CN104031906A - Screening and domestication method of bacteria colony for producing polyhydroxyalkanoate by using xylose - Google Patents
Screening and domestication method of bacteria colony for producing polyhydroxyalkanoate by using xylose Download PDFInfo
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Abstract
The invention provides a screening and domestication method of a bacteria colony for producing polyhydroxyalkanoate by using xylose, and aims at obtaining a mixed bacteria colony for producing PHA by using xylose at one time. A satiation hunger domestication mode of a PHA synthesis bacteria colony is adopted, a sludge sample is domesticated by taking xylose as a sole carbon source, and in the domestication process, a Nile blue staining method of the polyhydroxyalkanoate synthesis bacteria colony is performed to screen the bacteria colony and the bacteria colony is refilled to the bacteria colony domesticated by using xylose. The stable high-yielding PHA mixed bacteria colony is finally obtained, and the mixed bacteria colony system mainly contains four dominant strains, namely, gamma-Proteobacteria, Cellvibrio sp., Uncultured bacterium and Pseudomonas putida. The method is simple and effective and short in domestication time, the obtained bacteria colony has a relatively high capability of producing PHA by using xylose, and great significance and practical values in producing PHA by using a cellulose base raw material in a fermentation mode are achieved.
Description
Technical field
The present invention relates to a kind of screening and acclimation method that efficiently utilizes wood sugar to produce PHA flora.
Background technology
PHA (polyhydroxyalkanoates PHA) the synthetic carbon source storage property particle that is many bacteriums under the uneven condition of growth, is subject to people's favor as a kind of renewable, bio-degradable plastics.It,, except having the character close with conventional plastic, also has the characteristics such as physiologically acceptable, optical activity concurrently, becomes the favorable substitutes of plastics, aspect solution environmental pollution and energy shortage, has very large potentiality.At present, PHA mainly adopts sterile fermentation to produce, and comprises Alcaligenes, vinelandii, pseudomonas and recombination bacillus coli etc.But sterile fermentation need be used the high-quality substrates such as glucose, and the fermentating controling process such as sterilizing causes PHA production cost expensive, higher than the 5-10 of conventional plastic doubly [1].Fancy price has seriously hindered the market requirement and the use of PHA.
Cellulose biomass material is as renewable resources the abundantest on the earth, extensively be present in the various agriculture and forestry products such as stalk and processing waste thereof, but cellulose biomass compact structure is very difficult bioavailable, causes these renewable resourcess not obtain large-scale recycling.In lignocellulosic material, the content of wood sugar reaches 18%～30%, is second largest glucide of occurring in nature [2].Therefore many scholars are by artificial screening or build to obtain and utilize wood sugar to produce PHA bacterial strain, to utilizing wood sugar to produce PHA, for take Mierocrystalline cellulose, as raw material produces PHA, lay the foundation, thereby significantly reduce the production cost of PHA.But current single bacterium utilizes the ability of wood sugar product PJHAs still very limited, and maximum production only reaches 3g/l left and right [3,4], cannot meet industrial requirement far away.
Microorganism is generally present in nature with many cells system, and 99% microorganism can not pure culture.It is human being's production service for life that utilization is distributed widely in natural microorganism multicell; it is the economic model of a kind of low cost, high benefit; simultaneously with respect to single bacterium; the stability of mixed bacterium is high; robustness is good; can complete more complicated biological function [5] by the changeable environment of tolerance, therefore in fields such as medicine, food brewing, environment protection, Energy resources, be used widely.Mixed fungus fermentation is used open system simultaneously, without running costs such as sterilizings, so mixed bacterial utilizes wood sugar to produce the cost that PHA can significantly reduce PHA.But, the synthetic bacterial strain of PHA is lacked to suitable acclimation and screening pressure, it is long that traditional be satiated with food hungry pattern and anaerobism are supported well pattern acclimation period, and need to take small molecules fatty acid material as domestication substrate, therefore there is no at present the report that mixed bacterial utilizes wood sugar product PHA.
[1]K?Sudesh,K?Bhubalan,JA?Chuah,et?al.Synthesis?of?polyhydroxyalkanoate?from?palm?oil?and?some?new?applications.App?Microbiol?and?Biot,2010,89:1373-1386
[2] Xu Yong, Wang Xing, Zhu Junjun, bravely strong, remaining generation Yuan. the new outlet [J] that wood sugar high-performance bio transforms. Chinese biological engineering magazine (China Biotechnology), 2012,32 (005): 113-119
[3]FK?Young,JR?Kastner,SW?May.Microbial?Production?of?poly-β-hydroxybutyric?acid?from?d-xylose?and?lactose?by?Pseudomonas?cepacia.Appl?Environ?Microb,1994,60:4195-4198
[4]R?Li,Q?Chen,PG?Wang,et?al.A?novel-designed?Escherichia?coli?for?the?production?of?various?polyhydroxyalkanoates?from?inexpensive?substrate?mixture.Appl?Microbiol?Biot,2007,75:1103-1109
[5]K?Brenner,L?You,FH?Arnold.Engineering?microbial?consortia:a?new?frontier?in?synthetic?biology.Trends?Biotechnol,2008,26:483-489
Summary of the invention
In view of above-mentioned, the invention provides a kind of screening and acclimation method that efficiently utilizes wood sugar to produce PHA flora---the method for using the hungry pattern domestication of being satiated with food to screen in conjunction with Nile blue dyeing applies clear and definite screening pressure to flora.
