CN109486710B

CN109486710B - Method for continuously fermenting and culturing microorganisms by recycling wastewater and bacteria with self-flocculation and self-sedimentation characteristics used by method

Info

Publication number: CN109486710B
Application number: CN201811468572.7A
Authority: CN
Inventors: 陈国强; 凌晨
Original assignee: Beijing Micro Structure Factory Biotechnology Co ltd
Current assignee: Beijing Micro Structure Factory Biotechnology Co ltd
Priority date: 2018-12-03
Filing date: 2018-12-03
Publication date: 2022-10-21
Anticipated expiration: 2038-12-03
Also published as: CN109486710A

Abstract

The invention discloses a method for continuously fermenting and culturing microorganisms by recycling wastewater and bacteria with self-flocculation and self-sedimentation characteristics used by the method, wherein the bacteria with the self-flocculation and self-sedimentation characteristics belong to halophilic bacteria and can perform self-flocculation and self-sedimentation under the condition of salt concentration of not less than 0.08M. The invention utilizes the waste water generated by washing the self-settled bacterial thallus to continuously ferment and culture the microorganism, thereby not only reducing the energy consumption generated by centrifugation, waste water treatment and sterilization, but also improving the yield. The bacteria can become platform bacteria of the next-generation industrial microorganisms, and the continuous fermentation culture method for recycling the wastewater can be used for large-scale biological fermentation.

Description

Method for continuously fermenting and culturing microorganisms by recycling wastewater and bacteria with self-flocculation and self-sedimentation characteristics used by method

Technical Field

The invention belongs to the field of biotechnology application, and particularly relates to a method for continuously fermenting and culturing microorganisms by recycling waste water through salt and bacteria with self-flocculation and self-sedimentation characteristics used by the method.

Background

Large-scale biological fermentation in industry is subject to the current fermentation process, and compared with petrochemical industry, the cost is always too high. The reasons for the excessive costs are, on the one hand, the low yields of product; on the one hand, due to the large energy consumption in the production process, sterilization, stirring and complex downstream treatment account for a large proportion, and more waste water is generated to pollute the environment.

At present, the applicant develops a set of non-sterilization open continuous fermentation process based on halomonas, which does not need sterilization, thereby greatly reducing the process energy consumption in the production process. But the cost is still high due to the energy consumption and low yield caused by centrifugation in the downstream process, and salt-containing wastewater is generated in the production process, so that the environment is polluted, and the large-scale biological fermentation in the industry is still limited.

Disclosure of Invention

To solve one or more problems of the prior art, an aspect of the present invention is to provide a method for continuous fermentative culture of microorganisms using recycled wastewater, comprising:

s1: inoculating bacteria with self-flocculation and self-sedimentation characteristics into an initial fermentation medium for fermentation culture;

s2: after the first fermentation culture, separating first self-settling bacterial thalli from a first fermentation product, adding waste water generated by washing the first self-settling bacterial thalli into a first supernatant remained after separation, and continuing a second fermentation culture;

s3: after the second fermentation culture, separating second self-settling bacterial thalli from a second fermentation product, adding waste water generated by washing the second self-settling bacterial thalli into a second supernatant remained after separation, and continuing the next fermentation culture;

s4: repeating the step S3 for N times continuously, wherein N is a natural number;

s5: and combining the bacterial thalli washed in each step to obtain the harvested microorganism.

In the method for continuously fermenting and culturing the microorganisms by recycling the wastewater, the separation in the step S2 and/or the step S3 is obtained from the settled bacterial thalli from the lower layer of the fermentation product after standing and layering; wherein the standing and layering time can be 10-30min.

The bacterium having self-flocculating and self-precipitating properties is a halophilic bacterium, preferably a bacterium belonging to the genus Halomonas (Halomonas sp.).

The self-flocculation and self-sedimentation characteristics mean that bacteria can perform self-flocculation and self-sedimentation under the condition that the salt concentration is not less than 0.08M. The bacteria have reversible self-flocculation and self-sedimentation properties, and the phenomena of self-flocculation and self-sedimentation can gradually disappear under the condition that the salt concentration is lower than 0.08M.

The bacteria with the self-flocculation and self-sedimentation characteristics are bacteria of Halomonas after etf-alpha and etf-beta genes are knocked out.

The 16S rDNA sequence of the bacterium with the self-flocculation and self-sedimentation characteristics is shown as SEQ ID NO. 12 or SEQ ID NO. 13.

The bacteria with self-flocculation and self-sedimentation characteristics is Halomonas salina (Halomonas camphaniensis) LSKO with a preservation number of CGMCC No.16437.

In the method for continuously fermenting and culturing the microorganisms by recycling the wastewater, provided by the invention, the salt concentration in a fermentation culture system is not lower than 0.08mol/L; and/or

The fermentation culture is carried out under the conditions of stirring and aeration, wherein the initial rotation speed of the stirring is 200-250RPM, and the rotation speed is stabilized at 700-800RPM after the culture is carried out for 5-8 hours; the rate of aeration is 1vvm; the temperature of the fermentation culture is 20-45 ℃; the initial pH value of the fermentation culture system is 5.0-11.0; the initial dissolved oxygen of the fermentation culture system is 5-100%; and/or

The OD600 of the bacteria with self-flocculation and self-sedimentation characteristics in the initial fermentation medium in the step S1 is 2-4; and/or

The time of the first fermentation culture in the step S2 is 44-52h; and/or

The time of the second fermentation culture in the step S3 is 20-28h; and/or

Both the OD600 of the self-flocculating and self-settling bacteria in the culture system is 25-30 when the second fermentation culture is continued in the step S2 and when the next fermentation culture is continued in the step S3; and/or

