KR-20260064928-A - Culturing method for enhancing the growth of methanotroph

Abstract

The present invention relates to a culture method for promoting the growth of methanotrophic bacteria. By adding phosphate, copper salt, and molybdate, which were identified as major factors for the growth of methanotrophic bacteria through RSM experiments, to the culture medium components at optimal concentrations, the cell growth of methanotrophic bacteria can be promoted, thereby allowing methanotrophic bacteria to be cultured at high concentrations, and a large amount of PHB derived from methanotrophic bacteria can be obtained therefrom.

Inventors

• 김민식
• 민경선
• 이지예
• 조재환
• 박권우
• 문명훈
• 김선정
• 조영정
• 강응수
• 이수연
• 이준표

Assignees

• 한국에너지기술연구원

Dates

Publication Date

20260508

Application Date

20241030

Claims (10)

1. Methanogenic bacteria phosphate, copper salt and A culture method for promoting the growth of methanotrophic bacteria comprising the step of culturing in a medium containing molybdate.
2. A culture method according to claim 1, wherein the methanotrophic bacterium is Methylocystis parvus OBBP.
3. A culture method according to claim 1, wherein the phosphate is KH₂PO₄ or K₂HPO₄ .
4. A culture method according to claim 1, wherein the copper salt is copper sulfate pentahydrate ( CuSO₄ · 5H₂O ).
5. A culture method according to claim 1, wherein the molybdate is sodium molybdate dihydrate (Na₂MoO₄·2H₂O ) .
6. A culture method according to claim 1, wherein the medium contains 30 nM to 40 nM of phosphate.
7. A culture method according to claim 1, wherein the medium contains 70 μM to 80 μM of copper salt.
8. A culture method according to claim 1, wherein the medium comprises 5 μM to 15 μM of molybdate.
9. A culture method according to claim 1, wherein the method involves growing the methanotrophic bacteria until the concentration of the methanotrophic bacteria reaches an OD 600 of 4 to 6.
10. A method for producing microbial-derived PHB (polyhydroxybutyrate) using the culture method of claim 1.

Description

Culturing method for enhancing the growth of methanotrophs The present invention relates to a culture method for promoting the growth of methanotrophic bacteria. Methanogenic bacteria are bacteria capable of utilizing methane as their sole carbon and energy source in aerobic environments, and research utilizing this ability to convert methane gas into methanol or various high-value chemicals has recently been gaining attention. However, due to the slow growth rate of methanotrophic bacteria and limitations in genetic engineering technology, research on the production of useful chemicals through the genetic modification of methanotrophic bacteria remains in its early stages. Although major metabolic pathways such as the RuMP cycle and serine cycle have been identified through research on the metabolic processes of methanotrophic bacteria, the understanding of their physiological characteristics and metabolic regulation remains insufficient. Furthermore, there is a difficulty in that the methods for genetic manipulation, which are essential for the metabolic engineering improvement of methanotrophic bacteria, are significantly lacking compared to existing commercial strains, including E. coli. Additionally, the low growth rate of methanotrophic bacteria due to limited mass transfer of methane substrates is another factor hindering commercialization. Methane is a major component of natural gas and shale gas with confirmed reserves exceeding 187.4 trillion m³ (1,687 TOE), making the production of high-value products through biological conversion essential. To achieve this, many technical issues related to the research of methanotrophic bacteria must be resolved. Figure 1 shows the media components and concentrations used in the Plackett-Burman design. Figure 2 shows the Plackett-Burman design. Figure 3 shows the change in the growth curve of methanotrophic bacteria according to PBD conditions. Figure 4 shows the analysis of the PBD experiment results. Figure 5 shows the experimental design configuration using the Box-Behnken design technique. Figure 6 shows the change in the growth curve of methanotrophic bacteria according to RSM experimental conditions. Figure 7 is a reaction surface diagram for deriving RSM results for phosphate, CuSO₄ , and Na₂MoO₄ . Figure 8 shows the optimal composition for the growth of methanogenic bacteria. Figure 9 shows the growth and PHB production patterns of methanotrophic bacteria in the optimal RSM medium. The present invention will be described in detail below. The present invention relates to a culture method for promoting the growth of methanotrophic bacteria. The method of the present invention comprises the step of culturing methanotrophic bacteria in a medium containing phosphate, copper salt, and molybdate. The above-mentioned methanotrophic bacteria are genus Methylocystis, genus Methylomonas, genus Methylobacter, genus Methylococcus, genus Methylomicrobium, genus Methylosphaera, genus Methyllocaldum, genus Methyloglobus, genus Methylosarcina, genus Methyloprofundus, genus Methylothermus, genus Methylohalobius, genus Methylogaea, genus Methylomarinum, genus Methylovulum, genus Methylomarinovum, It may be a strain belonging to the genus Methylorubrum, Methyloparacoccus, Methylosinus, Methylocella, Methylocapsa, Methylofurula, Methylacidiphilum, or Methylacidimicrobium, for example, it may be a strain of the genus Methylocystis, and more specifically, it may be Methylocystis parvus OBBP. The phosphate salt in the above medium may be, for example, KH₂PO₄ or K₂HPO₄ , and the phosphate may be included in an amount of 30 nM to 40 nM , preferably 32 nM to 38 nM, more preferably 35 nM. The copper salt in the above medium may be, for example, copper sulfate pentahydrate ( CuSO₄ · 5H₂O ), and the copper salt may be included in an amount of 70 μM to 80 μM, preferably 74 μM to 78 μM, and more preferably 76 μM. The molybdate in the above medium may be, for example, sodium molybdate dihydrate ( Na₂MoO₄ · 2H₂O ), and the molybdate may be included in an amount of 5 μM to 15 μM, preferably 7 μM to 10 μM, more preferably 8 μM to 9 μM. The above medium may further include MgSO₄ , CaCl₂ · 2H₂O , FeEDTA, NaHCO₃ , or Trace metal solution as needed. The above culture may be carried out using means known in the art, such as methods and conditions. For example, W1 medium may be used as the base medium, and phosphate, copper salt, and molybdate may be added to achieve the optimal concentration. Additionally, it may be carried out while supplying oxygen and methane gas under a temperature of 30°C. The method of the present invention may involve growing the methanotrophic bacteria until the OD 600 concentration reaches 4 to 6. More specifically, based on an OD 600 after 96 hours of culture, an increase in the concentration of methanotrophic bacteria of 200% or more may be observed compared to using a conventional medium (W1 medium). The present invention relates to a method for producing PHB derive