CN102120973A - Halomonas strain and application thereof - Google Patents
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Abstract
本发明公开了一株盐单胞菌及其应用。该菌株为盐单胞菌(Halomonas sp.)TD01，其保藏编号为CGMCC NO.4353。实验表明，本发明菌株盐单胞菌(Halomonas sp.)TD01可在矿物培养基(MM)中高效积累聚羟基脂肪酸酯(PHA)，为PHA和PHBV的生物合成提供了良好的保证。本发明的盐单胞菌营养要求简单、发酵过程无需灭菌，可以连续进行，简单易于控制。本发明的几种利用嗜盐菌Halomonas TD01制备PHA的方法均不同程度的降低了生产成本，提高了PHA的产量，且得到的PHA的分子量可达500kDa以上，具有工业应用价值。The invention discloses a halomonas strain and application thereof. The bacterial strain is Halomonas sp. TD01, and its preservation number is CGMCC NO.4353. Experiments show that the strain Halomonas sp. TD01 of the present invention can efficiently accumulate polyhydroxyalkanoate (PHA) in mineral medium (MM), which provides a good guarantee for the biosynthesis of PHA and PHBV. The Halomonas bacterium of the invention has simple nutritional requirements, the fermentation process does not need to be sterilized, can be carried out continuously, and is simple and easy to control. Several methods for preparing PHA by using the halophilic bacteria Halomonas TD01 of the present invention reduce the production cost to varying degrees, increase the output of PHA, and the molecular weight of the obtained PHA can reach more than 500kDa, which has industrial application value.
Description
Technical field
The present invention relates to a strain salt Zymomonas mobilis and an application thereof.
Background technology
In recent years, along with rapid development of economy, society rapidly increases the demand of the energy and material.But, shortage of resources, serious social concern such as environmental pollutions etc. have day by day proposed challenge to the supply based on the traditional material of oil, and people press for the material that a kind of energy substitutes the sustainable development of petrochemical complex plastics.A class is the degradable plastics of raw material with the renewable resources and polyhydroxyalkanoate (PHA) comes to this.
A kind of thermoplastic granulates that can store as carbon source and the energy of the PHA synthetic that is many microorganisms under carbon, situation that nitrogen nutrition is unbalance.According to free carbon chain length difference be divided into short chain PHA (4-6C, scl-PHA) and middle long-chain PHA (〉=6C, mcl-PHA).3-hydroxybutyric acid 3-hydroxyl pentanoate copolymer (PHBV) and poly-(PHB) are a kind of among the short chain PHA.PHA has excellent biological compatibility because of it, biodegradability, and good characteristic such as piezoelectricity and be known as " environmentally friendly material " has become technical field of biological material active research focus the most in recent years.It extensively is applied to biodegradable wrapping material, medical embedded material, fields such as controlled slow releasing carrier of medication.
Though the development of PHA industrial chain is swift and violent over past ten years, the complicacy of PHA manufacturing processed makes its manufacturing cost high, can't compete with the traditional material based on oil.Major cause comprises material cost, energy consumption, downstream processing cost etc.So attempt to take a lot of methods to reduce the production cost of PHA at present, such as, come the good superior strain of acquired character by genetic engineering modified or pathways metabolism transformation, explore simple and effective downstream extraction purification process, utilize cheap starting material etc.
Continuously ferment: be meant with certain speed and in fermentor tank, add fresh culture, flow out nutrient solution with identical speed simultaneously, thereby make the liquid measure in the fermentor tank keep the constant fermenting process.
Summary of the invention
An object of the present invention is to provide a strain salt Zymomonas mobilis.
Salt Zymomonas mobilis provided by the present invention is salt Zymomonas mobilis (Halomonas sp.) TD01, and its deposit number is CGMCC NO.4353.
The application of salt Zymomonas mobilis (Halomonas sp.) TD01CGMCC NO.4353 in preparation PHA also belongs to protection scope of the present invention.
In the above-mentioned application, described PHA is PHB or PHBV.
Another object of the present invention provides the method for a kind of PHA of preparation.
The method for preparing PHA provided by the present invention comprises the steps: fermenting salt Zymomonas mobilis (Halomonas sp.) TD01CGMCC NO.4353, obtains PHA, and fermention medium that uses in the described fermentation and fermenting container are all sterilized or be all unsterilised.
In the above-mentioned method for preparing PHA, the initial temperature of described fermentation is 20 ℃-45 ℃, is specially 20 ℃ or 37 ℃ or 45 ℃; The initial pH value of the system of described fermentation is 5.0-11.0, is specially 5.0 or 9.0 or 11.0; The initial dissolved oxygen of the system of described fermentation is 5%-100%, is specially 5% or 50% or 100%.
In the above-mentioned method for preparing PHA, the method for described fermentation comprises the steps:
(1) salt Zymomonas mobilis (Halomonas sp.) TD01CGMCC NO.4353 is inserted initial fermention medium, carry out fermentation culture;
(2) on the basis of step (1), replenish sugared source of adding and nitrogenous source, continue fermentation culture;
(3) on the basis of step (2), stop to replenish the adding nitrogenous source, continue to replenish the sugared source of adding, continue fermentation culture.
In the above-mentioned method for preparing PHA, the time of fermentation culture described in described step (1) and the step (2) is 12 hours; And/or the time of fermentation culture is 24 hours in the described step (3);
In the above-mentioned method for preparing PHA, in the described step (1), per 1 liter of initial fermention medium is made up of sodium-chlor 20g-200g, glucose 5g-100g, yeast powder 0g-50g, ammonium chloride 0.5g-20g, urea 0g-20g, sal epsom 0.1g-5g, potassium primary phosphate 0.5g-10g, disodium hydrogen phosphate dodecahydrate 1g-20g, 1mL-30mL trace element I and 0.1mL-10mL trace element II and tap water, is settled to 1 liter with tap water;
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 2g-10g, two hydration calcium chloride 1g-5g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of micro-II is prepared as follows: with Zinc vitriol 50mg-200mg, four hydration Manganous chloride tetrahydrate 10mg-50mg, boric acid 100mg-500mg, cobalt chloride hexahydrate 50mg-400mg, Salzburg vitriol 3mg-30mg, Nickel dichloride hexahydrate 5mg-50mg, two molybdic acid hydrate sodium 10mg-50mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II.
