The effectiveness and role of phages in the disruption and inactivation of clinical P. aeruginosa biofilms.

Biofilms of P. aeruginosa are known to be resilient forms of survival of this opportunistic pathogen, both within the host and in natural or engineered environments. This study investigated the role of phages in the disruption and inactivation of clinical P. aeruginosa biofilms by previously isolated phages. All seven tested clinical strains formed biofilms in 56-80 h. Four previously isolated phages were effective in disrupting the formed biofilms when applied at multiplicity of infection (MOI) of 10, where phage cocktails had equivalent or worse performance than single phages. Phage treatments reduced the biofilms' biomass (cells and extracellular matrix) by 57.6-88.5% after 72 h of incubation. Biofilm disruption led to the detachment of 74.5-80.4% of the cells. The phages were also able to kill the cells from the biofilms, reducing the living cell counts by approximately 40.5-62.0% after a single treatment. A fraction of 24-80% of these killed cells were also lysed due to phage action. This study showed that phages can have a relevant role in disrupting, inactivating, and destroying P. aeruginosa biofilms, which can be used in the development of treatment processes to complement or replace antibiotics and/or disinfectants.

Oerskovia paurometabola can efficiently decolorize azo dye Acid Red 14 and remove its recalcitrant metabolite.

The biodegradation of dyes remains one of the biggest challenges of textile wastewater. Azo dyes are one of the most commonly employed dye classes, and biological treatment processes tend to generate recalcitrant aromatic amines, which are more toxic than the parent dye molecule. This study aimed to isolate bacterial strains with the capacity to degrade both the azo dye and the resulting aromatic amines towards the development of a simple and reliable treatment approach for dye-laden wastewaters. A mixed bacterial enrichment was first developed in an anaerobic-aerobic lab-scale sequencing batch reactor (SBR) fed with a synthetic textile wastewater containing the model textile azo dye Acid Red 14 (AR14). Eighteen bacterial strains were isolated from the SBR, including members of the Acinetobacter, Pseudomonas and Oerskovia genera, Oerskovia paurometabola presenting the highest decolorization capacity (91% after 24 h in static anaerobic culture). Growth assays supported that this is a facultative bacterium, and decolorization batch tests with 20-100 mg AR14 L-1 in a synthetic textile wastewater supplemented with yeast extract indicated that O. paurometabola has a high color removal capacity for a significant range of AR14 concentrations. In addition, a model typically used to describe biodegradation of xenobiotic compounds was adjusted to the results, to predict AR14 biodegradation time profiles at different initial concentrations. HPLC analysis confirmed that decolorization occurred through azo bond reduction under anaerobic conditions, the azo dye being completely reduced after 24 h of anaerobic incubation for the range of concentrations tested. Interestingly, partial (up to 63%) removal of one of the resulting aromatic amines (4-amino-naphthalene-1-sulfonic acid) was observed when subsequently subjected to aerobic conditions. Overall, this work showed the azo dye biodegradation potential of specific bacterial strains isolated from mixed culture bioreactors, reporting for the first time the decolorization capacity of an Oerskovia sp. with further biodegradation of a recalcitrant sulfonated aromatic amine metabolite.

Denitrification activity of polyphosphate accumulating organisms (PAOs) in full-scale wastewater treatment plants.

A comprehensive assessment of full-scale enhanced biological phosphorus removal (EBPR) plants (five plants, 19 independent tests) was undertaken to determine their effectiveness in terms of aerobic and anoxic P removal. By comparing parallel P uptake tests under only aerobic or under anoxic-aerobic conditions, results revealed that introducing an anoxic stage led to an overall P removal of on average 90% of the P removed under only aerobic conditions. This was achieved with negligible higher PHA and glycogen requirements, 30% lower overall oxygen consumption and with the simultaneous removal of nitrate, reducing up to an estimate of 70% of carbon requirements for simultaneous N and P removal. Varying fractions of denitrifying polyphosphate accumulating organisms (DPAOs), from an average of 25% to 84%, were found in different plants. No correlation was found between the DPAO fractions and EBPR configuration, season, or the concentration of any of the microbial groups measured via quantitative fluorescence in situ hybridisation. These included Type I and Type II Ca. Accumulibacter and glycogen accumulating organisms, suggesting that chemical batch tests are the best methodology for quantifying the potential of anoxic P removal in full-scale wastewater treatment plants.

