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Glycogen-accumulating organisms (GAOs) have been previously observed to be more competitive in anaerobic volatile fatty acid (VFA) uptake as compared to polyphosphate-accumulating organisms (PAOs) at high temperatures in enhanced biological phosphorus removal (EBPR) systems. However, previous evidence regarding the impact of temperature on GAOs has exclusively focused on Competibacter. Defluviicoccus vanus GAOs (DvGAOs) make up another very important GAO group and have been found in EBPR plants; however, their competitiveness at high temperature for anaerobic VFA uptake was previously unknown. The rate of uptake of VFAs by both DvGAOs reactors enriched with acetate or propionate was much lower than those of Candidatus Accumulibacter phosphatis PAOs operated under similar conditions, showing that PAOs displayed a competitive advantage at high temperature, unlike previous observations with Competibacter. This study provides new insight into our understanding of GAOs in EBPR processes, where the impact of temperature on PAO-GAO competition is highly dependent on the phylogenetic identity of the GAO present in the system. New strategies for promoting EBPR performance at high temperatures are proposed on the basis of the results of this work.

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.

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation. Funding The European Commission FP7 NeurOmics project; CHDI Foundation; the Medical Research Council UK; the Brain Research Trust; and the Guarantors of Brain.

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% and99%, 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.

Denitrifying sulfur conversion-associated enhanced biological phosphorous removal (DS-EBPR) system is not only a novel wastewater treatment process, but also an ideal model for microbial ecology in a community context. However, it exists the knowledge gap on the roles and interactions of functional microorganisms in the DS-EBPR system for carbon (C), nitrogen (N), phosphorus (P) and sulfur (S) bioconversions. We use genome-resolved metagenomics to build up an ecological model of microbial communities in a lab-scale DS-EBPR system with stable operation for more than 400 days. Our results yield 11 near-complete draft genomes that represent a substantial portion of the microbial community (39.4%). Sulfate-reducing bacteria (SRB) and sulfide-oxidizing bacteria (SOB) promote complex metabolic processes and interactions for C, N, P and S conversions. Bins 1-4 and 10 are considered as new potential polyphosphate-accumulating organisms (PAOs), in which Bins 1-4 can be considered as S-related PAOs (S-PAOs) with no previously cultivated or reported members. Our findings give an insight into a new ecological system with C, N, P and S simultaneous bioconversions and improve the understanding of interactions among SRB, SOB, denitrifiers and PAOs within a community context.

Sustainable development is driving a rapid focus shift in the wastewater and organic waste treatment sectors, from a “removal and disposal” approach towards the recovery and reuse of water, energy and materials (e.g. carbon or nutrients). Purple phototrophic bacteria (PPB) are receiving increasing attention due to their capability of growing photoheterotrophically under anaerobic conditions. Using light as energy source, PPB can simultaneously assimilate carbon and nutrients at high efficiencies (with biomass yields close to unity (1 g CODbiomass·g CODremoved−1)), facilitating the maximum recovery of these resources as different value-added products. The effective use of infrared light enables selective PPB enrichment in non-sterile conditions, without competition with other phototrophs such as microalgae if ultraviolet-visible wavelengths are filtered. This review reunites results systematically gathered from over 177 scientific articles, aiming at producing generalized conclusions. The most critical aspects of PPB-based production and valorisation processes are addressed, including: (i) the identification of the main challenges and potentials of different growth strategies, (ii) a critical analysis of the production of value-added compounds, (iii) a comparison of the different value-added products, (iv) insights into the general challenges and opportunities and (v) recommendations for future research and development towards practical implementation. To date, most of the work has not been executed under real-life conditions, relevant for full-scale application. With the savings in wastewater discharge due to removal of organics, nitrogen and phosphorus as an important economic driver, priorities must go to using PPB-enriched cultures and real waste matrices. The costs associated with artificial illumination, followed by centrifugal harvesting/dewatering and drying, are estimated to be 1.9, 0.3–2.2 and 0.1–0.3 $·kgdry biomass−1. At present, these costs are likely to exceed revenues. Future research efforts must be carried out outdoors, using sunlight as energy source. The growth of bulk biomass on relatively clean wastewater streams (e.g. from food processing) and its utilization as a protein-rich feed (e.g. to replace fishmeal, 1.5–2.0 $·kg−1) appears as a promising valorisation route.

