CN102925382A - Method for producing hydrocarbons for making fuel by using sea water as medium and special strain - Google Patents
Method for producing hydrocarbons for making fuel by using sea water as medium and special strain Download PDFInfo
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Abstract
The invention discloses a method for making fuel by using sea water as a medium and a special strain. The special strain is holomonas (Halomonas sp.)LS21, of which the collection number is CGMCCNo.6593. Experiments show that the Halomonas sp.LS21 and the recombinant strain thereof can produce hydrocarbons mainly comprising alkane, olefin and long-chain fatty acid by using natural sea water as a fermenting medium and using cheap carbon materials and nitrogen sources such as pre-treated cellulose, hemicellulose, waste oils and fats and kitchen waste as substrate. The separated hydrocarbons can be used as biofuel, and continuous fermentation without sterilization can be realized. The holomonas and the recombinant strain thereof have low requirement on nutrients. The fermentation process can go continuously without sterilization. The method is simple and easy to control and low in cost, and has a great prospect in industrial application.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method and special strain therefore that fuel is used hydrocarbon polymer of producing take seawater as medium.
Background technology
For centuries, conventional fossil energy is supporting the development of industrial civilization always.But because fossil energy is non-renewable, the precious resources under the earth accumulated in 1 years finally can approach exhaustion under human high strength exploitation and consumption.The problems such as the climate change that excessive use of while fossil resource brings, environmental pollution also will make the mankind pay huge cost.The power supply problem will become the important factor of restriction Future in China Sustainable Socioeconomic Development.
For guaranteeing independence and the sustainability of power supply, various countries have started a field energy source and chemical field new technology tide at present, namely use " cell factory and biosynthesizing " progressively to substitute traditional chemical technique.Utilize biotechnology take renewable biological source as the raw material production biofuel and various industrial chemicals and product wherein focus especially.Compare with traditional fossil oil, renewable, nontoxic, degradable characteristics that biofuel has, and can effectively reduce the discharging of obnoxious flavour and particulate matter, be the fossil oil substitute of high-quality, therefore be called again " green fuel ".
But at present the biofuel of listing is mainly derived from the external transesterification of triacylglycerol in the fermentation reaction of W-Gum and soybean, Semen Brassicae campestris, the plam oil, and its composition is ethanol, fatty acid methyl ester or ethyl ester etc.Thisly utilize food crop to produce with the people situation of striving grain, taking a large amount of arable lands for the method for raw material production biofuel; Traditional zymotechnique need to consume a large amount of fresh water as medium, produces in a large number not tractable waste water after the reaction, has not only consumed resource but also polluted environment.For practicality and the range of application that improves biofuel, put forth effort on exploitation new catalysis process and technical process, the using method of seeking more efficient saccharase and optimizing enzyme is expanded available raw material and the fermentation media scope becomes necessary.On this basis, by transforming the pathways metabolism of microorganism, can utilize cheap non-grain and oil class raw material such as Mierocrystalline cellulose etc., directly produce in vivo biofuel, thereby break away from a series of high energy consumption production process, reduce cost.
Summary of the invention
An object of the present invention is to provide a strain Halomonas (Halomonas sp.).
Halomonas provided by the invention (Halomonas sp.) LS 21, its deposit number is CGMCC No.6593.
The application of above-mentioned Halomonas (Halomonas sp.) LS 21 in preparation polyhydroxyalkanoate/3-hydroxybutyrate and/or lipid acid also is the scope of protection of the invention; In the above-mentioned application, described lipid acid is specially C16 lipid acid, is also referred to as palmitinic acid.
Can utilize the method for synthetic biology that above-mentioned Halomonas (Halomonas sp.) LS 21 is carried out gene recombination, modify its metabolic pathway, make this engineering strain on the basis that can utilize multiple carbon and nitrogen sources, express the genes involved of the enzyme of coding catalytic production hydrocarbon polymer.
Therefore, another object of the present invention provides a kind of recombinant bacterium.
Recombinant bacterium provided by the invention for the encoding gene of acyl group acyl carrier protein reductase enzyme and the encoding gene of acetaldehyde decarboxylase are all imported in the Host Strains, obtains recombinant bacterium.
In the above-mentioned recombinant bacterium, Host Strains is Halomonas (Halomonas sp.); Concrete Halomonas (Halomonas sp.) LS 21 that adopts in the embodiments of the invention;
Above-mentioned acyl group acyl carrier protein reductase enzyme and above-mentioned acetaldehyde decarboxylase are specifically all from cyanobacteria; Cyanobacteria is specially collection born of the same parents cyanobacterias (Synechocystis sp.) or beads cyanobacteria (Nostoc sp.) in the embodiments of the invention.
In the above-mentioned recombinant bacterium, the encoding gene of the encoding gene of acyl group acyl carrier protein reductase enzyme and acetaldehyde decarboxylase is all imported Host Strains import Host Strains for the fragment of the encoding gene of the encoding gene that will contain acyl group acyl carrier protein reductase enzyme and acetaldehyde decarboxylase; The nucleotides sequence of the fragment of the encoding gene of the described encoding gene that contains acyl group acyl carrier protein reductase enzyme and acetaldehyde decarboxylase is classified sequence 1 in the sequence table or the sequence 4 in the sequence table as.
Above-mentioned recombinant bacterium is following recombinant bacterium A or recombinant bacterium B:
Recombinant bacterium A will be for importing among Halomonas (Halomonas sp.) LS 21 from the encoding gene that contains acyl group acyl carrier protein reductase enzyme of collection born of the same parents cyanobacterias (Synechocystis sp.) and the fragment (sequence 1) of the encoding gene of acetaldehyde decarboxylase; The fragment that imports among concrete the present invention from the encoding gene of the encoding genes that contains acyl group acyl carrier protein reductase enzyme of collection born of the same parents cyanobacterias (Synechocystis sp.) and acetaldehyde decarboxylase imports among Halomonas (Halomonas sp.) LS 21 by recombinant vectors A, and this recombinant vectors A is for obtaining expressing acyl group acyl carrier protein reductase enzyme and acetaldehyde decarboxylase between XbaI that will the sequence 1 insertion vector pBBR1MCS1 in the sequence table and BamH I site.
