CN101892271A - Fermentation method for producing polyhydroxyalkanoate - Google Patents
Fermentation method for producing polyhydroxyalkanoate Download PDFInfo
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- CN101892271A CN101892271A CN2009100743910A CN200910074391A CN101892271A CN 101892271 A CN101892271 A CN 101892271A CN 2009100743910 A CN2009100743910 A CN 2009100743910A CN 200910074391 A CN200910074391 A CN 200910074391A CN 101892271 A CN101892271 A CN 101892271A
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- polyhydroxyalkanoate
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Abstract
The invention belongs to the technical field of polyhydroxyalkanoate production methods, and discloses a fermentation method for producing polyhydroxyalkanoate (PHA). The method is mainly characterized by comprising the following steps of: obtaining activated thalli from PHA producing strain-containing thalli suspension by seed culture, and taking the thalli as seeds for large-scale culture so as to obtain high-density thalli; regulating fermentation through metabolism to convert starch into the PHA which is greatly accumulated in thalli cells; and after the fermentation process is finished, separating the thalli by filtration, mechanically breaking walls of the obtained thalli, then performing centrifugal separation on the thalli to remove cell debris, adding an organic solvent into the PHA-containing supernate to perform extraction, adding methanol into the extract, and refining the deposited PHA to obtain a PHA product. The method for producing the PHA has the advantages of low production cost, short fermentation time and high product purity.
Description
Technical field
The invention belongs to the production method technical field of producing polyhydroxyalkanoate, relate in particular to a kind of fermentation method for producing polyhydroxyalkanoate.
Background technology
Polyhydroxyalkanoate (Polyhydroxyalkanoate is called for short PHA) is in the early development stage, is that the research of fermenting raw materials production PHA has bright future with glucose.
Polyhydroxyalkanoate is the general name of a class by microorganism synthetic biological degradability thermoplastic polyester.Excessive and certain nutritive substance lacks under the condition of (as limit nitrogen, limit phosphorus) in carbon source, and the eubolism approach of many microorganisms will be destroyed, and in its tenuigenin the PHA of certain structure of accumulation, as the stored substance of carbon and energy.When the energy in the environment lacks, microorganism these PHA can be stored things again katabolism fall, so that the vital movement energy needed to be provided.Therefore PHA has biological degradability and biocompatibility completely.Because PHA can be that carbon source through fermentation obtains with multiple material by multiple microorganism, its advantage is the carbon source cheapness, and the resulting polymers molecular weight can be up to 10
5More than, PHA can be degraded to water and carbonic acid gas fully in natural ecological environment, and environmental pollution is little, so cause people's extensive studies interest, the general structure of PHAs is formula as follows:
Along with the length of side chain R, on the side chain with the difference of functional group, the character of PHA can be changed to unbodied thermoplastic elastomer from the thermoplastics of high-crystallinity.Because PHA is with the excellent biological compatibility energy, the hot workability of biodegradability and plastics, therefore can be used as bio-medical material and biodegradable wrapping material simultaneously, become in recent years technical field of biological material one of active research focus the most.The information that PHA institute brings proves that the potentiality of biosynthesizing novel material almost are unlimited, and along with deepening continuously of research, also having more PHA can be synthesized out.PHA also has many high added value performances such as non-linear optical active, piezoelectricity, being separated by property of gas.Just because of PHA has compiled these good performances, make it except the biomaterial for medical purpose field, good application prospects is arranged in extensive fields such as wrapping material, jointing material, spray material and dress material, utensil class material, electronic product, durable consumer goods, agricultural prods, automated production, chemical mediator and solvents.Because the good performance of PHA and the diversity of product have attracted many enterprises to join in its research and development and the production.Rise the eighties and bring into use the biotechnology means, some characteristic genes are comprised that the pha synthesizing enzyme gene clone makes up engineering strain in intestinal bacteria, in order that simplify technology, improve PHA output, reduce cost, change the component structure and the function of synthetic polymer, to solve the difficult problem in producing, no matter all not to be better than R.eutrophus be substrate with glucose but a large amount of documents and materials all demonstrate the content of these engineering bacteria cell harvesting amounts, PHA or ferment strength, glucose concn and glucose applied the PHA output that method that stream adds obtains in the control fermentation also.Really supporting the Laplace bacillus mutant strain can synthesis of polyhydroxyl butyrate (PHB) or PHBV mixture.The PHBV mixture has better characteristic than PHB, utilizes and really supports the synthetic PHBV of Laplace bacillus, needs to add special prerequisite material such as propionic acid and valeric acid, these material price height, and also toxic to producing bacterium, have a strong impact on productive rate.It is substrate with glucose that ICI company utilizes Ralstonia eutrophus bacterial strain, and through the 100-1200h fermentation, cell concn can reach 100g/L, and PHB accounts for the 78-80% of dry cell weight.Fermenting process adds propionic acid, regulates and control to make the heavy PHV of product to reach 0-30% to fermenting process.Play that a lot of work have been done by the U.S. sixties but not industrialization always.1986-1992 Spain is that fermenting substrate is produced PHA with starch with Mediterranean Sea richly endowed bacillus (Haloferaxmediterranei), and output is lower, and PHA accounts for about 60% of cell concentration, and output only is 8g/L.