Achromobacter denitrificans PR1 has previously shown potential to degrade the antibiotic sulfamethoxazole, whereby sulfamethoxazole biotransformation was stimulated in the presence of biogenic substrates. This study examined the biotransformation kinetics of sulfamethoxazole and its two main conjugates, N4-acetyl-SMX and SMX-N1-Glucuronide, by activated sludge and activated sludge bioaugmented with A. denitrificans PR1. SMX biotransformation under both anoxic and aerobic conditions was tested, with and without the addition of acetate as growth substrate, to understand the range of applicable conditions for bioaugmentation purposes. Biological process models, such as the pseudo-first order kinetic and cometabolic models, were also applied and, following the estimation of kinetic parameters, could well describe data measured in bioaugmented and non-bioaugmented AS batch experiments under various test conditions. Experimental and modelling results suggest that (i) retransformation of the two conjugates to SMX in AS occurred under both aerobic and anoxic conditions, and (ii) biotransformation kinetics of SMX can vary significantly depending on redox conditions, e.g., SMX was biotransformed by AS only under aerobic conditions. Notably, SMX biotransformation was significantly enhanced when PR1 was bioaugmented in AS. Addition of acetate as biogenic substrate is not necessary, as PR1 was capable of enhancing the SMX biotransformation by using the carbon sources present in wastewater. Overall, bioaugmentation by means of A. denitrificans PR1 could be a viable strategy for enhancing SMX removal in AS wastewater treatment plants (WWTPs).

In this study, a sequencing batch reactor (SBR), operated with transient carbon availability (feast and famine) and different nutrients loads, was used to select cyanobacteria accumulating poly (3-hydroxyalkanoate) (PHB) and carbohydrates from a mixed wastewater-borne microbial culture. The SBR was operated with 12 h aerobic light and 12 h anaerobic dark phases, evaluating the effect of three different operational conditions consisting on; 1) carbon limitation, 2) carbon and phosphorus limitation and 3) phosphorus limitation. Once a steady state was reached in each operational period of the SBR, part of the biomass was collected and submitted to separate batch tests in order to investigate the maximum PHB and carbohydrates accumulation levels. Batch tests were performed during 24 h of illuminated aerobic condition and 24 h of dark anaerobic condition, while inorganic carbon was constantly present. During the SBR operation, inorganic carbon was mostly used for biomass and carbohydrate production, showing very low PHB accumulation levels (

The emission of the greenhouse gas nitrous oxide (N2O) can occur during biological nutrient removal. Denitrifying enhanced biological phosphorus removal (d-EBPR) systems are an efficient means of removing phosphate and nitrogen, performed by denitrifying polyphosphate-accumulating organisms (d-PAOs). The aim of this work was to study the effect of various combinations of electron acceptors, nitrate (NO3−), nitrite (NO2−), and N2O, on the denitrification pathway of a d-EBPR system. Batch tests were performed with different electron acceptor combinations, to explore the denitrification pathway. Reverse transcriptase-qPCR (RT-qPCR) and high-throughput sequencing, combined with chemical analysis, were used to study gene expression, microbial diversity, and denitrification kinetics. The potential for N2O production was greater than the potential for its reduction in most tests. A strong correlation was observed between the N2O reduction rate and the relative gene expression of nitrous oxide reductase per nitrite reductase (nosZ/(nirS + nirK)), suggesting that the expression of denitrifying marker genes is a strong predictor of the N2O reduction rate. The d-EBPR community maintained a core population with low variations throughout the study. Furthermore, phylogenetic analyses of the studied marker genes revealed that the organisms actively involved in denitrification were closely related to Thauera sp., Candidatus Accumulibacter phosphatis, and Candidatus Competibacter denitrificans. Moreover, Competibacter-related OTUs seem to be important contributors to the N2O reduction capacity of the system, likely scavenging the N2O produced by other organisms. Overall, this study contributes to a better understanding of the microbial biochemistry and the genetics involving biological denitrification removal, important to minimize N2O emissions in wastewater treatment plants.