Integrate the aminoacid sequence of the acyl group acyl carrier protein reductase enzyme in the born of the same parents cyanobacteria (Synechocystis sp.) as the sequence 2 in the sequence table; The nucleotides sequence of the gene of described acyl group acyl carrier protein reductase enzyme is classified in the sequence table sequence 1 as from 5 ' terminal the 1st-1023 Nucleotide; The aminoacid sequence of acetaldehyde decarboxylase is the sequence 3 in the sequence table; The nucleotides sequence of the gene of described acyl group acyl carrier protein reductase enzyme is classified in the sequence table sequence 1 as from 5 ' terminal the 1242nd-1937 Nucleotide.
Recombinant bacterium B is for importing among Halomonas (Halomonas sp.) LS21 from the fragment (sequence 4) of the encoding gene of the encoding gene that contains acyl group acyl carrier protein reductase enzyme of beads cyanobacteria (Nostoc sp.) and acetaldehyde decarboxylase; The fragment that imports among concrete the present invention from the encoding gene of the encoding gene that contains acyl group acyl carrier protein reductase enzyme of beads cyanobacteria (Nostoc sp.) and acetaldehyde decarboxylase imports among Halomonas (Halomonas sp.) LS 21 by recombinant vectors B, and this recombinant vectors B is for obtaining expressing acyl group acyl carrier protein reductase enzyme and acetaldehyde decarboxylase between XbaI that will the sequence 4 insertion vector pBBR1MCS1 in the sequence table and BamH I site.
The aminoacid sequence of the acyl group acyl carrier protein reductase enzyme in the beads cyanobacteria (Nostoc sp.) is the sequence 5 in the sequence table; The nucleotides sequence of the gene of described acyl group acyl carrier protein reductase enzyme is classified in the sequence table sequence 4 as from 5 ' terminal the 938th-1958 Nucleotide; The aminoacid sequence of acetaldehyde decarboxylase is the sequence 6 in the sequence table; The nucleotides sequence of the gene of described acyl group acyl carrier protein reductase enzyme is classified in the sequence table sequence 4 as from 5 ' terminal the 1st-696 Nucleotide.
Above-mentioned recombinant vectors also comprises the promotor that is operably connected to described nucleotide sequence.Described promotor is to have the promotor of regulating organoid specificity, tissue specificity, induction type, composing type or cell specific expression.In specific embodiments, described recombinant vectors comprises the sequence with following characteristics: (1) is operably connected to the adjusting sequence of described nucleotide sequence; (2) be operably connected to the selective marker of described nucleotide sequence; (3) be operably connected to the flag sequence of described nucleotide sequence; (4) be operably connected to the purification part of described nucleotide sequence; (5) be operably connected to the destination gene expression sequence of described nucleotide sequence.In certain embodiments, in the genome of described host cell, and the expression of this nucleotide sequence is subjected to regulate the control of promotor to described nucleotide sequence by stable integration.
The application of above-mentioned recombinant bacterium in preparation alkane also is the scope of protection of the invention.
In the above-mentioned application, described alkane is specially tridecane, pentadecane and/or heptadecane;
The 3rd purpose of the present invention provides a kind of fermention medium, and this fermention medium can be used to cultivate Halomonas (Halomonas sp.) LS 21 or above-mentioned recombinant bacterium.
Fermention medium provided by the invention is comprised of C source, N source and seawater;
Described C source is at least a in stalk cellulose vat liquor, oleic acid, animal tallow and the Zulkovsky starch;
Described N source is at least a in soybean protein and the whey-protein;
Described seawater is natural sea-water or artificial seawater;
Described stalk cellulose soak solution is prepared as follows: stalk in the NaOH aqueous solution soaking, is collected liquid and is the stalk cellulose soak solution.Specifically be prepared as follows: be that 1g/L, pH value are that 4 ℃ of 14 the NaOH aqueous solution soak 48h with stalk in concentration, collect liquid and be the stalk cellulose soak solution; Wherein stalk is the stalk after gas explosion is processed; Adopt in an embodiment of the present invention the maize straw after gas explosion is processed, concrete grammar is processed 0.5h for the maize straw with water content 10-35% carries out steam explosion under the condition of vapor pressure 1.0-1.5MPa, and oven dry obtains the stalk after gas explosion is processed.
Above-mentioned fermention medium is comprised of described stalk cellulose vat liquor, described oleic acid, described animal tallow, described Zulkovsky starch, described soybean protein, described whey-protein and described seawater;
The proportioning of described stalk cellulose vat liquor, described oleic acid, described animal tallow, described Zulkovsky starch, described soybean protein and described whey-protein is 150-400mL:5-20mL:3-10g:5-20g:1-4g:2g;
Wherein, described animal tallow is specially poultry fat; Described poultry fat further is specially pork fat;
The pH value of described fermention medium is 5.0-11.0, is specially 5.0 or 9.0 or 11.0;
The final concentration of NaCl is 20g/L to 50g/L in the described natural sea-water.
In the above-mentioned substratum, soybean protein is soybean protein powder, and whey-protein is lactalbumin powder.
In the above-mentioned fermention medium, the prescription of artificial seawater is Mocledon prescription, and is basically identical with the composition of natural sea-water, is widely used in culturing marine products, and it is used for configuring, and the final concentration of NaCl is specially 26.726g/L behind the fermention medium;
In the above-mentioned fermention medium, natural sea-water (NaCl content is about 26g/L) is used for configuring in the fermention medium post-fermentation and culture base contained leading ion content as follows: Na
+3537.73mg/L, Mg
2+543.97mg/L, Ca
2+210.33mg/L; K
+105.84mg/L; Cl
-7559.70mg/L, SO
2-591.75mg/L, Li
+193.0 μ g/L, Al
2+3.788 μ g/L, Br
-37.93 μ g/L; Natural sea-water is collected in harbour offshore seashore, PORT OF TIANJIN on August 2nd, 2012, seawater places sealing preservation in the polytetrafluoroethylplastic plastic bottle.
C source in the fermention medium of the present invention and N source include but are not limited to agricultural, forestry, the food wastes the like waste such as pretreated Mierocrystalline cellulose, hemicellulose, saturated and unsaturated longer chain fatty acid (grease), starch.These wastes have consisted of the main fermentation carbon and nitrogen sources except necessary trace element after the pre-treatment of different methods.
The 4th purpose of the present invention provides a kind of method for preparing polyhydroxyalkanoate/3-hydroxybutyrate and/or lipid acid.