In the above-mentioned method for preparing PHA, in described 1 liter of initial fermention medium, the quality of described sodium-chlor is specially 20g or 60g or 200g, the quality of described glucose is specially 5g or 30g or 100g, the quality of described yeast powder is specially 0g or 1g or 50g, the quality of described ammonium chloride is specially 0.5g or 2g or 20g, the quality of described urea is specially 0g or 3g or 20g, the quality of described sal epsom is specially 0.1g or 0.2g or 5g, the quality of described potassium primary phosphate is specially 0.5g or 1.5g or 10g, the quality of described disodium hydrogen phosphate dodecahydrate is specially 1g or 9.65g or 20g, the volume of described micro-I is specially 1mL or 10mL or 30mL, and the volume of described micro-II is specially 0.1mL or 1mL or 10mL;
In the above-mentioned method for preparing PHA, among described 1 liter of described micro-I, the quality of ferric ammonium citrate is that the quality of 2g or 5g or 10g, two hydration calcium chloride is 1g or 2g or 5g;
In the above-mentioned method for preparing PHA, among described 1 liter of micro-II, the quality of Zinc vitriol is 50mg or 100mg or 200mg, the quality of four hydration Manganous chloride tetrahydrates is 10mg or 30mg or 50mg, the quality of boric acid is 100mg or 300mg or 500mg, and the quality of cobalt chloride hexahydrate is 50mg or 200mg or 400mg, and the quality of Salzburg vitriol is 3mg or 10mg or 30mg, the quality of Nickel dichloride hexahydrate is 5mg or 20mg or 50mg, and the quality of two molybdic acid hydrate sodium is 10mg or 30mg or 50mg;
In the above-mentioned method for preparing PHA, the additional way of described sugared source and described nitrogenous source is for to add once every 4 hours;
In the above-mentioned method for preparing PHA, in the described step (2), the described sugared source of at every turn adding and the proportioning of described initial fermentation system are 60g: 2L-4L, are specially 60g: 3L; Described initial fermentation system is made up of the seed liquor of salt Zymomonas mobilis (Halomonas sp.) the TD01CGMCC NO.4353 of described initial fermention medium and inoculation.
In the above-mentioned method for preparing PHA, in the described step (2), the described nitrogenous source of at every turn adding and the proportioning of described initial fermentation system are 6g: 2L-4L, are specially 6g: 3L; Described initial fermentation system is made up of the seed liquor of salt Zymomonas mobilis (Halomonas sp.) the TD01CGMCC NO.4353 of described initial fermention medium and inoculation.
In the above-mentioned method for preparing PHA, in the described step (3), the described sugared source of at every turn adding and the proportioning of described initial fermentation system are 60g: 2L-4L, are specially 60g: 3L; Described initial fermentation system is made up of the seed liquor of salt Zymomonas mobilis (Halomonas sp.) the TD01CGMCC NO.4353 of described initial fermention medium and inoculation.
Described sugared source and described nitrogenous source are to add by the form of its aqueous solution, and described water is tap water;
In the above-mentioned method for preparing PHA, described sugared source is a glucose, and described nitrogenous source is an ammonium salt.
In the above-mentioned method for preparing PHA, described PHA is PHB or PHBV.
Another object of the present invention provides the another kind of method for preparing PHA.
Another kind provided by the present invention prepares the method for PHA, comprises the steps: fermenting salt Zymomonas mobilis (Halomonas sp.) TD01CGMCC NO.4353, obtains PHA, and fermention medium that uses in the described fermentation and fermenting container are all sterilized or be all unsterilised.
Above-mentioned another kind prepares in the method for PHA, and the initial temperature of described fermentation is 20 ℃-45 ℃, is specially 20 ℃ or 37 ℃ or 45 ℃; The initial pH value of the system of described fermentation is 5.0-11.0, is specially 5.0 or 9.0 or 11.0; The initial dissolved oxygen of the system of described fermentation is 5%-100%, is specially 5% or 50% or 100%.
Above-mentioned another kind prepares in the method for PHA, and described fermentation is for continuously fermenting.
Above-mentioned another kind prepares in the method for PHA, and described method of continuously fermenting comprises the steps:
1) the bacterial strain access is equipped with among the fermenting container I of fermention medium I, carries out fermentation culture;
2) on the basis of step 1), add sugared source and nitrogenous source, continue fermentation culture;
3) with step 2) in the fermentation logistics that obtains be added among the fermenting container II that fermention medium II is housed, fermentation culture is carried out in stream sugaring source in fermenting container II simultaneously; From fermenting container II, collect thalline, the thalline after promptly obtaining fermenting;
Described fermented product is all substances in the fermenting container;
Above-mentioned another kind prepares in the method for PHA, 1 liter of fermention medium I is made up of sodium-chlor 20g-200g, glucose 5g-100g, yeast powder 0g-50g, ammonium chloride 0.5g-20g, urea 0g-20g, sal epsom 0.1g-5g, potassium primary phosphate 0.5g-10g, disodium hydrogen phosphate dodecahydrate 0g-20g, 1mL-30mL trace element I and 0.1mL-10mL trace element II and tap water, is settled to 1 liter with tap water;
1 liter of fermention medium II is made up of sodium-chlor 20g-200g, glucose 5g-100g, yeast powder 0g-50g, sal epsom 0.1g-5g, potassium primary phosphate 0.5g-10g, disodium hydrogen phosphate dodecahydrate 0g-20g, 1mL-30mL trace element I and 0.1mL-10mL trace element II and tap water, is settled to 1 liter with tap water;
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 2g-10g, two hydration calcium chloride 1g-5g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of micro-II is prepared as follows: with Zinc vitriol 50mg-200mg, four hydration Manganous chloride tetrahydrate 10mg-50mg, boric acid 100mg-500mg, cobalt chloride hexahydrate 50mg-400mg, Salzburg vitriol 3mg-30mg, Nickel dichloride hexahydrate 5mg-50mg, two molybdic acid hydrate sodium 10mg-50mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II.
Above-mentioned another kind prepares in the method for PHA, among described 1 liter of fermention medium I, the quality of described sodium-chlor is specially 20g or 60g or 200g, the quality of described glucose is specially 5g or 30g or 100g, the quality of described yeast powder is specially 0g or 1g or 50g, the quality of described ammonium chloride is specially 0.5g or 2g or 20g, the quality of described urea is specially 0g or 3g or 20g, the quality of described sal epsom is specially 0.1g or 0.2g or 5g, the quality of described potassium primary phosphate is specially 0.5g or 1.5g or 10g, the quality of described disodium hydrogen phosphate dodecahydrate is specially 0g or 9.65g or 20g, the volume of described micro-I is specially 1mL or 10mL or 30mL, and the volume of described micro-II is specially 0.1mL or 1mL or 10mL;
Above-mentioned another kind prepares in the method for PHA, among described 1 liter of fermention medium II, the quality of described sodium-chlor is specially 20g or 60g or 200g, the quality of described glucose is specially 5g or 30g or 100g, the quality of described yeast powder is specially 0g or 1g or 50g, the quality of described sal epsom is specially 0.1g or 0.2g or 5g, the quality of described potassium primary phosphate is specially 0.5g or 1.5g or 10g, the quality of described disodium hydrogen phosphate dodecahydrate is specially 0g or 9.65g or 20g, the volume of described micro-I is specially 1mL or 10mL or 30mL, and the volume of described micro-II is specially 0.1mL or 1mL or 10mL;
Above-mentioned another kind prepares in the method for PHA, and among described 1 liter of described micro-I, the quality of ferric ammonium citrate is that the quality of 2g or 5g or 10g, two hydration calcium chloride is 1g or 2g or 5g;
Above-mentioned another kind prepares in the method for PHA, among described 1 liter of micro-II, the quality of Zinc vitriol is 50mg or 100mg or 200mg, the quality of four hydration Manganous chloride tetrahydrates is 10mg or 30mg or 50mg, the quality of boric acid is 100mg or 300mg or 500mg, the quality of cobalt chloride hexahydrate is 50mg or 200mg or 400mg, the quality of Salzburg vitriol is 3mg or 10mg or 30mg, the quality of Nickel dichloride hexahydrate is 5mg or 20mg or 50mg, and the quality of two molybdic acid hydrate sodium is 10mg or 30mg or 50mg.
