Comparison between kinetics of autochthonous marine bacteria in activated sludge and granular sludge systems at different salinity and SRTs.

Biological nutrient removal performances and kinetics of autochthonous marine biomass in forms of activated sludge and aerobic granular sludge were investigated under different salinity and sludge retention time (SRT). Both the biomasses, cultivated from a fish-canning wastewater, were subjected to stepwise increases in salinity (+2 gNaCl L-1), from 30 gNaCl L-1 up to 50 gNaCl L-1 with the aim to evaluate the maximum potential in withstanding salinity by the autochthonous marine biomass. Microbial marine species belonging to the genus of Cryomorphaceae and of Rhodobacteraceae were found dominant in both the systems at the maximum salinity tested (50 gNaCl L-1). The organic carbon was removed with a yield of approximately 98%, irrespective of the salinity. Similarly, nitrogen removal occurred via nitritation-denitritation and was not affected by salinity. The ammonium utilization rate and the nitrite utilization rate were approximately of 3.60 mgNH4-N gVSS-1h-1 and 10.0 mgNO2-N gVSS-1h-1, respectively, indicating a high activity of nitrifying and denitrifying bacteria. The granulation process did not provide significant improvements in the nutrients removal process likely due to the stepwise salinity increase strategy. Biomass activity and performances resulted affected by long SRT (27 days) due to salt accumulation within the activated sludge flocs and granules. In contrast, a lower SRT (14 days) favoured the discharge of the granules and flocs with higher inert content, thereby enhancing the biomass renewing. The obtained results demonstrated that the use of autochthonous-halophilic bacteria represents a valuable solution for the treatment of high-strength carbon and nitrogen saline wastewater in a wide range of salinity. Besides, the stepwise increase in salinity and the operation at low SRT enabled high metabolic activity and to avoid excessive accumulation of salt within the biomass aggregates, limiting their physical destructuration due to the increase in loosely-bound exopolymers.

Control strategy for filamentous sludge bulking: Bench-scale test and full-scale application.

Sludge bulking caused by the overgrowth of filamentous bacteria, especially Microthrix parvicella, has been observed in WWTPs worldwide during low-temperature periods. In this study, the impacts of sludge load on the in situ growth of M. parvicella and sludge settleability were first evaluated at 15 °C over a period of 500 d using a bench-scale anaerobic-anoxic-aerobic reactor fed with raw sewage from a full-scale WWTP. When the reactor was operated at a sludge load of 0.07 ±â€¯0.015 kg Chemical Oxygen Demand (COD) (kg MLSS·d)-1 for 120 d, the sludge volume index (SVI) increased gradually from 85 mL g-1 to 157 mL g-1, and the abundance of M. parvicella quantified by qPCR and FISH methods also increased from 0.42% to 4.63% and 1.56%-13.59%, respectively. When the sludge load was further reduced to 0.04 ±â€¯0.004 kg COD (kg MLSS·d)-1, the SVI value varied in a narrow range of 135-164 mL g-1 over a duration of 280 d, while the M. parvicella abundance increased to the maximum values of 10.13% (qPCR) and 18.53% (FISH), respectively. When the sludge load was increased to 0.12 ±â€¯0.016 kg COD (kg MLSS·d)-1, filamentous abundance and SVI were reduced to 1.06% (qPCR) and 105 mL g-1 within 100 d, suggesting that it might be possible to control the growth of M. parvicella by keeping the sludge load above 0.1 kg COD (kg MLSS·d)-1. The feasibility of the strategy was further validated in the same WWTP. It was found that the SVI and filamentous abundance in winter were successfully controlled for two successive years at below 120 mL g-1 and 7% (FISH), respectively, when the sludge load was maintained at 0.14 ±â€¯0.04 kg COD (kg MLSS·d)-1 by adjusting sludge discharge, proving that this sludge-load-based strategy could be an efficient approach to control filamentous bulking.

Factors affecting the growth of Microthrix parvicella: Batch tests using bulking sludge as seed sludge.

