阅读说明：本技术 一种提高微生物培养效果的方法及培养基 (Method and culture medium for improving microbial culture effect ) 是由 尹进 李明月 于 2021-10-26 设计创作，主要内容包括：本发明涉及微生物发酵技术领域,具体公开了一种提高微生物培养效果的方法及培养基。本发明通过向培养基中添加聚甘油脂肪酸酯,不仅提高了微生物对脂质碳源的利用率,还提高了发酵过程中微生物生物量的积累及次生代谢产物产量；同时,解决了现有菌种进行发酵测试时,发酵效果不稳定、重复性不高的问题,为优势菌的筛选提供了必要的技术支持。(The invention relates to the technical field of microbial fermentation, and particularly discloses a method and a culture medium for improving a microbial culture effect. According to the invention, the polyglycerol fatty acid ester is added into the culture medium, so that the utilization rate of the lipid carbon source by the microorganism is improved, and the accumulation of the biomass of the microorganism and the yield of secondary metabolites in the fermentation process are also improved; meanwhile, the problems of unstable fermentation effect and low repeatability in the fermentation test of the existing strains are solved, and necessary technical support is provided for screening of dominant bacteria.)

1. A method for improving the culture effect of microorganisms is characterized in that microorganism fermentation is carried out by utilizing a culture medium, wherein the culture medium takes lipid as a carbon source and is added with polyglycerol fatty acid ester; the improvement of the microbial culture effect comprises at least one item of (1) to (4):

(1) improving the utilization rate of the lipid carbon source by the microorganism;

(2) increasing the biomass of the microorganism;

(3) the yield of secondary metabolites of the microorganisms is improved;

(4) improving the fermentation stability of the microorganism.

2. The method according to claim 1, wherein the molar ratio of the lipid to the polyglycerin fatty acid ester in the medium is 12:1 to 61: 1.

3. The method according to claim 2, wherein the concentration of the lipid in the medium is 8 to 12g/L, and the concentration of the polyglycerin fatty acid ester is 0.6 to 3 g/L.

4. The method of claim 3, wherein the lipid is palm oil.

5. The method according to any one of claims 1 to 4, wherein the culture medium contains water and inorganic salts at the following concentrations:

1.57g/L ammonium sulfate, 5.66g/L sodium dihydrogen phosphate dodecahydrate, 1.5g/L potassium dihydrogen phosphate, 0.2g/L magnesium sulfate heptahydrate, 0.01g/L calcium chloride dihydrate, 0.02g/L ferrous sulfate heptahydrate, 0.0003g/L boric acid, 0.0002g/L cobalt chloride hexahydrate, 0.0001g/L zinc sulfate heptahydrate, 0.03g/L manganese chloride tetrahydrate, 0.03g/L sodium molybdate dihydrate, 0.02g/L nickel chloride hexahydrate, and 0.01g/L copper sulfate pentahydrate.

6. The method according to any one of claims 1 to 4, wherein the microorganism is selected from the group consisting of Eubacterium rolfsiiRalstonia eutrophaPseudomonas aeruginosaPseudomonas aeruginosa、Rhodococcus spRhodococcus opacusAnd Bacillus subtilisBacillus subtilisOne or more of (a).

7. A culture medium for improving the culture effect of microorganisms, which is characterized in that the culture medium takes lipid as a carbon source and is added with polyglycerol fatty acid ester; the improvement of the microbial culture effect comprises at least one item of (1) to (4):

(1) improving the utilization rate of the lipid carbon source by the microorganism;

(2) increasing the biomass of the microorganism;

(3) the yield of secondary metabolites of the microorganisms is improved;

(4) improving the fermentation stability of the microorganism.

8. The medium according to claim 7, wherein the molar ratio of the lipid to the polyglycerin fatty acid ester in the medium is 12:1 to 61: 1.

9. The medium according to claim 7 or 8, wherein the concentration of the lipid in the medium is 8 to 12g/L and the concentration of the polyglycerin fatty acid ester is 0.6 to 3 g/L.

