CN108660166A - The culture medium and its fermentation process of primary gram of Hall bacterium fermenting and producing PHA of sensitive plant - Google Patents

The culture medium and its fermentation process of primary gram of Hall bacterium fermenting and producing PHA of sensitive plant Download PDF

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Publication number
CN108660166A
CN108660166A CN201810551329.5A CN201810551329A CN108660166A CN 108660166 A CN108660166 A CN 108660166A CN 201810551329 A CN201810551329 A CN 201810551329A CN 108660166 A CN108660166 A CN 108660166A
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parts
fermentation
distilled water
hpo
glucose
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张利平
王鸿鹏
张进华
吕文达
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Baoding 100 Um Jie Biotechnology Co Ltd
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Baoding 100 Um Jie Biotechnology Co Ltd
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Abstract

The invention discloses a kind of fermentation mediums and its fermentation process of sensitive plant burkholderia fermenting and producing PHA, count in parts by weight, including：5~40 parts of glucose, 0.05~0.6 part of peptone, Na2HPO4‑.12H24~10 parts of O, MgSO4.7H20.05~0.6 part of O, KH2PO43~8 parts, K2HPO43~8 parts and 0.2~0.6 part of trace element solution, 1000 parts are settled to distilled water；Trace element solution：CuSO40.01~0.03 part, H3BO30.01~0.05 part, Na2MoO40.01~0.05 part, CaCl20.05~0.25 part, FeCl30.01~0.04 part, ZnSO40.01~0.04 part and 1000 parts of distilled water.The fermentation medium and fermentation process of the present invention substantially increases the yield of PHA, is conducive to carry out industrial production.

Description

The culture medium and its fermentation process of primary gram of Hall bacterium fermenting and producing PHA of sensitive plant
Technical field
The present invention relates to biological fermentation field more particularly to a kind of trainings of sensitive plant burkholderia fermenting and producing PHA Support base and its fermentation process.
Background technology
Poly- hydroxyl polyunsaturated fatty acid ester (Polyhydroxyalkanoate, PHA) is that one kind of Microbe synthesis is intracellular Polyester is a kind of natural polymeric biomaterial.PHA is a kind of energy that cell generates under the conditions of carbon nitrogen source imbalance storage Hide object, can synthesize by various bacteria perhaps, because have thermoplasticity, elasticity and biodegradability, biocompatibility, hydrophobicity, Barrier properties for gases, piezoelectricity, non-linear optical active and the peculiar property caused by different functional groups, and be applied to drop Plastics, slow-release material, tissue engineering material (biomaterial of human implantable), photoelectric material etc. are solved, therefore, it has become Technical field of biological material research hotspot the most active in recent years.
It is divided into two major classes according to composition PHA:One kind is short chain PHA (monomer C3-C5), and one kind is medium chain length PHA (monomer For C6-C14), short chain and middle long-chain copolymerization hydroxy fatty acid can be synthesized by having been reported bacterial strain these years.The production of PHA is undergone First generation PHA --- poly butyric ester (PHB), second generation PHA --- hydroxybutyric acid acid copolyesters (PHBV) and the third generation The production of PHA-hydroxybutyric acid sour copolyesters (PGBHHx), and forth generation PHA hydroxybutyric acids Hydroxyoctanoic acid (capric acid) co-polymeric acids [PH-BO (PHBD)] is still in the development phase.PHA structural diversifications, can be very square by changing strain, feed, fermentation process Just change the composition of PHA, and the performance diversification that composed structure diversity is brought makes it have apparent advantage in the application.
Currently, PHA is produced mainly by microbial fermentation, and production cost is high, low yield restricts always the extensive of PHA Using.In fermentation process, culture medium is to provide microorganism growth, breeding metabolism and the required nutrition of Product formation Substance source, while environmental factor necessary to growth and Product formation being also provided；Fermentation condition is control microorganism growth ring The key factor in border.The most important nutriment of growth is carbon source, nitrogen source, inorganic salts and trace element, therefore is filtered out The component and fermentation condition of growth Optimal compositions of fermentation medium become the prefered method that research improves PHA yield.
Invention content
It is an object of the invention to solve at least the above, and provide the advantages of at least will be described later.
This explanation is to provide a kind of Optimal Medium there are one purpose, can be by sending out sensitive plant burkholderia The optimization of ferment culture medium provides a kind of Optimal compositions of fermentation medium formula of high yield PHA.