Wherein 1 liter of the initial fermentation culture medium consists of 20g to 200g of sodium chloride, 5g to 100g of glucose, 0g to 50g of yeast powder, 0.5g to 20g of ammonium chloride, 0g to 20g of urea, 0.1g to 5g of magnesium sulfate, 0.5g to 10g of monopotassium phosphate, 1g to 20g of disodium hydrogen phosphate dodecahydrate, 1mL to 30mL of trace element I, 0.1mL to 10mL of trace element II and tap water, and the volume is fixed to 1 liter by the tap water;

wherein 1 liter of the trace element I is prepared according to the following method: mixing 2g-10g of ferric ammonium citrate, 1g-5g of calcium chloride dihydrate and 0.5mol/L of hydrochloric acid aqueous solution, and complementing the mixture to 1 liter by using 0.5mol/L of hydrochloric acid aqueous solution to obtain the trace element I;

wherein 1 liter of microelement II is prepared according to the following method: 50-200 mg of zinc sulfate heptahydrate, 10-50 mg of manganese chloride tetrahydrate, 100-500 mg of boric acid, 50-400 mg of cobalt chloride hexahydrate, 3-30 mg of copper sulfate pentahydrate, 5-50 mg of nickel chloride hexahydrate, 10-50 mg of sodium molybdate dihydrate and 0.5mol/L of hydrochloric acid aqueous solution are mixed, and the mixture is complemented to 1 liter by 0.5mol/L of hydrochloric acid aqueous solution to obtain the trace element II.

Another aspect of the present invention is to provide a bacterium having self-flocculating and self-settling properties, which belongs to halophilic bacteria and is capable of self-flocculating and self-settling at a salt concentration of not less than 0.08M.

The bacteria with self-flocculation and self-sedimentation characteristics provided by the invention belong to the genus Halomonas.

The bacteria with self-flocculation and self-precipitation characteristics is Halomonas LSKO with a preservation number of CGMCCNo.16437.

In another aspect of the present invention, there is provided a method for modifying a bacterium having self-flocculating and self-settling properties, comprising knocking out the etf-alpha and etf-beta genes of a bacterium belonging to the genus Halomonas.

Based on the technical scheme, the invention provides a method for continuously fermenting and culturing microorganisms by recycling wastewater based on bacteria with self-flocculation and self-sedimentation characteristics for the first time and the bacteria with self-flocculation and self-sedimentation characteristics for the method. The invention has the following beneficial effects:

1) The bacteria with the self-flocculation and self-sedimentation characteristics can perform self-flocculation and self-sedimentation under the condition that the salt concentration is not lower than 0.08M, so that a fermentation product does not need to be centrifugally separated in the continuous fermentation culture process, the separation of self-sedimentation bacteria and supernatant can be realized only by standing and layering, the separated bacteria are used for downstream treatment and product extraction, the salt-containing wastewater generated by washing the separated bacteria is added into the supernatant, and the supernatant can be used for the next round of continuous fermentation culture because part of unseparated suspended strains are contained, so that the energy consumption generated by centrifugation in the production process can be eliminated, the subsequent complicated sterilization and inoculation steps of a fermentation culture system can be omitted, the wastewater can be recycled in the whole fermentation culture process, and a large amount of water resources and cost are saved.

2) When the method for continuously fermenting and culturing microorganisms by recycling wastewater is used for producing Poly-3-hydroxybutyrate (PHB for short), compared with the traditional fermentation process for producing PHB, the method for continuously fermenting and culturing microorganisms by recycling wastewater provided by the invention has the advantages that the cell dry weight is increased from 0.45g/L/h of the traditional yield to 0.82g/L/h; the produced PHB is improved from 0.18g/L/h of the traditional yield to 0.33g/L/h, and the yield is greatly improved.

3) The bacterial strain with the self-flocculation and self-sedimentation characteristics is obtained from Halomonas LS21 and Halomonas TD01 by a genetic engineering technical means, can grow in a high-salt and high-alkali environment, and can perform self-flocculation and self-sedimentation under the condition that the salt concentration is not lower than 0.08M. The strain with the characteristic is used in the method for continuously fermenting and culturing the microorganism by recycling the wastewater, the application of the widened bacteria can be used for a continuous process of large-scale biological fermentation in industry, and the strain provided by the invention is expected to become a platform bacterium of the next-generation industrial microorganism.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a comparison graph of the strain morphology of Halomonas LSKO of the present invention and the starting strain Halomonas LS 21;

FIG. 2 is a diagram showing the state of the strain Halomonas LSKO of the present invention in self-flocculation and self-sedimentation with the lapse of culture time;

FIG. 3 is a graph showing the results of the self-flocculation and self-sedimentation tests of the strain Halomonas LSKO of the present invention at various sodium chloride concentrations.

Detailed Description

The invention provides a method for recycling continuous fermentation culture organisms of wastewater, aiming at the problems of energy consumption, lower yield, environmental pollution caused by wastewater discharge in the production process and the like generated by centrifugation of a downstream process in industrial large-scale biological fermentation. Therefore, the whole fermentation culture process can realize continuous fermentation culture without centrifugation and recycling of wastewater, thereby realizing the aim of microbial fermentation culture in industrial large-scale organisms.

Therefore, the invention further provides a method for continuously fermenting and culturing microorganisms by recycling wastewater by using the bacteria on the basis of screening or acquiring the bacteria with self-flocculation and self-sedimentation characteristics under a proper environment.

The present invention will be described in further detail with reference to specific examples.

The following disclosure provides many different embodiments, or examples, for implementing different aspects of the invention. Examples of various specific processes and materials are provided, but one of ordinary skill in the art will recognize the application of other processes and/or the use of other materials.

The present invention is described in detail below with reference to specific examples. The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, but the disclosure of the invention is not limited to the following embodiments.

The methods used in the following examples are conventional methods unless otherwise specified.

Example 1: bacteria having self-flocculating and self-settling properties and their identification

This example details the method of obtaining bacteria with self-flocculating and self-precipitating properties by means of genetic engineering.

