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A rapid methodology for intracellular storage polyphosphate (poly-P) identification and monitoring in enhanced biological phosphorus removal (EBPR) systems is proposed based on quantitative image analysis (QIA). In EBPR systems, 4',6-diamidino-2-phenylindole (DAPI) is usually combined with fluorescence in situ hybridization to evaluate the microbial community. The proposed monitoring technique is based on a QIA procedure specifically developed for determining poly-P inclusions within a biomass suspension using solely DAPI by epifluorescence microscopy. Due to contradictory literature regarding DAPI concentrations used for poly-P detection, the present work assessed the optimal DAPI concentration for samples acquired at the end of the EBPR aerobic stage when the accumulation occurred. Digital images were then acquired and processed by means of image processing and analysis. A correlation was found between average poly-P intensity values and the analytical determination. The proposed methodology can be seen as a promising alternative procedure for quantifying intracellular poly-P accumulation in a faster and less labour-intensive way.

The microbiology of denitrifying enhanced biological phosphorus removal systems has been a subject of much debate. The question has centred on the affinities of different types of Candidatus Accumulibacter PAOs, type I and type II, towards different electron acceptors such as oxygen, nitrate and nitrite. This study used a propionate anaerobic/anoxic/aerobic lab-scale sequencing batch reactor where a microbial culture was successfully enriched in Accumulibacter type I organisms (approx. 90%). The culture was able to take up phosphorus using nitrate, nitrite and oxygen as electron acceptors, although experiments with oxygen led to the fastest P removal rate. The phosphorus uptake to nitrogen consumed ratio (P/N ratio), when using both nitrate and nitrite, was shown to be affected by pH in the range of 7-8.2, achieving higher values for lower pH values (7.0-7.5). The effect of pH on P removal seems to follow a similar trend for both nitrate and nitrite. To our knowledge, this is the first study where the impact of pH in the phosphate removal stoichiometry using the three most significant electron acceptors is shown for such a high enrichment in Accumulibacter type I.

The application of membrane bioreactors (MBR) for wastewater treatment is growing worldwide due to their compactness and high effluent quality. However, membrane fouling, mostly associated to biological products, can reduce MBR performance. Therefore, it is important to monitor MBRs as close to real-time as possible to accelerate control actions for maximal biological and membrane performance. 2D-fluorescence spectroscopy is a promising on-line tool to simultaneously monitor wastewater treatment efficiency and the formation of potential biological fouling agents. In this study, 2D-fluorescence data obtained from the wastewater and the permeate of a MBR was successfully modelled using projection to latent structures (PLS) to monitor variations in the influent and effluent total chemical oxygen demand (COD). Analysis of the results also indicated that humic acids and proteins highly contributed to the measured COD in both streams. Nevertheless, this approach was not valid for other performance parameters of the MBR system (such as influent and effluent ammonia and phosphorus), which is usually characterised through a high number of analytical and operating parameters. Principal component analysis (PCA) was thus used to find possible correlations between these parameters, in an attempt to reduce the analytical effort required for full MBR characterisation and to reduce the time frame necessary to obtain monitoring results. The 3 first principal components, capturing 57% of the variance, indicated and confirmed expected relationships between the assessed parameters. However, this approach alone could not provide robust enough correlations to enable the elimination of parameters for process description (PCA loadings ≤ 0.5). Nevertheless, it is possible that the information captured by 2D-fluorescence spectroscopy could replace some of the analytical and operating parameters, since this technique was able to successfully describe influent and effluent total COD. It is thus proposed that combined modelling of 2D-fluorescence data and selected performance/operating parameters should be further explored for efficient MBR monitoring aiming at rapid process control.