Above-mentioned another kind prepares in the method for PHA, and described sugared source and described nitrogenous source are to add by the form of its aqueous solution, and described water is tap water.
Above-mentioned another kind prepares in the method for PHA, and described sugared source is a glucose, and described nitrogenous source is an ammonium salt.
Above-mentioned another kind prepares in the method for PHA, and described PHA is PHB or PHBV.
Another object of the present invention provides another method for preparing PHA.
Provided by the present invention another prepare the method for PHA, comprise the steps: fermenting salt Zymomonas mobilis (Halomonas sp) TD01CGMCC NO.4353, obtain PHA, fermention medium that uses in the described fermentation and fermenting container are all sterilized or are all unsterilised.
Above-mentioned another prepare in the method for PHA, the initial temperature of described fermentation is 20 ℃-45 ℃, is specially 20 ℃ or 37 ℃ or 45 ℃; The initial pH value of the system of described fermentation is 5.0-11.0, is specially 5.0 or 9.0 or 11.0;
Above-mentioned another prepare in the method for PHA, the fermention medium that uses in the described fermentation is following 1), 2), 3) or 4) shown in:
1), optimizes the MM-G substratum;
2), forms by optimizing MM-G substratum and propionic acid, propionic acid is 0.5g-20g: 1L with the proportioning of optimization MM-G substratum;
3), forms by optimizing MM-G substratum and valeric acid, valeric acid is 0.5g-20g: 1L with the proportioning of optimization MM-G substratum;
4) produce the waste liquid that organic acid produces;
1 liter of described optimization MM-G substratum is made up of glucose 10g-50g, sodium-chlor 30g-100g, yeast powder 0g-5g, ammonium chloride 0.5g-10g, sal epsom 0.1g-1g, potassium primary phosphate 0.5g-5g, disodium hydrogen phosphate dodecahydrate 3g-15g, micro-I 3mL-20mL, micro-II 0.5mL-3mL and water, and water is supplied volume to 1 liter;
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 2g-10g, two hydration calcium chloride 1g-5g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of micro-II is prepared as follows: with Zinc vitriol 50mg-200mg, four hydration Manganous chloride tetrahydrate 10mg-50mg, boric acid 100mg-500mg, cobalt chloride hexahydrate 50mg-400mg, Salzburg vitriol 3mg-30mg, Nickel dichloride hexahydrate 5mg-50mg, two molybdic acid hydrate sodium 10mg-50mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II.
Above-mentioned another prepare in the method for PHA, in described 1 liter of described optimization MM-G substratum, the quality of glucose is 10g or 30g or 50g, the quality of sodium-chlor is 30g or 60g or 100g, the quality of yeast powder is 0g or 1g or 5g, the quality of ammonium chloride is 0.5g or 2g or 10g, the quality of sal epsom is 0.1g or 0.2g or 1g, the quality of potassium primary phosphate is 0.5g or 1.5g or 5g, the quality of disodium hydrogen phosphate dodecahydrate is 3g or 9.65g or 15g, the volume of trace element is I 3mL or 10ml or 20mL, and the volume of micro-II is 0.5mL or 1ml or 3mL;
Among described 1 liter of described micro-I, the quality of ferric ammonium citrate is that the quality of 2g or 5g or 10g, two hydration calcium chloride is 1g or 2g or 5g;
Among described 1 liter of micro-II, the quality of Zinc vitriol is 50mg or 100mg or 200mg, the quality of four hydration Manganous chloride tetrahydrates is 10mg or 30mg or 50mg, the quality of boric acid is 100mg or 300mg or 500mg, the quality of cobalt chloride hexahydrate is 50mg or 200mg or 400mg, the quality of Salzburg vitriol is 3mg or 10mg or 30mg, and the quality of Nickel dichloride hexahydrate is 5mg or 20mg or 50mg, and the quality of two molybdic acid hydrate sodium is 10mg or 30mg or 50mg;
Above-mentioned another prepare in the method for PHA, described propionic acid is 0.5g: 1L or 3g: 1L or 20g: 1L with the proportioning of optimizing the MM-G substratum;
Above-mentioned another prepare in the method for PHA, described valeric acid is 0.5g: 1L or 3g: 1L or 20g: 1L with the proportioning of optimizing the MM-G substratum.
Above-mentioned another prepare in the method for PHA, described PHA is PHB or PHBV.
Experiment shows, bacterial strain salt Zymomonas mobilis of the present invention (Halomonas sp.) TD01 can be in mineral substratum (MM) efficient accumulating poly hydroxy fatty acid (PHA), for the biosynthesizing of PHA provides good assurance.Salt Zymomonas mobilis nutritional requirement of the present invention is simple, fermenting process simply is easy to control.
Of the present invention several utilize method that halophilic bacterium Halomonas TD01 prepares PHA or PHBV etc. all in various degree reduction production cost, improved the output of PHA, and the molecular weight of the PHA that obtains can reach more than the 500kDa, have industrial application value.Specific as follows:
1. salt Zymomonas mobilis of the present invention (Halomonas spp TD01) can accumulate PHB in a large number in the fermention medium that with glucose is carbon source;
2. fermentation bacterial strain of the present invention can produce PHBV when adding propionic acid or valeric acid as additive carbon in the fermention medium that with glucose is carbon source;
With the mixing acid waste liquid as culture medium culturing Halomonas TD01, be used to prepare PHA, realized the cyclic utilization of waste, further reduce the cost that Halomonas TD01 produces PHA;
4. for halophilic bacterium, high salt concentration is that growth is necessary, Halomonas TD01 growth simultaneously also relies on higher pH, and the production substratum of high salt and high pH can suppress the growth of other non-halophilic bacterium, therefore allow nothing sterilization production technique become possibility, reduce the energy consumption that sterilization process produces, reduce infringement to greatest extent the microorganism growth substratum, thereby from reducing cost to a great extent;
5. because halophilic bacterium is lived in the hypersaline environment, break so thalline is met water, direct dilute with water bacterium liquid (adding low quantity of surfactant) just can discharge the PHA particle after the fermentation ends, has avoided the conventional P HA leaching process of loaded down with trivial details costliness; The salt of producing in the substratum is recovered after fermentation ends, can reuse, and has reduced water consumption and wastewater flow rate, reduces cost.
In sum, bacterial strain of the present invention and application method thereof prepare significant to the low cost of PHA, lay a good foundation for the range of application that enlarges PHA, have excellent industrial application foreground.
Description of drawings
Fig. 1 is unsterilised not inoculation fermentation experiment blank.
Fig. 2 is whether microbiological contamination in PCR the verifies unsterilised fermenting process.
Fig. 3 is the unsterilised synoptic diagram that continuously ferments.
Fig. 4 cultivates the standard substance of production PHB and the color atlas of sample for utilizing the optimization culture media shaking vase.
Fig. 5 adds the standard substance of propionic acid product PHBV and the color atlas of sample for optimizing substratum.
Fig. 6 adds the standard substance of valeric acid product PHBV and the color atlas of sample for optimizing substratum.
Fig. 7 produces the standard substance of PHBV and the color atlas of sample for the waste liquid of producing the organic acid generation.
Fig. 8 is that bacterial strain prepares the standard substance of PHB and the color atlas of sample under unsterilised condition.
