Method for increasing PHA yield

Abstract

The invention relates to the field of PHA production, and discloses a method for increasing the yield of PHA, which is characterized by comprising the following steps: inoculating a PHA fermentation strain into a fermentation culture medium for fermentation under the condition that PHA can be produced by fermentation, and supplementing sulfur-containing acid radical ions in the fermentation process. By adopting the method, the number of thalli can be increased, higher PHA content can be obtained, and the growth of strains is not influenced.

Description

Technical Field

[0001] The present invention relates to the field of PHA production, and in particular to a method for increasing PHA yield. Background Art

[0002] Polyhydroxyalkanoate (PHA) is one of the bioplastics (biodegradable polymers) that has attracted great attention due to the increasing demand for environmentally compatible materials from renewable resources. Compared with traditional petroleum-based plastics, the biodegradability and structural diversity of PHA have opened up a wider range of application scenarios, such as packaging materials, medical fields (heart valves, sutures, etc.). PHA can be synthesized by microorganisms. However, as an intracellular product of microorganisms, PHA cannot be excreted from the body, which makes it necessary to use high-density fermentation to increase the amount of microorganisms to increase PHA production. At present, various difficulties have been encountered in the process of using microorganisms to produce PHA, including expensive growth conditions, huge energy requirements, large amounts of fresh water consumption and complex extraction processes, resulting in high costs and low yields, and unable to scale up.

[0003] Some researchers are also focusing on increasing cell volume through morphological engineering to improve PHA productivity. The current production intensity of PHA produced by fermentation of different strains and different substrates varies, among which the overall stable fermentation yield of the halophilic bacteria system is at the level of 72-78g / L. The combination optimization of carbon sources and nitrogen sources and the use of expensive organic nitrogen sources have improved the fermentation yield to a certain extent. Reports have shown that although limiting nitrogen sources has a positive effect on PHA accumulation, the lack of nitrogen sources greatly affects cell growth and shortens the exponential period of cell growth, which makes it difficult to achieve high PHA content and high cell dry weight by limiting nitrogen source culture. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems of low cell density and low PHA yield in the PHA production process in the prior art, and to provide a method for increasing PHA yield, which can significantly increase PHA content and cell biomass.

[0005] In order to achieve the above object, the present invention provides a method for increasing PHA production, characterized in that the method comprises inoculating PHA fermentation bacteria into a fermentation medium for fermentation under conditions capable of producing PHA by fermentation, and supplementing sulfur-containing acid radical ions during the fermentation process.

[0006] In the present invention, the addition of sulfur-containing acid radical ions during the fermentation process can increase the number of bacteria and obtain a higher PHA content without affecting the growth of the strain; in some preferred technical schemes, the addition of sulfate and / or sulfite during the fermentation process can more significantly increase the number of bacteria and obtain a higher PHA content without affecting the growth of the strain; in some preferred technical schemes, the addition of sulfur-containing acid radical ions and a nitrogen source during the fermentation process, and reasonable process control of the addition of sulfur-containing acid radical ions and the addition of nitrogen source during the fermentation process can further increase the bacterial density and PHA yield. DETAILED DESCRIPTION

[0007] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0008] In the present invention, the carbon-nitrogen ratio refers to the weight ratio of the mass of carbon atoms in the carbon source used to the mass of nitrogen atoms in the nitrogen source used, wherein the nitrogen atoms contained in the carbon source and the carbon atoms contained in the nitrogen source are not taken into account.

[0009] In order to achieve the above object, the first aspect of the present invention provides a method for increasing PHA production, characterized in that the method comprises inoculating PHA fermentation bacteria into a fermentation medium for fermentation under conditions capable of producing PHA by fermentation, and supplementing sulfur-containing acid radical ions during the fermentation process. The scheme can increase the number of bacteria and obtain a higher PHA content without affecting the growth of the strain.

[0010] Preferably, the PHA fermentation strain is Halomonas sp.; the Halomonas sp. may be a strain that can tolerate 40-80 g / L NaCl in the art. In a preferred embodiment of the present invention, the Halomonas sp. is a Halomonas sp. with a preservation number of CGMCC NO.4353, which is purchased from Tsinghua University and has been disclosed in CN102120973A.

[0011] Preferably, compared to 1L of fermentation medium, the inoculation amount of the PHA fermentation strain is 5×10 8 CFU-6×10 9 CFU, preferably 5×10 8 CFU-5×10 9 CFU.

[0012] In the present invention, when the PHA fermentation strain is inoculated, it can be inoculated in any form commonly used in the art, without particular limitation. Preferably, the PHA fermentation strain is inoculated in the form of a seed solution. According to the present invention, the inoculation amount of the fermentation strain may not be particularly limited. Preferably, compared to the fermentation medium, the inoculation amount of the PHA fermentation strain is 1-20% by volume, for example, 1% by volume, 5% by volume, 8% by volume, 10% by volume, 12% by volume, 14% by volume, 15% by volume, 16% by volume, 18% by volume, 20% by volume, and any range between any two values, more preferably 5-15% by volume.

[0013] According to the present invention, the OD of the seed solution 600 The value is preferably 3-5.

[0014] The seed solution can be obtained by conventional techniques in the art, for example, activating the low-temperature preserved strains and culturing them at least once, for example, 2-3 levels. The activation and culturing can be performed by conventional methods in the art, for example, activation can be performed on an activated solid plate and then cultured in a liquid seed culture medium.

[0015] The seed culture medium may contain 1-10 g / L yeast powder, 5-15 g / L peptone and 50-80 g / L sodium chloride, and may be sterilized by high temperature and high pressure. The solid plate may be added with 15-20 g / L agar on the basis of the seed culture medium. The pH of the seed culture medium is preferably 7.5-8.5.

[0016] The culture conditions preferably include: a temperature of 30-40°C and a time of 8-16 hours.

[0017] Preferably, the starting time of the sulfur-containing acid radical ion supplementation is when the growth of the PHA fermentation strain reaches the logarithmic phase. The starting time of the sulfur-containing acid radical ion supplementation can also be determined according to the demand of the PHA fermentation strain for sugar. Preferably, when the sugar content in the fermentation medium drops below 15g / L, preferably drops below 12g / L, and more preferably drops below 10g / L (for example, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, 10g / L and any range between any two values), the feeding is started.

[0018] In the present invention, the sugar content in the carbon source refers to the sugar content in the fermentation centrifugal supernatant measured by SBA-90 biosensor analyzer. The sugar content is the content of glucose.

[0019] Preferably, the sugar content in the fermentation broth is monitored in real time during the fermentation process.

[0020] Preferably, the end time of the sulfur-containing acid radical supplementation is when the PHA fermentation strain reaches the decay stage. In one embodiment of the present invention, another method can be used to determine the fermentation end point. Preferably, the method further includes: when the residual sugar content in the fermentation medium is less than 5g / L after the feeding is completed, the fermentation is terminated. 600 The growth condition is used to determine the end point of fermentation, such as the OD value of the fermentation liquid. 600 A decrease occurred in two consecutive measurements.

