CN111117906B

CN111117906B - Improved microbial culture method

Info

Publication number: CN111117906B
Application number: CN201811284552.4A
Authority: CN
Inventors: 陈国强; 刘絮
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2018-10-31
Filing date: 2018-10-31
Publication date: 2022-04-05
Anticipated expiration: 2038-10-31
Also published as: CN111117906A

Abstract

The present invention provides an improved method for culturing halophilic microorganisms, in which NaCl conventionally used for providing a high salinity environment for halophilic microorganisms is replaced with other sodium salts, either entirely or partially, whereby the chloride ion concentration in a fermentation system can be reduced, thereby reducing the corrosive effect of chloride ions of the fermentation system on fermentation equipment, facilitating subsequent wastewater treatment, and also reducing microbial contamination in open fermentation. The invention also relates to a culture medium for the above culture.

Description

Improved microbial culture method

Technical Field

The present invention relates to an improved method for cultivating halophilic microorganisms, in particular halophilic bacteria.

Background

1. Halophilic bacteria fermentation

In the field of production of desired products such as Polyhydroxyalkanoate (PHA) by microbial fermentation, the use of halophilic microbial fermentation has advantages such as open fermentation without sterilization, high cell density, high yield, etc., and thus has attracted considerable attention in the production field. In the open fermentation of halophilic microorganisms, high-concentration NaCl (for example, 60g/L NaCl) is often added to a commonly used culture medium, which helps to maintain a high extracellular osmotic pressure and provide relatively suitable external conditions for halophilic bacteria, so as to avoid the influence of other bacteria and achieve the purpose of open fermentation.

2. Corrosion of metals and adverse effects of chloride ions from salt-containing environments

The metal is easy to generate electrochemical corrosion in a salt-containing solution, and the corrosion is hydrogen evolution corrosion or oxygen absorption corrosion according to the acid-base condition of the solution, and the specific expression is as follows: under acidic condition, hydrogen ions get electrons to generate hydrogen, and under neutral and alkaline environment, water and oxygen get electrons to generate hydroxide radicals. In electrochemical etching, high salt concentrations lead to higher solution conductivity, thereby further enhancing the etching effect.

Meanwhile, in actual production, generally adopted fermentation equipment is made of stainless steel, the stainless steel can resist corrosion mainly because corrosion-resistant elements such as chromium, nickel and the like are doped in the stainless steel, and the chromium and the nickel can enable the stainless steel to form a compact oxidation film in an oxidation medium, so that the surface of the stainless steel is passivated, and the stainless steel is prevented from reacting with oxygen, thereby avoiding or reducing further corrosion. And the chloride ions in the liquid culture medium have strong activation performance, which can destroy the oxide film on the metal surface and prevent the stainless steel surface from further forming a film. In fact, chloride ions have an initiating effect on the pitting corrosion of stainless steel and the effect increases as its concentration increases, but after reaching a high concentration, it levels off.

3. Problems caused by high-chloride ion fermentation on downstream wastewater treatment

The problem of high salinity wastewater treatment is mainly that in such hypertonic conditions, microorganisms are difficult to survive, resulting in the difficult acclimatization and stabilization of the microbial activated sludge used for wastewater treatment. Meanwhile, in order to reach the discharge standard of urban sewage, the ion concentration in the urban sewage is also reduced to a certain value. However, if a medium with a high sodium chloride concentration is used, it is difficult to handle it not only biologically, but also chloride ion as an anion, which is a relatively difficult ion to handle and separate. Most chlorine salts are soluble and have very high solubility, making them difficult to precipitate out and easy to convert into toxic and difficult to utilize products such as hypochlorite.

Although it has been noted that the use of high concentrations of NaCl in the culture medium causes the above-mentioned problems, up to now no satisfactory alternative culture medium has been found which reduces the NaCl concentration, even without using NaCl, on the one hand due to cost considerations; on the other hand, some studies have found that when NaCl in a conventional medium is replaced by other salts, the growth of microorganisms can be slowed down, the dry weight can be reduced, and the final product synthesis is not facilitated.

Therefore, the development of a medium and a culture method which reduce the use of NaCl, even without NaCl, is of great significance for the fermentation and production of halophilic microorganisms.