Summary of the invention
The technical problem to be solved in the present invention just provides the fermentation method for producing polyhydroxyalkanoate that a kind of production cost is low, fermentation time is short, product purity is high.
For addressing the above problem, the technical solution used in the present invention is: this method comprises the following steps:
The first step: extract polyhydroxyalkanoate and produce bacterium,
The active sludge that will contain corn starch wastewater adds in the sbr reactor device, and temperature is controlled at 45-55 ℃, and pH value is controlled at 5-6, and hydrolytic acidification time is 22-26 hour, and stirs 10 minutes, places naturally then, treats that throw out all precipitates, and obtains the thalline suspension;
Second step: obtain the high-density thalline
To contain thalline suspension that polyhydroxyalkanoate produces bacterium and acquire thalline after the activation, and the thalline that obtains will be used for large scale culturing as seed, to obtain the high-density thalline through seed culture.
The 3rd step: utilize polyhydroxyalkanoate to produce monomer and polymer that bacterium produces polyhydroxyalkanoate,
Above-mentioned limpid solution is joined in the fermentor tank that has W-Gum according to 15% ratio, and temperature is controlled at 45-50 ℃, and pH value is controlled at 5-7, ferments 2026 hours, obtains fermented liquid;
The 4th step: the extraction of polyhydroxyalkanoate
With above-mentioned filtering fermentation liquor, and with the throw out lyophilize, and carry out mechanical breaking-wall method, and adopt 10000-13000 rev/min to descend centrifugal 2-5 minute, add organic solvent extraction, add methyl alcohol then, filter, drying precipitate is got final product.
Its additional technical feature is: the temperature in the above-mentioned the first step sbr reactor device is controlled at 48 ℃, and pH value is controlled at 5.7, and hydrolytic acidification time is 24 hours, and stirs 10 minutes, places naturally then, treats that throw out all precipitates, till solution is limpid.
Fermentation method for producing polyhydroxyalkanoate provided by the present invention compared with prior art has the following advantages: because these microorganisms producing bacterium can utilize cheap carbon source such as glucose, vegetables oil or starch etc.The accumulation rate of PHA preferably accounts for more than 80% of dry cell weight, and the ratio that carbon source is converted into PHA is preferably in more than 50%, and the extraction yield of PHA is preferably in more than 90%, and cell culture density is more than 150g/L, and fermentation time is less than 48 hours.Reduced production cost, fermentation time shortens short and has improved product purity.
Embodiment
Be described in further details below in conjunction with the structure and the use principle of accompanying drawing fermentation method for producing polyhydroxyalkanoate of the present invention.
Embodiment 1,
The first step: extract polyhydroxyalkanoate and produce bacterium,
The active sludge that will contain corn starch wastewater adds in the sbr reactor device, and temperature is controlled at 45 ℃, and pH value is controlled at 6, and hydrolytic acidification time is 26 hours, and stirs 10 minutes, places naturally then, treats that throw out all precipitates, and obtains the thalline suspension;
Second step: obtain the high-density thalline
To contain thalline suspension that polyhydroxyalkanoate produces bacterium and acquire thalline after the activation, and the thalline that obtains will be used for large scale culturing as seed, produce bacterium to obtain the high-density polyhydroxyalkanoate through seed culture.
The 3rd step: utilize polyhydroxyalkanoate to produce monomer and polymer that bacterium produces polyhydroxyalkanoate,
Above-mentioned limpid solution is joined in the fermentor tank that has W-Gum according to 15% ratio, and temperature is controlled at 45 ℃, and pH value is controlled at 7, ferments 26 hours, obtains fermented liquid;
The 4th step: the extraction of polyhydroxyalkanoate
With above-mentioned filtering fermentation liquor, and with the throw out lyophilize, and carry out mechanical breaking-wall method, and adopt 13000 rev/mins to descend centrifugal 5 minutes, add organic solvent extraction, add methyl alcohol then, filter, drying precipitate is got final product.