Sludge bulking caused by the overgrowth of filamentous bacteria, particularly Microthrix parvicella, is one of the challenges for the stable operation of municipal wastewater treatment plants (WWTPs). The driving forces for the development of sludge bulking, however, have not been well understood because of the extremely low growth rate of M. parvicella. In this study, batch experiments were performed using bulking sludge (sludge volume index (SVI), around 185mLg-1) from a full-scale WWTP as the seed sludge to investigate the influences of carbon source, anaerobic/aerobic alternation condition and temperature on the growth of M. parvicella. The qPCR results showed that the use of oleic acid as carbon source, anaerobic/aerobic alternation treatment and low temperature (13°C) were favorable conditions for maintaining the dominance of M. parvicella in the tested activated sludge. Under these conditions, the SVI values remained at comparatively high values of 170.5mLg-1, 162.5mLg-1 and 129.5mLg-1 after operation for approximately two months, and the relative abundances of M. parvicella were 36.7%, 9.74% and 34.07%, respectively, in comparison with the initial values of 33.04%, 29.29% and 54.66%. However, the relative abundances of M. parvicella decreased to 0.86-4.44%, 0.7% and 4.94%, respectively, under the conditions of other carbon sources, aerobic-only treatment and a temperature of 20°C. The FISH analysis gave a similar result. This study was performed with mixed sludge under controlled operating conditions, which provided a valuable information for the pure culture of M. parvicella and further investigations on its physiology and metabolism.

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.

Effect of culture residence time on substrate uptake and storage by a pure culture of Thiothrix (CT3 strain) under continuous or batch feeding.

A pure culture of the filamentous bacterium Thiothrix, strain CT3, was aerobically cultured in a chemostat under continuous acetate feeding at three different culture residence times (RT 6, 12 or 22 d) and the same volumetric organic load rate (OLR 0.12gCOD/L/d). Cells cultured at decreasing RT in the chemostat had an increasing transient response to acetate spikes in batch tests. The maximum specific acetate removal rate increased from 25 to 185mgCOD/gCOD/h, corresponding to a 1.8 to 8.1 fold higher respective steady-state rate in the chemostat. The transient response was mainly due to acetate storage in the form of poly(3-hydroxybutyrate) (PHB), whereas no growth response was observed at any RT. Interestingly, even though the storage rate also decreased as the RT increased, the storage yield increased from 0.41 to 0.50 COD/COD. This finding does not support the traditional view that storage plays a more important role as the transient response increases. The transient response of the steady-state cells was much lower than in cells cultured under periodic feeding (at 6 d RT, from 82 to 247mgCOD/gCOD/h), with the latter cells showing both storage and growth responses. On the other hand, even though steady-state cells had no growth response and their storage rate was also less, steady-state cells showed a higher storage yield than cells cultured under dynamic feeding. This suggests that in Thiothrix strain CT3, the growth response is triggered by periodic feeding, whereas the storage response is a constitutive mechanism, independent from previous acclimation to transient conditions.

A logistic model for the remediation of filamentous bulking in a biological nutrient removal wastewater treatment plant.

Biological nutrient removal (BNR) systems across the globe frequently experience bulking and foaming episodes, which present operational challenges such as poor sludge settling due to excessive filamentous bacteria. A full-scale BNR plant treating primarily domestic wastewater was monitored over a period of 1 year to investigate filamentous bacterial growth response under various plant operating parameters. Identification of filamentous bacteria by conventional microscopy and fluorescent in situ hybridisation indicated the dominance of Eikelboom Type021N, Thiothrix spp., Eikelboom Type 1851 and Eikelboom Type 0092. A cumulative logit model (CLM) was applied to elucidate significant relationships between the filamentous bacteria and plant operational parameters. The model could predict the potential abundance of dominant filamentous bacteria in relation to wastewater treatment plant operational parameters. Data obtained from the model corroborated with previous findings on the dominance of most filaments identified, except for Type 0092, which exhibited some unique traits. With further validation, the model could be successfully applied for identifying specific parameters which could contribute towards filamentous bulking, thus, providing a useful tool for regulating specific filamentous growth in full-scale wastewater treatment plants.

In situ groundwater and sediment bioremediation: barriers and perspectives at European contaminated sites.