1. The inventor uses Halomonas LS21 (CGMCC, address: no. 3 of Xilu No.1 of Beijing Korean district, north Chen) which is preserved in China general microbiological culture Collection center (CGMCC for short) by the applicant at 9.20 days 2012, with the preservation number of CGMCC No.6593, and is classified and named as Halomonas sp) as an initial strain (disclosed in patent document CN 102925382A), obtains a genetic engineering strain by the following genome editing means, and verifies that the strain has the characteristics of self flocculation and self sedimentation:

1.1. construction of knock-out plasmid pRE112-etf

pLS1-F (SEQ ID NO: 1) and pLS1-R (SEQ ID NO: 2) shown in Table 1 below were used as primers, pRE112-6I-SceI (obtained from Fu X, tanD, ai***lg, wuQ, chenJ, chen G. (2014). Development of Halomonas TD01as a host for open production of chemicals, metabolic engineering, 23) was used as a template, and the PCR system shown in Table 2 below and the following PCR program were used with Q5 enzyme: pre-denaturation at 95 ℃ for 5 minutes; then denaturation at 95 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 3 minutes, and 30 cycles; then extending for 10 minutes after 72 ℃, carrying out PCR reaction, and amplifying to obtain a gene knockout plasmid Backbone Backbone, wherein the nucleotide sequence is shown as SEQ ID NO. 7, and the sequence length is 5228bp.

pLS2-F (SEQ ID NO: 3) and pLS2-R (SEQ ID NO: 4) shown in Table 1 below were used as primers, the genome of Halomonas LS21 was used as a template, and the PCR system shown in Table 4 below and the following PCR program were used with pfu enzyme: pre-denaturation at 95 ℃ for 5 minutes; then denaturation at 95 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 30 cycles; then extending for 10 minutes after 72 ℃, carrying out PCR reaction, and amplifying to obtain the etf-alpha and etf-beta gene homology arms HA-1 of halomonas LS21, wherein the nucleotide sequence is shown as SEQID NO. 8, and the sequence length is 586bp. pLS3-F (SEQ ID NO: 5) and pLS3-R (SEQ ID NO: 6) shown in Table 1 below were used as primers, the LS21 genome of Halomonas was used as a template, and the PCR system shown in Table 2 below and the following PCR program were performed using Q5 enzyme: pre-denaturation at 95 ℃ for 5 minutes; then denaturation at 95 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 30 cycles; then extending for 10 minutes at 72 ℃, carrying out PCR reaction, and amplifying to obtain etf-alpha and etf-beta gene homologous arms HA-2 of halomonas LS21, wherein the nucleotide sequence is shown as SEQ ID NO. 9, and the sequence length is 493bp.

According to the Gibson assembly method (Gibson DG, young, chuangRY, venterJC, hutchison CA, smith HO. (2009). Enzymetic assembly of DNA molecules up to segmented cloned plasmids. Nature Methods,6 (5): 343-345.), the plasmid Backbone Backbone, the homologous arms HA-1 and HA-2 are connected to obtain a knockout plasmid, which is named as pRE112-etf.

Table 1: primers and sequences thereof

Table 2: PCR System (50. Mu.L System)

1.2. Knockout of etf-alpha and etf-beta genes of halomonas LS21

According to the related gene knockout method in Fu X, tanD, ai***lg, wuQ, chenJ, chenG. (2014). Development of Halomonas TD01as a host for open production of chemicals.Metabolic engineering,23, the etf-alpha and etf-beta genes are knocked out by using the gene knockout plasmid pRE112-etf constructed in the step 1.1, and recombinant bacteria are obtained.

1.3. Molecular identification

Sequencing the etf position on the genome of the recombinant bacterium obtained in the step 1.2, and taking the recombinant bacterium with the etf position deleted by 864bp on the genome as a positive recombinant bacterium, namely a bacterium strain with the etf-alpha and etf-beta gene knocked out, wherein the positive recombinant bacterium is named as halomonas (Halomonas camphaniensis) LSKO (hereinafter referred to as halomonas LSKO or LSKO).

The 16S rDNA sequence of the above strain Halomonas LSKO was detected using the 16S-F (SEQ ID NO: 10) and 16S-R (SEQ ID NO: 11) primers shown in Table 1 above to obtain a sequence shown in SEQ ID NO:12 as the 16S rDNA sequence of Halomonas LSKO.

The Halomonas LSKO strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address is No. 3 Siro 1 of Beijing market and south morning district), the preservation number is CGMCC No.16437, and the Halomonas is classified and named as Canpanani (Halomonascamphanensis) in 2018, 9 and 6 days.

1.4. Morphological identification

Taking LSKO bacterial liquid preserved by freezing a 15% glycerol tube, diluting the bacterial liquid concentration to 104cfu/mL, taking 100 mu L of the LSKO bacterial liquid, coating the 100 mu L of the LSKO bacterial liquid on an LB (LB) flat plate (the components are the same as those of a halomonas LS21 morphological identification culture medium), culturing the LB flat plate in an incubator at 37 ℃ for 48 hours, and observing the strain morphology by using a scanning electron microscope, wherein the strain halomonas LSKO and the starting strain halomonas LS21 are basically not different in strain morphology, namely the bacterial liquid is observed to be in a short rod shape under the microscope.

1.5. Self flocculation and self settling property test

The LSKO strain is subjected to gram stain identification, and the result is gram-negative bacteria. Meanwhile, the surface Zeta potential (data for representing the surface charge) of the LSKO strain is measured, and as shown in table 3 below, the results show that the surface charge of the strain LSKO is reduced relative to the original strain halomonas LS21, and the mutual repulsion is reduced due to the reduction of the surface charge, so that the LSKO strain is easier to adsorb together for self-flocculation. And the flocculation effect becomes more and more obvious as the culture time is prolonged, as shown in fig. 2, the self-flocculation condition of the LSKO strain along with the culture time is shown, and the result shows that the self-flocculation strains are more and the diameter of the generated self-flocculation thallus agglomerate is larger and larger as the culture time is prolonged.

Table 3: zeta potential comparison of Halomonas LSKO strain and LS21 strain at different salt concentrations

The inventors also tested the relationship between self-flocculation and self-sedimentation of Halomonas LSKO and the salt concentration in the solution. Wherein self-flocculation and self-sedimentation means that cells can settle due to gravity after self-flocculation, and the sedimentation degree is% = (bacterial liquid total OD 600-supernatant OD600 after sedimentation)/bacterial liquid total OD600 (Zhao, ning et al. "flocculation yeast microorganism is a purification host to be engineered for fuel ethanol production from bacterial microorganism". Biotechnology journal 9.3 (2014): 362-371.).

