Relationship Between the Dry Weight and the Biofilm Surface

Supplementary Material 2

Relationship between the dry weight and the biofilm surface

Sampling collection

For both Créteil and Daumesnil Lake, epilithic biofilms were removed from rock surface by scraping 20cm2 biofilm area with a sterile syringe-toothbrush sampler. Epilithic biofilms were stored in a sterile container that waspreviously weighed.

Epiphytic biofilms were collected by harvesting submerged leaves of Phragmites australis (the base attached to the stem) in Créteil Lake and Myriophyllum spicatum within Daumesnil Lake. Epiphytic biofilms were stored in sterile containers. After collection, biofilm samples were placed at 4°C until return to the laboratory,where they were immediately frozen at –20°C in order to be lyophilized.

Surface determination

Dry epilithic biofilms were weighed to quantifying the biofilm mass harvested by scraping 20cm2 of rocks.

For both P. australis and M. spicatum, six replicates of 40mg of dry leaves (mass used for DNA extraction) were placed between two glass plates with a size marker. Pictures taken from these replicates were analyzed using the area calculation tool in ImageJ (Schneider et al. 2012). Regarding P. australis, we assumed that mycobacteriacan only grow on the outer part of the leaves. Thus the area measured by ImageJ corresponds to biofilm area where mycobacteria can grow (Fig. 1). In contrast with M. spicatum,weconsidered that leaves are composed of a multitude of small cylindric objects (Fig. 1). Consequently, to assess the area of cylinders we used the formula: AreaCylinder= pi × diameter × height. However, only a limited area of the cylinders was visible (Fig. 1), this area (in green) corresponding to the parameter: diameter × height. Thus, to determine the whole surface cylinders,representing the biofilm area, we multiplied the visible surface areaby pi.

Figure 1 Diagram illustrating the methodology to determine biofilm areas on P. australis and M. spicatum.

Correlation between dry mass and surface

To determine the relationship between the dry mass of epilithic biofilms and the surface where mycobacteria could proliferate, the following equationwas used for each sample:

(1)

where SurfEpilithic is the surface; M is the dry mass of epilithic biofilm used for the DNA extraction (50mg); SurfScraped is the total surface scrapped during the sample collection, m0 is the mass of the empty container and m1 the mass of the container with dry biofilm.

Measures of biofilm area were compiled in the Table 1. Thus, we admitted that 40 mg of dry P. australisleaves from this plant corresponds to 9.5 cm2 of biofilm surface whereas 40 mg of dry M. spicatum leaves corresponds to 37.6 cm2 of biofilm surface.

Table 1 Biofilm area (in cm2) measured from 40 mg of dry P. australis and M. spicatum leaves

Replicates / Phragmites / Myriophyllum
1 / 10.27 / 35.17
2 / 8.42 / 37.22
3 / 9.88 / 37.34
4 / 8.39 / 38.17
5 / 9.66 / 39.07
6 / 10.11 / 38.45
Mean (cm2) / 9.46 / 37.57
Standard deviation (cm2) / 0.77 / 1.25
Coefficient of variation (%) / 8.11 / 3.31

Reference

Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9: 671–675.