This study aims at investigating the denitrification kinetics in two separate enriched cultures of denitrifying polyphosphate accumulating organisms (dPAO) and denitrifying glycogen accumulating organisms (dGAO) and compare their N2O accumulation potential under different conditions. Two sequencing batch reactors were inoculated to develop dPAO and dGAO enriched microbial communities separately. Seven batch tests with different combinations of electron acceptors (nitrate, nitrite and/or nitrous oxide) were carried out with the enriched biomass from both reactors. Results indicate that in almost all batch tests, N2O accumulated for both cultures, however dPAOs showed a higher denitrification capacity compared to dGAOs due to their higher nitrogen oxides reduction rates. Additionally, the effect of the simultaneous presence of several electron acceptors in the reduction rates of the different nitrogen oxides was also assessed in dPAOs and dGAOs This study was funded by the Spanish Government (MINECO) (CTM 2011-27163 and CTM 2015-66892-R), the European Commission (FP7-PEOPLE-2011-CIG 303946) and the Portuguese Fundação para a Ciência e Tecnologia (PTDC/AACAMB/12058/2010, UID/Multi/04378/2013 , PhD grant SFRH/BD/74515/2010). Spanish and Portuguese Governments are acknowledged for Acciones Integradas (PRIAIBPT- 2011-1232) and Luso-Espanhola action E-61/12. The European Commission is also acknowledged through COST action ES1202 (Water 2020). M. Pijuan and A. Ribera-Guardia acknowledge the Ramon y Cajal research fellowship (RYC-2009-04959) and the FPIPhD grant (BES-2012-052753) respectively provided by the Spanish Government

Glycogen accumulating organisms (GAOs) are thought to compete with polyphosphate accumulating organisms (PAOs) for the often-limiting carbon sources available in wastewater, deteriorating enhanced biological phosphorus removal (EBPR) performance at high temperatures. Fermentation liquids are often used to provide an additional carbon source supply in EBPR processes, where butyrate is known to be an important volatile fatty acid (VFA) produced in sludge fermentation. Nevertheless, the impact of butyrate on the PAO-GAO competition is not well understood especially at high temperature. The results of this study demonstrate that butyrate, as a supplemental or sole carbon source, could be promising for EBPR in tropical climates. When the carbon source was gradually changed from acetate to butyrate, a substantial PAO population was found under both conditions, despite a substantial shift in the abundance of Candidatus Accumulibacter phosphatis (decreased from 37.4% to 13.9%) to Rhodocyclaceae (increased from 2.0% to 14.5%), where both organisms likely played an important role in P-removal. Thus, a relatively stable P removal performance was realized throughout the whole operation period. Nevertheless, butyrate had a negative impact on GAOs. The biomass concentration and microbial diversity continually decreased in the GAO reactor, and Candidatus Competibacter phosphatis reduced from 27.3% to 6.2%, where the dominant population was replaced by Zoogloea. With the addition of butyrate as carbon source, the total amount of synthesized PHAs reduced in both PAO and GAO cultures and the composition of PHA was greatly changed. The presence of a novel PHA fraction (PHH) may disturb the microbial activity in the aerobic phase, where the GAO culture was more severely affected. Glycogen cycling also seemed to be limited in both reactors. This could reduce the GAO metabolism in both cultures and favor PAOs and P removal. Furthermore, the biomass growth rate of the PAO culture was higher than that of GAO when fed with butyrate, which also provides PAO a competitive advantage. All the above results indicate that butyrate could not be well metabolized by GAOs, but could provide PAOs a competitive advantage. Thus, mixed VFAs (i.e. acetate, propionate and butyrate) are likely to favor PAOs over GAOs in EBPR processes operated in warm climates, where the impact of substantial butyrate fractions represents an advantage towards successful process operation. Nanyang Technological University The authors are grateful for the financial support provided by the Advanced Environmental Biotechnology Centre (AEBC) of Nanyang Environment and Water Research Institute (NEWRI), Nanyang Technological University, Singapore, and the Research Fund for the Doctoral Program of Singapore and Interdisciplinary Graduate School of Nanyang Technological University.

