kind of new category bacterial strains with molten algae ability and its right
The application of phaeocystis globosa.
Background technique
Red tide refers to that certain phytoplanktons, protozoan or bacterium happen suddenly under certain environmental conditions in ocean water body
Property proliferation and aggregation, cause a certain range and in a period of time the phenomenon that water color-changing.The harm of red tide is very big, is mainly reflected in
Below three aspect: first, the destruction that red tide balances marine ecology.There is high chlorophyll, Gao Rong in red tide early period of origination, water body
Oxygen, high chemical oxygen consumption (COC) are solved, causes ambient ocean environment acutely to change, destroys the ecological balance of water body.Second, red tide is to sea
The destruction of foreign fishery and aquatic resources.Red tide early period of origination, algae bio mass propagation cause economic algae to change colour or rot,
The mucus secreted out of attaches to fish gill portion and interferes its breathing, causes fish hypoxia death.Some red tide plankton vivo biodistribution poison
Element can cause fishes and shrimps mussel poisoning dead；Later period occurs for red tide, after algae mortality, discharges pernicious gas and toxin, leads
Fish and other marine organisms are caused because of anoxic or to be poisoned to death.Third, harm of the red tide to human health.Some red tide planktons poison
Element is enriched with by food chain in fishes and shrimps body of shellfish, if the mankind ingest accidentally, gently if be poisoned, seriously can lead to death.Such as:
In April, 2011 Qinzhou City rhinoceros foot, a phaeocystis globosa red tide occurring of three ma gulf sea areas, cause a large amount of fish dead.Extensively
Though the western coastal red tide occurred at present is still with respect to its scale for Deposits in Eastern Coastal China and adjacent Guangdong Pearl River estuary
It is small, but harmfulness has begun and appears, and the speed of frequency is also being accelerated.If do not carry out early cope with the following more risk,
Scale, every technical research that the red tide of harm occurs prepare, once seriously break out, it will be to the Guangxi marine eco-environment and society
Economic development causes serious influence and loss.
Mainly there are physical method, chemical method and bioanalysis at present for the control of red tide.Although physical method takes effect quickly simultaneously
And will not cause damages to ecological environment, but very likely benthon is adversely affected, and can not be fundamentally
Administer red tide.Chemical method is current most efficient method in comparison, but it is easy to cause secondary pollution to environment, can not
Targetedly only red tide plankton is had an impact, non-red tide plankton can be also resulted in a certain degree while killing red tide plankton
On injury.Biological method is it is emphasised that the entirely management of the ecosystem, and wherein algae-lysing bacterium is as aquatic ecosystem biology
The important component of population structure and function is played to prevention and treatment wawter bloom and red tide and to the biomass balance for maintaining algae to Guan Chong
The effect wanted.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of new category bacterial strain with molten algae ability and its to spherical brown capsule
The application of algae.
In order to solve the above technical problems, the invention adopts the following technical scheme:
A kind of new category bacterial strain, deposit number is CCTCC NO:M 2019404, classification naming Mesobaculum
littorinae M0103。
Above-mentioned new category bacterial strain, 16S rRNA gene have the base sequence of sequence table SEQ .ID.NO.1.
The above-mentioned new bacterial strain that belongs to is in the application administered in red tide or wawter bloom.
The above-mentioned new bacterial strain that belongs to is in the application of molten algae aspect.
The above-mentioned new fermentation liquid for belonging to bacterial strain is used to prepare molten algae medicament.
Above-mentioned new category bacterial strain is for inhibiting or killing phaeocystis globosa.
The above-mentioned new fermentation liquid for belonging to bacterial strain is used to prepare phaeocystis globosa inhibitor or killing agent.
The above-mentioned new cultural method for belonging to bacterial strain, which is inoculated in culture medium, under conditions of 28-30 DEG C, pH4-8.5
It is cultivated.
