Figure. 3: Comparison of biofilm formed at ALI () and LLI ()
assessed by CFU/mL and the percentage of the attached to the total.
(n=18, mean ± SEM) ** p<0.01 (using unpaired t-tests).
No significant differences were observed between the biofilms grown on
ALI and LLI models when comparing the total CFU number. However, when
comparing the proportion of attached bacteria relative to the total
(attached + detached), a significantly higher proportion of biofilm
formation was observed for biofilms growing on ALI than LLI. While the
overall volume of biofilm in ALI and LLI were similar, biofilms grown on
ALI are more resilient and more difficult to detach from the membrane.
Conversely, biofilms grown on LLI are more vulnerable. Hence the
percentage of detached bacteria is greater. Also, the variability for
biofilms grown on LLI is wider, suggesting that these biofilms are more
heterogeneous.
Biofilms can adapt their architecture to cope with different
hydrodynamic conditions and nutrient availability [24]. The
interface on which the biofilm grows specifically determines the
availability and permeability of nutrients and thus will have an impact
on the morphology and properties of the biofilm. Several studies have
attempted to investigate the properties of biofilm formed on a
particular interface. Wu et al. [25] conducted interfacial
rheological measurements on ALI biofilms produced by Escherichia
coli and found that hydrophobic curli fibres associated with the
bacteria improve strength, viscoelasticity, and resistance to ALI
biofilms. Another study by Rühsa et. at. [26] measured the
transient elasticity and viscosity of the biofilm formed at the
water-oil interface using interfacial rheology and pendant drop
tensiometry and found that the ability to form biofilms against oil
improved with increasing hydrophobicity. However, these studies are
unable to provide comparative studies on the effect of the interface
type using a single platform to control the variables, as most of the
instruments are designed to investigate only one specific interface.
While microfluidic instruments have been developed to examine the effect
of external forces such as the hydrodynamic shear force on the growth of
biofilm [27], most of these devices are irreversibly bonded, which
severely undermine the flexibility and potential of incorporating other
end-point analysis approaches such as the approaches we adopted in our
study. The dual-chamber microreactor presented in this study overcome
these challenges by having a design that can be disassembled and with
the capabilities to incorporate multiple interfaces with interchangeable
membrane while keeping other variables (e.g., dynamic flow conditions)
constant between experiments.
The morphology of 48h-old ALI and LLI PAO1 biofilms were also visualized
using CLSM and SEM (Fig.4). The 3D reconstruction of z-stack CLSM images
(Fig. 4 A and D) show that a layer of biofilm was formed over the
membrane and the thickness of the biofilm was approximately 5μm (Fig. 4
B and E). Mushroom-shaped bacterial aggregates dispersed across the
membrane were observed in SEM images in both ALI and LLI models for PAO1
biofilms (Fig. 4 C and F), and their shapes are more distinct in the ALI
compared to the LLI. This is in good agreement with Moller, S., et al.
[5] which demonstrated that biofilms
grown under nutrient depletion are highly structured, and the
’mushroom-shaped’ microcolonies are interspersed between open water
channels. In our study, biofilms have been grown in static conditions
with a finite amount of culture media supplied, resulting in nutritional
exhaustion conditions towards the later stages of bacteria growth.
Moreover, in comparison to ALI culture, LLI culture has more nutrients
coming from the top chamber. Thus, the mushroom-shaped microcolonies
showed in our SEM images in both the LLI and ALI models, and the
difference between them corroborated Moller ’s
[5] discoveries.
Biofilms have also been known to have open water channels that
facilitate the diffusion of nutrients and waste products between the
external environment and biofilm and within the biofilm matrix
[8]. These channels are also evident
in our CLSM images (black space unstained with green and red) and SEM
images (black holes in the biofilm). Comparing Fig. 4 B and E, the
structure of the LLI model biofilm is more dispersed, while the ALI
model biofilm is more aggregated. Also, more water channel holes can be
seen in the LLI model biofilm SEM images. We hypothesize that the media
transmission occurring in the LLI model is higher than the ALI model,
enabling more water channels to be formed to facilitate the transport of
nutrients and waste. Differences in the structure of the biofilm matrix
could contribute to the differences in mechanical properties, which has
been demonstrated through the scattered structures (LLI model biofilm),
making it more vulnerable to disruption of physical forces.
Consequently, the adhesion strength between the biofilm and the
underlaid surface and the cohesive force within the biofilm is weaker
for the LLI biofilm relative to the ALI biofilm. This further supports
the findings in Fig. 3 that LLI biofilms are more susceptible to
physical removal.