Microcultivation of bactera single cells entrapped in alginate microbeads

20 Pages·2013·2.65 MB·English

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