Fig. 9 prepares PHB for bacterial strain continuously ferments under unsterilised condition the standard substance and the color atlas of sample.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The standard substance that use among the following embodiment are as follows:
The PHB standard substance are available from Sigma-Aldrich, and catalog number is 363502-10G.
The phenylformic acid standard substance are bought from State Standard Matter Research Centre, and production code member is GBW (E) 130035.
The PHBV standard substance are bought from Ningbo Tianan Biological Material Co., Ltd., and production code member is ENMAT-Y1000, and through further purifying, nucleus magnetic resonance (NMR) detects purity and each component concentration.The PHBV method of purification: PHBV is dissolved in chloroform, and centrifugal (5000 rev/mins, 5 minutes) remove precipitation; Chloroform splashes into excess ethyl alcohol mutually, centrifugal (5000 rev/mins, 5 minutes) collecting precipitation; 3-5 time so repeatedly.
The separation of embodiment 1, bacterial strain and evaluation
One, separates
From the Aydingkol of salt lake, Xinjiang, obtain soil sample and water sample, therefrom isolated strains.Well-grown bacterium colony is carried out fourier infrared (FI-IR) to be detected it and whether accumulates PHA.Obtain 10 strains and can produce the halophilic bacterium of PHA, select wherein a strain well-grown, the higher bacterial strain called after TD01 of PHA accumulation volume.
The LB substratum of separation and purification bacterial classification: sodium-chlor 60g/L, peptone 10g/L, yeast powder 1g/L, all the other components are water, the pH value is about 8.0;
Judge whether to accumulate the substratum of PHA for adding solid glucose mineral (MM-G) substratum of 2% agar.The MM-G substratum:
1 liter of MM-G substratum is prepared as follows: 10g glucose, 60g sodium-chlor, 0.5g yeast powder, 2g ammonium sulfate, 0.2g sal epsom, 1.5g potassium primary phosphate, 9.65g disodium hydrogen phosphate dodecahydrate, 10ml trace element I and 1ml trace element II are mixed, supply volume to 1 liter with deionized water.
1 liter of micro-I is prepared as follows: with ferric ammonium citrate 5g, two hydration calcium chloride 2g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid;
1 liter of micro-II is prepared as follows: with Zinc vitriol 100mg, four hydration Manganous chloride tetrahydrate 30mg, boric acid 300mg, cobalt chloride hexahydrate 200mg, Salzburg vitriol 10mg, Nickel dichloride hexahydrate 20mg, two molybdic acid hydrate sodium 30mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid.
Two. identify
1, identification of morphology
Activated spawn TD01, incubated overnight is diluted to 10
-4, to get 100mL and coat LB flat board (the same isolation medium of component), 37 ℃ of incubators were cultivated 48 hours, and observe colonial morphology: bacterium colony is rounded, the about 1mm of diameter, the edge is smooth, and is moistening, and white is translucent.Utilize the microscopic examination thalli morphology simultaneously, 100 times of amplifications, it is shaft-like that thalline is.
2, Physiology and biochemistry is identified
Bacterial strain is carried out gramstaining identify that the result is a Gram-negative bacteria.
3, Molecular Identification
Detect the 16S rDNA sequence of this bacterial strain, the primer of amplification 16S rDNA sequence is universal primer: 16F5 '-AGAGTTTGATCCTGGCTCAG-3 ', 16R 5 '-ACGGCTACCTTGTTACGACT-3 '.The sequence that records is shown in SEQ ID NO:1.
This sequence is compared in NCBI, and this bacterial strain and Halomonas sp.G29 similarity reach 99% as a result.
Comprehensive above qualification result is salt Zymomonas mobilis (Halomonas sp.) with this identification of strains.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 19th, 2010 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.4353.Strain name is TD01, classification called after salt Zymomonas mobilis (Halomonas sp.).
Three, Performance Detection
Bacterial strain Halomonas TD01 performance is detected, and substratum is LB, and 37 ℃ of following 200 rev/mins of shaking tables of condition were cultivated 48 hours, measured its OD600 and dry cell weight.
Change sodium chloride concentration in the substratum, carry out the experiment of sodium chloride concentration gradient.
Change culture temperature, carry out the thermograde experiment.
3 repetitions are established in experiment.The result shows that bacterial strain Halomonas TD01 is the very wide moderate halophilic bacterium of a kind of subject range, can grow under 10-200g/L sodium-chlor condition, the suitableeest sodium chloride concentration is 40-80g/L; Maximum growth temperature is 45 ℃, and optimum growth temperature is 30-40 ℃; Minimum growth pH is 5.0, and the highest growth pH is 11.0, and optimal pH is 7.5-9.5.
(1) utilizes the optimization culture media shaking vase to cultivate and produce PHB
The method that adopts single-factor variable and orthogonal test to combine is carried out the growth working condition optimization of bacterial strain TD01.Through optimization, consider each substrate cost simultaneously, obtain the optimal conditions of this bacteria growing and production PHA.
Optimal conditions is: 37 ℃ of temperature, pH value 9.0;
Method 1:
Optimize the MM-G substratum for 1 liter: be made up of 30g glucose, 60g sodium-chlor, 1g yeast powder, 2g ammonium chloride, 0.2g sal epsom, 1.5g potassium primary phosphate, 9.65g disodium hydrogen phosphate dodecahydrate, 10ml trace element I, 1ml trace element II and deionized water, deionized water is supplied volume to 1 liter.
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 5g, two hydration calcium chloride 2g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of described micro-II is prepared as follows: with Zinc vitriol 100mg, four hydration Manganous chloride tetrahydrate 30mg, boric acid 300mg, cobalt chloride hexahydrate 200mg, Salzburg vitriol 10mg, Nickel dichloride hexahydrate 20mg, two molybdic acid hydrate sodium 30mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II;
Optimizing the MM-G substratum for 1 liter is prepared as follows:
Substrate: 30g glucose, 60g sodium-chlor, 1g yeast powder, the 870mL dissolved in distilled water was sterilized 20 minutes for 121 ℃;
Glucose: 30g/L glucose, be mixed with 50 times of mother liquors, sterilized 20 minutes for 115 ℃;
Component I: 2g/L ammonium chloride, 0.2g/L sal epsom, be mixed with 50 times of mother liquors, sterilized 20 minutes for 121 ℃;
Component I I:1.5g/L potassium primary phosphate, 9.65g/L disodium hydrogen phosphate dodecahydrate are mixed with 50 times of mother liquors, sterilize 20 minutes for 121 ℃;
Trace element: micro-I 10ml/L, micro-II 1ml/L, be mixed with 50 times of mother liquors, mother liquor pH value is adjusted to 4.0～5.0, sterilizes 20 minutes for 121 ℃, and micro-I and micro-II preparation are the same;
Substrate, glucose, component I, component I I, trace element be sterilization separately respectively, after the cooling, gets 20mL glucose mother liquid, 20mL component I mother liquor, 20mL component I I mother liquor and 20mL trace element mother liquor respectively and adds substrate, re-adjustment pH to 9.0.
Shaking table is training English board HYG-A type, Taicang experimental installation factory, granary, Jiangsu, China.
