Simon Jon McIlroy

MiDAS: the field guide to the microbes of activated sludge.

The Microbial Database for Activated Sludge (MiDAS) field guide is a freely available online resource linking the identity of abundant and process critical microorganisms in activated sludge wastewater treatment systems to available data related to their functional importance. Phenotypic properties of some of these genera are described, but most are known only from sequence data. The MiDAS taxonomy is a manual curation of the SILVA taxonomy that proposes a name for all genus-level taxa observed to be abundant by large-scale 16 S rRNA gene amplicon sequencing of full-scale activated sludge communities. The taxonomy can be used to classify unknown sequences, and the online MiDAS field guide links the identity to the available information about their morphology, diversity, physiology and distribution. The use of a common taxonomy across the field will provide a solid foundation for the study of microbial ecology of the activated sludge process and related treatment processes. The online MiDAS field guide is a collaborative workspace intended to facilitate a better understanding of the ecology of activated sludge and related treatment processes—knowledge that will be an invaluable resource for the optimal design and operation of these systems. Database URL: http://www.midasfieldguide.org

Ecology of the Microbial Community Removing Phosphate from Wastewater under Continuously Aerobic Conditions in a Sequencing Batch Reactor

ABSTRACT All activated sludge systems for removing phosphate microbiologically are configured so the biomass is cycled continuously through alternating anaerobic and aerobic zones. This paper describes a novel aerobic process capable of decreasing the amount of phosphate from 10 to 12 mg P liter−1 to less than 0.1 mg P liter−1 (when expressed as phosphorus) over an extended period from two wastewaters with low chemical oxygen demand. One wastewater was synthetic, and the other was a clarified effluent from a conventional activated sludge system. Unlike anaerobic/aerobic enhanced biological phosphate removal (EBPR) processes where the organic substrates and the phosphate are supplied simultaneously to the biomass under anaerobic conditions, in this aerobic process, the addition of acetate, which begins the feed stage, is temporally separated from the addition of phosphate, which begins the famine stage. Conditions for establishing this process in a sequencing batch reactor are detailed, together with a description of the changes in poly-β-hydroxyalkanoate (PHA) and poly(P) levels in the biomass occurring under the feed and famine regimes, which closely resemble those reported in anaerobic/aerobic EBPR processes. Profiles obtained with denaturing gradient gel electrophoresis were very similar for communities fed both wastewaters, and once established, these communities remained stable over prolonged periods of time. 16S rRNA-based clone libraries generated from the two communities were also very similar. Fluorescence in situ hybridization (FISH)/microautoradiography and histochemical staining revealed that “Candidatus Accumulibacter phosphatis” bacteria were the dominant poly(P)-accumulating organisms (PAO) in both communities, with the phenotype expected for PAO. FISH also identified large numbers of betaproteobacterial Dechloromonas and alphaproteobacterial tetrad-forming organisms related to Defluviicoccus in both communities, but while these organisms assimilated acetate and contained intracellular PHA during the feed stages, they never accumulated poly(P) during the cycles, consistent with the phenotype of glycogen-accumulating organisms.

A metabolic model for members of the genus Tetrasphaera involved in enhanced biological phosphorus removal

Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to ‘Candidatus Accumulibacter phosphatis’ is unclear, although they may occupy different ecological niches in EBPR communities. The genomes of four Tetrasphaera isolates (T. australiensis, T. japonica, T. elongata and T. jenkinsii) were sequenced and annotated, and the data used to construct metabolic models. These models incorporate central aspects of carbon and phosphorus metabolism critical to understanding their behavior under the alternating anaerobic/aerobic conditions encountered in EBPR systems. Key features of these metabolic pathways were investigated in pure cultures, although poor growth limited their analyses to T. japonica and T. elongata. Based on the models, we propose that under anaerobic conditions the Tetrasphaera-related PAOs take up glucose and ferment this to succinate and other components. They also synthesize glycogen as a storage polymer, using energy generated from the degradation of stored polyphosphate and substrate fermentation. During the aerobic phase, the stored glycogen is catabolized to provide energy for growth and to replenish the intracellular polyphosphate reserves needed for subsequent anaerobic metabolism. They are also able to denitrify. This physiology is markedly different to that displayed by ‘Candidatus Accumulibacter phosphatis’, and reveals Tetrasphaera populations to be unusual and physiologically versatile PAOs carrying out denitrification, fermentation and polyphosphate accumulation.

