Phage-host associations in a full-scale activated sludge plant during sludge bulking.

Sludge bulking, a notorious microbial issue in activated sludge plants, is always accompanied by dramatic changes in the bacterial community. Despite large numbers of phages in sludge systems, their responses to sludge bulking and phage-host associations during bulking are unknown. In this study, high-throughput sequencing of viral metagenomes and bacterial 16S rRNA genes were employed to characterize viral and bacterial communities in a sludge plant under different sludge conditions (sludge volume index (SVI) of 180, 132, and 73 ml/g). Bulking sludges (SVI > 125 ml/g) taken about 10 months apart exhibited similar bacterial and viral composition. This reflects ecological resilience of the sludge microbial community and indicates that changes in viral and bacterial populations correlate closely with each other. Overgrowth of "Candidatus Microthrix parvicella" led to filamentous bulking, but few corresponding viral genotypes were identified. In contrast, sludge viromes were characterized by numerous contigs associated with "Candidatus Accumulibacter phosphatis," suggesting an abundance of corresponding phages in the sludge viral community. Notably, while nitrifiers (mainly Nitrosomonadaceae and Nitrospiraceae) declined significantly along with sludge bulking, their corresponding viral contigs were identified more frequently and with greater abundance in the bulking viromes, implying that phage-mediated lysis might contribute to the loss of autotrophic nitrifiers under bulking conditions.

The potential role of 'Candidatus Microthrix parvicella' in phosphorus removal during sludge bulking in two full-scale enhanced biological phosphorus removal plants.

We investigated the bacterial community compositions and phosphorus removal performance under sludge bulking and non-bulking conditions in two biological wastewater treatment systems (conventional A²/O (anaerobic/anoxic/aerobic) and inverted A²/O (anoxic/anaerobic/aerobic) processes) receiving the same raw wastewater. Sludge bulking resulted in significant shift in bacterial compositions from Proteobacteria dominance to Actinobacteria dominance, characterized by the significant presence of filamentous 'Candidatus Microthrix parvicella'. Quantitative real-time polymerase chain reaction (PCR) analysis revealed that the relative abundance of 'Candidatus Accumulibacter phosphatis', a key polyphosphate-accumulating organism responsible for phosphorus removal, with respect to 16s rRNA genes of total bacteria was 0.8 and 0.7%, respectively, for the conventional and inverted A²/O systems when sludge bulking occurred, which increased to 8.2 and 12.3% during the non-bulking period. However, the total phosphorus removal performance during the bulking period (2-week average: 97 ± 1 and 96 ± 1%, respectively) was not adversely affected comparable to that during the non-bulking period (2-week average: 96 ± 1 and 96 ± 1%, respectively). Neisser staining revealed the presence of large polyphosphate granules in 'Candidatus Microthrix parvicella', suggesting that this microbial group might have been responsible for phosphorus removal during the sludge bulking period when 'Candidatus Accumulibacter phosphatis' was excluded from the 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.

Quantification of pathogenic microorganisms and microbial indicators in three wastewater reclamation and managed aquifer recharge facilities in Europe.

Managed Aquifer Recharge (MAR) is becoming an attractive option for water storage in water reuse processes as it provides an additional treatment barrier to improve recharged water quality and buffers seasonal variations of water supply and demand. To achieve a better understanding about the level of pathogenic microorganisms and their relation with microbial indicators in these systems, five waterborne pathogens and four microbial indicators were monitored over one year in three European MAR sites operated with reclaimed wastewater. Giardia and Cryptosporidium (oo)cysts were found in 63.2 and 36.7% of the samples respectively. Salmonella spp. and helminth eggs were more rarely detected (16.3% and 12.5% of the samples respectively) and Campylobacter cells were only found in 2% of samples. At the Belgian site advanced tertiary treatment technology prior to soil aquifer treatment (SAT) produced effluent of drinking water quality, with no presence of the analysed pathogens. At the Spanish and Italian sites amelioration of microbiological water quality was observed between the MAR injectant and the recovered water. In particular Giardia levels decreased from 0.24-6.14 cysts/L to 0-0.01 cysts/L and from 0.4-6.2 cysts/L to 0-0.07 cysts/L in the Spanish and Italian sites respectively. Salmonella gene copies and Giardia cysts were however found in the water for final use and/or the recovered groundwater water at the two sites. Significant positive Spearman correlations (p<0.05, r(s) range: 0.45-0.95) were obtained, in all the three sites, between Giardia cysts and the most resistant microbial markers, Clostridium spores and bacteriophages.