Disclosure of Invention

The inventors of the present invention have conducted a great deal of experimental studies to solve the above problems. As a result, it was found that when NaCl used in the culture of halophilic microorganisms is replaced in whole or in part with other sodium salts (such as one or more of sodium sulfate, sodium phosphate, sodium citrate, sodium acetate, sodium gluconate, sodium nitrate, and sodium carbonate), the growth of halophilic microorganisms is good and not reduced as conventionally expected, and the product synthesis is not reduced. By adopting the culture scheme, the NaCl dosage in the culture medium is reduced, so that the corrosion of a fermentation system on fermentation equipment caused by the existence of NaCl can be reduced, and the chloride ion concentration in the fermentation system is reduced, thereby being beneficial to the subsequent wastewater treatment. Furthermore, the inventors have also surprisingly found that the use of other sodium salts such as Na₂SO₄When the fermentation is carried out in an open state instead of NaCl, the fermentation is carried outSo as to more effectively avoid the generation of mixed bacteria.

Based on the above, the present invention relates to the following aspects.

In one aspect, the invention relates to a method of culturing a halophilic microorganism, wherein a sodium salt and a carbon source are added to a basal medium for culturing said halophilic microorganism, wherein the sodium salt is a sodium salt other than NaCl, or a combination of NaCl and other sodium salts.

Specifically, the aforementioned halophilic microorganisms may be halophilic bacteria, halophilic archaea and algae. More specifically, the halophilic microorganism may be a halophilic bacterium, preferably a bacterium of the genus Halomonas (Halomonas), more preferably Halomonas bluephasegenes, Halomonas camphaniensis or a combination of both. For example, Halomonas bluephagene TD01, Halomonas campniensis LS21 or a combination thereof with a preservation number of CGMCCNo.4353.

Specifically, the sodium salt may be one or more or all of sodium sulfate, sodium phosphate, sodium citrate, sodium acetate, sodium gluconate, sodium nitrate and sodium carbonate, or a combination of these salts and sodium chloride; the sodium salt is preferably selected from: sodium sulfate, sodium phosphate, a combination of sodium sulfate and sodium chloride, a combination of sodium phosphate and sodium sulfate, a combination of sodium sulfate, sodium phosphate, sodium citrate, sodium acetate, and sodium gluconate; more preferably sodium sulphate.

When the sodium salt added is sodium sulfate or contains sodium sulfate therein, the concentration of sodium sulfate may be in the range of 5g/L to 100g/L, preferably 5g/L to 80g/L, for example 10 to 60 g/L.

When the sodium salt added is or includes sodium phosphate, the concentration of sodium phosphate may be in the range of 5g/L to 100g/L, preferably 5g/L to 80g/L, for example 10 to 60g/L, such as 55 g/L.

In particular, the carbon source mentioned above (which may also be understood herein as a substrate for the synthesis of the product) may be glucose, gluconic acid, gluconate ester or a combination thereof, preferably glucose. The concentration of the carbon source may be in the range of about 1-100g/L, about 1-90g/L, about 1-80g/L, about 1-70g/L, or about 1-60 g/L; preferably, the concentration may be in the range of about 3-60g/L, about 3-50g/L, or about 3-40 g/L; more preferably about 5 to 60g/L, about 10 to 60g/L, about 20 to 40g/L, and specifically may be, for example, about 10g/L, about 15g/L, about 20g/L, about 25g/L, about 30g/L, about 35g/L, and the like.

The above-mentioned basal medium means a medium containing nutrients which can be used to support the growth of the microorganism of the present invention. The above-mentioned basic medium may be a medium conventionally used in the art for culturing microorganisms, such as mineral medium, LB medium, MM medium or beef extract peptone, etc., or a medium modified according to the intended purpose on the basis of these media. That is, one skilled in the art can routinely select an appropriate basal medium as long as it is capable of allowing the growth of the microorganism.

According to the method, the NaCl concentration adopted in the halophilic microorganism fermentation culture is reduced, so that open fermentation of halophilic microorganisms is ensured, the corrosion effect of a fermentation system on fermentation equipment caused by the existence of chloride ions is reduced, the chloride ion concentration in the fermentation system is reduced, the subsequent wastewater treatment is facilitated, and the mixed bacteria pollution can be reduced. Therefore, the method can reduce chloride ions in the fermentation medium, further reduce the concentration of the chloride ions in the wastewater, reduce the cost of wastewater treatment and realize a more environment-friendly production mode.