Firstly, washing the halomonas LSKO thallus twice by using 40g/L sodium chloride solution and culturing the halomonas LSKO thallus for 48 hours by using an LB culture medium, adding the obtained bacterial liquid into 4 test tubes in equal amount for heavy suspension, standing for 5 minutes, and observing that the self-flocculation and self-sedimentation phenomena appear in the 4 test tubes as shown in the A frame in figure 3; centrifuging 4 test tubes in A in figure 3, respectively, washing the obtained precipitated thallus twice with 0.02M sodium chloride solution, resuspending, standing for 5 minutes, and observing that no self-flocculation or self-sedimentation occurs in 4 test tubes as shown in B in figure 3; the 4 tubes in panel B of FIG. 3 were centrifuged, and the resulting precipitated cells were washed twice with equal volumes of 0.02M, 0.04M, 0.08M and 0.1M NaCl solutions, resuspended, and left to stand for 5 minutes, as shown in panel C of FIG. 3, it was observed that no self-flocculation and self-sedimentation occurred in the 0.02M and 0.04M NaCl solutions, but in the 0.08M and 0.1M NaCl solutions, the phenomena of self-flocculation and self-sedimentation stably existed, and it was also verified that the self-flocculation and self-sedimentation occurred in the cells were reversible. In combination with the data in the above table 3, zeta potential of LSKO should not be higher than 17.3 + -0.8 mV, and when the Zeta potential is higher than the Zeta potential, the self-flocculation and self-sedimentation phenomena of LSKO disappear.

The present inventors designed another test tube experiment by first washing LSKO cells cultured in LB medium twice with 0.1M sodium chloride solution for 48 hours to obtain a bacterial solution, adjusting OD600 of the bacterial solution to 4, then placing 6ml of the bacterial solution in a test tube with a diameter of 1cm, mixing them uniformly with Votex, and then standing. The results show that the bacteria solution in the test tube can achieve 95% of self-sedimentation degree in 20 seconds, and the sedimentation effect is higher than that of Zymomonamobilis reported in the prior art (Zhao, ning et al, "flocculation catalysis from Zymomonamobilis a purification host to be engineered for fusehanol production from lipocyte biological." Biotechnology Journal 9.3 (2014): 362-371.).

The inventors designed another fermenter test in which, after culturing the strain LSKO in a fermenter with a salt concentration of 0.1M for 48 hours, the OD600 of the culture broth was measured and the stirring and aeration were stopped. The results show that the fermenter broth can achieve 80% self-settling in 1 minute, which has not been reported before. Based on the above test results, the strain LSKO has very excellent self-flocculation and self-sedimentation effects.

2. The inventor uses Halomonas TD01 (which is preserved in China general microbiological culture Collection center (CGMCC for short, address: no. 3 of Suzuku-Lu No.1 of North Chen West Lu in the south facing area of Beijing, china institute of microbiology, postal code 100101) by the applicant at 11/19/2010, the preservation number is CGMCC No.4353, the strain name is TD01, and the classification name is Halomonas (Halomonas sp.) as an original strain (disclosed in patent document CN 102120973A), obtains a positive recombinant strain by the method for obtaining the Halomonas LSKO, and the strain is named as Halomonas (Halomonas phageminesiensis) TDKO.

The 16S rDNA sequence of the strain Halomonas TDKO was detected using the 16S-F (SEQ ID NO: 10) and 16S-R (SEQ ID NO: 11) primers shown in Table 1 above to obtain a sequence shown in SEQ ID NO:13 as the 16S rDNA sequence of Halomonas TDKO.

By adopting the same method, the inventor verifies the self-flocculation and self-sedimentation characteristics of the halomonas TDKO strain, obtains the results similar to the results of the halomonas LSKO strain, namely, good self-flocculation and self-sedimentation effects can be realized under the condition of proper salt concentration, and the self-flocculation and self-sedimentation characteristics are reversible, so that the description is omitted.

Other strains having self-flocculating and self-settling properties obtained according to the method of example 1 are also within the scope of the present application. Other strains with self-flocculating and self-precipitating properties obtained by other methods known to the skilled person, such as physical mutagenesis, chemical mutagenesis methods etc. also belong to the present application.

Example 2: method for producing poly-3-hydroxybutyrate by culturing microorganisms by using continuous fermentation culture method of recycling wastewater (PHB)

In order to verify the feasibility and effectiveness of the method for continuous fermentative culture of microorganisms using recycled wastewater according to the present invention, in this example, the halopmonas LSKO having self-flocculation and self-sedimentation characteristics obtained in example 1 above was used as an example in the following method for continuous fermentative culture of microorganisms using recycled wastewater, which was further used for the production of PHB using the microorganisms cultured by fermentation.

The process of continuously fermenting and culturing the microorganisms by recycling the wastewater comprises the following steps:

s1: inoculating bacteria with self-flocculating and self-settling characteristics (the example uses Halomonas LSKO) into an initial fermentation medium for fermentation culture;

s2: after the first fermentation culture, separating first self-settling bacterial thalli from a first fermentation product, adding waste water generated by washing the first self-settling bacterial thalli into the residual first supernatant, and continuing a second fermentation culture;

s3: after the second fermentation culture, separating second self-settling bacterial thalli from a second fermentation product, adding the waste water generated by washing the second self-settling bacterial thalli into the residual second supernatant, and continuing the next fermentation culture;

s4: repeating the step S3 for N times continuously, wherein N is a natural number (0, 1, 2, \8230;);

Further, PHB was produced using the S5-harvested LSKO cell of the microorganism Halomonas salina.

The fermentation culture process of the Halomonas LSKO is carried out under the conditions of stirring and aeration, the stirring speed of the initial fermentation culture is 200-250RPM, and the stirring speed is stabilized at 700-800RPM after the culture is about 5-8 hours; the aeration rate during the culture was 1vvm. In addition, the whole fermentation culture process is stabilized at the temperature of 20-45 ℃, the pH value of the culture system is 5.0-11.0, and acid and alkali can be supplemented in the culture process to maintain the pH condition. The concentration of sodium chloride in the culture system is maintained at 0.08mol/L or more during the culture, and the concentration of sodium chloride in the culture system is stabilized at about 0.08mol/L in this example.

