CN1107686C - Process for preparing polyhydroxyl alkanoic acid - Google Patents
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Abstract
The present invention discloses a production method for polyhydroxyl alkanoic acid, which is characterized in that round brown azotobacter chroococcum G-3 strains and large bacillus megatherium G-6 strains are mixed to culture and to produce the polyhydroxyl alkanoic acid. The present invention eliminates capsular polysaccharide substances generated in the process of azotobacter cultivation and enhances the biomass of the strains, and thereby, the ratio yield of PHA is increased; the production cost is reduced; the biomass reaches 53 g/l as much as possible; the PHA amount reaches 42.4 g/l; the cost of the PHA is reduced to 4 dollars per kilo and is a quarter of the lowest cost reported in the world at present, namely 16 dollars per kilo.
Description
The present invention relates to a kind of production method of poly (hydroxyalkanoate).
20th century, the synthetic plastics product was more and more close with daily life along with the development of petrochemical industry, made synthetic plastics industry become a big leading industry of human social economy at present, had brought certain prosperity to human society.Yet the waste of chemosynthesis plastics but becomes a kind of " white garbage " that can't handle on the earth, brings serious pollution for human limited living environment.Therefore, seek the biodegradable plastics of a kind of ideal and replace the petroleum chemistry synthetic plastics, just seem very important.With microorganisms producing degradability plastics----poly (hydroxyalkanoate) (being called for short PHA), be an analog thermoplastic material of instead of chemical synthetic plastics.
The reserve substance of the many microorganisms of PHA synthetic cell internal carbon source and energy under non-equilibrium growth conditions, its general molecular formula can be expressed as
M=1 wherein, 2 or 3, in most of the cases m=1 is the beta-hydroxy alkanoic acid.N is monomeric number, R then represents side chain, mostly be the positive alkanoic acid of different chain length, also can be side chain, undersaturated or be with substituent alkanoic acid, that R among the PHA of most microorganisms is that promptly poly-β-polyhydroxybutyrate (poly-β-hydroxybutyrate the is called for short PHB) PHB of methyl distributes is the widest, discovery the earliest, be the main member among the PHA, concentrate on PHB about the molecular structure of PHA and the big quantity research of PHA particle situation aspect.In PHB, n=600-2500, molecular weight are 6000-250000.Fusing point is 180 ℃, and the PHB molecule shows that through x-ray analysis PHB is made up of a plurality of D type 3HB unit, is right hand α-Luo Xuanjiegou, and pitch is 0.596nm, and every circle is made up of two beta-hydroxy-butanoic acids.Because the difference of R base, found at present more than the 90 kind of PHA that different monomers is formed, number according to carbon atom in the monomer can be divided into PHA short chain PHA, (short-chain-length PHA, scl PHA), monomer is that the hydroxy fatty acid that contains 3-5 carbon atom is PHB and P (HB-CO-HV) (multipolymer of poly-beta-hydroxy-butanoic acid and beta-hydroxy valeric acid); Another kind of is medium chain PHA (medium-chain-lengthPHA, mcl PHA), and monomer is the hydroxy fatty acid that contains 6-14 carbon atom.
PHA except that have with the similar physico-chemical property of chemosynthesis plastics, also have degradation property fully, its goods are the same with chemical synthetic plastics, have broad application prospects in industrial and agricultural production.But the waste of different is PHA goods not only can be degraded fully, eliminates " white pollution ", and degradation product also can be used as agricultural fertilizer, and agricultural development is of great benefit to.Simultaneously, PHA is except that having fully degradation property, and good characters such as its good biocompatibility, optical activity, piezoelectricity, nontoxicity and natural sex are also given in biosynthesizing.Therefore, it also is that medically medical treatment is implanted and the degradability medical macromolecular materials of the tool application prospect of used in tissue engineering.Therefore, the development research of PHA causes the concern of world technology circle always.
External at present research to this product, genetic background, the pathways metabolism of in theory microorganism being synthesized PHA, regulatory mechanism, the molecular structure of PHA, physico-chemical property etc. have been studied clear.And from application, the strain excellent of several plant heights product PHA and the small-scale production technology of these bacterial strains have also been obtained.Britain, Korea S, the U.S. also have product to come out in addition.From production cost, the production cost of PHA is too high at present, on dominating the market, can't compete mutually with the chemosynthesis plastics.Therefore, reduce the production cost of PHA, just become the key subject of this area research at present.