In another aspect, the invention also relates to a culture medium for use in the above method. The medium is obtained by adding a carbon source and a sodium salt to a basal medium for culturing the halophilic microorganism, the carbon source, the basal, the sodium salt being as defined above.

In particular, the culture medium of the invention may be selected from the following:

(1) a basal medium added with sodium salts except NaCl and a carbon source;

(2) and (3) a basal medium added with a combination of NaCl and other sodium salts and a carbon source.

Wherein the type and concentration of sodium salts, and the formulation and type of basal medium are as defined above.

Drawings

FIG. 1 is a result of examination of the fermentation system in example 6 in which NaCl was used as a sodium salt for fermentation by bacterial 16s rRNA sequencing, showing that a small amount of mixed bacteria was present in the system;

FIG. 2 sequencing of 16s rRNA of bacteria using Na as in example 6₂SO₄The results of the tests carried out on the fermentation system in which the sodium salt was fermented showed that no microbial contamination was present in the system.

Detailed Description

As used herein, a "halophilic microorganism" refers to a microorganism that requires a salt concentration for growth and that grows best in an environment with a salt concentration. The halophilic microorganism may be halophilic bacteria, halophilic archaea and algae. In some embodiments of the invention, the halophilic microorganism may be a halophilic bacterium, preferably a bacterium of the genus Halomonas (Halomonas), more preferably Halomonas bluephagesis, Halomonas campniensis, or a combination of both, such as Halomonas bluephagesis TD01, Halomonas campniensis LS21 with accession number cgmccno.4353, or a combination thereof.

The dry cell weight (CDW, g/L) referred to herein is the ratio of the mass of ice-dried biomass to the volume of fermentation product.

PHA as referred to herein means polyhydroxyalkanoate, which can be classified into homopolymers and copolymers according to monomer composition. Depending on the number of carbon atoms of the monomer, the PHA of the present invention may be a short chain PHA (i.e., a hydroxy fatty acid monomer C3-C5) or a medium-long chain PHA (i.e., a hydroxy fatty acid monomer C6-C16), but is not limited thereto. In some embodiments of the invention, the PHA may be a homopolymer, including but not limited to polyhydroxypropionate, polyhydroxybutyrate, polyhydroxyvalerate, and the like, for example, poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), poly-3-hydroxypropionate (P3HP), or poly-3-hydroxyvalerate (P3HV), and the like. In some embodiments of the invention, the PHA may be a copolymer such as, but not limited to, a dimer, a trimer, and the like, for example, the copolymer may be a copolymer of a hydroxypropionate ester and a hydroxybutyrate ester; copolymers of a hydroxy propionate and a hydroxy valerate; a copolymer of hydroxybutyrate and hydroxyvalerate; hydroxy propionate, hydroxy butyrate, hydroxy valerate, and the like. More specifically, in some embodiments of the invention, the PHA can be poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB3HV), or poly (3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB4HB3HV), combinations thereof, or the like. In some embodiments of the invention, the PHA may be P3HB (i.e., a poly-beta-hydroxybutyrate or a poly-3-hydroxybutyrate). In other embodiments of the invention, the PHA may be P3HB3HV (i.e., a 3-hydroxybutyrate 3-hydroxyvalerate copolymer).

The P3HB content (wt%) referred to herein is the mass percentage of P3HB to the mass of the ice-dried cells participating in the esterification, where the mass of P3HB is the total mass of 3HB obtained after the esterification.

As referred to herein, a "basal medium" is a liquid or solid or semi-solid that covers an organism in a culture vessel, such as a petri dish or the like, and contains nutrients to nourish and support the cells. According to actual requirements, the culture medium may be additionally added with other substances such as antibiotics for resistance selection, sodium salts with high salinity required for maintaining the growth of specific microorganisms such as halophilic bacteria, carbon sources for metabolism of microorganisms to synthesize products, and the like, as appropriate. The skilled person can select a suitable basal medium depending on the microorganism to be cultured. For example, the basal medium may be MM medium, LB medium, or the like.