Above-mentioned cultural method, comprising the following steps:
(1) inclined-plane culture: M0103 strain is inoculated in solid slope culture medium, is cultivated for 24 hours under the conditions of 28 DEG C；
(2) primary culture: cultured inclined-plane is taken under aseptic condition, takes a ring in 100mL fluid nutrient medium with oese
In, 28 DEG C, shaking table 180rpm is cultivated for 24 hours, obtains primary culture bacterium solution；
(3) expand culture: primary culture bacterium solution being inoculated in 500mL fluid nutrient medium, inoculum concentration 5%, is shaken by 28 DEG C
Bed 180rpm cultivates 72h, obtains after expanding culture bacterium solution.
The formula of fluid nutrient medium are as follows: yeast extract powder 2.0g/L, malt extract powder 2.0g/L, glucose 2.0g/L, sea
Salt 26.0g/L, 121 DEG C of high-temp steam sterilizing 20min；Solid slope culture medium is that final concentration 1.3% is added in fluid nutrient medium
Agar powder.
Inventor's isolated algae-lysing microorganism of the invention in a coarse shore spiral shell for North Sea gold bay mangrove, it
Belong to acetobacter section (Acetobacteraceae), be new category bacterial strain, deposit number is CCTCC NO:M 2019404, classification life
Entitled Mesobaculum littorinae M0103.The bacterial strain screening is simple, is easy culture, and inventor has also set up corresponding training
The method of supporting.Studies have shown that M0103 bacterial strain algicidal effect is good, fermentation liquid has Beibu Bay advantage algae phaeocystis globosa
Have and inhibit well and killing effect, for 24 hours under treatment conditions molten algae rate up to 90% or more.Since M0103 bacterial strain comes from the North Sea
Sea area snail, thus also have the characteristics that ecological security is good, it not will cause secondary pollution in use process.The discovery of the bacterial strain
Enrich China is controlling spherical palm fibre except algae effect stability, efficient, environmental-friendly advantage using microbial resources
It has a good application prospect, has also been established for the exploitation of wawter bloom prevention and treatment biological agent with research field solid in terms of capsule algae red tide
Basis, will be well worth doing in the harmful algal bloom event that water body in large is broken out from now on.
Detailed description of the invention
Fig. 1 is based on effective publication bacterium neighbouring in 16S rRNA gene sequencing building bacterial strain M0103 and red Bacteriaceae
The N-J phylogenetic tree of strain.
Preservation information explanation
Mesobaculum littorinae M0103, deposit number be CCTCC NO:M 2019404, preservation date:
On May 29th, 2019, preservation address are as follows: the Chinese Wuhan Wuhan University, postcode 430072, depositary institution: Chinese Typical Representative culture
Object collection.
Specific embodiment
In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or says according to commodity
Bright book selection.
Tranquillimonas alkanivorans A34TIt buys to Germany Microbiological Culture Collection Center (DSMZ).
The separation preparation of embodiment 1, new strains M0103 of the present invention
Coarse shore spiral shell tissue leachate is taken, by its dilution spread in ISP4 solid medium, 30 DEG C are cultivated 21 days, picking
Single colonie, then scribing line purifying obtain.
It send to China typical culture collection center preservation, deposit number is CCTCC NO:M 2019404.
The appearance features of embodiment 2, new strains M0103 of the present invention
The single colonie for taking bacterial strain M0103 is transferred on ISP2+2.6% sea salt solid medium (agar), in 30 DEG C of constant temperature
48h is cultivated in incubator, observes the forms such as size, color, edge, protrusion, smoothness, viscosity, the transparency of its bacterium colony respectively
Feature.
The results show that bacterial strain M0103 forms neat in edge, microprotrusion, light on ISP2+2.6% sea salt solid medium
It is sliding, opaque, the circular colonies of off-white color, diameter about 2-3mm.
The growth characteristics of embodiment 3, new strains M0103 of the present invention
Picking cultivates fresh cultured object for 24 hours on ISP2+2.6% sea salt solid medium, is inoculated into R2A Liquid Culture
Base, 37 DEG C of shaking table culture 36h, as seed.
The composition of ISP2+2.6% sea salt solid medium: yeast extract powder 2.0g/L, malt extract powder 2.0g/L, grape
Sugared 2.0g/L, sea salt 26.0g/L, 1.4% agar powder, deionized water 1000mL.
