CN113801810B

CN113801810B - Halomonas strain and application thereof

Info

Publication number: CN113801810B
Application number: CN202110929333.2A
Authority: CN
Inventors: 沈宏伟; 吕金艳; 银会娟; 司徒卫; 余柳松; 何世琪
Original assignee: Zhuhai Medfa Biotechnology Co ltd
Current assignee: Zhuhai Medfa Biotechnology Co ltd
Priority date: 2021-08-13
Filing date: 2021-08-13
Publication date: 2022-06-24
Anticipated expiration: 2041-08-13
Also published as: CN113801810A; WO2023016058A1

Abstract

The invention discloses a halomonas and application thereof. The Halomonas is Halomonas lutescens MDF-9, and the preservation number of the Halomonas is GDMCC NO. 61850. The halomonas can efficiently accumulate Polyhydroxyalkanoate (PHA) in the culture medium, and provides good guarantee for the biosynthesis of the PHA. The fermentation process of the halomonas does not need sterilization, can be continuously carried out, and is simple and easy to control. The concentration of sodium chloride is low and is 10-20 g/L in the fermentation process, the wastewater in the later period is easy to treat, the nutrient components of a culture medium used for fermentation are simple, toxic and harmful metal compounds are not used, and the method is safer and more environment-friendly. The method for preparing PHA by using halophilic bacteria (Halomonas lutescens MDF-9) reduces the production cost, improves the yield of PHA and has industrial application value.

Description

Halomonas strain and application thereof

Technical Field

The invention relates to the field of microbial fermentation, in particular to a halomonas and application thereof.

Background

The degradation speed of the chemical-based plastic is slow, the period is long, the chemical-based plastic is more difficult to treat particularly after micro-plastic is formed, and the environment is seriously polluted when the chemical-based plastic is used in large quantity. Therefore, the development of the bio-based degradable plastic is beneficial to the development of environmental health.

Polyhydroxyalkanoates (PHA) is a biological material that is synthesized and stored intracellularly by microorganisms under conditions of excess carbon source and limitation of other nutrients. The monomer is divided into short-chain PHA (4-6C, scl-PHA) and medium-long-chain PHA (more than or equal to 6C, mcl-PHA) according to the difference of the carbon chain length of the monomer. Poly- β -hydroxybutyrate (PHB) is one of the short chain PHAs. PHA has the characteristics of degradability, biocompatibility, diversity of texture and the like, and has wide application prospects in the fields of chemical products, medical implant materials, drug sustained-release carriers and the like.

Although the PHA industry chain has developed rapidly over the last decade, the complexity of the PHA manufacturing process makes its manufacturing cost prohibitive and not competitive with petroleum-based traditional materials. The main reasons include raw material costs, energy consumption, downstream processing costs, etc. Therefore, many methods have been attempted to reduce the production cost of PHA, such as obtaining high-yielding bacteria with excellent properties by genetic engineering or metabolic pathway modification, exploring simple and effective downstream extraction and purification methods, and using inexpensive raw materials. The prior art discloses some halomonas, which can realize PHA production under non-sterilization conditions, effectively reduce production cost and eliminate aseptic operation complexity. But there are also significant drawbacks: (1) the concentration of NaCl is as high as 20-200 g/L, so that the later-stage wastewater is difficult to treat; (2) cobalt chloride and nickel chloride which are toxic and harmful substances are used in the culture process; (3) the medium composition is complex. Therefore, there is a need for a halomonas and a fermentation process that can produce PHA under low salt concentration and no harmful substances in fermentation conditions.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a Halomonas and application thereof, wherein the strain is Halomonas (Halomonas lutescens) MDF-9 with the preservation number of GDMCC NO. 61850. The halomonas can efficiently accumulate Polyhydroxyalkanoate (PHA) in the culture medium, and provides good guarantee for the biosynthesis of the PHA.

The invention provides a Halomonas (Halomonas lutescens MDF-9), and the preservation number of the Halomonas is GDMCC NO. 61850.

The invention also provides application of the halomonas in preparation of polyhydroxyalkanoate.

Further, the polyhydroxyalkanoate is poly-beta-hydroxybutyric acid.