Technical scheme of the present invention is as follows:
The active sludge that is rich in mixed bacterial of take is raw material, and the hungry domestication pattern of being satiated with food of utilizing mixed bacterial to produce PHA is tamed, and in domestication process, samples, and uses Nile blue staining to carry out the screening of the synthetic flora of PHA; After the PHA high yield flora enrichment culture that screening obtains, be recycled in the system of taming, the flora in domestication is carried out to disturbance; The mixed thalline system that domestication finishes that rear use obtains carries out PHA fermentative production, carries out PHA extraction after fermentation ends.
The described hungry domestication pattern of being satiated with food is to comprise charging, ventilation, and four steps of sedimentation and draining are a culture cycle.
Described Nile blue screening method is that the cell suspension of getting concentration gradient is evenly applied on screening culture medium flat board, and dark culturing is placed under 365nm UV-light irradiates, and selects the flat board that fluorescent grain is many to wash plate, is inoculated in domestication system after enrichment culture.
Domestication substratum is: wood sugar 1800mg/L, ammonium chloride 170mg/L, dipotassium hydrogen phosphate 90mg/L, potassium primary phosphate 35mg/L, magnesium sulfate 600mg/L, EDTA100mg/L, calcium chloride 70mg/L, micro-2mL/L, wherein trace element consists of: Iron trichloride hexahydrate 1000mg/L, boric acid 150mg/L, CoCL2 6H2O 150mg/L, tetrahydrate manganese chloride 120mg/L, Zinc Sulphate Heptahydrate 120mg/L, Sodium Molybdate Dihydrate 60mg/L, cupric sulfate pentahydrate 30mg/L, potassiumiodide 30mg/L; PH6.6～6.7.
Screening culture medium: peptone 10g/L, extractum carnis 5g/L, sodium-chlor 2g/L, Nile blue final concentration 50mg/L, agar 20g/L; PH7.5.
Fermention medium: wood sugar 20g/L, ammonium chloride 2g/L, dipotassium hydrogen phosphate 3.3g/L, magnesium sulfate 1.2g/L, micro-10mL/L; PH8.
In the mixed thalline system obtaining, main advantage bacterial strain is γ-Proteobacteria, Cellvibrio sp., Uncultured bacterium and Pseudomonas putida.
Advantage of the present invention is to use the mode one step acquisition that the hungry pattern of being satiated with food is tamed and Nile blue dyeing screening combines to utilize wood sugar to produce PHA mixed bacterial, make full use of the quick validity of screening, the advantage that has retained again domestication stability, obtain fast the stabilising system of synthetic PHA, avoided the loaded down with trivial details of conventional P HA syllogic production technique.The method is simply effective, and the domestication time is short, and gained flora has the ability of utilizing more by force wood sugar to produce PHA, to realizing with cellulose base fermenting raw materials production PHA, is also significant and practical value.
Accompanying drawing explanation
Fig. 1: mixed bacterial compositional analysis.1 is γ-Proteobacteria, and 2 is Cellvibrio sp., and 3 is Uncultured bacterium, and 4 is Pseudomonas putida.
Fig. 2: xylose utilization and PHA resultant curve figure.
Embodiment
Basic step is as follows:
A) with the active sludge that is rich in mixed bacterial for setting out, use for reference the hungry domestication pattern of being satiated with food that mixed bacterial produces PHA and tame.A culture cycle, comprising: charging, ventilation, sedimentation, four steps of draining.Each cycle of feed finger starts to add fresh wood sugar domestication substratum, and draining refers to that each periodic duty finishes rear discharge section mixed-culture medium;
B) in domestication process, sample, use Nile blue staining to carry out the screening of the synthetic flora of PHA: to get the cell suspension that is diluted to suitable concn gradient and be evenly applied on screening culture medium flat board, dark culturing, be placed under UV-light and irradiate, select the flat board that fluorescent grain is many to wash plate, after enrichment culture, be inoculated in domestication system;
C) after the PHA high yield flora enrichment culture that screening obtains, be recycled in the system of taming, the flora in domestication is carried out to disturbance;
E) the mixed thalline system that domestication finishes that rear use obtains take wood sugar and carries out PHA fermentative production as carbon source, carries out PHA extraction and determination of yield after fermentation ends.