R2A solid medium: yeast powder 0.5g/L, tryptone 0.5g/L, casamino acid 0.5g/L, glucose
0.5g/L, soluble starch 0.5g/L, dipotassium hydrogen phosphate 0.3g/L, epsom salt 0.05g/L, Sodium Pyruvate 0.3g/L, fine jade
Rouge 13.0g/L, deionized water 1000mL.
1. growth temperature
Culture is inoculated in ISP2+2.6% sea salt solid medium, is respectively placed in 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30
DEG C, 37 DEG C, 40 DEG C and 45 DEG C of cultures, each temperature gradient do three it is parallel, observe growing state after 7 and 14 days respectively.Knot
Fruit shows that bacterial strain M0103 growth temperature range is 15-35 DEG C, 28-30 DEG C of optimum temperature.
2. growing NaCl tolerance
Culture is inoculated in the R2A solid medium of the sodium chloride containing 0-15%, concentration gradient 1%, 28 DEG C of trainings respectively
It supports, each NaCl concentration gradient does three in parallel, respectively in 7 and 14 days observation growing states.The results show that bacterial strain M0103
NaCl tolerance is 1-14%, optimal salinity 2-3%.
3. growing pH range
It is 4.0-12.0, gradient 0.5 by R2A Liquid Culture keynote pH.By the M0103 seed cultivated by 2% inoculation
Amount access, 30 DEG C of cultures, respectively in 7 and 14 days observation growing states.The results show that the pH range of bacterial strain M0103 growth is
4.0-8.5, optimal pH 7.0.
4. illumination
Culture is inoculated in ISP2+2.6% sea salt solid medium, is individually positioned in illumination and dark culturing case,
28 DEG C of cultures, each factor does three in parallel, respectively in 3 and 7 days observation growing states.The results show that the growth of bacterial strain M0103
The influence not being illuminated by the light, it is insensitive to light.
The physio-biochemical characteristics of embodiment 4, new strains M0103 of the present invention
Bacterial strain M0103 and relevant bacterial strain are carried out using API ZYM and API 50CH identification systems (bioM é rieux)
Physiological and biochemical property identification.Wherein,
API ZYM is the micro method system an of sxemiquantitative, is aimed at designed by research enzyme activity.It can system and quickly
Study the activity of 19 kinds of enzymes in ground.Experimental procedure is as follows:
1) sample preparation of samples: is diluted in minimum volume: can use 2mL sterile distilled water or other such as common physiological saline
Buffer is not needed.A bacteria suspension is prepared, turbidity is in McFarland NO.5-6-.From inclined-plane or centrifugation meat soup culture
The pure culture of object can be used to prepare bacteria suspension.
2) preparation of test bar: prepare a culture plate and lid.Remember sample number in the side of disk.A plastics can be used
Wash bottle, packing about 5ml distilled water is in being able to maintain certain humidity in culture plate, when in order to cultivate.It is taken out from the packaging of sealing
API ZYM test bar, sets in culture plate.
3) inoculation of test bar: being inoculated with suction pipe, and 2 drops sample (65 μ L) are accessed in each cup of test bar.
4) after inoculation, plastic closure, 37 DEG C of culture 4h test bar culture: are put on pallet.All determination condition (time,
Temperature, culture medium, suspension concentration) it to be consistent.The test bar of inoculation is kept in dark place.
5) the result observation of test bar: after culture, 1 drop ZYM A reagent and 1 is added to drip ZYM B reagent.Add lustre to after five minutes, such as
Fruit is the positive, and test bar is placed in 10s (second, similarly hereinafter) under an intense light source (1000 watts of light bulbs), light bulb is placed on 4s on cup.This
It is the yellow to eliminate extra strong orchid in cup.Negative reaction becomes colorless after exposure.
6) record reaction: 0-5 marks corresponding color depth.0 is equivalent to negative reaction, and 5 be most strong reaction.2-4 is two
Intensity between person.From micro- millimole concentration (nM) approximate known to color depth: 1 quite discharges 5 micro- millimole concentration
(5nM), 2 are equivalent to 10nM, and 3 be 20nM, and 4 be 30nM, and 5 be 40nM or higher.
It the results are shown in Table 1.
