Fast DNA-analyses for surveillance of microbial communities in full-scale deammonification tanks: Potential for control and troubleshooting.

The partial nitritation/anammox process is a popular process for sidestream nitrogen removal, but the process is sensitive to disturbances and requires extensive surveillance and monitoring for optimal performance. We followed two parallel sidestream full-scale deammonification reactors treating digester centrate for a year with high time-resolution of both online sensor data and microbial community as measured by Nanopore DNA sequencing. DNA surveillance revealed system disturbances and allowed for detection of process and equipment upsets, and it facilitated remediating operational actions. Surveillance of anammox bacteria (Ca. Brocadia) revealed unexpected variations, and the composition and dynamics of the flanking community indicated causes for occasional process disturbances with poor nitrogen removal. Monitoring the ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) could potentially allow reactor operation with increased dissolved oxygen (DO), yielding higher ammonia conversion while keeping NOB in control. The use of fast and frequent DNA sequencing (sampling 3-5 times a week, analysed once per week) was an important supplement, and in many cases superior, to the online sensor data for process surveillance, understanding and control.

Full-scale activated sludge transplantation reveals a highly resilient community structure.

Well-functioning and stable microbial communities are critical for the operation of activated sludge (AS) wastewater treatment plants (WWTPs). Bioaugmentation represents a potentially useful approach to recover deteriorated systems or to support specific AS processes, but its application in full-scale WWTPs is generally problematic. We conducted a massive transplantation (in one day) exchanging AS from a donor to a recipient full-scale WWTP with similar process type (biological removal of nitrogen and phosphorus) and performance, but with differences in microbial community structure. The treatment performance in the recipient plant was not compromised and the effluent quality remained stable. The AS community structure of the recipient plant was initially very similar to the donor AS, but it almost completely restored the pre-transplantation structure approximately 40 days after transplantation, corresponding to 3 times the solid retention time. Most of the unique species of donor AS added to recipient AS disappeared quickly, although some disappeared more slowly the following months, indicating some survival and potentially a time limited function in the recipient plant. Moreover, the addition in higher abundance of most species already present in the recipient AS (e.g., the polyphosphate accumulating organisms) or the reduction of the abundance of unwanted bacteria (e.g., filamentous bacteria) in the recipient plant was not successful. Moreover, we observed similar abundance patterns after transplantation for species belonging to different functional guilds, so we did not observe an increase of the functional redundancy. Investigations of the microbial community structure in influent wastewater revealed that for some species the abundance trends in the recipient plant were closely correlated to their abundance in the influent. We showed that a very resilient microbial community was responsible for the outcome of the transplantation of AS at full-scale WWTP, potentially as a consequence of mass-immigration from influent wastewater. The overall results imply that massive transplantation of AS across different WWTPs is not a promising strategy to permanently solve operational problems. However, by choosing a compatible AS donor, short term mitigation of serious operational problems may be possible.

Candidatus Amarolinea and Candidatus Microthrix Are Mainly Responsible for Filamentous Bulking in Danish Municipal Wastewater Treatment Plants.

Filamentous bulking is a common serious operational problem leading to deteriorated sludge settling that has long been observed in activated sludge biological wastewater treatment systems. A number of bacterial genera found therein possess filamentous morphology, where some have been shown to be implicated in bulking episodes (e.g., Ca. Microthrix), the impact of many others is still not clear. In this study we performed a survey of 17 Danish municipal wastewater treatment plants (WWTPs) with nutrient removal using 16S rRNA amplicon sequencing over a period of 13 years, where all known filamentous bacteria from 30 genera were analyzed. The filamentous community constituted on average 13 ± 6%, and up to 43% of total read abundance with the same genera common to all plants. Ca. Microthrix and several genera belonging to phylum Chloroflexi were among the most abundant filamentous bacteria. The effect of filamentous bacteria on sludge settling properties was analyzed using measurements of the diluted sludge volume index (DSVI). Strong positive correlations with DSVI were observed only for Ca. Microthrix and Ca. Amarolinea, the latter being a novel, recently characterized genus belonging to the phylum Chloroflexi. The bulking potential of other filamentous bacteria was not significant despite their presence in many plants. Low phylogenetic diversity was observed for both Ca. Microthrix and Ca. Amarolinea, making physiological characterization of individual species and potential development of control strategies more feasible. In this study we show that, despite the high diversity of filamentous phylotypes in Danish WWTPs, only few of them were responsible for severe bulking episodes.

A Critical Assessment of the Microorganisms Proposed to be Important to Enhanced Biological Phosphorus Removal in Full-Scale Wastewater Treatment Systems.