Below in conjunction with drawings and Examples, the present invention is described in detail.
1. substratum configuration
Domestication substratum: wood sugar 1800mg/L, ammonium chloride 170mg/L, dipotassium hydrogen phosphate 90mg/L, potassium primary phosphate 35mg/L, magnesium sulfate 600mg/L, EDTA100mg/L, calcium chloride 70mg/L, micro-2mL/L, wherein trace element consists of: Iron trichloride hexahydrate 1000mg/L, boric acid 150mg/L, CoCL2 6H2O 150mg/L, tetrahydrate manganese chloride 120mg/L, Zinc Sulphate Heptahydrate 120mg/L, Sodium Molybdate Dihydrate 60mg/L, cupric sulfate pentahydrate 30mg/L, potassiumiodide 30mg/L; PH6.6～6.7.
Screening culture medium: peptone 10g/L, extractum carnis 5g/L, sodium-chlor 2g/L, Nile blue (final concentration 50mg/L), agar 20g/L; PH7.5.
Fermention medium: wood sugar 20g/L, ammonium chloride 2g/L, dipotassium hydrogen phosphate 3.3g/L, magnesium sulfate 1.2g/L, micro-10mL/L; PH8.
2. wood sugar produces the domestication of PHA mixed bacterial
(1) will be rich in right amount mixed bacterial active sludge and be placed in 1L large beaker, add water to 800mL, and be placed under magnetic stirring apparatus 300rpm and stir, use air pump ventilation, keep air flow 500mL/min aeration 24h.
(2) use for reference the hungry domestication pattern of being satiated with food that mixed bacterial produces PHA, culture cycle of 12h, comprising: charging 0.5h, ventilation 10h, sedimentation 1h, draining 0.5h.Each cycle of feed finger starts to add substratum 400mL, and draining refers to that each periodic duty finishes the mixed-culture medium of rear discharge 1/2 water and 1/10.
(3) while starting to cultivate, front 2 cycles add initial medium, then according to the ratio of initial medium and domestication substratum 1:3,1:1 and 3:1, increase gradually the ratio of domestication substratum, finally with domestication substratum, active sludge are tamed completely.3. wood sugar produces PHA mixed bacterial screening re-injection
(1) the mixed bacterial 1mL getting in domestication process is placed in centrifuge tube, get 100 μ L and join in the centrifuge tube that fills 900 μ L physiological saline and mix, method the like preparation 10
-2, 10
-3, 10
-4the cell suspension of concentration gradient.
(2) get 100 μ L10
-4the cell suspension of concentration gradient is evenly applied on screening culture medium flat board, is placed in 35 ℃ of incubators, dark culturing 48h.
(3) treat to grow on flat board bacterium colony and be placed under 365nm UV-light and irradiate, select the flat board that fluorescent grain is many to wash plate, be then inoculated in domestication substratum, 37 ℃, 200rpm cultivates.
(4), during the system charging in domestication, cultured mixed bacterial is recycled into and in beaker, continues domestication and cultivate.
4. domestication finishes the evaluation of rear mixed bacterial culture composition
Mixed bacterial is carried postgenome and is used PCR-DGGE technology to mix the separation of bacterium band, then order-checking, as shown in Figure 1, in final mixed bacterial, dominant strain is γ-Proteobacteria, Cellvibrio sp., Uncultured bacterium and Pseudomonas putida to sequencing result.
5.PHA fermentative production
Domestication finishes the final mixed bacterial obtaining of rear use, adds fermention medium to carry out PHA fermentative production.Period sampling measuring xylose utilization rate and corresponding PHA output in fermenting process.Result as shown in Figure 2, obtains maximum PHA output and accounts for dry cell weight 31%, reaches 1.5g/l.
Claims (7)
1. screening and an acclimation method that utilizes wood sugar to produce PHA flora, it is characterized in that: the active sludge that is rich in mixed bacterial of take is raw material, the hungry domestication pattern of being satiated with food of utilizing mixed bacterial to produce PHA is tamed, in domestication process, sample, use Nile blue staining to carry out the screening of the synthetic flora of PHA; After the PHA high yield flora enrichment culture that screening obtains, be recycled in the system of taming, the flora in domestication is carried out to disturbance; The mixed thalline system that domestication finishes that rear use obtains carries out PHA fermentative production, carries out PHA extraction after fermentation ends.
2. screen as claimed in claim 1 and acclimation method, it is characterized in that the described hungry domestication pattern of being satiated with food is to comprise charging, ventilation, four steps of sedimentation and draining are a culture cycle.
3. power is screened and acclimation method as claimed in claim 1, it is characterized in that described Nile blue screening method is that the cell suspension of getting concentration gradient is evenly applied on screening culture medium flat board, dark culturing is placed under 365nm UV-light irradiates, select the flat board that fluorescent grain is many to wash plate, after enrichment culture, be inoculated in domestication system.