Bacterial strain Halomonas TD01 is inoculated in the LB substratum, in 37 ℃, 200 rev/mins shaking tables, cultivates 12h, obtain seed culture fluid;
Seed culture fluid inserted by 5% inoculum size (being 5mL) be equipped with in the 500mL triangular flask that 100mL optimizes the MM-G substratum, under 37 ℃, pH9.0,200 rev/mins of conditions (amplitude is 30mm), shaking table was cultivated 48 hours, obtained fermented liquid.
Take out 10000 rev/mins of centrifugal 10 minutes collection thalline of 25mL fermented liquid, with deionized water wash twice, freezing ice is done.Thalline was weighed and is calculated dry cell weight (CDW) after ice was done.
Get the dried back of 30mg ice thalline and carry out esterification, gas-chromatography (GC) detects PHA content.
Esterification process is: gets 30mg ice dry mycelium in the esterification pipe, adds 2mL chloroform, 2mL esterifying liquid mixing, and covered and enclosed, esterification is 4 hours in 100 ℃ of baking ovens.After being cooled to room temperature, add 1mL distilled water, the mixing that fully vibrates, standing demix.After treating chloroform and water separating fully, get chloroform and carry out gas chromatographic analysis mutually.1 liter of esterifying liquid collocation method: 1g phenylformic acid, the 30mL vitriol oil are dissolved in 970mL methyl alcohol, obtain esterifying liquid.
The standard specimen material of getting 10mg simultaneously carries out esterification.
Gas-chromatography (GC) is analyzed: according to the Hewlett Packard of Hewlett-Packard Corporation 6890 (HP, USA) specification sheets of gas chromatograph operation gas chromatograph.Setting the column cap temperature is 140 ℃, and the sampler temperature is 200 ℃, and detector temperature is 220 ℃, and column head pressure is 0.25Mpa, and the temperature programming condition is: 140 ℃ 1 minute, be warming up to 220 ℃ with 20 ℃/minute speed, and kept 1 minute in this temperature.The sample feeding amount is 1 μ l, and sample introduction uses goes up the microsyringe that Hai'an booth microsyringe factory produces.
Under as above GC conditions, retention time 2.5 ± 0.1min of 3HB in the standard substance, the benzoic retention time of interior mark is 3.7 ± 0.1min.The retention time 2.4min of 3HB in the sample, the benzoic retention time of interior mark is 3.7min.The collection of illustrative plates of standard substance is shown in Fig. 4 A, and the collection of illustrative plates of sample is shown in Fig. 4 B.
3 repetitions are established in experiment, and the result takes the mean.
As a result, this thalline can synthesize PHB, and PHB is formed by the 3HB monomer polymerization.
Cultivate after 48 hours, somatic cells dry weight (CDW) reaches the 7.4g/L fermented liquid, and the PHB quality accounts for 68.5% of thalline weight.
Method 2:
Basic identical described in method and the method 1, different is that used substratum is formed and the fermentation condition difference, specific as follows:
Optimize the MM-G substratum for 1 liter: be made up of 10g glucose, 30g sodium-chlor, 0.5g ammonium chloride, 0.1g sal epsom, 0.5g potassium primary phosphate, 3g disodium hydrogen phosphate dodecahydrate, 3mL trace element I, 0.5mL trace element II and deionized water, deionized water is supplied volume to 1 liter.
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 2g, two hydration calcium chloride 1g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of micro-II is prepared as follows: with Zinc vitriol 50mg, four hydration Manganous chloride tetrahydrate 10mg, boric acid 100mg, cobalt chloride hexahydrate 50mg, Salzburg vitriol 3mg, Nickel dichloride hexahydrate 5mg, two molybdic acid hydrate sodium 10mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II.
The starting condition of fermentation is as follows: temperature is 20 ℃, and the pH value is 5.0.
3 repetitions are established in experiment, and the result takes the mean.
As a result, cultivate after 48 hours, somatic cells dry weight (CDW) reaches the 3.3g/L fermented liquid, and the PHB quality accounts for 23.6% of thalline weight.
Method 3:
Basic identical described in method and the method 1, different is that used substratum is formed and the fermentation condition difference, specific as follows:
Optimize the MM-G substratum for 1 liter: be made up of 50g glucose, 100g sodium-chlor, 5g yeast powder, 10g ammonium chloride, 1g sal epsom, 5g potassium primary phosphate, 15g disodium hydrogen phosphate dodecahydrate, 20mL trace element I, 3mL trace element II and deionized water, deionized water is supplied volume to 1 liter.
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 10g, two hydration calcium chloride 5g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of micro-II is prepared as follows: with Zinc vitriol 200mg, four hydration Manganous chloride tetrahydrate 50mg, boric acid 500mg, cobalt chloride hexahydrate 400mg, Salzburg vitriol 30mg, Nickel dichloride hexahydrate 50mg, two molybdic acid hydrate sodium 50mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II.
The starting condition of fermentation is as follows: temperature is 45 ℃, and the pH value is 11.0.
3 repetitions are established in experiment, and the result takes the mean.
As a result, cultivate after 48 hours, somatic cells dry weight (CDW) reaches the 2.8g/L fermented liquid, and the PHB quality accounts for 33.9% of thalline weight.
(2) add propionic acid or valeric acid and produce PHBV as additive carbon
1, adds propionic acid
Method 1:
Method 1 is basic identical in method and the experiment (one), and different is that used substratum is made up of optimization MM-G substratum and propionic acid in experiment (one) method 1, and the proportioning of the optimization MM-G substratum in propionic acid and experiment (one) method 1 is 3g: 1L.
3 repetitions are established in experiment, and the result takes the mean.
Under as above GC conditions, the retention time 2.7min of 3HB in the standard substance, the retention time of 3HV is 3.1min in the standard substance, the benzoic retention time of interior mark is 3.8min.The retention time 2.7min of 3HB in the sample, the retention time of 3HV is 3.1min in the sample, the benzoic retention time of interior mark is 3.8min.
The collection of illustrative plates of standard substance is shown in Fig. 5 A, and the collection of illustrative plates of sample is shown in Fig. 5 B.
The result: cultivated 48 hours, dry cell weight CDW is the 5.4g/L fermented liquid; Bacterium produces PHBV, and wherein the 3HV ratio is 11%.The PHBV quality accounts for 58.9% of thalline weight.
Method 2:
3 repetitions are established in experiment, and the result takes the mean.
The result: cultivated 48 hours, dry cell weight CDW is the 3.8g/L fermented liquid; Bacterium produces PHBV, and wherein the 3HV ratio is 6.7%.The PHBV quality accounts for 45.7% of thalline weight.
Method 3:
3 repetitions are established in experiment, and the result takes the mean.
The result: cultivated 48 hours, dry cell weight CDW is the 2.6g/L fermented liquid; Bacterium produces PHBV, and wherein the 3HV ratio is 12.5%.The PHBV quality accounts for 31.5% of thalline weight.
2, add valeric acid
Method 1:
Method 1 is basic identical in method and the experiment (one), and different is that used substratum is made up of optimization MM-G substratum and valeric acid in experiment (one) method 1, and the proportioning of the optimization MM-G substratum in valeric acid and experiment (one) method 1 is 3g: 1L.
3 repetitions are established in experiment, and the result takes the mean.