[0021] In the present invention, the term "reaching the logarithmic phase" refers to the fermentation liquid OD 600 Begins to increase significantly (OD 600 The increase is greater than 10 per hour, and the growth curve shows an exponential trend. "Arrival of the decline phase" refers to the fermentation liquid OD 600 The increase started slowly (OD 600 If the increase is less than 5% per hour or even decreases, it can be determined by turbidity measurement (turbidimetry): the optical density is measured by ultraviolet spectrophotometer at a wavelength of 600 nanometers to draw the growth curve of the PHA fermentation strain, thereby determining the time to reach the logarithmic phase or the decay phase.

[0022] In the present invention, OD 600 It refers to the absorbance of the fermentation broth at a wavelength of 600nm in a spectrophotometer.

[0023] Preferably, the OD of the fermentation broth is monitored in real time during the fermentation process. 600 .

[0024] Preferably, the amount of sulfur-containing acid radical ions supplemented is such that when the growth of the PHA fermentation strain reaches the logarithmic phase to the cell death phase (at this time, the fermentation liquid OD 600When the temperature reaches 400-500 °C, the concentration of the sulfur-containing acid radical ion is maintained at 6-200 mmol / L relative to 1 L of fermentation medium, for example, it can be 6 mmol / L, 8 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 1mmol / L, 2mmol / L, 3mmol / L, 4mmol / L, 5mmol / L, 6mmol / L, 7mmol / L, 8mmol / L, 9mmol / L, 10mmol / L, 110mmol / L, 120mmol / L, 130mmol / L, 140mmol / L, 150mmol / L, 160mmol / L, 170mmol / L, 180mmol / L, 190mmol / L, 200mmol / L, and any range composed of any of the above values ​​and any value in the range, more preferably 6-120mmol / L. The inventors of the present invention found during the experiment that when the concentration of sulfur-containing acid radical ions is maintained within the above range relative to 1L fermentation medium, it is beneficial to increase the number of bacteria and obtain a higher PHA content. If the concentration is too high, cell growth is inhibited, and the cell dry weight and PHA content no longer increase; if the concentration is too low, the cells grow slowly, the PHA accumulation and cell dry weight increase slowly, and the fermentation cycle is significantly extended.

[0025] In order to further increase the number of bacteria and PHA content, preferably, relative to 1L of fermentation medium, the supplementary amount of the sulfur-containing acid radical ion is 60-400mmol, for example, it can be 60mmol, 80mmol, 100mmol, 120mmol, 140mmol, 250mmol, 160mmol, 170mmol, 180mmol, 200mmol, 220mmol, 240mmol, 250mmol, 260mmol, 280mmol, 300mmol, 320mmol, 340mmol, 350mmol, 360mmol, 380mmol, 400mmol, and any range composed of any of the above numerical values ​​and any value in the range, more preferably 120-300mmol, and further preferably 170-200mmol.

[0026] In the present invention, preferably, the sulfur-containing acid radical ions are sulfate and / or sulfite. The sulfate and / or sulfite are derived from salts that can ionize sulfate and / or sulfite in water.

[0027] In some preferred embodiments of the present invention, the sulfate ion is derived from at least one of sulfate and bisulfate, and more preferably at least one of ammonium sulfate, ammonium bisulfate, calcium sulfate, calcium bisulfate, magnesium sulfate, magnesium bisulfate, sodium sulfate, sodium bisulfate, potassium sulfate and potassium bisulfate;

[0028] In some preferred embodiments of the present invention, the sulfite ion is derived from at least one of sulfite and bisulfite, preferably at least one selected from ammonium sulfite, ammonium bisulfite, calcium sulfite, calcium bisulfite, magnesium sulfite, magnesium bisulfite, sodium sulfite, sodium bisulfite, potassium sulfite and potassium bisulfite.

[0029] In the present invention, when the sulfur-containing acid radical ions are added to the fermentation system during the fermentation culture process, the sulfur-containing acid radical ions can be added once before the start of fermentation, and can be added to the culture medium, or after the seed liquid is inoculated into the fermentation culture medium, the sulfur-containing acid radical ions can be added before the start of fermentation; the sulfur-containing acid radical ions can also be added to the fermentation system after fermentation. The addition method of adding to the fermentation system after fermentation can be a one-time addition or a two-stage supplement.

[0030] In order to further increase the bacterial count and PHA content, preferably, the sulfur-containing acid radical ions can be supplemented by adding them once or in two stages during the fermentation culture process.

[0031] More preferably, the one-time supplement is to supplement sulfur-containing acid ions in the fermentation system when the growth of the PHA fermentation strain reaches the logarithmic phase or when the sugar content in the fermentation medium drops to below 15 g / L (preferably drops to below 12 g / L, more preferably drops to 10 g / L). Relative to 1 L of fermentation medium, the supplementary amount of sulfur-containing acid ions is 60-400 mmol, for example, it can be 60 mmol, 80 mmol, 100 mmol, 120 mmol, 140 mmol, 250 mmol, 160 mmol, 170 mmol, 180 mmol, 200 mmol, 220 mmol, 240 mmol, 250 mmol, 260 mmol, 280 mmol, 300 mmol, 320 mmol, 340 mmol, 350 mmol, 360 mmol, 380 mmol, 400 mmol, and any range composed of any of the above numerical values ​​and any value in the range, more preferably 120-300 mmol, and further preferably 170-200 mmol.

[0032] More preferably, the two-stage supplementation includes a first-stage supplementation and a second-stage supplementation. The first-stage supplementation is: when the growth of the PHA fermentation strain reaches the logarithmic phase or when the sugar content in the fermentation medium drops to below 15 g / L (preferably drops to below 12 g / L, more preferably drops to 10 g / L), sulfur-containing acid radical ions are added to the fermentation system, and the supplementation amount of sulfur-containing acid radical ions relative to 1L fermentation medium is 20-140 mmol, for example, it can be 20 mmol, 30 mmol, 40 mmol, 50 mmol, 60 mmol, 70 mmol, 80 mmol, 90 mmol, 100 mmol, 110 mmol, 120 mmol, 130 mmol, 140 mmol and any range composed of any of the above values ​​and any value in the range, more preferably 30-90 mmol, and further preferably 60-70 mmol; the second The stage supplementation is as follows: after the first stage supplementation, sulfur-containing acid radical ions are added to the fermentation system. Relative to 1L fermentation medium, the supplementation amount of sulfur-containing acid radical ions is 40-300mmol, for example, it can be 40mmol, 50mmol, 60mmol, 70mmol, 80mmol, 90mmol, 100mmol, 110mmol, 115mmol, 120mmol, 125mmol, 130mmol, 150mmol, 160mmol, 180mmol, 200mmol, 220mmol, 240mmol, 260mmol, 280mmol, 300mmol and any range composed of any of the above numerical values ​​and any value in the range, more preferably 80-250mmol, and further preferably 115-130mmol.