4. power is screened and acclimation method as claimed in claim 1, it is characterized in that taming substratum is: wood sugar 1800mg/L, ammonium chloride 170mg/L, dipotassium hydrogen phosphate 90mg/L, potassium primary phosphate 35mg/L, magnesium sulfate 600mg/L, EDTA100mg/L, calcium chloride 70mg/L, trace element 2mL/L, wherein trace element consists of: Iron trichloride hexahydrate 1000mg/L, boric acid 150mg/L, CoCL2 6H2O 150mg/L, tetrahydrate manganese chloride 120mg/L, Zinc Sulphate Heptahydrate 120mg/L, Sodium Molybdate Dihydrate 60mg/L, cupric sulfate pentahydrate 30mg/L, potassiumiodide 30mg/L; PH6.6～6.7.
5. power is screened and acclimation method as claimed in claim 1, it is characterized in that screening culture medium: peptone 10g/L, extractum carnis 5g/L, sodium-chlor 2g/L, Nile blue final concentration 50mg/L, agar 20g/L; PH7.5.
6. power is screened and acclimation method as claimed in claim 1, it is characterized in that fermention medium: wood sugar 20g/L, ammonium chloride 2g/L, dipotassium hydrogen phosphate 3.3g/L, magnesium sulfate 1.2g/L, micro-10mL/L; PH8.
7. power screening and acclimation method as claimed in claim 1, in the mixed thalline system that it is characterized in that obtaining, main advantage bacterial strain is γ-Proteobacteria, Cellvibrio sp., Uncultured bacterium and Pseudomonas putida.
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| CN201410302267.6A CN104031906B (en) | 2014-06-27 | A kind of screening utilizing xylose to produce PHA flora and acclimation method |
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| CN201410302267.6A CN104031906B (en) | 2014-06-27 | A kind of screening utilizing xylose to produce PHA flora and acclimation method |
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| CN104031906B CN104031906B (en) | 2016-11-30 |
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Cited By (6)
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| CN104694453A (en) * | 2015-03-08 | 2015-06-10 | 北京工业大学 | Method for screening and enriching halophilic poly PHA (phytohemagglutinin) mixed bacteria by using glucose as substrate |
| CN106636291A (en) * | 2016-11-09 | 2017-05-10 | 天津大学 | Double-bacteria system construction method for PHA accumulation |
| CN106754471A (en) * | 2016-11-21 | 2017-05-31 | 天津大学 | A kind of method for producing PHA pseudomonad Primary Studies in activated sludge |
| CN109097429A (en) * | 2018-09-15 | 2018-12-28 | 天津科技大学 | A kind of wooden carbon source domestication sewage mixed bacteria synthesis poly 3-hydroxy butyrate/3- hydroxyl pentanoate copolymer method |
| CN110317839A (en) * | 2019-07-08 | 2019-10-11 | 哈尔滨工业大学 | A method of regulating and controlling mixed bacterial polyhydroxyalkanoates synthesising stability using sodium chloride |
| CN113073127A (en) * | 2020-01-03 | 2021-07-06 | 陈宁远 | Method for screening PHA (polyhydroxyalkanoate) producing bacteria |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694453A (en) * | 2015-03-08 | 2015-06-10 | 北京工业大学 | Method for screening and enriching halophilic poly PHA (phytohemagglutinin) mixed bacteria by using glucose as substrate |
| CN106636291A (en) * | 2016-11-09 | 2017-05-10 | 天津大学 | Double-bacteria system construction method for PHA accumulation |
| CN106636291B (en) * | 2016-11-09 | 2020-09-08 | 天津大学 | Double-bacterium system construction method for PHA accumulation |
| CN106754471A (en) * | 2016-11-21 | 2017-05-31 | 天津大学 | A kind of method for producing PHA pseudomonad Primary Studies in activated sludge |
| CN109097429A (en) * | 2018-09-15 | 2018-12-28 | 天津科技大学 | A kind of wooden carbon source domestication sewage mixed bacteria synthesis poly 3-hydroxy butyrate/3- hydroxyl pentanoate copolymer method |
| CN110317839A (en) * | 2019-07-08 | 2019-10-11 | 哈尔滨工业大学 | A method of regulating and controlling mixed bacterial polyhydroxyalkanoates synthesising stability using sodium chloride |
| CN110317839B (en) * | 2019-07-08 | 2020-12-11 | 哈尔滨工业大学 | Method for regulating and controlling synthesis stability of mixed flora polyhydroxyalkanoate by using sodium chloride |
| CN113073127A (en) * | 2020-01-03 | 2021-07-06 | 陈宁远 | Method for screening PHA (polyhydroxyalkanoate) producing bacteria |
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