Under as above GC conditions, the retention time 2.7min of 3HB in the standard substance, the retention time of 3HV is 3.1min in the standard substance, the benzoic retention time of interior mark is 3.8min.The retention time 2.7min of 3HB in the sample, the retention time of 3HV is 3.0min in the sample, the benzoic retention time of interior mark is 3.6min.
The collection of illustrative plates of standard substance as shown in Figure 6A, the collection of illustrative plates of sample is shown in Fig. 6 B.
The result: cultivated 48 hours, dry cell weight CDW is the 6.5g/L fermented liquid; Bacterial strain produces PHBV, and wherein the 3HV ratio is 30%.PHBV accounts for 64.2% of thalline weight.
Method 2:
3 repetitions are established in experiment, and the result takes the mean.
The result: cultivated 48 hours, dry cell weight CDW is the 3.1g/L fermented liquid; Bacterium produces PHBV, and wherein the 3HV ratio is 12%.The PHBV quality accounts for 56.9% of thalline weight.
Method 3:
3 repetitions are established in experiment, and the result takes the mean.
The result: cultivated 48 hours, dry cell weight CDW is the 2.7g/L fermented liquid; Bacterium produces PHBV, and wherein the 3HV ratio is 33.6%.The PHBV quality accounts for 48.1% of thalline weight.
(3) the industrial acid mixture waste liquid is produced PHBV as substratum
Basic identical in method and the experiment (one), different is the waste liquid that used substratum produces for the production organic acid, and the pH value of this waste liquid is transferred to 9.0.
This mixing acid waste liquid main component is acetate, propionic acid, valeric acid, lactic acid, succsinic acid etc.
3 repetitions are established in experiment, and the result takes the mean.
Under as above GC conditions, the retention time 2.7min of 3HB in the standard substance, the retention time of 3HV is 3.1min in the standard substance, the benzoic retention time of interior mark is 3.8min.The retention time 2.4min of 3HB in the sample, the retention time of 3HV is 3.0min in the sample, the benzoic retention time of interior mark is 3.7min.
The collection of illustrative plates of standard substance is shown in Fig. 7 A, and the collection of illustrative plates of sample is shown in Fig. 7 B.
The result: cultivate after 48 hours, its dry cell weight (CDW) is the 1.70g/L fermented liquid; Bacterial strain produces PHBV, and wherein the 3HV ratio is 40%.PHBV accounts for 46% of thalline weight.
When leavening temperature changes arbitrary temperature between 20 ℃-45 ℃ into when (concrete as 20 ℃ or 45 ℃), somatic cells dry weight and PHB output and above-mentioned no significant difference;
When the pH of fermentation system value changes arbitrary value between 5.0-11.0 into when (concrete as 5.0 or 11.0), somatic cells dry weight and PHB output and above-mentioned no significant difference.
(1) experimental group
Method 1:
1, culture of seed liquid
Firsts and seconds seed liquor substratum is the LB substratum, and 1 liter of LB substratum consists of: 60g sodium-chlor, 10g peptone, 5g yeast powder, complement to 1 liter with distilled water, and sterilized 20 minutes for 121 ℃.
Get 20 μ l and guarantee that at-80 ℃ of glycerine salt Zymomonas mobilis (Halomonas sp.) the TD01CGMCC No.4353 that deposits inserts the 20mL primary seed solution, incubated overnight under 37 ℃, 200 rev/mins conditions obtains the first order seed nutrient solution.The first order seed nutrient solution inserts by 5% inoculum size and is equipped with in the 500mL triangular flask of 100mL secondary seed solution, meets 3 bottles of 300mL altogether, cultivates about 12 hours, and obtains the secondary seed nutrient solution for 37 ℃, 200 rev/mins.
2, fermentation
1 liter the fermentation initial medium by sodium-chlor 60g, glucose 30g, yeast powder 1g, ammonium chloride 2g, urea 3g, sal epsom 0.2g, potassium primary phosphate 1.5g, disodium hydrogen phosphate dodecahydrate 9.65g, 10mL trace element I, 1mL trace element II and tap water are formed, and are settled to 1 liter with tap water.
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 5g, two hydration calcium chloride 2g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of micro-II is prepared as follows: with Zinc vitriol 100mg, four hydration Manganous chloride tetrahydrate 30mg, boric acid 300mg, cobalt chloride hexahydrate 200mg, Salzburg vitriol 10mg, Nickel dichloride hexahydrate 20mg, two molybdic acid hydrate sodium 30mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II.
The preparation of feeding medium during fermentation substratum:
1 liter of supplemented medium I:600g glucose, 60g ammonium chloride is settled to 1 liter with tap water.
1 liter of supplemented medium II:600g glucose is settled to 1 liter with tap water.
The fermentation starting condition: temperature is 37 ℃, and the pH value is 9.0, and DO is set to 50%, and the air air flow is 3 liters/minute, DO and rotating speed coupling mutually, 200 rev/mins of minimum speeds, 800 rev/mins of maximum speeds.
The 6L fermentor tank (Bioflo 3000, New Brunswick, NJ, USA) in preparation 2.7L fermentation initial medium, do not sterilize, institute's water is a tap water.After fermentation starting condition (temperature, pH, DO) debugging is finished, insert 300mL secondary seed nutrient solution, begin fermentation.Ferment after 12 hours, added 100mL supplemented medium I every 4 hours; Ferment after 24 hours, added 100mL supplemented medium II every 4 hours.Ferment after 48 hours, stop fermentation.All substances in fermenting container note is made fermented liquid.
3, detect dry cell weight and PHB content
Meter during from the fermentation beginning took out 20mL fermented liquid centrifugal (10000 rev/mins, 10 minutes) every 4 hours and collects thalline, and freezing ice is done, and was used to calculate dry cell weight and GC and analyzed.Dry cell weight and GC analytical procedure are with consistent described in the embodiment 2.
Ferment after 48 hours, collect thalline, freezing ice is done, and extracts PHB with chloroform extraction method, and GPC (SpectraSystem P2000equipped with Shi-madzu HSG 60column) detects the molecular weight of the PHB that produces.
Chloroform extraction method extracts PHB: get 1g ice and do the back thalline and place the esterification pipe, add the 5mL chloroform according to 1: 5 ratio, in 100 ℃ of baking ovens 4 hours.After the cooling, add 5mL distilled water, mixing is placed in the centrifuge tube, 5000 rev/mins centrifugal 5 minutes, extract lower floor's organic phase, splash in the excessive ice-cold alcohol (being preset in 4 ℃ of refrigerators), 5000 rev/mins of white precipitates (being PHB) that collection in centrifugal 5 minutes is separated out, oven dry.
Under as above GC conditions, the retention time 2.5min of 3HB in the standard substance, the benzoic retention time of interior mark is 3.7min.The retention time 2.4min of 3HB in the sample, the benzoic retention time of interior mark is 3.7min.
The collection of illustrative plates of standard substance is shown in Fig. 8 A, and the collection of illustrative plates of sample is shown in Fig. 8 B.
3 repetitions are established in experiment, and the result takes the mean.
As a result, after 48 hours, the somatic cells dry weight is the 78.32g/L fermented liquid, and the PHB quality accounts for 67.48% of thalline weight.The weight-average molecular weight of synthetic PHB is 800000.
