SCIENCE CHINA

Physics, Mechanics & Astronomy

•Article• September 2019 Vol. 62 No. 9: 994611
https://doi.org/10.1007/s11433-019-9393-x

Tensile properties of individual multicellular Bacillus subtilis fibers

1,2 1 1 1,2*
Xuan Ye , Tao Wang , Zhuo Zhuang , and XiDe Li
1
Applied Mechanics Laboratory, Department of Engineering Mechanics, Tsinghua University, Beijing 100084, China;
2
Center for Nano and Micro Mechanics, Tsinghua University, Beijing 100084, China

Received January 30, 2019; accepted March 14, 2019; published online April 20, 2019

Microfibers formed by Bacillus subtilis (B. subtilis) have attracted interest because of their potential for use as biodegradable
fibers. In this work, an efficient method based on the micro-liquid bridge method (LBM) is proposed to investigate the
mechanical properties and the deformation evolution in individual fibers. For the first time, tensile testing of fibers of this type
containing several cells is conducted in a scanning electron microscope (SEM) chamber and the in situ deformation evolution of
the fibers and the septa is observed. Experimental results show that these fibers are almost broken at the positions of the septa at
low humidity, but also show that their fracture morphologies are different. At high humidity, local necking deformation occurs at
the septum position. To explore the deformation mechanism of an individual bacterial fiber with a diameter of several hundred
nanometers under different humidity conditions, we use the finite element method (FEM) to analyze the tensile deformation
behavior of these fibers when their septa are at various separation levels. The numerical results indicate that weak interactions
among the septa lead to the dispersion of both the fibrous tensile strength and the modulus. These results may be helpful in
understanding the deformation mechanism, thus leading to further improvements in the mechanical performance of these fibers.
Bacillus subtilis, mechanical properties, micro-liquid bridge method, finite element method

PACS number(s): 46.80.+j, 62.20.-x, 62.25.+g, 68.08.-p

Citation: X. Ye, T. Wang, Z. Zhuang, and X. D. Li, Tensile properties of individual multicellular Bacillus subtilis fibers, Sci. China-Phys. Mech. Astron. 62,
994611 (2019), https://doi.org/10.1007/s11433-019-9393-x

1 Background lengths of approximately 2 µm, these new fibers can be up to

With the rapid development of micro- and nano-science and
technology, it is of great research significance to study the
mechanical properties of micro- and nano-scale materials
and structures, and to predict and analyze the mechanical
properties and reliability of microelectromechanical systems
(MEMS) and devices [1-3]. The Bacillus subtilis (B. subtilis)
fiber has recently appeared as a new type of biological ma-
terial with nontoxic, nonpolluting and biodegradable char-
acteristics. An individual B. subtilis fiber is a chain of
connected cells produced by inhibiting the cell wall separa-
tion of B. subtilis. Unlike wild-type B. subtilis cells with
*Corresponding author (email: lixide@mail.tsinghua.edu.cn)
several hundred microns in length [4]. Additionally, these
bacterial fibers can be extracted from a culture solution to
form a bacterial thread (composed of multiple fibers ar-
ranged in parallel) with a length of a few centimeters and a
diameter of a few millimeters. Performing an in-depth study
of the mechanical behavior of the individual B. subtilis fiber
will be highly important in promoting the application of
these fibers as engineering materials.
However, the small-scale dimensions of the individual B.
subtilis fibers range from a few micrometers up to a hundred
micrometers, making measurement of their mechanical re-
sponse and performance challenging. Therefore, it is of
major theoretical and practical importance to provide reliable
measurement methods and analytical models that can char-

© Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019 phys.scichina.com link.springer.com
 X. Ye, et al. Sci. China-Phys. Mech. Astron.
acterize the different factors influencing their deformations,
including the fiber microstructures, their elastic and viscoe-
lastic parameters, and environmental parameters. At the cell
scale, Touhami et al. [5], Hoh and Schoenenberger [6], and
Zhao et al. [7] obtained the average spring constant of the
cell wall of different cells, while Vadillo-Rodriguez et al. [8-
10] measured the viscoelastic parameters using atomic force
microscopy (AFM). Tuson et al. [11] proposed a method
called “cell length analysis of mechanical properties”
(CLAMP) to measure the mechanical properties of a bac-
terium in vivo. Amir et al. [12] applied a controllable hy-
drodynamic force to bend the B. subtilis cells and thus
obtained the stresses that regulate the cell wall growth me-
chanism. At the bacterial thread scale, Thwaites et al. [13-15]
stretched bacterial threads that contained 20000 parallel
bacterial fibers under various relative humidities using an
instrument similar to standard machines (e.g., the Instron
material testing system) and obtained the elastic modulus of
the threads. Recently, Zhang et al. [16] measured the dy-
namic mechanical properties of B. subtilis threads using a
cantilever-probe system.
While the mechanical behavior of the cells or the bacterial
threads has been widely measured experimentally, as de-
scribed above, previous studies involving AFM, CLAMP,
and hydrodynamic force have mainly focused on studies of
the mechanical properties of the cell walls or the membranes
of a single cell, indicating that they are unsuitable for me-
chanical testing of individual multicellular fibers containing
several to hundreds of mutant cells. In addition, the test re-
sults for bacterial threads are not suitable for prediction of
the mechanical properties of the cell walls or the individual
cellular fibers. It thus remains challenging to manipulate and
measure the properties of the individual fibers quantitatively
because of their nanoscale diameters and high slenderness
ratios.
Recently, we performed experiments that involved
stretching individual fibers with an average diameter of
0.7 µm and a length range of 25.7-254.3 µm (where the fi-
bers contained tens of bacterial cells up to several hundreds)
using an in-house-built multiscale material testing system
under an optical microscope (OM) [17, 18]. We found that
the mechanical properties of the fibers were strongly de-
pendent on the relative humidity. The tensile strengths of the
fibers were highly dispersed, which we speculated to be
caused by the septa. Regrettably, we could not clearly ob-
serve the fibers’ deformation evolution due to the spatial
resolution limitation of the OM.
In order to finely measure the deformation and reveal the
evolution process, in particular the local deformation me-
chanism of the septa, the B. subtilis fibers containing several
cells were uniaxially stretched in the SEM. A novel method
called the liquid bridge method (LBM) was proposed in this
paper for the clamping and alignment of multicellular B.
September (2019) Vol. 62 No. 9 994611-2
subtilis fibers. We observed the deformation evolution pro-
cesses of the fibers in real time and found various fracture
morphologies at the septum positions under low humidity
conditions and necking evolution at the septum positions for
the fibers under high humidity conditions. In particular, by
considering the complex associational behaviors between the
biostructure and the fiber’s mechanical properties, along
with the failure processes under low and high humidity, we
applied the finite element method (FEM) to analyze the
tensile deformation behavior of these fibers when their septa
were at various separation levels.
2 Experiments and results
Test system. Uniaxial tensile tests of the fibers were per-
formed on our in-house-built multiscale material testing
system (MMTS) in an SEM (Quanta 450 FEG, FEI, Hills-
boro, OR, USA); the system was composed of a microscale
material testing module (m-MTM) and a nanoscale material
testing module (n-MTM) [17]. The n-MTM contained a pair
of symmetrical wedge-grip units and a specimen connection
and support unit (SCSU), which was manufactured on a si-
licon wafer using well-known silicon deep etching technol-
ogy. In the SCSU, the n-MTM was available in two sizes,
with specimen spacing of 10 μm and 100 μm. In the uniaxial
tensile tests, we first fixed the symmetrical wedge-grip unit
to the support stages of the m-MTM and then installed the
specimen on the clamping ends of the SCSU. Depending on
the measurement method and the measurement environment
used, the specimen clamping method could include gluing,
van der Waals interactions, electron beam-induced deposi-
tion (EBID), and capillary adhesion. Next, the double sup-
port bars of the SCSU were cut off using a small rotating
diamond saw, and the m-MTM provided an external load and
a force sensing function to achieve the required uniaxial
stretching or compression of the specimen. The complete