Understanding the microbiology of phosphorus (P) removal is considered essential to knowledge-based optimization of enhanced biological P removal (EBPR) systems. Biological P removal is achieved in these systems by promoting the growth of organisms collectively known as the polyphosphate accumulating organisms (PAOs). Also considered important to EBPR are the glycogen accumulating organisms (GAOs), which are theorized to compete with the PAOs for resources at the expense of P removal efficiency. Numerous studies have sought to identify the PAOs and their GAOs competitors, with several candidates proposed for each over the last few decades. The current study collectively assessed the abundance and diversity of all proposed PAOs and GAOs in 18 Danish full-scale wastewater treatment plants with well-working biological nutrient removal over a period of 9 years using 16S rRNA gene amplicon sequencing. The microbial community structure in all plants was relatively stable over time. Evidence for the role of the proposed PAOs and GAOs in EBPR varies and is critically assessed, in light of their calculated amplicon abundances, to indicate which of these are important in full-scale systems. Bacteria from the genus Tetrasphaera were the most abundant of the PAOs. The "Candidatus Accumulibacter" PAOs were in much lower abundance and appear to be biased by the amplicon-based method applied. The genera Dechloromonas, Microlunatus, and Tessaracoccus were identified as abundant putative PAO that require further research attention. Interestingly, the actinobacterial Micropruina and sbr-gs28 phylotypes were among the most abundant of the putative GAOs. Members of the genera Defluviicoccus, Propionivibrio, the family Competibacteraceae, and the spb280 group were also relatively abundant in some plants. Despite observed high abundances of GAOs (periodically exceeding 20% of the amplicon reads), P removal performance was maintained, indicating that these organisms were not outcompeting the PAOs in these EBPR systems. Phylogenetic diversity within each of the PAOs and GAOs genera was observed, which is consistent with reported metabolic diversity for these. Whether or not key traits can be assigned to sub-genus level clades requires further investigation.

"Candidatus Propionivibrio aalborgensis": A Novel Glycogen Accumulating Organism Abundant in Full-Scale Enhanced Biological Phosphorus Removal Plants.

Enhanced biological phosphorus removal (EBPR) is widely used to remove phosphorus from wastewater. The process relies on polyphosphate accumulating organisms (PAOs) that are able to take up phosphorus in excess of what is needed for growth, whereby phosphorus can be removed from the wastewater by wasting the biomass. However, glycogen accumulating organisms (GAOs) may reduce the EBPR efficiency as they compete for substrates with PAOs, but do not store excessive amounts of polyphosphate. PAOs and GAOs are thought to be phylogenetically unrelated, with the model PAO being the betaproteobacterial "Candidatus Accumulibacter phosphatis" (Accumulibacter) and the model GAO being the gammaproteobacterial "Candidatus Competibacter phosphatis". Here, we report the discovery of a GAO from the genus Propionivibrio, which is closely related to Accumulibacter. Propionivibrio sp. are targeted by the canonical fluorescence in situ hybridization probes used to target Accumulibacter (PAOmix), but do not store excessive amounts of polyphosphate in situ. A laboratory scale reactor, operated to enrich for PAOs, surprisingly contained co-dominant populations of Propionivibrio and Accumulibacter. Metagenomic sequencing of multiple time-points enabled recovery of near complete population genomes from both genera. Annotation of the Propionivibrio genome confirmed their potential for the GAO phenotype and a basic metabolic model is proposed for their metabolism in the EBPR environment. Using newly designed fluorescence in situ hybridization (FISH) probes, analyses of full-scale EBPR plants revealed that Propionivibrio is a common member of the community, constituting up to 3% of the biovolume. To avoid overestimation of Accumulibacter abundance in situ, we recommend the use of the FISH probe PAO651 instead of the commonly applied PAOmix probe set.

Integrative microbial community analysis reveals full-scale enhanced biological phosphorus removal under tropical conditions.

Management of phosphorus discharge from human waste is essential for the control of eutrophication in surface waters. Enhanced biological phosphorus removal (EBPR) is a sustainable, efficient way of removing phosphorus from waste water without employing chemical precipitation, but is assumed unachievable in tropical temperatures due to conditions that favour glycogen accumulating organisms (GAOs) over polyphosphate accumulating organisms (PAOs). Here, we show these assumptions are unfounded by studying comparative community dynamics in a full-scale plant following systematic perturbation of operational conditions, which modified community abundance, function and physicochemical state. A statistically significant increase in the relative abundance of the PAO Accumulibacter was associated with improved EBPR activity. GAO relative abundance also increased, challenging the assumption of competition. An Accumulibacter bin-genome was identified from a whole community metagenomic survey, and comparative analysis against extant Accumulibacter genomes suggests a close relationship to Type II. Analysis of the associated metatranscriptome data revealed that genes encoding proteins involved in the tricarboxylic acid cycle and glycolysis pathways were highly expressed, consistent with metabolic modelling results. Our findings show that tropical EBPR is indeed possible, highlight the translational potential of studying competition dynamics in full-scale waste water communities and carry implications for plant design in tropical regions.

Complete Genome Sequence of the Bacterium Aalborg_AAW-1, Representing a Novel Family within the Candidate Phylum SR1.

Here, we present the complete genome sequence of the candidate phylum SR1 bacterium Aalborg_AAW-1. Its 16S rRNA gene is only 85.5% similar to that of the closest relative, RAAC1_SR1, and the genome of Aalborg_AAW-1 consequently represents the first of a novel family within the candidate phylum SR1.

'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.