Evaluating the effects of strain selection on the attenuation of Bacillus subtilis spores through saturated porous media

Abstract

Increasingly stringent water quality regulations concerning microbiological parameters govern the use of groundwater resources that are vulnerable to mixing with surface waters. These drinking water sources are at higher risk for infiltration by pathogenic microorganisms, including the oocysts of the human enteroparasite Cryptosporidium spp. Cryptosporidium can cause severe gastroenteritis in humans, and the characteristics of Cryptosporidium oocysts, including low infectious dose, high resistance to inactivation, and long survival in the environment pose a significant risk to public health if present in treated drinking water. Bacillus subtilis is widely used as a surrogate for biocolloid transport in saturated porous media, and recognized as a conservative indicator for the transport of Cryptosporidium parvum oocysts during filtration. However, no study has directly compared the transport of spores from different strains within the Bacillus subtilis species. Strain variability has the potential to impact retention in porous media based on differences in size and electrophoretic mobility. The transport behaviour of four strains of Bacillus subtilis (wild-type and laboratory type, subspecies subtilis and subspecies spizizenii; 1.9 to 2.9µm diameter) is contrasted in this research to two sizes of fluorescent polystyrene microspheres (1.1µm and 4.5µm diameter) through packed saturated crushed quartz sand. A peristaltic pump was used to introduce (bio)colloids into the duplicate column apparatus at a loading rate of 0.1m/day. (Bio)colloid removal was assessed and compared by constructing breakthrough curves of normalized concentrations and box-and-whisker diagrams of percent removal of Bacillus subtilis strains. Under unfavourable conditions minimal reduction (<0.22log10) in effluent spore concentration was observed over the column depth of 15cm. In favourable attachment conditions up to 0.69 log10 reduction was observed but the sampling schedule employed was insufficient to clearly identify a pseudo steady-state plateau. An analysis of variance was used to determine the statistical significance of spore strain, subspecies, and type. A significant difference between the four strains was observed at the lower ionic strength, with spore subspecies and type affecting spore removal in unfavourable conditions (p < 0.05). Some sensitivity to settling and laboratory storage suggests that standardized sample handling procedures are required. Differences observed here between the strains of Bacillus subtilis spores indicate that riverbank filtration performance assessments and drinking water treatment plant process demonstrations may benefit from a recommended strain for use.