It is excellent by the fermentation condition to second order fermentation it is a still further object of the present invention to provide a kind of fermentation condition of optimization Change, the optimal conditions of fermentation of high yield PHA when second order fermentation is provided.
In order to realize these purposes and other advantages according to this explanation, a kind of sensitive plant burkholderia hair is provided Ferment produces the culture medium and its fermentation process of PHA, including：
It counts in parts by weight, including following components：
5~40 parts of glucose, 0.05~0.6 part of peptone, Na2HPO4.12H24~10 parts of O, MgSO4.7H2O 0.05~ 0.6 part, KH2PO43~8 parts, K2HPO43~8 parts and 0.2~0.6 part of trace element solution, 1000 are settled to distilled water Part；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01~0.03 part, H3BO30.01~0.05 part, Na2MoO40.01~0.05 part, CaCl20.05~ 0.25 part, FeCl30.01~0.04 part, ZnSO40.01~0.04 part and 1000 parts of distilled water.
Preferably, it counts in parts by weight, including following components：
8~35 parts of glucose, 0.1~0.4 part of peptone, Na2HPO4.12H24.5~8 parts of O, MgSO4.7H2O 0.15~ 0.4 part, KH2PO43~8 parts, K2HPO43~8 parts and 0.2~0.6 part of trace element solution, 1000 are settled to distilled water Part；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01~0.03 part, H3BO30.01~0.05 part, Na2MoO40.01~0.05 part, CaCl20.05~ 0.25 part, FeCl30.01~0.04 part, ZnSO40.01~0.04 part and 1000 parts of distilled water.
Preferably, it counts in parts by weight, including following components：
10~25 parts of glucose, 0.15~0.35 part of peptone, Na2HPO4.12H25~6.8 parts of O, MgSO4.7H2O 0.15~0.35, KH2PO43~8 parts, K2HPO43~8 parts and 0.2~0.6 part of trace element solution, are settled to distilled water 1000 parts；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01~0.03 part, H3BO30.01~0.05 part, Na2MoO40.01~0.05 part, CaCl20.05~ 0.25 part, FeCl30.01~0.04 part, ZnSO40.01~0.04 part and 1000 parts of distilled water.
Preferably, it counts in parts by weight, including following components：
13~18 parts of glucose, 0.25~0.3 part of peptone, Na2HPO4.12H25.5~6.2 parts of O, MgSO4.7H2O 0.25~0.3 part, KH2PO43~8 parts, K2HPO43~8 parts and 0.2~0.6 part of trace element solution, are settled to distilled water 1000 parts；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01~0.03 part, H3BO30.01~0.05 part, Na2MoO40.01~0.05 part, CaCl20.05~ 0.25 part, FeCl30.01~0.04 part, ZnSO40.01~0.04 part and 1000 parts of distilled water.
Preferably, it counts in parts by weight, including following components：
15 parts of glucose, 0.3 part of peptone, Na2HPO4.12H25.7 parts of O, MgSO4.7H20.3 part of O, KH2PO43~8 Part, K2HPO43~8 parts and 0.2~0.6 part of trace element solution, 1000 parts are settled to distilled water；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01~0.03 part, H3BO30.01~0.05 part, Na2MoO40.01~0.05 part, CaCl20.05~ 0.25 part, FeCl30.01~0.04 part, ZnSO40.01~0.04 part and 1000 parts of distilled water.
Preferably, it is inoculated with and a certain amount of lives through strain in the fermentation medium as described in any one of claim 1-5 The secondary seed culture solution progress fermented and cultured certain time for changing and expanding the sensitive plant Burkholder strain of culture is sent out Zymotic fluid.
Sensitive plant Burkholder thalline is collected from the zymotic fluid, for extracting PHA.
Preferably, the condition of fermented and cultured is：24 DEG C~30 DEG C, 160~220rpm of rotating speed of temperature, inoculum concentration 5%~ 20%, the initial pH value of fermentation medium is 6.5~7.5, fermentation time 48h.
Preferably, further comprising the steps of：
In the period of preceding 30h since the fermented and cultured or the preceding 40h since the fermented and cultured, continue to the fermentation It ventilates in culture medium, ventilatory capacity 300L/h.
Within the period that fermentation time is 30h~40h, grape is added one or more times into the fermentation medium Sugar, the total addition level of glucose are the initial glucose amount being twice in the fermentation medium.