In the step S1, halomonas LSKO is subjected to fermentation culture in a fermentation tank, wherein an initial fermentation medium (the components of the medium will be described below) is previously contained in the fermentation tank, the initial dissolved oxygen is 5% -100%, and the initial inoculation amount of the Halomonas LSKO is OD600 of 2-4.

The 1 liter of initial fermentation culture medium consists of 20 to 200g of sodium chloride, 5 to 100g of glucose, 0 to 50g of yeast powder, 0.5 to 20g of ammonium chloride, 0 to 20g of urea, 0.1 to 5g of magnesium sulfate, 0.5 to 10g of monopotassium phosphate, 1 to 20g of disodium hydrogen phosphate dodecahydrate, 1 to 30mL of trace element I, 0.1 to 10mL of trace element II and tap water, and the volume is fixed to 1 liter by the tap water;

wherein 1 liter of the trace element I is prepared according to the following method: mixing 2g-10g of ferric ammonium citrate, 1g-5g of calcium chloride dihydrate and 0.5mol/L of hydrochloric acid aqueous solution, and supplementing the mixture to 1 liter by using 0.5mol/L of hydrochloric acid aqueous solution to obtain the trace element I;1 l of trace element II was prepared as follows: 50-200 mg of zinc sulfate heptahydrate, 10-50 mg of manganese chloride tetrahydrate, 100-500 mg of boric acid, 50-400 mg of cobalt chloride hexahydrate, 3-30 mg of copper sulfate pentahydrate, 5-50 mg of nickel chloride hexahydrate, 10-50 mg of sodium molybdate dihydrate and 0.5mol/L of hydrochloric acid aqueous solution are mixed, and the mixture is complemented to 1 liter by 0.5mol/L of hydrochloric acid aqueous solution to obtain the trace element II.

In the steps S2 and S3, when the first round of fermentation of the halomonas LSKO for 48 hours in the initial fermentation medium is finished, stirring and ventilation are stopped, a large amount of thalli self-flocculation and self-sedimentation are observed after the fermentation medium is kept still for about 5 minutes, the fermentation product can be observed to be obviously layered after the fermentation medium is kept still for about 30 minutes, the lower layer of self-sedimentation thalli can be taken out from the fermentation tank by opening a valve at the bottom of the fermentation tank, and supernatant (containing part of unseparated suspended thalli) is left in the fermentation tank, so that the settled thalli can be separated from the fermentation product without centrifugation. And washing the separated thalli to be used for extracting PHB, and adding salt-containing wastewater generated by washing the thalli into a supernatant liquid left in a fermentation tank to supplement fermentation liquor for continuous second round fermentation culture. When the second round of fermentation culture is started, the OD600 of the halopmonas LSKO in the fermentation tank is about 25-30, and the concentration of the thallus is higher than that of the thallus inoculated during the initial fermentation culture, so that in the step S3, the yield similar to that of the first round of culture can be achieved by the second round of fermentation culture within about 24 hours, and after 24 hours of culture, the operation of separating the self-settling halopmonas LSKO thallus from the bottom of the fermentation tank after standing and layering and the subsequent next round of fermentation culture operation are repeated, so that the continuous fermentation culture process is realized, wastewater can be recycled in the whole fermentation process, centrifugation is not needed, the time consumption of the thallus separating process is short, and sterilization and inoculation in the subsequent steps are not needed.

This example continued the continuous fermentative cultivation of microorganisms for a total of 4 rounds. PHB was produced using the Halomonas LSKO cells obtained in each round (see Jiang, X.R., yao, Z.H., & Chen, G.Q. (2017.) Controlling cell volume for efficacy PHB production by microorganisms, metabolic engineering,44, 30-37.).

The following table 4 shows the comparison of the productivity of the microorganisms obtained for the production of PHB using the continuous fermentative cultivation method based on Halomonas LSKO with the productivity obtained using the conventional process (see Jiang, X.R., yao, Z.H., & Chen, G.Q. (2017). Controllingcell volume for efficacy PHB production by halomonas. Metabolic engineering,44, 30-37.), and the results show that the productivity using the continuous fermentative cultivation method with recycling of waste water is significantly improved: the data in Table 4 show that the dry cell weight (CDW) is increased from 0.45g/L/h to 0.82g/L/h in the conventional process; PHB is improved to 0.33g/L/h from 0.18g/L/h in the traditional process, which shows that the yield can be improved by the continuous fermentation culture method of recycling wastewater based on the Halomonas LSKO. And the produced wastewater can be recycled in the whole processes of fermentation culture and PHB production, and the operations of centrifugal separation of thalli and subsequent sterilization and inoculation are omitted, so that not only are resources saved, but also the energy consumption is reduced, and the production cost is obviously reduced.

Table 4: comparison of PHB production rate produced by continuous fermentation culture method of recycling wastewater and PHB production rate produced by traditional method

Note: the yield calculation in table 4 above is: the yield (unit g/L/h) is obtained by dividing the final harvested cell dry weight and the yield of PHB (unit g/L) by the time consumed by the production process.

The method for continuously fermenting and culturing the microorganisms by recycling the wastewater is also suitable for other existing or modified microorganisms with self-flocculation and self-sedimentation characteristics, and can also complete the processes of recycling the wastewater and continuously fermenting and culturing the microorganisms on a large scale. The microorganism to be cultured is not limited to one capable of producing PHB.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Qinghua university

<120> a method for continuous fermentation culture of microorganisms by recycling wastewater and bacteria having self-flocculation and self-sedimentation characteristics used therein

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

aattgctgct gagagcagtc ttcatgcagt tcacttacac cg 42

<210> 2

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aacccaagga ggcctcatta cgggccctat cacttattca g 41

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

taatgaggcc tccttgggtt 20

<210> 4

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcgctgctgt tccaccat 18

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gatcacggca tgttagcaat ctt 23