[0033] In order to further increase the bacterial count and PHA content, preferably, the fermentation medium contains a carbon source, a nitrogen source, phosphate, magnesium salt and sodium salt.

[0034] Preferably, the carbon source is glucose. In the fermentation medium, in order to further increase the number of bacteria and the PHA content, the content of the carbon source can be selected in a wider range. Preferably, relative to 1L of the fermentation medium, the content of the carbon source is 5-50g, for example, 5g, 10g, 15g, 20g, 25g, 30g, 35g, 40g, 45g, 50g and any range between any two values, more preferably 15-30g.

[0035] In the present invention, the nitrogen source is an organic nitrogen source and / or an inorganic nitrogen source. The organic nitrogen source may be an organic nitrogen source conventionally used in the art, for example, it may be at least one of urea, corn steep liquor, enzymatic corn steep liquor, yeast powder, yeast extract and peptone; the inorganic nitrogen source may be an inorganic nitrogen source conventionally used in the art, for example, it may be an ammonium salt, for example, it may be selected from at least one of ammonium chloride, ammonium nitrate, diammonium phosphate, ammonium sulfate and ammonium sulfite.

[0036] In the fermentation medium, the content of the nitrogen source can be selected within a wide range. Preferably, relative to 1 L of fermentation medium, the molar amount of nitrogen element in the nitrogen source is 10-225 mmol, more preferably 85-170 mmol.

[0037] In order to further increase the bacterial count and PHA content, preferably, the nitrogen source in the fermentation medium is an organic nitrogen source, more preferably urea.

[0038] In some preferred embodiments, the content of urea is 1-10 g, for example, it can be 1 g, 2 g, 4 g, 6 g, 8 g, 10 g, and any range between any two values, more preferably 2-6 g.

[0039] Preferably, the carbon-nitrogen ratio in the fermentation medium is 5-10:1, for example, it can be 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 and any range between any two values, more preferably 6-8:1.

[0040] The phosphate may be a phosphate conventionally used in the PHA fermentation process, for example, a sodium salt of phosphate, a potassium salt of phosphate, preferably, the phosphate is dipotassium hydrogen phosphate and / or disodium hydrogen phosphate. Preferably, relative to 1L of fermentation medium, the content of dipotassium hydrogen phosphate is 2-5g, for example, it may be 2g, 3g, 4g, 5g and any range between any two values, the content of disodium hydrogen phosphate is 2-10g, for example, it may be 2g, 4g, 6g, 8g, 10g and any range between any two values, more preferably, the content of dipotassium hydrogen phosphate is 3-4g, and the content of disodium hydrogen phosphate is 5-7g.

[0041] The magnesium salt may be any conventional magnesium salt, but does not include magnesium salt of phosphate. Preferably, the magnesium salt is magnesium sulfate. Preferably, the content of magnesium sulfate is 0.1-0.5 g relative to 1 L of fermentation medium, for example, 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g and any range between any two values, more preferably 0.2-0.3 g.

[0042] The sodium salt may be any conventional sodium salt, but does not include sodium salt of phosphate. Preferably, the sodium salt is sodium chloride. In the fermentation medium, the content of the sodium salt may be selected in a wide range. Preferably, relative to 1L of the fermentation medium, the content of the sodium salt is 8-30 g, for example, 8 g, 10 g, 12 g, 14 g, 16 g, 18 g, 20 g, 22 g, 24 g, 26 g, 28 g, 30 g, and any range between any two values, more preferably 12-18 g.

[0043] According to a preferred embodiment of the present invention, the fermentation medium comprises urea, glucose, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and sodium chloride.

[0044] According to a preferred embodiment of the present invention, relative to 1L of fermentation medium, the content of glucose is 5-50g, the content of urea is 1-10g, the content of dipotassium hydrogen phosphate is 2-5g, the content of disodium hydrogen phosphate is 4-8g, the content of magnesium sulfate is 0.1-0.5g, and the content of sodium chloride is 8-30g.

[0045] According to a more preferred embodiment of the present invention, relative to 1L of fermentation medium, the content of glucose is 20-40g, the content of urea is 2-6g, the content of dipotassium hydrogen phosphate is 3-4g, the content of disodium hydrogen phosphate is 5-7g, the content of magnesium sulfate is 0.2-0.3g, and the content of sodium chloride is 12-18g.

[0046] The fermentation of the present invention can be carried out in an open environment.

[0047] According to the present invention, the temperature of PHA fermentation can be its conventional fermentation temperature, preferably, the temperature is 32-40°C, for example, it can be 32°C, 33°C, 34°C, 35°C, 35.5°C, 36°C, 36.5°C, 37°C, 37.5°C, 38°C, 38.5°C, 39°C, 40°C and any range between any two values, more preferably, the temperature is 35-39°C.

[0048] According to the present invention, the pH of PHA fermentation can be its conventional fermentation pH, preferably, the pH value is 7-11, for example, it can be 7, 7.5, 8, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 9, 9.5, 10, 10.5, 11 and any range between any two values, more preferably 7.5-9. Wherein, the pH value can be adjusted using conventional alkali, and the pH stability of the fermentation liquid is controlled by adding alkali during the fermentation process, which will not be repeated here.

[0049] According to the present invention, the dissolved oxygen content of PHA fermentation can be its conventional fermentation dissolved oxygen content, preferably, the dissolved oxygen content is 1-40%, for example, it can be 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% and any range between any two values, more preferably 5-40%.

[0050] Preferably, the fermentation is carried out under ventilation conditions, and the ventilation rate is 1-2vvm, for example, 1vvm, 1.1vvm, 1.2vvm, 1.3vvm, 1.4vvm, 1.5vvm, 1.6vvm, 1.7vvm, 1.8vvm, 1.9vvm, 2vvm and any range between any two values, more preferably 1.2-1.8vvm

[0051] Preferably, the fermentation conditions include: temperature of 32-40° C., pH of 7.5-9, and dissolved oxygen of 5-40%.

[0052] The fermentation conditions of the PHA may further include stirring, wherein the stirring speed may be selected in a wide range, preferably, based on a 2-7L fermenter, the stirring speed is 400-1000rpm, for example, 400rpm, 450rpm, 500rpm, 550rpm, 600rpm, 650rpm, 700rpm, 750rpm, 800rpm, 850rpm, 900rpm, 950rpm, 1000rpm and any range between any two values. It should be understood that those skilled in the art may adjust the speed according to the volume of the fermenter and the actual fermentation conditions.

[0053] In order to further increase the number of bacteria and the PHA content, preferably, the method further comprises supplementing a nitrogen source during the fermentation process. The nitrogen source is an organic nitrogen source and / or an inorganic nitrogen source. In order to further increase the number of bacteria and the PHA content, the nitrogen source is an organic nitrogen source.