experimental system is shown in Figure 1.
Liquid bridge method. This material is moisture-sensi-
tive and thus we naturally think of clamping using capillary
force [19, 20]. However, because the previously developed
liquid drop method (LDM) [18] under an OM is relatively
complex and is not suitable for clamping of the test specimen
in an SEM environment, the LBM has been proposed here to
provide the clamping connection for the multicellular fibers.
The LBM implementation process is described as follows
(see Figure 2). First, a small number of freeze-dried multi-
cellular fibers is added to deionized water to obtain a sus-
pension with an appropriate concentration. Then, a tiny drop
of the fiber suspension is transferred using a dropper pipette
into the gap between the clamping ends of the n-MTM
(Figure 2(b) and (f)). At this stage, a liquid bridge is formed
in the gap by the adhesion between the liquid and the solid
 X. Ye, et al. Sci. China-Phys. Mech. Astron.
Figure 1 (Color online) Experimental system. (a) Entire system under
OM. (b) Nanoscale material testing module observed in an SEM [17].
(the clamping ends of the n-MTM), along with the cohesion
of the liquid itself. The flow characteristics in the liquid
bridge between two rods pulled apart have been investigated
[21]. In our experiment, as the liquid evaporates, it should
create a similar flow effect which then accounts for the
straightening of the fibers along the axial direction of the
liquid bridge. At the beginning, we can obtain a fiber with
ends that adhered to the clamping ends of the n-MTM by
surface tension (Figure 2(c) and (g)). To ensure that only a
single fiber was clamped, we need to control the con-
centration of the suspension and verified it by the observa-
tion in the SEM. It was worth mentioning that the entire
process took only 15 s. Subsequently, the entire experimental
system remained stationary until the liquid at the clamping
ends completely evaporated and the fiber was dried (Figure 2
(d) and (h)). At this time, the fibers adhered to the clamping
ends by the combination of the adhesion which was similar
to the colloid from the culture medium and van der Waals
force. As the water evaporated, the fiber would strongly
Figure 2 (Color online) LBM procedure. (a) Ultrasonically cleaned clamping ends of the n-MTM viewed under the OM. (b) The moment at which a tiny
drop of the fiber suspension was transferred to the clamping ends of the n-MTM. (c) Fiber caught by the clamping ends of the n-MTM under capillary force.
(d) A dried individual fiber. (e)-(h) Schematic diagrams corresponding to Figure 2(a)-(d), respectively.
September (2019) Vol. 62 No. 9 994611-3
adhere to the clamping ends through the interface interaction
between them. In the experiments, we did not observe the
slippage or peeling off between the specimens and the
clamping ends. During this process, the fibers may be in a
pre-stretched state. In order to eliminate the initial tensile
stress generated by the LBM, we first relaxed the fiber to
release the initial tensile stress generated during the drying
process using the fine-tuning function of the m-MTM, then
stretched the fiber at a loading speed of 1.0 μm/s. The entire
stretching process was recorded by video recording. The
elongation of the fiber was obtained by analyzing the SEM
image sequences, and the load was calculated from the de-
flection of the tip of the force-sensing probe based on the
beam theory [18]; considering that the end of the probe was
fixed on one clamping end of the MTS, the deflection of the
tip was entirely caused by the tension of the fiber.
From these experiments, we also found that the flat
clamping ends of the n-MTM were suitable for clamping
fibers with lengths of 30-500 μm (Figure 3(a)), while for
shorter fibers (e.g., with a length of 10 μm), sharp clamping
ends should be used (Figure 3(b)). This is because a rela-
tively short and thick liquid bridge is formed between the
two flat clamping ends when the gap between the clamping
ends is small (Figure 3(c)), and the fibers in the liquid bridge
will be broken or pulled off at the clamping ends because of
the large capillary force that is provided in such a situation.
With regard to the sharp clamping ends, when the gap is
small, a slender liquid bridge can be formed to realize
clamping of shorter fibers (Figure 3(d)). It should be noted
that this does not mean that the sharp clamping ends are only
suitable for short fibers; these ends are suitable for clamping
of fibers with lengths in the range from 10 to 500 μm.
The LBM is suitable for use with moisture-sensitive low-
dimensional materials (e.g., one-dimensional materials with
lengths ranging from a few microns to hundreds of microns
and diameters on the micron and submicron scales). When
compared with the LDM, the LBM is easier and more effi-
cient, can be performed without the aid of a manipulator, and
avoids complex procedures such as picking up, transferring
and clamping samples.
 X. Ye, et al. Sci. China-Phys. Mech. Astron. September (2019) Vol. 62 No. 9 994611-4