Method provided by the invention for the above-mentioned Halomonas that ferments (Halomonas sp.) LS 21, namely obtains polyhydroxyalkanoate/3-hydroxybutyrate and/or lipid acid.In the aforesaid method, described lipid acid is C16 lipid acid.
In the aforesaid method, after fermentation, also comprise tunning is collected thalline, esterification, obtain polyhydroxyalkanoate/3-hydroxybutyrate and/or lipid acid.
The 5th purpose of the present invention provides a kind of method for preparing alkane.
Method provided by the invention comprises the steps: the above-mentioned recombinant bacterium that ferments, and namely obtains alkane; Described alkane is specially tridecane, pentadecane and/or heptadecane.
In the above-mentioned method for preparing alkane, after fermentation, comprise the steps: to collect tunning with methyl alcohol or hexanaphthene extraction, collect organic phase and obtain alkane.
Prepare the method for polyhydroxyalkanoate/3-hydroxybutyrate and/or lipid acid or prepare in the method for alkane above-mentioned, the substratum that fermentation is adopted is above-mentioned fermention medium.
In the aforesaid method, the temperature of described fermentation is 20 ℃-45 ℃, is specially 20 ℃ or 42 ℃ or 45 ℃; The rotating speed of described fermentation is the 100rpm(rotation radius: 15mm); Described fermentation time is 24 to 96 hours.
In above-mentioned fermenting process, for Halomonas (Halomonas sp.) LS 21, comprise wild strain and gene recombination bacterial strain, high salt concentration and alkaline pH environment are that its growth is necessary, the artificial seawater of using among the present invention provides the salt concentration of average 35g/L, to the used alkali treatment method of the pre-treatment of waste the pH value of substratum is maintained about 11, the high-alkali production growth conditions of high salt like this can suppress the growth of other non-halophilic bacterium, make without the sterilization production technique and become possibility, greatly reduce the required energy consumption of sterilization process, thereby reduced cost.
Of the present invention experimental results show that, the wild Halomonas of one strain (Halomonas sp.) LS 21 of the autonomous screening of the present invention, and take it as the basis, by genetic engineering modified structure recombinant bacterium, this wild mushroom and recombinant bacterium all can be in the fermention mediums with natural sea-water or artificial seawater configuration, production take chief component as alkane, the hydrocarbon polymer of alkene and longer chain fatty acid, this hydrocarbon can be used as biofuel and uses after separating; And fermenting process can be realized unsterilised continuously fermenting.Halomonas among the present invention and recombinant bacterial strain nutritional requirement thereof are simple, fermenting process need not sterilization and can carry out continuously, and be simple and easy to control, low-cost, have a good prospects for commercial application.
Definition
In this manual, used abbreviation gene title or polypeptide title to quote, this abbreviation gene or polypeptide title represent the kind of gene or polypeptide.These genes comprise coding phase homopolypeptide and the portion gene with identical physiological function homeopeptide.Polypeptide comprises all polypeptide with identical activity (isozyme).
The ncbi database that the accession number that this paper quotes is safeguarded from NIH (National Institute of Health, U.S.A) (NCBI (National Center for BiotechnologyInformation)).Unless otherwise mentioned, accession number is according to providing in the database in May, 2012.
Hydrocarbon polymer as herein described includes but are not limited to alkane, alkene, lipid acid, ester, aldehyde and Fatty Alcohol(C12-C14 and C12-C18) etc.Wherein alkane represents only to contain the hydrocarbon polymer of single C-C, and its carbonatoms is at least about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 carbon.
Above-mentioned bacterial strains LS21 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 20th, 2012 and (is called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.6593, and Classification And Nomenclature is Halomonas (Halomonas sp.).
Description of drawings
Fig. 1 is the growing state (frame in) of wild-type LS21 in many carbon sources substratum of seawater preparation
Fig. 2 is LS21 at stalk cellulose is the GC analysis chart (in the frame) of accumulation PHA in the substratum of sole carbon source
Fig. 3 is LS21 in the substratum of artificial seawater preparation in the accumulation PHA(solid box) and the GC analysis chart (in the dotted line frame) of the lipid acid of C16
Fig. 4 contains the GC collection of illustrative plates that the restructuring LS21 that expresses the acyl group acyl carrier protein reductase enzyme derive from collection born of the same parents cyanobacteria and acetaldehyde decarboxylase produces alkane
Fig. 5 contains the GC collection of illustrative plates that the restructuring LS21 that expresses the acyl group acyl carrier protein reductase enzyme derive from the beads cyanobacteria and acetaldehyde decarboxylase produces alkane
Fig. 6 is C11, C13, C15, C17 alkane standard substance GC collection of illustrative plates
Fig. 7 produces the GC collection of illustrative plates of the compound comparison of alkane and alkane standard substance for restructuring LS21B
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Isolation identification and the growth characteristics of embodiment 1, Halomonas (Halomonas sp.) LS 21
One, bacterial strain LS 21 separates
From Da Bancheng salt lake, Xinjiang, obtain soil sample and water sample, the isolated strains therefrom take Mierocrystalline cellulose as sole carbon source.Obtain 5 strains and can produce the bacterial classification that utilizes Mierocrystalline cellulose to be the sole carbon source growth, select wherein a strain well-grown and accumulabile PHA bacterial strain called after LS 21.
The making of Mierocrystalline cellulose substratum: with stalk cellulose soak solution filtered through gauze, the residual stalk of elimination, add in proportion NaCl(40g/L) and component I and micro-mixing solutions, with NaOH solution adjust pH to 10, behind Precipitation, centrifugal 5min under 5000rpm keeps supernatant liquor as the stalk cellulose substratum.
Stalk cellulose soak solution: take by weighing stalk (water content 10～35% maize straws are carried out steam explosion the process 0.5h oven dry acquisition) 6g after gas explosion is processed under the condition of vapor pressure 1.0～1.5MPa, add the NaOH aqueous solution (1g/L that 500mL configures with deionized water, pH=14), place 4 ℃ of lower 48h of immersion, collect liquid and obtain the stalk cellulose soak solution.
