Method for producing PHB using high-density culture of thermophilic Cardimonas thermodepolymerans

Abstract

The present invention relates to a method for producing PHB using high-concentration culture of the thermophilic Caldimonas thermodepolymeranth. The present invention confirmed that the thermophilic bacterium Caldimonas thermodepolymeranth strain was cultured at a high concentration, and that by restricting the nitrogen source during the static period after high-concentration culture, the C / N ratio was increased, thereby increasing the accumulation of PHB within the high-concentration cultured cells and increasing the PHB production yield.

Description

Technology Field

[0001] The present invention relates to a method for producing PHB using high-concentration culture of the thermophilic Caldimonas thermodepolymeran. Background Technology

[0002] Plastics are widely used in everything from industrial materials to disposable consumables due to their excellent lightweight, processability, and cost-effectiveness, as well as their superior durability, which includes being heat-stable, rust-resistant, and resistant to decomposition by light or moisture. However, since most plastics currently in use exist semi-permanently in the natural environment without decomposing, there is an urgent need to address the disposal of discarded plastics. As an alternative to these petrochemical-based plastics, research on biodegradable bioplastics is actively underway, focusing on biodegradable polyhydroxyalkanoates (PHA, etc.) derived from biomass using microorganisms.

[0003] Polyhydroxyalkanoates are recombinant E. coli ( E. coli ) or Lastonia utropha ( Ralstonia eutropha It is a biodegradable polyester industrially produced by ). According to recent research Methylocystis hirsutaAlternatively, the production of polyhydroxyalkanoates from methane or CO2 using cyanobacteria has been reported. Poly(3-hydroxybutyrate, PHB) is the most studied biopolyester among many types of microorganisms and possesses biocompatibility and biodegradability, making it suitable for various applications in the biomedical field. However, the utilization of PHAs containing PHB is currently limited due to high production costs. The high production cost of PHAs is attributed to low carbon conversion efficiency, and to reduce production costs, strategies such as 1) production of PHAs using alternative carbon sources, 2) regulation of PHA synthesis pathways, and 3) simplification of the fermentation process are being studied.

[0004] Meanwhile, thermophilic microorganisms typically refer to strains that grow optimally at temperatures of 45°C or higher. High-temperature processes utilizing thermophilic strains offer many industrial advantages, such as preventing contamination by mesophilic microorganisms, promoting the diffusion of organic compounds, increasing reaction rates, and achieving high thermal energy efficiency due to the reduced need for cooling. One of the thermophilic strains, *Caldimonas thermodepolymerance* ( Caldimonas thermodepolymerans It is a thermophilic bacterium that grows optimally at 50-55°C, isolated from activated sludge in Egypt in 2003, utilizing its degradation activity against polymers with theoester bonds such as 3-hydroxybutyrate and 3-mercaptopropionate copolymers. C. thermodepolymerans It possesses not only polyester degradation activity but also the ability to synthesize PHB. This strain is capable of growth and PHB synthesis using various carbon sources as substrates, and it exhibits the characteristic of preferentially metabolizing xylose over glucose and utilizing it with higher efficiency. Although extensive research on high-concentration cell culture has been conducted over the past few decades, studies utilizing thermophilic strains remain limited, and C. thermodepolymerans There is no research utilizing it.

[0005] Accordingly, the inventors established optimal conditions for culturing the thermophilic bacterium *Caldimonas thermodepolymeranus* at a high concentration and confirmed the PHB yield, thereby confirming that cultivation at high temperatures is possible without a separate sterilization process and that the energy used for cooling the fermenter can be minimized, thus completing the present invention. The problem to be solved

[0006] The objective of the present invention is to provide a step of inoculating a Caldimonas thermodepolymerans strain into a culture medium;

[0007] A step of culturing by adding a supply solution to a medium inoculated with the above-mentioned Caldimonas thermodepolymerance strain; and

[0008] The present invention provides a method for high-concentration cell culture of a Caldimonas thermodepolymeranus strain, comprising the step of continuously culturing by increasing the amount of feed solution added when the biomass of the cultured strain exceeds a reference value.