The invention also provides a method for preparing the polyhydroxyalkanoate, which comprises the following steps: fermenting Halomonas lutescens MDF-9 (GDMCC NO. 61850) to obtain polyhydroxyalkanoate, wherein the fermentation culture medium and the fermentation container used in the fermentation are not sterilized. The non-sterilization is to save the flow and the cost, and can better embody the value of the invention, and the sterilization can also realize the technical effect of the invention.

Further, the concentration of sodium chloride in the fermentation medium is 10-20 g/L. The fermentation medium disclosed by the invention is low in salt concentration, and is more favorable for treating wastewater.

Further, the pH value of the fermentation is 8-10, the stirring speed is 50-800 rpm, the ventilation volume is 0.1-2 vvm, and the fermentation temperature is 25-40 ℃. The yield of PHA is higher under the above fermentation conditions.

Further, the fermentation method comprises the following steps:

(1) inoculating Halomonas lutescens MDF-9 (Halomonas lutescens MDF-9) GDMCC NO.61850 into a fermentation culture medium for fermentation culture;

(2) when the concentration of sugar in the fermentation liquor is detected to be lower than 10g/L in the fermentation process, adding sugar to maintain the concentration at 30 g/L;

(3) and (5) continuing culturing for 30-40 h, and ending fermentation when the sugar concentration in the fermentation solution is lower than 0.2 g/L.

Further, the fermentation medium comprises the following components:

carbon source: any one or more of glucose, fructose and sodium gluconate; the strain of the invention not only can use glucose as a carbon source, but also can use fructose and sodium gluconate.

Nitrogen source: corn steep liquor dry powder, yeast powder, tryptone, urea, ammonium sulfate and ammonium chloride;

other nutrient components: potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate;

sodium chloride;

the fermentation medium does not contain cobalt chloride and nickel chloride. Cobalt chloride and nickel chloride are toxic metal compounds, and may be sensitized by inhalation and skin contact. Has extremely high toxicity to aquatic organisms and may have long-term adverse effects on the water environment. Therefore, the culture medium without cobalt chloride and nickel chloride is safer and more environment-friendly.

Further, the total concentration of the carbon source is 90-150 g/L; 1-8% of corn steep liquor dry powder, 0.5-5% of yeast powder, 0.1-3% of tryptone, 0.1-3% of urea, 0.1-3% of ammonium sulfate and 0.1-3% of ammonium chloride; the content of the monopotassium phosphate is 0.1-2%, the content of the disodium hydrogen phosphate is 0.05-1%, the content of the magnesium sulfate is 0.05-1%, and the content is in a mass-volume ratio.

Further, the polyhydroxyalkanoate is poly-beta-hydroxybutyric acid.

In summary, compared with the prior art, the invention achieves the following technical effects:

1. the concentration of sodium chloride is low in the fermentation process and is 10-20 g/L, and the later-stage wastewater is easy to treat.

2. The fermentation nutrient components are simple, and toxic and harmful metal compounds are not used.

3. After the strain is cultured in a fermentation tank by using the fermentation medium, the dry weight of the strain can reach 198.5g/L, and the content of PHA can reach 79.5%.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a morphological diagram of a single colony after plating a plate with the strain of example 1 of the present invention.

FIG. 2 is an image of microscopic observation of the strain of example 1 of the present invention before fermentation.

FIG. 3 is a microscopic image of the strain of example 1 of the present invention after fermentation.

FIG. 4 is an FTIR spectrum of a Sigma PHB standard of the present invention in example 1.

FIG. 5 is an FTIR spectrum of a PHB sample prepared in example 1 of the present invention.

FIG. 6 is a GC spectrum of a standard sample in example 1 of the present invention.

FIG. 7 is a GC spectrum of a sample tested in example 1 of the present invention.

FIG. 8 is an FTIR spectrum of a PHB sample prepared in example 2 of the present invention.

FIG. 9 is a GC map of a PHB sample prepared using medium 1 without sterilization in example 2 of the present invention.

FIG. 10 is a GC map of a PHB sample prepared using medium 2 without sterilization in example 2 of the present invention.

FIG. 11 is a GC map of a PHB sample prepared using medium 3 without sterilization in example 2 of the present invention.

FIG. 12 is a GC map of a PHB sample prepared using medium 4 without sterilization in example 2 of the present invention.