An examination of the mechanisms for stable foam formation in activated sludge systems

Screening pure cultures of 65 mycolic acid producing bacteria (Mycolata) isolated mainly from activated sludge with a laboratory based foaming test revealed that not all foamed under the conditions used. However, for most, the data were generally consistent with the flotation theory as an explanation for foaming. Thus a stable foam required three components, air bubbles, surfactants and hydrophobic cells. With non-hydrophobic cells, an unstable foam was generated, and in the absence of surfactants, cells formed a greasy surface scum. Addition of surfactant converted a scumming population into one forming a stable foam. The ability to generate a foam depended on a threshold cell number, which varied between individual isolates and reduced markedly in the presence of surfactant. Consequently, the concept of a universal threshold applicable to all foaming Mycolata is not supported by these data. The role of surfactants in foaming is poorly understood, but evidence is presented for the first time that surfactin synthesised by Bacillus subtilis may be important.

Identification of active denitrifiers in full-scale nutrient removal wastewater treatment systems

Denitrification is essential to the removal of nitrogen from wastewater during treatment, yet an understanding of the diversity of the active denitrifying bacteria responsible in full-scale wastewater treatment plants (WWTPs) is lacking. In this study, stable-isotope probing (SIP) was applied in combination with microautoradiography (MAR)-fluorescence in situ hybridization (FISH) to identify previously unrecognized active denitrifying phylotypes in a full-scale WWTP with biological N and P removal. Acknowledging that different denitrifiers will have specific carbon source preferences, a fully (13)C-labelled complex substrate was used for SIP incubations, under nitrite-reducing conditions, in order to maximize the capture of the potentially metabolically diverse denitrifiers likely present. Members of the Rhodoferax, Dechloromonas, Sulfuritalea, Haliangium and Thermomonas were represented in the 16S rRNA gene clone libraries from DNA enriched in (13)C, with FISH probes optimized here for their in situ characterization. FISH and MAR confirmed that they were all active denitrifiers in the community. The combined approach of SIP and MAR-FISH represents an excellent approach for identifying and characterizing an un-described diversity of active denitrifiers in full-scale systems.

'Candidatus Competibacter'-lineage genomes retrieved from metagenomes reveal functional metabolic diversity

The glycogen-accumulating organism (GAO) ‘Candidatus Competibacter’ (Competibacter) uses aerobically stored glycogen to enable anaerobic carbon uptake, which is subsequently stored as polyhydroxyalkanoates (PHAs). This biphasic metabolism is key for the Competibacter to survive under the cyclic anaerobic-‘feast’: aerobic-‘famine’ regime of enhanced biological phosphorus removal (EBPR) wastewater treatment systems. As they do not contribute to phosphorus (P) removal, but compete for resources with the polyphosphate-accumulating organisms (PAO), thought responsible for P removal, their proliferation theoretically reduces the EBPR capacity. In this study, two complete genomes from Competibacter were obtained from laboratory-scale enrichment reactors through metagenomics. Phylogenetic analysis identified the two genomes, ‘Candidatus Competibacter denitrificans’ and ‘Candidatus Contendobacter odensis’, as being affiliated with Competibacter-lineage subgroups 1 and 5, respectively. Both have genes for glycogen and PHA cycling and for the metabolism of volatile fatty acids. Marked differences were found in their potential for the Embden–Meyerhof–Parnas and Entner–Doudoroff glycolytic pathways, as well as for denitrification, nitrogen fixation, fermentation, trehalose synthesis and utilisation of glucose and lactate. Genetic comparison of P metabolism pathways with sequenced PAOs revealed the absence of the Pit phosphate transporter in the Competibacter-lineage genomes—identifying a key metabolic difference with the PAO physiology. These genomes are the first from any GAO organism and provide new insights into the complex interaction and niche competition between PAOs and GAOs in EBPR systems.