Preferably, further comprising the steps of：
Fermentation time be 30h~40h period in, once added into the fermentation medium glucose it Before, it is passed through that cold air is primary, and cold air temperature is set as 0 DEG C~4 DEG C into the fermentation medium, the duration for being passed through cold air is 10 ~30s is passed through the finish time of cold air and is spaced 3~5min between being carved at the beginning of adding glucose.
Alternatively,
Within the period that fermentation time is 30h~40h, it is passed through cold air several times into the fermentation medium, and divide Cold air is repeatedly passed through with addition glucose several times successively alternately, wherein be passed through every time finish time of cold air with it is next At the beginning of secondary addition glucose carve between be spaced 3~5min, every time add glucose finish time be passed through next time it is cold 35~45min is spaced between being carved at the beginning of gas.
Preferably, further include slant medium and seed culture medium, count in parts by weight, the slant medium includes Following components：
KH2PO40.1~0.4 part, K2HPO40.1~0.4 part, 0.03~0.25 part of NaCl, 3~15 parts of mannitol, ferment 0.5~5.5 part of female powder, MgSO4.7H21000 parts of 0.05~0.45 part of O, 5~20 parts of agar and distilled water, PH 6.5~7.0.
It counts in parts by weight, the seed culture medium includes following components：
1000 parts of 1~6 part of beef extract, 5~20 parts of peptone, 1~8 part of NaCl and distilled water, PH7~7.2.
The present invention includes at least following advantageous effect：
The present invention by glucose in sensitive plant burkholderia basal medium formulation, peptone, Na2HPO4.12H2O、MgSO4.7H2Tetra- kinds of components of O carry out the horizontal orthogonal test of four factor three, are obtained according to orthogonal experiments Sensitive plant burkholderia production PHA Optimal compositions of fermentation medium be：15 parts of glucose, 0.3 part of peptone, Na2HPO4.12H25.7 parts of O, MgSO4.7H20.3 part of O, KH2PO45.3 parts, K2HPO45.3 parts and trace element solution 0.3 Part, it is settled to 1000 parts with distilled water；It counts in parts by weight, the trace element solution, including following components：CuSO4 0.01 Part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO40.02 part and distilled water 1000 parts.Filtering out optimal conditions of fermentation by single factor test is：28 DEG C, rotating speed 200rpm, inoculum concentration 20%, initial pH value 7.2, Add the amount for being doubled in Optimal compositions of fermentation medium glucose into fermentation medium respectively in fermentation 32h and 36h, fermentation time is 48h.Sensitive plant burkholderia carries out fermented and cultured using Optimal compositions of fermentation medium and optimal conditions of fermentation so that PHA is produced Amount has 4.19g/L to improve to 5.43g/L.
Part is illustrated to embody by further advantage, target and the feature of the present invention by following, and part will also be by this The research and practice of invention and be understood by the person skilled in the art.
Description of the drawings
Fig. 1 is the different temperatures of invention to sensitive plant burkholderia cell density OD600The influence of value and PHA yield；
Fig. 2 is the different rotating speeds of invention to sensitive plant burkholderia cell density OD600The influence of value and PHA yield；
Fig. 3 is the different pH sensitive plants burkholderias of invention to cell density OD600The influence of value and PHA yield；
Fig. 4 is the different vaccination amount of invention to sensitive plant burkholderia cell density OD600The shadow of value and PHA yield It rings.
Specific implementation mode
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art with reference to specification text Word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein are not precluded one or more The presence or addition of a other elements or combinations thereof.
Embodiment 1
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
1) actication of culture：By the sensitive plant burkholderia of freezen protective in being activated on slant medium, picking single bacterium It falls and is inoculated in seed culture medium described in 50mL, 28 DEG C, culture is used as primary seed solution afterwards for 24 hours, by first order seed described in 50mL Liquid is transferred in the seed culture medium described in 150mL, and culture is used as secondary seed solution afterwards for 24 hours.
The inclined-plane culture based formulas is：KH2PO40.25 part, K2HPO40.25 part, 0.1 part of NaCl, mannitol 10 Part, 3 parts of yeast powder, MgSO4.7H21000 parts of 0.2 part of O, 14 parts of agar and distilled water, PH6.5~7.0.