10. The culture medium of claim 9, wherein the culture medium comprises: water and 10g/L of palm oil, 0.6-3 g/L of decaglycerol monolaurate, 1.57g/L of ammonium sulfate, 5.66g/L of sodium dihydrogen phosphate dodecahydrate, 1.5g/L of potassium dihydrogen phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.01g/L of calcium chloride dihydrate, 0.02g/L of ferrous sulfate heptahydrate, 0.0003g/L of boric acid, 0.0002g/L of cobalt chloride hexahydrate, 0.0001g/L of zinc sulfate heptahydrate, 0.03g/L of manganese chloride tetrahydrate, 0.03g/L of sodium molybdate dihydrate, 0.02g/L of nickel chloride hexahydrate, and 0.01g/L of copper sulfate pentahydrate.

Technical Field

The invention relates to the technical field of microbial fermentation, in particular to a method and a culture medium for improving the culture effect of microorganisms.

Background

Polyhydroxyalkanoates (PHA) is a high molecular polymer composed of 100 to 30000 same or different hydroxy fatty acid monomers. PHA is a biopolymer material that has developed rapidly over 20 years due to its combination of good biocompatibility, biodegradability and thermal processability of plastics. Therefore, the composite material can be used as a biomedical material and a biodegradable packaging material at the same time. In addition, PHA has many high value-added properties including nonlinear optical properties, piezoelectric properties, and gas barrier properties. Thus, PHA has become the most active research hotspot in the field of biomaterials in recent years. PHA is an intracellular polyester synthesized by many microorganisms, and at present, the production method thereof is mainly microbial fermentation. Various methods for producing PHA using microorganisms utilizing oils and fats as a carbon source have been described in the prior art. However, the yield of PHA is still to be further improved.

Factors affecting PHA production mainly include growth and metabolic activity of the bacterial species themselves and the influence of culture conditions on bacterial species activity. Therefore, the selection of strains and optimization of culture conditions are the focus of current research. On the one hand, the shake flask test commonly used for strain screening is used as the first step of screening, but the shake flask is found to have low stability in experiments, and the same strain can still show different activities in a plurality of shake flasks even if the same culture solution is used, so that a large error is generated. On the other hand, regardless of the culture conditions, the utilization of the carbon source by the bacterial species is an important factor affecting the PHA production. In the prior art, various schemes are tried to solve the problem of utilization rate of lipid carbon sources by microorganisms, but most of the schemes can enhance the vegetative reproduction of the microorganisms, increase the number of thalli or the propagation speed, but cannot realize the improvement of the yield of PHA.

For example, the technical solution of using gum arabic as additive shows great difference in multiple cultivations, some culture bottles even precipitate; although casein and phosphate are adopted as additives to obtain good effect in the initial stage of fermentation, the pH value and ionic strength of a culture system are changed along with the fermentation, the emulsification effect is influenced, and the fermentation stability and the microbial activity are reduced; attempts to use tween series as additives did not improve PHA accumulation.

Therefore, the utilization rate of the lipid by the microorganisms is improved, the fermentation level of the strain in multi-batch culture is stabilized, and the method has important significance for screening dominant bacteria.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a method and a culture medium for improving the culture effect of microorganisms, and the method and the culture medium can effectively improve the utilization rate of lipid carbon sources by the microorganisms, improve the biomass of the microorganisms, improve the yield of secondary metabolites of the microorganisms and improve the stability of the multiple fermentation effect of the microorganisms.

The research of the invention shows that in the process of carrying out microbial culture by taking lipid as a main carbon source or a unique carbon source, the conditions of unstable effect, insufficient utilization rate of the carbon source, insufficient accumulation of microbial biomass and low yield of secondary metabolites frequently occur among multiple times of culture, but the reasons are various, and various factors also have mutual influence. Through further research, the addition of polyglycerol fatty acid ester substances to the culture medium has a positive and effective effect on solving the problems.

In order to solve the problems and avoid the situations, the invention adopts a plurality of substances as additives to carry out comparison and screening through a large number of objective experiments, and three additives with better comprehensive effects, namely the liquid detergent, the polyglycerol fatty acid ester and the saponin, are screened out according to reference indexes such as the emulsification effect, the stability and the homogeneity of the mixture after the additives are added and the like. Furthermore, by combining the experimental comparison of biomass accumulation level and secondary metabolite yield, the finally screened polyglycerol fatty acid ester can be more favorable for improving the utilization rate of the lipid carbon source by the microorganisms, shows good stability in multi-batch fermentation experiments, and is not obviously influenced by the addition concentration.