FIG. 13 is a GPC chart of PHB sample prepared using medium 1 without sterilization in example 2 of the present invention.

FIG. 14 is a GPC chart of PHB sample prepared using medium 2 without sterilization in example 2 of the present invention.

FIG. 15 is a GPC chart of PHB sample prepared using medium 3 without sterilization in example 2 of the present invention.

FIG. 16 is a GPC chart of PHB sample prepared using medium 4 without sterilization in example 2 of the present invention.

FIG. 17 shows the results of gas chromatography for PHB samples prepared under culture condition 1 without sterilization in example 3 of the present invention.

FIG. 18 shows the results of gas chromatography for PHB samples prepared under culture condition 2 without sterilization in example 3 of the present invention.

FIG. 19 shows the results of gas chromatography for PHB samples prepared under culture condition 3 without sterilization in example 3 of the present invention.

FIG. 20 shows the results of gas chromatography of PHB samples prepared under culture condition 4 without sterilization in example 3 of the present invention.

FIG. 21 shows the result of electrophoresis of the 486bp product in example 4.

FIG. 22 shows the result of electrophoresis of the 432bp product in example 4.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, shall fall within the scope of protection of the present invention.

The invention provides a Halomonas (Halomonas lutescens MDF-9) which can accumulate PHA in cells.

Firstly, culture conditions are as follows: the concentration of sodium chloride is 10-20 g/L, the pH value is 8-10, the ventilation volume is 0.1-2 vvm, and the temperature is 25-40 ℃.

II, culture medium composition:

carbon source: any one or more of glucose, fructose and sodium gluconate, and the total concentration is 90-150 g/L.

Nitrogen source: 1-8% of corn steep liquor dry powder, 0.5-5% of yeast powder, 0.1-3% of tryptone, 0.1-3% of urea, 0.1-3% of ammonium sulfate and 0.1-3% (m/v) of ammonium chloride.

10-20 g/L of sodium chloride.

Other nutrient components: 0.1-2% of monopotassium phosphate, 0.05-1% of disodium hydrogen phosphate and 0.05-1% (m/v) of magnesium sulfate.

Thirdly, the method for producing PHA by using the halomonas of the invention is as follows:

In particular, the method comprises the following steps of,

the halomonas is inoculated into a liquid culture medium, wherein the components of the culture medium comprise one or two or three of the carbon sources, the sugar concentration is 90-150 g/L, the sodium chloride is 10-20 g/L, the corn steep liquor dry powder is 1-8%, the yeast powder is 0.5-5%, the tryptone is 0.1-3%, the urea is 0.1-3%, the ammonium sulfate is 0.1-3%, the ammonium chloride is 0.1-3% (m/v), the potassium dihydrogen phosphate is 0.1-2%, the disodium hydrogen phosphate is 0.05-1%, and the magnesium sulfate is 0.05-1% (m/v).

Culturing for 30-40 h under the conditions that the pH is 8-10, the ventilation volume is 0.1-2 vvm, the stirring speed is 50-800 rpm, and the temperature is 25-40 ℃, when the sugar concentration in the fermentation liquor is lower than 10g/L, adding 1500-800 g/L glucose and 0.5-10 g/L ammonium chloride solution to maintain the sugar concentration at 10-50 g/L, and when the sugar concentration is finished, the sugar concentration is lower than 0.2 g/L.

The 3 media used in the following examples were a plate solid medium used to coat the strain and 2 liquid media. The seed culture medium and the fermentation culture medium are used for shaking culture and 5L fermentation tank culture of the halomonas of the invention respectively. They are composed in detail as follows:

plate solid medium: 0.5-1% of yeast powder; 0.5-1% of tryptone; 1-2% of sodium chloride, 18g/L of agar powder and pH of 9.0.

Seed culture medium: 30-50 g/L of glucose solution; 10-20 g/L of sodium chloride, 1-8% of corn steep liquor dry powder, 0.5-5% of yeast powder, 0.1-3% of tryptone, 0.1-3% of urea, 0.1-3% of ammonium sulfate, 0.1-3% (m/v) of ammonium chloride, 0.1-2% of monopotassium phosphate, 0.05-1% of disodium hydrogen phosphate and 0.05-1% (m/v) of magnesium sulfate. The pH of the culture medium is adjusted to 8.0-10 by sodium hydroxide, and the volume of the culture medium is 50mL (250mL triangular flask).