The seed culture based formulas is：1000 parts of 3 parts of beef extract, 10 parts of peptone, 5 parts of NaCl and distilled water, PH7 ~7.2.
2) Spawn incubation：It is packed into fermentation tank by fermentative medium formula configuration fermentation medium 4.5L, temperature is waited for after sterilizing It is down to room temperature, the second order fermentation culture solution is inoculated in by inoculum concentration 5% in fermentation tank.
The fermentative medium formula is：20 parts of glucose, 0.4 part of peptone, Na2HPO4.12H27.6 parts of O, MgSO4.7H20.4 part of O, KH2PO45.3 parts, K2HPO45.3 parts and 0.3 part of trace element solution, are settled to distilled water 1000 parts；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01 part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO4 0.02 part and 1000 parts of distilled water.
3) fermented and cultured：Temperature is set to 24 DEG C, 26 DEG C, 28 DEG C and 30 DEG C, rotating speed 180rpm, and initial pH value 6.8 is led to Tolerance is 300L/h, and fermented and cultured 48h terminates.After fermentation different temperatures is measured respectively using ultraviolet specrophotometer to issue Zymotic fluid cell density OD600Value, the results are shown in Figure 1.
4) thalline is collected, PHA is extracted.Drying measures PHA yield under different temperatures, and the results are shown in Figure 1.
As shown in Figure 1, sensitive plant Burkholder fermented liquid cell density OD600Value and PHA yield are increased with temperature It is in up-trend, is begun to decline after 28 DEG C, therefore select optimum fermentation temp for 28 DEG C.
Embodiment 2
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
Fermentative medium formula is：15 parts of glucose, 0.3 part of peptone, Na2HPO4.12H25.7 parts of O, MgSO4.7H2O 0.3 part, KH2PO45.3 parts, K2HPO45.3 parts and 0.3 part of trace element solution, 1000 parts are settled to distilled water；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01 part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO4 0.02 part and 1000 parts of distilled water.
Fermented and cultured：Temperature is set as 28 DEG C.
Other operating procedures are the same as the embodiment 1.
Sensitive plant burkholderia fermenting and producing PHA, it is 5.26g/L that drying weighing, which measures PHA yield, than existing skill Art is not optimised fermentation condition and culture medium improves 25.5%.
Embodiment 3
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
1) with the step 1) of embodiment 1
2) with the step 2) of embodiment 1
3) fermented and cultured：At 28 DEG C, rotating speed is set to 160rpm, 180rpm, 200rpm and 220rpm, initial pH value 6.8, ventilatory capacity 300L/h, fermented and cultured 48h terminates.Different turn is measured respectively using ultraviolet specrophotometer after fermentation The lower zymotic fluid cell density OD of speed600Value, the results are shown in Figure 2.
4) thalline is collected, PHA is extracted.Drying measures PHA yield under different rotating speeds, and the results are shown in Figure 2.
As shown in Figure 2, sensitive plant Burkholder fermented liquid cell density OD600Value increases as rotating speed increases, but Cell density variation is little after 200rpm；PHA yield is in increase trend as rotating speed increases, and is begun to decline after 200rpm, Therefore the rotating speed that selects most preferably to ferment is 200rpm.
Embodiment 4
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
Fermentative medium formula is：15 parts of glucose, 0.3 part of peptone, Na2HPO4.12H25.7 parts of O, MgSO4.7H2O 0.3 part, KH2PO45.3 parts, K2HPO45.3 parts and 0.3 part of trace element solution, 1000 parts are settled to distilled water；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01 part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO4 0.02 part and 1000 parts of distilled water.
Fermented and cultured：Rotating speed is 200rpm.
Other operating procedures are the same as the embodiment 3.
Sensitive plant burkholderia fermenting and producing PHA, it is 5.21g/L that drying weighing, which measures PHA yield, than existing skill Art is not optimised fermentation condition and culture medium improves 24.3%.
Embodiment 5
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
1) with the step 1) of embodiment 1
2) with the step 2) of embodiment 1
3) fermented and cultured：At 28 DEG C, rotating speed 200rpm, initial pH are modulated to 6.5,6.8,7.2 and 7.5 respectively, ventilatory capacity Terminate for 300L/h, fermented and cultured 48h.Zymotic fluid bacterium under different pH is measured respectively using ultraviolet specrophotometer after fermentation Volume density OD600Value, the results are shown in Figure 3.