The present study focuses on investigating the effects of biological compartment conditions on the transport of nitrate and perchlorate in an Ion Exchange Membrane Bioreactor (IEMB). In this hybrid process, the transport depends not only on the membrane properties but also on the biological compartment conditions. The experiments were planned according to the Plackett-Burman statistical design in order to cover a broader range of experimental conditions, under which a previously developed mechanistic transport model was not able to predict correctly the transport fluxes of the target pollutants. Using Principal Component Analysis, it was possible to identify not only the concentrations of target (nitrate and perchlorate) and of major driving counter-ion (chloride) but also those of some biomedium components (e.g. ammonia, ethanol and sulphate) as variables that affect the transport rate of micropollutants across the membrane. These conclusions are based on the loadings of the two first principal components that describe 84% of the data variance. The present study also revealed that the hydraulic retention time and the hydrodynamic conditions in the biocompartment have a minor contribution to the micropollutants transport. The results obtained are important for process optimization purposes.

Pharmaceuticals and personal care products (PPCPs) are becoming increasingly recognised as important micropollutants to be monitored in wastewater treatment plants (WWTPs), since WWTP effluents represent an important point source to natural aquatic systems. In this study, the abundance of 65 PPCPs was analysed in 5 Portuguese WWTPs during the spring and autumn. Due to the fact that analytical approaches normally used to quantify the abundance of these compounds are labour intensive and require various specific procedures, this study proposes a set of simplified analytical methods for the quantification of pharmaceutically active compounds (PhACs) and polycyclic musks in liquid and sludge samples. The analytical methods were validated using influent wastewater matrices, showing comparable limits of detection and quantification as literature values for most PPCPs, with the exception of the estrogenic compounds. The PhAC concentrations detected in the WWTP survey were in the range of 0.050-100 µg L(-1) in the influent and up to 50 µg L(-1) in the effluent, where the non-steroidal anti-inflammatory drugs (NSAIDs) were the most abundant and frequently detected group. Some musks were detected up to 11.5 µg L(-1) in the influent and 0.9 µg L(-1) in the effluent, and adsorbed in the sludge up to 22.6 µg g(-1).

Biological nutrient removal (BNR) plants can reduce both carbon and oxygen requirements by increasing the fraction of phosphorus (P) removed by denitrifying polyphosphate accumulating organisms (DPAOs). Contrasting findings have been reported in literature concerning whether or not PAOs and DPAOs are different microorganisms. In this study, quantitative fluorescence in situ hybridisation (FISH) measurements from different EBPR sludges support the hypothesis that PAOs and DPAOs are phyogenetically different. This experimental evidence is discussed within the context of literature findings and suggestions for future research concerning the identity of PAOs and DPAOs are proposed. Further, this paper discusses the different methodologies available for assessing the DPAO fraction through chemical analytical techniques, where the relative fraction estimated is highly dependent on the methodology employed. Thus, we recommend an alteration to previously proposed methods in order to calculate the DPAO fraction through anaerobic-anoxic and anaerobic-aerobic batch tests. This information is expected to be valuable in studies focussed on optimising the amount of phosphorus removal achieved with simultaneous denitrification.

A reactor was successfully enriched (90% as shown by Fluorescence in situ Hybridization) in Defluviicoccus vanus-related organisms presenting a Glycogen Accumulating Organisms (GAO) phenotype. Initial batch tests were performed using anaerobic/aerobic conditions to assess the capacity of different carbon sources utilization frequently abundant in wastewater: acetate, propionate, butyrate, valerate and glucose. Acetate and propionate were totally consumed in the anaerobic phase as well as butyrate and valerate, though these last ones with a very low consumption rate. All substrates were converted to polyhydroxyalkanoates (PHA). Glucose had a very slight anaerobic consumption but failed to disclose a typical GAO phenotype. In aerobic conditions, again all carbon sources were readily consumed except for glucose, with acetate and propionate having the higher consumption rates. Therefore, glucose seems not be used by this type of organisms. Acetate and propionate consumption rates indicated that these GAOs could reveal good competition advantages in EBPR systems where these carbon sources are available, especially propionate. Volatile Fatty Acid (VFA) uptake in aerobic phase and consequential PHA production indicate these organisms as possible candidates for PHA production., (IWA Publishing 2008.)