[0009] Another objective of the present invention is the step of inoculating a strain of *Caldimonas thermodepolymeranus* into a culture medium;

[0010] A step of culturing the above strain until the early exponential phase;

[0011] A step of adding a supply solution to the strain cultured up to the initial exponential growth phase and culturing it up to the exponential phase;

[0012] A step of culturing the strain cultured up to the logarithmic growth phase by increasing the amount of supply solution added until the stationary phase;

[0013] The present invention provides a method for producing PHB using a Caldimonas thermodepolymeranus strain, comprising the step of reducing the amount of added supply solution and restricting the nitrogen source in the strain cultured up to the above static period. means of solving the problem

[0014] To achieve the above objective, the present invention comprises the step of inoculating a Caldimonas thermodepolymerans strain into a culture medium;

[0015] A step of culturing by adding a supply solution to a medium inoculated with the above-mentioned Caldimonas thermodepolymerance strain; and

[0016] A method for high-concentration cell culture of a Caldimonas thermodepolymeranus strain is provided, comprising the step of continuously culturing by increasing the amount of feed solution added when the biomass of the cultured strain is above a reference value.

[0017] In addition, the present invention comprises the step of inoculating a strain of *Caldimonas thermodepolymeranus* into a culture medium;

[0018] A step of culturing the above strain until the early exponential phase;

[0019] A step of adding a supply solution to the strain cultured up to the initial exponential growth phase and culturing it up to the exponential phase;

[0020] A step of culturing the strain cultured up to the logarithmic growth phase by increasing the amount of supply solution added until the stationary phase;

[0021] A method for producing PHB using a Caldimonas thermodepolymeranus strain is provided, comprising the step of culturing the strain cultured up to the above static period by reducing the amount of added supply solution and restricting the nitrogen source. Effects of the invention

[0022] The present invention confirmed that by culturing the thermophilic bacterium *Caldimonas thermodepolymerance* strain at a high concentration and restricting the nitrogen source during the static period after high concentration cultivation, the C / N ratio is increased, thereby increasing the accumulation of PHB within the high-concentration cultured cells and increasing the PHB production yield, which can be usefully utilized in related industries. Brief explanation of the drawing

[0023] Figure 1 is a figure showing the culture conditions, nutrients, and strain growth according to culture time in high-concentration culture of the Caldimonas thermodepolymeranse strain of the present invention. Figure 2 is a figure showing the PHB content and growth ratio according to culture time in high-concentration culture of the Caldimonas thermodepolymerance strain of the present invention. Figure 3 is a figure showing the culture conditions, nutrient source, and strain growth according to culture time in the PHB accumulation culture of the Caldimonas thermodepolymerance strain of the present invention. Figure 4 is a figure showing the PHB content, carbon source, nitrogen source, and growth ratio according to culture time in the PHB accumulation culture of the Caldimonas thermodepolymerance strain of the present invention. Specific details for implementing the invention

[0024] Embodiments of the present invention will be described in detail below with reference to the attached drawings. In the following description, detailed descriptions of technologies well known to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such descriptions would unnecessarily obscure the essence of the present invention. Additionally, the terminology used in this specification is used to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs.

[0025] Therefore, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0026] The present invention comprises the step of inoculating a Caldimonas thermodepolymerans strain into a culture medium;

[0027] A step of culturing by adding a supply solution to a medium inoculated with the above-mentioned Caldimonas thermodepolymerance strain; and

[0028] A method for high-concentration cell culture of a Caldimonas thermodepolymeranus strain is provided, comprising the step of continuously culturing by increasing the amount of feed solution added when the biomass of the cultured strain is above a reference value.

[0029] According to one embodiment of the present invention, the medium may be a mineral salt medium (mineral salt media, MSM).

[0030] According to one embodiment of the present invention, the feed solution may comprise 500 g / L xylose, 5 g / L Fe(III)NH4-Citrate, 5 g / L MgSO4, 2.5 g CaCl2, and 25 mL / L microelement solution.

[0031] According to one embodiment of the present invention, the trace element solution may comprise 50 g / L EDTA, 13.8 g / L FeCl3·6H2O, 0.84 g / L ZnCl2, 0.13 g / L CuCl2·2H2O, 0.1 g / L CoCl2·6H2O, 0.016 g / L MnCl2·6H2O, and 0.1 g / L H3BO3.

[0032] According to one embodiment of the present invention, the culture may be performed under conditions where the dissolved oxygen (DO) setting value is 25 to 35%.

[0033] According to one embodiment of the present invention, the biomass reference value may be a dry cell weight (DCW) of 5 to 6 g / L.

[0034] According to one embodiment of the present invention, the supply solution may be supplied at a rate of 15 to 17 ml / h when the biomass is below the biomass reference value.

[0035] According to one embodiment of the present invention, increasing the amount of the supply solution added may be done at a rate of 32 ml / h.

[0036] According to one embodiment of the present invention, increasing the amount of the supply solution added may involve adding an additional 75 to 85 ml of xylose solution, and the xylose solution may be a solution with a concentration of 495 to 505 g / L.