The enzyme activity of the API ZYM test strips of 1 M0103 of table is reacted
Note: "+" is the positive, and " w " is weakly positive, and "-" is feminine gender.
API 50CH is a standardized identification systems, it is using 50 biochemical tests come the carbon hydrate to microorganism
The metabolism of object is studied.Experimental procedure is as follows:
1) preparation of samples: the bacterium colony that picking grows late log phase on a small quantity is placed in 10mL API 50CHL fluid nutrient medium,
A bacteria suspension is prepared, turbidity is in McFarland NO.5-6.
2) preparation of test bar: prepare a culture plate and lid.Remember sample number in the side of disk.A plastics can be used
Wash bottle dispenses a small amount of distilled water in culture plate, keeps humidity in incubation.API 50CH examination is taken out from the packaging of sealing
Item is tested, is set in culture plate.
3) inoculation of test bar: being inoculated with suction pipe, is accessed appropriate sample in each cup of test bar, is filled it up with the portion of pipe
Point, bacteria suspension is partially not added on pipe, anaerobic environment can be kept in such pipe, two drop paraffin oils are added and are sealed.
4) test bar culture: after inoculation, putting plastic closure on pallet, 37 DEG C of cultures 12 and for 24 hours.All determination conditions
(time, temperature, culture medium, suspension concentration) will be consistent.The test bar of inoculation is kept in dark place.
5) the result observation of test bar: during strip is incubated for, acid is produced in the tubule in strip under anaerobic environment, PH changes
Become and make indicator color change in culture medium, fermentation thus can be observed.Any activity is free of in strip in first hole
Ingredient is controlled in this, as feminine gender.
6) record reaction: with positive (+), weakly positive (w), negative (-) explains each test, result is recorded in
As a result (table 2) on record.
The API 50CH test strips of 2 M0103 of table utilize sole carbon source acid-producing result
| Substrate component | As a result | Substrate component | As a result | Substrate component | As a result |
| Control | - | Inositol | - | D- melezitose | + |
| Mannitol | - | Mannitol | - | D- gossypose | + |
| Erythritol | - | Sorbierite | - | Starch | - |
| D-arabinose | w | Methyl D-mannopyranose glycosides | + | Glycogen | - |
| L-arabinose | + | Methyl-D-glucopyranose glycosides | + | Xylitol | - |
| D-ribose | + | N-Acetyl-D-glucosamine | w | D- gentiobiose | + |
| D- xylose | + | Amarogentin | w | The Toulon D- sugar | + |
| L- xylose | w | Arbutin | + | D- lyxose | + |
| D- adonite | - | Aesculin ironic citrate | + | D-Tag | + |
| Methyl-β-D xylopyranose glucosides | + | Salicin | + | D- fucose | + |
| D- galactolipin | + | D- cellobiose | + | L-fucose | + |
| D-Glucose | + | D-Maltose | + | D-arabitol | + |
| D-Fructose | + | D- lactose | + | L- arabite | w |
| D-MANNOSE | + | D- melibiose | + | K-IAO | w |
| L- sorbose | + | D- sucrose | + | 2 ketone group K-IAOs | - |
| L- rhamnose | - | D- trehalose | + | 5 ketone group K-IAOs | + |
| Melampyrin | - | Inulin | - |
As it can be seen that the major physiological biochemical character of bacterial strain M0103 from Tables 1 and 2: aerobic bacterium；It can urease-producing, oxidation
Enzyme and catalase；Beta galactosidase is positive, alkaline phosphatase, lipoid esterase (C8), acid phosphatase, beta galactose glycosides
Enzyme, alpha-glucosidase are positive；Esterase (C4), leucine arylaminopeptidase, cystine arylaminopeptidase, alpha-galactoside
Enzyme, beta-glucosidase are in weakly positive；Lipoidase (C14), valine arylaminopeptidase, Alpha-Fucosidase, trypsase,
Chymotrypsin, N- acetyl-glucosaminidase, alpha-Mannosidase, β-glycuronide enzyme, naphthols-AS-BI- phosphoric acid hydrolysis
Enzyme is negative；Tween20, Tween40 and Tween80 are not hydrolyzed, aesculin is hydrolyzed；It is unable to hydrocellulose；D- can be utilized
Arabinose, L-arabinose, D-ribose, D- xylose, L- xylose, methyl-β-D xylopyranose glucosides, D- galactolipin, D- grape
Sugar, D-Fructose, D-MANNOSE, L- sorbose, methyl D-mannopyranose glycosides, methyl-D-glucopyranose glycosides, N- acetyl Portugal
Grapes glucosamine, amarogentin, arbutin, aesculin ironic citrate, salicin, D- cellobiose, D-Maltose, D- lactose, D- are close
Disaccharides, D- sucrose, D- trehalose, D- melezitose, D- gossypose, D- gentiobiose, the Toulon D- sugar, D- lyxose, D-Tag,
D- fucose, L-fucose, D-arabitol, L- arabite, K-IAO, 5 ketone group K-IAOs etc. are used as carbon source
Produce acid；Mannitol, D- adonite, D- adonite, L- rhamnose, melampyrin, erythritol, inositol, chrysanthemum are not utilized
The sole carbon sources such as powder, starch, 2 ketone group K-IAOs, glycogen, xylitol, mannitol, sorbierite produce acid.