In the technology and economic problems assessment of Britain's ICI Imperial Chemical Industries eighties (ICI) with regard to industrial-scale production PHB, choose vinelandii, methylotrophic bacteria and alcaligenes eutrophus as first-selected bacterial strain, but in assessment, because of methylotrophic bacteria PHB productive rate medium, the PHB molecular weight is little, extract difficulty, so abandoned; Though productive rate height in the vinelandii PHB born of the same parents, molecular weight is big again, owing to also produce polysaccharide in its growth, and the ratio productive rate that has reduced PHB also is eliminated; Finally selected alcaligenes eutrophus.Because this bacterial strain is PHB content height not only, molecular weight is big, and can utilize the carbon source of various less expensives.Henceforth really support and produce the main bacterial strain that alkali bar mattress just becomes whole world development research production PHB, so the whole world has also started the research heat of one alcaligenes eutrophus.Research through more than ten years, people are starting strain with the alcaligenes eutrophus, genetic background, pathways metabolism, regulatory mechanism, molecular structure, physico-chemical property to PHB have been studied clear, the strain excellent of PHB is produced in a strain that makes alcaligenes eutrophus become tool application prospect at present, but its productive rate does not still surpass 80%.
Page.W.J is at Applied and Environmental Microbiology (1992,58:2866-2873) having studied the employing vinelandii in the magazine is main carbon source with glucose, with valeric acid, enanthic acid, n-nonanoic acid etc. is the synthetic P (HB-CO-HV) of auxiliary carbon source, with beet sirup and valeric acid is carbon source, 38h obtains 19-22g P (HB-CO-HV), HV component 8.5-23mol% to 40h in 2.5 liters of fermentor tanks.Page.W.J is at Appliedand Environmental Microbiology (1993,59:4236-4244) having studied the employing vinelandii in the magazine is carbon source with glucose, adds 0.1% fish peptone, in 2.5 liters of fermentor tanks behind the 47h, PHB accounts for dry cell weight 79%, reaches 32 grams per liters; The garden beet molasses reach 66% for carbon source 36h, and PHB is 22 grams per liters.
The purpose of this invention is to provide the height accumulation production method of a kind of PHA, and make the PHA of accumulation be easy to extract, to overcome the deficiency that the prior art productive rate is low, cost is high.
The microorganism of using
The microorganism that is used for producing poly (hydroxyalkanoate) of the present invention is: 1. azotobacter chroococcum (Azotobacter chroococcum) G-3 (hereinafter to be referred as the G-3 bacterial strain), its on May 2nd, 1999 in China's typical culture collection center preservation, deposit number is CCTCC NO:M99006.2. bacillus megaterium (Bacillus megaterium) G-6 (hereinafter to be referred as the G-6 bacterial strain), by other people in China's typical culture collection center preservation, deposit number is CCTCC AB92075, this bacterial strain also can obtain from Shaanxi Institute of Microbiology.
The G-3 bacterial strain is transformed in mutagenesis
With 30 ℃ of azotobacter chroococcum bacterium liquid of cultivating 16h, centrifugal removal supernatant liquor, use PH=6.0,0.1mol/L phosphoric acid buffer washing thalline 2 times, be made into 108 cells/ml bacteria suspension with this damping fluid again, it is 600 μ g/ml that adding chemical mutagen nitrosoguanidine (NTG) liquid makes final concentration, 30 ℃ of water bath processing 30min, intermittently shake, the centrifugal NTG that removes, wash 1 time with phosphoric acid buffer again, be resuspended in the damping fluid of same volume, get in the small test tube of 1ml suspension adding 50 * 5min, test tube is placed laser beam the place ahead, shine bacteria suspension from the side, laser power 20mW, wavelength go into=632.8nm, expand bundle spot diameter 0.3cm, irradiation distance 30cm, irradiation time 15min, postradiation bacterium liquid make suitably dilution back coating plate, cultivate 28-32h for 30 ℃, choose single bacterium colony and connect the inclined-plane, after above-mentioned mutagenesis repeats repeatedly, obtain a strain by primary dcreening operation and multiple sieve at last and produce the more weak mutant strain of pod membrane, be numbered vinelandii G-3 bacterial strain, this mutant strain is except that above-mentioned characteristic, also show the glucose utilization ability is weakened, and growth and breeding speed is very fast in sucrose medium, utilizes the very strong characteristic of sucrose ability.