Component I: 1.5g/L potassium primary phosphate, 9.65g/L disodium hydrogen phosphate dodecahydrate, be mixed with 50 times of mother liquors, sterilized 20 minutes for 121 ℃;
The trace element mixing solutions: micro-I 10mL/L, micro-II 1mL/L, be mixed with 50 times of mother liquors, mother liquor pH value is adjusted to 4.0-5.0, sterilizes 20 minutes for 121 ℃;
Trace element I and the preparation of micro-II are with lower;
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 5g, CALCIUM CHLORIDE DIHYDRATE 2g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of micro-II is prepared as follows: with Zinc vitriol 100mg, four hydration Manganous chloride tetrahydrate 30mg, boric acid 300mg, cobalt chloride hexahydrate 200mg, Salzburg vitriol 10mg, Nickel dichloride hexahydrate 20mg, two molybdic acid hydrate sodium 30mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II.
Substrate, components I, compositionⅱ, trace element be separately sterilization respectively, after the cooling, gets respectively 20mL components I mother liquor, 20mL compositionⅱ mother liquor and 20mL trace element mother liquor and adds substrate.
In actual culturing process, can in above-mentioned substratum, add again certain density microbiotic to keep the stability of plasmid, such as 100 μ g/mL penbritins, the paraxin of 50 μ g/mL sulphuric acid kanamycins and 34 μ g/mL etc.
Isolation medium: sodium-chlor 60g/L, peptone 10g/L, yeast powder 1g/L, all the other components are water, the pH value is about 8.0.
Two, identification of strains
1, identification of morphology
Get 15% glycerine pipe freezing LS21 bacterium liquid, bacterial concentration is diluted to 10
4Cfu/mL gets 100 μ L and coats LB flat board (the same isolation medium of component), and 37 ℃ of incubators were cultivated 48 hours, observe colonial morphology: bacterium colony is rounded, diameter 1-2mm, the smooth of the edge, moistening, canescence is translucent, and microscopically is observed thalline and is rod-short.
2, Physiology and biochemistry is identified
The LS21 bacterial strain is carried out gramstaining identify, the result is Gram-negative bacteria.
3, Molecular Identification
Detect the 16S rDNA sequence of LS21 bacterial strain, the primer of amplification 16S rDNA sequence is universal primer: 16F5 '-AGAGTTTGATCCTGGCTCAG-3 ', 16R 5 '-ACGGCTACCTTGTTACGACT-3 '.The sequence that records is shown in the sequence 7 in the sequence table.This sequence is compared in NCBI, and this bacterial strain and Halomonas sp. similarity reach 99% as a result.
Comprehensive above qualification result is Halomonas (Halomonas sp.) with the LS21 identification of strains.
Above-mentioned bacterial strains LS21 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 20th, 2012 and (is called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.6593, and Classification And Nomenclature is Halomonas (Halomonas sp.).
Embodiment 2, Halomonas (Halomonas sp.) LS 21CGMCC No.6593 utilize seawater to be the application in the medium fermentation
1, seawater preparation fermention medium
Utilize seawater and changing food waste namely to save again energy-conserving and environment-protective of cost, therefore can adopt it as culture medium raw material, based on above-mentioned purpose, development research utilizes seawater to be the fermention medium of medium: can adopt natural sea-water, also can adopt identical with its component or very approximate artificial seawater prescription (Mocledon prescription) configuration simulation; Moisture accounts for 79% in the meal kitchen industry organic waste, crude fat is about 29% in the dry material, crude protein accounts for 20%, starch accounts for 31%, can adopt mixed C source, N source and the simulation of corresponding proportioning.
Natural sea-water (NaCl content is about 26g/L) is used for configuring in the fermention medium post-fermentation and culture base contained leading ion content as follows: Na
+3537.73mg/L, Mg
2+543.97mg/L, Ca
2+210.33mg/L; K
+105.84mg/L; Cl
-7559.70mg/L, SO
2-591.75mg/L, Li
+193.0 μ g/L, Al
2+3.788 μ g/L, Br
-37.93 μ g/L; Natural sea-water is collected in harbour offshore seashore, PORT OF TIANJIN on August 2nd, 2012, seawater places sealing preservation in the polytetrafluoroethylplastic plastic bottle.Compare with following artificial seawater, principal element content is without significant difference.
The prescription of artificial seawater is the Mocledon prescription, adds following component (being the final concentration in fermention medium A) in every liter of deionized water: sodium-chlor (NaCl) 26.726g/L, magnesium chloride (MgCl
2) 2.26g/L, sal epsom (MgSO
4) 3.248g/L, calcium chloride (CaCl
2) 1.153g/L, sodium bicarbonate (NaHCO
3) 0.198g/L, Repone K (KCl) 0.721g/L, Sodium Bromide (NaBr) 0.058g/L, boric acid (H
3BO
3) 0.058g/L, water glass (Na
2SiO
3) 0.0024g/L, water glass (Na
2Si
4O
9) 0.0015g/L, phosphoric acid (H
3PO
4) 0.002g/L, chlordeneization two aluminium (Al
2Cl
6) 0.013g/L, ammonia (NH
3) 0.002g/L, lithium nitrate (LiNO
3) 0.0013g/L.
Every 1L fermention medium A(utilizes artificial seawater to be medium) be prepared as follows: stalk cellulose vat liquor, 10mL oleic acid, 5g pork fat (commercially available pork that 200mL embodiment 1 is obtained, get fatty tissue, should fat homogenate become pasty state), 10g Zulkovsky starch, 2g soybean protein powder and 2g lactalbumin powder mix, with artificial seawater polishing volume, use the pH value to 11 of the NaOH solution regulation system of 5M.
Every 1L fermention medium B(utilizes natural sea-water to be medium) be prepared as follows: only the artificial seawater among the fermention medium A is replaced with natural sea-water.
Every 1L contains micro-fermention medium and is prepared as follows: stalk cellulose vat liquor, 10mL oleic acid, 5g pork fat, 10g Zulkovsky starch, 2g soybean protein powder, 2g lactalbumin powder and 20mL that 200mL embodiment 1 is obtained are mixed by the micro-mixing solutions that embodiment 1 prepares, with artificial seawater polishing volume, the pH value is 11.
2, fermentation
1) dry cell weight detects
3 groups of fermentations:
A organizes (LS 21seawater): the triangular flask of each 500mL 60mL fermention medium A that packs into, access Halomonas (Halomonas sp.) LS 21CGMCC No.6593,42 ℃, 100rpm(rotation radius 15mm) bottom fermentation was cultivated 3 days, obtained fermented liquid.
D group: the triangular flask of each 500mL 60mL fermention medium B that packs into, access Halomonas (Halomonas sp.) LS 21CGMCC No.6593,42 ℃, 100rpm(rotation radius 15mm) bottom fermentation was cultivated 3 days, obtained fermented liquid.
