Identity and physiology of glycogen accumulating organisms in activated sludge

Abstract

Glycogen accumulating organisms (GAOs) form part of the core microbial component of the activated sludge community of both industrial and domestic wastewater treatment plants. A plethora of research has focused on the activity of GAOs in enhanced biological phosphorus removal (EBPR) domestic wastewater treatment plants due to their competition for carbon with the polyphosphate accumulating organisms (PAOs) at the expense of EBPR efficiency. However, in industrial systems such as winery wastewater (WWW), where GAOs are known to proliferate and cause settling problems, limited research has focused on their identification and ecophysiology. Initial metagenomics and fluorescence in situ hybridization (FISH) surveys of WWW treatment plants revealed that Defluviicoccus-related GAOs belonging to cluster II (DF2) and III (DF3) appeared as highly dominant with abundances reaching up to 52% of the bacterial population. GAOs belonging to the Competibacteraceae (CPB_S18 and CPB_S60) were also observed in notable numbers along with putative GAOs belonging to the Actinobacteria (Micropruina spp. and Nakamurella spp.) and Gammaproteobacteria CCM19a. Previous investigations have suggested that high carbon loads along with low nitrogen levels are a combination of factors that enhance the proliferation of GAOs. In order to elucidate if the low nitrogen levels of WWW are the underlying cause of DF2 proliferation, incubations under different COD:N ratios using ¹³C - acetate and ¹⁵N - urea were performed. Cell substrate assimilation was quantified using an innovative technique, FISH-NanoSIMS, revealing that low (100:1) or null nitrogen concentrations enhanced DF2 carbon uptake while ratios of 60:1 and 20:1 reduced carbon uptake. Nitrogen dosing at COD:N ratios of 60:1 or higher was demonstrated as a feasible strategy for controlling the excessive DF2 growth in WWW treatment plants. The unique filamentous morphology of DF3 is important as its proliferation leads to severe bulking issues. Although previously reported as abundant in industrial and domestic activated sludge, limited research has focused on understanding the physiology of these organisms. The first genome of a filamentous DF3 was extracted from a WWW activated sludge metagenome. Annotation revealed interesting metabolic features that help to understand the competitiveness and abundance of this microorganism in WWW activated sludge. The genetic potential to cycle trehalose through glycogen, nitrogen fixation, hydrogenase activity and urea uptake appear as adaptive strategies of DF3 to the WWW nitrogen limited environment. In a recent 16S rRNA survey of EBPR plants, Micropruina spp. were identified as the most abundant GAO, yet little is known about their ecophysiology. To further elucidate the ecophysiology of this putative GAO observed in WWW and EBPR treatment plants, genomic and metabolomic studies were made in pure culture Micropruina glycogenica str. Lg2T [T superscript] and compared to the in situ physiology of the genus using state-of-the-art single cell techniques. Micropruina spp. were observed to take up carbon substrates under anaerobic conditions, which were partly fermented to lactate, acetate, propionate and ethanol, and partly stored as glycogen for aerobic use. This physiology is markedly different from the classical GAO model, suggesting a need to reconsider current understanding of the GAO phenotype. Metagenomics analyses revealed a codominance of filamentous Thiothrix spp. and GAOs in a particular WWW treatment plant with bulking issues. In an attempt to understand this problem, a further experiment based on raw wastewater feeding was performed. Raw feeding reduced the Thiothrix spp. population and improved settling, therefore, direct feeding is proposed as a control method for industrial plants with surge/anaerobic lagoons in order to manage the bulking problems caused by Thiothrix spp..