4) thalline is collected, PHA is extracted.Drying measures PHA yield under different pH, and the results are shown in Figure 3.
From the figure 3, it may be seen that sensitive plant Burkholder fermented liquid cell density OD600Value and PHA yield are increased with pH value It is in increase trend, begins to decline after pH7.2, therefore the pH that selects most preferably to ferment is 7.2.
Embodiment 6
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
Fermentative medium formula is：15 parts of glucose, 0.3 part of peptone, Na2HPO4.12H25.7 parts of O, MgSO4.7H2O 0.3 part, KH2PO45.3 parts, K2HPO45.3 parts and 0.3 part of trace element solution, 1000 parts are settled to distilled water；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01 part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO4 0.02 part and 1000 parts of distilled water.
Fermented and cultured：Initial pH is set as 7.2.
Other operating procedures are the same as the embodiment 5.
Sensitive plant burkholderia fermenting and producing PHA, it is 5.30g/L that drying weighing, which measures PHA yield, than existing skill Art is not optimised fermentation condition and culture medium improves 26.4%.
Embodiment 7
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
1) with the step 1) of embodiment 1
2) Spawn incubation：It is packed into fermentation tank by fermentative medium formula configuration fermentation medium 4.5L, temperature is waited for after sterilizing It is down to room temperature, the second order fermentation culture solution is inoculated in by inoculum concentration 5%, 10%, 20% and 25% in fermentation tank respectively.
The fermentative medium formula is：20 parts of glucose, 0.4 part of peptone, Na2HPO4.12H27.6 parts of O, MgSO4.7H20.4 part of O, KH2PO45.3 parts, K2HPO45.3 parts and 0.3 part of trace element solution, are settled to distilled water 1000 parts；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01 part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO4 0.02 part and 1000 parts of distilled water.
3) fermented and cultured：At 28 DEG C, rotating speed 200rpm, initial pH7.2, ventilatory capacity 300L/h, fermented and cultured 48h is tied Beam.Zymotic fluid cell density OD under different vaccination amount is measured respectively using ultraviolet specrophotometer after fermentation600Value, as a result As shown in Figure 4.
4) thalline is collected, PHA is extracted.Drying measures PHA yield under different temperatures, and the results are shown in Figure 4.
As shown in Figure 4, sensitive plant Burkholder fermented liquid cell density OD600Value and PHA yield add with inoculum concentration Be in greatly increase trend, increasing inoculum concentration again after inoculum concentration 20% then begins to decline, therefore select most preferably to ferment inoculum concentration for 20%.
Embodiment 8
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
Spawn incubation：The second order fermentation culture solution is inoculated in by inoculum concentration 20% in fermentation tank respectively.
Fermentative medium formula is：15 parts of glucose, 0.3 part of peptone, Na2HPO4.12H25.7 parts of O, MgSO4.7H2O 0.3 part, KH2PO45.3 parts, K2HPO45.3 parts and 0.3 part of trace element solution, 1000 parts are settled to distilled water；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01 part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO4 0.02 part and 1000 parts of distilled water.
Other operating procedures are identical as the embodiment 7.
Sensitive plant burkholderia fermenting and producing PHA, it is 5.35g/L that drying weighing, which measures PHA yield, than existing skill Art is not optimised fermentation condition and culture medium improves 27.7%.
Embodiment 9
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
1) with the step 1) of embodiment 1
2) Spawn incubation：It is packed into fermentation tank by fermentative medium formula configuration fermentation medium 4.5L, temperature is waited for after sterilizing It is down to room temperature, the second order fermentation culture solution is inoculated in by inoculum concentration 20% in fermentation tank.
The fermentative medium formula is：5 parts of glucose, 0.05 part of peptone, Na2HPO4.12H24 parts of O, MgSO4.7H20.05 part of O, KH2PO45.3 parts, K2HPO45.3 parts and 0.3 part of trace element solution, are settled to distilled water 1000 parts；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01 part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO4 0.02 part and 1000 parts of distilled water.
3) fermented and cultured：At 28 DEG C, rotating speed 200rpm, initial pH7.2, ventilatory capacity 300L/h, fermented and cultured 48h is tied Beam.
4) thalline is collected, PHA is extracted.
Burkholderia fermenting and producing PHA, it is 4.20g/L that drying weighing, which measures PHA yield, more unexcellent than the prior art Change fermentation condition and culture medium improves 0.2%.