Figure 3 (Color online) (a) Long fiber with length of 203.6 μm clamped
at the flat clamping ends. (b) Short fiber with length of 28.4 μm clamped at
the sharp clamping ends. (c) Short and thick liquid bridge formed between
the flat clamping ends. (d) Slender liquid bridge formed between the sharp
clamping ends. Figure 4 (Color online) Stress-strain curves of B. subtilis fibers under
low and high humidity conditions. The mutation in the tensile curve derives
from the change in humidity (there are only two humidity states in this
Experimental results. Previous studies have shown that study).
the B. subtilis fibers are sensitive to humidity, but we cannot
establish a tunable humidity environment within the chamber
of a conventional SEM. Therefore, we regulated each B.
subtilis fiber’s dryness to simulate the specimen under low or
high humidity conditions. The method used to obtain a
specimen with low humidity is to naturally dry the fiber for
20 min after evaporation of the liquid bridge. In contrast, to
obtain the mechanical properties of fibers under high hu-
midity conditions, we performed the tensile tests in the SEM
vacuum chamber immediately after evaporation of the liquid
bridge. At this time, the fiber cells still contain water and it Figure 5 Tensile testing of B. subtilis fibers when using (a) flat clamping
will not be lost immediately during the short-time experi- ends and (b) sharp clamping ends in an SEM chamber.
ments. If the specimen is kept in high vacuum for a long
time, water will gradually lose. Figure 4 shows the typical showed significant elongation both in parts of the cell wall
tensile testing results obtained when using flat and sharp and at the septum positions, and ultimately the fibers necked
clamping ends in the SEM (see Figure 5). The tensile results at the septum positions (Figure 6(d)-(e)).
show that the multicellular fibers have profoundly different
mechanical properties that depend on the different humid-
ities of the fibers (there are only two humidity states in this 3 Finite element simulations of stretched B.
study because of the difficulty of controlling the humidity in subtilis fiber
the SEM) (see Figure 4). For the fibers with low humidity,
the experimental stress-strain curves were fitted using a The fibrous mechanical parameters described above, such as
linear spring model and had an average elastic modulus of the elastic modulus and the viscosity coefficient, are given
(8.7±1.2) GPa and fracture stress of (86.0±22.3) MPa. At based on theoretical models by combining the experimental
high humidity, these fibers were viscoelastic materials with a test results, which assume that the B. subtilis fibers are one-
certain degree of nonlinearity during the initial loading stage. dimensional continuous hollow cylinders. In fact, these fi-
We obtained an average elastic modulus E of (62.7±11.7) MPa bers exhibit a continuous cylindrical wall with invagination
and an average viscosity η of (83.4±32.7) MPa·s based on at the septum positions at varying separation levels. These
fitting of the experimental stress-strain curves using the theoretical models cannot adequately characterize the way in
Kelvin-Voigt model [18]. For the fibers with low humidity, which the septa affect the deformation and fracture of these
fracture only occured at the septum position, although the fibers. In particular, it is not possible to provide adequate
fracture morphologies of the cross-sections of multicellular information on the inelastic deformation evolution and the
fibers vary and include vertical fracture (Figure 6(a)), micro- fracture mechanism of the B. subtilis fibers using the ex-
curved fracture (Figure 6(b)) and large-curved fracture perimentally measured stress-strain curves alone. Here, we
(Figure 6(c)). For fibers with high humidity, viscoelasticity use the FEM to simulate the uniaxial stretching process of
dominated the fiber deformation process. These fibers the B. subtilis fibers.
 X. Ye, et al. Sci. China-Phys. Mech. Astron. September (2019) Vol. 62 No. 9 994611-5