The B group (adds trace element, LS 21seawater+MM): the triangular flask of each 500mL 60mL that packs into contains micro-fermention medium, access Halomonas (Halomonas sp.) LS 21CGMCC No.6593,42 ℃, the 100rpm(rotation radius is 15mm) bottom fermentation cultivated 3 days, obtained fermented liquid.
Get and respectively organize 10000 rev/mins of centrifugal 10 minutes collection thalline of 25mL fermented liquid, use the deionized water washed twice, freezing ice is done.Thalline was weighed and is calculated dry cell weight (CDW) after ice was done.Each processing establish 3 parallel.
Partial results as shown in Figure 1, A group (LS 21seawater) is grown in the fermention medium A that does not add trace element, CDW can reach 4.06g/L; B group (adding trace element, LS 21seawater+MM) is grown in the fermention medium that adds trace element, and CDW can reach 4.65g/L.
And the D group is grown in fermention medium B, and CDW result and A group are without significant difference.
Can find out from the above results, trace element can not grown by remarkably influenced LS21, still can finely grow at the fermention medium A and the fermention medium B that do not add trace element, and in addition, natural sea-water and artificial seawater can remarkably influenced LS21 growths.
Therefore, be in cost consideration, adopt fermention medium A or fermention medium B to carry out following research.
2) LS 21 can produce the PHA(polyhydroxyalkanoate in the substratum of sole carbon source)
3-hydroxybutyrate (3-hydroxybutyric acid, 3HB) is the monomer among the PHA, can be used for detecting whether produce PHA.
Every 1L control medium (stalk cellulose is the substratum of sole carbon source): will add in proportion NaCl(40g/L by the stalk cellulose soak solution that embodiment 1 obtains) and by the component I among the embodiment 1 reach by the micro-mixing solutions among the embodiment 1, NaOH solution with 5M transfers to 10 with the pH value, behind Precipitation, centrifugal 5min under 5000rpm keeps supernatant liquor as the stalk cellulose substratum.
C organizes (stalk cellulose is sole carbon source): the triangular flask of each 500mL 60mL control medium of packing into, and access Halomonas (Halomonas sp.) LS 21CGMCC No.6593,42 ℃, the 100rpm bottom fermentation was cultivated 3 days.Get 10000 rev/mins of centrifugal 10 minutes collection thalline of 25mL fermented liquid, use the deionized water washed twice, freezing ice is done, and C group CDW is 3.17g.
Thalline carries out esterification after getting 30mg ice that C group obtains and doing, and gas-chromatography (GC) detects PHA content, each processing establish 3 parallel.Esterification process is: gets 30mg ice dry mycelium in the esterification pipe, adds 2mL chloroform, 2mL esterifying liquid mixing, and covered and enclosed, esterification is 4 hours in 100 ℃ of baking ovens.After being cooled to room temperature, add 1mL distilled water, the mixing that fully vibrates, standing demix., get chloroform and carry out mutually gas chromatographic analysis with after water separates fully until chloroform.1L esterifying liquid collocation method: 1g phenylformic acid, the 30mL vitriol oil are dissolved in 970mL methyl alcohol, obtain esterifying liquid.The standard specimen material of getting simultaneously 10mg carries out esterification.Standard substance are 3-hydroxybutyrate (3-hydroxybutyric acid, 3HB), in be designated as phenylformic acid Benzoic Acid(available from chemical reagent Beijing company limited of traditional Chinese medicines group; Catalog number: 30018760).
Gas-chromatography (GC) analysis: according to Shimadzu company GC-2014(SHIMADZU, Japan) the specification sheets operation gas chromatograph of gas chromatograph.Setting the column cap temperature is 80 ℃, and the sampler temperature is 240 ℃, and detector temperature is 250 ℃, column head pressure is 0.25MPa, the temperature programming condition is: 80 ℃ 1.5 minutes, be warming up to 140 ℃ with 35 ℃/minutes speed, be warming up to 280 ℃ and kept 2 minutes in this temperature by 40 ℃/minutes speed again.Use the automatic sampler sample introduction, the sample feeding amount is 1 μ l.
The result is shown in Figure 2, under as above GC conditions, and the retention time 2.5 ± 0.1min of standard substance 3-hydroxybutyrate (3-hydroxybutyric acid, 3HB), the benzoic retention time of interior mark is 3.7 ± 0.1min.The retention time 2.4min of 3HB in the sample, the benzoic retention time of interior mark is 3.7min; Can find out, fermentation culture LS21 has produced 3HB, and the content that the C group produces 3HB is 27.19 ± 6.07% of dry cell weight, thereby proves that it can accumulate PHA.
Can find out, LS 21 can produce 3HB and PHA in stalk cellulose is the substratum of sole carbon source.
3) fermentation produces PHA and C16 lipid acid in the fermention medium of mixed carbon source
Thalline carried out esterification after the 30mg ice of getting respectively above-mentioned A group (fermention medium A), B group (containing micro-fermention medium) and D group (fermention medium B) was done, gas-chromatography (GC) detection 3HB content, each processing establish 3 parallel.Gas-chromatography (GC) analysis: according to Shimadzu company GC-2014(SHIMADZU, Japan) the specification sheets operation gas chromatograph of gas chromatograph.Setting the column cap temperature is 80 ℃, and the sampler temperature is 240 ℃, and detector temperature is 280 ℃, column head pressure is 0.25MPa, the temperature programming condition is: 80 ℃ 2.5 minutes, be warming up to 140 ℃ with 35 ℃/minutes speed, be warming up to 280 ℃ and kept 2 minutes in this temperature by 30 ℃/minutes speed again.Use the automatic sampler sample introduction, the sample feeding amount is 1 μ l.
Standard substance are that standard substance are 3-hydroxybutyrate (3-hydroxybutyric acid, 3HB), in be designated as phenylformic acid Benzoic Acid(available from chemical reagent Beijing company limited of traditional Chinese medicines group; Catalog number: 30018760); Palmitinic acid (lipid acid that is called again C16) is available from Tokyo chemical reagents corporation (TCI TOKYO CHEMICAL INDUSTRY Co.LTD).