Oryzisolibacter propanilivorax gen. nov., sp. nov., a propanil-degrading bacterium.

Strain EPL6T, a Gram-negative, motile, short rod was isolated from a propanil and 3,4-dichloroaniline enrichment culture produced from rice paddy soil. Based on the analyses of the 16S rRNA gene sequence, strain EPL6T was observed to be a member of the family Comamonadaceae, sharing the highest pairwise identity with type strains of the species Alicycliphilus denitrificans K601T (96.8 %) and Melaminivora alkalimesophila CY1T (96.8 %). Strain EPL6T was able to grow in a temperature range of 15-37 °C, pH 6-9 and in the presence of up to 4 % (w/v) NaCl and tested positive for catalase and oxidase reactions. The major respiratory quinone was Q8. The genomic DNA had a G+C content of 69.4±0.9 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol, and the major fatty acid methyl esters comprised C16 : 0, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c/iso-C15 : 0 2-OH). Comparison of the genome sequence of strain EPL6T and of its closest neighbours, Melaminivora alkalimesophila CY1T and Alicycliphilus denitrificans K601T, yielded values of ANI ≤84.1 % and of AAI ≤80.3 %. Therefore, the genetic, phylogenetic, phenotypic and chemotaxonomic characteristics support the classification of this organism into a new taxon. Considering the genetic divergence of strain EPL6T from the type strains of the closest species, which belong to distinct genera, we propose a new genus within the family Comamonadaceae, named Oryzisolibacter propanilivorax gen. nov., sp. nov., represented by the isolate EPL6T as the type strain of the species (=LMG 28427T=CECT 8927T).

Impact of sludge retention time on MBR fouling: role of extracellular polymeric substances determined through membrane autopsy.

The impact of sludge retention time (SRT) on the biofouling of a membrane bioreactor (MBR) by extracellular polymeric substances (EPS) was investigated. The MBR was operated at 60 and 20 d SRT. The gel layer (recovered through optimized membrane autopsy methods) and the cake layer were analyzed for their content and profile of EPS proteins and polysaccharides. The change to a shorter SRT led to decreased membrane filterability, concomitant with a higher expression of EPS proteins in the cake layer, which were identified as being mainly related with biosynthesis and stress functions. The gel layer was more substantial in internal fibers, with polysaccharides being the major component in this layer. With the decrease in SRT (and filterability decrease), the overall polysaccharide content and sugar variety increased. In conclusion, SRT impacted not only on the quantity but also the composition of EPS molecules, and both were shown to be important in biofouling.

Impact of biogenic substrates on sulfamethoxazole biodegradation kinetics by Achromobacter denitrificans strain PR1.

Pure cultures have been found to degrade pharmaceutical compounds. However, these cultures are rarely characterized kinetically at environmentally relevant concentrations. This study investigated the kinetics of sulfamethoxazole (SMX) degradation by Achromobacter denitrificans strain PR1 at a wide range of concentrations, from ng/L to mg/L, to assess the feasibility of using it for bioaugmentation purposes. Complete removal of SMX occurred for all concentrations tested, i.e., 150 mg/L, 500 µg/L, 20 µg/L, and 600 ng/L. The reaction rate coefficients (kbio) for the strain at the ng/L SMX range were: 63.4 ± 8.6, 570.1 ± 15.1 and 414.9 ± 124.2 L/g[Formula: see text]·day), for tests fed without a supplemental carbon source, with acetate, and with succinate, respectively. These results were significantly higher than the value reported for non-augmented activated sludge (0.41 L/(g [Formula: see text]·day) with hundreds of ng/L of SMX. The simultaneous consumption of an additional carbon source and SMX suggested that the energetic efficiency of the cells, boosted by the presence of biogenic substrates, was important in increasing the SMX degradation rate. The accumulation of 3-amino-5-methylisoxazole was observed as the only metabolite, which was found to be non-toxic. SMX inhibited the Vibrio fischeri luminescence after 5 min of contact, with EC50 values of about 53 mg/L. However, this study suggested that the strain PR1 still can degrade SMX up to 150 mg/L. The results of this work demonstrated that SMX degradation kinetics by A. denitrificans PR1 compares favorably with activated sludge and the strain is a potentially interesting organism for bioaugmentation for SMX removal from polluted waters.