Currently, the feast and famine (FF) regime is the most widely applied strategy to select for polyhydroxyalkanoate (PHA) accumulating organisms in PHA production systems with mixed microbial cultures. As an alternative to the FF regime, this work studied the possibility of utilizing a permanent feast regime as a new operational strategy to select for PHA accumulating photosynthetic mixed cultures (PMCs). The PMC was selected in an illuminated environment and acetate was constantly present in the mixed liquor to guarantee a feast regime. During steady-state operation, the culture presented low PHA accumulation levels, likely due to low light availability, which resulted in most of the acetate being used for biomass growth (Yx/s of 0.64 ± 0.18 Cmol X/Cmol Acet). To confirm the light limitation on the PMC, SBR tests were conducted with higher light availability, at similar levels as would be expectable from natural sunlight. In this case, the Yx/s reduced to 0.11 ± 0.01 Cmol X/Cmol Acet and the culture presented a PHB production yield on acetate of 0.67 ± 0.01 Cmol PHB/Cmol Acet, leading to a maximum PHB content of 60%. Unlike other studied PMCs, the PMC was capable of simultaneous growth and PHB accumulation continuously throughout the cycle. Thus far, 60% PHA content is the maximum value ever reported for a PMC, a result that prospects the utilization of feast regimes as an alternative strategy for the selection of PHA accumulating PMCs. Furthermore, the PMC also presented high phosphate removal rates, delivering an effluent that complies with phosphate discharge limits. The advantages of selecting PMCs under a permanent feast regime are that no aeration inputs are required; it allows higher PHA contents and phosphate removal rates in comparison to FF-operated PMC systems; and it represents a novel means of integrating wastewater treatment with resource recovery in the form of PHA.

An indigenous plant growth-promoting bacterium isolated from Peganum Harmala rhizosphere in the arid ecosystem was found to solubilize and accumulate phosphates. This isolate was identified as Pseu...

Achromobacter denitrificans strain PR1, previously found to harbour specific degradation pathways with high sulfamethoxazole (SMX) degradation rates, was bioaugmented into laboratory-scale membrane bioreactors (MBRs) operated under aerobic conditions to treat SMX-containing real domestic wastewater. Different hydraulic retention times (HRTs), which is related to reaction time and loading rates, were considered and found to affect the SMX removal efficiency. The availability of primary substrates was important in both bioaugmented and non-bioaugmented activated sludge (AS) for cometabolism of SMX. High HRT (24 h) resulted in low food to microorganism ratio (F/M) and low SMX removal, due to substrate limitation. Decrease in HRT from 24 h to 12 h, 6 h and finally 4 h led to gradual increases in primary substrates availability, e.g. organic compounds and ammonia, resulted in increased SMX removal efficiency and degradation rate, and is more favorable for high-rate wastewater treatment processes. After inoculation into the MBRs, the bioaugmentation strain was sustained in the reactor for a maximum of 31 days even though a significant decrease in abundance was observed. The bioaugmented MBRs showed enhanced SMX removal, especially under SMX shock loads compared to the control MBRs. The results of this study indicate that re-inoculation is required regularly after a period of time to maintain the removal efficiency of the target compound. (c) 2018 Elsevier B.V.

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.

The occurrence of pharmaceutical compounds in wastewater treatment plants and surface waters has been detected worldwide, constituting a potential risk for aquatic ecosystems. Adult zebrafish, of both sexes, were exposed to three common pharmaceutical compounds (atenolol, ketoprofen and diclofenac) and their UV photolysis by-products over seven days. The results show that diclofenac was removed to concentrations < LOD after 5 min of UV irradiation. The oxidative stress response of zebrafish to pharmaceuticals and their photolysis by-products was evaluated through oxidative stress enzymes (glutathione-S-transferase, catalase, superoxide dismutase) and lipid peroxidation. Results suggest that the photolysis by-products of diclofenac were more toxic than those from the other compounds tested, showing an increase in GST and CAT levels, which are also supported by higher MDA levels. Overall, the toxicity of waters containing atenolol and ketoprofen was reduced after the parent compounds were transformed by photolysis, whereas the toxicity increased significantly from the by-products generated through diclofenac photolysis. Therefore, diclofenac photolysis would possibly necessitate higher irradiation time to ensure that the associated by-products are completely degraded to harmless form(s).

A novel Phototrophic - Enhanced Biological Phosphorus Removal (Photo-EBPR) system, consisting of a consortium of photosynthetic organisms and polyphosphate accumulating organisms (PAOs), was studied in this work. A sequencing batch reactor was fed with a mixture of acetate and propionate (75%–25%) and subjected to dark/light cycles in order to select a photo-EBPR system containing PAOs and photosynthetic organisms, the latter likely providers of oxygen to the system. The results from the selection period (stage 1) showed that the photo-EBPR culture was capable of performing P release in the dark and P uptake in the presence of light, under limited oxygen concentrations. During the optimization period, the aeration period, which was initially provided at the end of the light phase, was gradually reduced until a non-aerated system was achieved, while the light intensity was increased. After optimization of the operational conditions, the selected consortium of photosynthetic organisms/PAOs showed high capacity of P removal in the light phase in the absence of air or other electron acceptor. A net P removal of 34 ± 3 mg-P/L was achieved, with a volumetric P removal rate of 15 ± 2 mg-P/L.h, and 79 ± 8% of P removal from the system. Also, in limiting oxygen conditions, the P uptake rate was independent of the PHA consumption, which demonstrates that the organisms obtained energy for P removal from light. These results indicated that a photo-EBPR system can be a potential solution for P removal with low COD/P ratios and in the absence of air, prospecting the use of natural sunlight as illumination, which would reduce the costs of EBPR operation regarding aeration.