Fermentation medium: 90-150 g/L of glucose solution; 10-20 g/L of sodium chloride, 1-8% of corn steep liquor dry powder, 0.5-5% of yeast powder, 0.1-3% of tryptone, 0.1-3% of urea, 0.1-3% of ammonium sulfate, 0.1-3% (m/v) of ammonium chloride, 0.1-2% of monopotassium phosphate, 0.05-1% of disodium hydrogen phosphate and 0.05-1% (m/v) of magnesium sulfate. Adjusting the pH value of the culture medium to 8-10 by using sodium hydroxide.

The fermentation tank with 5L proves that the fermentation is carried out for 30-40 h, the dry weight of the thallus in the fermentation liquor at the fermentation end is 90-200 g/L, and the PHA content in the dry thallus is 50-80%.

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

EXAMPLE 1 isolation and purification of the Strain

First, strain isolation

100. mu.L of Qinghai lake water was applied to a separation and purification solid medium (medium components: yeast powder 10g/L, tryptone 5g/L, sodium chloride 35g/L, agar powder 18g/L, pH 9.0), and static culture was carried out at 33 ℃ for 24 hours.

Secondly, strain purification

And (3) picking out single colonies growing in the solid culture medium, diluting by 100-10000 times with 35g/L saline, coating on the solid culture medium, and statically culturing for 24 hours at 33 ℃. Repeating the above steps until the single colony grown on the solid culture medium has consistent shape, and completing the strain purification.

Thirdly, identification of the strains

The colony morphology is shown in FIG. 1, and is circular, white, slightly convex in the middle, complete in the edge and opaque. The morphology of the cells after gram-staining was observed with a microscope, and the morphology of the cells before fermentation was shown in FIG. 2, which indicates short rods, no spores, and gram-negative cells. The shape of the cells at the late stage of fermentation is shown in FIG. 3, and it is seen that the cells are white and transparent in the middle, colored at both ends, and have flagella and are capable of movement. The 16S rDNA sequence of the strain is detected, and the detected sequence is shown in SEQ ID NO. 1. The sequence is aligned in NCBI database, and the similarity of the strain and Halomonas lutescens Q1U reaches 99%. Combining the above identification results, the strain was identified as Halomonas (Halomonas lutescens). The strain has been deposited in Guangdong province microorganism culture collection center (GDMCC for short, address: No. 59 building 5 of Mitsui 100, Mitsui, Guangzhou city, and postal code 510070, Guangdong province microbiological research institute) at 8.2.8.1.2021. The accession number is GDMCC NO. 61850. The strain is named as MDF-9 and classified and named as Halomonas lutescens.

Verification experiment for PHA (polyhydroxyalkanoate) production by strains

The purified strain of the present invention was inoculated into a seed medium (seed medium composition: glucose 90 g/L; sodium chloride 35g/L, corn steep liquor dry powder 1%, yeast powder 0.5%, urea 0.1%, ammonium sulfate 0.1%, ammonium chloride 0.1%, potassium dihydrogen phosphate 0.3%, disodium hydrogen phosphate 0.1%, magnesium sulfate 0.1%, (m/v) sodium hydroxide was added to adjust the pH of the medium to 9.0, the volume of the medium was 30mL (250mL Erlenmeyer flask), and shake flask culture was used to verify whether intracellular PHA was synthesized or not, culture conditions were 35 ℃, 200rpm, and culture time was 40 h.

FTIR verification of intracellular metabolites of Halomonas lutescens

Centrifuging 30mL of fermentation liquor after fermentation is finished at 8000rpm for 5min, removing supernatant, then adding 25mL of deionized water, shaking uniformly, then 8000rpm for 5min, removing supernatant, then adding 5mL of deionized water, then adding 10mL of trichloromethane, fully mixing and shaking for 60min, centrifuging at 8000rpm for 5min, taking a lower trichloromethane phase, removing trichloromethane by rotary evaporation, collecting obtained white powder, and drying at 80 ℃ for 24 h. FTIR analysis was performed and the results are shown in FIG. 5.