Embodiment 10
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
1) with the step 1) of embodiment 1
2) Spawn incubation：It is packed into fermentation tank by fermentative medium formula configuration fermentation medium 4.5L, temperature is waited for after sterilizing It is down to room temperature, the second order fermentation culture solution is inoculated in by inoculum concentration 20% in fermentation tank.
The fermentative medium formula is：20 parts of glucose, 0.5 part of peptone, Na2HPO4.12H27.6 parts of O, MgSO4.7H20.3 part of O, KH2PO45.3 parts, K2HPO45.3 parts and 0.3 part of trace element solution, are settled to distilled water 1000 parts；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01 part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO4 0.02 part and 1000 parts of distilled water.
3) fermented and cultured：At 28 DEG C, rotating speed 200rpm, initial pH7.2, ventilatory capacity 300L/h, fermented and cultured 48h is tied Beam.
4) thalline is collected, PHA is extracted.
Burkholderia fermenting and producing PHA, it is 4.79g/L that drying weighing, which measures PHA yield, more unexcellent than the prior art Change fermentation condition and culture medium improves 14.3%.
Embodiment 11
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
1) with the step 1) of embodiment 1
2) Spawn incubation：It is packed into fermentation tank by fermentative medium formula configuration fermentation medium 4.5L, temperature is waited for after sterilizing It is down to room temperature, the second order fermentation culture solution is inoculated in by inoculum concentration 20% in fermentation tank.
The fermentative medium formula is：25 parts of glucose, 0.4 part of peptone, Na2HPO4.12H29.5 parts of O, MgSO4.7H20.3 part of O, KH2PO45.3 parts, K2HPO45.3 parts and 0.3 part of trace element solution, are settled to distilled water 1000 parts；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01 part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO4 0.02 part and 1000 parts of distilled water.
3) fermented and cultured：At 28 DEG C, rotating speed 200rpm, initial pH7.2, ventilatory capacity 300L/h, fermented and cultured 48h is tied Beam.
4) thalline is collected, PHA is extracted.
Sensitive plant burkholderia fermenting and producing PHA, it is 4.63g/L that drying weighing, which measures PHA yield, than existing skill Art is not optimised fermentation condition and culture medium improves 10.5%.
Embodiment 12
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
1) with the step 1) of embodiment 1
2) Spawn incubation：It is packed into fermentation tank by fermentative medium formula configuration fermentation medium 4.5L, temperature is waited for after sterilizing It is down to room temperature, the second order fermentation culture solution is inoculated in by inoculum concentration 20% in fermentation tank.
The fermentative medium formula is：40 parts of glucose, 0.6 part of peptone, Na2HPO4.12H210 parts of O, MgSO4.7H20.6 part of O, KH2PO45.3 parts, K2HPO45.3 parts and 0.3 part of trace element solution, are settled to distilled water 1000 parts；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01 part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO4 0.02 part and 1000 parts of distilled water.
3) fermented and cultured：At 28 DEG C, rotating speed 200rpm, initial pH7.2, ventilatory capacity 300L/h, fermented and cultured 48h is tied Beam.
4) thalline is collected, PHA is extracted.
Sensitive plant burkholderia fermenting and producing PHA, it is 4.22g/L that drying weighing, which measures PHA yield, than existing skill Art is not optimised fermentation condition and culture medium improves 0.7%, effect unobvious.
Embodiment 13
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
1) with the step 1) of embodiment 1
2) Spawn incubation：It is packed into fermentation tank by fermentative medium formula configuration fermentation medium 4.5L, temperature is waited for after sterilizing It is down to room temperature, the second order fermentation culture solution is inoculated in by inoculum concentration 20% in fermentation tank.
The fermentative medium formula is：15 parts of glucose, 0.3 part of peptone, Na2HPO4.12H25.7 parts of O, MgSO4.7H20.3 part of O, KH2PO45.3 parts, K2HPO45.3 parts and 0.3 part of trace element solution, are settled to distilled water 1000 parts；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01 part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO4 0.02 part and 1000 parts of distilled water.
3) fermented and cultured：At 28 DEG C, rotating speed 200rpm, initial pH7.2, ventilatory capacity 300L/h, fermentation time 36h, It is passed through 0 DEG C of cold air 20s, it is to be twice in the amount of the fermentation medium glucose that interval 5min, which adds glucose total amount, cultivates 48h Terminate.
