Enhanced biological phosphorus removal (EBPR) from wastewaters is a complex process requiring anaerobic/aerobic conditions and specific wastewater characteristics. It relies on polyphosphate accumulating organisms (PAOs) storing excess amounts of polyphosphate. Carbon transformations involving accumulation and utilisation of polyβ- hydroxyalkanoates (PHAs) and glycogen also are central to EBPR. The primary objective of this project was to use non-culture dependent techniques, particularly 168 rDNA cloning and fluorescence in situ hybridisation (FISH) to investigate particular microbial populations in various EBPR activated sludge communities.
Identification of Polyphosphate Accumulating Organisms. A number of sludges from laboratory-scale sequencing batch reactors (SBRs) performing EBPR were used to generate clone libraries of 16S rRNA genes. Clones belonging to the betaproteobacteria were targetted for the design of FISH probes because there were pre-existing indicators this group of bacteria could be the PAOs. The specific group of betaproteobacteria targetted by the FISH probes were closely related to Rhodocyclus and Propionibacter. This discovery was simultaneously made by another research group who called the target PAOs "Candidatus Accumulibacter phosphatis", or simply Accumulibacter. Sequential FISH and polyphosphate staining provided direct evidence that cells binding the PAO probes contained polyphosphate and were indeed true PAOs. The newly designed PAO probes were also used in FISH experiments to quantify the PAOs in a number of laboratory-scale sludges. The number of PAO-probe positive cells was directly correlated with P-removal efficiency (sludge P content) in each of these sludges.
A laboratory-scale SBR was operated for EBPR in order to provide samples for PAO quantification by FISH and for a range of additional investigations. Inconsistent EBPR performance was achieved during this SBR operation, although diversity of the microbial community was successfully reduced and PAOs were enriched. Micromanipulation was employed in an attempt to isolate PAOs from the SBR. Although no pure culture of a PAO was generated, some descriptive aspects of the microbial community was gained by transmission electron microscopy (TEM).
Identification of Glycogen Accumulating Organisms. Two laboratory-scale sludges (called Q and T sludges) operated with deteriorated EBPR performance were investigated for the presence of glycogen accumulating organisms (GAOs). GAOs have the ability to carry out the same reactions as PAOs, but they do not release or accumulate P under the anaerobic/aerobic cycling conditions. GAOs are presumed to compete with PAOs for carbon in the form of volatile fatty acids (VFAs) and their presence leads to deterioration of EBPR. 16S rDNA clone libraries were prepared from DNA extracted from the Q and T sludges. Representative sequences were phylogenetically analysed and probes targetting each of these groups were designed. All determined sequences in the Q sludge were at least 99.7% identical, forming a novel cluster in the gammaproteobacteria radiation. The T sludge contained clones from the Acidobacteria subphylum-4, candidate bacterial phylum OPIO, the Cytophaga-Flavobacterium subphylum of Bacteriodetes, alphaproteobacteria and gammaproteobacteria radiation with 95% sequence identity to the gammaproteobacteria sequences from the Q sludge.
The populations' targetted by eight probes designed from the clonal sequences were investigated for the GAO phenotype and quantified in fixed Q and T sludge samples. FISH and post-FISH chemical staining were used to determine that bacteria from the novel gammaproteobacteria cluster (named "Candidatus Competibacter phosphatis") were GAOs in Q (92% of all bacteria) and T (28% of all bacteria) sludges and in two fullscale activated sludges. These GAO clone sequences were also found to be very similar to sequences from three previously reported molecular analyses of sludges being investigated for EBPR,
Investigation of PAOs and GAOs in a full-scale system. This study involved the long-term application at the Noosa Wastewater Treatment Plant of FISH probes including the designed PAO and GAO probes and the comparison of the abundance of a range of microorganisms with process performance indicators according to chemical analytical and operational data. One important outcome was the refinement of the area-ratio method of quantifying bacterial populations, in particular the proportion of the Accumulibacter PAOs at Noosa. As with the lab-scale processes, a correlation between the Accumulibacter PAOs and EBPR performance was discovered. The Accumulibacter PAOs comprised 16% of all bacteria during good P removal periods but only 7% during poor EBPR. The combination of FISH and Sudan Black B or Nile blue A staining for PHA permitted the partial to complete identification of three potential competitors to the
Other microorganisms in EBPR. Microscopic investigation of EBPR sludges revealed a number of microorganisms in the bacterial community other than PAOs or GAOs. Actinobacterial sequences were frequently found amongst sequences from EBPR sludge clones. These Actinobacteria were investigated in a number of laboratory-scale sludges. FISH probes were designed from the cloned Actinobacterial 16S rRNA gene sequences to specifically target two populations closely related to Terrabacter tumescens. These targetted Actinobacteria were not observed in high abundance in any laboratory- or fullscale EBPR sludges. Nor did they contain polyphosphate or PHA. One Actinobacterial population targetted by the designed probe 'TerB', was detected in almost all EBPR sludges examined, but its function, if any, in EBPR is not known.
Portuguese EBPR study. A laboratory scale SBR operating for EBPR and fed a mixture of volatile fatty acids (VFAs) showed stable and efficient EBPR capacity over a four year period. P, PHA and glycogen cycling consistent with classical anaerobic/aerobic EBPR were demonstrated with the order of anaerobic VF A uptake being propionate, acetate then butyrate. Two main microorganisms were observed to be responsible for anaerobic/aerobic P and PHA transformations. FISH and post-FISH chemical staining for intracellular polyphosphate and PHA were used to determine that Accumulibacter was the most abundant PAO, comprising 53% of the bacterial community. Also by these methods, Competibacter was determined to be the primary GAO in this EBPR sludge, comprising 13% of the bacteria.
Outcomes. Major findings from this work were that:
• a PAO (Accumulibacter) was identified and determined to play a large role in P removal in all examined EBPR systems
• Accumulibacter was abundant in all full-scale EBPR sludges examined, and in particular one full-scale system (NoosaWTP) results suggested a strong link between this PAO and EBPR; and
• a GAO was identified, was observed to be present in most EBPR sludges and dominated in a number of deteriorated EBPR systems.
The dynamic relationship between PAO and GAOs is suspected to be a key factor in EBPR stability. These research results are currently being used in a study of this relationship, by attempting to combine bacterial population data with chemical operational data in a range of full-scale EBPR systems in order to provide an absolute model of the EBPR process. This model could then provide a true understanding of the key EBPR operational parameters, and how modifications of these parameters could be used to optimise EBPR performance in various systems.