The FTIR spectrum of the PHB standard product from Sigma is shown in FIG. 4, and the C ═ O peak (1723-. As shown in FIG. 5, the PHB characteristic peak at 1723-1740 was found to preliminarily confirm that the strain of the present invention can indeed synthesize PHA at a content of 46% (w/w).

(II) GC verification of Halomonas lutescens intracellular metabolite

The detection steps of intracellular products in the strain are as follows:

(1) digestion

(a) 2mL of liquid A (prepared from 15mL of concentrated sulfuric acid, 485mL of methanol and 0.5g of benzoic acid) and 2mL of chloroform were added to the digestion cup, and after mixing, the dry microbial cells (containing about 100mg of dry microbial cells) after fermentation were added.

(b) And (4) putting the digestion cup into a digestion instrument, wherein the temperature is 100 ℃, and the digestion time is 3-4 hours.

(c) And after digestion is finished, cooling the digestion cup to room temperature, and taking out the digestion cup. Adding 1mL of pure water into the digestion cup, oscillating for 1-2 min, and standing for 0.5 h.

(2) Preparing a standard substance:

preparation of 3HB standard: weighing 50mg of PHB standard sample, and carrying out sample introduction pretreatment according to the PHA digestion step.

(3) Gas chromatography detection:

a detector: FID, column temperature 140 ℃, sample introduction temperature 200 ℃, detector temperature 220 ℃, temperature programming, 10 ℃/min, temperature rise to 220 ℃, capillary column SP-2560, sample introduction amount 0.5 mu L.

GC spectra of the standard sample and the detection sample are shown in FIGS. 6 and 7. As can be seen from the standard sample, the peak at the retention time of about 3.5min is 3-hydroxybutyrate (3HB), and the peak at about 5.2min is a benzoic acid standard, so that the intracellular metabolite of the strain is a polymer of 3-hydroxybutyrate, namely poly (3-hydroxybutyrate) ester, namely PHB, according to the result of FTIR verification.

Example 2 method for producing PHA Using Halomonas of the present invention in a fermenter

Firstly, strain activation

Taking strains in a refrigerator at 4 ℃ in a laboratory, picking single colonies by using an inoculating loop, streaking and inoculating the strains on a solid plate, and culturing for 24 hours by using a solid plate culture medium. The above operations are repeated, and the plate is inoculated for the second stage and cultured for 24 h.

Second, preparation of seed liquid

First-stage bacterial liquid: and (3) taking the secondary plate, selecting a single colony, inoculating the single colony in a seed culture medium, and culturing the culture solution for 12 hours at the temperature of 25-40 ℃ and the rpm of 150-220 by using a shaking table.

Secondary bacterial liquid: the first-order bacterial liquid 500. mu.L (1% inoculum size) was taken, inoculated into a seed medium, and the culture liquid was cultured for 12 hours at 220rpm on a shaker at 37 ℃.

Third, fermentation tank culture

30 minutes before inoculation, a fermentation medium is prepared, the seed solution is inoculated into a 5L fermentation tank according to the inoculation amount of 1-20%, 4 kinds of fermentation media (a culture medium 1, a culture medium 2, a culture medium 3 and a culture medium 4) with different proportions are used, as shown in Table 1, the pH is adjusted and maintained to be 9.0, the ventilation amount is 0.5vvm, the stirring speed is 500rpm, the temperature is 32 ℃, and when the concentration of sugar in the fermentation liquor is detected to be lower than 10g/L in the fermentation process, the sugar is supplemented to maintain the concentration of the sugar at 30 g/L. Culturing for 30-40 h, and finishing fermentation when the sugar concentration in the fermentation solution is lower than 0.2 g/L. The fermentation culture medium and the fermentation container used in the whole fermentation process do not need to be sterilized.

