CN103627774A - Method for producing polyhydroxyalkanoate by virtue of mixed cultivation of two types of bacteria - Google Patents
Method for producing polyhydroxyalkanoate by virtue of mixed cultivation of two types of bacteria Download PDFInfo
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- CN103627774A CN103627774A CN201310572845.3A CN201310572845A CN103627774A CN 103627774 A CN103627774 A CN 103627774A CN 201310572845 A CN201310572845 A CN 201310572845A CN 103627774 A CN103627774 A CN 103627774A
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- polyhydroxyalkanoate
- fermentation
- pha
- bacteria
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Abstract
The invention relates to a method for producing polyhydroxyalkanoate by virtue of the combined fermentation of two wild fungi. The method comprises the following steps: respectively inoculating bacteria 1175 (bacillus) and WN-H41 (burkholderia) into a fermentation medium; culturing two bacteria seeds for 18 hours; firstly inoculating bacteria 1175, fermenting and culturing for 24 hours; then inoculating WN-H41, continuously fermenting for 48 hours (the total inoculation amount is 10%, 1175/WN-H41=1/2) and ending fermentation. According to the method, the extraction ratio of the polyhydroxyalkanoate (PHA) is above 40% under the condition that the culture medium is not completely optimized, the polyhydroxyalkanoate is milk white, and through gel permeation chromatography (GPC) detection, Mn is 1.12*10<5>, and the PDI is 1.16. Compared with a pure bacterium fermentation method, the method has certain advantages that the selection range of substrates is wider, the sterile requirements are reduced, the product variety is increased, and the like. The method provides thinking for the development and utilization of biodegradable plastics.
Description
Technical field
The present invention relates to a kind of method of utilizing two strain wild mushroom combined ferments to produce polyhydroxyalkanoate (PHA)-biodegradable plastic.
Background technology
Plastics have become the fourth-largest mainstay industry in national economy.Along with the continuous enhancing of demand, conventional plastic is depended on fossil oil unduly, and after using, the drawback of difficult recovery, difficult degradation day by day highlights.Meanwhile, Xinjiang needs a large amount of plastic sheeting for farm uses and water saving tubing every year as a big agricultural province, and these products mostly are chemosynthesis product at present, if can partly or entirely substitute them by biodegradable plastic, the farming to later and plant growth is very favourable.This just requires the green plastic of researcher development of new.
Polyhydroxyalkanoate (PHA) is the storage characteristics polyester granulate of a quasi-microorganism accumulation.The reserve substance in unbalanced growth condition lower eyelid self-energy and carbon source, fully biodegradable under natural condition.Under aerobic conditions, PHA can degradable Cheng Shui and CO
2.Under anaerobic, the PHA generation methane that can be degraded by microorganisms.As a kind of potential environmentally friendly bio-degradable plastics, it has the character similar to polypropylene (PP), therefore becomes the focus of Recent study.
The synthetic fermenting process of present stage PHA is to utilize purebred microorganism, genetic engineering bacterium mostly, raw materials for production are mainly the organic substrates such as propionic acid that glucose or price are more expensive, valeric acid, the searching of superior strain, the selection of substrate, the sterilizing of equipment and purification of products are all restricting the reduction of PHA production cost.In order to offset high production cost, Application Areas is also mainly confined to the high value added products such as medical devices, cosmetics of super quality packing box.Research now shows, utilizes mixed fermentation with various bacterium to produce PHA and more has superiority, no matter be to economic aspect or environment aspect.Utilize at present the report of microorganism mixed fermentation production PHA also less, or the simple active sludge of domestication that utilizes ferment.
Summary of the invention
The method that the object of the present invention is to provide a kind of production polyhydroxyalkanoate (PHA), this method can be enriched the production of PHA, reduces aseptic requirement, makes the PHA superior performance of producing.
The present invention is achieved by the following technical programs:
By experiment, from Xinjiang physical environment, filter out the bacterial strain of synthetic PHA, through form observation and gene order comparison, determine kind.
Two kinds of bacterium (WN-H
41 and 1175) mixing fermentation culture, optimization for fermentation technology.
Separating-purifying product, analyzes its structure, does the mensuration of molecular weight, thermal characteristics and mechanical property.Mixed fermentation product and sterile fermentation product compare.
Advantage of the present invention: produce at present PHA and mainly utilize pure bacterium or recombinant bacterium.Mixed fermentation is the acclimated activated sludges that are limited to also more.The method of two kinds of wild mushroom combined ferment product PHA is compared with the fermentation of pure bacterium/recombinant bacterium, and the tolerance of miscellaneous bacteria is eager to excel, and substrate is selected wide; Compare with active sludge fermentation, target bacterial classification is clearer and more definite, is beneficial to follow-up metabolism research.
Embodiment
Below in conjunction with embodiment, the present invention is described further,
The mixed fermentation of 1: two strain bacterium of embodiment
All select the seed inoculation of 18h fermentation, fermention medium is poor nitrogen substratum.Mixed fermentation first inoculates 1175, after fermentation 24h, inoculates WN-H
41, (1175/WN-H
41=1/2, total inoculum size is 10%), 100mL/250mL Erlenmeyer flask, 30 ℃, 100r/min, total fermentation time 72h.
The comparison of table 1 sterile fermentation and mixed fermentation
As can be seen from the test results, on original substratum, WN-H
41 & 1175 mixed fermentation dry cell weights are respectively WN-H
44.38 and 1.46 times of 1 and 1175 dry cell weights; Product amount is respectively WN-H
41 and 1175 2.76 and 0.92 times.Illustrate that these two kinds of bacterium mixed culture antagonistic action can not occur each other, the mesostate of a contrary strain bacterium may also can promote the growth of another strain bacterium.And product amount and 1175 single bacterium fermentations obviously do not reduce, reducing part may be because can flow distribution.