Impact of sludge retention time on the fine composition of the microbial community and extracellular polymeric substances in a membrane bioreactor.

Membrane bioreactors (MBRs) are an advanced technology for wastewater treatment whose wide application has been hindered by rapid fouling of the membranes. MBRs can be operated with long sludge retention time (SRT), a crucial parameter impacting microbial selection in the reactor. This also affects filtration performance, since a major fouling agent are the extracellular polymeric substances (EPS). In this study, the impact of the SRT on the ecophysiology of the MBRs and, consequently, on membrane fouling was evaluated. A MBR was operated under a SRT of 60 days followed by a SRT of 20 days. A comprehensive analysis of the microbial community structure and EPS proteins and polysaccharide profiles of the mixed liquor and cake layer was carried out throughout both operation periods. The results of this study showed that the imposition of a shorter SRT led to a shift in the dominant bacterial populations. The mixed liquor and cake layer communities were very different, with Actinomycetales order standing out in the cake layer at SRT of 20 days. Overall, higher EPS concentrations (particularly proteins) were found at this SRT. Furthermore, EPS profiles were clearly affected by the SRT: it was possible to correlate a group of soluble EPS proteins with the SRT of 60 days, and a lower sludge age led to a lower diversity of polysaccharide sugar monomers, with an increase of glucose and galactose in the cake layer. This study improves our knowledge regarding the molecular reasons for fouling, which may contribute to improve MBR design and operation.

Patulibacter medicamentivorans sp. nov., isolated from activated sludge of a wastewater treatment plant.

A Gram-positive, aerobic, non-motile, non-endospore-forming rod-shaped bacterium with ibuprofen-degrading capacity, designated strain I11(T), was isolated from activated sludge from a wastewater treatment plant. The major respiratory quinone was demethylmenaquinone DMK-7, C18 : 1 cis9 was the predominant fatty acid, phosphatidylglycerol was the predominant polar lipid, the cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid and the G+C content of the genomic DNA was 74.1 mol%. On the basis of 16S rRNA gene sequence analysis, the closest phylogenetic neighbours of strain I11(T) were Patulibacter ginsengiterrae CECT 7603(T) (96.8 % similarity), Patulibacter minatonensis DSM 18081(T) (96.6 %) and Patulibacter americanus DSM 16676(T) (96.6 %). Phenotypic characterization supports the inclusion of strain I11(T) within the genus Patulibacter (phylum Actinobacteria). However, distinctive features and 16S rRNA gene sequence analysis suggest that is represents a novel species, for which the name Patulibacter medicamentivorans sp. nov. is proposed. The type strain is I11(T) ( = DSM 25962(T) = CECT 8141(T)).

Polyhydroxyalkanoates production from ethanol- and lactate-rich fermentate of confectionary industry effluents.

Polyhydroxyalkanoate (PHA) production has been the focus of considerable research to increase productivities and reduce production costs. In this study, a fermented confectionary industry wastewater was used as feedstock for mixed microbial culture PHA production. The feedstock was dominated by lactate and ethanol (60-90 % of all soluble fermentation products). The culture selection reactor was inoculated with municipal activated sludge and was operated at an organic loading rate (OLR) of 100 Cmmol·L-1·d-1, achieving a robust PHA-accumulating enrichment, which produced up to 52.6 ± 0.4 wt% of PHA in accumulation assays. An OLR increase in the culture selection stage to 150 Cmmol·L-1·d-1 led to a PHA content of 59.1 ± 0.6, a yield of 0.93 ± 0.01 Cmol-PHA·Cmol-S-1 and a productivity of 0.93 ± 0.01 g-PHA L-1·h-1. A correlation analysis of the impact of ethanol concentrations from 3.19 to 20.3 Cmmol·L-1 in the reactor showed that ethanol inhibited PHA production rate and yield and the consumption of other carbon sources available. Microbial community analysis revealed the increase of Amaricoccus genus during the bioreactor operation time, a known PHA accumulator. The produced polymer was poly(3-hydroxybutyrate) with an average molecular weight of 4.3 × 105 Da and a polydispersity index of 1.88.