TABLE 14 different fermentation media components and their corresponding cell dry weights, PHA contents and weight average molecular weights

After fermentation, the dry weight of the cells was analyzed by the following method:

(1) taking 50ml of centrifuge tube, drying for two hours at 80 ℃, weighing in a dryer to normal temperature, and counting M0;

(2) accurately measuring 20mL of fermentation liquor, and putting the fermentation liquor into a 50mL centrifuge tube after weighing;

(3) symmetrically placing into a centrifuge according to the weight, and centrifuging at 8000rpm for 5 min;

(4) removing the supernatant, adding 20mL of pure water into the thalli, and performing vortex oscillation until all the thalli are in a suspension state;

(5) centrifuging again at 8000rpm for 5min, and repeating washing for 3 times;

(6) placing the centrifuged thallus into a forced air drying oven at 80 ℃, and drying for 24 h;

(7) taking out and weighing, and measuring as M1;

(8) drying in a forced air drying oven at 80 deg.C for 2 hr, taking out, and weighing as M2;

(9) drying in a forced air drying oven at 80 deg.C for 2 hr, taking out, and weighing as M3;

(10) CDW was calculated with substantially no change in M1, M2, and M3.

The method for extracting PHB in the bacteria comprises the following steps:

taking 30mL of fermentation liquor after fermentation of No. 1-4 culture medium is finished, centrifuging at 8000rpm for 5min, removing supernatant, adding 25mL of deionized water, shaking uniformly, shaking at 8000rpm for 5min, removing supernatant, adding 5mL of deionized water, adding 10mL of trichloromethane, mixing fully, shaking for 60min, centrifuging at 8000rpm for 5min, taking a lower trichloromethane phase, removing trichloromethane by rotary evaporation, and collecting obtained white powder, namely PHB. FTIR analysis was performed, as shown in FIG. 8. The peak of PHB characteristic of 1723-1740 in FIG. 8 is shown to confirm that the strain of the present invention can indeed synthesize PHB.

The detection steps of the PHB content in the strain are as follows:

(1) digestion

(b) And (4) putting the digestion cup into a digestion instrument, wherein the temperature is 100 ℃, and the digestion time is 3-4 h.

(2) Preparing a standard substance:

3HB Standard preparation (PHB polymerized from 3HB monomers): weighing five PHB standard samples, and carrying out sample injection pretreatment according to the PHA digestion step.

(3) Gas chromatography detection:

a detector: FID, column temperature 140 ℃, sample introduction temperature 200 ℃, detector temperature 220 ℃, temperature programming is adopted, 10 ℃/min, temperature is raised to 220 ℃, capillary column SP-2560, and sample introduction amount is 0.5 mu L.

Through the above analysis and detection, the dry weight of the cells fermented using medium 1 was 93.3g/L, and the PHA content was 69.5%. The dry weight of the cells fermented in medium 2 was 112.7g/L, and the PHA content was 62%. The dry weight of the cells fermented in medium 3 was 105.5g/L, and the PHA content was 55.1%. The dry weight of the cells fermented in medium 4 was 198.5g/L, and the PHA content was 75.1%. The results of GC mapping are shown in FIG. 9 (Medium 1), FIG. 10 (Medium 2), FIG. 11 (Medium 3), and FIG. 12 (Medium 4).

Molecular weight detection and analysis method

The instrument comprises the following steps: shimadzu LC 20AD

A detector: RID-10A

The sample pretreatment method comprises the following steps: sealing at 100 deg.C for 15min

Analysis conditions were as follows:

the results of the molecular weight test are shown in FIG. 13 (Medium 1), FIG. 14 (Medium 2), FIG. 15 (Medium 3), and FIG. 16 (Medium 4). The weight average molecular weight of PHB obtained with medium 1 was 104003Da, that of PHB obtained with medium 2 was 90126Da, that of PHB obtained with medium 3 was 121083Da and that of PHB obtained with medium 4 was 83168 Da. The PHA with different molecular weights is required for different purposes, and the PHA with different molecular weights can be prepared by using different culture medium components in the method disclosed by the invention, so that the method is suitable for multiple purposes.