[0038] In addition, the present invention comprises the step of inoculating a strain of *Caldimonas thermodepolymeranus* into a culture medium;

[0039] A step of culturing the above strain until the early exponential phase;

[0040] A step of adding a supply solution to the strain cultured up to the initial exponential growth phase and culturing it up to the exponential phase;

[0041] A step of culturing the strain cultured up to the logarithmic growth phase by increasing the amount of supply solution added until the stationary phase;

[0042] A method for producing PHB using a Caldimonas thermodepolymeranus strain is provided, comprising the step of culturing the strain cultured up to the above static period by reducing the amount of added supply solution and restricting the nitrogen source.

[0043] The term “early exponential phase” used in the present invention refers to the early part of the exponential phase in the growth curve of microorganisms, signifying the point in time when the rate of cell division and proliferation increases. In one embodiment of the present invention, the range of the early exponential phase is defined from the point in time when biomass reaches 0.5 g DCW / L to the point in time when biomass reaches 9 g DCW / L, but is not limited thereto.

[0044] The term “exponential phase” used in the present invention refers to the section of the microorganism growth curve where the cells of the microorganism divide and proliferate at a maximum rate, signifying a period of balanced growth where the rate of increase in the number of microorganism cells and the rate of increase in the total mass of the microorganism cells are equal. In one embodiment of the present invention, it is defined as the period from when the biomass reaches 9 g DCW / L to when it reaches 50 g DCW / L, but is not limited thereto.

[0045] The term “stationary phase” as used in the present invention refers to a period in the growth curve of microorganisms where there is no increase or decrease in the total number of cells, and where logarithmic growth is reduced due to factors that limit population growth, such as depletion of nutrients, accumulation of growth-inhibiting substances, or lack of space. In one embodiment of the present invention, it is defined as the period after the point where the biomass reaches 50 g DCW / L, but is not limited thereto.

[0046] According to one embodiment of the present invention, the initial logarithmic growth phase may have a dry cell weight (DCW) of 0.4 to 0.6 g / L to 8.5 to 9.5 g / L.

[0047] According to one embodiment of the present invention, adding a feed solution to the strain cultured up to the initial logarithmic growth stage may be done by adding the feed solution at a rate of 15 to 17 ml / h.

[0048] According to one embodiment of the present invention, the logarithmic growth group may have a cell dry weight of 8.5 to 9.5 g / L to 49 to 51 g / L.

[0049] According to one embodiment of the present invention, increasing the amount of the supply solution added may be adding the supply solution at a rate of 47 to 49 ml / h.

[0050] According to one embodiment of the present invention, the cell dry weight may exceed 49 to 51 g / L.

[0051] According to one embodiment of the present invention, reducing the amount of the supply solution added may be done sequentially to conditions of 39 to 41, 31 to 33, and 15 to 17 ml / h.

[0052] According to one embodiment of the present invention, limiting the nitrogen source may involve substituting the nitrogen source, and substituting the nitrogen source may involve increasing the carbon nitrogen ratio (C / N ratio).

[0053] According to one embodiment of the present invention, the nitrogen source may include ammonia, and the ammonia solution may be substituted with sodium hydroxide (NaOH).

[0054] According to one embodiment of the present invention, the PHB may be produced in an amount of 31 to 32 g / L.

[0056] The present invention will be explained in more detail below through examples. These examples are merely for the purpose of explaining the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0058] <Example 1> Preparation for the culture of *Caldimonas thermodepolymerans*

[0059] The thermophilic strain of the present invention, *Caldimonas thermodepolymerans* (German Biological Resource Center, DSMA 15344), was cultured. Specifically, the *Caldimonas thermodepolymerans* strain was inoculated onto agar mineral salt media (MSM) containing 20 g / L of xylose as a carbon source and cultured for 24 hours. Subsequently, the cultured colony was streaked and cultured for 24 hours, and the incubation process after streaking was repeated once. Afterward, a single cultured colony was inoculated into a 500 ml flask containing 200 ml of MSM liquid medium and cultured at 50°C and 200 rpm. Subculturing was performed a total of two times at 12-hour intervals after culture. A cultured Caldimonas thermodepolymerance strain was mixed with a 40% (w / v) glycerol solution in a 1:1 volume ratio and stored at -80℃ to prepare a glycerol stock.

[0060] In addition, for high-concentration culture in a bioreactor, culture was performed in an MSM liquid medium containing 20 g / L xylose as a carbon source. The feed solution contained 500 g / L xylose, 5 g / L Fe(III)NH4-Citrate, 5 g / L MgSO4, 2.5 g CaCl2, and 25 mL / L microelement solution. The specific composition of the MSM medium and microelement solution is shown below.