This paper contains a critical examination of the current application of environmental biotechnologies in the field of bioremediation of contaminated groundwater and sediments. Based on analysis of conventional technologies applied in several European Countries and in the US, scientific, technical and administrative barriers and constraints which still need to be overcome for an improved exploitation of bioremediation are discussed. From this general survey, it is evident that in situ bioremediation is a highly promising and cost-effective technology for remediation of contaminated soil, groundwater and sediments. The wide metabolic diversity of microorganisms makes it applicable to an ever-increasing number of contaminants and contamination scenarios. On the other hand, in situ bioremediation is highly knowledge-intensive and its application requires a thorough understanding of the geochemistry, hydrogeology, microbiology and ecology of contaminated soils, groundwater and sediments, under both natural and engineered conditions. Hence, its potential still remains partially unexploited, largely because of a lack of general consensus and public concerns regarding the lack of effectiveness and control, poor reliability, and possible occurrence of side effects, for example accumulation of toxic metabolites and pathogens. Basic, applied and pre-normative research are all needed to overcome these barriers and make in situ bioremediation more reliable, robust and acceptable to the public, as well as economically more competitive. Research efforts should not be restricted to a deeper understanding of relevant microbial reactions, but also include their interactions with the large array of other relevant phenomena, as a function of the truly variable site-specific conditions. There is a need for a further development and application of advanced biomolecular tools for site investigation, as well as of advanced metabolic and kinetic modelling tools. These would allow a quicker evaluation of the bioremediation potential of a site, and in turn a preliminary assessment of the technical feasibility of the chosen bioprocess which could replace or at least reduce the need for time-consuming and expensive field tests. At the same time, field tests will probably remain unavoidable for a detailed design of full scale remedial actions and the above reported tools will in any event be useful for a better design and a more reliable operation.

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.

Different activity levels of Dehalococcoides mccartyi revealed by FISH and CARD-FISH under non-steady and pseudo-steady state conditions.

A mixed culture capable of dechlorinating perchloroethylene (PCE) to ethene was analyzed under non steady and pseudo-steady state conditions. Dehalococcoides mccartyi, considered to be the primary dechlorinating bacterium able to completely degrade chlorinated hydrocarbons to non toxic ethene, could be detected by CAtalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) since the beginning of culture operation but highlighted by conventional FISH only during active PCE dechlorination to ethene and vinyl chloride (VC). Data generated from FISH and CARD-FISH analyses were compared to those generated from applying PCR-based techniques directed at defining cell abundances (Real Time PCR, qPCR) and assessing cell activities (Reverse Transcription qPCR, RT-qPCR) of Dehalococcoides strains involved in the PCE reductive dechlorination (RD) process. qPCR targeting reductive dehalogenase genes coding for enzymes involved in the individual steps of the RD process, showed that Dehalococcoides strains carrying the tceA gene dominated the community. This observation was consistent with PCE conversion products detected under pseudo-steady state (ethene and VC production), since this tceA gene is known to be associated to strains capable of reducing chlorinated solvents beyond 1,2-cis-dichloroethene (cis-DCE). In line with the FISH data, Dehalococcoides 16S rRNA and tceA genes were expressed only during pseudo-steady state conditions when PCE was converted completely to the final metabolic product ethene. Furthermore, Dehalococcoides cell abundances estimated by CARD-FISH correlated positively with their 16SrRNA gene copy numbers quantified by qPCR. This is consistent with the ability of both these methods to estimate total Dehalococcoides cell numbers including those with low metabolic activities. Thus, this study shows that application potential of FISH analysis to quantify rapidly and efficiently only active dechlorinators in complex communities.

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.

Conductive magnetite nanoparticles accelerate the microbial reductive dechlorination of trichloroethene by promoting interspecies electron transfer processes.

Playing your part: Conductive magnetite nanoparticles accelerate the microbial reductive dechlorination of trichloroethene (TCE), an ubiquitous and toxic subsurface contaminant. The stimulatory effect most likely results from the nanoparticles promoting the establishment of interspecies electron transfer (IET) processes between non-dechlorinating and dechlorinating microorganisms.

Study of performances, stability and microbial characterization of a Sequencing Batch Biofilter Granular Reactor working at low recirculation flow.