Enhanced bioremediation of methyl tert-butyl ether (MTBE) by microbial consortia obtained from contaminated aquifer material.

A microcosm study was carried out to evaluate the potential for biodegradation of methyl tert-butyl ether (MTBE) impacting groundwater at a former oil refinery site located in Naples (SW Italy). A screening of aerobic, anaerobic and co-metabolic aerobic conditions was carried out by triplicate batch reactors, using contaminated soil and groundwater from the study site. All microcosms were amended with ammonium and phosphate salts and, if aerobic, they were supplied with excess oxygen throughout the static incubation period of 6 months. Propane, pentane and n-hexane were selected as the primary substrates for co-metabolic treatments. After the initial lag phase (40-60d), quantitative MTBE decay was repeatedly observed in the aerobic set amended only with nitrogen and phosphorus and further fed with MTBE, thus suggesting that the indigenous soil bacteria have the ability to degrade MTBE. All other treatments, i.e., anaerobic and co-metabolic aerobic, resulted unsuccessful after incubation extending up to 190d. Bacterial consortia in the active microcosms were later enriched and further studied through second and third generation batch reactors with no soil, operated under continuous mixing for 5-7 months. MTBE degradation rate progressively increased with reactor operating time, following a zero order kinetics in the concentration range 1-10mgL(-1) and leading to a residual concentration of less than 10microgL(-1). The calculated maximum biodegradation rate was 20mg(MTBE)g(VSS)(-1)h(-1). An accumulation of nitrite ions also occurred after long operating times, thus inhibiting MTBE degradation. This effect was minimized by replacing ammonium with nitrate. Identified degradation intermediates were tert-butyl alcohol and tert-butyl formate. Fluorescent in situ hybridization was applied for a preliminary microbiological screening of the consortia, suggesting that the detected cocci (about 0.5 and 1.5microm diameter, respectively) and long bacilli with a narrow diameter might be as yet undescribed species.

Analysis of the microbial community structure and function of a laboratory scale enhanced biological phosphorus removal reactor.

A laboratory scale sequencing batch reactor (SBR) operating for enhanced biological phosphorus removal (EBPR) and fed with a mixture of volatile fatty acids (VFAs) showed stable and efficient EBPR capacity over a four-year-period. Phosphorus (P), poly-beta-hydroxyalkanoate (PHA) and glycogen cycling consistent with classical anaerobic/aerobic EBPR were demonstrated with the order of anaerobic VFA uptake being propionate, acetate then butyrate. The SBR was operated without pH control and 63.67 +/- 13.86 mg P l-1 was released anaerobically. The P% of the sludge fluctuated between 6% and 10% over the operating period (average of 8.04 +/- 1.31%). Four main morphological types of floc-forming bacteria were observed in the sludge during one year of in-tensive microscopic observation. Two of them were mainly responsible for anaerobic/aerobic P and PHA transformations. Fluorescence in situ hybridization (FISH) and post-FISH chemical staining for intracellular polyphosphate and PHA were used to determine that 'Candidatus Accumulibacter phosphatis' was the most abundant polyphosphate accumulating organism (PAO), forming large clusters of coccobacilli (1.0-1.5 micro m) and comprising 53% of the sludge bacteria. Also by these methods, large coccobacillus-shaped gammaproteobacteria (2.5-3.5 micro m) from a recently described novel cluster were glycogen-accumulating organisms (GAOs) comprising 13% of the bacteria. Tetrad-forming organisms (TFOs) consistent with the 'G bacterium' morphotype were alphaproteobacteria, but not Amaricoccus spp., and comprised 25% of all bacteria. According to chemical staining, TFOs were occasionally able to store PHA anaerobically and utilize it aerobically.

Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating microorganisms.

Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus (P) removal. Considerable effort has been devoted to understanding the biochemical nature of enhanced biological phosphorus removal (EBPR) and it has been shown that intracellular polymer storage plays an important role in PAO's metabolism. The storage capacity of PAOs gives them a competitive advantage over other microorganisms present that are not able to accumulate internal reserves. Intracellular polymers stored by PAOs include polyphosphate (poly-P), polyhydroxyalkanoates (PHAs) and glycogen. Staining procedures for qualitative visualization of polymers by optical microscopy and combinations of these procedures with molecular tools for in situ identification are described here. The strengths and weaknesses of widely used polymer quantification methods that require destruction of samples, are also discussed. Finally, the potential of in vivo nuclear magnetic resonance (NMR) spectroscopy for on-line measurement of intracellular reserves is reported.