Polyglycerol fatty acid ester (PGFE), abbreviated as polyglycerol ester, is an ester of polyglycerol and fatty acid. In the present invention, the polyglycerin fatty acid ester is selected from: at least one of dimer to decaglycerol stearate series, dimer to decaglycerol oleate series, dimer to decaglycerol palmitate series, dimer to decaglycerol myristate series, dimer to decaglycerol laurate series, polyglycerol polyricinoleate series, dimer glycerol dipolyhydroxystearate, trimer glycerol diisostearate, lactic acid monoglyceride, succinic acid monoglyceride, citric acid monoglyceride, oleic acid monoglyceride, caprylic acid capric acid monoglyceride, diacetyl tartaric acid monoglyceride/diglyceride.

In the specific embodiment of the present invention, decaglycerol monolaurate is taken as an illustrative example, and it is understood that the above alternative species of polyglycerol fatty acid esters may be substituted equivalently in practice due to the same or similar properties as decaglycerol monolaurate.

Experimental research shows that the polyglycerol fatty acid ester is added into a culture medium taking lipid as a carbon source, and the mixture can still keep good uniformity after high-temperature sterilization and shows good stability in multiple fermentations. The culture medium containing the polyglycerol fatty acid ester is used for fermenting the strains, so that the grease can be more effectively utilized, higher biomass is obtained, and the accumulation of secondary metabolite PHA is improved. In the embodiment of the invention, the secondary metabolite is PHA.

In a first aspect, the present invention provides a method for improving the culture effect of microorganisms, wherein the microorganism fermentation is carried out by using a culture medium, wherein the culture medium takes lipid as a carbon source and is added with polyglycerol fatty acid ester; the improvement of the microbial culture effect comprises at least one item of (1) to (4):

(1) improving the utilization rate of the lipid carbon source by the microorganism;

(2) increasing the biomass of the microorganism;

(3) the yield of secondary metabolites of the microorganisms is improved;

(4) improving the fermentation stability of the microorganism.

Preferably, the medium is a liquid medium.

Further, the molar ratio of the lipid to the polyglycerol fatty acid ester in the culture medium is 12: 1-61: 1, and typical non-limiting examples of the lipid to the polyglycerol fatty acid ester include 61:1, 56:1, 52:1, 48:1, 42:1, 36:1, 28:1, 24:1, 18:1 and 12: 1.

Wherein the lipid is vegetable oil, animal oil or kitchen waste oil.

The vegetable oil is selected from one or more of palm oil, peanut oil, soybean oil, sesame oil, olive oil, sunflower oil, rapeseed oil, corn oil, cottonseed oil, tung oil, ebony oil, pine seed oil, castor oil and barbadosnut seed oil.

The animal oil is selected from one or more of fish oil, chicken oil, beef tallow, mutton fat and lard.

Preferably, the concentration of the lipid in the culture medium is 8-12 g/L, and the concentration of the polyglycerol fatty acid ester is 0.6-3 g/L. Typically, without limitation, the lipid concentration may be 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, and the polyglycerol fatty acid ester concentration may be 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 1.2g/L, 1.4g/L, 1.6g/L, 1.8g/L, 2g/L, 2.2g/L, 2.4g/L, 2.6g/L, 2.8g/L, 3 g/L.

In a particular embodiment of the invention, the lipid is, by way of illustration, a vegetable oil, more particularly palm oil.

Still further, the medium also contains water and inorganic salts at the following concentrations: 1.57g/L ammonium sulfate, 5.66g/L sodium dihydrogen phosphate dodecahydrate, 1.5g/L potassium dihydrogen phosphate, 0.2g/L magnesium sulfate heptahydrate, 0.01g/L calcium chloride dihydrate, 0.02g/L ferrous sulfate heptahydrate, 0.0003g/L boric acid, 0.0002g/L cobalt chloride hexahydrate, 0.0001g/L zinc sulfate heptahydrate, 0.03g/L manganese chloride tetrahydrate, 0.03g/L sodium molybdate dihydrate, 0.02g/L nickel chloride hexahydrate, and 0.01g/L copper sulfate pentahydrate.