Embodiment 2,
The first step: extract polyhydroxyalkanoate and produce bacterium,
The active sludge that will contain corn starch wastewater adds in the sbr reactor device, and temperature is controlled at 55 ℃, and pH value is controlled at 5, and hydrolytic acidification time is 22 hours, and stirs 10 minutes, places naturally then, treats that throw out all precipitates, and obtains the thalline suspension;
Second step: obtain the high-density thalline
To contain thalline suspension that polyhydroxyalkanoate produces bacterium and acquire thalline after the activation, and the thalline that obtains will be used for large scale culturing as seed, produce bacterium to obtain the high-density polyhydroxyalkanoate through seed culture.
The 3rd step: utilize polyhydroxyalkanoate to produce monomer and polymer that bacterium produces polyhydroxyalkanoate,
Above-mentioned limpid solution is joined in the fermentor tank that has W-Gum according to 15% ratio, and temperature is controlled at 50 ℃, and pH value is controlled at 5, ferments 20 hours, obtains fermented liquid;
The 4th step: the extraction of polyhydroxyalkanoate
With above-mentioned filtering fermentation liquor, and with the throw out lyophilize, and carry out mechanical breaking-wall method, and adopt 10000 rev/mins to descend centrifugal 2 minutes, add organic solvent extraction, add methyl alcohol then, filter, drying precipitate is got final product.
Embodiment 3,
The first step: extract polyhydroxyalkanoate and produce bacterium,
The active sludge that will contain corn starch wastewater adds in the sbr reactor device, and temperature is controlled at 48 ℃, and pH value is controlled at 5.5, and hydrolytic acidification time is 24 hours, and stirs 10 minutes, places naturally then, treats that throw out all precipitates, and obtains the thalline suspension;
Second step: obtain the high-density thalline
To contain thalline suspension that polyhydroxyalkanoate produces bacterium and acquire thalline after the activation, and the thalline that obtains will be used for large scale culturing as seed, produce bacterium to obtain the high-density polyhydroxyalkanoate through seed culture.
The 3rd step: utilize polyhydroxyalkanoate to produce monomer and polymer that bacterium produces polyhydroxyalkanoate,
Above-mentioned limpid solution is joined in the fermentor tank that has W-Gum according to 15% ratio, and temperature is controlled at 47 ℃, and pH value is controlled at 6, ferments 24 hours, obtains fermented liquid;
The 4th step: the extraction of polyhydroxyalkanoate
With above-mentioned filtering fermentation liquor, and with the throw out lyophilize, and carry out mechanical breaking-wall method, and adopt 12000 rev/mins to descend centrifugal 3 minutes, add organic solvent extraction, add methyl alcohol then, filter, drying precipitate is got final product.
Embodiment 4,
The first step: extract polyhydroxyalkanoate and produce bacterium,
The active sludge that will contain corn starch wastewater adds in the sbr reactor device, and temperature is controlled at 50 ℃, and pH value is controlled at 5.8, and hydrolytic acidification time is 25 hours, and stirs 10 minutes, places naturally then, treats that throw out all precipitates, and obtains the thalline suspension;
Second step: obtain the high-density thalline
To contain thalline suspension that polyhydroxyalkanoate produces bacterium and acquire thalline after the activation, and the thalline that obtains will be used for large scale culturing as seed, produce bacterium to obtain the high-density polyhydroxyalkanoate through seed culture.
The 3rd step: utilize polyhydroxyalkanoate to produce monomer and polymer that bacterium produces polyhydroxyalkanoate,
Above-mentioned limpid solution is joined in the fermentor tank that has W-Gum according to 15% ratio, and temperature is controlled at 48 ℃, and pH value is controlled at 6.5, ferments 23 hours, obtains fermented liquid;
The 4th step: the extraction of polyhydroxyalkanoate
With above-mentioned filtering fermentation liquor, and with the throw out lyophilize, and carry out mechanical breaking-wall method, and adopt 11000 rev/mins to descend centrifugal 4 minutes, add organic solvent extraction, add methyl alcohol then, filter, drying precipitate is got final product.
Claims (2)
1. fermentation method for producing polyhydroxyalkanoate, it is characterized in that: this method comprises the following steps:
The first step: extract polyhydroxyalkanoate and produce bacterium,
The active sludge that will contain corn starch wastewater adds in the sbr reactor device, and temperature is controlled at 45-55 ℃, and pH value is controlled at 5-6, and hydrolytic acidification time is 22-26 hour, and stirs 10 minutes, places naturally then, treats that throw out all precipitates, and obtains the thalline suspension;
Second step: obtain the high-density thalline
To contain thalline suspension that polyhydroxyalkanoate produces bacterium and acquire thalline after the activation, and the thalline that obtains will be used for large scale culturing as seed, produce bacterium to obtain the high-density polyhydroxyalkanoate through seed culture.