[0061] - mineral salt media: 9 g / L of Na2HPO4·12H2O, 1.5 g / L of KH2PO4, 1.0 g / L of NH4Cl, 0.2 g / L of MgSO4·7H2O, 0.02 g / L of CaCl2·2H2O, 0.0012 g / L of Fe(III-C) and 5 g / L of NH extract

[0062] - trace element solution: 50 g / L EDTA, 13.8 g / L FeCl3·6H2O, 0.84 g / L ZnCl2, 0.13 g / L CuCl2·2H2O, 0.1 g / L CoCl2·6H2O, 0.016 g / L MnCl2·6H2O, 0.016 g / L H

[0064] <Example 2> High-concentration culture of *Caldimonas thermodepolymeranus*

[0065] For high-concentration cell culture, nutrients must be continuously supplied while maintaining the substrate concentration within the reactor; furthermore, as oxygen consumption increases with increasing cell concentration, the dissolved oxygen level in the medium must also be kept constant. Accordingly, to culture the *Caldimonas thermodepolymeranus* strain at a high concentration, 2 L of MSM was added to a 5 L biofermenter (BioCNS, Korea) equipped with a microsparger and a 6-blade impeller, and the *Caldimonas thermodepolymeranus* strain was cultured at 50°C. Additionally, to maintain a constant oxygen concentration, the dissolved oxygen (DO) setting was set to 30%. The feed solution was continuously introduced into the culture medium using a peristaltic pump at a constant flow rate in each section. The pH was set to 7.0 and adjusted using 2M hydrochloric acid solution (HCl) and 5N ammonia solution (NH4OH), with the 5N ammonia solution used for pH adjustment and as a nitrogen source. As cell growth increased, the amount of oxygen consumed increased, causing the DO to decrease; therefore, to maintain the DO set value, the impeller stirring speed was adjusted to 300 to 1400 rpm. When the stirring speed reached the maximum value, the flow rate of the supplied air was adjusted to 1 to 4.8 L / min using mass flow control (Brooks, Hatfield, PA).

[0066] The supply of the feed solution was carried out in two stages; it was supplied at 16 ml / h from the start of culture until the biomass (dry cell weight, DCW) reached 5.5 DCW g / L, and additionally, 80 ml of xylose solution at a concentration of 500 g / L was added through the sample inlet, after which the supply rate was adjusted to 32 ml / h. The culture results are shown in Table 1.

[0067] Maximum specific growth rate (h -1 ) Maximum PHB content (wt%) Maximum biomass (g / L) Maximum production PHB (g / L) Maximum volumetric productivity (g / L / h) 0.50 21.3 50.2 9.6 0.36

[0069] As a result, as shown in Figures 1 and 2 and Table 1, the maximum specific growth rate was at culture 0.50 h -1 It was confirmed that the maximum biomass was 50.2 g / L. The PHB content in the biomass was 19.2 wt%, and the produced PHB was 9.6 g / L. The maximum PHB content was 21.3 wt%, and the maximum volumetric productivity was 0.36 g / L / h.

[0071] <Example 3> Induction of PHB accumulation through control of substrate and nitrogen supply

[0072] PHA containing PHB accumulates in the form of granules within the cell as a carbon and energy storage source when carbon is oversupplied and nutrients such as nitrogen, phosphorus, and sulfur are deficient. Accordingly, high-concentration cell growth was induced and the nitrogen source was removed to induce the accumulation of PHB. Specifically, the DO setting value and air flow rate were the same as the conditions of Example 2 above, and the stirring speed was adjusted to 300 to 1250 rpm. The concentration of residual xylose in the medium was checked at each sampling time using high-performance liquid chromatography to calculate the substrate consumption rate, and based on this, the feed solution was supplied at 16 ml / h during the early exponential phase (0.5 g DCW / L), 48 ml / h during the exponential phase (9–18 g DCW / L), and sequentially lowered to 40, 32, and 16 ml / h during the stationary phase (50 g DCW / L) according to the growth stage to prevent the concentration of xylose in the medium from being depleted or excessively accumulated.

[0073] Upon reaching the static stage, the supply of additional nitrogen sources was restricted by replacing the 5N ammonia solution, which serves as an alkaline agent for pH adjustment and an additional nitrogen source, with 5N sodium hydroxide. The culture results are shown in Table 2.