It is to be understood that in the foregoing method, the microorganism is a microorganism capable of fermentative metabolism using a lipid as a carbon source. Preferably, the microorganism is selected from among Eubacterium rolfsii (R) ((R))Ralstonia eutropha) Pseudomonas aeruginosaPseudomonas aeruginosa）、Rhodococcus (A)Rhodococcus opacus) And Bacillus subtilis (B.) (Bacillus subtilis) One or more of (a).

More specifically, in some embodiments of the invention, the microorganism is a eubacterium reuteri, in particularRalstonia eutrophaH16. The specific fermentation method comprises the following steps: the microorganism was inoculated to the aforementioned medium to an initial OD of 0.05, followed by culture at 220 rpm at 30 ℃ for 48 hours.

Still further, in order to better improve the culture effect of the microorganism, an activation step may be added before the fermentation step, and the medium used in the activation step may be an activation medium conventionally used in the art, including water, yeast extract, peptone, fructose, and sodium chloride. The activation method may also be any method conventionally used in the art, such as streaking and then shake culture.

In some embodiments, when Eubacterium reuteri is used as the fermentation strain, the medium of the streaked culture contains water and 5g/L yeast extract, 10g/L peptone, 3g/L fructose, 10g/L sodium chloride and 1.5g/L agar powder; the culture medium for shake culture contains water and 5g/L yeast extract, 10g/L peptone, 3g/L fructose and 10g/L sodium chloride.

In some embodiments, the conditions of the streaking culture include a culture at 30 ℃ for 48h, and the conditions of the shake culture include a culture at 30 ℃ and 220 rpm for 8 h.

In a specific embodiment of the invention, the method for preparing PHA is further provided, which takes palm oil as a carbon source and ferments the eumycete rolfsii in the presence of decaglycerol monolaurate to obtain fermentation liquor containing PHA.

In a second aspect, the present invention provides a culture medium for improving the effect of culturing microorganisms, wherein the culture medium uses lipid as a carbon source and is added with polyglycerol fatty acid ester; the improvement of the microbial culture effect comprises at least one item of (1) to (4):

(1) improving the utilization rate of the lipid carbon source by the microorganism;

(2) increasing the biomass of the microorganism;

(3) the yield of secondary metabolites of the microorganisms is improved;

(4) improving the fermentation stability of the microorganism.

Further, the molar ratio of the lipid to the polyglycerol fatty acid ester in the medium is 12:1 to 61: 1.

Preferably, the concentration of the lipid in the culture medium is 8-12 g/L, and the concentration of the polyglycerol fatty acid ester is 0.6-3 g/L.

More specifically, the culture medium comprises: water and 10g/L of palm oil, 0.6-3 g/L of decaglycerol monolaurate, 1.57g/L of ammonium sulfate, 5.66g/L of sodium dihydrogen phosphate dodecahydrate, 1.5g/L of potassium dihydrogen phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.01g/L of calcium chloride dihydrate, 0.02g/L of ferrous sulfate heptahydrate, 0.0003g/L of boric acid, 0.0002g/L of cobalt chloride hexahydrate, 0.0001g/L of zinc sulfate heptahydrate, 0.03g/L of manganese chloride tetrahydrate, 0.03g/L of sodium molybdate dihydrate, 0.02g/L of nickel chloride hexahydrate, and 0.01g/L of copper sulfate pentahydrate.

The invention has the beneficial effects that:

according to the invention, by adding the specific additive into the fermentation medium, the utilization rate of the lipid carbon source and the yield of the secondary metabolite of the microorganism are improved, the problems of low stability and low repeatability of the existing fermentation test method are solved, the fermentation level of the microorganism in multi-batch culture is stabilized, and necessary and good technical support is provided for screening the dominant strain.

Drawings

FIG. 1 is a graph showing the visual observation of the emulsified state of the emulsion after the addition of varying concentrations of decaglycerol monolaurate; the subscripted concentrations in the figures represent the decaglycerol monolaurate/medium, and the bottles are labeled as the volume of addition of the decaglycerol monolaurate mother liquor per bottle, and the subsequent references to this section are based on the subscripted concentrations in the figures.

FIG. 2 shows the emulsion state observed by optical microscopy after the addition of varying concentrations of decaglycerol monolaurate; the subscripted concentrations indicate the decaglycerol monolaurate/medium observed at 10 and 40 times objective respectively.

