Waste stabilization pond ecology :a molecular approach

Abstract

Waste Stabilization Ponds (WSPs) are used to treat wastewater largely in developing countries, though their ecology is not well understood. Past studies have used taxonomic microscopy methods to assess the photosynthetic organisms vital for WSPs functioning. There has been little use of molecular methods based on evolutionary classification in this field. This thesis sets out to develop molecular methods to study the ecology of these systems. Efficient, non-biased DNA extraction is vital for reliable molecular analysis. Commercially available DNA extraction kits were tested for efficiency when used on WSP samples. Qiagen’s Blood and Tissue kit was recommended for use. The proportion of non-photosynthetic to photosynthetic organisms making up the WSP community was investigated. Fluorescence in Situ Hybridisation (FISH) and flow cytometry based methods were devised to this end. FISH had low efficiency due to variable algal cell wall permeability. Flow cytometry proved to be an effective way to sort photosynthetic organisms from non-photosynthetic, though fixation of samples reduced efficiency. Flow cytometric counting and a PCR and DGGE approach optimised to assess microalgae and cyanobacteria were used in two case studies. The first showed a significant difference between the community found in two pond systems in Brazil, one fed with domestic wastewater and the other with mixed industrial - domestic wastewater. The second assessed the effects of engineered baffles on communities in facultative ponds (in Colombia) across the diurnal cycle. The baffled pond had lower diversity, but more of the species identified were photosynthetic. The PCR-DGGE based method was compared to traditional microscopy techniques with the help of a taxonomic specialist. Little agreement between the methods was seen at species level. The molecular analysis, including the primers chosen and the available database sequences favoured the Chlorophyceae and the cyanobacteria, the microscopy methods favoured the larger Euglenophyceae and other microalgae with morphologically distinct characteristics.