4) thalline is collected, PHA is extracted.
Sensitive plant burkholderia fermenting and producing PHA, it is 5.38g/L that drying weighing, which measures PHA yield, than existing skill Art is not optimised fermentation condition and culture medium improves 28.4%.
Embodiment 14
The fermentation process of primary gram of Hall bacterium fermenting and producing PHA culture medium of sensitive plant, including following key step：
1) with the step 1) of embodiment 1
2) Spawn incubation：It is packed into fermentation tank by fermentative medium formula configuration fermentation medium 4.5L, temperature is waited for after sterilizing It is down to room temperature, the second order fermentation culture solution is inoculated in by inoculum concentration 20% in fermentation tank.
The fermentative medium formula is：15 parts of glucose, 0.3 part of peptone, Na2HPO4.12H25.7 parts of O, MgSO4.7H20.3 part of O, KH2PO45.3 parts, K2HPO45.3 parts and 0.3 part of trace element solution, are settled to distilled water 1000 parts；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01 part, H3BO30.01 part, Na2MoO40.01 part, CaCl20.1 part, FeCl30.02 part, ZnSO4 0.02 part and 1000 parts of distilled water.
3) fermented and cultured：At 28 DEG C, rotating speed 200rpm, initial pH7.2, ventilatory capacity 300L/h, fermentation time 36h, It is passed through 0 DEG C of cold air 20s for the first time, waits for that being spaced 5min after being passed through cold air for the first time adds a glucose, then be spaced 40min is passed through 0 DEG C of cold air 20s for the second time, waits for being spaced 5min after being passed through cold air for the second time, then adds a glucose, and two Secondary added glucose total amount is to be twice in the amount of the fermentation medium glucose, and culture 48h terminates.
4) thalline is collected, PHA is extracted.
Sensitive plant burkholderia fermenting and producing PHA, it is 5.43g/L that drying weighing, which measures PHA yield, than existing skill Art is not optimised fermentation condition and culture medium improves 29.5%.Present embodiment is the best fermentation condition and fermented and cultured of the present invention Base.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited In specific details and legend shown and described herein.

Claims (10)

1. a kind of fermentation medium of sensitive plant burkholderia fermenting and producing PHA, which is characterized in that it counts in parts by weight, Including following components：
5~40 parts of glucose, 0.05~0.6 part of peptone, Na2HPO4.12H24~10 parts of O, MgSO4.7H2O 0.05~0.6 Part, KH2PO43~8 parts, K2HPO43~8 parts and 0.2~0.6 part of trace element solution, 1000 parts are settled to distilled water；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01~0.03 part, H3BO30.01~0.05 part, Na2MoO40.01~0.05 part, CaCl20.05~0.25 part, FeCl30.01~0.04 part, ZnSO40.01~0.04 part and 1000 parts of distilled water.
2. the fermentation medium of sensitive plant burkholderia fermenting and producing PHA as described in claim 1, which is characterized in that It counts in parts by weight, including following components：
8~35 parts of glucose, 0.1~0.4 part of peptone, Na2HPO4.12H24.5~8 parts of O, MgSO4.7H2O 0.15~0.4 Part, KH2PO43~8 parts, K2HPO43~8 parts and 0.2~0.6 part of trace element solution, 1000 parts are settled to distilled water；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01~0.03 part, H3BO30.01~0.05 part, Na2MoO40.01~0.05 part, CaCl20.05~0.25 part, FeCl30.01~0.04 part, ZnSO40.01~0.04 part and 1000 parts of distilled water.
3. the fermentation medium of sensitive plant Burkholder fermenting and producing PHA as described in claim 1, which is characterized in that press Parts by weight meter, including following components：
10~25 parts of glucose, 0.15~0.35 part of peptone, Na2HPO4.12H25~6.8 parts of O, MgSO4.7H2O 0.15~ 0.35、KH2PO43~8 parts, K2HPO43~8 parts and 0.2~0.6 part of trace element solution, 1000 parts are settled to distilled water；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01~0.03 part, H3BO30.01~0.05 part, Na2MoO40.01~0.05 part, CaCl20.05~0.25 part, FeCl30.01~0.04 part, ZnSO40.01~0.04 part and 1000 parts of distilled water.