The general formulation of MM medium was: 0.1-2 ‰ (NH)₄)₂SO₄Or urea, 0.1-1 MgSO₄，5‰-10‰Na₂HPO₄·12H₂O，0.5‰-2‰KH₂PO₄Not more than 0.1% of other trace elements (Fe (III) -NH)₄-Citrate，CaCl₂·2H₂O，ZnSO₄·7H₂O，MnCl₂·4H₂O，H₃BO₃，CoCl₂·6H₂O，CuSO₄·5H₂O，NiCl₂·6H₂O，NaMoO₄·2H₂Trace O (pH adjusted to about 9.0));

preferably: 0.1% (NH)₄)₂SO₄Or 0.2% urea, 0.02% MgSO₄，1.0％Na₂HPO₄·12H₂O，0.15％KH₂PO₄Not more than 0.1% of other trace elements (Fe (III) -NH)₄-Citrate，CaCl₂·2H₂O，ZnSO₄·7H₂O，MnCl₂·4H₂O，H₃BO_3，CoCl₂·6H₂O，CuSO₄·5H₂O，NiCl₂·6H₂O，NaMoO₄·2H₂O (pH adjusted to about 9.0)).

The general formulation of LB liquid medium is: 4-6g/L yeast extract, 8-12g/L peptone, 8-12g/L NaCl, and the balance of distilled water (pH adjusted to 7.0-7.2); preferably: 5g/L yeast extract, 10g/L peptone, 10g/L NaCl, and the balance distilled water (pH adjusted to 7.0-7.2).

The medium for culturing the halophilic microorganisms of the present invention is obtained by adding a carbon source, which can be utilized by the halophilic microorganisms to synthesize a desired product, and a sodium salt for providing the halophilic microorganisms with a high salinity environment suitable for their growth, on the basis of a basic medium.

As used herein, a "carbon source" is a nutrient that provides the microorganisms with the carbon elements necessary for growth and reproduction. In the present invention, a "carbon source" is a source for the halophilic microorganisms of the present invention to synthesize PHA, and thus, may be used interchangeably herein with "substrate". The carbon source may be glucose, gluconic acid, gluconate ester or a combination thereof. Preferably, the present invention uses glucose as a carbon source. The gluconate mentioned above may be any one or more gluconate salts as long as it can be used as a carbon source for the microorganism to which the present invention relates for polymer production, for example, sodium gluconate, potassium gluconate, calcium gluconate, etc. The concentration of glucose, gluconic acid, gluconate, or gluconate as the carbon source may be appropriately adjusted by those skilled in the art according to the culture conditions and microorganisms used, and may be in the range of about 1-100g/L, about 1-90g/L, about 1-80g/L, about 1-70g/L, or about 1-60 g/L; preferably, the concentration may be in the range of about 3-60g/L, about 3-50g/L, or about 3-40 g/L; more preferably about 5 to 60g/L, about 10 to 60g/L, about 20 to 40g/L, and specifically may be, for example, about 10g/L, about 15g/L, about 20g/L, about 25g/L, about 30g/L, about 35g/L, and the like.

The aforementioned "sodium salts" used to maintain a suitably high salinity environment for the halophilic microorganisms may be one or more or all of sodium sulfate, sodium phosphate, sodium citrate, sodium acetate, sodium gluconate, sodium nitrate, sodium carbonate, and combinations of these salts with sodium chloride. The case where the sodium salt is simply sodium chloride is excluded in the present invention. In some embodiments of the invention, the "sodium salt" may be selected from sodium sulfate, sodium phosphate, a combination of sodium sulfate and sodium chloride, a combination of sodium phosphate and sodium sulfate, a combination of sodium sulfate, sodium phosphate, sodium citrate, sodium acetate, sodium gluconate. For example, in some embodiments of the invention, the added sodium salt is:

①Na₂SO₄+NaCl

②Na₂SO₄

③Na₃PO₄

④Na₂SO₄+Na₃PO₄

mixed sodium salt (sodium sulfate, sodium phosphate, sodium citrate, sodium acetate, sodium gluconate)

In each of the above cases, the total concentration of sodium salt added may be in the range of 10g/L to 100 g/L.

Examples

The invention is further illustrated by the following specific examples. It will be understood that these embodiments are described by way of example only and to aid understanding of the invention, and are not intended to limit the invention to these embodiments.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The bacteria used in the examples are Halomonas bluephagene TD01, which was deposited at the China general microbiological culture Collection center at 19.2010, 11.53 with the collection accession number CGMCC No.4353 and classified and named Halomonas sp.TD01 (also known as Halomonas bluephagene TD01, see XB Chen et al (2017), Construction of Halomonas bluephagene capable of being used as high cell density growth for efficacy PHA production. Appl Microbiol biotechnol. volume 244, Part 1,534 pages 541); it is described in patent application publication No. CN 102120973A; the bacteria are available to the public from the university of Qinghua.