The biological characteristics of above-mentioned azotobacter chroococcum G-3: vinelandii G-3 grows vigorous on nitrogen-free agar, and bacterium colony is circular, convexity, and from the beginning of being oyster white, not sticking, rough, bacterium colony is a light coffee color when making always, bacterium colony is more sticking, and wrinkle is arranged.The original vinelandii of the viscosity ratio of G-3 bacterial strain fermentation liquor are low, and produce more than 10 hours evenings of the former bacterial strain of time ratio of pod membrane, and thalline is easily separated.
Technical scheme of the present invention is to adopt azotobacter chroococcum (Azotobacter chroococcum) G-3 bacterial strain and bacillus megaterium (Bacillus megaterium) G-6 bacterial strain mixed culture to produce poly (hydroxyalkanoate), and the G-6 bacterial strain mainly is that the polysaccharide that utilizes the G-3 bacterial strain to accumulate in culturing process is grown.
It is too high because of concentration of reduced sugar in the nutrient solution that mixed culture can solve the G-3 bacterial strain, viscosity is excessive and effect is checked in metabolism that cause, because azotobacter chroococcum can produce the main ingredient of polysaccharide material as pod membrane in the growth and breeding process, adopting azotobacter chroococcum to carry out in batches in the feeding glucose fermentation process, above-mentioned pod membrane substance accumulation increased when benefit sugar amount was high, the oxygen and the nutritive substance transmission of fermentation system will be influenced, to cause feedback repression but also raw sugar is too high, the continuation propagation that suppresses bacterium, and bacillus megaterium G-6 is in the growth and breeding process, can produce a kind of glycanase by self Metabolic activity is secreted into outside the born of the same parents, this enzyme can become the Polyose degradation in the environment monose or disaccharide, as the nutritive substance of sporeformer self and utilize, and unnecessary sugar is accumulated in external form with PHA as carbon source.The G-3 bacterial strain is utilizing a large amount of glycan materials that produce under the situation of sucrose, can be used as the required carbon source of G-6 strain growth, and the G-6 bacterial strain has reduced the viscosity of fermented liquid when utilizing the sucrose material to breed as nutritive substance, improved the mass transfer of fermented liquid, remove the feedback inhibition of biochemical reaction for the G-3 bacterial strain, improved the utilization ratio of sucrose.
The thalline concentration of reduced sugar increases with the increase of the biomass of thalline in the fermenting process, but at the cultivation initial stage, 10h begins to mend the biomass increase that sugar does not have influence on thalline, but when yeast culture when concentration of reduced sugar reaches 6% during to 28h, mending sugar can only increase the viscosity of fermented liquid, fermentation can't go on, at this moment the biomass of thalline can only reach about 20g/L, if at this moment insert the G-6 bacterial strain, just can utilize reducing sugar to reduce fermentation broth viscosity by the bacterial strain breeding, benefit sugar can further be gone on, promote thalli growth to increase the biomass of culturing process.
Bacillus megaterium G-6 bacterial strain suitable culture condition.Experiment divides two groups to be carried out, and first group is inserted the G-6 bacterial strain in the BM liquid nutrient medium; Second group with the G-3 bacterial strain after the culture sterilization that is cultured to 26h under the liquid vinelandii substratum, insert the G-6 bacterial strain, two groups are all carried out shaking table and cultivate under 30 ℃ of conditions, and survey the OD that it respectively organizes culture at regular intervals
600Value, by table 1 as seen, the G-6 bacterial strain is in the deactivation nutrient solution of G-3 bacterial strain, first group of well-grown of ratio under the situation that no any carbon source material adds, be that the G-6 bacterial strain is more suitable in carry out growth and breeding in the G-3 strain cultured solution, its carbon source may be the G-3 bacterial strain is formed pod membrane outside born of the same parents a polysaccharide fraction.