4. the fermentation medium of sensitive plant Burkholder fermenting and producing PHA as described in claim 1, which is characterized in that press Parts by weight meter, including following components：
13~18 parts of glucose, 0.25~0.3 part of peptone, Na2HPO4.12H25.5~6.2 parts of O, MgSO4.7H2O 0.25~ 0.3 part, KH2PO43~8 parts, K2HPO43~8 parts and 0.2~0.6 part of trace element solution, 1000 parts are settled to distilled water；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01~0.03 part, H3BO30.01~0.05 part, Na2MoO40.01~0.05 part, CaCl20.05~0.25 part, FeCl30.01~0.04 part, ZnSO40.01~0.04 part and 1000 parts of distilled water.
5. the fermentation medium of sensitive plant burkholderia fermenting and producing PHA as described in claim 1, which is characterized in that It counts in parts by weight, including following components：
15 parts of glucose, 0.3 part of peptone, Na2HPO4.12H25.7 parts of O, MgSO4.7H20.3 part of O, KH2PO43~8 parts, K2HPO43~8 parts and 0.2~0.6 part of trace element solution, 1000 parts are settled to distilled water；
It counts in parts by weight, the trace element solution, including following components：
CuSO40.01~0.03 part, H3BO30.01~0.05 part, Na2MoO40.01~0.05 part, CaCl20.05~0.25 part, FeCl30.01~0.04 part, ZnSO40.01~0.04 part and 1000 parts of distilled water.
6. a kind of fermentation process of fermenting and producing PHA, which is characterized in that include the following steps：
It is inoculated in the fermentation medium as described in any one of claim 1-5 a certain amount of through actication of culture and expansion culture The secondary seed culture solution of sensitive plant Burkholder strain carries out fermented and cultured certain time acquisition zymotic fluid；
Sensitive plant Burkholder thalline is collected from the zymotic fluid, for extracting PHA.
7. the fermentation process of fermenting and producing PHA as claimed in claim 6, which is characterized in that the condition of fermented and cultured is：Temperature 24 DEG C~30 DEG C, 160~220rpm of rotating speed, the initial pH value of inoculum concentration 5%~20%, fermentation medium is 6.5~7.5, hair The ferment time is 48h.
8. the fermentation process of fermenting and producing PHA as claimed in claim 6, which is characterized in that further comprising the steps of：
In the period of preceding 30h since the fermented and cultured or the preceding 40h since the fermented and cultured, continue to the fermented and cultured Blowing air in base, blowing air amount are 300L/h；
It is primary into the fermentation medium or add glucose several times within the period that fermentation time is 30h~40h, The total addition level of glucose is the initial glucose amount being twice in the fermentation medium.
9. the fermentation process of fermenting and producing PHA as claimed in claim 8, which is characterized in that further comprising the steps of：
Within the period that fermentation time is 30h~40h, before once adding glucose into the fermentation medium, to It is primary that cold air is passed through in the fermentation medium, cold air temperature is set as 0 DEG C~4 DEG C, be passed through cold air duration be 10~ 30s is passed through the finish time of cold air and is spaced 3~5min between being carved at the beginning of adding glucose；
Alternatively,
Within the period that fermentation time is 30h~40h, it is passed through cold air several times into the fermentation medium, and several times Cold air is passed through with addition glucose several times successively alternately, wherein be passed through finish time of cold air every time and add next time It is spaced 3~5min between being carved at the beginning of adding glucose, add the finish time of glucose every time and is passed through cold air next time 35~45min is spaced between start time.
10. the fermentation process of fermenting and producing PHA as claimed in claim 6, which is characterized in that further include slant medium and kind Sub- culture medium, is counted in parts by weight, and the slant medium includes following components：
KH2PO40.1~0.4 part, K2HPO40.1~0.4 part, 0.03~0.25 part of NaCl, 3~15 parts of mannitol, yeast powder 0.5 ~5.5 parts, MgSO4.7H21000 parts of 0.05~0.45 part of O, 5~20 parts of agar and distilled water, PH 6.5~7.0；
It counts in parts by weight, the seed culture medium includes following components：
1000 parts of 1~6 part of beef extract, 5~20 parts of peptone, 1~8 part of NaCl and distilled water, PH7~7.2.
CN201810551329.5A 2018-05-31 2018-05-31 The culture medium and its fermentation process of primary gram of Hall bacterium fermenting and producing PHA of sensitive plant Pending CN108660166A (en)

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