CN104087525B - A kind of novel biosurfactant production seawater fermented type bacterial strain - Google Patents
A kind of novel biosurfactant production seawater fermented type bacterial strain Download PDFInfo
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- CN104087525B CN104087525B CN201410211268.XA CN201410211268A CN104087525B CN 104087525 B CN104087525 B CN 104087525B CN 201410211268 A CN201410211268 A CN 201410211268A CN 104087525 B CN104087525 B CN 104087525B
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Abstract
Traditional zymotic typically uses freshwater resources.The invention discloses the seawater fermented type bacterial strain of a kind of novel biosurfactant production, can well utilize natural sea-water to carry out fermenting and producing rhanolipid as biosurfactant as fermentation water and recycled wood materials as single carbon source.By coastal freshwater resources lack and water quality not good enough area set up seawater fermentation enterprise, it is possible to resolve the conventional fermentation industry dependence to freshwater resources.Bacterial strain of the present invention is sieved again by seawater enrichment culture, hyper salemia flat board primary dcreening operation, synthetic media and seawater shake flask fermentation is verified and obtained isolated and purified, and by bio-chemical characteristics and Molecular Identification analysis, determine that this seawater fermented type bacterial strain is pseudomonas aeruginosa (Pseudomonas aeruginosa), being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 25th, 2014, deposit number is: CGMCC No.9091.This invention bacterial strain is utilizing the advantage in seawater fermentation, gained crude product rhamnolipid can be used for zonal marketing, will play potential application prospect in fields such as agricultural, petrochemical industry, environmental improvement, soil remediation, cosmetics industry, pharmaceutical industry and food service industrys.
Description
Technical field
The invention belongs to field of fermentation engineering, be specifically related to a strain seawater fermented type bacterial strain and utilizing natural sea-water as sending out
Ferment water and recycled wood materials carry out fermenting and producing rhanolipid as biosurfactant as single carbon source.
Background technology
Most of surfactants in the market are essentially from petroleum-based chemical products, and its production process needs to disappear
Consuming substantial amounts of oil product, and easily cause environmental pollution, the acquisition of biosurfactant not there is problems in that.Raw
Thing surfactant rhamnolipid is a kind of novel anionic biosurfactant obtained by fermentable cometabolism,
There is the features such as biodegradable, hypotoxicity and environment friendly.And because of rhamnolipid have good emulsibility, wetability,
Foaming characteristic, has potential application in agricultural, petrochemical industry, environmental protection, cosmetics industry, pharmaceutical industry and food service industry etc.
Prospect.
Fermentation water and inorganic salts cost are the important component parts of fermentation enterprise production cost.Containing rich in natural sea-water
Rich inorganic salts mineral element, can compensate for part inorganic ion in fermentation water.Traditional zymotic water mostly is fresh water, at present sea
Water desalinating cost is higher, therefore lacks at fresh water, and the best Coastal beach area of water quality utilizes novel seawater fermentation strain to produce
Rhanolipid as biosurfactant acquires a special sense.
The cost of fermenting carbon source raw material is also the important component part of Fermentation Engineering production cost, and at present waste oil, fried
The recycled wood materials such as Cargo Oil are the most well utilized, and have become serious environmental contaminants on the contrary or have been reclaimed unreasonably
Dining table, the health of harm people are returned in utilization.Therefore setting up utilizes the natural sea-water as fermentation water and recycled wood materials to be
The fermentation enterprise of single carbon source through fermentation biosurfactant production, both can solve the traditional industry fermentation dependence to freshwater resources,
The process being alternatively recycled wood materials provides new approaches.
Summary of the invention
It is an object of the invention to provide a strain seawater fermented type bacterial strain, be used for utilizing natural sea-water as fermentation water and to give up
Abandoning edible oil and fat is that single carbon source through fermentation produces rhanolipid as biosurfactant, can lack at fresh water, the edge that water quality is the best
Haiti district set up seawater fermentation enterprise, and use this bacterial strain produce gained crude product, can be used for zonal marketing.
The seawater fermented type bacterial strain that the present invention provides is the pseudomonas aeruginosa with sea water resistance salinity, and strain name is Z-
2, its Classification And Nomenclature is pseudomonas aeruginosa (pseudomonas aeruginosa), and is preserved on April 25th, 2014
(address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China Committee for Culture Collection of Microorganisms's common micro-organisms center
Institute of Microorganism, Academia Sinica), deposit number is: CGMCC No.9091.