Partial results as shown in Figure 3, to be LS21 do not add the artificial seawater substratum of trace element at fermention medium A(to A) in can accumulate the lipid acid (A group) of 3HB and C16, B is LS21 can accumulate 3HB and C16 in containing micro-fermention medium lipid acid (B group); Under as above GC conditions, the retention time 2.299min of 3-hydroxybutyrate in the standard substance (3-hydroxybutyric acid, 3HB), the benzoic retention time of interior mark is 3.915min; The retention time 2.300min of 3HB in the sample, the benzoic retention time of interior mark is 3.908min; Can find out, fermentation LS21 can accumulate a small amount of 3HB in fermention medium A, and can accumulate the lipid acid (chromatographic peak in the dotted line frame) of the C16 that accounts for dry cell weight 27.37%.
The content that the A group produces 3HB is 9.17 ± 1.01% of dry cell weight; The content that the B group produces 3HB is 4.34 ± 0.42% of dry cell weight.
The result who detects the D group is that LS21 is at fermention medium B(natural sea-water preparation substratum) in also can accumulate the lipid acid of 3HB, PHA and C16, the content that produces 3HB is organized without significant difference with A.
Embodiment 2, utilize LS21 preparation to produce the recombinant bacterium of alkane
One, utilize LS21 and the preparation of collection born of the same parents cyanobacteria genes involved to produce the recombinant bacterium A of alkane
Be documented in Accumulation of poly-β-hydroxybutyrate in cyanobacterium Synechocystissp.PCC6803 by importing collection born of the same parents cyanobacterias (Synechocystis sp.) PCC6803(, Wu Qingyu etc., Bioresource Technology, 76 volumes, the 2nd phase, 2001, P85-90, the public can obtain from Tsing-Hua University).The acyl group acyl carrier protein reductase enzyme (acyl-acyl carrier protein reductase) of bacterial strain coding and the gene of acetaldehyde decarboxylase (aldehyde decarbonylase) allos in LS21 produce alkane.The acyl group acyl carrier protein reductase enzyme (sll0208 of collection born of the same parents cyanobacteria PCC6803; NP442147), the nucleotides sequence of its encoding gene is classified in the sequence table sequence 1 as from 5 ' terminal the 1st-1023 Nucleotide, and its aminoacid sequence is the sequence 2 in the sequence table; Acetaldehyde decarboxylase (the sllO209 of collection born of the same parents cyanobacteria PCC6803; NC_000911.1), the nucleotides sequence of its encoding gene is classified in the sequence table sequence 1 as from 5 ' terminal the 1242nd-1937 Nucleotide, and its aminoacid sequence is the sequence 3 in the sequence table.
1, makes up recombinant bacterium LS21A
Integrate the total DNA of born of the same parents cyanobacteria PCC6803 as template with what extract; use respectively primer 6803-F:gtggTCTAGAcctcccccccagcaacttagactag(restriction enzyme site to be XbaI enzyme cutting site (capitalization part)) be BamH I restriction enzyme site (capitalization part) with primer 6803-R:ccggGGATCCccagcagcattgagctttgataatt(restriction enzyme site) amplification; obtain the approximately PCR product of 2kb; through order-checking; this PCR product is the fragment that contains acyl group acyl carrier protein reductase enzyme and acetaldehyde decarboxylase; has the Nucleotide shown in the sequence 1 in the sequence table; wherein sequence 1 is acyl group acyl carrier protein reductase enzyme encoding gene from 5 ' terminal the 1st-1023 Nucleotide in the sequence table, and sequence 1 is acetaldehyde decarboxylase encoding gene from 5 ' terminal the 1242nd-1937 Nucleotide.
After pcr amplification finishes, use the method for solution recovery purifying, purified pcr product.
With purified pcr product after XbaI and Bam I enzyme are cut, be documented in Kovach with the carrier pBBR1MCS1(that cuts through same enzyme, M.E.Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carring different antibiotic-resistant cecassettes.Gene166 (1995) 175 – 176, the public can obtain from Tsing-Hua University) connect, obtain recombinant plasmid called after pBBR1MCS1-A, through order-checking, this recombinant plasmid is the carrier that obtains between the XbaI of the 1 insertion pBBR1MCS1 of sequence in the sequence table and BamH I restriction enzyme site.
Recombinant plasmid pBBR1MCS1-A is transformed into intestinal bacteria S17-1(is documented in Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.M.Bierman et al., GENE, 116 volumes, the 1st phase, 1992, P43-49, the public can obtain from Tsing-Hua University) the competent cell, obtain positive colony in the LB flat board screening that contains 25 μ g/mL paraxin.Extract the plasmid of positive colony, be pBBR1MCS1-A, will contain the positive colony called after S17-1/pBBR1MCS1-A of this plasmid.
Picking S17-1/pBBR1MCS1-A bacterium colony, in liquid Lb substratum, be cultured to logarithmic phase, S17-1/pBBR1MCS1-A and wild-type Halomonas (Halomonas sp.) LS 21(embodiment 1 are obtained) cultivate altogether, engage conversion, obtain recombinant bacterium LS21A.
Specific as follows: get respectively donor bacterium S17-1/pBBR1MCS1-A and recipient bacterium wild-type Halomonas (Halomonas sp.) LS 21 each 1mL of bacterium liquid, 6000rpm, 1.5min is centrifugal, removes supernatant; Use respectively more common LB and salt adding LB(NaCl 35g/L, pH10) clean respectively donor bacterium and recipient bacterium once, resuspension; Get 400ul donor bacterium and recipient bacterium and mix, 6000rpm, 1.5min is centrifugal, removes supernatant; With 100ulLB liquid with the mixture resuspension, with gained bacterium drop at the uncomfortable pH of LB(NaCl 20g/L added with antibiotic not) on the flat board, forward leaves standstill and cultivates 6h in 37 ℃ of constant incubators after bacterium liquid dries up; With 1mL liquid LB(NaCl 35g/L, pH10) the bacterium liquid on the flat board is washed, mixing, 6000rpm, 1.5min is centrifugal, gets supernatant; With 500ul LB(NaCl 35g/L, pH10) with the thalline resuspension, dilute 10 times, 100 times after, get respectively stoste, 10
-1Times liquid and 10
-2Times each 200ul of liquid evenly coats LB(NaCl 35g/L, pH10, ammonification benzyl microbiotic) on the flat board, cultivate 24h for 37 ℃.After joint transforms and finishes, choose the transformant that obtains the mould resistance of chlorine and identify correct (primer is 6803-F and 6803-R, and the product that obtains 2Kb is correct transformant) through PCR, called after recombinant bacterium LS21A.