FIG. 3 is a visual observation of the emulsion state after different concentrations of detergent are added; the concentrations indicated in the figure indicate the amounts of detergent/culture medium, and the bottles are indicated by the volume of detergent stock solution added per bottle, and the subsequent references to these parts are based on the concentrations indicated in the figure.

FIG. 4 shows the emulsion state observed by an optical microscope after adding different concentrations of detergent; the subscript concentrations indicate the detergent/medium, observed at 10-fold objective and 40-fold objective, respectively.

FIG. 5 is a visual observation of the emulsified state of the emulsion after different concentrations of saponin were added; the concentrations indicated in the figure represent the saponin/medium, and the bottles are marked with the addition volume of the saponin mother liquor in each bottle, and the subsequent reference to this part is based on the concentrations indicated in the figure.

FIG. 6 shows the emulsion state observed by optical microscope after different concentrations of saponin are added; the subscripted concentrations indicate saponin/medium, all observed under a 10-fold objective.

Detailed Description

The invention provides a method and a culture medium for improving the application of polyglycerol fatty acid ester in improving the culture effect of microorganisms. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be obtained by conventional purchasing approaches.

The microorganism is a group of single-cell or multi-cell organisms with small individual and simple structure, and comprises bacteria, fungi, actinomycetes and the like. The microorganism of the present invention is a microorganism capable of using a lipid as a carbon source. Mainly comprises bacteria using lipid as a carbon source. For example, the microorganisms of choice include Eubacterium reuteri (R) ((R))Ralstonia eutropha) Pseudomonas aeruginosaPseudomonas aeruginosa）、Rhodococcus (A)Rhodococcus opacus) And Bacillus subtilis (B.) (Bacillus subtilis) At least one of (1). In a particular embodiment of the invention, in bacteriaRalstonia eutrophaH16 doesAre exemplary illustrations.

The lipid of the invention refers to an ester or a derivative thereof generated by dehydration condensation of fatty acid and alcohol, and is insoluble in water and easily soluble in an organic solvent. Lipids include lipids (triglycerides) and lipids (phospholipids, sugar esters and sterols). In general, fats and oils which are liquid at ordinary temperatures are referred to as oils, and fats and oils which are solid are referred to as fats. Substantially all lipids can serve as a carbon source for microbial growth. However, in the embodiment of the present invention, it is preferable that the oil or fat which is liquid at room temperature be used as a carbon source for the microbial fermentation. In the specific embodiment of the present invention, palm oil in grease is taken as an example.

The polyglycerin fatty acid ester of the present invention: is an ester formed by esterifying polyglycerol and fatty acid at high temperature. In a specific embodiment of the present invention, decaglycerol monolaurate is exemplified.

The main component of the detergent is a surfactant. In the embodiment of the invention, the detergent containing various components such as C10-16 alcohol polyoxyethylene ether sodium sulfate, C8-16 alkyl glucoside, C10-16 sodium alkyl benzene sulfonate, cocamidopropyl amine oxide, polyoxyethylene sorbitan monolaurate and the like is taken as an illustration.

The saponin comprises sapogenin and sugar, wherein the saponin with the sapogenin being triterpenes is called triterpenoid saponin. In a specific embodiment of the present invention, quinapride is used as an illustration.

The invention is further illustrated by the following examples:

example 1 comparison of mixtures after addition of different additives (preliminary experiments)

1. Preparing and sterilizing additive mother liquor:

polyglycerin fatty acid ester: 3g of decaglycerol monolaurate is weighed, 10 mL of water is added, and the mixture is heated at 121 ℃; sterilizing at high temperature for 20 min.

Liquid detergent: 1g of the detergent was weighed, 10 mL of water was added, and the mixture was filtered through a 0.22 μm filter to sterilize the mixture.

Quini-saponins: weighing 3g of quinapride, adding 15 mL of water, and carrying out temperature control at 121 ℃; sterilizing at high temperature for 20 min.

2. The preparation process of the emulsion mixture comprises the following steps:

weighing corresponding additives, dissolving in water, sterilizing aqueous solution of polyglycerol fatty acid ester and quinizarin at 121 deg.C for 15 min, and filtering with 0.22 μm filter membrane for sterilization. The sterilized lipid and the corresponding additive aqueous solution were added to the fermentation medium in a certain amount, and directly shaken in a shaker at 220 rpm, thereby obtaining the corresponding emulsion mixture.