Activated sludge was submitted to aerobic dynamic substrate feeding for the production of biodegradable plastics. Two sequencing batch reactors were operated with acetate or propionate as sole carbon substrates. With acetate a homopolymer of polyhydroxybutyrate (PHB) was obtained and with propionate a copolymer of hydroxybutyrate and hydroxyvalerate P(HB/HV) was produced. Three main morphotypes were identified in both sludges: two belong to the Alphaproteobacteria class and the third to the Betaproteobacteria class. Bacilli belonging to Betaproteobacteria were shown by FISH analysis, applied in combination with Nile Blue post-staining, to be the main responsible for PHAs storage. The latter were affiliated to Azoarcus genus within Betaproteobacteria.

A method for non-mechanistic and non-linear modelling of complex biological processes is presented, using the example of the extractive membrane bioreactor (EMB). The model is based on artificial neural networks (ANN), which are able to predict the state of the process from a combination of reactor operational parameters and natural fluorescence fingerprints. Current as well as historic process operation is included in the ANN input vector, in order to account for lag-times within the reactor system and for biofilm dynamics that are dependent on process history. The model is especially relevant for practitioners, as it does not require assumptions on underlying process mechanisms, and it relies on routinely available operational data and on an easy-to-install, non-invasive, in-situ, on-line monitoring method. Moreover, it focuses on the prediction of overall process performance parameters, which are of immediate relevance in practice. The developed model was able to predict the process state very well. Sensitivity analysis revealed that the main impact on process performance stems from process operation rather than the physiological state of the biological culture, and that in the EMB configuration employed process operation history decisively impacts on the process outcome.

In this work, sludge was submitted to aerobic dynamic substrate feeding. Two sequencing batch reactors were operated, with acetate or propionate as carbon substrates. When acetate was used the system only produced a homopolymer of polyhydroxybutyrate (PHB). In order to maximize the PHB production, tests with different concentrations of acetate and ammonia were preformed. The best results (67.2% of PHB by cell dry weight) were obtained for 0.7 Nmmol/l of ammonia and 180 Cmmol/l of acetate. The PHB cell content was further improved by pulse addition of substrate, three times 60 Cmmol/l of acetate, reaching a value of 78.5%. Propionate can be used as a precursor for hydroxyvalerate. In conjunction with other substrates, it allows for the formation of copolymers, which present better processing properties on commercial applications. Tests with different concentrations of propionate and ammonia were performed. Under the operating conditions used, the maximum PHA accumulated inside cells was 34.8%, with 30 Cmmol/l of propionate and no ammonia.

Natural fluorescence as a method to monitor biofilm processes was studied, using the example of an extractive membrane bioreactor for the degradation of 3-chloro-4 methylaniline and 1,2-dichloroethane. Non-invasive, on-line, in-situ 2D fluorometry monitoring was employed to elicit biofilm process status. The fluorescence fingerprints were deconvoluted in a pattern recognition approach using artificial neural networks (ANN) through association with key process performance parameters.

In the present work, sludge from a stable and efficient phosphorus removal process was used to evaluate the change in the metabolism when the reactor operation was modified from anaerobic/aerobic to aerobic dynamic substrate feeding. The change in operational conditions allowed the population to modify the metabolism of phosphorus and polyhydroxyalkanoates (PHA) accumulation. Under aerobic dynamic feeding, phosphorus accumulation was almost hindered while the amount of PHA accumulation increased significantly. After ten days of operation under dynamic substrate feeding, the reactor operation turned back to anaerobic/aerobic cycles and the metabolism of phosphorus was progressively recovered.

Biological phosphorus removal from water surfaces is an important process for the control of eutrophication. Even though much attention has been devoted to understanding the metabolism of bacteria and the effect of operational parameters on the efficiency of the phosphorus removal process, certain aspects are still unclear. The aim of this work was to evaluate the influence of pH control on the stability and efficiency of two sequenced batch reactors (SBR): one operated with pH control (pH 7.0) and the other without pH control (pH raised from around 7.8 to 8.5). The reactor operated without pH control showed higher efficiency on phosphorus removal and stability than the reactor with pH control. Based on the kinetics of both reactors it could be inferred that a different population developed in both systems.