2, fermentation recombinant bacterium LS21A produces alkane
The fermention medium A(that restructuring LS21A is inoculated into embodiment 2 preparations utilizes artificial seawater to be medium) in, 42 ℃, 100rpm(rotation radius 15mm) cultivated 3 days.Get the 15mL culture, centrifugal 10 minutes, 10000rpm extract supernatant liquor and thalline respectively with methyl alcohol, wherein somatic cells group is resuspended in methyl alcohol, and supersound process behind the vortex is extracting behind the broken thalline, after centrifugal 1 minute, collect supernatant liquor with 10000rpm.
Supernatant liquor is transferred to new GC sample bottle and analyze by GC.Chromatographic column is HP-5 capillary column (column length 30m, internal diameter 0.25mm).(temperature in remains on 300 ℃ behind the GC/MS post to 1 μ l, and column temperature remains on 100 ℃, continues 3 minutes without split stream sampling.With 20 ℃/minute speed temperature is risen to 320 ℃.Column temperature remains on 320 ℃ of column temperatures 5 minutes.The flow velocity of carrier gases helium is 1.5mL/ minute.
Standard substance be undecane (CAS:1120-21-4), tridecane (CAS:629-50-5), pentadecane (CAS:629-62-9), heptadecane (CAS:629-78-7) available from Tokyo chemical reagents corporation (TCI TOKYO CHEMICAL INDUSTRY Co.LTD), in be designated as phenylformic acid Benzoic Acid(available from chemical reagent Beijing company limited of traditional Chinese medicines group; Catalog number: 30018760);
Result such as Fig. 4 and shown in Figure 6, Fig. 4 contain the GC collection of illustrative plates that the restructuring LS21 that expresses the acyl group acyl carrier protein reductase enzyme derive from collection born of the same parents cyanobacteria and acetaldehyde decarboxylase produces alkane; Fig. 6 is C11, C13, C15, C17 alkane standard substance GC collection of illustrative plates;
Under as above GC conditions,
Undecane, tridecane, pentadecane, margaric retention time are respectively 4.660min, 6.895min, 9.065min, 11.456min and 13.188min in the standard substance, tridecane, pentadecane and margaric retention time are respectively 6.891min, 9.053min and 11.442min in the sample, can find out, the retention time at product peak is with consistent with standard substance, illustrate that restructuring LS21A has produced tridecane, pentadecane and heptadecane, and generation is the tridecane of 1.48 μ l/mL, the pentadecane of 0.87 μ l/mL and the heptadecane of 0.66 μ l/mL.
Adopting the fermention medium B(that restructuring LS21A is inoculated into embodiment 2 preparation that uses the same method utilizes natural sea-water to be medium) in, the result has also produced tridecane, pentadecane and heptadecane, and the amount that produces and fermentation results in fermention medium A are without significant difference.
Two, utilize the preparation of LS21 and beads cyanobacteria genes involved to produce the recombinant bacterium B of alkane
Be documented in Characterization of phytochelatin synthase-like protein encoded by alr0975from a prokaryote by importing beads cyanobacteria (Nostoc sp.) PCC 7120(, Nostocsp.PCC7120, Naoki Tsuji et al., Biochemical and Biophysical Research Communications, 315 volumes, the 3rd phase, 2004, P751-755, the public can obtain from Tsing-Hua University).The gene of bacterial strain coding acyl group acyl carrier protein reductase enzyme (acyl – acyl carrier protein reductase) and acetaldehyde decarboxylase (aldehyde decarbonylase) allos in LS21 produces alkane.The acyl group acyl carrier protein reductase enzyme (alr5283 of beads cyanobacteria PCC7210; NP 489323.1, and the nucleotides sequence of its encoding gene is classified sequence 4 in the sequence table as from 5 ' terminal 938-1958 position Nucleotide, and its aminoacid sequence is the sequence 5 in the sequence table; Beads cyanobacteria PCC7210 acetaldehyde decarboxylase (alr5284; NP-489324), the nucleotides sequence of its encoding gene is classified sequence 4 in the sequence table as from 5 ' terminal 1-696 position Nucleotide, and its aminoacid sequence is the sequence 6 in the sequence table.
1, makes up recombinant bacterium LS21B
Take the total DNA of beads cyanobacteria PCC7210 that extracts as template; use respectively primer 7120-F (cgTCTAgagttagatcgccaagcacttgttt; restriction enzyme site is XbaI) and primer 7120-R (ccccGGATCCagttagggattaggtattgggtatt; restriction enzyme site is the BamH I) amplification; obtain the approximately PCR product of 2kb; through order-checking; this PCR product is the fragment that contains acyl group acyl carrier protein reductase enzyme and acetaldehyde decarboxylase; has the Nucleotide shown in the sequence 4 in the sequence table; wherein sequence 4 is acyl group acyl carrier protein reductase enzyme encoding gene from 5 ' terminal the 938th-1958 Nucleotide in the sequence table, and sequence 4 is acetaldehyde decarboxylase encoding gene from 5 ' terminal the 1st-696 Nucleotide.
After pcr amplification finishes, use the method for solution recovery purifying, purified pcr product.
With purified pcr product after XbaI and BamH I enzyme are cut, be connected with the carrier pBBR1MCS1 that cuts through same enzyme, obtain recombinant plasmid called after pBBR1MCS1-B, through order-checking, this recombinant plasmid is the carrier that obtains between the XbaI of the 4 insertion pBBR1MCS1 of sequence in the sequence table and BamH I restriction enzyme site.
Recombinant plasmid pBBR1MCS1-B is transformed in the intestinal bacteria S17-1 competent cell, obtains positive colony in the LB flat board screening that contains 25 μ g/mL paraxin.Extract the plasmid of positive colony, be pBBR1MCS1-B, will contain the positive colony called after S17-1/pBBR1MCS1-B of this plasmid.
Picking S17-1/pBBR1MCS1-B bacterium colony, in liquid Lb substratum, be cultured to logarithmic phase, S17-1/pBBR1MCS1-B and wild-type Halomonas (Halomonas sp.) LS 21(embodiment 1 obtained) cultivate altogether, engage conversion, obtain recombinant bacterium LS21B, method is the same.After joint transforms and finishes, choosing the transformant that obtains the mould resistance of chlorine and identify correct (primer is 7120-F and 7120-R, and the product that obtains 2Kb is correct transformant) through PCR, is the LS21B that recombinates.