And bacterial strain M0103 is in alkaline phosphate enzyme, valine arylamine enzyme, cystine arylamine enzyme, acid phosphatase, naphthols-
AS-BI- phosphohydrolase, beta galactoside enzyme and alpha-gluconase activity enzyme the isoreactivity close bacterial strain with homology
Tranquillimonas alkanivorans A34TRelated enzyme activity have significant difference.
Embodiment 5, new strains M0103 of the present invention 16S rRNA gene PCR amplification and sequencing and 16S rRNA
Phylogenetic character.
1. extracting genomic DNA
M0103 is seeded to ISP2+2.6% sea salt solid medium, grows to late log phase, one bacterium colony of picking adds
Enter in 100ul 10%Chelex-100 solution, 100 DEG C of heating 10min (dividing, similarly hereinafter), after cooling, 5000rmp (revolving speed) centrifugation
10min takes liquid upper layer as template DNA.
The PCR amplification and sequencing of 2.16S rRNA gene
Amplification and sequencing primer are bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCA-3') and 1492R
(5'-GGTTACCTTGTTACGACTT-3'), PCR reaction condition: 94 DEG C of initial denaturation 8min, 94 DEG C of denaturation 50s, 55 DEG C are annealed
45s, 72 DEG C of extension 90s, totally 30 recycle, and after circulation terminates, 72 DEG C of extensions maintain 10min.Amplified production is solidifying through 1% agarose
Gel electrophoresis detection after Gel Logic 2200Pro gel imager test strip band is qualified, entrusts Shanghai Mei Ji biological medicine technology
Co., Ltd's Guangzhou Branch is sequenced.
Sequencing result is arranged through DNA Star software, utilizes EzTaxon server (http://www.eztaxon.org/)
And the website Blast is compared online；The sequence of homology highest bacterial strain is chosen as reference subject, with MEGA5.0 software,
Genealogical tree, the value of the confidence of Boostrap1000 each branch of detection, to each bacterial strain are constructed using Neighbor-Joining method
Phylogenetic is analyzed.
Sequencing result shows that the 16S rRNA gene order length of bacterial strain M0103 is 1458bp.Its 16S rRNA gene
Nucleotide sequence such as sequence is base sequence shown in SEQ.ID.NO.1.
16S rRNA sequence is drawn into evolutionary relationship tree using software MEGA version5.0.software package.
It is calculated using maximum-likelihood, and by neighbor-joining, maximum-parsimony and carried out in terms of verifying
It calculates, bootstrap is set as 1000 circulations.As a result as shown in Figure 1.Homology analysis shows that bacterial strain M0103 belongs to
Mesobaculum sp., Tranquillimonas alkanivorans A34TGather in a branch, 16S rRNA's is homologous
Property highest, similitude 95.05%.It can be defined as when the 16S rRNA homology of usual two bacterial strains is less than 96% different
Belong to, but polyphase sort research thinks that the determination that one belongs to or plants should not need other physiology only according to a single standard
The analysis of the indexs such as biochemical indicator, chemical feature analysis and full-length genome identification, just can determine that the classification position of bacterial strain M0103.
