Online Resource 2: Cell wall fragment formation in pure cultures of
Escherichia coli
Title of paper:
SOM genesis - Microbial biomass as a significant
source
Journal:
Biogeochemistry
Authors:
Anja Miltner, Petra Bombach, Burkhard Schmidt-Brücken, Matthias Kästner
Affiliation and contact details of corresponding author:
Anja Miltner ()
UFZ - Helmholtz-Centre for Environmental Research, Department of Environmental
Biotechnology, Permoserstr. 15, 04318 Leipzig, Germany
e-mail: anja.miltner@ufz.de
phone: ++49/341/235-1763
fax: ++49/341/235-1471
Cell wall fragments in pure cultures of Escherichia
coli
Materials and Methods
The general hypothesis of the paper is that organic particles of 100 - 500 nm size derived from
cell wall fragments contribute significantly to soil organic matter. If the fragments observed in
the scanning electron micrographs of soil samples actually originate from bacterial cells, they
should also be found in pure cultures of bacteria.
We therefore prepared SEM micrographs from pure cultures of Escherichia coli K12, of both
intact cells and cell fragments prepared using ultrasonic disruption. Briefly, cells were grown
on mineral medium containing 2 g/l glucose as the sole C source. The exact composition of
the medium is described by Kindler et al. (2006). Cells were grown overnight until they
reached the stationary phase. The culture was divided in two parts. In the first part, the cells
were left intact. They were only centrifuged (5000 g, 10 min) and washed with distilled water
to remove residual medium. The other part was fragmented by a 10 min treatment with an
ultrasonic probe. The cell wall fragments were separated from the soluble cell contents by
centrifugation (13,000 g, 30 min) and washed with distilled water. Both samples were then
fixed with glutaraldehyde, prepared for SEM using an increasing acetone series and criticalpoint drying and analysed by SEM as described in Online Resource 1.
Results and Discussion
The SEM micrograph of the intact stationary phase cells (Fig 1a) shows the typical
morphology of the E. coli cells. In addition, abundant small particles are visible which are
normally not shown since most scientists prefer to present plain cells in such micrographs.
The particles are attached to the cell surfaces or distributed in the voids between the cells.
These particles look highly similar to those observed in soil and in groundwater microcosms.
As the cells were washed to remove residual mineral medium, these particles must originate
from cell debris produced during growth of the bacteria. In the present stationary phase
culture, presumably a high number of cells already had died and partially disintegrated when
the samples were taken.
For reference, cell wall fragments from the same culture produced by ultrasonic treatment
were studied by SEM (Fig. 1b). The sonication intensity was pre-checked for maximum cell
disruption and protein release. As a result, no more intact cells were found in the SEM
micrographs. Instead, the samples completely consisted of patchy material similar to the one
observed in the sample with the intact cells, providing additional evidence that the patches are
cell wall fragments.
Fig. 1
SEM micrographs of pure culture samples: a) intact cells, b) ultrasonically disrupted cells
Reference:
Kindler R, Miltner A, Richnow H-H, Kästner M (2006) Fate of gram-negative bacterial biomass in soil mineralization and contribution to SOM. Soil Biol Biochem 38:9 - 24.