Biofilm development on two distinct rock substrata was investigated both in vitro and in a subsurface flow wastewater treatment wetland in order to determine the effect of hydrophobicity on initial bacterial adsorption, tertiary biofilm development and microbial population structure. Two commonly used wetland rock types, slag (a hydrophobic by-product of the steel smelter industry) and greywacke (a more hydrophilic sedimentary rock) were evaluated. In vitro investigations of initial microbial adsorption trends showed that the more hydrophobic slag displayed rapid bacterial adsorption rates compared to greywacke. Mean microbial adsorption rates of a mixed wetland bacterial population over 5 hours, described using a first order kinetics model, were 1.3 × 10-12 m/sec for slag and consistently lower at 8.7 × 10-13 m/sec for greywacke. Pristine rock studs of the two substrata were also exposed to wetland microbial communities during a six week field trial using confocal scanning laser microscopy to determine tertiary biofilm structure and fluorescent in situ hybridisation to investigate bacterial populations. During the first five weeks of growth CSLM analysis revealed that 75% of biofilms on slag were thicker and had greater coverage compared with those grown on greywacke. After six weeks of growth over 50% of the tertiary biofilms were structurally very similar on both rock types and only 25% of those grown on slag were larger than those on greywacke. In situ hybridisation analysis of bacterial populations revealed very little difference in population structure between biofilms grown on slag and those grown on greywacke. Eubacteria were present as a very high proportion of total bacteria throughout biofilm development (74.3%). The beta subgroup was the most populous of the Proteobacteria (31.4%) followed by the gamma subgroup (13.4%)and the alpha subgroup (1.3%).
The results of this study suggest that slag, as a more hydrophobic substratum, promotes the initial adsorption of bacteria during early biofilm growth and better supports mature biofilm structures when used in wetlands. This study has implications for the design and construction of wastewater treatment wetlands.