The impact of pH on the anaerobic and aerobic metabolism of Tetrasphaera-enriched polyphosphate accumulating organisms.

Members of the genus Tetrasphaera are putative polyphosphate accumulating organisms (PAOs) that have been found in greater abundance than Accumulibacter in many full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plants worldwide. Nevertheless, previous studies on the effect of environmental conditions, such as pH, on the performance of EBPR have focused mainly on the response of Accumulibacter to pH changes. This study examines the impact of pH on a Tetrasphaera PAO enriched culture, over a pH range from 6.0 to 8.0 under both anaerobic and aerobic conditions, to assess its impact on the stoichiometry and kinetics of Tetrasphaera metabolism. It was discovered that the rates of phosphorus (P) uptake and P release increased with an increase of pH within the tested range, while PHA production, glycogen consumption and substrate uptake rate were less sensitive to pH changes. The results suggest that Tetrasphaera PAOs display kinetic advantages at high pH levels, which is consistent with what has been observed previously for Accumulibacter PAOs. The results of this study show that pH has a substantial impact on the P release and uptake kinetics of PAOs, where the P release rate was >3 times higher and the P uptake rate was >2 times higher at pH 8.0 vs pH 6.0, respectively. Process operational strategies promoting both Tetrasphaera and Accumulibacter activity at high pH do not conflict with each other, but lead to a potentially synergistic impact that can benefit EBPR performance.

Step-by-step strategy for protein enrichment and proteome characterisation of extracellular polymeric substances in wastewater treatment systems.

Extracellular polymeric substances (EPS) are keys in biomass aggregation and settleability in wastewater treatment systems. In membrane bioreactors (MBR), EPS are an important factor as they are considered to be largely responsible for membrane fouling. Proteins were shown to be the major component of EPS produced by activated sludge and to be correlated with the properties of the sludge, like settling, hydrophobicity and cell aggregation. Previous EPS proteomic studies of activated sludge revealed several problems, like the interference of other EPS molecules in protein analysis. In this study, a successful strategy was outlined to identify the proteins from soluble and bound EPS extracted from activated sludge of a lab-scale MBR. EPS samples were first subjected to pre-concentration through lyophilisation, centrifugal ultrafiltration or concentration with a dialysis membrane coated by a highly absorbent powder of polyacrylate-polyalcohol, preceded or not by a dialysis step. The highest protein concentration factors were achieved with the highly absorbent powder method without previous dialysis step. Four protein precipitation methods were then tested: acetone, trichloroacetic acid (TCA), perchloric acid and a commercial kit. Protein profiles were compared in 4-12 % sodium dodecyl sulphate polyacrylamide gel electrophoresis gels. Both acetone and TCA should be applied for the highest coverage for soluble EPS proteins, whereas TCA was the best method for bound EPS proteins. All visible bands of selected profiles were subjected to mass spectrometry analysis. A high number of proteins (25-32 for soluble EPS and 17 for bound EPS) were identified. As a conclusion of this study, a workflow is proposed for the successful proteome characterisation of soluble and bound EPS from activated sludge samples.

Microbial population analysis of nutrient removal-related organisms in membrane bioreactors.

Membrane bioreactors (MBR) are an important and increasingly implemented wastewater treatment technology, which are operated at low food to microorganism ratios (F/M) and retain slow-growing organisms. Enhanced biological phosphorus removal (EBPR)-related organisms grow slower than ordinary heterotrophs, but have never been studied in detail in MBRs. This study presents a comprehensive analysis of the microorganisms involved in EBPR in pilot- and full-scale MBRs, using fluorescence in situ hybridization (FISH), as well as an overall assessment of other relevant microbial groups. The results showed that polyphosphate accumulating organisms (PAOs) were present at similar levels in all studied MBRs (10% ± 6%), even those without a defined anaerobic zone. Glycogen accumulating organisms were also detected, although rarely. The FISH results correlated well with the observed P removal performance of each plant. The results from this study suggest that a defined anaerobic zone is not necessarily required for putative PAO growth in MBRs, since polyphosphate storage may provide a selective advantage in fulfilling cell maintenance requirements in substrate-limited conditions (low F/M).