2, fermentation recombinant bacterium LS21B produces alkane
The fermention medium A(that restructuring LS21B is inoculated into embodiment 2 preparations utilizes artificial seawater to be medium) in, 42 ℃, 100rpm(rotation radius 15mm) cultivated 3 days.Get the 15mL culture, centrifugal 10 minutes, 10000rpm extract upper cleer and peaceful thalline respectively with methyl alcohol, and wherein somatic cells group is resuspended in methyl alcohol, and supersound process behind the vortex is extracting behind the broken thalline, with 10000rpm after centrifugal 1 minute.
Supernatant liquor is transferred to new GC sample bottle and analyze by GC.Chromatographic column is HP-5 capillary column (column length 30m, internal diameter 0.25mm).(temperature in remains on 300 ℃ behind the GC/MS post to 1 μ l, and column temperature remains on 100 ℃, continues 3 minutes without split stream sampling.With 20 ℃/minute speed temperature is risen to 300 ℃.Column temperature remains on 300 ℃ of column temperatures 5 minutes.The flow velocity of carrier gases helium is 1.5mL/ minute.The retention time at product peak and compare to confirm consistence with standard substance.
Standard substance be undecane (CAS:1120-21-4), tridecane (CAS:629-50-5), pentadecane (CAS:629-62-9), heptadecane (CAS:629-78-7) available from Tokyo chemical reagents corporation (TCI TOKYO CHEMICAL INDUSTRY Co.LTD), in be designated as phenylformic acid Benzoic Acid(available from chemical reagent Beijing company limited of traditional Chinese medicines group; Catalog number: 30018760);
Result such as Fig. 5-shown in Figure 7, Fig. 5 contain the GC collection of illustrative plates that the restructuring LS21B that expresses the acyl group acyl carrier protein reductase enzyme that derives from the beads cyanobacteria and acetaldehyde decarboxylase produces alkane; Fig. 6 is C11, C13, C15, C17 alkane standard substance GC collection of illustrative plates; Fig. 7 produces the GC collection of illustrative plates of the compound comparison of alkane and alkane standard substance for restructuring LS21B;
Under as above GC conditions, undecane, tridecane, pentadecane, margaric retention time are respectively 4.660min, 6.895min, 9.065min, 11.456min and 13.188min in the standard substance; Tridecane, pentadecane and margaric retention time are respectively 6.884min, 9.042min and 11.436min in the sample; Can find out, the retention time at product peak illustrate that restructuring LS21B has produced tridecane, pentadecane and heptadecane, and generation is the tridecane of 1.72 μ l/mL, the pentadecane of 0.92 μ l/mL and the heptadecane of 0.73 μ l/mL with consistent with standard substance.
Adopting the fermention medium B(that restructuring LS21A is inoculated into embodiment 2 preparation that uses the same method utilizes natural sea-water to be medium) in, the result has also produced tridecane, pentadecane and heptadecane, and the amount that produces and fermentation results in fermention medium A are without significant difference.
Claims (10)
1. Halomonas (Halomonas sp.) LS 21, its deposit number is CGMCC No.6593.
2. the application of Halomonas claimed in claim 1 (Halomonas sp.) LS 21 in preparation polyhydroxyalkanoate/3-hydroxybutyrate and/or lipid acid; Described lipid acid is specially C16 lipid acid.
3. a recombinant bacterium for acyl group acyl carrier protein reductase enzyme encoding gene and acetaldehyde decarboxylase encoding gene are all imported in the Host Strains, obtains recombinant bacterium.
4. recombinant bacterium according to claim 3, it is characterized in that: described Host Strains is Halomonas (Halomonas sp.); Described Halomonas (Halomonas sp.) is specially Halomonas claimed in claim 1 (Halomonas sp.) LS 21;
Described acyl group acyl carrier protein reductase enzyme and described acetaldehyde decarboxylase are specifically all from cyanobacteria;
Described cyanobacteria further is specially collection born of the same parents cyanobacterias (Synechocystis sp.) or beads cyanobacteria (Nostoc sp.).
5. claim 3 or the 4 described recombinant bacteriums application in preparation alkane; Described alkane is specially tridecane, pentadecane and/or heptadecane.
6. a fermention medium is comprised of C source, N source and seawater;
Described C source is at least a in stalk cellulose vat liquor, oleic acid, animal tallow and the Zulkovsky starch;
Described N source is at least a in soybean protein and the whey-protein;
Described seawater is natural sea-water or artificial seawater;
Described stalk cellulose soak solution is prepared as follows: stalk in the NaOH aqueous solution soaking, is collected liquid and is the stalk cellulose soak solution.
7. fermention medium according to claim 6 is characterized in that:
Described fermention medium is comprised of described stalk cellulose vat liquor, described oleic acid, described animal tallow, described Zulkovsky starch, described soybean protein, described whey-protein and described seawater;
The proportioning of described stalk cellulose vat liquor, described oleic acid, described animal tallow, described Zulkovsky starch, described soybean protein and described whey-protein is 150-400ml:5-20ml:3-10g:5-20g:1-4g:2g;
Described animal tallow is specially poultry fat; Described poultry fat further is specially pork fat;
The pH value of described fermention medium is 5.0-11.0, is specially 5.0 or 9.0 or 11.0;
The final concentration of NaCl is 20g/L to 50g/L in the described natural sea-water.
8. a method for preparing polyhydroxyalkanoate/3-hydroxybutyrate and/or lipid acid for the Halomonas claimed in claim 1 that ferments (Halomonas sp.) LS 21, namely obtains polyhydroxyalkanoate/3-hydroxybutyrate and/or lipid acid; Described lipid acid is specially C16 lipid acid.
9. method for preparing alkane, comprise the steps: to ferment claim 3 or 4 described recombinant bacteriums namely obtain alkane;
Described alkane is specially tridecane, pentadecane and/or heptadecane.
10. it is characterized in that according to claim 8 or 9 described methods:
The substratum that described fermentation is adopted is claim 6 or 7 described fermention mediums;
The temperature of described fermentation is 20 ℃-45 ℃, and the temperature of described fermentation is specially 20 ℃ or 42 ℃ or 45 ℃;
The rotating speed of described fermentation is 100rpm(rotation radius 15mm);
Described fermentation time is 24 to 96 hours.
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