First group in table 1 and reduced time as a result (h) 4.5 9 12 15 18 first groups of 1.8 2.4 2.8 3.0 2.9 (OD of second group
600Value) second group 14 18 20 22 18.8
Annotate: the PH that inserts the G-6 bacterial strain in second group of experiment is 6.5, mends NH simultaneously
4NO
3
Separately and the growth of mixed culture G-3 bacterial strain and G-6 bacterial strain and the accumulation of PHA.The G-3 bacterial strain is that the liquid selective medium (is nitrogenous source with 0.1% peptone) of carbon source is cultured to 26h at 2% sucrose earlier, mends 1.5g sucrose therebetween at twice, inserts 10%G-6 bacterial strain inoculum again, adds 0.05%NH simultaneously
4NO
3Be cultured to 42h with 0.05% peptone, contrast is respectively the G-3 bacterial strain under the same terms and the single culture of G-6 bacterial strain.The result is as shown in table 2 below.
Table 2 independent and the growth of mixed culture G-3 bacterial strain and G-6 bacterial strain and accumulation strain culturing time biomass PHA content (%) Y of PHA
X/SRSG-3 42 14.8 80 0.37 3.2%G-6 16 1.2 21 0.05 1.5%G-3+G-6 42 17.9 81 0.45 0.24%
Annotate: Y
X/SBe Yg biomass/g sucrose, RS: reducing sugar
Fig. 1 is the mixed culture cellular form;
Fig. 2 is a G-6 bacterial strain single culture thing cellular form;
Fig. 3 is a G-3 bacterial strain single culture thing cellular form;
By table 2, Fig. 1, Fig. 2 and Fig. 3 as seen, Mixed culture is conducive to the conversion ratio that the G-3 bacterial strain improves biomass and PHA. The mixed culture cellular morphology (Fig. 1) of G-6 bacterial strain is than the cellular morphology of the independent cultivation of G-6 grow good (Fig. 2); And the G-3 bacterial strain when Mixed culture owing to reduce polysaccharide material to the inhibitory action of its breeding, and the viscosity of zymotic fluid descends, mass transfer effect changes, be conducive to its growth and breeding, therefore as seen, also good than independent cultivation G-3 strain growth, illustrate that this two strains bacterium has good, stable compatibility, can set up a kind of mutualistic symbiosis relation.
In the sucrose nutrient solution of G-3 bacterial strain access 2%, under 30 ℃, mixing speed 200rpm, cultivate 26h after, access G-6 bacterial strain, the access amount is 15%, and adds simultaneously 0.05% peptone and 0.05%NH4NO
3, add 30% sucrose to above-mentioned mixed liquor discontinuous, survey residual sugar and reach 0.2-0.1%, to put tank and collect thalline, centrifugal, the dry dry mycelium that gets extracts PHA.
The extraction of PHA can be adopted following three kinds of methods:
1、CHCl
3-H
2The O extraction method
Zymotic fluid is through 4000rpm, after 20min is centrifugal, collecting cell, washing secondary, absolute ethyl alcohol wash-out once, 90 ℃ are dried to constant weight, go in the refluxing extraction device, add the CHCl of 80 times of volumes3Solution 55 ℃ of lower stirrings 8 hours, adds a small amount of distilled water, vigorous stirring 1 hour, and membrane filtration is collected organic phase, and reduced pressure concentration reclaims CHCl3, concentrate precipitates with cold methanol, filters, abandons filtrate, the dry PHA sterling that gets of filtration product.
2、NaClO-CHCl
3The two-phase extraction method
Zymotic fluid is through 4000rpm, after 20min is centrifugal, collecting cell, washing secondary, absolute ethyl alcohol wash-out once, 90 ℃ are dried to constant weight, add the NaClO-CHCl of 25 times of volumes in the thalline3Solution stirs under 55 ℃, 4000rpm, and 30min is centrifugal, be divided into three layers, use the pipette, extract organic phase, in organic phase, add water, vigorous stirring 30min, staticly make layering, filter, get organic phase, reduced pressure concentration, concentrate precipitates with cold methanol, filter, abandon filtrate, natural drying, get the PHA sterling.
3, SDS-NaClO method
After the fermentation ends, through 100 ℃ of heat treated after 1 minute, cooling is 55 ℃ rapidly, wash three times, then carry out freezing processing, collect thalline, in thalline, add 1% (W/V) SDS solution, water-bath 15min under 55 ℃ of stirrings, with 3 times of volume water washings, centrifugal 20 minutes of 4000rpm, 55 ℃ of lower 30%NaClO (V/V) effect 30min that add use 10 times of volume water washings immediately, the centrifugal 20min of 4000rpm, must precipitate and again use 3 times of volume water washings, 4000rpm is after centrifugal 20 minutes, dry PHA white powder.
