Study of the tensile properties of individual multicellular fibres generated by Bacillus subtilis

Abstract

Multicellular fibres formed by Bacillus subtilis (B. subtilis) are attracting interest because of their potential application as degradable biomaterials. However, mechanical properties of individual fibres remain unknown because of their small dimensions. Herein, a new approach is developed to investigate the tensile properties of individual fibres with an average diameter of 0.7 μm and a length range of 25.7-254.3 μm. Variations in the tensile strengths of fibres are found to be the result of variable interactions among pairs of microbial cells known as septa. Using Weibull weakest-link model to study this mechanical variability, we predict the length effect of the sample. Moreover, the mechanical properties of fibres are found to depend highly on relative humidity (RH), with a brittle-ductile transition occurring around RH = 45%. The elastic modulus is 5.8 GPa in the brittle state, while decreases to 62.2 MPa in the ductile state. The properties of fibres are investigated by using a spring model (RH < 45%) for its elastic behaviour, and the Kelvin-Voigt model (RH > 45%) for the time-dependent response. Loading-unloading experiments and numerical calculations demonstrate that necking instability comes from structural changes (septa) and viscoelasticity dominates the deformation of fibres at high RH.

Figure 1. Tensile tests under various humidity levels on B. subtilis multicellular fibres with different genes deleted.

(a) Force–displacement curves of B. subtilis multicellular fibres with sigD, lytE, and lytD deleted. These studies were conducted at 22 ± 2 °C with relative humidity RH = 28 ± 8%. (b) Stress–strain curves corresponding to (a). (c) Stress–strain curves of B. subtilis multicellular fibres with sigD and lytE deleted. These studies were conducted at room temperature with low RH (<45%). (d,e) Tensile tests of B. subtilis multicellular fibres with sigD, lytE, and lytD deleted under various RH (24–60%). When RH < 45%, the stress and strain have an approximately linear relation, which is a typical property of elastic–brittle materials. When RH > 45%, the stress–strain curves exhibit viscoelastic behaviour and necking failure.

Figure 2

(a,b) Scanning electron micrographs of the fracture cross-sections of multicellular fibres at low RH. Fracture occurs at the positions of the septa. (c,d) Transmission electron micrographs of septa at various separation levels.

Figure 3. Scanning electron micrographs show ductile deformation of typical multicellular fibres at high RH.

Viscoelasticity gradually dominates deformation of the fibres at high RH. The fibre elongates significantly both in the bacterial cells and at the positions of septa, and necking failure occurs.

Figure 4. Mechanical models for B. subtilis multicellular fibres at low and high RH.

(a) Schematic diagram of cellular indention developed in previous studies. The AFM tip first approaches the outer layer of the cell envelope in the short-axis direction, and then transfers the pressure load from the outer layer to the inner layer. (b) Tensile test of individual fibre in this study. The load is along the long-axis direction of the B. subtilis cell. The cell wall and plasma membrane are simultaneously stretched. (c) Spring model is used to describe the elastic properties of the fibre at low RH; this shows a stress–strain curve of a typical sample and the corresponding linear fitting result at RH = 26%. (d) Kelvin–Voigt model is used to describe the tensile mechanical behaviour of the fibre at high RH; this shows the parameter fitting for a typical sample at RH = 52%. In the inset, the model is shown as a parallel combination of a spring and a dashpot.

Figure 5. Relationship between fracture load and sample length of multicellular fibres generated from B. subtilis with sigD and lytE genes, and with sigD, lytE and lytD genes deleted, measured at room temperature and low RH (<45%).

Figure 6. Modified Weibull distribution of the fibres’ average tensile strength.

(a,b) Tensile strength at low RH. Weibull distribution parameters can be obtained by fitting the experimental data to the linear function in equation (6) using the least-squares method. For multicellular fibres with three genes deleted, their Weibull modulus m and average tensile strength are 2.1 and 63.3 MPa, respectively; for multicellular fibres with two genes deleted, they are 1.8 and 82.5 MPa, respectively. (c) Tensile strength at high RH. The parameters m and are 1.7 and 33.7 MPa, respectively, for fibres with three genes deleted.

Figure 7. Materials.

(a) Photograph of B. subtilis 168 (left) and B. subtilis multicellular fibres (right). (b) SEM image of the disorderly-stacked fibres. (c) An individual chain-like fibre composed of bacterial cells connected by septa.

Figure 8. Testing platform and measurement method.

(a)Tensile testing system. ①m-MTS system. ②m-MTS control unit. ③Micro/nanomanipulator. ④Micro/nanomanipulator control unit. ⑤ Optical microscope. ⑥ Microscope light source. The inset shows an enlarged view of the selected area. (b) A reliable structure for uniaxial tensile testing of an individual fibre is formed. ⑦ A 100-μm-diameter tungsten wire fixed on the 5 × 5 mm² transparent plate. ⑧ Force-sensing probe. ⑨ Individual B. subtilis multicellular fibre. (c) Schematic of the tensile testing.

Figure 9. Procedure of the liquid drop method (LDM).

(a) A drop of fibre suspension is transferred to the surface of the transparent plate to cover the fixed tungsten wire. (b) One end of a fibre is adhered to the tungsten wire by capillary force (denoted by the red arrow), while the other end is free (denoted by the black arrow). (c) A sensing probe controlled by the micro/nanomanipulator moves to the free end of the selected fibre and clamps it with the aid of the capillary force. (d) A reliable uniaxial tension structure of the individual fibre is formed.

Figure 10. Process of manipulating the probe.

(a)The sensing probe is moved to the free end of a fibre. (b,c) The probe is lifted until the tip is detached from the liquid surface. (d) The whole system is kept stationary until the suspension evaporates. (e) The probe is lowered to recover the fibre in the horizontal plane.