Embodiment 2: the GPC determining molecular weight of mixed fermentation product
To WN-H
41,1175 and WN-H
4three kinds of products that 1 & 1175 fermentations are purified have all carried out the analysis of infiltration gel chromatography, and result is as following table 2
The GPC of product is extracted in table 2 sterile fermentation and mixed fermentation
From test result, can find out WN-H
4the number-average molecular weight of 1 & 1175 mixed fermentation synthetic products compares WN-H
4the number-average molecular weight of 1 and 1175 independent fermentation synthetic products is a large order of magnitude all, and dispersity is only 1.16.R.Rai has gathered the character of multiple mcl-PHAs product in article, and wherein Mn is at 2.52*10
4to 1.91*10
5between, PDI is between 1.7 to 4.4.The minimum value that the PDI of our mixed fermentation resulting polymers is enumerated much smaller than R.Rai, illustrates that the relative molecular weight homogeneity of this polymkeric substance is very good.
Claims (5)
1. utilize two strain wild mushroom combined ferments to produce a method for polyhydroxyalkanoate (PHA)-biodegradable plastic, it is characterized in that, select two kinds of clear and definite bacteriums 1175 (bacillus) and WN-H
4polyhydroxyalkanoate is produced in 1 (Burkholderia genus) mixed fermentation.
2. a kind of method of utilizing two strain wild mushroom combined ferments to produce polyhydroxyalkanoate (PHA)-biodegradable plastic according to claim 1, is characterized in that bacterium 1175 and WN-H
41 vaccination ways meets WN-H after first meeting 1175,24h
41.
3. a kind of method of utilizing two strain wild mushroom combined ferments to produce polyhydroxyalkanoate (PHA)-biodegradable plastic according to claim 1, is characterized in that bacterium 1175 and WN-H
4total inoculum size of 1 is the inoculative proportion (1175/WN-H of 10%, two strain bacterium
41) be 1/2.
4. purification of products method according to claim 1, is characterized in that, the first ultrasonic extraction 20min of freeze drying cell, then use 40 ℃ of extracting 12h of methyl alcohol-chloroform (v/v=1/2) solution.
5. purification of products method according to claim 1, is characterized in that, 4 layers of filtered through gauze twice of the complete use of extracting, and filtrate is separated out product with cold methanol.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310572845.3A CN103627774A (en) | 2013-11-18 | 2013-11-18 | Method for producing polyhydroxyalkanoate by virtue of mixed cultivation of two types of bacteria |
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|---|---|---|---|
| CN201310572845.3A CN103627774A (en) | 2013-11-18 | 2013-11-18 | Method for producing polyhydroxyalkanoate by virtue of mixed cultivation of two types of bacteria |
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| CN103627774A true CN103627774A (en) | 2014-03-12 |
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| CN201310572845.3A Pending CN103627774A (en) | 2013-11-18 | 2013-11-18 | Method for producing polyhydroxyalkanoate by virtue of mixed cultivation of two types of bacteria |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660166A (en) * | 2018-05-31 | 2018-10-16 | 保定百恩杰生物科技有限公司 | The culture medium and its fermentation process of primary gram of Hall bacterium fermenting and producing PHA of sensitive plant |
| CN108949842A (en) * | 2018-07-20 | 2018-12-07 | 中南大学 | A kind of resource utilization method of lignocellulosic acid system pretreatment waste liquid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8030021B2 (en) * | 2008-06-24 | 2011-10-04 | The Board Of Trustees Of The Leland Standford Junior University | Use of selection pressures to enable microbial biosynthesis of polyhydroxyalkanoates from anaerobic degradation products |
-
2013
- 2013-11-18 CN CN201310572845.3A patent/CN103627774A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8030021B2 (en) * | 2008-06-24 | 2011-10-04 | The Board Of Trustees Of The Leland Standford Junior University | Use of selection pressures to enable microbial biosynthesis of polyhydroxyalkanoates from anaerobic degradation products |
Non-Patent Citations (3)
| Title |
|---|
| BINGXIN ZHENG: "Isolation and Characterization of Poly(3-hydroxybutyrate)-Producing Bacteria from Aerobic Sludge", 《APPL BIOCHEM BIOTECHNOL》, vol. 175, 11 October 2014 (2014-10-11), pages 421 - 427, XP035428889, DOI: 10.1007/s12010-014-1271-x * |
| 郑冰心: "PHA生产菌株的选育及其混合发酵条件的优化", 《CNKI优秀硕士学位论文全文库》, 31 December 2014 (2014-12-31) * |
| 郑冰心: "利用响应面法优化聚羟基丁酸混菌发酵培养基", 《石河子大学学报(自然科学版）》, vol. 32, no. 5, 31 May 2014 (2014-05-31) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660166A (en) * | 2018-05-31 | 2018-10-16 | 保定百恩杰生物科技有限公司 | The culture medium and its fermentation process of primary gram of Hall bacterium fermenting and producing PHA of sensitive plant |
| CN108949842A (en) * | 2018-07-20 | 2018-12-07 | 中南大学 | A kind of resource utilization method of lignocellulosic acid system pretreatment waste liquid |
| CN108949842B (en) * | 2018-07-20 | 2020-11-10 | 中南大学 | Resource utilization method of lignocellulose acid method pretreatment waste liquid |
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Application publication date: 20140312 |