Characterization of conventional microbial culture methods and development of methods for isolating and culturing of uncultured microbes

• Project/Area Number: 19H03086
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 41050:Environmental agriculture-related
• Research Institution: University of Tsukuba
• Principal Investigator: Aoyagi Hideki 筑波大学, 生命環境系, 教授 (00251025)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000); Fiscal Year 2021: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2020: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2019: ¥7,150,000 (Direct Cost: ¥5,500,000、Indirect Cost: ¥1,650,000)
• Keywords: 微生物培養法 / 寒天平板培地 / フラスコ振盪培養 / ダークマター微生物 / 未培養微生物 / 培養用シート / 前処理 / ナノ粒子

Outline of Research at the Start

従来の微生物培養法では、自然界に存在する微生物の1%程度しか培養できない事が明らかとなり、限界が指摘されている。残された99%の未培養微生物（Dark matter 微生物）の活用を目指し、環境ゲノム解析が盛んに行われているが、塩基配列だけでは分からない未知の機能の解明や実利用を行う場合、未培養微生物の単離培養が必須となり、従来の微生物培養法の問題点を排除した新規培養法の開発が求められている。本研究では、申請者が微生物のスクリーニング実験の過程で偶然、見出した新奇現象を解析し、解析結果から新たな方法論（培養条件や培養法）を提案し、多様な未培養微生物の培養化とライブラリー化をおこなう。

Outline of Final Research Achievements

The characteristics of representative culture methods for microbes (shake flask culture method and the agar plate culture method) were analyzed. The behavior of the gas and liquid phases during shake flask culture of microbes were analyzed by using originally constructed systems. The cultured microbes were affected by various factors associated with the sampling operation. The analysis of the agar plate culture method elucidated the changes in various factors and characteristics that occur in the plate culture medium, and the problems associated with the conventional method were clarified.

Academic Significance and Societal Importance of the Research Achievements

自然界に存在する微生物の1%程度しか培養できない”という課題は、様々な微生物関連分野で共通の問題である。本研究で得られた成果は、従来の微生物培養法の特性を解析、把握することで得られる新たな知見をベースに、多様な未培養微生物の培養化に貢献できる方法論を提案しており国内外の大学、研究機関や微生物関連産業で広く使用でき、学術・産業面のイノベーション創出に貢献できる。

Research Products

• [Journal Article] 振盪培養中の間欠的な培養栓の開封やフラスコ気相部への通気が培養微生物群集構造に及ぼす影響 (2020)
  • Author(s): 高橋 将人、青柳 秀紀
  • Journal Title: 生物工学会誌 Volume: 98 Pages: 71-71
  • Open Access
• [Journal Article] Biosynthesis of silver nanoparticles mediated by extracellular pigment from Talaromyces purpurogenus and their biomedical applications. (2019)
  • Author(s): S. Bhatnagar, T. Kobori, D. Ganesh, K. Ogawa, H. Aoyagi
  • Journal Title: Nanomaterials Volume: 9 Issue: 7 Pages: 1042-1042
  • DOI: 10.3390/nano9071042
  • NAID: 120007133017
  • Peer Reviewed / Open Access
• [Journal Article] 従来の振盪フラスコ培養法の解析と利用 (2019)
  • Author(s): 高橋将人、青柳秀紀
  • Journal Title: 生物工学会誌 Volume: 97 Pages: 559-562
  • Open Access
• [Journal Article] 特集 未培養微生物(微生物ダークマター)の 培養，解析，利用に関する研究開発の最前線と展望 (2019)
  • Author(s): 青柳秀紀、青井議輝
  • Journal Title: 生物工学会誌 Volume: 97 Pages: 546-546
  • Open Access
• [Journal Article] A High Throughput Isolation Method for Phosphate-Accumulating Organisms (2019)
  • Author(s): A. Anand, H. Aoyagi
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 18083-18083
  • DOI: 10.1038/s41598-019-53429-2
  • NAID: 120007132934
  • Peer Reviewed / Open Access
• [Presentation] 微生物の振盪フラスコ培養中における培養栓の換気能評価とその応用 (2019)
  • Author(s): 高橋将人、青柳秀紀
  • Organizer: 第71回日本生物工学会大会
• [Presentation] pH 変動培養による有用金属回収微生物の新規スクリーニング法の開発 (2019)
  • Author(s): 高野力、村山晃一、青柳秀紀
  • Organizer: 第71回日本生物工学会大会
• [Presentation] セルロースメンブランを用いた植物生育促進細菌の評価・培養法の開発 (2019)
  • Author(s): 大矢裕之、藤原優人、中村真由、青柳秀紀
  • Organizer: 第71回日本生物工学会大会
• [Presentation] マイクロ波（非熱効果）が微生物の生理活性に及ぼす影響の解析（第2 報） (2019)
  • Author(s): 松田将典、堀越智、青柳秀紀
  • Organizer: 第71回日本生物工学会大会
• [Presentation] 模擬微小重力環境が腸内有用細菌の諸特性に及ぼす影響の解析と利用 (2019)
  • Author(s): 増田亜理沙、青柳秀紀
  • Organizer: 第71回日本生物工学会大会
• [Presentation] ITO 電極システムと試料の前処理を活用した新規微生物培養化法の開発 (2019)
  • Author(s): 名取暁、小山純弘、青柳秀紀
  • Organizer: 第71回日本生物工学会大会
• [Presentation] 微生物培養による平板培地のpH変化挙動解析と利用 (2019)
  • Author(s): 原田玲奈、青柳秀紀
  • Organizer: 第71回日本生物工学会大会
• [Presentation] Characteristics of the conventional microbial cell culture method: Problems and solutions (2019)
  • Author(s): Hideki Aoyagi
  • Organizer: The 31th Annual Meeting of the Thai Society for Biotechnology and International Conference (TSB 2019) “Bioinnovation for sustainability”
  • Int'l Joint Research / Invited
• [Presentation] Effect of low shear and modeled-microgravity condition on the physiological activities of enteric useful bacteria and its application (2019)
  • Author(s): Arisa Masuda, Hideki Aoyagi
  • Organizer: The 31th Annual Meeting of the Thai Society for Biotechnology and International Conference (TSB 2019) “Bioinnovation for sustainability”
  • Int'l Joint Research
• [Presentation] Isolation of bacteria from microbial flora in soy sauce brewery (2019)
  • Author(s): Daisuke Matsuzaki, Hideki Aoyagi
  • Organizer: The 31th Annual Meeting of the Thai Society for Biotechnology and International Conference (TSB 2019) “Bioinnovation for sustainability”
  • Int'l Joint Research
• [Presentation] Microbial community structure analysis in Acer palmatum bark and isolation of novel bacteria belonging to Abditibacteriota (former Candidate division FBP) (2019)
  • Author(s): Kazuki Kobayashi, Hideki Aoyagi
  • Organizer: The 31th Annual Meeting of the Thai Society for Biotechnology and International Conference (TSB 2019) “Bioinnovation for sustainability”
  • Int'l Joint Research
• [Presentation] Monitoring the pH changes on the agar plate medium during cultivation of microbes and its application (2019)
  • Author(s): Reina Harada, Hideki Aoyagi
  • Organizer: The 31th Annual Meeting of the Thai Society for Biotechnology and International Conference (TSB 2019) “Bioinnovation for sustainability”
  • Int'l Joint Research