A quantitative study on the formation of Pseudomonas aeruginosa biofilm

The effect of temperature on P. aeruginosa biofilm formation was evaluated with respect to three parameters—the mass of biofilm
formed, the production of extracellular polysaccharide and the adhesion force. Quantification
of biofilm biomass was done using the dye crystal violet (Merritt et al. 2011]). Biofilm biomass formed at temperatures 28, 33, 37 and 42°C were 1.25 (O.D), 2.20
(O.D) 2.80 (O.D) and 2.30 (O.D) respectively (Table 1). The results show that biofilm biomass was highest for biofilm sample formed at
37°C. This is due to increased production of extracellular matrix material by P. aeruginosa cells at 37°C.

Table 1. Biofilm formation evaluated at different growth temperature supporting information

Adhesion force of biofilm is a measure of adhesiveness of biofilm (probe-biofilm interaction
force). Adhesion force of biofilm grown at 28, 33, 37 and 42°C were 4.7, 5.4, 10.8
and 6.6 nN respectively (Table 1).

The results indicate that biofilm adhesion force was highest for biofilm sample formed
at 37°C. This is due to increased production (and accumulation) of extracellular matrix
material by P. aeruginosa cells at 37°C (Ashwin et al. 2014]; Oh et al. 2007], 2009]; Fang et al. 2000]). The increased production of extracellular polymeric material is due to higher metabolic
rate of P. aeruginosa cells at 37°C.

Extracellular polysaccharides play an important role in biofilm formation since it
is only when extracellular polysaccharides are produced that cell aggregation and
biofilm formation occur. This is because the extracellular polysaccharides act as
‘cement’ in holding the cells together (Sutherland 2001]).The amount of extracellular polysaccharide produced by P. aeruginosa cells grown at 28, 33, 37 and 42°C were 890, 1,210, 1,570 and 1,240 µg respectively
(Table 1). The results show that extracellular polysaccharide production was highest for biofilm
sample formed at 37°C.

The higher amount of calcium and magnesium ions incorporated in the biofilm (formed
at 37°C: Table 1) suggests that biofilm (formed at 37°C) would have a higher mechanical stability
(than biofilms grown at 28, 33 and 42°C). This is because calcium and magnesium ions
have been reported to bind with negatively charged polysaccharides and have also been
implicated in cell–cell binding mechanisms (Korstgens et al. 2001]; Lattner et al. 2003]).

Surface topography of biofilm was characterized by evaluating its surface roughness.
Surface roughness of biofilm is an important parameter that is characteristic of growth
conditions and has been reported to have an effect on the rate of diffusion of nutrients,
thickness of dissolved oxygen boundary layer and mass transfer resistance (Zhang et
al. 1994]). Surface roughness of biofilm was evaluated using parameter R
_a
. This parameter measures the arithmetic average of deviation about the mean profile
line (Ashwin et al. 2014]). Table 1 also shows that biofilm sample grown at 28°C had the highest surface roughness, although
no specific trend was observed. This indicates that growth temperature has a significant
effect on the surface topography of P. aeruginosa biofilm.

The aforementioned results show that P. aeruginosa biofilm (strain MTCC 2297) formed at 37°C produce higher amount of extracellular
polysaccharide and also have a higher biomass, higher adhesion force and greater mechanical
stability than biofilms formed at other temperatures (in the temperature range 28–42°C).