EXAMPLE 3 exploration for PHA production under different culture conditions

This example uses medium 4 from example 2, the same procedure but with only 4 set of comparative experiments on the culture conditions, to verify whether different culture conditions have an effect on the yield of PHB product. The 4 different culture conditions were as follows:

culture conditions 1: pH 8.0, aeration 0.1vvm, temperature 25 deg.C

Culture conditions 2: pH 10, aeration 2vvm, temperature 40 deg.C

Culture conditions 3: pH 8.8, aeration 1vvm, temperature 32 deg.C

Culture conditions 4: pH 9.2, aeration 0.8vvm, temperature 37 deg.C

The gas chromatography results of the products under the culture conditions 1 to 4 are shown in FIG. 17 (Medium 1), FIG. 18 (Medium 2), FIG. 19 (Medium 3), and FIG. 20 (Medium 4). The peak at a retention time of about 3.5min in the figure was 3-hydroxybutyric acid (3HB), and PHB was polymerized from a 3HB monomer. As a result, the peak at a retention time of about 3.5min was higher and the area was larger in FIG. 20. Therefore, PHB with a higher content could be obtained under culture condition 4.

Example 4 detection of undesired bacteria

From the start of the fermentation, the fermentation broth was diluted to 10 at 4-hour intervals ^-4100 mu L of the culture medium is taken and coated on an LB plate (prepared with the same plate solid culture medium), the culture is carried out in an incubator at 37 ℃ for 24 hours, the colony morphology is observed, 60 colonies are randomly selected for PCR verification, and whether the fermentation is infected with bacteria or not under the non-sterilization condition is determined.

The two sequences selected were highly conserved sequences of Halomonas lutescens MDF-9. Wherein, the two pairs of primers are respectively:

F5’-GCACACCAAGTCACATCGTCCAG-3’

r5 '-CGGCGTGTAGCTGGCGAGCTTGG-3' with a fragment length of 486bp

F5’-CAGACTTGACGCGGTCGGCAATT-3’

R5 '-CGCTCCGTTTACGGTAGTGTTGT-3' and the fragment length is 432 bp.

The results of PCR detection are shown in FIGS. 21 and 22. In the figure, A is a sample taken after 12 hours of fermentation, B is a sample taken after 48 hours of fermentation, and 60 samples are taken for PCR verification. M is marker, C is positive control using Halomonas lutescens MDF-9 pure strain as template, and 1-10 are partial sampling samples. The colony morphology and the PCR verification result show that other bands are not amplified except the band of the target fragment, which indicates that the fermentation process is not infected with bacteria, and indicates that the non-sterile fermentation is feasible, so that the energy consumption in the production process is greatly reduced, the cost is reduced, and the fermentation process is simplified.

For halophilic bacteria, high salt concentration is necessary for growth, while the growth of Halomonas lutescens MDF-9 depends on higher pH value, and the high salt and high pH culture medium can inhibit the growth of other non-halophilic bacteria, thus making a non-sterilization production process possible. However, the NaCl concentration of the components of the culture medium disclosed in the prior art is 20-200 g/L, the NaCl concentration of the culture medium disclosed in the invention is only 10-20 g/L, and the culture method is more beneficial to the growth of other non-halophilic bacteria under the condition of lower salt concentration than under the condition of higher salt concentration, but the culture method disclosed in the invention can also realize non-sterile fermentation, reduce the energy consumption generated in the sterilization process, and furthest reduce the damage to the microbial culture medium, thereby reducing the cost to a greater extent. The low salt concentration also leads to simpler post-treatment of wastewater and can save the production cost.

The culture medium has simple components, does not contain cobalt chloride and nickel chloride, and can be sensitized by the inhalation of the cobalt chloride and the nickel chloride and the contact of skin. Has extremely high toxicity to aquatic organisms and may have long-term adverse effects on the water environment. Therefore, the culture medium without cobalt chloride and nickel chloride is safer and more environment-friendly.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

