1. STRUCTURAL INVESTIGATION OF AQUEOUS
SOLUTIONS OF Bacillus subtilis BIOFILM COMPONENTS
E. Benigar,a
I. Dogsa,b
I. Kralj Cigić,a
A. Zupančič Valant,a
S. Sretenovic,b
D. Stopar,b
A. Jamnik,a
M. Tomšič.a,*
a
Chair of Physical Chemistry, Faculty of Chemistry and Chemical Technology,
University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia
*
Matija.Tomsic@fkkt.uni-lj.si
b
Chair of Microbiology, Biotechnical Faculty
University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia
MOTIVATION and PROSPECTS
Bacillus subtilis (BS) biofilms are composed of sticky gel-like EPS-matrix
(polysaccharide levan, proteins, nucleic acids and other biopolymers). In pure
aqueous system non-ionic levan exhibits rather peculiar non-gelling behavior
up to high concentrations (60 wt. %). However, BS biofilm forms a weak hydro-
gel only up to 8 wt. % of major polymeric component levan. Our goal was to ex-
plore these differences in behavior of BS levan from the structural point of view
and to study the effect of minor biofilm components (nucleic acids, proteins,
simple electrolytes) and bacterial cells on structure and dynamics of levan
solutions.
The results obtained for BS levan were compared to the ones obtained for
commercially available levans from Zymomonas mobilis (ZM) and Erwinia
herbicola (EH). Levan samples and polymeric mixtures were investigated by
rheological, small-angle X-ray scattering (SAXS), static and dynamic light
scattering (SLS and DLS), density and sound velocity measurements, as well
as by microscopy. One of the aims was also to compare the structural proper-
ties of native and synthetic biofilm. Determination of polymer branching after
per-O-methylation analysis and basic chemical and spectrophotometric analy-
sis of the purity of the original levan samples was also performed.
EXPERIMENTAL
Materials. BS levan was isolated and purified directly from BS biofilm. DNA
was isolated from BS cells. ZM and EH levans as well as protein collagen were
purchased from Sigma Aldrich.
SAXS Experiments were performed on a modified Kratky camera with
modern focusing multilayer optics at wavelength of CuK
line.
SLS and DLS Experiments were performed on 3D-DLS Spectrometer in a
3D-cross-correlation mode at λ=632.8 nm.
Dynamic Rheological Experiments were performed on Anton Paar rheome-
ters Physica MCR 302 and 301 with cone-plate geometry (with cone: r=60
mm, angle 0.52° and truncation 62 μm.
Biofilm Growth and Isolation of Levan:
BS culture growth medium 24 h old biofilm raw biofilm isolated EPS isolated levan
RESULTS and CONCLUSIONS
The macroscopic rheological results
showed Newtonian-like behavior of
aqueous solutions of levan at low con-
centrations but at the same time point-
ed out the strongly elastic character of
these solutions. This elastic character
was supported by the DLS results
showing strong nondiffusive relaxation
processes which could be explained by
the strong intramolecular interactions
of levan at such low concentrations.
The latter also manifest themselves in
the presence of large levan particles
that were confirmed by microscopy.
The extent of branching and the hydra-
tion of the levan samples of different
origin were also investigated, but they
did not show any considerable mutual
differences. Structural details on the
nanoscale were obtained by SAXS.
The rheological results showed that
the presence of DNA in levan solution
caused significant increase of its vis-
cosity and a strong pseudoplastic char-
acter, while the presence of collagen
similarly caused a significant increase
of viscosity and pseudoplastic behav-
ior that was accompanied also by the
notable increase in the rigidity of the
system. These results therefore indi-
cate that from the structural point of
view levan in these samples has the
role of a filling agent, long flexible DNA
molecules obviously effectively inter-
connect the levan moieties in the
system and introduce a strong pseudo-
plastic behavior, but less flexible pseu-
doplastic collagen fibrils significantly
contribute to the structural rigidity of
the system.
BS, ZM, EH LEVANS SYNTHETIC BIOFILM BS
Figure 1. Viscosity curves - levans. Figure 2. Amplitude sweeps - levans.
Figure 3. Angle dependent DLS results - levans.
Figure 4. Hydration number - levans.
0.1 1 10 100 1000
1E-3
0.01
0.1
1
10
Medium:
SYM electrolytes
sEPS
nBF2
sBF
SYM
nBF1
.γ [1/s]
η[Pas]
Figure 7. Viscosity curves - L, L+D, L+C, sEPS. Figure 8. Viscosity curves - sEPS, sBF, nBF1, nBF2.
Figure 9. Amplitude sweeps - L, L+D, L+C, sEPS.
Figure 10. Amplitude sweeps - sEPS, sBF, nBF1, nBF2.
0.1 1 10 100 1000
1E-3
0.01
0.1
1
10
100
η[Pas]
.
γ [1/s]
1% levan
8% levan
EH
ZM
BS
EH
ZM
BS
1 10 100 1000 10000
0.01
0.1
1
10
100
1000
1 % levan - G'
1 % levan - G''
8 % levan - G'
8 % levan - G''
EH
ZM
BSZMEH
BS
G',G''[Pa]
γ [%]
(a) Solvent
2 μm
(b) BS
2 μm
(c) ZM
2 μm
(d) EH
2 μm
Figure 5. Microscopy images - levans.
Figure 6. Molecular distribution of 2 % levans in space. Figure 11. SAXS curves - L, L+D, L+C, sEPS.
Figure 12. SAXS curves - sEPS, sBF, nBF.
Figure 12. Microscopy images - L, L+D, L+C, sEPS.
8 µm
(a) L
8 µm
(b) L+D
8 µm
(c) L+C
8 µm
(d) L+D+C
-4.4 -4.2 -4.0 -3.8
3
4
5
6
7
8
ln(1/τc
)
ln(q)
2.000 % BS, slope = 4.41
0.010 % BS, slope = 4.36
1.000 % ZM, slope = 4.22
0.001 % ZM, slope = 4.36
0.100 % EH, slope = 4.35
0.001 % EH, slope = 4.35
DIFFUSIVE CHARACTER: slope = 2
SLOWER NONDIFFUSIVE: slope > 2
0 20 40 60 80
5.6
5.8
6.0
6.2
6.4
6.6
6.8
γ [mg/mL]
BS
ZM
EH
nh
0.1 1 10 100 1000
1E-3
0.01
0.1
1
10
Medium:
SYM electrolytes
L+D+C (= sEPS)
L+C
L+D
SYM
L
.γ [1/s]
η[Pas]
0.1 1 10 100
0.1
1
10
Medium: SYM electrolytes
G'
G''
L+D+C (= sEPS)
L+C
L+D
γ [%]
G',G''[Pa]
0.1 1 10 100
0.1
1
10
Medium: SYM electrolytes
G'
G''
sEPS
nBF2
sBF
nBF1
γ [%]
G',G''[Pa]
0.1 1
0.01
0.1
L+C
Medium:
SYM electrolytes
L+D+C(= sEPS)
L
L+D
q [nm-1
]
I(q)[cm-1
]
0.1 1
0.01
0.1
1
Medium:
SYM electrolytes
nBF
sEPS
sBF
q [nm-1
]
I(q)[cm-1
]