[0054] Preferably, the nitrogen source is fed using a feed medium.

[0055] Preferably, the nitrogen source in the feed medium is an organic nitrogen source and / or an inorganic nitrogen source, more preferably an organic nitrogen source, and further preferably urea.

[0056] The inventors of the present invention have found that the use of urea in fermentation medium and feed medium instead of enzymatic corn steep liquor, corn steep liquor, yeast powder, yeast extract, peptone and other composite organic nitrogen sources can further increase bacterial biomass and PHA production. In particular, enzymatic corn steep liquor and corn steep liquor contain a large amount of impurities and have a strong color, which requires a large amount of water resources to separate the bacterial liquid after the strain fermentation. The use of urea can make the bacterial liquid separation simpler and easier, and consume less water resources for bacterial cleaning.

[0057] Preferably, the inorganic nitrogen source is an ammonium salt, more preferably at least one selected from ammonium chloride, ammonium nitrate, diammonium hydrogen phosphate, ammonium sulfate and ammonium sulfite.

[0058] Preferably, the feed medium further comprises a carbon source, more preferably, the carbon source is glucose.

[0059] Preferably, the amount of feed medium added is such that the content of carbon source in the fermentation medium is less than 20 g / L, for example, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L and any range consisting of any two values ​​and any value in the range, preferably 5-10 g / L.

[0060] Preferably, the feed medium comprises a carbon source and a nitrogen source.

[0061] Preferably, the carbon-nitrogen ratio in the feed medium is (20-30):1, for example, it can be 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, and any range between any two values ​​and any value in the range.

[0062] The carbon source content in the feed medium can be selected within a wide range. In order to reduce the volume of the feed medium, the carbon source concentration can be controlled at a high level. Preferably, the carbon source content in the feed medium is above 500 g / L.

[0063] Preferably, the inorganic nitrogen source is an ammonium salt, more preferably at least one selected from ammonium chloride, ammonium nitrate, diammonium phosphate, ammonium sulfate and ammonium sulfite. The amount of the inorganic nitrogen source can be calculated according to the amount of the carbon source and the carbon-nitrogen ratio requirements, which will not be repeated here.

[0064] According to a preferred embodiment of the present invention, the feeding method is a two-stage feeding, wherein a first feeding is performed using a first feeding medium, and then a second feeding is performed using a second feeding medium.

[0065] Preferably, the carbon-nitrogen ratio of the first feed medium is 5-10:1, for example, it can be 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and any range composed of any two values, and any value in the range. The supplement amount of the first feed medium is 10-25% by volume of the fermentation medium, for example, it can be 10% by volume, 15% by volume, 20% by volume, 25% by volume, and any range composed of any two values, and any value in the range.

[0066] Preferably, the first feed medium contains 550-650 g / L of carbon source, for example, it can be 550 g / L, 570 g / L, 590 g / L, 610 g / L, 630 g / L, 650 g / L, and any range composed of any two values ​​and any value in the range.

[0067] Preferably, the first feed medium further comprises dipotassium hydrogen phosphate and disodium hydrogen phosphate and magnesium sulfate.

[0068] Preferably, relative to 1 L of the first feed medium, the content of dipotassium hydrogen phosphate is 1-5 g, the content of disodium hydrogen phosphate is 5-10 g, and the content of magnesium sulfate is 0.1-1.5 g.

[0069] More preferably, relative to 1 L of the first feed medium, the content of dipotassium hydrogen phosphate is 3-4 g, the content of disodium hydrogen phosphate is 7-8 g, and the content of magnesium sulfate is 0.5-1 g.

[0070] Preferably, the carbon-nitrogen ratio of the second feed medium is 40-60:1, for example, it can be 40:1, 42:1, 44:1, 46:1, 48:1, 50:1, 52:1, 54:1, 56:1, 58:1, 60:1 and any range between any two values ​​and any value in the range, more preferably (50-60):1.

[0071] Preferably, the supplement amount of the second feed medium is 45-65 volume % of the fermentation medium, for example, it can be 45 volume %, 50 volume %, 55 volume %, 60 volume %, 65 volume % and any range composed of any two values ​​and any value in the range.

[0072] Preferably, the second feed medium contains 750-850 g / L of carbon source, for example, it can be 750 g / L, 770 g / L, 790 g / L, 810 g / L, 830 g / L, 850 g / L, and any range composed of any two values ​​and any value in the range.

[0073] Preferably, the starting time of the nitrogen source supplement is the logarithmic phase of the growth of the halophilic mononas. The timing of the nitrogen source supplement can also be determined according to the demand of the PHA fermentation strain for sugar. Preferably, when the carbon source content in the fermentation medium drops below 15g / L, preferably drops below 12g / L, and more preferably drops below 10g / L (for example, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, 10g / L and any range between any two values), the nitrogen source is supplemented.

[0074] Preferably, the nitrogen source supplementation is terminated when the growth of the halophilic monas reaches the decay phase. In one embodiment of the present invention, another method can be used to determine the fermentation endpoint. Preferably, the method further comprises: when the carbon source content (such as glucose content) in the fermentation medium is lower than 5g / L after the feeding is completed, the fermentation is terminated. 600 The growth condition is used to determine the end point of fermentation, such as the OD value of the fermentation liquid. 600 A decrease occurred in two consecutive measurements.

[0075] Preferably, under the above fermentation conditions, the fermentation is stopped 2 hours after the second feeding is completed.

[0076] Preferably, the fermentation is a continuous fermentation or a batch fermentation.

[0077] Under the preferred conditions of the present invention, the bacterial biomass and PHA yield can be further increased.

[0078] According to the present invention, the method further comprises extracting PHA from the fermentation broth obtained from the fermentation. The method for extracting PHA can be a conventional method in the art. For example, reference document: Chung A, Liu Q, Ouyang SP, et al. Microbial production of 3-hydroxydodecanoic acid by phaoperon and fadBA knockout mutant of Pseudomonas putida KT2442 harboring tesB gene [J]. Applied Microbiology & Biotechnology, 2009, 83 (3): 513-519.

[0079] The present invention also relates to the use of the aforementioned sulfur-containing acid radical ions in increasing bacterial biomass and / or increasing PHA yield.

[0080] Unless otherwise specified, the materials and reagents used in the examples of the present invention can be obtained from commercial sources. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0081] Preparation of seed culture medium: 1L culture medium contains 60g sodium chloride, 10g peptone, and 5g yeast extract powder. Adjust pH to 8, stir evenly, divide into conical bottles, put into autoclave at 115℃, and sterilize for 20 minutes. For solid plate, add 18g agar powder to the seed culture medium.

[0082] In the following examples, the carbon content of the nitrogen source was neglected when calculating the carbon-nitrogen ratio.