Novel Bacteriophages Show Activity against Selected Australian Clinical Strains of Pseudomonas aeruginosa.

Multi-drug resistant (MDR) clinical strains of Pseudomonas aeruginosa are the most prevalent bacteria in the lungs of patients with cystic fibrosis (CF) and burn wounds and among the most common in immunocompromised hospital patients in Australia. There are currently no promising antibiotics in the pipeline being developed against these strains. Phage therapy, which uses viruses known as bacteriophages to infect and kill pathogenic bacteria, could be a possible alternative treatment. To this end, we isolated and characterised four novel phages against Australian clinical strains of P. aeruginosa isolated from patients with cystic fibrosis, from infected blood and joint aspirate in Southeast Queensland, Australia. Activated sludge was enriched for phages using the clinical strains, and four bacteriophages were isolated. The phages were able to cause lysis in a further three identified clinical isolates. Morphology showed that they were all tailed phages (of the order Caudovirales), two belonging to the family Myoviridae and the others assigned to the Podoviridae and Siphoviridae. Their genomes were sequenced to reveal a doubled stranded DNA topology with genome sizes ranging from 42 kb to 65 kb. In isolating and characterising these novel phages, we directed our efforts toward the development and use of these phages as candidates for phage therapy as an alternative strategy for the management or elimination of these pathogenic strains. Here we describe novel phage candidates for potential therapeutic treatment of MDR Australian clinical isolates of P. aeruginosa.

Dynamics of Microbial Communities in Phototrophic Polyhydroxyalkanoate Accumulating Cultures.

Phototrophic mixed cultures (PMC) are versatile systems which can be applied for waste streams, valorisation and production of added-value compounds, such as polyhydroxyalkanoates (PHA). This work evaluates the influence of different operational conditions on the bacterial communities reported in PMC systems with PHA production capabilities. Eleven PMCs, fed either with acetate or fermented wastewater, and selected under either feast and famine (FF) or permanent feast (PF) regimes, were evaluated. Overall, results identified Chromatiaceae members as the main phototrophic PHA producers, along with Rhodopseudomonas, Rhodobacter and Rhizobium. The findings show that Chromatiaceae were favoured under operating conditions with high carbon concentrations, and particularly under the PF regime. In FF systems fed with fermented wastewater, the results indicate that increasing the organic loading rate enriches for Rhodopseudomonas, Rhizobium and Hyphomicrobiaceae, which together with Rhodobacter and Chromatiaceae, were likely responsible for PHA storage. In addition, high-sugar feedstock impairs PHA production under PF conditions (fermentative bacteria dominance), which does not occur under FF. This characterization of the communities responsible for PHA accumulation helps to define improved operational strategies for PHA production with PMC.

Electrochemical oxidation processes for PFAS removal from contaminated water and wastewater: fundamentals, gaps and opportunities towards practical implementation.

Electrochemical oxidation (EO) is emerging as one of the most promising methods for the degradation of recalcitrant per- and poly-fluoroalkyl substances (PFASs) in water and wastewater, as these compounds cannot be effectively treated with conventional bio- or chemical approaches. This review examines the state of the art of EO for PFASs destruction, and comprehensively compares operating parameters and treatment performance indicators for both synthetic and real contaminated water and wastewater media. The evaluation shows the need to use environmentally-relevant media to properly quantify the effectiveness/efficiency of EO for PFASs treatment. Additionally, there is currently a lack of quantification of sorption losses, resulting in a likely over-estimation of process' efficiencies. Furthermore, the majority of experimental results to date indicate that short-chain PFASs are the most challenging and need to be prioritized as environmental regulations become more stringent. Finally, and with a perspective towards practical implementation, several operational strategies are proposed, including processes combining up-concentration followed by EO destruction.

Evaluation of continuous and intermittent trickling strategies for the removal of hydrogen sulfide in a biotrickling filter.

