title: Development of a systematic feedback isolation approach for targeted strains from mixed culture systems.
abstract: Elucidation of functions of bacteria in a mixed culture system (MCS) such as composting, activated sludge system is difficult, since the system is complicating with many unisolated bacteria. Here, we developed a systematic feedback isolation strategy for the isolation and rapid screening of multiple targeted strains from MCS. Six major strains (Corynebacterium sphenisci, Bacillus thermocloacae, Bacillus thermoamylovorans, Bacillus smithii, Bacillus humi, and Bacillus coagulans), which are detected by denaturing gradient gel electrophoresis (DGGE) analysis in our previous study on MCS for l-lactic acid production, were targeted for isolation. Based on information of suitable cultivation conditions (e.g., media, pH, temperature) from the literature, feedback isolation was performed to form 136 colonies. The following direct colony matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was optimised as the second screening to narrow down 20 candidate colonies from similar spectra patterns with six closest type strains. This step could distinguish bacteria at the species level with distance similarity scores ≥0.55 corresponding to 16S rRNA gene sequence similarity ≥98.2%, suggesting that this is an effective technique to minimize isolates close to targeted type strains. Analysis of 16S rRNA gene sequences indicated that two targeted strains and one strain related to the target had successfully been isolated, showing high similarities (99.5-100%) with the sequences from the DGGE bands, and that the other candidates were affiliated with three strains that were closely related to the target species. This study proposes a new method for systematic feedback isolation that may be useful for isolating targeted strains from MCS for further investigation.
authors: Laboratory of Soil and Environmental Microbiology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Bioresources and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; Department of Microbiology, National College, Affiliated to Tribhuvan University, NIST-Higher Education Program, Khusibu, Nayabazar, Kathmandu, P.O. Box: 8659, Nepal.
authors: Pramod
authors: P
authors: Poudel
authors: Laboratory of Soil and Environmental Microbiology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Bioresources and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; Laboratory of Microbial Environmental Protection, Tropical Microbiology Unit, Center for International Education and Research of Agriculture, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: tashiro@agr.kyushu-u.ac.jp.
authors: Yukihiro
authors: Y
authors: Tashiro
authors: Japan Eco-Science (Nikkan Kagaku) Co. Ltd., 11-2 Shiomigaokacho, Chuo-ku, Chiba 260-0034, Japan; Graduate School of Advanced Integration Science, Chiba University, 1-33 Yayoi-cho, Chiba 263-8522, Japan; Department of Biochemistry and Integrative Medical Biology, Keio School of Medicine, Shinanomachi 35, Shinjuku-ku, Tokyo 160-8582, Japan; Sermas Co. Ltd., Ichikawa Minami 2-8-8, Ichikawa, Chiba 272-0033, Japan.
authors: Hirokuni
authors: H
authors: Miyamoto
authors: Miroku Co. Ltd., Iwaya 706-27, Kitsuki, Oita 873-0006, Japan.
authors: Hisashi
authors: H
authors: Miyamoto
authors: Laboratory of Soil and Environmental Microbiology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Bioresources and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors: Yuki
authors: Y
authors: Okugawa
authors: Laboratory of Soil and Environmental Microbiology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Bioresources and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; Laboratory of Microbial Environmental Protection, Tropical Microbiology Unit, Center for International Education and Research of Agriculture, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors: Kenji
authors: K
authors: Sakai
mesh_terms: D001407:Bacillus / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification* / Q000378:metabolism; D003352:Corynebacterium / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification* / Q000378:metabolism; D046508:Culture Techniques / Q000379:methods*; D005246:Feedback; D019344:Lactic Acid / Q000096:biosynthesis; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D012336:RNA, Ribosomal, 16S; D019344:Lactic Acid
keywords: Direct colony matrix assisted laser desorption ionization-time of flight mass spectrometry; Feedback information; Mixed culture system; Systematic feedback isolation; Targeted strains