Figure 6 (Color online) Typical failures of B. subtilis fibers. (a)-(c) Scanning electron micrographs of fracture cross-sections of multicellular fibers with
low humidity, demonstrating that the B. subtilis fibers fracture at the septum positions with various fracture morphologies. (d)-(e) Scanning electron
micrographs of fibers with high humidity that have necked at the septum positions.

3.1 Cell division of B. subtilis and formation of a sep-

tum

Typical transmission electron microscopy (TEM) images

that illustrate the cell division process of B. subtilis are
shown in Figure 7, while a corresponding schematic diagram
is shown in Figure 8. Before cell division, the rod-shaped B.
subtilis is elongated to approximately twice its cell length by
inserting a new peptidoglycan (indicated by the red lines in
Figure 8) into the lateral cell wall. The septum (indicated by
the blue lines in Figure 8) then begins to grow inwards at the
middle of the double-length cell cylinder until the mother
cell synthesizes a complete septum that contains two flat
parallel plates with a thin interface layer between them
(Figure 7(a)-(c)). Finally, the mother cell splits into two
daughter cells under the action of hydrolase [22] (Figure
7(d)-(f)). In this study, an individual multicellular fiber is a
chain of cells that is connected through these septa by in-
hibiting cell separation [23, 24]. In addition, the septa of the
same multicellular fiber can have different separation levels.
3.2 Finite element models of fibers at varying separa-
tion levels
Previous studies have shown that cells exhibit different
mechanical behaviors at various stages in their physiological
processes [25]. To study the mechanical properties and the
deformation evolution of these multicellular fibers with septa
at different separation levels, we established four three-di-
mensional finite element models, as shown in Figure 9(a)-
(d), which corresponded to the initial (Figure 7(c)), early
(Figure 7(d)), middle (Figure 7(e)), and late (Figure 7(f))
stages of the cell separation process, respectively. The geo-
metric sizes of these fibers were obtained from the TEM
images shown in Figure 7(c)-(f). Considering that an actual
fiber was connected via the septa at various separation levels,
we also established a typical fibrous model that contained
Figure 7 Typical TEM images of the B. subtilis cell division process. (a)
New peptidoglycan is synthesized, and the cylindrical part of the cell is
elongated. (b) A portion of a septum is synthesized in the middle of the
mother cell. (c) A complete septum containing two flat parallel plates with
a thin interface layer between them is synthesized. (d) The mother cell
begins to divide under the action of hydrolase. (e) Two daughter cells with
partially connected ends. (f) The daughter cells when almost entirely se-
parated [18].
several septa at different separation levels (from left to right:
initial stage, late stage, late stage, and initial stage), as illu-
strated in Figure 9(e).
3.3 Material properties
The fracture behavior of the fibers with low humidity was
studied using the elastic-fracture constitutive and extended
finite element method (XFEM) [26, 27]. The average elastic
modulus for the cell wall from the experiments is 8.7 GPa.
The intermediate interface layer is set to be softer than the
cell wall (by 0.2 times) [28]. Because the fiber structure near
the septum is similar to a circumferentially crack-notched
round bar [29], we use the stress intensity factor K I to de-
scribe the fracture behavior of an individual fiber, which can
be calculated as follows:
D F
K I = 1.72 1.27 , (1)
d d 3/2
 X. Ye, et al. Sci. China-Phys. Mech. Astron.
Figure 8 (Color online) Schematic of B. subtilis cell division process
corresponding to the images in Figure 7.
Figure 9 (Color online) Fibrous models for septa at the (a) initial, (b)
early, (c) middle, and (d) late separation stages in the cell separation pro-
cess. (e) Fibrous model containing several septa at different separation
stages (from left to right: initial stage, late stage, late stage, and initial
stage).
where D, d , and F denote the diameters of the cylinder and
the septum and the fracture force, respectively. The calcu-
lated average fracture toughness is K IC = 33.6 kN/m3/2 and
the corresponding energy release rate is G IC = 0.2 N/m. Un-
der high humidity conditions, the viscoelastic constitutive
relationship (the Kelvin-Voigt model) was used to study the
deformation behavior of the fibers. The elastic modulus and
the viscosity coefficient of the cell wall are 62.7 MPa and
83.4 MPa·s respectively. The finite element calculations are
then performed using uniaxial displacement loading (the
details of the finite element model is shown in Appendix).
3.4 Numerical results
For the fibers with low humidity, the experimental results
show that the multicellular fibers all fracture at the septa,
while the fracture morphologies at the cross-sections of the
multicellular fibers differ, as shown in Figure 6(a)-(c). We
speculate that it is related to the various separation levels of
the septa. Here, the numerical results (Figure 10) again in-
