Polyphosphates and poly-β-hydroxybutyrate granules identification through quantitative image analysis in enhanced biological phosphorus removal systems

Abstract

Enhanced biological phosphorus removal (EBPR) is a widely implemented technique for having the potential to cheaply and reliably remove phosphate from wastewater treatment processes, than traditional chemical methods. EBPR is performed by operating the system sequentially with anaerobic and aerobic conditions. Several studies were already performed ranging from different strategies for the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) to modeling both types of bacterial activities. Until now, slight attention has been given to the development of newer, faster, simpler, and better suited monitoring techniques for this type of system. This work is focused on the development of image analysis techniques for polyphosphates and poly-β-hydroxybutyrate granules in EBPR systems since off-line analyses are labor intensive and not able to be performed in full-scale plants. A lab-scale sequencing batch reactor fed with synthetic wastewater containing volatile fatty acids (VFAs) and orthophosphate was used. The reactor had a working volume of 4 L and was operated with a cycle time of 6 h consisting of 2 h anaerobic, 3 h aerobic, 50 min settling and decanting, and 5 min anaerobic idle periods. In each cycle, 2 L of synthetic wastewater was fed to the reactor in the first 5 min of the anaerobic period, resulting in a hydraulic retention time (HRT) of 12 h. The pH was controlled during both the anaerobic and aerobic periods around 7, and the temperature was controlled at 30 ºC in order to provide selective advantages to GAOs over PAOs. The ratio between chemical oxygen demand (COD) and P in the feed was kept at 10 (gCOD/g P). Biomass samples were collected at the end of the anaerobic and aerobic phases and fixed with phosphate buffer saline solution (PBS) and ethanol. Two fluorescence staining methods were used: (1) DAPI for poly-P identification; and (2) nile blue for poly-β-hydroxybutyrate granules. So far, promising results were achieved regarding the type of images achieved by these fluorescence staining methods and the image analysis procedures still under development