Separation screening and the authentication step of the seawater fermented type bacterial strain of described novel biosurfactant production be: takes coastal
Beach plant rhizosphere soil sample or mud sample, utilize seawater fermentation medium enrichment culture, hyper salemia flat board primary dcreening operation, synthesis in laboratory
Culture medium sieves again verifies that with seawater shake flask fermentation isolated and purified bacterial strain forms size and surface tension according to zymotic fluid oil extraction circle
Mensuration, finishing screen is selected a strain and natural sea-water can be utilized to be that single carbon source through fermentation is raw as fermentation water and recycled wood materials
The bacterial strain of biosurfactant production rhamnolipid, is numbered Z-2；Then according to morphological feature, the physiological and biochemical index of bacterium colony,
And 16SrDNA sequencing result carries out tetraploid rice analysis discovery with the sequence in GenBank, bacterial strain Z-2 is identified as vacation
Zygosaccharomyces (Pseudomonas sp.), named pseudomonas aeruginosa Z-2.
The process using the prepared crude product rhamnolipid of this bacterial strain seawater fermentation is as follows:
(1) preparation of fermentation seed liquid: the pseudomonas aeruginosa Z-2 inclined plane inoculating hidden of going bail for is in the high salt seed of triangular flask
In culture medium, and triangular flask being placed in constant-temperature table shaken cultivation, temperature is 30 DEG C of rotating speed 180r min-1, after incubated overnight i.e.
Obtain seed liquor.
(2) preparation of zymotic fluid: take the Z-2 bacterial strain seed liquor obtained in (1), be seeded to seawater by the inoculum concentration of 3%
In fermentation medium, 180r min-1, under the conditions of 30 DEG C, after shaken cultivation 3d, i.e. obtain zymotic fluid.
(3) separation of fermentation crude product: the zymotic fluid (2) prepared, under the conditions of 4 DEG C, is centrifuged 20min with 8000rpm,
Remove thalline, take supernatant be concentrated on a rotary evaporator original volume 1/3 after, add concentrated hydrochloric acid and also regulate zymotic fluid pH
To about 2.0, after the heavy filtration of acid, then spin concentration on a rotary evaporator, until the appearance of thick crude product, obtain and slightly produce
Thing rhamnolipid.
(4) further, the medium component in described process (1) and (2): high salt seed culture medium (g/L): peptone
10, beef extract 5, NaCl30, pH7.4.Seawater fermentation medium: the natural sea-water 1000ml after Medium speed filter paper filtration, adds yeast
Powder 1g, fried Cargo Oil 3% (V/V), natural ph.
Compared with the prior art this invention, has obvious beneficial effect, knowable to technique scheme: the sea separated
Water by fermentation type bacterial strain pseudomonas aeruginosa Z-2 has the feature of good sea water resistance salinity, and natural sea-water can be utilized as fermentation
It is that single carbon source through fermentation produces rhanolipid as biosurfactant, and salinity and cultivation bar on an equal basis with water and recycled wood materials
Under part, seawater fermentation is substantially suitable in synthetic media fermentation in yield, and part seawater fermentation yield relatively synthetic media is sent out
Ferment can exceed 35%, and therefore on production cost and yield, seawater fermented type bacterial classification the most substantially overcomes and yields poorly and too rely on
The defect of freshwater resources fermentation.In addition the fermentation crude product rhamnolipid of the present invention can replace as the novel of chemical surfactant
Dai Pin, for oil exploitation, environmental improvement, soil remediation etc. for the highest industry of fermented product purity requirement.Of course through
Further product purification, it is also to have potential application prospect at aspects such as cosmetics industry, pharmaceutical industry and food.
Accompanying drawing explanation
Fig. 1 is that bacterial strain Z-2 seawater of the present invention fermentation utilizes recycled wood materials and the design sketch of emulsification occurs.
Fig. 2 is the growth curve in high salt broth bouillon of bacterial strain Z-2 of the present invention.
Fig. 3 is the phylogenetic tree of bacterial strain Z-2 of the present invention.
Fig. 4 is that bacterial strain Z-2 of the present invention is at different fermentations culture medium tunning character and the comparison diagram of yield.
Detailed description of the invention
Below by specific embodiment, the present invention will be further described.
Embodiment 1: the screening separating step of novel biosurfactant production seawater fermented type bacterial strain
(1) sampling
The following sample of main collection: soil sample or mud sample pick up from Coastal beach.Water sample picks up from the coastal seawater near harbour.Adopt
Take back at once after collection and be stored in 4 DEG C.