The Sequencing Batch Biofilter Granular Reactor (SBBGR) is a promising wastewater treatment technology characterized by high biomass concentration in the system, good depuration performance and low sludge production. Its main drawback is the high energy consumption required for wastewater recirculation through the reactor bed to ensure both shear stress and oxygen supply. Therefore, the effect of low recirculation flow on the long-term (38 months) performance of a laboratory scale SBBGR was studied. Both the microbial components of the granules, and their main metabolic activities were evaluated (heterotrophic oxidation, nitrification, denitrification, fermentation, sulphate reduction and methanogenesis). The results indicate that despite reduced recirculation, the SBBGR system maintained many of its advantageous characteristics.

Genome sequence of "Candidatus Microthrix parvicella" Bio17-1, a long-chain-fatty-acid-accumulating filamentous actinobacterium from a biological wastewater treatment plant.

"Candidatus Microthrix" bacteria are deeply branching filamentous actinobacteria which occur at the water-air interface of biological wastewater treatment plants, where they are often responsible for foaming and bulking. Here, we report the first draft genome sequence of a strain from this genus: "Candidatus Microthrix parvicella" strain Bio17-1.

Analytical solution for the modeling of the natural time-dependent reduction of waterborne viruses injected into fractured aquifers.

We propose an analytical solution in order to explain the processes that determine the fate and behavior of the viruses during transport in a fractured aquifer at Salento (Italy). The calculations yield the efficiency of filtration in fractures at a site near Nardò (Southern Italy) in reducing the numbers of enteric viruses (i.e., Enteroviruses and Norovirus) in secondary municipal effluents that have been injected in the aquifer over the period 2006-2007. The model predicted, by a theoretical expression, the time-dependent rate of virus reduction, which was in good agreement with field data. The analytical solution yields the achievable "Log reduction credits" for virus reduction in wells located at the setback distances that are usually adopted in local drinking water regulations. The resulting new analytical formula for the time-dependent reduction of viruses during subsurface transport can easily be applied in health risk-based models used to forecast the spread of waterborne diseases and provides appropriate criteria (i.e., distances) needed to meet standards for the quality of drinking water derived from undisinfected groundwater.

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.

Quantitative PCR monitoring of antibiotic resistance genes and bacterial pathogens in three European artificial groundwater recharge systems.

Aquifer recharge presents advantages for integrated water management in the anthropic cycle, namely, advanced treatment of reclaimed water and additional dilution of pollutants due to mixing with natural groundwater. Nevertheless, this practice represents a health and environmental hazard because of the presence of pathogenic microorganisms and chemical contaminants. To assess the quality of water extracted from recharged aquifers, the groundwater recharge systems in Torreele, Belgium, Sabadell, Spain, and Nardò, Italy, were investigated for fecal-contamination indicators, bacterial pathogens, and antibiotic resistance genes over the period of 1 year. Real-time quantitative PCR assays for Helicobacter pylori, Yersinia enterocolitica, and Mycobacterium avium subsp. paratuberculosis, human pathogens with long-time survival capacity in water, and for the resistance genes ermB, mecA, blaSHV-5, ampC, tetO, and vanA were adapted or developed for water samples differing in pollutant content. The resistance genes and pathogen concentrations were determined at five or six sampling points for each recharge system. In drinking and irrigation water, none of the pathogens were detected. tetO and ermB were found frequently in reclaimed water from Sabadell and Nardò. mecA was detected only once in reclaimed water from Sabadell. The three aquifer recharge systems demonstrated different capacities for removal of fecal contaminators and antibiotic resistance genes. Ultrafiltration and reverse osmosis in the Torreele plant proved to be very efficient barriers for the elimination of both contaminant types, whereas aquifer passage followed by UV treatment and chlorination at Sabadell and the fractured and permeable aquifer at Nardò posed only partial barriers for bacterial contaminants.

Structure analysis and performance of a microbial community from a contaminated aquifer involved in the complete reductive dechlorination of 1,1,2,2-tetrachloroethane to ethene.