4, the detection of assorted bacterium
Meter during from the fermentation beginning was got fermented liquid every 4 hours and is diluted to 10
-4, to get 100 μ l and coat LB flat board (preparing same isolation medium), 37 ℃ of incubators were cultivated 24 hours, observed colonial morphology, selected 70 bacterium colonies and carried out the PCR checking, determined the whether microbiological contamination of unsterilised condition bottom fermentation.
Two sections highly conserved sequences that sequence is Halomonas TD01 choosing.Wherein, two pairs of primers are respectively:
F15’-GCCAACACGGTCAACATCCTCG-3’，
R15 '-CGGCGTTGAGCTGGAGCTGTTG-3 ', fragment length are 442bp;
F25’-CCAGATTGACGCGGCTGGCAAT-3’，
R25 '-CGCTGCCTTGTACGGTGATGTTG-3 ', PCR fragment length are 277bp.
The PCR detected result as shown in Figure 2.Among the figure, A is fermentation sampling in 12 hours sample, and B is fermentation sampling in 48 hours sample, gets 70 samples altogether and carries out the PCR checking.M is marker, C be Halomonas TD01 purebred do template over against photograph, 1～7 is the part sample of taking a sample.
Colonial morphology and PCR checking result show that not microbiological contamination shows that unsterilised fermentation is feasible in the fermenting process, and the energy consumption that this has greatly reduced production process has reduced cost, has simplified fermenting process.
(2) blank group (unsterilised do not connect bacterium)
Basic identical in fermentation process and the experiment (one), different is not insert bacterial strain Halomonas TD01.
Fermented 48 hours, and in the fermenting process, got fermented liquid every 4 hours and detect OD
600, get the centrifugal collection thalline of fermented liquid simultaneously, ice is done the back and is calculated dry cell weight.
3 repetitions are established in experiment, and the result takes the mean.The result as shown in Figure 1.Result's demonstration, after 48 hours, fermented liquid OD
600Have only 3.0, the varied bacteria growing of 1.4g/L fermented liquid is arranged.Compare with Halomonas TD01 fermenting process, assorted bacterium influence can be ignored, and thinking does not have microbiological contamination.
Method 2:
Basic identical described in method and the method 1, different is fermentation initial medium and fermentation condition difference, specific as follows:
1 liter of fermentation initial medium is by sodium-chlor 20g, glucose 5g, and ammonium chloride 0.5g, sal epsom 0.1g, potassium primary phosphate 0.5g, disodium hydrogen phosphate dodecahydrate 1g, 1mL trace element I, 0.1mL trace element II and tap water are formed, and are settled to 1 liter with tap water.
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 2g, two hydration calcium chloride 1g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of micro-II is prepared as follows: with Zinc vitriol 50mg, four hydration Manganous chloride tetrahydrate 10mg, boric acid 100mg, cobalt chloride hexahydrate 50mg, Salzburg vitriol 3mg, Nickel dichloride hexahydrate 5mg, two molybdic acid hydrate sodium 10mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II.
The starting condition of fermentation is as follows: temperature is 20 ℃, and the pH value is 5.0, and DO is set to 5%.
Through 48 hours, the somatic cells dry weight was 34.62g/L, and PHB content accounts for 54.35% of thalline weight, and not detecting simultaneously has varied bacteria growing.
Method 3:
Basic identical described in method and the method 1, different is fermentation initial medium and fermentation condition difference, specific as follows:
1 liter the fermentation initial medium by sodium-chlor 200g, glucose 100g, yeast powder 50g, ammonium chloride 20g, urea 20g, sal epsom 5g, potassium primary phosphate 10g, disodium hydrogen phosphate dodecahydrate 20g, 30mL trace element I, 10mL trace element II and tap water are formed, and are settled to 1 liter with tap water.
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 10g, two hydration calcium chloride 5g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of micro-II is prepared as follows: with Zinc vitriol 200mg, four hydration Manganous chloride tetrahydrate 50mg, boric acid 500mg, cobalt chloride hexahydrate 400mg, Salzburg vitriol 30mg, Nickel dichloride hexahydrate 50mg, two molybdic acid hydrate sodium 50mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II.
The starting condition of fermentation is as follows: temperature is 45 ℃, and the pH value is 11.0, and DO is set to 100%.
Through 48 hours, the somatic cells dry weight was 35.46g/L, and PHB content accounts for 53.36% of thalline weight, and not detecting simultaneously has varied bacteria growing.
Fermented liquid described in the present embodiment refers to all substances in the fermentor tank.
Method 1:
1, culture of seed liquid
Firsts and seconds seed liquor substratum is the LB substratum, and 1 liter of LB substratum consists of: 60g sodium-chlor, 10g peptone, 5g yeast powder, complement to 1 liter with distilled water, and sterilized 20 minutes for 121 ℃.
Get 20 μ l and guarantee that at-80 ℃ of glycerine salt Zymomonas mobilis (Halomonas sp.) the TD01CGMCC No.4353 that deposits inserts the 20mL primary seed solution, incubated overnight under 37 ℃, 200 rev/mins conditions obtains the first order seed nutrient solution.The first order seed nutrient solution inserts by 5% inoculum size and is equipped with in the 500mL triangular flask of 100mL secondary seed solution, meets 3 bottles of 300mL altogether, cultivates about 12 hours, and obtains the secondary seed nutrient solution for 37 ℃, 200 rev/mins.
2, fermentation
Fermentation system is as shown in Figure 3: 2.7L fermentation initial medium I is housed in first fermentor tank, 1L fermentation initial medium II is housed in second fermentor tank.
1 liter of fermentation initial medium I is by sodium-chlor 60g, glucose 30g, yeast powder 1g, ammonium chloride 2g, urea 3g, sal epsom 0.2g, potassium primary phosphate 1.5g, disodium hydrogen phosphate dodecahydrate 9.65g, 10mL trace element I, 1mL trace element II and tap water are formed, and are settled to 1 liter with tap water.
1 liter of fermentation initial medium II is by sodium-chlor 60g, glucose 30g, and sal epsom 0.2g, potassium primary phosphate 1.5g, disodium hydrogen phosphate dodecahydrate 9.65g, 10mL trace element I, 1mL trace element II and tap water are formed, and are settled to 1 liter with tap water.
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 5g, two hydration calcium chloride 2g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of micro-II is prepared as follows: with Zinc vitriol 100mg, four hydration Manganous chloride tetrahydrate 30mg, boric acid 300mg, cobalt chloride hexahydrate 200mg, Salzburg vitriol 10mg, Nickel dichloride hexahydrate 20mg, two molybdic acid hydrate sodium 30mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II.
The preparation of feeding medium during fermentation substratum:
1 liter of supplemented medium I:600g glucose, 60g ammonium chloride is settled to 1 liter with tap water.
1 liter of supplemented medium II:600g glucose is settled to 1 liter with tap water.
Initial fermentation condition in the fermenting process in two fermentor tanks is all as follows: temperature is 37 ℃, and the pH value is 9.0, and DO is set to 50%, and the air air flow is 3 liters/minute, DO and rotating speed coupling mutually, 200 rev/mins of minimum speeds, 800 rev/mins of maximum speeds.
First 6L fermentor tank (Bioflo 3000, New Brunswick, NJ, USA) in preparation 2.7L fermentation initial medium, do not sterilize, institute's water is a tap water.After fermentation starting condition (temperature, pH, DO) debugging is finished, insert 300mL secondary seed nutrient solution, begin fermentation.Ferment after 8 hours, beginning flow feeding substratum I, flow velocity is set to 1ml/min.
