Development of a systematic feedback isolation approach for targeted strains from mixed culture systems
| Autor: | Hisashi Miyamoto, Kenji Sakai, Pramod Poudel, Hirokuni Miyamoto, Yuki Okugawa, Yukihiro Tashiro |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
biology Strain (chemistry) 030106 microbiology Bioengineering Bacillus Corynebacterium biology.organism_classification 16S ribosomal RNA Isolation (microbiology) Applied Microbiology and Biotechnology Microbiology Feedback 03 medical and health sciences 030104 developmental biology Mixed culture Culture Techniques RNA Ribosomal 16S Bacillus coagulans Lactic Acid Gene Bacteria Temperature gradient gel electrophoresis Biotechnology |
| Zdroj: | Journal of bioscience and bioengineering. 123(1) |
| ISSN: | 1347-4421 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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