An anaerobic microcosm set up with aquifer material from a 1,1,2,2-tetrachloroethane (TeCA) contaminated site and amended with butyrate showed a complete TeCA dechlorination to ethene. A structure analysis of the microbial community was performed by fluorescence in situ hybridization (FISH) with already available and on purpose designed probes from sequences retrieved through 16S rDNA clone library construction. FISH was chosen as identification tool to evaluate in situ whether the retrieved sequences belong to primary bacteria responsible for the biodegradative reactions. FISH probes identified up to 80% of total bacteria and revealed the absence or the marginal presence of known TeCA degraders and the abundance of two well-known H(2)-utilizing halorespiring bacteria, Sulfurospirillum (32.4 +/- 8.6% of total bacteria) and Dehalococcoides spp. (14.8 +/- 2.8), thereby providing a strong indication of their involvement in the dechlorination processes. These results were supported by the kinetic and thermodynamic analysis which provided indications that hydrogen was the actual electron donor for TeCA dechlorination. The specific probes, developed in this study, for known dechlorinators (i.e., Geobacter, Dehalobacter, and Sulfurospirillum species) represent a valuable tool for any future in situ bioremediation study as well as a quick and specific investigation tool for tracking their distribution in the field.

Identity, abundance and ecophysiology of filamentous bacteria belonging to the Bacteroidetes present in activated sludge plants.

Filamentous members of the Bacteroidetes are commonly observed in activated sludge samples originating from both municipal and industrial wastewater treatment plants (WWTP), where they occasionally can cause bulking. Several oligonucleotide 16S rRNA-targeted probes were designed to target filaments with a needle-like appearance similar to Haliscomenobacter hydrossis. The design of these probes was based on an isolate and a sequence obtained from a micromanipulated filament. The abundance of filamentous Bacteroidetes was determined in 126 industrial samples applying already published and the newly developed probes. Small populations were found in 62 % of the WWTP investigated. However, only relatively few WWTP (13 %) contained large populations of filamentous Bacteroidetes potentially responsible for bulking incidences. The identity of the most abundant filamentous Bacteroidetes with H. hydrossis morphology could be detected by probes CFB719, SAP-309 and the newly designed probe HHY-654. A comprehensive study on the ecophysiology of probe-defined Bacteroidetes populations was conducted on Danish and Czech samples. The studies revealed that they were specialized bacteria involved in degradation of sugars, e.g. glucose and N-acetylglucosamine, and may participate in the conversion of lipopolysaccharides and peptidoglycan liberated by decaying cells. Many surface-associated exo-enzymes were excreted, e.g. chitinase, glucuronidase, esterase and phosphatase, supporting conversion of polysaccharides and possibly other released cell components. The role of filamentous bacteria with a H. hydrossis-like morphology in the activated sludge ecosystem is discussed.

Microbial characterisation of polyhydroxyalkanoates storing populations selected under different operating conditions using a cell-sorting RT-PCR approach.

The identity of polyhydroxyalkanoates (PHA) storing bacteria selected under aerobic dynamic feeding conditions, using propionate as carbon source (reactor P), was determined by applying reverse transcriptase-polymerase chain reaction (RT-PCR) on micromanipulated cells and confirmed by fluorescence in situ hybridisation (FISH). Four genera, Amaricoccus, Azoarcus, Thauera and Paraccoccus were detected, the latter only rarely present. All the biomass was involved in PHA storage as shown by Nile Blue staining. By quantitative FISH, their specific amount was determined in this and two other systems using acetate as the carbon substrate (sequencing batch reactor [SBR] A and A1). SBR A and reactor P had the same sludge retention time (SRT, 10 days), while reactor A1 was operated with the SRT of 1 day and the double organic loading rate (OLR). Systems fed with acetate (41.1 +/- 2.2 and 49.4 +/- 1.4% total Bacteria, for A and A1, respectively) became enriched in Thauera independently on the SRT and OLR, while it was only present in a minor amount when propionate was used as a substrate (1.9 +/- 0.2% total Bacteria). Amaricoccus was present in both reactors operated at 10 days SRT, favoured in the one fed with propionate (61.4 +/- 1.9% total bacteria), and almost completely removed at the SRT of 1 day. Azoarcus cells were found in all the analysed systems (3.9 +/- 0.3, 23.3 +/- 1.5 and 45.9 +/- 1.5 for P, A and A1, respectively), while Paracoccus was scarcely present.