Treat that first ferment tank is after 24 hours (being that bacterium reaches the logarithmic growth after date), fermentation broth stream in first jar is added second jar of importing (but the fermented liquid that remains in first fermentor tank is 3L), it is 1ml/min that flow velocity is set to, while flow feeding medium ii in second jar, flow velocity is set to 1ml/min.
Treat second jar fermentation after 24 hours, from second jar, take out fermented liquid (is 2L but remain the interior fermented liquid of second fermentor tank), from the fermented liquid that takes out, collect thalline, be used to extract PHA.Whole system is continuously fermented and was continued for 2 weeks.
3, detect dry cell weight and PHB content
Meter when beginning from fermenting took out 20mL fermented liquid centrifugal (10000 rev/mins, 10 minutes) every 12 hours and collects thalline, used deionized water wash 2 times, and freezing ice is dried, was used to calculate dry cell weight and GC and analyzed.Dry cell weight and GC analytical procedure are with consistent described in the embodiment 2.
Ferment after 2 weeks, collect thalline, freezing ice is done, and extracts PHB with chloroform extraction method, and GPC (Spectra SystemP2000equipped with Shi-madzu HSG 60column) detects the molecular weight of the PHB that produces.The PHA extracting method is with consistent described in the embodiment 2.
Under as above GC conditions, the retention time 2.5min of 3HB in the standard substance, the benzoic retention time of interior mark is 3.7min.The retention time 2.4min of 3HB in the sample, the benzoic retention time of interior mark is 3.7min.
The collection of illustrative plates of standard substance is shown in Fig. 9 A, and the collection of illustrative plates of sample is shown in Fig. 9 B.
3 repetitions are established in experiment, and the result takes the mean.
As a result, first jar entered stationary phase after 48 hours, and the somatic cells dry weight maintains about 70g/L, and the PHB quality accounts for 50% of thalline weight; The weight-average molecular weight of synthetic PHB is about 500000.As a child entered stationary phase for second jar 48, the somatic cells dry weight maintains about 60g/L, and the PHB quality accounts for 70% of thalline weight; The weight-average molecular weight of synthetic PHB is about 800000.
4, the detection of assorted bacterium
Meter during from the fermentation beginning was got fermented liquid every 1 day and is diluted to 10
-4, to get 100 μ l and coat LB flat board (preparing same isolation medium), 37 ℃ of incubators were cultivated 24 hours, observed colonial morphology, and each flat board is selected 10 bacterium colonies and is carried out the PCR checking, determines the whether microbiological contamination of unsterilised condition bottom fermentation.
Two sections highly conserved sequences that sequence is Halomonas TD01 choosing.Wherein, two pairs of primers are respectively:
F15’-GCCAACACGGTCAACATCCTCG-3’，
R15 '-CGGCGTTGAGCTGGAGCTGTTG-3 ', fragment length are 442bp;
F25’-CCAGATTGACGCGGCTGGCAAT-3’，
R25 '-CGCTGCCTTGTACGGTGATGTTG-3 ', PCR fragment length are 277bp.
The PCR detected result as shown in Figure 2.Among the figure, A is fermentation sampling in 12 hours sample, and B is fermentation sampling in 48 hours sample, gets 70 samples altogether and carries out the PCR checking.M is marker, C be Halomonas TD01 purebred do template over against photograph, 1～7 is the part sample of taking a sample.
Colonial morphology and PCR checking result show that not microbiological contamination shows that unsterilised continuously fermenting is feasible in the fermenting process, and the energy consumption that this has greatly reduced production process has reduced cost, has simplified fermenting process.
Method 2:
Basic identical described in method and the method 1, different is that used initial fermention medium is formed and the fermentation condition difference, specific as follows:
1 liter of fermentation initial medium I is by sodium-chlor 20g, glucose 5g, and ammonium chloride 0.5g, sal epsom 0.1g, potassium primary phosphate 0.5g, disodium hydrogen phosphate dodecahydrate 1g, 1mL trace element I, 0.1mL trace element II and tap water are formed, and are settled to 1 liter with tap water.
1 liter of fermentation initial medium II is by sodium-chlor 20g, glucose 5g, and sal epsom 0.1g, potassium primary phosphate 0.5g, disodium hydrogen phosphate dodecahydrate 1g, 1mL trace element I, 0.1mL trace element II and tap water are formed, and are settled to 1 liter with tap water.
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 2g, two hydration calcium chloride 1g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of micro-II is prepared as follows: with Zinc vitriol 50mg, four hydration Manganous chloride tetrahydrate 10mg, boric acid 100mg, cobalt chloride hexahydrate 50mg, Salzburg vitriol 3mg, Nickel dichloride hexahydrate 5mg, two molybdic acid hydrate sodium 10mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II.
Initial fermentation condition in the fermenting process in two fermentor tanks is all as follows: temperature is 20 ℃, and the pH value is 5.0, and DO is set to 5%.
3 repetitions are established in experiment, and the result takes the mean.
The result: after 2 weeks, first jar of somatic cells dry weight maintains 35g/L, and the PHB quality on average accounts for 50% of thalline quality; Second jar of somatic cells dry weight maintains 30g/L, and the PHB quality on average accounts for 65% of thalline quality, and not detecting simultaneously has varied bacteria growing.
Method 3:
Basic identical described in method and the method 1, different is that used initial fermention medium is formed and the fermentation condition difference, specific as follows:
1 liter of fermentation initial medium I is by sodium-chlor 200g, glucose 100g, yeast powder 50g, ammonium chloride 20g, urea 20g, sal epsom 5g, potassium primary phosphate 10g, disodium hydrogen phosphate dodecahydrate 20g, 30mL trace element I, 10mL trace element II and tap water are formed, and are settled to 1 liter with tap water.
1 liter of fermentation initial medium II is by sodium-chlor 200g, glucose 100g, and sal epsom 5g, potassium primary phosphate 10g, disodium hydrogen phosphate dodecahydrate 20g, 30mL trace element I, 10mL trace element II and tap water are formed, and are settled to 1 liter with tap water.
1 liter of described micro-I is prepared as follows: with ferric ammonium citrate 10g, two hydration calcium chloride 5g and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-I;
1 liter of micro-II is prepared as follows: with Zinc vitriol 200mg, four hydration Manganous chloride tetrahydrate 50mg, boric acid 500mg, cobalt chloride hexahydrate 400mg, Salzburg vitriol 30mg, Nickel dichloride hexahydrate 50mg, two molybdic acid hydrate sodium 50mg and 0.5mol/L combined, complement to 1 liter with the 0.5mol/L aqueous hydrochloric acid, obtain described micro-II.
Initial fermentation condition in the fermenting process in two fermentor tanks is all as follows: temperature is 45 ℃, and the pH value is 11.0, and DO is set to 100%.
3 repetitions are established in experiment, and the result takes the mean.
As a result, through 2 weeks, first jar of somatic cells dry weight maintains 40g/L, and the PHB quality accounts for 55% of thalline weight; Second jar of somatic cells dry weight maintains about 30g/L, and the PHB quality accounts for 65% of thalline weight, and not detecting simultaneously has varied bacteria growing.
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