Shear stress affects the architecture and cohesion of Chlorella vulgaris biofilms

Abstract

The architecture of microalgae biofilms has been poorly investigated, in particular with respect to shear stress, which is a crucial factor in biofilm-based reactor design and operation. To investigate how microalgae biofilms respond to different hydrodynamic regimes, the architecture and cohesion of Chlorella vulgaris biofilms were studied in flow-cells at three shear stress: 1.0, 6.5 and 11.0 mPa. Biofilm physical properties and architecture dynamics were monitored using a set of microscopic techniques such as, fluorescence recovery after photobleaching (FRAP) and particle tracking. At low shear, biofilms cohesion was heterogeneous resulting in a strong basal (close to the substrate) layer and in more loose superficial ones. Higher shear (11.0 mPa) significantly increased the cohesion of the biofilms allowing them to grow thicker and to produce more biomass, likely due to a biological response to resist the shear stress. Interestingly, an acclimation strategy seemed also to occur which allowed the biofilms to preserve their growth rate at the different hydrodynamic regimes. Our results are in accordance with those previously reported for bacteria biofilms, revealing some general physical/mechanical rules that govern microalgae life on substrates. These results may bring new insights about how to improve productivity and stability of microalgae biofilm-based systems.

Figure 1

Structural parameters of C. vulgaris biofilms grown in flow-cells at three hydrodynamic shears (1.0, 6.5 and 11.0 mPa). Biovolume (a), roughness (b), mean (c) and max thickness (d) are reported as function of time. The biovolume curves were fitted using the logistic function (dashed lines). The results are reported as the mean and standard deviation of at least three independent biological replicates. Error bars represent the standard deviation (n ≥ 3).

Figure 2

Vertical profile of growth rate in the biofilms formed by C. vulgaris grown at three hydrodynamic shears: 1.0, 6.5 and 11.0 mPa. The growth rate for each layer was calculated as the slope of the linear regression between the coverage of cells in each layer over time. The data are reported as the mean of at least three independent biological replicates (n ≥ 3).

Figure 3

Attenuated light in the biofilms of C. vulgaris grown at three different hydrodynamic shears (1.0, 6.5 and 11.0 mPa). In (a) the attenuated light is shown as a function of time. The relationships among the light attenuated in the biofilms and the structural parameters biovolume (b), mean thickness (c) and maximal thickness (d) of the biofilms are also reported. The data represent the mean of at least three independent biological replicates (n ≥ 3).

Figure 4

Max intensity projections of representative CLSM stacks acquired during the erosion test. The biofilms grown at three shears (1, 6.5 and 11.0 mPa) were subjected to four (9, 18, 36 and 71 mPa) increasing shears (10 min for each shear) and the biovolume of the biofilms still present in the flow cell was measured after each step by CLSM in order to test the cohesiveness of C. vulgaris biofilms. Images are shown for the biofilms grown at 1 and 11.0 mPa and the brightness has been adjusted for visual purposes.

Figure 5

Erosion test performed on the biofilms grown at three different hydrodynamic regimes (1.0, 6.5 and 11.0 mPa). The biofilms were subjected to an increasing set of shears (9.0, 18, 36, and 71 mPa), each step lasted 10 min. The biovolume detached at each shear was calculated with respect to the initial biovolume (pre-erosion test). The results are the mean and standard deviation of at least three independent biological replicates (n ≥ 3). Bars with different letters represent statistically different means (p < 0.05) as determined by pair-wise comparisons after two-way ANOVA considering both the growth shear stress and the shear stress applied during the erosion stress as factors.

Figure 6

Vertical profiles of cell coverage in the biofilms during the erosion test. Panel (a) shows the vertical profile before the erosion test. Panels (b–d) show the vertical distribution of cells after the application of the erosion test for the biofilm grown at three hydrodynamic shears: 1.0, 6.5 and 11.0 mPa, respectively. The biofilms were subjected to an increasing set of shears (9.0, 18, 36, and 71 mPa), each step lasted for 10 min. Error bars represent the standard deviation (n ≥ 3).