Halomonas (Halomonas campeniensis) LS 21: is a gram-negative halophilic bacterium screened by the laboratory, and has very good industrial production application prospect. It is disclosed in "Haitao Yue et al A search-based open and connected processes for polyhydroxyakanoates processes by recombinant microorganisms LS21 growth in mixed substrates Biotechnologies for Biofuels 2014,7(1): 1-12", which is publicly available from the university of Qinghua.

The specific formulation of MM medium for culturing Halomonas bluephagenes TD01 and Halomonas camphaniensis LS21 is as follows:

0.5g/L of urea; MgSO (MgSO)₄ 0.2g/L；Na₂HPO₄·12H₂O 9.65g/L；KH₂PO₄1.5 g/L; and add up<0.1g/L of Fe (III) -NH₄-Citrate，CaCl₂·2H₂O，ZnSO₄·7H₂O，MnCl₂·4H₂O，H₃BO₃，CoCl₂·6H₂O，CuSO₄·5H₂O，NiCl₂·6H₂O，NaMoO₄·2H₂O。

Adding 30g/L of glucose as a fermentation substrate and 60g/L of NaCl into the culture medium to provide a high-salt environment required by halophilic bacteria; and the pH was adjusted to 9.0.

The method for detecting the content of Polyhydroxyalkanoate (PHA) by gas chromatography comprises the following steps:

setting the furnace temperature to 80 ℃, the injector temperature to 200 ℃, the detector temperature to 220 ℃, the column head pressure to 0.25Mpa, and the temperature programming conditions as follows: the temperature was held at 80 ℃ for 1.5 minutes, raised to 140 ℃ at a rate of 30 ℃/min, then raised to 220 ℃ at a rate of 40 ℃/min and held at this temperature for 0.5 minutes. The amount of the sample to be introduced was 1. mu.l, and a microsyringe manufactured by Agilent was used.

Gas phase sample preparation: taking 40-60mg of stem cells of a sample to be detected (bacterial liquid is taken to be centrifuged at 10000rpm at normal temperature for 10 minutes, and after the obtained cell sediment is washed once by water, the stem cells are dried by ice to obtain the stem cells, and the homopolymer is produced in the cells), adding 2ml of chloroform and 2ml of esterification solution (3% (v/v) of concentrated sulfuric acid and 2g/L of benzoic acid in pure methanol are taken as internal standards) into an esterification tube, covering and sealing the esterification tube, and heating the esterification tube at 100 ℃ for 4 hours. After cooling, 1ml of distilled water was added thereto, the mixture was sufficiently shaken and allowed to stand, and after the chloroform phase and the aqueous phase were completely separated, 1. mu.l of the chloroform phase in the lower layer was taken out and injected into a gas chromatograph (Hewlett Packard6890, HP) to conduct chromatography. The gas chromatograph was operated according to the specifications for the HP Hewlett Packard6890 gas chromatograph.

Preparation of a standard sample: 10-20mg of standard sample is put into an esterification tube, 2ml of chloroform and 2ml of esterification solution are added, and esterification is carried out at 100 ℃ after capping and sealing.

And (4) analyzing results: taking a standard sample as a reference, if an esterified sample (a sample to be detected) of a cell to be detected has an obvious peak at a standard sample, calculating the mass of each monomer according to the peak area, and then calculating the molar ratio according to the mass fraction of each monomer; the specific gravity (wt%) of the polymer contained in the dry cell weight can be calculated from the amount of the sample added.

Example 1 use of Na₂SO₄Mixing NaCl as sodium salt for fermentation

To reduce NaCl usage, the inventors tried Na₂SO₄A partial or complete replacement of NaCl provided a suitable high salinity environment, while experiments using NaCl only as the sodium salt served as a control. In this experiment, Halomonas bluephagesis TD01 was used as a fermentation strain for fermentation culture, the MM medium described above was used as a basal medium, and sodium salt and glucose were added. The culture conditions are shown in Table 1, using a shake flask experiment. Three replicates were set for each set of experiments and the results were averaged. The results are shown in Table 1 (wherein the percentage of sodium salt is mass fraction).