SEQUENCE LISTING

<110> Zhuhaimaide Producer science and technology Co., Ltd

<120> one strain of halomonas and application thereof

<130> 20210813

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1422

<212> DNA

<213> Halomonas lutescens

<400> 1

ggtctaccat gcagtcgagc ggtagcacag agaagcttgc ttcttgggtg acgagcggcg 60

gacgggtgag taatgcatag gaatctgccc gatagtgggg gataacctgg ggaaacccag 120

gctaataccg catacgtcct acgggagaaa gggggcttcg gctcccgcta ttggatgagc 180

ctatgtcgga ttagctagtt ggtgaggtaa aggctcacca aggcgacgat ccgtagctgg 240

tctgagagga tgatcagcca catcgggact gagacacggc ccgaactcct acgggaggca 300

gcagtgggga atattggaca atgggcgcaa gcctgatcca gccatgccgc gtgtgtgaag 360

aaggccttcg ggttgtaaag cactttcagc gaggaagaac gcctagcggt taatacccgc 420

taggaaagac atcactcgca gaagaagcac cggctaactc cgtgccagca gccgcggtaa 480

tacggagggt gcaagcgtta atcggaatta ctgggcgtaa agcgcgcgta ggtggcttga 540

taagccggtt gtgaaagccc cgggctcaac ctgggaacgg catccggaac tgtcaagcta 600

gagtgcagga gaggaaggta gaattcccgg tgtagcggtg aaatgcgtag agatcgggag 660

gaataccagt ggcgaaggcg gccttctgga ctgacactga cactgaggtg cgaaagcgtg 720

ggtagcaaac aggattagat accctggtag tccacgccgt aaacgatgtc gaccagccgt 780

tgggtgccta gcgcactttg tggcgaagtt aacgcgataa gtcgaccgcc tggggagtac 840

ggccgcaagg ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg 900

gtttaattcg atgcaacgcg aagaacctta cctactcttg acatcctgcg aacttgtgag 960

agatcacttg gtgccttcgg gaacgcagag acaggtgctg catggctgtc gtcagctcgt 1020

gttgtgaaat gttgggttaa gtcccgtaac gagcgcaacc cttgtcctta tttgccagca 1080

cgtaatggtg ggaactctaa ggagactgcc ggtgacaaac cggaggaagg tggggacgac 1140

gtcaagtcat catggccctt acgagtaggg ctacacacgt gctacaatgg ccggtacaaa 1200

gggttgccaa ctcgcgagag tgagctaatc ccgaaaagcc ggtctcagtc cggatcggag 1260

tctgcaactc gactccgtga agtcggaatc gctagtaatc gtgaatcaga atgtcacggt 1320

gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgggagtgg actgcaccag 1380

aagtggttag cctaacgcaa gagggcgatc accacggttg tt 1422

Claims

1. Halomonas lutescens MDF-9, wherein the preservation number of the Halomonas lutescens is GDMCC NO: 61850, and the 16S rDNA sequence of the Halomonas luteus is shown in SEQ ID NO. 1.

2. The use of Halomonas purorum according to claim 1 in the preparation of poly-beta-hydroxybutyrate.

3. A method of preparing a poly β -hydroxybutyrate, comprising the steps of:

fermenting Halomonas nivalens according to claim 1 to obtain poly-beta-hydroxybutyrate, wherein a fermentation medium and a fermentation vessel used in the fermentation are not sterilized; the concentration of sodium chloride in the fermentation medium is 10-20 g/L; the fermentation medium does not contain cobalt chloride and nickel chloride.

4. The method according to claim 3, wherein the pH value of the fermentation is 8 to 10, the stirring speed is 50 to 800rpm, the aeration rate is 0.1 to 2vvm, and the fermentation temperature is 25 to 40 ℃.

5. A method according to any one of claims 3 to 4, wherein the fermentation method comprises the steps of:

(1) inoculating the Halomonas muddy flesh into a fermentation culture medium for fermentation culture;

(2) when the concentration of sugar in fermentation liquor is detected to be lower than 10g/L in the fermentation process, adding sugar to maintain the concentration at 30 g/L;

(3) and (5) continuing culturing for 30-40 h, and ending fermentation when the sugar concentration in the fermentation liquid is lower than 0.2 g/L.

6. The method of claim 5, wherein the fermentation medium consists of:

carbon source: any one or more of glucose, fructose and sodium gluconate;

sodium chloride.

7. The method according to claim 6, wherein the total concentration of the carbon source is 90-150 g/L;

the content of the corn steep liquor dry powder is 1-8%, the content of the yeast powder is 0.5-5%, the content of the tryptone is 0.1-3%, the content of the urea is 0.1-3%, the content of the ammonium sulfate is 0.1-3%, and the content of the ammonium chloride is 0.1-3%;

the content of the monopotassium phosphate is 0.1-2%, the content of the disodium hydrogen phosphate is 0.05-1%, the content of the magnesium sulfate is 0.05-1%, and the content is a mass-volume ratio.