The emulsion comprises: water, additives (the specific concentration is shown in Table 1), 10g/L palm oil, 1.57g/L ammonium sulfate, 5.66g/L sodium dihydrogen phosphate dodecahydrate, 1.5g/L potassium dihydrogen phosphate, 0.2g/L magnesium sulfate heptahydrate, 0.01g/L calcium chloride dihydrate, 0.02g/L ferrous sulfate heptahydrate, 0.0003g/L boric acid, 0.0002g/L cobalt chloride hexahydrate, 0.0001g/L zinc sulfate heptahydrate, 0.03g/L manganese chloride tetrahydrate, 0.03g/L sodium molybdenum dihydrate, 0.02g/L nickel chloride hexahydrate, and 0.01g/L copper sulfate pentahydrate.

3. And (3) detecting the state of the emulsion mixture:

after shaking for 30 min in a shaker at 220 rpm, the corresponding emulsion mixture was visually observed and examined by light microscopy. The size and the number of oil drops on the liquid surface are mainly observed through visual observation, and the larger the oil drops are, the larger the number is, the poorer the effect is; otherwise, the effect is good. The smaller the oil drop is during microscope detection, the more the quantity is, the better the effect is; otherwise, the effect is poor. The specific test results are shown in fig. 1-6, and the observed effects are shown in table 1.

TABLE 1 concentrations and effects of various additives used

As shown in fig. 1-6 and table 1, the three additives can achieve good effects after reaching reasonable concentrations, and the aqueous solutions of the three additives generate uniform liquid without precipitation after high-temperature sterilization (polyglycerol fatty acid ester and quinasaponin) or filtration sterilization (detergent); meanwhile, the components are not sensitive to factors such as pH, ionic strength and the like, and cannot be influenced by pH change in the fermentation process; finally, the three additives are all low in cost to use.

Example 2 Effect of additives on lipid carbon Source utilization

This example uses the fungus cultured by RocheRalstonia eutropha H16 is an example for explaining the effect of additives on the utilization rate of lipid carbon source, and the specific steps are as follows:

seed culture: culturing fungi of RocheRalstonia eutropha H16 was streaked onto solid plates of seed medium and cultured at 30 ℃ for 48H. Then selecting a single clone to inoculate into a 10 mL bacteria shaking tube filled with 3 mL seed culture medium, and culturing for 12 h-16 h under the conditions of 30 ℃ and 220 rpm to obtain turbid bacterial liquid; then transferring the bacterial liquid into a 100 mL conical flask filled with 10 mL seed culture medium according to the initial OD value of 0.1, and culturing for 8h at 30 ℃ and 220 rpm; at this time, the bacterial suspension was transferred again to a new 150 mL Erlenmeyer flask containing 15 mL of seed medium at an initial OD of 0.1, and cultured at 30 ℃ and 220 rpm for 12 to 16 hours. The seed medium comprises water and: 5g/L yeast extract, 10g/L peptone, 3g/L fructose and 10g/L sodium chloride (solid medium added with 1.5g/L agar powder).

Fermentation culture: the seed solution was transferred to a 250 mL conical flask containing 30 mL of fermentation medium (directly added with carbon source and additives) at an initial OD of 0.05, and subjected to fermentation culture at 220 rpm at 30 ℃ for 48 hours. After the shake flask fermentation is finished, centrifuging to collect cells, washing with 30% ethanol, drying, weighing to obtain dry cell weight, and detecting by gas chromatography to obtain corresponding PHA content.

The fermentation medium comprises water and: carbon source (fructose or palm oil), additive (no, decaglycerol monolaurate, detergent or saponin), 1.57g/L ammonium sulfate, 5.66g/L sodium dihydrogen phosphate dodecahydrate, 1.5g/L potassium dihydrogen phosphate, 0.2g/L magnesium sulfate heptahydrate, 0.01g/L calcium chloride dihydrate, 0.02g/L ferrous sulfate heptahydrate, 0.0003g/L boric acid, 0.0002g/L cobalt chloride hexahydrate, 0.0001g/L zinc sulfate heptahydrate, 0.03g/L manganese chloride tetrahydrate, 0.03g/L sodium molybdate dihydrate, 0.02g/L nickel chloride hexahydrate and 0.01g/L copper sulfate pentahydrate.