[0074] Maximum specific growth rate (h -1 ) Maximum PHB content (wt%) Maximum biomass (g / L) Maximum production PHB (g / L) Maximum volumetric productivity (g / L / h) 0.57 51.6 63.1 31.9 1.30

[0076] As a result, as shown in Figures 3, 4 and Table 2, the maximum specific growth rate was 0.57 h -1 It was confirmed that after nitrogen source restriction, the carbon-to-nitrogen ratio (C / N ratio) increased from 57.4 to 1931, and the PHB content increased to a maximum of 51.6 wt%. The maximum biomass was 63.1 g / L, of which the PHB content was 50.6 wt%, the maximum PHB production was 31.9 g / L, and the maximum volumetric productivity was 1.30 g / L / h.

[0078] Accordingly, the present invention confirmed that by culturing the thermophilic bacterium *Caldimonas thermodepolymerance* strain at a high concentration and restricting the nitrogen source during the static period after high concentration cultivation, the C / N ratio was increased, thereby increasing the accumulation of PHB in the cells cultured at a high concentration and increasing the PHB production yield.

Claims

Claim 1 A method for high-concentration cell culture of a Caldimonas thermodepolymerans strain, comprising: a step of inoculating a Caldimonas thermodepolymerans strain into a culture medium; a step of adding a feed solution to the culture medium inoculated with the Caldimonas thermodepolymerans strain and culturing; and a step of continuously culturing by increasing the amount of feed solution added when the biomass of the cultured strain is above a reference value. Claim 2 A method according to claim 1, wherein the medium is a mineral salt medium (mineral salt media, MSM). Claim 3 A method according to claim 1, wherein the feed solution comprises 500 g / L xylose, 5 g / L Fe(III)NH4-Citrate, 5 g / L MgSO4, 2.5 g CaCl2, and 25 mL / L microelement solution. Claim 4 The method according to claim 3, wherein the trace element solution comprises 50 g / L EDTA, 13.8 g / L FeCl3·6H2O, 0.84 g / L ZnCl2, 0.13 g / L CuCl2·2H2O, 0.1 g / L CoCl2·6H2O, 0.016 g / L MnCl2·6H2O, and 0.1 g / L H3BO3. Claim 5 A method according to claim 1, wherein the culture is performed under conditions where the dissolved oxygen (DO) setting value is 25 to 35%. Claim 6 A method according to claim 1, wherein the biomass reference value is a dry cell weight (DCW) of 5 to 6 g / L. Claim 7 A method according to claim 1, wherein the supply solution is supplied at a rate of 15 to 17 ml / h when the biomass is below the biomass reference value. Claim 8 In claim 1, the method of increasing the amount of the supply solution added is to add it at a rate of 32 ml / h. Claim 9 A method according to claim 1, wherein increasing the amount of the supply solution added is to add an additional 75 to 85 ml of xylose solution. Claim 10 In claim 9, the method wherein the xylose solution is a solution with a concentration of 495 to 505 g / L. Claim 11 A method for producing PHB using a Caldimonas thermodepolymeranus strain, comprising: a step of inoculating a Caldimonas thermodepolymeranus strain into a culture medium; a step of culturing the strain to an early exponential phase; a step of adding a feed solution to the strain cultured to the early exponential phase and culturing it to an exponential phase; a step of increasing the amount of feed solution added to the strain cultured to the exponential phase and culturing it to a stationary phase; and a step of decreasing the amount of feed solution added to the strain cultured to the stationary phase and culturing it while restricting the nitrogen source. Claim 12 In claim 11, the method wherein the initial logarithmic growth phase has a dry cell weight (DCW) of 0.4 to 0.6 g / L to 8.5 to 9.5 g / L. Claim 13 In claim 11, the method of adding a feed solution to the strain cultured up to the initial logarithmic growth stage is to add the feed solution at a rate of 15 to 17 ml / h. Claim 14 In claim 11, the method wherein the logarithmic growth group has a cell dry weight of 8.5 to 9.5 g / L to 49 to 51 g / L. Claim 15 In claim 11, the method of increasing the amount of the feed solution added is to add the feed solution at a rate of 47 to 49 ml / h. Claim 16 In claim 11, the method wherein the above-mentioned settling apparatus has a cell dry weight exceeding 49 to 51 g / L. Claim 17 A method according to claim 11, wherein reducing the amount of the supply solution added is sequentially reduced to conditions of 39 to 41, 31 to 33, and 15 to 17 ml / h. Claim 18 In Clause 11, the method of limiting the above nitrogen source is to substitute the nitrogen source. Claim 19 In paragraph 18, the method of replacing the above nitrogen source is to increase the carbon nitrogen ratio (C / N ratio). Claim 20 In paragraph 18, the method wherein the nitrogen source comprises ammonia. Claim 21 In claim 20, the above nitrogen source is replaced with sodium hydroxide (NaOH). Claim 22 In claim 11, the method wherein the PHB is produced at 31 to 32 g / L.

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