[0083] The PHA fermentation bacteria is Halomonas sp., and its preservation number is CGMCCNO.4353.

[0084] Corn syrup was purchased from Shandong Zhenhua Biotechnology Co., Ltd.

[0085] The sugar content was determined using the SBA-90 biosensor analyzer method.

[0086] The method for determining the biomass of Halomonas in the fermentation broth is to take 25-45 ml of the fermentation broth and centrifuge (8000 rpm, 10 min) to retain the precipitate, wash it twice with sterile water, use a vacuum freeze dryer to dry the washed precipitate for 48 hours and then weigh it.

[0087] Reference for PHA extraction: Chung A, Liu Q, Ouyang SP, et al. Microbial production of 3-hydroxydodecanoic acid by phaoperon and fadBA knockout mutant of Pseudomonas putida KT2442 harboring tesB gene[J]. Applied Microbiology&Biotechnology, 2009, 83(3): 513-519.

[0088] The following examples were carried out in a 5L fermenter with an initial liquid volume of 3L.

[0089] Preparation Example 1

[0090] Halophilic mononas were inoculated into a seed culture medium and cultured at 37°C and 200 rpm for primary activation until OD600 reached about 4 to obtain a primary seed solution; the primary seed solution was inoculated into a seed culture medium at an inoculum amount of 10 volume %, and cultured at 37°C and 200 rpm for secondary activation until OD600 reached about 5 to obtain a secondary seed solution, thereby obtaining a fermentation seed solution, wherein the content of Halophilic mononas in the seed solution was 5×10 8 -5×10 9 CFU / mL.

[0091] In the following embodiments and comparative examples, when the sugar content in the fermentation system drops below 10 g / L for the first time, the growth of the PHA fermentation strain reaches the logarithmic phase. After the addition of the first feed medium is completed, that is, 20 hours after the fermentation begins, the second feed medium begins to be fed; when the second feed medium is added, the growth of the PHA fermentation strain reaches the decay phase.

[0092] Example 1

[0093] Configuration of fermentation medium: 3L fermentation medium contains: 45g sodium chloride, 10.5g dipotassium hydrogen phosphate, 18.6g disodium hydrogen phosphate, 0.75g magnesium sulfate, 100g glucose, 11g urea. Adjust pH to 8.5.

[0094] The first feed medium: glucose was used as the carbon source at a concentration of 600 g / L, urea was used as the nitrogen source, the carbon-nitrogen ratio was 25:1, 3.3 g / L dipotassium hydrogen phosphate, 7.7 g / L disodium hydrogen phosphate and 0.8 g / L magnesium sulfate were added, and sodium sulfate was added to make the concentration of sodium sulfate 0.37 mol / L. The volume ratio of the first feed medium to the fermentation medium was 18:100.

[0095] The second feed medium: glucose was used as the carbon source at a concentration of 800 g / L, urea was used as the nitrogen source, the carbon-nitrogen ratio was 54:1, and sodium sulfate was added to make the concentration of sodium sulfate 0.2 mol / L. The volume ratio of the second feed medium to the fermentation medium was 60:100.

[0096] The seed solution prepared in Preparation Example 1 was inoculated into the fermentation medium at an inoculum amount of 10 volume %, and fermented at 37° C. and a ventilation rate of 1.5 vvm, while the pH during the fermentation process was controlled at about 8. In addition, the rotation speed was controlled at about 900 rpm during the fermentation process.

[0097] During the fermentation process, the sugar content and pH of the fermentation system were monitored in real time. When the sugar content in the fermentation system dropped below 10 g / L for the first time, the first feed medium was added. When the first feed medium was finished, the second feed medium was added. The feed flow acceleration was such that the sugar content in the fermentation broth was maintained at 5-10 g / L. After the second feed medium was finished, the fermentation was continued for 2 hours to terminate the fermentation.

[0098] The biomass of Halomonas and PHA production in the fermentation broth are shown in Table 1.

[0099] Example 2

[0100] Configuration of fermentation medium: 3L fermentation medium contains: 45g sodium chloride, 10.5g dipotassium hydrogen phosphate, 18.6g disodium hydrogen phosphate, 0.75g magnesium sulfate, 100g glucose, 11g urea. Adjust pH to 8.5.

[0101] First feed medium: add ammonium sulfate as nitrogen source and make the concentration of ammonium sulfate reach 0.37 mol / L, use glucose as carbon source, the concentration is 600 g / L, the carbon-nitrogen ratio is 23:1, and add 3.3 g / L dipotassium hydrogen phosphate, 7.7 g / L disodium hydrogen phosphate, and 0.8 g / L magnesium sulfate. The volume ratio of the first feed medium to the fermentation medium is 18:100.

[0102] The second feed medium: glucose was used as the carbon source at a concentration of 800 g / L, with a carbon-nitrogen ratio of 54:1, and ammonium sulfate was added as the nitrogen source to a concentration of 0.22 mol / L. The volume ratio of the second feed medium to the fermentation medium was 60:100.

[0103] The seed solution prepared in Preparation Example 1 was inoculated into the fermentation medium at an inoculum of 10% by volume, and fermented at 37° C. and a ventilation rate of 1.5 vvm, while the pH during the fermentation process was controlled at about 8. In addition, the rotation speed was controlled at about 900 rpm during the fermentation process.

[0104] During the fermentation process, the sugar content and pH of the fermentation system were monitored in real time. When the sugar content in the fermentation system dropped below 10 g / L for the first time, the first feed medium was added. When the first feed medium was finished, the second feed medium was added. The feed flow acceleration was such that the sugar content in the fermentation broth was maintained at 5-10 g / L. After the second feed medium was finished, the fermentation was continued for 2 hours to terminate the fermentation.

[0105] The biomass of Halomonas and PHA production in the fermentation broth are shown in Table 1.

[0106] Example 3

[0107] Configuration of fermentation medium: 3L fermentation medium contains: 45g sodium chloride, 10.5g dipotassium hydrogen phosphate, 18.6g disodium hydrogen phosphate, 0.75g magnesium sulfate, 100g glucose, 11g urea. Adjust pH to 8.5.

[0108] First feed medium: ammonium sulfite was added to supplement sulfite ions, so that the concentration of ammonium sulfite in the first feed medium reached 0.37 mol / L, glucose was used as the carbon source, the concentration was 600 g / L, ammonium sulfite was used as the nitrogen source, the carbon-nitrogen ratio was 23:1, and 3.3 g / L dipotassium hydrogen phosphate, 7.7 g / L disodium hydrogen phosphate, and 0.8 g / L magnesium sulfate were added. The volume ratio of the first feed medium to the fermentation medium was 18:100.

[0109] Second feed medium: ammonium sulfite was added to supplement sulfite ions so that the concentration of ammonium sulfite in the first feed medium reached 0.22 mol / L. Glucose was used as the carbon source and ammonium sulfite as the nitrogen source. The volume ratio of the second feed medium to the fermentation medium was 60:100.