The experiments were divided into the following groups according to the difference between the carbon source and the additive, and the concentrations of the additive in the test groups containing the additive were the ones that showed the better concentration in example 1 (pre-experiment):

the carbon source is fructose, and the concentration of the fructose in the culture medium is 25 g/L; no additives are contained.

② the carbon source is palm oil, the concentration of which in the culture medium is 10 g/L; no additives are contained.

③, the carbon source is palm oil, and the concentration of the carbon source in the culture medium is 10 g/L; the additive was decaglycerol monolaurate, which was present in the medium at a concentration of 0.6 g/L.

Fourthly, the carbon source is palm oil, and the concentration of the carbon source in the culture medium is 10 g/L; the additive was decaglycerol monolaurate, which was present at a concentration of 1.5g/L in the medium.

Fifthly, the carbon source is palm oil, and the concentration of the carbon source in the culture medium is 10 g/L; the additive was decaglycerol monolaurate, which was present in the culture medium at a concentration of 3 g/L.

Sixthly, the carbon source is palm oil, and the concentration of the palm oil in the culture medium is 10 g/L; the additive is liquid detergent, and the concentration of the liquid detergent in the culture medium is 0.2 g/L.

Seventhly, the carbon source is palm oil, and the concentration of the palm oil in the culture medium is 10 g/L; the additive is liquid detergent, and the concentration of the liquid detergent in the culture medium is 1 g/L.

The carbon source is palm oil, and the concentration of the palm oil in the culture medium is 10 g/L; the additive is quinasaponin, and the concentration of the additive in the culture medium is 2 g/L.

Ninthly, the carbon source is palm oil, and the concentration of the carbon source in the culture medium is 10 g/L; the additive is quinasaponin, and the concentration of the additive in the culture medium is 4 g/L.

The results of the measurements of the dry cell weight, the PHA content of the metabolite, the PHA titer and the like after fermentation of each group are shown in Table 2 below. The dry cell weight is obtained by centrifugally collecting thalli from fermentation liquor, drying and weighing the thalli and then calculating; the PHA content and the PHA titer are obtained by calculation after the detection by a gas phase method.

Wherein carbon source utilization (%) = amount of actually produced PHA (PHA titer)/amount of carbon source theoretically convertible to PHA × 100%.

Wherein PHA content (wt%) = PHA production/cell dry weight × 100%.

Table 2 test results of each parameter of the experimental groups

The results show that in the first group to the second group, the carbon sources used in the first group are different, palm oil is not used as the carbon source, and 25g/L fructose is used as the carbon source, so that the first group can be regarded as a positive control of the experiment; group II directly uses 10g/L palm oil as carbon source, without any additive, and can be used as negative control of the experiment. In addition, palm oil was used as a carbon source in all experimental groups, corresponding emulsifiers were added, the amounts of different additives were the concentrations that showed the best effect in example 1 (preliminary experiment), and at other concentrations, the respective additives failed to achieve the effect better than that described in table 2.

As can be seen from Table 2, after fermentation, all the experimental groups added with polyglycerol fatty acid ester and 1g/L detergent had cell dry weight, PHA content and PHA titer far higher than those of the negative control, and were equivalent to the positive control level. The results show that when a proper amount of polyglycerol fatty acid ester and detergent are added, microorganisms can grow normally, the utilization rate of the carbon source palm oil is obviously improved, the SD value of each experimental group is small, and the culture effect is stable. The tested group added with the liquid detergent shows good effect, but the effect is greatly fluctuated under different concentrations, which is not beneficial to large-scale practice, so the polyglycerol fatty acid ester is the most preferable scheme. This method is significantly superior to other methods that have been developed in view of the nature of the additive itself and the fermentation results.

The experimental groups added with 0.2g/L of liquid detergent and saponin are lower than the positive control in terms of cell dry weight, PHA content and PHA titer. This indicates that the type of additives and the concentration of certain additives can have a crucial effect on the fermentation performance when the microorganism is fermenting using lipid carbon sources.

In conclusion, the additive is screened, so that the uniformity and stability of the culture medium are improved, the utilization rate of the lipid carbon source by the microorganism is improved, the stability and repeatability of microbial growth and fermentation are effectively improved, and a good technical support is provided for screening of dominant bacteria.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.