Table 1:

the results show that, when NaCl used to provide a high salinity environment was partially used with Na, as compared with experiment 1-1, which used NaCl alone as the sodium salt₂SO₄When replaced, the growth and product synthesis of the TD01 bacterium were not substantially affected. Therefore, the corrosion effect of the fermentation system on the fermentation equipment due to the existence of chloride ions can be reduced, and the concentration of the chloride ions in the fermentation system is reduced, so that the subsequent wastewater treatment is facilitated.

Example 2 use of Na₂SO₄Replacing NaCl as sodium salt to carry out fermentation

The experiment was carried out according to the materials and conditions described in example 1, with the difference that only Na was used₂SO₄Without using NaCl as the sodium salt. Meanwhile, an experiment using only NaCl as a sodium salt was taken as a control. Three replicates were set for each set of experiments and the results were averaged. The results are shown in Table 2.

Table 2:

the results show that NaCl used to provide a high salinity environment was treated with Na, as compared to experiment 1-1, which used NaCl alone as the sodium salt₂SO₄When replaced, the growth and product synthesis of the TD01 bacterium were not substantially affected. Therefore, the corrosion effect of the fermentation system on the fermentation equipment due to the existence of chloride ions can be reduced, and the concentration of the chloride ions in the fermentation system is reduced, so that the subsequent wastewater treatment is facilitated. Furthermore, the inventors have found that lower concentrations of Na, whether assayed from cell dry weight or product content, are used₂SO₄Experiment 2-2 of (a) was able to achieve an experimental effect comparable to experiment 2-3 with higher concentrations of NaCl (experiment 2-2 yielded even higher yields of P3 HB). This means that the concentration of sodium salt in the fermentation system can be reduced, thereby reducing the cost and reducing the environmental pressure of the fermentation waste liquidForce.

Example 3 use of Na₃PO₄Replacing NaCl as sodium salt to carry out fermentation

The experiment was carried out according to the materials and conditions described in example 1, with the difference that Na was used₃PO₄Replacing NaCl as sodium salt to ferment, Na₃PO₄The concentration was 55g/L (equivalent to the amount of Na ions provided by 60g/L NaCl).

The results demonstrate that sodium phosphate can indeed replace sodium chloride for fermentation. The bacteria grew well and PHA yield was comparable to that of fermentation with NaCl, in other words with Na₃PO₄NaCl is replaced as sodium salt for fermentation, so that the corrosion effect of a fermentation system on fermentation equipment caused by the existence of chloride ions can be reduced under the condition of not influencing the synthesis of a target product, and the concentration of the chloride ions in the fermentation system is reduced, thereby being beneficial to the subsequent wastewater treatment.

Example 4 use of Na₃PO₄With Na₂SO₄The combination is fermented as sodium salt

The experiment was carried out according to the materials and conditions described in example 1, with the difference that Na was used₃PO₄Mixed Na₂SO₄As sodium salt, fermentation was conducted while controlling the total amount at about 60 g/L. The results show that Na₃PO₄With Na₂SO₄When the combination was used as a sodium salt for fermentation, the bacteria grew well and the PHA yield was comparable to that when fermentation was carried out with NaCl providing an equimolar amount of Na ions.

Example 5 fermentation Using Mixed sodium salts

The experiment was carried out according to the materials and conditions described in example 1, with the difference that Na₂SO₄、Na₃PO₄Sodium citrate, sodium acetate, sodium gluconate. The total concentration was controlled at about 60 g/L.

The results show (see table 3, percentage of sodium salt is mass fraction), when the above-mentioned combination of sodium salts is used for fermentation, the bacteria growth is good, and at a certain ratio, better fermentation effect can be obtained compared with the single use of NaCl.

Table 3:

example 6 fermentation Using Halomonas camphaniensis LS21 with Mixed sodium salt

The experiment was carried out using Halomonas camphaniensis LS21 as the strain, and the materials and conditions in example 5 were followed, with the total concentration of sodium salt being controlled at 40g/L, while a control experiment was carried out using NaCl salt as the sodium salt which provides an equimolar amount of Na ion.

Table 4:

the experimental result shows that, for the strain LS21, the combination of NaCl and other sodium salts does not adversely affect the synthesis amount of P3HB, and through the replacement, the corrosion effect of the fermentation system on the fermentation equipment caused by the existence of chloride ions can be reduced, and the concentration of the chloride ions in the fermentation system is reduced, so that the subsequent wastewater treatment is facilitated.

