Microcultivation of bactera single cells entrapped in alginate microbeads PDF
Preview Microcultivation of bactera single cells entrapped in alginate microbeads
Microcultivation of bacteria single cells entraped in alginate microbeads Rosa M. Aragão Börner PhD student Department of Biotechnology Lund University Endophytes for plant protection: the state of the art. Berlin, 27-29th May 2013 Why still to cultivate in omics era? Genomic Assigned potential function Still need of Cultivation efforts: • Provide reference strains • Broaden our view in the area of basic research • Gives new organisms for Genomes online database novel metabolites of commercial interest Organisms sequenced during the Human Microbiome Project are highlighted in blue. Science / AAAS 328, 994- 999 (2010). Enrichment a ir e t c a b r e b m u n g o L Serial dilutions Time Streaking on solid medium Unculturables Not-yet-cultured Single cell Microcolony Weaver et al. 1988 Bio Technol Manome et al. 2001 FEMS Microbiol Lett Akselband et al. 2005 J. Microbiol Methods For isolation, cultivation and diversity studies in a hightroughput method Zengler et al., 2002 PNAS 99: 15681 Aoi et al., 2009 Appl Env Microb 75:3826 3D Support Mechanical Stability Exchange of Protection from environment quorum sensing Protection from competitors signals Keeps ”communication” Nutrients accesibility Exchange of nutrients Exclusion of toxic metabolites Alginate as entrapment material Alginate Biocompatible Inexpensive Mild process Li et al 2012 Energy 38:249 Chavari et al Probiotics 2012DOI: 10.5772/50046 Alginate beads – the classics Alginate solution + cells Cryopreservation nozzle Immobilization of cells for of algi or plant cell fermentation lines CaCl2 solution 500 µm – >1 cm Tend to be teardrop shaped Polymerization by extrusion http://usmplanktonlab.blogspot.se, http://www.bashanfoundation.org, http://www.teriin.org/ Entrapment using microemulsion method Oil soluble H+ acid cells alginate O CaCO Ca C O 3 Homogenization Mixing with Emulsion Emulsion poured in 50 O + 2x106 cells hydrophobic formation and mM CaCl2 solution to phase beads gelation separate beads from oil and to complete gelation Mix matrix Emulsion formation Acid addition Alginate microbead formation Poncelet et al. 1992 Appl Microbiol Biotechnol Entrapment using microemulsion method Advantage • Safe • Simple Phase separation Cleaning of the Few nm - mm • Low cost microbeads with 50 mM Tris-HCl/CaCl 2 • Possible to adapt in any laboratory condition Initial cell load Poisson probability Bead size 20 – 80 µm 2 x 106 cells/ml 60 – 80% Weaver et al. 1988 Bio Technol
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