dicate that the fibers fracture at the septum positions under
September (2019) Vol. 62 No. 9 994611-6
Figure 10 (Color online) Calculated results for multicellular fibers with
septa at the (a) initial, (b) early, (c) middle, (d) late, and (e) multiple
different separation stages of the cell separation process under low hu-
midity conditions. The fibers all break at the septum positions, which is
consistent with the experimental results shown in Figure 6(a)-(c).
uniaxial tension and the calculated fracture morphologies of
the fibers are consistent with the experimental results, which
indicate that the septa are weak connections. The simulated
load-displacement curves and the stress-strain curves of the
fibers at the different separation stages can also be obtained
(Figure 11). We find that the fibrous stress-strain curves and
the corresponding mechanical properties (i.e., the elastic
modulus and the fracture strength) have high dispersibility
because of the local structural changes (septa) that occur in
the fibers.
The simulation results for the five fibrous models with
high humidity are shown in Figure 12, illustrating that these
fibers are elongated both in parts of the cell wall and at the
septum positions, while the necking instability also occurs at
the septum positions. For the fibers in the later separation
stages (Figure 12(c) and (d)), the necking at the septum
positions is more obvious. For the fiber that contains several
septa at different separation levels (initial-late-late-initial),
the calculated tensile result (Figure 12(e)) is close to the
experimental results (Figure 6(d) and (e)), which shows that
an uneven distribution of the septa accelerates necking fail-
ure of the multicellular fiber.
The simulated stress-strain curves of multicellular fibers
under high humidity condition with multiple different se-
paration stages can also be obtained (Figure 13). The stress-
strain curves of these fibers still show wide dispersion be-
cause of the different separation levels of the septa within
these fibers.
4 Discussion
In this study, a liquid bridge was formed between the two
clamping ends of the n-MTM (Figure 14) by the adhesion of
the liquids to the side surfaces of the clamping ends. The side
surface of the liquid presented a concave meniscus surface
because of the adhesion of the solids (i.e., the clamping ends)
at both ends and the surface tension of the liquid. This
concave meniscus surface caused a pressure difference be-
 X. Ye, et al. Sci. China-Phys. Mech. Astron.
Figure 11 (Color online) Numerical results for multicellular fibers at the
initial, early, middle, late, and multiple different separation stages under
low humidity conditions. The wide dispersion of the stress-strain curves is
caused by the different separation levels of the septa within the fibers.
Figure 12 (Color online) Numerical results for multicellular fibers at (a)
initial, (b) early, (c) middle, (d) late, and (e) multiple different separation
stages under high humidity conditions.
Figure 13 (Color online) Calculated stress-strain curves for fibers with
multiple different separation levels under high humidity conditions.
tween the interior and the exterior of the liquid. According to
the Laplace-Young equation [30, 31], the hydrostatic pres-
sure difference is given by
p = 2 cos / D 0, (2)
where γ is the liquid surface energy, θ is the contact angle,
and D0 is the distance between the clamping ends.
Considering that the capillary force between two plates is
September (2019) Vol. 62 No. 9 994611-7
Figure 14 (Color online) Schematic of a liquid bridge between two
clamping ends.
composed of two parts, where one part represents the action
of the negative pressure in the liquid along the direction of
the liquid bridge axis in the wetted region of the solid, while
the other part represents the action of the surface tension on
the three-phase line along the liquid bridge axis, the capillary
force between the two plates separated by a liquid bridge is
then given by
f = A p + lsin
= 2 Lt cos / D 0 + 2 (L + t )sin , (3)
in which A is the area over which the upper and lower par-
allel plate surfaces are wetted by the liquid, l is the wetted
area perimeter, L is the wetted length, and t is the wetted
thickness.
For the liquid bridge that is formed between the two silicon
plates (i.e., the clamping ends), the water surface energy γ is
0.072 N/m, and θ is 10.0° (with an oxide thickness of ap-
proximately 3 nm) [32]. We assume that the wetted length L
is equal to the length of the clamping ends of the SCSU, and
that the wetted thickness t is equal to the thickness of the
clamping ends. Because the SCSU is manufactured on a si-
licon wafer using well-known silicon deep etching technol-
ogy, t is known to be 25 μm. For the flat clamping ends, L is
80 μm, and thus the value of the capillary force is 5.5 μN
when D0 is equal to 100 μm, and the value of capillary force
is 31.0 μN when D0 is equal to 10 μm. For the sharp
clamping ends, L is 4 μm, and the capillary force is thus
0.9 μN when D0 is equal to 100 μm, and the value is 2.1 μN
when D0 is equal to 10 μm. We thus obtain capillary force
ranges of 5.5-31.0 μN for the flat clamping ends and