(2) enrichment culture
In the shaking flask added with seawater fermentation medium, add soil sample or mud sample, transfer again in newly after 30 DEG C of shaken cultivation 3d
Seawater fermentation medium, altogether enrichment culture 3 times, causes the microorganism that recycled wood materials can be utilized to do sole carbon source to obtain richness
Collection.
(3) primary dcreening operation
Taking pregnant solution in (2) to cultivate at hyper salemia plate streaking or dilution spread, picking transparent circle is more greatly and clearly
After single bacterium colony meets 30 DEG C of inclined-plane cultivation 1-2d, 4 DEG C save backup.
(4) multiple sieve
Taking the inclined-plane of each bacterial strain in (3), connect high salt seed culture medium, after activated overnight, the amount by 3% accesses synthesis and cultivates
In base, 180r min-1, under the conditions of 30 DEG C after shaken cultivation 3d, take zymotic fluid and carry out oil extraction circle and surface tension test.
(5) seawater shake flask fermentation checking
From (4), select the multiple sieve preferable shaking flask of fermentation, take 1.5mL zymotic fluid (containing bacterial classification) to 50mL seawater fermented and cultured
In base, 180r min-1, under the conditions of 30 DEG C after shaken cultivation 3d, take zymotic fluid and carry out oil extraction circle, capillary mensuration.
Measuring surface tension and use surface tension instrument, oil extraction circle measures the method for size and is: in 90mm culture dish tegillum,
Adding 20mL deionized water, dropping 100uL diesel oil forms oil film, then adds the zymotic fluid of 10uL at oil film center.If oil extraction
Loop diameter exceedes the diameter of culture dish tegillum, then add after zymotic fluid being diluted 10 times.Take that oil extraction circle is relatively big and surface tension
The bacterial strain preservation substantially reduced, is numbered Z-2.
Further, described in step (2), (3) and (4), medium component is as follows:
High salt seed culture medium (g/L): peptone 10, beef extract 5, NaCl30, pH7.4.
Seawater fermentation medium (SM): the seawater 1000ml after Medium speed filter paper filtration, adds dusty yeast 1g, fried Cargo Oil 3% (V/
V), pH is nature.
Synthetic media (CM, g/L): NaNO32.5, K2HPO44, KH2PO44, MgSO40.2, CaCl20.02, KCl1,
NaCl30, dusty yeast 1, fried Cargo Oil 3% (V/V), pH is adjusted to about 6.5.
Hyper salemia flat board uses the de-fiber sheep blood adding 5% in meat peptone culture medium to prepare.High salt meat peptone Media Components
For (g/L): peptone 10, beef extract 3, sodium chloride 30, Japanese isinglass 15, deionized water 1000mL, pH is adjusted to about 7.4.Join
During blood plate processed, meat peptone medium sterilization is taken out after being cooled to about 50 DEG C, after adding the de-fiber aseptic sheep blood mixing of 5%
Topple over rapidly culture dish, after cooling, i.e. obtain blood plate.
Embodiment 2: the qualification of seawater fermented type bacterial strain Z-2
Seawater fermented type bacterial strain Z-2 carries out Physiology and biochemistry identify and 16S rDNA Molecular Identification, and combined bacterium
Morphological feature and physiological and biochemical property determine the kind of this seawater fermented type bacterial strain.
(1) seawater fermented type bacterial strain Z-2 cellular morphology, physiological and biochemical property
According to " common bacteria system identification handbook ", carrying out including form microscopy, producing gemma, Gram's staining, purulence is blue or green
Element, produce indoles, reduction nitrate, glucose oxidative fermentation, catalase, gelatin liquefaction, Starch Hydrolysis, citrate,
Every experiments such as methyl red, acetyl methyl carbinol (V-P), malonate.Measurement result such as table 1 below:
The cellular morphology of table 1 seawater fermented type bacterial strain Z-2 and physiological and biochemical property
Note :+: utilize or be positive；-: negative reaction.