Parameter detection and analysis method in the sweat
1, Determination of Reducing Sugars. Use 721 spectrophotometers, use the fixed sugared method of 3,5-dinitrosalicylic acid colorimetric.
2, sucrose-determination method. The Roe colorimetric method.
3, the determination method of biomass.
A, cell concentration: the zymotic fluid dilution is rear with the 721 spectrophotometric instrumentation 600nm OD of place values.
B, dry cell weight: zymotic fluid is through centrifugal (4000rpm/min), and centrifugal washing is 1-2 time again, the centrifugal wash-out of ethanol once, 90 ℃ of freeze-day with constant temperature, to constant weight, the cooling after weighing.
4, PHA Determination on content method uses the sulfuric acid degraded to measure the PHA method.
The making of A, PHA uv-absorbing (209nm) typical curve
Take by weighing series standard PHB (sigma product), after 100 ℃ of 15 minutes vitriol oils degradeds, uv-absorbing, drawing standard curve are measured in the constant gradient dilution at the 209nm place.
The mensuration of B, sample
Take by weighing dry mycelium, add 100 ℃ of effects of the vitriol oil after 15 minutes, the uv-absorbing of its 209nm is surveyed in dilution,
Through looking into the concentration that typical curve draws PHA, calculate the percentage composition that PHA accounts for dry cell weight at last.
The present invention has eliminated the capsular polysaccharide material that produces in the vinelandii culturing process, has improved the biomass of thalline, and then increases the ratio productive rate of PHA, has reduced production cost.Use this mixed culturing method to produce PHA, biomass is that biomass reaches 53g/L at most, and the PHA amount reaches 42.4g/L, and the transformation efficiency of sugar is stabilized in about 0.34, and solved azotobacter chroococcum fermentative production PHA and produced polysaccharide, increased viscosity and reduce the ratio yield issues of PHA.The G-6 bacterial strain does not produce gemma in mixed culture, and the intracellular PHA particle is big many during than pure culture, and the PHA molecular weight is big, reaches 1,000,000 orders of magnitude, and the PHA particle do not have tunicle outward, and the mature cell wall is easily broken, and this just makes the back operation extract and becomes easy.Possible by analysis reason is that the G-3 bacterial strain provides rich nutrient substances for the G-6 bacterial strain in mixed culture, during mixed culture unlimited prolongation the growth logarithmic phase of G-6 bacterial strain, make the G-6 bacterial strain can't form gemma, form the PHA particle in vivo and the carbon source material of surplus stored.
Embodiments of the invention:
1, the production of P (HB-CO-HV)
G-3, G-6 bacterial strain are inserted in the 2% sucrose fermention medium shake-flask culture 24h respectively.
Get the above-mentioned culture of G-3 bacterial strain, the amount with 10% inserts in the fermentor tank, and adds 0.05% peptone and 0.05%NH simultaneously
4NO
3, 30 ℃, 200rpm continue to cultivate, and carry out 3 times with the sucrose liquid of 30% concentration therebetween and intermittently mend sugar, to 34h, add 0.5% valeric acid in fermentor tank, and mend sugar once, continue to be cultured to 42h and put jar, collect thalline, adopt continuous-flow centrifugation, wet thallus.
Above-mentioned wet thallus is through 100 ℃ of heat treated after 1 minute, be cooled to 55 ℃ rapidly, wash three times, carry out freezing treatment (20 ℃) then, collect thalline, add 1% (W/V) SDS solution in thalline, water-bath 15min under 55 ℃ of stirrings is with 3 times of volume water washings, centrifugal 20 minutes of 4000rpm, 55 ℃ add 30%NaClO (V/V) effect 30min down, use 10 times of volume water washings immediately, the centrifugal 20min of 4000rpm, must precipitate and use 3 times of volume water washings once more, behind centrifugal 20 minutes of the 4000rpm, dry P (HB-CO-HV) white powder that gets calculates its productive rate and accounts for 86% of stem cell weight.
2, the production of PHB
G-3, G-6 bacterial strain are inserted in the 2% sucrose fermention medium shake-flask culture 24h respectively.