Metabolic model for the filamentous 'Candidatus Microthrix parvicella' based on genomic and metagenomic analyses

‘Candidatus Microthrix parvicella’ is a lipid-accumulating, filamentous bacterium so far found only in activated sludge wastewater treatment plants, where it is a common causative agent of sludge separation problems. Despite attracting considerable interest, its detailed physiology is still unclear. In this study, the genome of the RN1 strain was sequenced and annotated, which facilitated the construction of a theoretical metabolic model based on available in situ and axenic experimental data. This model proposes that under anaerobic conditions, this organism accumulates preferentially long-chain fatty acids as triacylglycerols. Utilisation of trehalose and/or polyphosphate stores or partial oxidation of long-chain fatty acids may supply the energy required for anaerobic lipid uptake and storage. Comparing the genome sequence of this isolate with metagenomes from two full-scale wastewater treatment plants with enhanced biological phosphorus removal reveals high similarity, with few metabolic differences between the axenic and the dominant community ‘Ca. M. parvicella’ strains. Hence, the metabolic model presented in this paper could be considered generally applicable to strains in full-scale treatment systems. The genomic information obtained here will provide the basis for future research into in situ gene expression and regulation. Such information will give substantial insight into the ecophysiology of this unusual and biotechnologically important filamentous bacterium.

The microbiology of phosphorus removal in activated sludge processes - the current state of play.

This review discusses critically what we know and would like to know about the microbiology of phosphorus (P) removal in activated sludge systems. In particular, the description of the genome sequences of two strains of the polyphosphate accumulating organism found in these processes, Candidatus ‘Accumulibacter phosphatis’, allows us to address many of the previously unanswered questions relating to how these processes behave, and to raise new questions about the microbiology of P removal. This article attempts to be deliberately speculative, and inevitably subjective, but hopefully at the same time useful to those who have an active interest in these environmentally very important processes.

Filamentous Bacterium Eikelboom Type 0092 in Activated Sludge Plants in Australia Is a Member of the Phylum Chloroflexi

ABSTRACT Molecular data show that the filamentous bacterium Eikelboom type 0092, frequently seen in Australian activated sludge plants, is a member of the phylum Chloroflexi. Fluorescence in situ hybridization (FISH) probes designed against cloned 16S rRNA sequences from a full-scale enhanced biological phosphate removal-activated sludge plant community, where this was a dominant filament morphotype, suggest that it can exist as two variants, differing in their trichome diameter. When applied to samples from several treatment plants in eastern Australia, each FISH probe targeted only the type 0092 filament morphotype against which it was designed. The patterns of FISH signals generated with both were consistent with the ribosomes not being evenly distributed but arranged as intracellular aggregates. The FISH survey data showed that these two variants appeared together in most but not all of the plants examined. None stained positively for intracellular presence of either poly-β-hydroxyalkanoates or polyphosphate.

Extracting nucleic acids from activated sludge which reflect community population diversity

Critical to most studies in molecular microbial ecology is the application of DNA/RNA extraction methods which can reveal the true level of population biodiversity present in samples from the community under investigation. Activated sludge communities have been studied extensively using molecular methods, but rarely have the nucleic acid isolation methods applied been assessed for their ability to achieve this. This study compares eight published RNA and DNA extraction protocols and one commercially available DNA isolation kit for their capacity to provide high quality nucleic acids that reflect the community composition. Each method was assessed on the basis of nucleic acid yield, purity and integrity, and the ability to provide PCR amplifiable RNA and DNA from known marker populations that varied in their resistance to nucleic acid extraction. Only three consistently provided DNA from each of the marker populations known to be present in the samples from fluorescence in situ hybridisation analysis. The failure of the other methods emphasises the need to validate all DNA/RNA extraction protocols. It is recommended that several validated extraction methods be used and the extracts pooled to further minimise any risk of bias.