0.9-2.1 μN for sharp clamping ends, where the latter range is
much smaller.
From eq. (3), if D0 remains constant and L decreases, the
capillary force between the two silicon plates separated by
the liquid bridge is then also reduced. This explains why the
flat clamping ends cannot clamp the short specimen well
when compared with the sharp champing ends. Because of
the excessive capillary force between the flat clamping ends,
the fibers in the liquid bridge are broken (previous experi-
mental results indicate that the maximum breaking load of an
individual fiber is 12 μN). If L remains constant and D0 de-
creases, the capillary force is then increased, which explains
 X. Ye, et al. Sci. China-Phys. Mech. Astron.
why the specimen cannot be clamped when the two flat
clamping ends are relatively closely spaced.
The mechanical properties of the multicellular B. subtilis
fibers may be affected by different factors, including in-
dividual organisms, changes in the loading rates, the biolo-
gical pretreatment methods used, and in particular, the
moisture contents and saturability of the cells in the fibers
and the retained septa with various separation levels, which
changes the composition and structure of cells (or the ske-
leton of cells). The experimental results indicated that the
humidity seriously affected the mechanical properties of the
fibers because of the presence of multiple hydrogen bonds
between the peptides of the peptidoglycan contained in the
fibers, which made the polymer networks rigid under dry
conditions. As the degree of hydration increased, water then
competed for the hydrogen bond sites, which made the net-
works more flexible and resulted in reductions in both the
elastic modulus and the strength of the fibers. Both the ex-
perimental and computational results reveal that the septa
among the pairs of microbial cells are the weak links in the
multicellular fibers where failures are most likely to occur,
and the failure strength is dependent on the strength of the
weakest septum. On one hand, we hope to perform in-situ
stretching of single-cell and dual-cell in SEM to observe the
deformation evolution process of cell cylinder and the septa
in real time and reveal their failure mechanism. On the other
hand, for this type of fiber with diameter at such a small
length scale, the size effect and surface effect also have a
certain influence on the mechanical properties [33, 34].
5 Conclusions
We performed direct stretching of individual B. subtilis fi-
bers in situ in an SEM chamber using the new developed
liquid bridge method. By taking the effects of the bios-
tructure into account, a finite element analysis revealed the
reason for the wide dispersion in the fiber tensile behavior
under low and high humidity conditions. The numerical re-
sults showed that the different degrees of separation of the
septa contribute to changes in both the elastic modulus and
the strength in these fibers, while the weakest septum
dominated the mechanical properties of the individual fibers.
We expect that our findings, at the level of the individual
bacterial fiber, could provide a mechanism to improve fi-
brous mechanical performance in engineering applications.
To be used as mass-produced materials, the weak septa in
these fibers must be reinforced locally to enhance the load-
bearing capacity.
This work was supported by the National Natural Science Foundation of
China (Grant Nos. 11872035, 11472151, 11632010, and 11227202). The
authors thank Prof. Guo-Qiang Chen and Dr. Liang Zhao for synthesizing
September (2019) Vol. 62 No. 9 994611-8
the B. subtilis fibers and providing the TEM images of B. subtilis cell di-
vision.
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Appendix The details of finite element model
A1 Elastic model at low humidity
As mentioned above, B. subtilis fibers show obvious brittle
fracture characteristics at low humidity. In order to better
understand their mechanical behavior and whole deforma-
tion and fracture process, we established finite element
models of different cell separation stages and their combi-
nations. The profile of a typical finite element model is
shown in Figure a1. The following material parameters are
Figure a1 (Color online) The finite element model of a B. subtilis fiber
under a tension load.
September (2019) Vol. 62 No. 9 994611-9
used: the Youngʼs modulus of the cell wall is 8.7 GPa,
Poissonʼs ratio is 0.45, and fracture energy is G IC = 0.2 N/m.
The Youngʼs modulus of the interface layer is 0.2 times that
of the cell wall. We adopt XFEM technology to simulate the
crack propagation in the commercial finite element code
Abaqus.
A2 Viscoelastic model at high humidity
B. subtilis fibers exhibit viscoelastic behavior at high hu-
midity. They are stretched to produce large deformations
accompanied by necking. We established a finite element
model with the same geometric parameters and boundary
conditions as those in the previous section. The viscoelastic
constitutive model (Kelvin-Voigt model) was used to capture
the large deformation and necking phenomena. The corre-
sponding Young’s modulus is 62.7 MPa and viscosity para-
meter η is 83.4 MPa·s.