[0110] The seed solution prepared in Preparation Example 1 was inoculated into the fermentation medium at an inoculum amount of 10 volume %, and fermented at 37° C. and a ventilation rate of 1.5 vvm, while the pH during the fermentation process was controlled at about 8. In addition, the rotation speed was controlled at about 900 rpm during the fermentation process.

[0111] During the fermentation process, the sugar content and pH of the fermentation system were monitored in real time. When the sugar content in the fermentation system dropped below 10 g / L for the first time, the first feed medium was added. When the first feed medium was finished, the second feed medium was added. The feed flow acceleration was such that the sugar content in the fermentation broth was maintained at 5-10 g / L. After the second feed medium was finished, the fermentation was continued for 2 hours to terminate the fermentation.

[0112] The comparison of Halomonas biomass, PHA yield and raw material cost per unit product in the fermentation broth is shown in Table 1.

[0113] Example 4

[0114] Compared with Example 1, the difference is that sulfate is not added to the feed medium, and sulfate is added before the first feed medium is fed. The specific steps are as follows:

[0115] Configuration of fermentation medium: 3L fermentation medium contains: 45g sodium chloride, 10.5g dipotassium hydrogen phosphate, 18.6g disodium hydrogen phosphate, 0.75g magnesium sulfate, 100g glucose, 11g urea. Adjust pH to 8.5.

[0116] The first feed medium: glucose was used as the carbon source at a concentration of 600 g / L, urea was used as the nitrogen source, the carbon-nitrogen ratio was 25:1, and 3.3 g / L dipotassium hydrogen phosphate, 7.7 g / L disodium hydrogen phosphate, and 0.8 g / L magnesium sulfate were added. The volume ratio of the first feed medium to the fermentation medium was 18:100.

[0117] The second feed medium: glucose is used as the carbon source at a concentration of 800 g / L, urea is used as the nitrogen source, the carbon-nitrogen ratio is 54:1, and the volume ratio of the second feed medium to the fermentation medium is 60:100.

[0118] The seed solution prepared in Preparation Example 1 was inoculated into the fermentation medium at an inoculum amount of 10 volume %, and fermented at 37° C. and a ventilation rate of 1.5 vvm, while the pH during the fermentation process was controlled at about 8. In addition, the rotation speed was controlled at about 900 rpm during the fermentation process.

[0119] During the fermentation process, the sugar content and pH of the fermentation system were monitored in real time. When the sugar content in the fermentation system dropped below 10 g / L for the first time, 0.56 mol of sodium sulfate was added to the fermenter to make the sulfate ion concentration in the fermenter reach 0.19 mol / L. After the addition of sodium sulfate, the first feed medium was added. When the first feed medium was finished, the second feed medium was added. The feed flow acceleration was such that the sugar content in the fermentation liquid was maintained at 5-10 g / L. After the second feed medium was finished, the fermentation was continued for 2 hours to end.

[0120] The biomass of Halomonas and PHA production in the fermentation broth are shown in Table 1.

[0121] Example 5

[0122] The method of Example 1 was followed, except that sodium sulfate was not added to the second feed medium.

[0123] Configuration of fermentation medium: 3L fermentation medium contains: 45g sodium chloride, 10.5g dipotassium hydrogen phosphate, 18.6g disodium hydrogen phosphate, 0.75g magnesium sulfate, 100g glucose, 11g urea. Adjust pH to 8.5.

[0124] First feed medium: sodium sulfate was added to make the concentration of sodium sulfate reach 0.37 mol / L, glucose was used as carbon source, the concentration was 600 g / L, urea was used as nitrogen source, the carbon-nitrogen ratio was 25:1, and 3.3 g / L dipotassium hydrogen phosphate, 7.7 g / L disodium hydrogen phosphate, and 0.8 g / L magnesium sulfate were added. The volume ratio of the first feed medium to the fermentation medium was 18:100.

[0125] The second feed medium: glucose was used as the carbon source at a concentration of 800 g / L, urea was used as the nitrogen source, and the carbon-nitrogen ratio was 54:1.

[0126] The seed solution prepared in Preparation Example 1 was inoculated into the fermentation medium at an inoculum of 10% by volume, and fermented at 37° C. and a ventilation rate of 1.5 vvm, while the pH during the fermentation process was controlled at about 8. In addition, the rotation speed was controlled at about 900 rpm during the fermentation process.

[0127] During the fermentation process, the sugar content and pH of the fermentation system were monitored in real time. When the sugar content in the fermentation system dropped below 10 g / L for the first time, the first feed medium was added. When the first feed medium was finished, the second feed medium was added. The feed flow acceleration was such that the sugar content in the fermentation broth was maintained at 5-10 g / L. After the second feed medium was finished, the fermentation was continued for 2 hours to terminate the fermentation.

[0128] The biomass of Halomonas and PHA production in the fermentation broth are shown in Table 1.

[0129] Example 6

[0130] The method of Example 1 was followed, except that the concentration of sulfate ions in the first feed medium and the fermentation medium was 0.2 mol / L; the concentration of sulfate ions in the second feed medium and the fermentation medium was 0.15 mol / L. The biomass of Halomonas in the fermentation broth and the PHA yield are shown in Table 1.

[0131] Example 7

[0132] The method of Example 1 was followed, except that the concentration of sulfate ions in the first feed medium and the fermentation medium was 0.45 mol / L; the concentration of sulfate ions in the second feed medium and the fermentation medium was 0.35 mol / L. The biomass of Halomonas in the fermentation broth and the PHA yield are shown in Table 1.

[0133] Example 8

[0134] The method of Example 1 was followed, except that the concentration of sulfate ions in the first feed medium and the fermentation medium was 0.12 mol / L; the concentration of sulfate ions in the second feed medium and the fermentation medium was 0.07 mol / L. The biomass of Halomonas in the fermentation broth and the PHA yield are shown in Table 1.

[0135] Example 9

[0136] The method of Example 1 was followed, except that the concentration of sulfate ions in the first feed medium and the fermentation medium was 0.7 mol / L; the concentration of sulfate ions in the second feed medium and the fermentation medium was 0.4 mol / L. The biomass of Halomonas in the fermentation broth and the PHA yield are shown in Table 1.

[0137] Example 10

[0138] The method of Example 1 is followed, except that the nitrogen sources of the first feed medium and the second feed medium are both yeast extract powder.

[0139] The biomass of Halomonas and PHA production in the fermentation broth are shown in Table 1.

[0140] Embodiment 11

[0141] The method of Example 1 is followed, except that the nitrogen sources of the first feed medium and the second feed medium are both ammonium chloride.

[0142] The biomass of Halomonas and PHA production in the fermentation broth are shown in Table 1.