The 3rd step: utilize polyhydroxyalkanoate to produce monomer and polymer that bacterium produces polyhydroxyalkanoate,
Above-mentioned limpid solution is joined in the fermentor tank that has W-Gum according to 15% ratio, and temperature is controlled at 45-50 ℃, and pH value is controlled at 5-7, ferments 20-26 hour, obtains fermented liquid;
The 4th step: the extraction of polyhydroxyalkanoate
With above-mentioned filtering fermentation liquor, and with the throw out lyophilize, and carry out mechanical breaking-wall method, and adopt 10000-13000 rev/min to descend centrifugal 2-5 minute, add organic solvent extraction, add methyl alcohol then, filter, drying precipitate is got final product.
2. fermentation method for producing polyhydroxyalkanoate according to claim 1, it is characterized in that: the temperature in the above-mentioned the first step sbr reactor device is controlled at 48 ℃, pH value is controlled at 5.7, hydrolytic acidification time is 24 hours, and stirred 10 minutes, naturally place then, treat that throw out all precipitates, till solution is limpid.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111206058A (en) * | 2020-03-03 | 2020-05-29 | 北京化工大学 | Method for producing polyhydroxyalkanoate by using acetic acid or butyric acid |
| CN111349662A (en) * | 2020-04-29 | 2020-06-30 | 吉林中粮生化有限公司 | Method for preparing PHA (polyhydroxyalkanoate) by fermenting PHA fermentation process wastewater |
| CN111500650A (en) * | 2020-06-30 | 2020-08-07 | 中粮营养健康研究院有限公司 | Method for efficiently producing PHA |
| CN111518847A (en) * | 2020-04-29 | 2020-08-11 | 中粮营养健康研究院有限公司 | Method and system for treating polyhydroxyalkanoate fermentation liquor by using chromatographic adsorption method and application of obtained fermentation waste liquor |
| CN111518848A (en) * | 2020-03-06 | 2020-08-11 | 中科院过程工程研究所南京绿色制造产业创新研究院 | Preparation method for preparing polyhydroxyalkanoate from small molecular alcohol |
| CN111647151A (en) * | 2020-06-10 | 2020-09-11 | 珠海麦得发生物科技股份有限公司 | Efficient full-automatic secondary PHA purification process |
| CN113354802A (en) * | 2021-05-26 | 2021-09-07 | 清华大学 | High-purity extraction method of polyhydroxyalkanoate |
| TWI740573B (en) * | 2020-07-13 | 2021-09-21 | 國立暨南國際大學 | Polyhydroxyalkanoate extraction system |
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2009
- 2009-05-20 CN CN2009100743910A patent/CN101892271A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111206058A (en) * | 2020-03-03 | 2020-05-29 | 北京化工大学 | Method for producing polyhydroxyalkanoate by using acetic acid or butyric acid |
| CN111206058B (en) * | 2020-03-03 | 2022-05-31 | 北京化工大学 | Method for producing polyhydroxyalkanoate by using acetic acid or butyric acid |
| CN111518848A (en) * | 2020-03-06 | 2020-08-11 | 中科院过程工程研究所南京绿色制造产业创新研究院 | Preparation method for preparing polyhydroxyalkanoate from small molecular alcohol |
| CN111349662A (en) * | 2020-04-29 | 2020-06-30 | 吉林中粮生化有限公司 | Method for preparing PHA (polyhydroxyalkanoate) by fermenting PHA fermentation process wastewater |
| CN111518847A (en) * | 2020-04-29 | 2020-08-11 | 中粮营养健康研究院有限公司 | Method and system for treating polyhydroxyalkanoate fermentation liquor by using chromatographic adsorption method and application of obtained fermentation waste liquor |
| CN111518847B (en) * | 2020-04-29 | 2022-06-21 | 中粮营养健康研究院有限公司 | Method and system for treating polyhydroxyalkanoate fermentation liquor by using chromatographic adsorption method and application of obtained fermentation waste liquor |
| CN111647151A (en) * | 2020-06-10 | 2020-09-11 | 珠海麦得发生物科技股份有限公司 | Efficient full-automatic secondary PHA purification process |
| CN111500650A (en) * | 2020-06-30 | 2020-08-07 | 中粮营养健康研究院有限公司 | Method for efficiently producing PHA |
| US11155483B1 (en) | 2020-06-30 | 2021-10-26 | Nutrition & Health Research Institute, COFCO Corporation | Method for efficiently producing PHA |
| TWI740573B (en) * | 2020-07-13 | 2021-09-21 | 國立暨南國際大學 | Polyhydroxyalkanoate extraction system |
| CN113354802A (en) * | 2021-05-26 | 2021-09-07 | 清华大学 | High-purity extraction method of polyhydroxyalkanoate |
| CN113354802B (en) * | 2021-05-26 | 2022-04-22 | 清华大学 | High-purity extraction method of polyhydroxyalkanoate |
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Application publication date: 20101124 |