Example 7 fermenter experiment

The fermentation culture was carried out using Halomonas bluephagene TD01 as a fermentation strain, the MM medium as a basal medium, and 20g/L glucose and Na as additives₂SO₄10g/L, a 1L fermenter was used, the volume of the fermentation broth being 600mL, pH9.0, and temperature 37 ℃. The control group was run under the same experimental conditions except that 10g/L NaCl was used as the sodium salt. The results are shown in Table 3.

Table 5:

the halophilic bacteria fermentation has the advantages that open fermentation can be carried out without additional sterilization, meanwhile, various air filter membranes and complex sterile operation are not needed in the fermentation process to reduce the possibility of bacterial contamination, and the production cost, the labor and the material resources are greatly saved. Conventional open fermentation relies on a high salt, high pH environment to inhibit the growth of other infectious microbes.

Whether the bacteria exist can be determined by performing PCR reaction on the fermented bacteria liquid by using a universal primer of the 16s rRNA of the bacteria and performing DNA sequencing on the obtained fragment. Sequencing detection is carried out by Biotechnology Limited of Beijing Rui Boxing, ABI3730XL automatic sequencer is used for sequencing, a fluorescence-labeled Sanger sequencing method is adopted, bases of 4 dideoxynucleotides (ddNTPs) are respectively labeled by different fluorescence, 4 fluorophores on DNA fragments with different lengths are excited by laser when passing through a capillary, the fluorescence emits fluorescence with different colors, the fluorescence is identified by a CCD detection system and is directly translated into a DNA sequence, and thereby, 16s sequence data can be obtained. The 16s sequence is a characteristic sequence of bacteria, and can be compared with a database to judge which bacteria is the 16s sequence, when only one bacteria exists in a fermentation liquid, the 16s sequencing should have a single peak, and the sequencing quality is high. When the bacteria are polluted, various bacteria in the bacteria can amplify signals when the DNA is amplified by PCR, so that different sequencing signals exist at the same site during sequencing, and a double peak appears in the result.

Sequencing Using the universal primers for bacterial 16s rRNA, surprisingly, found that Na was used₂SO₄The production of hetero-bacteria (less hetero-peaks) was well avoided when the fermentation was carried out as sodium salt, in contrast to the production of hetero-bacteria (more hetero-peaks) in the late stage of the fermentation system when NaCl was used as sodium salt (see FIGS. 1 and 2), thus demonstrating that when Na was used at a low concentration₂SO₄When the sodium salt is used for fermentation, the production of mixed bacteria can be more effectively avoided compared with the conventional NaCl, which has significant meaning for open fermentation.

Claims

1. A method of culturing a halophilic microorganism, wherein a sodium salt is added in a concentration suitable for maintaining a high salinity environment required by said halophilic microorganism, and a carbon source utilized by said halophilic microorganism as a substrate for the synthesis of polyhydroxyalkanoate, in a basal medium for culturing said halophilic microorganism, wherein said halophilic microorganism is Halomonas bluephagesis or Halomonas camphaniensis, wherein said sodium salt is selected from the group consisting of: sodium sulfate; sodium phosphate; a combination of sodium phosphate and sodium sulfate; sodium sulfate, sodium phosphate, sodium citrate, sodium acetate and sodium gluconate, wherein the basic culture medium is an MM culture medium.

2. The method according to claim 1, wherein the halophilic microorganism is Halomonas bluephagene TD01 or Halomonas camphaniensis LS21 with a preservation number of CGMCC No. 4353.

3. The process of claim 1 or 2, wherein the sodium salt is sodium sulfate.

4. The method of claim 3, wherein the concentration of the sodium salt added is from 5g/L to 100 g/L.

5. Use of a culture medium supplemented with a sodium salt and a carbon source, the sodium salt being in a concentration suitable for maintaining a high salinity environment required by a halophilic microorganism, on the basis of a basal medium, wherein the sodium salt is selected from the group consisting of: sodium sulfate; sodium phosphate; a combination of sodium phosphate and sodium sulfate; a combination of sodium sulphate, sodium phosphate, sodium citrate, sodium acetate, sodium gluconate, wherein the concentration of sodium sulphate is in the range of 5g/L to 100g/L when the added sodium salt is sodium sulphate or is comprised therein, wherein the halophilic microorganism is Halomonas bluephaseensis or Halomonas camphaniensis, wherein the basal medium is MM medium.

6. The use of claim 5, wherein the sodium salt is sodium sulfate.