Biotrickling filter (BTF) is a widely applied bioreactor for odour abatement in sewer networks. The trickling strategy is vital for maintaining a sound operation of BTF. This study employed a lab-scale BTF packed with granular activated carbon at a short empty bed residence time of 6 s and pH 1-2 to evaluate different trickling strategies, i.e., continuous trickling (different velocities) and intermittent trickling (different trickling intervals), in terms of the removal of hydrogen sulfide (H2S), bed pressure drop, H2S oxidation products and microbial community. The H2S removal performance decreased with the trickling velocity (∼3.6 m/h) in BTF. In addition, three intermittent trickling strategies, i.e., 10-min trickling per 24 h, 8 h, and 2 h, were investigated. The H2S elimination capacity deteriorated after about 2 weeks under both 10-min trickling per 24 h and 8 h. For both intermittent (10-min trickling per 2 h) and continuous trickling, the BTF exhibited nearly 100 % H2S removal for inlet H2S concentrations<100 ppmv, but intermittent BTF showed better removal performance than continuous trickling when inlet H2S increased to 120-190 ppmv. Furthermore, the bed pressure drops were 333 and 3888 Pa/m for non-trickling and trickling periods, respectively, which makes intermittent BTF save 83 % energy consumption of the blower compared with continuous tirckling. However, intermittent BTF exhibited transient H2S breakthrough (<1 ppmv) during trickling periods. Moreover, elemental sulfur and sulfate were major products of H2S oxidation and Acidithiobacillus was the dominant genus in both intermittent and continuous trickling BTF. A mathematical model was calibrated for the intermittent BTF and a sensitivity analysis was performed on the model. It shows mass transfer parameters determine the H2S removal. Overall, intermittent trickling strategy is promising for improving odour abatement performance and reducing the operating cost of the BTF.

Diclofenac biotransformation in the enhanced biological phosphorus removal process.

Diclofenac is a pharmaceutical active compound frequently detected in wastewater and water bodies, and often reported to be persistent and difficult to biodegrade. While many previous studies have focussed on assessing diclofenac biodegradation in nitrification and denitrification processes, this study focusses on diclofenac biodegradation in the enhanced biological phosphorus removal (EBPR) process, where the efficiency of this process for diclofenac biodegradation as well as the metabolites generated are not well understood. An enrichment of Accumulibacter polyphosphate accumulating organisms (PAOs) was operated in an SBR for over 300 d, and acclimatized to 20 µg/L of diclofenac, which is in a similar range to that observed in domestic wastewater influents. The diclofenac biotransformation was monitored in four periods of stable operation and linked to the microbial community and metabolic behaviour in each period. Nitrification was observed in two of the four periods despite the addition of a nitrification inhibitor, and these periods were positively correlated with increased diclofenac biodegradation. Interestingly, in two periods with excellent phosphorus removal (>99%) and no nitrification, different levels of diclofenac biotransformation were observed. Period 2, enriched in Accumulibacter Type II achieved more significant diclofenac biotransformation (3.4 µg/gX), while period 4, enriched in Accumulibacter Type I achieved lower diclofenac biotransformation (0.4 µg/gX). In total, 23 transformation products were identified, with lower toxicity than the parent compound, enabling the elucidation of multiple metabolic pathways for diclofenac biotransformation. This study showed that PAOs can contribute to diclofenac biotransformation, yielding less toxic transformation products, and can complement the biodegradation carried out by other organisms in activated sludge, particularly nitrifiers.

Biotrickling filter for the removal of volatile sulfur compounds from sewers: A review.

Volatile sulfur compounds (VSCs) were identified as the dominant priority odorants emitted from sewers, including hydrogen sulfide (H2S), methyl mercaptan (MM), dimethyl disulfide (DMDS) and dimethyl sulfide (DMS). Biotrickling filter (BTF) is a widely-applied technology for odour abatement in sewers because of its relatively low operating cost and efficient H2S removal. The authors review the mechanisms and performance of BTF for the removal of these four VSCs, and discuss the key influencing factors including of empty bed residence time (EBRT), pH, temperature, nutrients, water content, trickling operation and packing materials. Besides, measures to improve the VSCs removal in BTF are proposed in the context of key influencing factors. Finally, the review assesses the new challenges of BTF for sewer emissions treatment, namely with respect to the performance of BTF for greenhouse gases (GHG) treatment.