The content of fatty acid feature of the new microbe of the present invention of embodiment 6
The measurement of the total fatty acid content of bacterial strain M0103.
Configure following solution: Reagent Isosorbide-5-Nitrae 5g sodium hydroxide is dissolved in 150mL methanol and 150mL distilled water；
Reagent2,190mL concentrated hydrochloric acid, 275mL methanol are dissolved in 135mL distilled water；Reagent 3,200mL n-hexane and 200mL second
Ether is uniformly mixed；Reagent 4,10.8g sodium hydroxide are dissolved in 900mL distilled water；Reagent 5, saturated sodium chloride solution,
40g sodium chloride+100mL distilled water.
1) appropriate bacterial cultures is taken, is placed in 8mL screw socket glass tube, 1 solution of 1mL Reagent is added, tightens spiral shell
Lid, boiling water bath 5min take out oscillation 5-10s, tighten blind nut once again, continue boiling water bath 25min；
2) after sample cell is cooling, 2 solution of 2mL Reagent is added, covers tightly and vibrates 5-10s, subsequent accurately control 80 ±
1 DEG C of water-bath 10min, ice bath are cooling；This step needs strict temperature control and time, in case carboxylic acid and ring type fatty acid are by broken
It is bad；
3) 3 solution of 1.25mL Reagent is added in cooling sample cell, quick oscillation 10min or so discards lower layer
Water phase；
4) 4 solution of 2mL Reagent and 5 solution of 4mL Reagent, quick oscillation 5min are added in remaining organic phase
Left and right, takes upper organic phase to set spare in gas-chromatography sample bottle.
6890 gas chromatograph of HP and mass spectrograph combination analysis, commission South China Botanical Garden Chinese Academy of Sciences are tested.
The results show that the main cell fatty acid of bacterial strain M0103 is C18:1ω 7c and C16:0, percentage composition is respectively
63.87% and 20.12%.Meet the main fatty acid type of acetobacter section.And with similar strain fatty acid species and content
On, it is variant, judge that same similar strain belongs to variety classes with this.
The G+C mol% content characteristics of the new microbe of the present invention of embodiment 7
Shanghai Major Biological Medical Technology Co., Ltd. is entrusted to carry out genome sequencing.The results show that strain to be tested
The G+C mol% of M0103 is 68.2mol%.
Algicidal effect of the 8 various concentration strain to be tested M0103 of embodiment to phaeocystis globosa
Phaeocystis globosa condition of culture: phaeocystis globosa (Phaeocystis globosa) is trained with f/2 culture medium
It supports, is incubated in 20 DEG C of illumination boxs, 100 μm of ol photonsm of intensity of illumination-2·s-1, light dark period 14h:10h
(light: dark).
The phaeocystis preparation of molten algae experiment: the algae solution of 1000mL logarithmic growth phase is taken, is crossed and is filtered out by the film in 20 μm of apertures
Utricule is removed, it is unicellular to leave phaeocystis, with 4 × 104Cells/mL frustule concentration is that initial concentration is tested.
Molten algae efficiency calculation method: molten algae efficiency (%)=(1- experimental group frustule concentration/control group frustule concentration)
× 100%.Wherein the frustule concentration of phaeocystis globosa passes through microscopic counting with 0.1mL water sample tally.
The fermentation process of M0103:
(1) strain: the strain to be tested M0103 is used；
(2) inclined-plane culture: the strain in step (1) is inoculated in solid slope culture medium, in 28 DEG C of culture 48h；
(3) primary culture: taking cultured inclined-plane under aseptic condition, a ring thallus is taken to be inoculated in the training of 100mL sea salt liquid
It supports in base, 28 DEG C, shaking table 180rpm is cultivated for 24 hours, obtains primary culture bacterium solution；
(4) expand culture: primary culture bacterium solution being inoculated in 500mL aforesaid liquid culture medium, inoculum concentration 5%, 28
DEG C, shaking table 180rpm cultivates 72h, obtains after expanding culture bacterium solution, collects fermentation liquid.