[0143] Example 12

[0144] The method of Example 1 was followed, except that the carbon-nitrogen ratio in the first feed medium was 50:1, and the sodium sulfate concentration was 0.2 mol / L; the carbon-nitrogen ratio in the second feed medium was 70:1, and the sodium sulfate concentration was 0.15 mol / L. The biomass of Halomonas in the fermentation broth and the PHA yield are shown in Table 1.

[0145] Embodiment 13

[0146] The method of Example 1 was followed, except that the carbon-nitrogen ratio in the first feed medium was 10:1, and the sodium sulfate concentration was 0.45 mol / L; the carbon-nitrogen ratio in the second feed medium was 20:1, and the sodium sulfate concentration was 0.35 mol / L. The biomass of Halomonas in the fermentation broth and the PHA yield are shown in Table 1.

[0147] Embodiment 14

[0148] The method of Example 1 is followed, except that sodium sulfate is not added to the first feed medium. The specific steps are as follows:

[0149] Configuration of fermentation medium: 3L fermentation medium contains: 45g sodium chloride, 10.5g dipotassium hydrogen phosphate, 18.6g disodium hydrogen phosphate, 0.75g magnesium sulfate, 100g glucose, 11g urea. Adjust pH to 8.5.

[0150] The first feed medium: glucose was used as the carbon source at a concentration of 600 g / L, urea was used as the nitrogen source, the carbon-nitrogen ratio was 25:1, and 3.3 g / L dipotassium hydrogen phosphate, 0.8 g magnesium sulfate and 7.7 g / L disodium hydrogen phosphate were added. The volume ratio of the first feed medium to the fermentation medium was 18:100.

[0151] Second feed medium: sodium sulfate was added to make the concentration of sodium sulfate reach 0.32 mol / L, glucose was used as carbon source with a concentration of 800 g / L, urea was used as nitrogen source, and the carbon-nitrogen ratio was 54: 1. The volume ratio of the second feed medium to the fermentation medium was 60:100.

[0152] The seed solution prepared in Preparation Example 1 was inoculated into the fermentation medium at an inoculum of 10% by volume, and fermented at 37° C. and a ventilation rate of 1.5 vvm, while the pH during the fermentation process was controlled at about 8. In addition, the rotation speed was controlled at about 900 rpm during the fermentation process.

[0153] During the fermentation process, the sugar content and pH of the fermentation system were monitored in real time. When the sugar content in the fermentation system dropped below 10 g / L for the first time, the first feed medium was added. When the first feed medium was finished, the second feed medium was added. The feed flow acceleration was such that the sugar content in the fermentation broth was maintained at 5-10 g / L. After the second feed medium was finished, the fermentation was continued for 2 hours to terminate the fermentation.

[0154] The biomass of Halomonas and PHA production in the fermentation broth are shown in Table 1.

[0155] Embodiment 15

[0156] The method of Example 1 was followed, except that sodium thiosulfate was used instead of sodium sulfate in the feed medium. The biomass of Halomonas and the PHA yield in the fermentation broth are shown in Table 1.

[0157] Example 16

[0158] The method of Example 1 was followed, except that sodium bisulfite was used instead of sodium sulfate in the feed medium. The biomass of Halomonas and the PHA yield in the fermentation broth are shown in Table 1.

[0159] Embodiment 17

[0160] The method of Example 1 was followed, except that 50% sodium sulfate and 50% sodium sulfite were used to replace the sodium sulfate in the feed medium. The biomass of Halomonas and the PHA yield in the fermentation broth are shown in Table 1.

[0161] Comparative Example 1

[0162] The method of Example 1 is followed, except that sodium sulfate is not added to the first feed medium and the second feed medium. The specific steps are as follows:

[0163] Configuration of fermentation medium: 3L fermentation medium contains: 45g sodium chloride, 10.5g dipotassium hydrogen phosphate, 18.6g disodium hydrogen phosphate, 0.75g magnesium sulfate, 100g glucose, 11g urea. Adjust pH to 8.5.

[0164] The first feed medium: glucose was used as the carbon source at a concentration of 600 g / L, urea was used as the nitrogen source, the carbon-nitrogen ratio was 25:1, and 3.3 g / L dipotassium hydrogen phosphate and 7.7 g / L disodium hydrogen phosphate were added. The volume ratio of the first feed medium to the fermentation medium was 18:100.

[0165] The second feed medium: glucose was used as the carbon source at a concentration of 800 g / L, urea was used as the nitrogen source, and the carbon-nitrogen ratio was 54: 1. The volume ratio of the second feed medium to the fermentation medium was 60:100.

[0166] The seed solution prepared in Preparation Example 1 was inoculated into the fermentation medium at an inoculum of 10% by volume, and fermented at 37° C. and a ventilation rate of 1.5 vvm, while the pH during the fermentation process was controlled at about 8. In addition, the rotation speed was controlled at about 900 rpm during the fermentation process.

[0167] During the fermentation process, the sugar content and pH of the fermentation system were monitored in real time. When the sugar content in the fermentation system dropped below 10 g / L for the first time, the first feed medium was added. When the first feed medium was finished, the second feed medium was added. The feed flow acceleration was such that the sugar content in the fermentation broth was maintained at 5-10 g / L. After the second feed medium was finished, the fermentation was continued for 2 hours to terminate the fermentation.

[0168] The biomass of Halomonas and PHA production in the fermentation broth are shown in Table 1.

[0169] Comparative Example 2

[0170] The method of Comparative Example 1 was followed, except that the nitrogen source in the feed medium was yeast extract powder.

[0171] The biomass of Halomonas and PHA production in the fermentation broth are shown in Table 1.

[0172] Comparative Example 3

[0173] The method of Comparative Example 1 is followed, except that the nitrogen source in the feed medium is ammonium chloride.

[0174] The biomass of Halomonas and PHA production in the fermentation broth are shown in Table 1.

[0175] Table 1

[0176] serial number Biomass (g / L) PHA yield (g / L) Example 1 141 115.6 Example 2 132 108.24 Example 3 125 102.5 Example 4 125 102.5 Example 5 111 91 Example 6 122 100 Example 7 121 99.22 Example 8 107 87.74 Example 9 113 92.7 Example 10 124 101.7 Embodiment 11 122 100 Example 12 109 90 Embodiment 13 92 73 Embodiment 14 110 77 Embodiment 15 75 62 Example 16 117 96 Embodiment 17 120 98.4 Comparative Example 1 98 76 Comparative Example 2 97 72 Comparative Example 3 94 67

[0177] It can be seen from the results in Table 1 that the technical solution of the present invention is used to supplement nitrogen sources and sulfate ions and / or sulfite ions during the appropriate bacterial growth period for the production of PHA by Halomonas. Compared with the currently commonly used production methods, the biomass and PHA yield are high, the proportion of PHA in the biomass is increased, and the production cost of PHA is greatly reduced.