A review of the biotransformations of priority pharmaceuticals in biological wastewater treatment processes.

Wastewater effluent discharges have been considered as one of the main sources of synthetic chemicals entering into the aquatic environment. Even though they occur at low concentrations, pharmaceutically active compounds (PhACs) can have an impact on ecological toxicity that affects aquatic organisms. Moreover, new regulations in development toward preserving water quality reinforces the increasing need to monitor and abate some PhACs in wastewater treatment plants (WWTPs), where they are typically only partially eliminated. Unlike most previous reviews, we have focussed on how the main biological and chemical molecular factors impact the biotransformations of key PhACs in biological WWTP processes. Biotransformations have been found to be an important contributor towards the removal of PhACs from WWTP effluents. This review paper critically assesses these aspects and the recent advances that have been achieved in wastewater treatment processes for biodegradation of 7 PhACs; namely the non-steroidal anti-inflammatory drug (NSAID) diclofenac (DCF); the macrolide antibiotics azithromycin (AZM), erythromycin (ERY) and clarithromycin (CLR); the two natural estrogens estrone (E1) and 17ß-estradiol (E2), and the synthetic estrogen 17α-ethinylesradiol (EE2). These represent the micropollutants of the EU Watch list in Decision 2015/495/EU that are most relevant to WWTPs due to their frequent detection. The metabolic pathways, transformation products and impact of relevant factors to biological WWTP processes is addressed in this review. The biokinetics of PhAC biodegradation in different engineered bioprocesses is also discussed. Promising technologies and operational strategies that are likely to have a high impact on controlling PhAC releases are highlighted and future research needs are also proposed.

Accumulibacter diversity at the sub-clade level impacts enhanced biological phosphorus removal performance.

Accumulibacter is a well-known group of organisms, typically considered to be polyphosphate accumulating organisms (PAOs), but potentially capable of glycogen accumulating organism (GAO) metabolism under limiting influent phosphate levels. Metabolic features of Accumulibacter are typically linked to its phylogenetic identity at the Type or clade level, though it is unclear the extent to which Accumulibacter diversity can correlate with its capacity to perform P removal. This paper investigates the fine-scale diversity of Accumulibacter and its link with enhanced biological phosphorus removal (EBPR) performance under various operating conditions, to understand the conditions and community structure leading to successful and unsuccessful EBPR operation. For this purpose, the organic carbon feeding rate and total organic carbon concentration were varied during three distinct operational periods, where influent phosphate was never limiting. Accumulibacter was always the dominant microbial group (>80% of all bacteria according to quantitative fluorescence in situ hybridisation - FISH) and low levels of Competibacter and other GAOs were consistently observed (<15% of all bacteria). Steady state was achieved in each of the three periods, with average phosphorus removal levels of 36%, 99% and >99%, respectively. Experimentally determined stoichiometric activity supported the expression of a mixed PAO/GAO metabolism in the first steady state period and the typical PAO metabolism in the other two steady state periods. FISH quantification and amplicon sequencing of the polyphosphate kinase (ppk1) functional gene indicated that Accumulibacter clade IIC was selected in the first steady state period, which shifted to clade IA after decreasing the carbon feeding rate in steady state period 2, and finally shifted back to clade IIC in the third steady state period. Fine-resolution Ppk-based phylogenetic analysis revealed three different clusters within Accumulibacter clade IIC, where clusters IICii and IICiii were linked to poor EBPR performance in period 1, and cluster IICi was linked to good EBPR performance in period 3. This study shows that the deterioration of EBPR processes through GAO activity at non-limiting P concentrations can be linked to organisms that are typically classified as PAOs, not only to known GAOs such as Competibacter. Intra-clade phylogenetic diversity within Accumulibacter showed that some clusters actually behave similarly to GAOs even without influent phosphate limitation. This study highlights the need to closely re-examine traditional interpretations regarding the link between the microbial community composition and identity with the performance and metabolism of EBPR systems.