Liquid Culture based formulas described in step (3) and (4): yeast extract powder 2.0g/L, malt extract powder 2.0g/L,
Glucose 2.0g/L, sea salt 26.0g/L, deionized water 1000mL, 121 DEG C of high-temp steam sterilizing 20min；Step (2) is described solid
Body slant medium is the agar powder that final concentration 1.3% is added in aforesaid liquid culture medium.
By the product of algae-lysing bacterium M0103 fermented and cultured with final concentration of 0.1%, 0.5%, 1%, 2% bacterium of volume
The ratio of fermentation culture medium and algae solution is co-cultured.Blank control is set, and control group is to be added 0.1%, 0.5%, 1%, 2%
Phase algae solution while volume sterile ISP2+2.6% sea salt fluid nutrient medium.Experimental group and control group are respectively provided with 3 repetitions.?
0h, 6h, it samples for 24 hours with 48h, takes 2mL culture solution every time, carry out the counting of frustule concentration, and calculate molten algae efficiency (result is listed in
Table 3).
3 M0103 of table is in the molten phycomycete of various concentration to the molten algae efficiency of phaeocystis globosa
| Handle time/sample | 0h (%) | 6h (%) | (%) for 24 hours | 48h (%) |
| 0.1% | 0.27±0.12 | 16.25±7.13 | 27.40±10.10 | 50.78±9.72 |
| 0.5% | 3.51±1.52 | 23.67±5.22 | 54.33±6.92 | 79.33±8.83 |
| 1% | 2.89±1.08 | 40.54±11.35 | 73.98±4.50 | 87.22±1.23 |
| 2% | 5.77±1.93 | 38.77±8.05 | 91.41±4.14 | 98.04±1.25 |
As known from Table 3, bacterial strain M0103 fermentation liquid is added to the spherical shape palm fibre in logarithmic phase growth period with 0.1% initial bacteria concentration
In capsule algae, molten algae rate is 50.78% after 48h；When with the addition of 0.5% initial bacteria concentration, algicidal effect is 54.33%, 48h for 24 hours
It is 79.33%；When with the addition of 1.0% initial bacteria concentration, algicidal effect is 73.98%, 48h 87.22% for 24 hours；It is dense with 2.0%
When degree addition, alga-lysing activity is up to 91.41%, 48h up to 98.04% for 24 hours.As it can be seen that M0103 is to phaeocystis globosa with higher
Molten algae efficiency.
Sequence table
<110>Guangxi Academy Of Sciences
<120>the new category bacterial strain with molten algae ability and its application to phaeocystis globosa
<160> 3
<170> SIPOSequenceListing 1.0
<210> 3
<211> 1458
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tagagtttga tcctggctca gaacgaacgc tggcggcagg cttaacacat gcaagtcgag 60
cgaagacttc ggtcttagcg gcggacgggt gagtaacacg tgggaacatg cccttcactg 120
caggatagtc ccgggaaact gggtttaata ctgcatacgc ccttcggggg aaagatttat 180
cggtgaagga ttggcccgcg ctggattagg tagttggtgg ggtaatggcc taccaagccg 240
acgatccata gctggtttga gaggatgatc agccacactg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt ggggaatctt agacaatggg cgcaagcctg atctagccat 360
gccgcgtgag tgacgaaggc cttagggtcg taaagctctt tcgctgggga agataatgac 420
tgtacccagt aaagaaaccc cggctaactc cgtgccagca gccgcggtaa tacggagggg 480
gttagcgttg ttcggaatta ctgggcgtaa agcgcgcgta ggcggattag tcagtcagag 540
gtgaaatccc agggctcaac cctggaactg cctttgatac tgctagtctt gagttcgaga 600
gaggtgagtg gaactccgag tgtagaggtg aaattcgtag atattcggaa gaacaccagt 660
ggcgaaggcg gctcactggc tcgatactga cgctgaggtg cgaaagcgtg gggagcaaac 720
aggattagat accctggtag tccacgccgt aaacgatgaa tgccagacgt cgggcagcat 780
gctgttcggt gtcacaccta acggattaag cattccgcct ggggagtacg gtcgcaagat 840
taaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 900
agcaacgcgc agaaccttac caacccttga catggatatc gtagttccga gagatcggtt 960
cgtcagttcg gctggatatc acacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag 1020
atgttcggtt aagtccggca acgagcgcaa cccacatcct tagttgccag cagttcggct 1080
gggcactcta gggaaactgc ccgtgataag cgggaggaag gtgtggatga cgtcaagtcc 1140