Get the above-mentioned culture of G-3 bacterial strain, the amount with 10% inserts in the fermentor tank, and adds 0.05% peptone and 0.05%NH simultaneously
4NO
3, 30 ℃, 200rpm continue to cultivate, and carry out 3 times with the sucrose liquid of 30% concentration therebetween and intermittently mend sugar, continue to be cultured to 42h and put jar, collect thalline, adopt continuous-flow centrifugation, wet thallus.
Adopt the extracting method of embodiment 1 to extract PHB.
The qualitative detection of A, sample
Use the Fx-90Q nuclear magnetic resonance analyser to survey
1The H spectrum.
Testing conditions: solvent: CDCl
3Temperature: 55 ℃; Standard: TMS; Spectrum width: 100HZ; Observed frequency: 89.55MHZ; Observe biasing: 54.3KHZ; Repetition time: 1 second; Add up 16 * 15 times.
Test sample is P (HB-CO-HV) product among the PHA, and the ratio of HB and HV is 74: 26.
The molecular weight determination of B, sample
Adopt viscosimetry (Ubbelohde viscometer).
Experiment condition: strength of solution: C=0.25mg/ml; Temperature: 30 ℃; Solvent: trichloromethane, elution time t
0=45 seconds; Solution: elution time t=124 second (four times average)
Calculate PHB molecular weight M=1.5 * 10
6
The mensuration of C, fusing point and degree of crystallinity
Adopt differential scanning instrument (DERKW-ELMER DSCT) mensuration PHB fusing point to be: 166.35 ℃; PHB degree of crystallinity is 57%.
3, the wet thallus with embodiment 1 or 2 gained passes through freezing pre-treatment (20 ℃), dry again, directly with this dry mycelium injection moulding or mold pressing processing minaudiere and non-returnable container bag, make the cost of PHA reduce to 4 dollars of per kilograms, have only existing report 16 dollars of world's least cost per kilograms 1/4, and products obtained therefrom meets application requiring through detecting, and its detected result sees the following form.
| Test item | Inspecting standard | Assay | The individual event evaluation | |
| Minaudiere | Heat-drawn wire ℃ | 80 | 95 | Qualified |
| Tensile strength (vertically) Mpa | ≥10 | 11.1 | Qualified | |
| Tensile strength (laterally) Mpa | ≥10 | 11.1 | Qualified | |
| Embrittlement temperature (40 ℃) | Nothing is split | Nothing is split | Qualified | |
| Fall down test (1m, 3 times) | Crack-free | Crack-free | Qualified | |
| Disposable packaging box | Tensile strength (vertically) Mpa | ≥8 | 11.5 | Qualified |
| Tensile strength (laterally) MPa | ≥8 | 11.5 | Qualified | |
| Elongation at break (vertically) % | ≥20 | 24 | Qualified | |
| Elongation at break (laterally) % | ≥20 | 26 | Qualified | |
| Angle tear strength (vertically) KN/m | ≥150 | 200 | Qualified | |
| Angle tear strength (laterally) KN/m | ≥150 | 200 | Qualified | |
| Melt flow rate (MFR) (10min) | 2-20 | 3 | Qualified |
Above detected result is detected by the detection of Xi'an product quality supervision.
Claims (5)
1, a kind of production method of poly (hydroxyalkanoate) is characterized in that: adopt azotobacter chroococcum (Azotobacterchroococcum) G-3 bacterial strain and bacillus megaterium (Bacillus megaterium) G-6 bacterial strain mixed culture to produce poly (hydroxyalkanoate).
2, the production method of poly (hydroxyalkanoate) according to claim 1 is characterized in that: the G-3 bacterial strain inserts in 2% the sucrose nutrient solution, under 30 ℃, stirring velocity 200rpm, cultivate 26h after, insert the G-6 bacterial strain.
3, the production method of poly (hydroxyalkanoate) according to claim 2 is characterized in that: the access amount of G-6 bacterial strain is 15%.
4, according to the production method of claim 2 or 3 described poly (hydroxyalkanoate)s, it is characterized in that: the G-6 bacterial strain inserts adds 0.05% peptone and 0.05%NH simultaneously
4NO
3
5, the production method of poly (hydroxyalkanoate) according to claim 4 is characterized in that: add 30% sucrose to the mixed solution discontinuous.
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