[0178] Among them, the biomass of Example 13 is relatively low, but compared with Comparative Example 1, after supplementing sulfate ions, the proportion of PHA content in biomass is still increased, indicating that the implementation of the method provided by the present invention within the preferred range is more conducive to increasing PHA yield and biomass.

[0179] In Example 15, compared with Example 1, both the biomass and PHA yield decreased, indicating that adding the preferred sulfur-containing acid radical ions of the present invention is more conducive to increasing PHA yield and biomass.

[0180] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for increasing PHA production, characterized in that: The method comprises the following steps: inoculating PHA fermentation bacteria into a fermentation medium for fermentation under conditions capable of producing PHA by fermentation, and supplementing sulfur-containing acid radical ions during the fermentation process.

2. The method according to claim 1, wherein: The PHA fermentation bacteria is Halomonas; Preferably, compared to 1L of fermentation medium, the inoculation amount of the PHA fermentation strain is 4×10 8 CFU-6×10 9 CFU, preferably 5×10 8 CFU-5×10 9 CFU; More preferably, the PHA fermentation strain is inoculated in the form of a seed solution, and the inoculation amount of the PHA fermentation strain is 1-20% by volume, preferably 5-15% by volume, compared to the fermentation medium, and the OD of the seed solution is 600 The value is 3-5.

3. The method according to claim 1 or 2, wherein: The starting time for the supplementation of the sulfur-containing acid radical ions is when the growth of the PHA fermentation bacteria reaches the logarithmic phase; and / or, The timing for ending the supplementation of the sulfur-containing acid radical ions is when the PHA fermentation bacteria reaches the decay stage; Preferably, the amount of the sulfur-containing acid radical ions supplemented is such that, when the growth of the PHA fermentation strain reaches the logarithmic phase to the cell death phase, the concentration of the sulfur-containing acid radical ions relative to 1L of fermentation medium is 6-200mmol / L, more preferably 6-120mmol / L; Preferably, relative to 1L of fermentation medium, the amount of the sulfur-containing acid ion supplement is 60-400mmol, more preferably 120-300mmol, and further preferably 170-200mmol; Preferably, the sulfur-containing acid ions are sulfate and / or sulfite; More preferably, the sulfate ion is derived from at least one of sulfate and bisulfate, and further more preferably at least one of ammonium sulfate, ammonium bisulfate, calcium sulfate, calcium bisulfate, magnesium sulfate, magnesium bisulfate, sodium sulfate, sodium bisulfate, potassium sulfate and potassium bisulfate; And / or, the sulfite ion is derived from at least one of sulfite and bisulfite, preferably at least one selected from ammonium sulfite, ammonium bisulfite, calcium sulfite, calcium bisulfite, magnesium sulfite, magnesium bisulfite, sodium sulfite, sodium bisulfite, potassium sulfite and potassium bisulfite.

4. The method according to claim 1 or 2, wherein: The fermentation medium contains a carbon source, an organic nitrogen source, phosphate, a magnesium salt and a sodium salt; Preferably, the carbon-nitrogen ratio in the fermentation medium is 5-10:1; Preferably, the fermentation medium contains 5-50 g / L of carbon source, more preferably 15-30 g / L of carbon source; Preferably, the carbon source is glucose; Preferably, the organic nitrogen source is urea; Preferably, the phosphate is dipotassium hydrogen phosphate and / or disodium hydrogen phosphate; Preferably, the magnesium salt is magnesium sulfate and / or magnesium chloride; Preferably, the sodium salt is sodium chloride; Preferably, the fermentation conditions include: temperature of 32-40°C, pH of 7-11, dissolved oxygen of 1-40%; And / or, the fermentation is carried out under ventilation conditions with a ventilation rate of 1-2 vvm.

5. The method according to claim 4, wherein: The fermentation medium comprises urea, glucose, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and sodium chloride; More preferably, relative to 1L of fermentation medium, the content of glucose is 5-50g, the content of urea is 1-10g, the content of dipotassium hydrogen phosphate is 2-5g, the content of disodium hydrogen phosphate is 2-10g, the content of magnesium sulfate is 0.1-0.5g, and the content of sodium chloride is 8-30g; Further preferably, relative to 1L of fermentation medium, the content of glucose is 20-40g, the content of urea is 2-6g, the content of dipotassium hydrogen phosphate is 3-4g, the content of disodium hydrogen phosphate is 5-7g, the content of magnesium sulfate is 0.2-0.3g, and the content of sodium chloride is 12-18g.

6. The method according to claim 1 or 2, wherein: The method further comprises supplementing a feed medium containing a nitrogen source during the fermentation process; Preferably, the nitrogen source is an organic nitrogen source and / or an inorganic nitrogen source; Preferably, the feed medium further comprises a carbon source; More preferably, the carbon source is glucose; More preferably, the organic nitrogen source is at least one of urea, urea, corn steep liquor, enzymatic corn steep liquor, yeast powder, yeast extract and peptone; More preferably, the inorganic nitrogen source is an ammonium salt, more preferably at least one selected from ammonium chloride, ammonium nitrate, diammonium hydrogen phosphate, ammonium sulfate and ammonium sulfite; Preferably, the nitrogen source supplementation is started when the growth of the fermentation strain reaches the logarithmic phase; and / or, The nitrogen source supplementation ends when the growth of the halophilic monas reaches the decay phase; Preferably, the nitrogen source is supplemented in a two-stage feeding manner.

7. The method according to claim 6, wherein: The two-stage feeding comprises a first feeding using a first feeding medium and then a second feeding using a second feeding medium; Preferably, the carbon-nitrogen ratio of the first feed medium is (20-30):1; Preferably, the supplement amount of the first feed medium is 10-25% by volume of the fermentation medium; Preferably, the first feed medium comprises 550-650 g / L of a carbon source; More preferably, the first feed medium further comprises dipotassium hydrogen phosphate, disodium hydrogen phosphate and magnesium sulfate; More preferably, relative to 1L of the first feed medium, the content of dipotassium hydrogen phosphate is 1-5g, the content of disodium hydrogen phosphate is 5-10g, and the content of magnesium sulfate is 0.1-1.5g; Further preferably, relative to 1 L of the first feed medium, the content of dipotassium hydrogen phosphate is 3-4 g, the content of disodium hydrogen phosphate is 7-8 g, and the content of magnesium sulfate is 0.5-1 g.

8. The method according to claim 7, wherein: The carbon-nitrogen ratio of the second feed medium is (40-60):1, more preferably (50-60):1; Preferably, the supplement amount of the second feed medium is 45-65% by volume of the fermentation medium; Preferably, the second feed medium comprises 750-850 g / L of a carbon source.

9. The method according to claim 1 or 2, wherein: The fermentation is continuous fermentation or intermittent fermentation.

10. The method according to claim 1 or 2, wherein: The method further comprises: when the carbon source content in the fermentation medium is lower than 5 g / L after the sulfur-containing acid radical ions are supplemented, the fermentation is terminated.