tcatggccct tacgggttgg gctacacacg tgctacaatg gcatctacag tgggttaatc 1200
ccaaaaagat gtctcagttc ggattgaggt ctgcaactcg accccatgaa gtcggaatcg 1260
ctagtaatcg cggaacagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc 1320
cgtcacacca tgggagttgg gtttacccga aggcggtgcg ccaacctttt ggaggcagcc 1380
gaccacggta agctcagcga ctggggtgaa gtcgtaacaa ggtagccgta ggggaacctg 1440
cggctggatc acctcctt 1458
<210> 1
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
agagtttgat cctggctca 19
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggttaccttg ttacgactt 19
Claims (10)
1. a kind of new category bacterial strain, deposit number is CCTCC NO:M 2019404, classification naming Mesobaculum
littorinae M0103。
2. new category bacterial strain according to claim 1, it is characterised in that its 16S rRNA gene has sequence table
The base sequence of SEQ.ID.NO.1.
3. newly belonging to bacterial strain described in claim 1 in the application administered in red tide or wawter bloom.
4. newly belonging to bacterial strain described in claim 1 in the application of molten algae aspect.
5. the fermentation liquid for newly belonging to bacterial strain described in claim 1 is used to prepare molten algae medicament.
6. newly belonging to bacterial strain described in claim 1 for inhibiting or killing phaeocystis globosa.
7. the fermentation liquid for newly belonging to bacterial strain described in claim 1 is used to prepare phaeocystis globosa inhibitor or killing agent.
8. newly belonging to the cultural method of bacterial strain described in claim 1, it is characterised in that: the bacterium is inoculated in culture medium, in 28-30
DEG C, it is cultivated under conditions of pH4-8.5.
9. cultural method according to claim 8, it is characterised in that the following steps are included:
(1) inclined-plane culture: M0103 strain is inoculated in solid slope culture medium, is cultivated for 24 hours under the conditions of 28 DEG C；
(2) primary culture: taking cultured inclined-plane, take a ring in 100mL fluid nutrient medium with oese under aseptic condition, and 28
DEG C, shaking table 180rpm is cultivated for 24 hours, obtains primary culture bacterium solution；
(3) expand culture: primary culture bacterium solution being inoculated in 500mL fluid nutrient medium, inoculum concentration 5%, 28 DEG C, shaking table
180rpm cultivates 72h, obtains and expands culture bacterium solution.
10. cultural method according to claim 9, it is characterised in that: the formula of the fluid nutrient medium are as follows: yeast extracts
Powder 2.0g/L, malt extract powder 2.0g/L, glucose 2.0g/L, sea salt 26.0g/L, 121 DEG C of high-temp steam sterilizing 20min；Institute
Stating solid slope culture medium is that the agar powder of final concentration 1.3% is added in fluid nutrient medium.
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Cited By (2)
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| CN111621431A (en) * | 2019-12-25 | 2020-09-04 | 广西科学院 | Micromonospora aureofaciens with algae-lysing capability and application thereof to Karenia mikimotoi |
| CN116376707A (en) * | 2022-10-18 | 2023-07-04 | 南京灿辰微生物科技有限公司 | Penicillium oxalicum 0710-26 and application thereof |
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| CN111621431B (en) * | 2019-12-25 | 2022-05-10 | 广西科学院 | Micromonospora aureofaciens with algae-lysing capability and application thereof to Karenia mikimotoi |
| CN116376707A (en) * | 2022-10-18 | 2023-07-04 | 南京灿辰微生物科技有限公司 | Penicillium oxalicum 0710-26 and application thereof |
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| CN110272850B (en) | 2022-05-17 |
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