(2) the 16S rDNA sequence analysis of seawater fermented type bacterial strain Z-2
Utilize Hangzhou Xin Jie company DNA of bacteria kit extract STb gene, with extract STb gene as template, with
Universal primer P1 (27F): 5 '-AGAGTTTGATCCTGGCTCAG-3 ' of 16SrDNA and P2 (1492R): 5 '-
TACGGYTACCTTGTTACGACTT-3 ' expands, reactant liquor 50 μ L, containing 1 × PCRBuffer, and 200 μm ol/LdNTP,
1.5mmol/LMgCl2 .6 μm ol/L primer, 50ng template DNA, 2.5U Taq enzyme.Amplification condition is 95 DEG C of denaturations 5min,
94 DEG C of 1min, 50 DEG C of 90s, 72 DEG C of 2min, 35 circulations, finally it is incubated 10min in 72 DEG C.Check order by the raw work bioengineering in Shanghai
Co., Ltd completes, and surveyed total order is classified as:
Embodiment 3: the determination of seawater fermented type bacterial strain Z-2
The 16SrDNA sequencing result of this bacterial strain is carried out tetraploid rice with the sequence in GenBank, through BLAST comparison,
With pseudomonas (Pseudomonas sp.), there is the highest homology.Relevant 16SrDNA sequence is transferred from GenBank,
Use the softwares such as ClustalX and Mega5 to carry out Phylogenetic Analysis, use adjacent method constructing system chadogram (Fig. 3).Comprehensively
The bio-chemical characteristics result (table 1) of this bacterial strain and Molecular Identification cluster analysis result, it can be assumed that seawater fermented type bacterial strain Z-2
For pseudomonas aeruginosa.Its Classification And Nomenclature is pseudomonas aeruginosa (pseudomonas aeruginosa), and is preserved in
(address: in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, state's Microbiological Culture Collection administration committee common micro-organisms center
Institute of microbiology of the academy of sciences of state), deposit number is: CGMCC No.9091.
Embodiment 4: seawater fermented type bacterial strain Z-2 fermentation performance in seawater fermentation medium and synthetic media.
The seawater fermented type bacterial strain Z-2 learning from else's experience isolated and purified, activated overnight in inoculation high salt seed culture medium, by activation well
Seed liquor inoculate seawater fermentation medium and synthetic media respectively by 3% amount, and under same culture conditions after cultivation 3d,
The zymotic fluid taking two kinds of culture mediums respectively surveys oil extraction circle and capillary size, and the height of fermentation yield.By Fig. 4's
Lateral comparison, it appeared that seawater (Jiangsu great Feng Gang and Chen Jiagang) at two used by this patent, in terms of seawater fermentation the most relatively
Synthetic media fermentation is preponderated, and particularly Chen Jia port seawater fermentation yield exceeds 35% than the fermentation yield of synthetic media.
Therefore on production cost and yield, seawater fermented type bacterial classification substantially overcome yield poorly and too rely on freshwater resources fermentation lack
Fall into.For in a certain sense, this microbial inoculum exploitation being beneficial to this seawater fermented type bacterial strain Z-2 and utilization and extention.
Claims (2)
1. a novel biosurfactant production seawater fermented type bacterial strain, it is characterised in that described bacterial strain is verdigris vacation list
Born of the same parents bacteria strain Z-2 (Pseudomonas aeruginosa Z-2), depositary institution: China Committee for Culture Collection of Microorganisms
Common micro-organisms center, deposit number is: CGMCC No.9091.
A kind of novel biosurfactant production seawater fermented type bacterial strain the most according to claim 1, it is characterised in that
The 16S rDNA sequence of described seawater fermented type bacterial strain Z-2 is as follows:
。
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| CN104357363B (en) * | 2014-11-19 | 2018-01-26 | 沈阳化工大学 | Pseudomonas strains and its method for preparing compound surfactant |
| CN105002122B (en) * | 2015-08-17 | 2017-03-29 | 类延乐 | A kind of culture medium of screening and culturing rhamnolipid producing strains |
| CN105132466A (en) * | 2015-08-23 | 2015-12-09 | 盐城师范学院 | Method for production of surfactants by means of fermentation of paper-making wastewater |
| CN106497818B (en) * | 2015-09-07 | 2019-06-25 | 粮华生物科技（北京）有限公司 | Pseudomonas aeruginosa and microbial inoculum and their applications in degraded oil and/or oil product |
| CN112410038A (en) * | 2019-08-21 | 2021-02-26 | 深圳市万卉园景观工程有限公司 | Saline-alkali soil conditioner and preparation method and application thereof |
| CN110616243A (en) * | 2019-09-23 | 2019-12-27 | 深圳市仙湖植物园管理处 | Rhamnolipid fermentation medium and preparation method and application thereof |
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