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gactgctctc agcagcaatt 20

<210> 7

<211> 5228

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aattgctgct gagagcagtc ttcatgcagt tcacttacac cgcttctcaa cccggtacgc 60

accagaaaat cattgatatg gccatgaatg gcgtttgaat tgtgattaaa aaggcaactt 120

tatgcccatg caacagaaac tataaaaaat acagagaatg aaaagaaaca gatagatttt 180

ttagttcttt aggcccgtag tctgcaaatc cttttatgat tttctatcaa acaaaagagg 240

aaaatagacc agttgcaatc caaacgagag tctaatagaa tgaggtcgaa aagtaaatcg 300

cgcgggtttg ttactgataa agcaggcaag acctaaaatg tgtaaagggc aaagtgtata 360

ctttggcgtc accccttaca tattttaggt ctttttttat tgtgcgtaac taacttgcca 420

tcttcaaaca ggagggctgg aagaagcaga ccgctaacac agtacataaa aaaggagaca 480

tgaacgatga acatcaaaaa gtttgcaaaa caagcaacag tattaacctt tactaccgca 540

ctgctggcag gaggcgcaac tcaagcgttt gcgaaagaaa cgaaccaaaa gccatataag 600

gaaacatacg gcatttccca tattacacgc catgatatgc tgcaaatccc tgaacagcaa 660

aaaaatgaaa aatatcaagt tcctgagttc gattcgtcca caattaaaaa tatctcttct 720

gcaaaaggcc tggacgtttg ggacagctgg ccattacaaa acgctgacgg cactgtcgca 780

aactatcacg gctaccacat cgtctttgca ttagccggag atcctaaaaa tgcggatgac 840

acatcgattt acatgttcta tcaaaaagtc ggcgaaactt ctattgacag ctggaaaaac 900

gctggccgcg tctttaaaga cagcgacaaa ttcgatgcaa atgattctat cctaaaagac 960

caaacacaag aatggtcagg ttcagccaca tttacatctg acggaaaaat ccgtttattc 1020

tacactgatt tctccggtaa acattacggc aaacaaacac tgacaactgc acaagttaac 1080

gtatcagcat cagacagctc tttgaacatc aacggtgtag aggattataa atcaatcttt 1140

gacggtgacg gaaaaacgta tcaaaatgta caattaccct gttatcccta ccaccacgat 1200

ctcggcgtcg cccgccatga tcgcgttggc ggccagcatc acggccttca ggcccgagcc 1260

gcacaccttg ttgatggtca tggccggcac catcgccggc aggccggcct tgatcgcggc 1320

ctggcgtgcg gggttctggc ccgaaccggc ggtcagcacc tggcccatga tgacttcgct 1380

cacctgctcc ggcttgacgc cggcgcgctc cagcgcggcc ttgatgacca cggcacccag 1440

tagggataac agggtaatgg taccattacc ctgttatccc tagtgttgac ggtcacgccc 1500

ttggtcgcca cttcctgcgc cagtgccatg gtgaagccat gcaggccggc cttggcggtg 1560

gagtagttgg tctggccgaa ctggcccttc tgcccgttca ccgacgagat gttgacgatg 1620

cggccccagc cacggtcggc catgccgtcg atcacctgct tggtgacgtt gaacagcgag 1680

gtcaggttgg tgtcgatcac cgcatcccag tcggcgcggg tcatcttgcg gaacataggg 1740

ataacagggt aatgtgcaca ttaccctgtt atccctaaac tgcacatggc cctcgtcgac 1800

aaagacgtcg aggatgcccg tgtcggcaaa gtccagcagc gtggtcagca gcgtgacgct 1860

ggcggccggg tgctcgccgc gcgcggccag caccgccagc gcggtcgaga caatggtgcc 1920

gcccacgcag aagccgagca cgttgatctt gtcctggccg ctgatgtcgc gcgcgacttc 1980

gatggcgcgg atggccgcgt gctcgatgta gtcgtcccag gtgctgctag ggataacagg 2040

gtaatcatat gaactatata aaagcaggca aatggctaac cgtattccta accttttgaa 2100

ttcgatcgct agtttgtttt gactccatcc attagggctt ctaaaacgcc ttctaaggcc 2160

atgtcagccg ttaagtgttc ctgtgtcact gaaaattgct ttgagaggct ctaagggctt 2220

ctcagtgcgt tacatccctg gcttgttgtc cacaaccgtt aaaccttaaa agctttaaaa 2280

gccttatata ttcttttttt tcttataaaa cttaaaacct tagaggctat ttaagttgct 2340

gatttatatt aattttattg ttcaaacatg agagcttagt acgtgaaaca tgagagctta 2400

gtacgttagc catgagagct tagtacgtta gccatgaggg tttagttcgt taaacatgag 2460

agcttagtac gttaaacatg agagcttagt acgtgaaaca tgagagctta gtacgtacta 2520

tcaacaggtt gaactgctgg atcgatcctt tttgtccggt gttgggttga aggtgaagcc 2580

ggtcggggcc gcagcggggg ccggcttttc agccttgccc ccctgcttcg gccgccgtgg 2640

ctccggcgtc ttgggtgccg gcgcgggttc cgcagccttg gcctgcggtg cgggcacatc 2700

ggcgggcttg gccttgatgt gccgcctggc gtgcgagcgg aacgtctcgt aggagaactt 2760

gaccttcccc gtttcccgca tgtgctccca aatggtgacg agcgcatagc cggacgctaa 2820

cgccgcctcg acatccgccc tcaccgccag gaacgcaacc gcagcctcat cacgccggcg 2880

cttcttggcc gcgcgggatt caacccactc ggccagctcg tcggtgtagc tctttggcat 2940

cgtctctcgc ctgtcccctc agttcagtaa tttcctgcat ttgcctgttt ccagtcggta 3000

gatattccac aaaacagcag ggaagcagcg cttttccgct gcataaccct gcttcggggt 3060

cattatagcg attttttcgg tatatccatc ctttttcgca cgatatacag gattttgcca 3120

aagggttcgt gtagactttc cttggtgtat ccaacggcgt cagccgggca ggataggtga 3180

agtaggccca cccgcgagcg ggtgttcctt cttcactgtc ccttattcgc acctggcggt 3240

gctcaacggg aatcctgctc tgcgaggctg gccggctacc gccggcgtaa cagatgaggg 3300

caagcggatg gctgatgaaa ccaagccaac caggaagggc agcccaccta tcaaggtgta 3360

ctgccttcca gacgaacgaa gagcgattga ggaaaaggcg gcggcggccg gcatgagcct 3420

gtcggcctac ctgctggccg tcggccaggg ctacaaaatc acgggcgtcg tggactatga 3480

gcacgtccgc gagctggccc gcatcaatgg cgacctgggc cgcctgggcg gcctgctgaa 3540

actctggctc accgacgacc cgcgcacggc gcggttcggt gatgccacga tcctcgccct 3600

gctggcgaag atcgaagaga agcaggacga gcttggcaag gtcatgatgg gcgtggtccg 3660

cccgagggca gagccatgac ttttttagcc gctaaaacgg ccggggggtg cgcgtgattg 3720

ccaagcacgt ccccatgcgc tccatcaaga agagcgactt cgcggagctg gtgaagtaca 3780

tcaccgacga gcaaggcaag accgagcgcc tgggtcacgt gcgcgtcacg aactgcgagg 3840

caaacaccct gcccgctgtc atggccgagg tgatggcgac ccagcacggc aacacccgtt 3900

ccgaggccga caagacctat cacctgctgg ttagcttccg cgcgggagag aagcccgacg 3960

cggagacgtt gcgcgcgatt gaggaccgca tctgcgctgg gcttggcttc gccgagcatc 4020

agcgcgtcag tgccgtgcat cacgacaccg acaacctgca catccatatc gccatcaaca 4080

agattcaccc gacccgaaac accatccatg agccgtatcg ggcctaccgc gccctcgctg 4140

acctctgcgc gacgctcgaa cgggactacg ggcttgagcg tgacaatcac gaaacgcggc 4200

agcgcgtttc cgagaaccgc gcgaacgaca tggagcggca cgcgggcgtg gaaagcctgg 4260

tcggctggat cgggccctaa atacctgtga cggaagatca cttcgcagaa taaataaatc 4320

ctggtgtccc tgttgatacc gggaagccct gggccaactt ttggcgaaaa tgagacgttg 4380

atcggcacgt aagaggttcc aactttcacc ataatgaaat aagatcacta ccgggcgtat 4440

tttttgagtt atcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg 4500

gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt 4560

cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga 4620

ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga 4680

tgaatgctca tccggaattc cgtatggcaa tgaaagacgg tgagctggtg atatgggata 4740

gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga 4800

gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt 4860

acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag 4920

ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct 4980

tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc 5040

tggcgattca ggttcatcat gccgtctgtg atggcttcca tgtcggcaga atgcttaatg 5100

aattacaaca gtactgcgat gagtggcagg gcggggcgta atttttttaa ggcagttatt 5160

ggtgccctta aacgcctggt gctacgcctg aataagtgat agggcccgta atgaggcctc 5220

cttgggtt 5228

<210> 8

<211> 586

<212> DNA

<213> Halomonas sp

<400> 8

taatgaggcc tccttgggtt ggcgctgcac tacccgtggc gccgctggaa gcctggatat 60

ccagcgccct ctgccgcggg ttgccctggc actggccgag tgcgctgagc cggtggatga 120

gacgcggcat gcggccgagc gccagacctt ggcagcgcca attcccacca ctctatcgcg 180

cattgaagat ctggggcagg tggcagtgga tcccgctggc gtggcgttgg ccgaggctga 240

attcatcctc tctggtggca atggtgttaa acagtgggac gccttccacc atgctgcgaa 300

agtactaggc gctaccgaag gggcctcgcg tgtcgcggtg gatgacggct ttatggctcg 360

tgatcggcag gtaggtgcaa ccggcacctg ggtaaccgcc cgagtctata tggcggtggg 420

tatttcaggc gcgatccagc acctacaggg cattcagcgc tgcgacaagg tggtggcaat 480

caatctcgat ccggggtgcg acatgatcaa acgcgcagac ctggcggtga taggcgacag 540

cacgcaaatt cttgctgcgt tagtggcgat ggtggaacag cagcga 586

<210> 9

<211> 493

<212> DNA

<213> Halomonas sp

<400> 9

gatcacggca tgttagcaat cttaaagccc gccctctgtt ggcgggcttc ttgttatcta 60

ttgtttaatc tctgagtctt tattttcgaa gcctttacgt tttaagtctt tacgttctct 120

gtcgttatct tcttctatgt tgttatgacc tacttgccag taaagcctgg ataaagccgc 180

aggcgctgtc tttccaggtg taaacccatc caggcgtgta gtactagtac cgtgatgacg 240

atggccccgc ccagtagcgt tgatgccgtc ggcgtctcgc ctagtagcca ccaaacccat 300

agtgtgccca atgcggtttc caccaggtaa aacagtgcca cctcggtaga cggcaggtaa 360

cgggtggcgc tattgatcag caccgtggcc aggggcatct gcactagccc catcagtgcc 420

agcacgccat aacgctcggc atcaagcgcc agcggcgagg ccatgggcag agcaattgct 480

gctgagagca gtc 493

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

agagtttgat catggctcag 20

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tacggctacc ttgttacgac tt 22

<210> 12

<211> 1530

<212> DNA

<213> Halomonas sp

<400> 12

tgaagagttt gatcatggct cagattgaac gctggcggca ggcctaacac atgcaagtcg 60

agcggtaaca ggggtagctt gctacccgct gacgagcggc ggacgggtga gtaatgcata 120

ggaatctgcc cgatagtggg ggataacctg gggaaaccca ggctaatacc gcatacgtcc 180

tacgggagaa agggggctcc ggctcccgct attggatgag cctatgtcgg attagctagt 240

tggtgaggta atggctcacc aaggcaacga tccgtagctg gtctgagagg atgatcagcc 300

acatcgggac tgagacacgg cccgaactcc tacgggaggc agcagtgggg aatattggac 360

aatgggcgaa agcctgatcc agccatgccg cgtgtgtgaa gaaggccttc gggttgtaaa 420

gcactttcag cgaggaagaa cgcctagtgg ttaataccca ttaggaaaga catcactcgc 480

agaagaagca ccggctaact ccgtgccagc agccgcggta atacggaggg tgcaagcgtt 540

aatcggaatt actgggcgta aagcgcgcgt aggtggcttg ataagccggt tgtgaaagcc 600

ccgggctcaa cctgggaacg gcatccggaa ctgtcaagct agagtgcagg agaggaaggt 660

agaattcccg gtgtagcggt gaaatgcgta gagatcggga ggaataccag tggcgaaggc 720

ggccttctgg actgacactg acactgaggt gcgaaagcgt gggtagcaaa caggattaga 780

taccctggta gtccacgccg taaacgatgt cgaccagccg ttgggtgcct agcgcacttt 840

gtggcgaagt taacgcgata agtcgaccgc ctggggagta cggccgcaag gttaaaactc 900

aaatgaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gatgcaacgc 960

gaagaacctt acctactctt gacatcctgc gaacttgtga gagatcactt ggtgccttcg 1020

ggaacgcaga gacaggtgct gcatggctgt cgtcagctcg tgttgtgaaa tgttgggtta 1080

agtcccgtaa cgagcgcaac ccttgtcctt atttgccagc gggtaatgcc gggaactcta 1140

aggagactgc cggtgacaaa ccggaggaag gtggggacga cgtcaagtca tcatggccct 1200

tacgagtagg gctacacacg tgctacaatg gccggtacaa agggttgcga gctcgcgaga 1260

gtcagctaat cccgaaaagc cggtctcagt ccggatcgga gtctgcaact cgactccgtg 1320

aagtcggaat cgctagtaat cgtgaatcag aatgtcacgg tgaatacgtt cccgggcctt 1380

gtacacaccg cccgtcacac catgggagtg gactgcacca gaagtggtta gcttaacctt 1440

cgggaaagcg atcaccacgg tgtggttcat gactggggtg aagtcgtaac aaggtagccg 1500

taggggaacc tgcggctgga tcacctcctt 1530

<210> 13

<211> 1528

<212> DNA

<213> Halomonas sp

<400> 13

tgaagagttt gatcatggct cagattgaac gctggcggca ggcctaacac atgcaagtcg 60

agcggtaaca ggggtagctt gctacccgct gacgagcggc ggacgggtga gtaatgcata 120

ggaatctgcc cggtagtggg ggataacctg gggaaaccca ggctaatacc gcatacgtcc 180

tacgggagaa agggggctcc ggctcccgct attggatgag cctatgtcgg attagctagt 240

tggtgaggta atggctcacc aaggcaacga tccgtagctg gtctgagagg atgatcagcc 300

acatcgggac tgagacacgg cccgaactcc tacgggaggc agcagtgggg aatattggac 360

aatgggggca accctgatcc agccatgccg cgtgtgtgaa gaaggccttc gggttgtaaa 420

gcactttcag cgaggaagaa cgcctagtgg ttaataccca ttaggaaaga catcactcgc 480

agaagaagca ccggctaact ccgtgccagc agccgcggta atacggaggg tgcaagcgtt 540

aatcggaatt actgggcgta aagcgcgcgt aggtggcttg ataagccggt tgtgaaagcc 600

ccgggctcaa cctgggaacg gcatccggaa ctgtcaagct agagtgcagg agaggaaggt 660

agaattcccg gtgtagcggt gaaatgcgta gagatcggga ggaataccag tggcgaaggc 720

ggccttctgg actgacactg acactgaggt gcgaaagcgt gggtagcaaa caggattaga 780

taccctggta gtccacgccg taaacgatgt cgaccagccg ttgggtgcct agcgcacttt 840

gtggcgaagt taacgcgata agtcgaccgc ctggggagta cggccgcaag gttaaaactc 900

aaatgaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gatgcaacgc 960

gaagaacctt acctactctt gacatcctgc gaacttgtga gagatcactt ggtgccttcg 1020

ggaacgcaga gacaggtgct gcatggctgt cgtcagctcg tgttgtgaaa tgttgggtta 1080

agtcccgtaa cgagcgcaac ccttgtcctt atttgccagc gggtaatgcc gggaactcta 1140

aggagactgc cggtgacaaa ccggaggaag gtggggacga cgtcaagtca tcatggccct 1200

tacgagtagg gctacacacg tgctacaatg gccggtacaa agggttgcga gctcgcgaga 1260

gtcagctaat cccgaaaagc cggtctcagt ccggatcgga gtctgcaact cgactccgtg 1320

aagtcggaat cgctagtaat cgtgaatcag aatgtcacgg tgaatacgtt cccgggcctt 1380

gtacacaccg cccgtcacac catgggagtg gactgcacca gaagtggtta gcctaacgca 1440

agagggcgat caccacggtg tggttcatga ctggggtgaa gtcgtaacaa ggtagccgta 1500

ggggaacctg cggctggatc acctcctt 1528

Claims

1. A method for continuously fermenting and culturing microorganisms by recycling waste water is characterized in that,

the method comprises the following steps:

s3: after the second fermentation culture, separating second self-settling bacterial thalli from a second fermentation product, adding waste water generated by washing the second self-settling bacterial thalli into the second supernatant remained after separation, and continuing the next fermentation culture;

s5: combining the bacterial thalli washed in each step into harvested microorganisms,

the bacteria with the characteristics of self flocculation and self sedimentation are Halomonas salina (Halomonas camphaniensis) LSKO with the preservation number of CGMCCNo.16437.

2. The method of claim 1,

the separation in step S2 and/or step S3 is obtained from the settled bacterial biomass from the lower layer of the still layered fermentation product.

3. The method of claim 2, wherein the standing layering time is 10-30min.

4. The method of claim 1,

the self-flocculation and self-sedimentation characteristics mean that bacteria can perform self-flocculation and self-sedimentation under the condition that the salt concentration is not less than 0.08M.

5. The method of any one of claims 1 to 4,

the salt concentration in the fermentation culture system is not lower than 0.08mol/L; and/or

The bacteria with self-flocculating and self-settling properties in step S1 have an OD600 of 2-4 in the initial fermentation medium; and/or

The time of the first fermentation culture in the step S2 is 44-52h; and/or

The time of the second fermentation culture in the step S3 is 20-28h.

6. A bacterium having self-flocculating and self-settling properties,

the bacteria can perform self flocculation and self sedimentation under the condition that the salt concentration is not lower than 0.08M; the bacterium is Halomonas salina (Halomonascamphanensis) LSKO with the preservation number of CGMCC No.16437.