Polyhydroxyalkanoate (PHA) production via mixed microbial cultures (MMCs) can potentially decrease process operational costs as compared to conventional pure culture techniques. However, the volumetric productivity of PHA by MMCs must be augmented to increase its cost competitiveness. For this purpose, a three-stage bioreactor system was operated in this study, with (i) anaerobic fermentation of molasses, (ii) culture selection, and (iii) PHA accumulation and harvesting stages. In stage2, bioreactor operation with pH control at 8 led to twice the biomass concentration (up to 8 g VSS L-1, where VSS is the volatile suspended solids) as compared to operation without pH control (maximum pH 9). No loss in the specific PHA storage efficiency was observed (PHA content up to 57.5% and PHA storage rate up to 0.27 Cmol PHA Cmol X-1 h(-1), where X is the active biomass), thereby resulting in twice the volumetric PHA production rate. The limited biomass growth at the higher pH level was not due to nutrient limitation, but likely to a shift in the microbial community. It is hypothesized that the increased enrichment of Azoarcus at pH 8 led to higher PHA productivity. pH control in the culture selection stage can lead to enhanced PHA production from MMCs, improving the viability of the process.

This work studied the possibility of operating a viable polyhydroxyalkanoate (PHA) producing photosynthetic mixed culture (PMC) under dark/light periods without aeration. The culture was subjected to a feast and famine regime, being fed in the dark phase and entering into famine during the light phase. Throughout consecutive feast and famine dark/light periods, the PMC became enriched in PHA accumulating organisms, where non-PHA producing algae that can grow under continuous illumination were out-competed. The very low algae levels enabled greater light and carbon source availability for PHA accumulating bacteria, leading to higher metabolic rates and PHA levels. The PMC reached a PHA content of 30% PHA/VSS, and doubled its specific PHA production rate in relation to PMCs operated previously under continuous illumination. This new process takes a further step towards operating a more cost effective PMC system for PHA production, opening up the possibility for direct sunlight utilization in the future.

For the first time, a mixed photosynthetic culture (MPC) consisting of a consortium of bacteria and algae was investigated for its capacity to accumulate polyhydroxyalkanoates (PHA). The culture was subjected to a feast and famine regime in an illuminated environment without supplying oxygen or any other electron acceptor. The MPC accumulated PHA during the feast phase and consumed it in the famine phase, where the PHA consumption was made possible due to oxygen production by algae. The internal cycling of carbohydrates was also observed, which was likely linked to bacterial glycogen being used as an additional source of energy for acetate uptake during the feast phase, and restored in the famine phase via PHA degradation. The MPC reached a PHA content of 20%, with a PHA storage yield per acetate similar to aerobic systems, opening up the possibility of a new sunlight-driven PHA production process without the need for aeration.

Tetrasphaera and Candidatus Accumulibacter are two abundant polyphosphate accumulating organisms in full-scale enhanced biological phosphorus removal (EBPR) systems. However, little is known about the metabolic behaviour and ecological niche that each organism exhibits in mixed communities. In this study, an enriched culture of Tetrasphaera and Ca. Accumulibacter was obtained using casein hydrolysate as sole carbon source. This culture was able to achieve a high phosphorus removal efficiency (>99%), storing polyphosphate while consuming amino acids anaerobically. Microautoradiography and fluorescence in situ hybridisation confirmed that more than 90% Tetrasphaera cells were responsible for amino acid consumption while Ca. Accumulibacter likely survived on fermentation products. Tetrasphaera performed the majority of the P removal (approximately 80%) in this culture, and batch tests showed that the metabolism of some carbon sources could actually lead to anaerobic orthophosphate (Pi) uptake (9.0 ± 2.1 mg-P/L) through energy generated by fermentation of glucose and amino acids. This anaerobic Pi uptake may lead to lower net Pi release to C uptake ratios and reduce the Pi needed to be removed aerobically in WWTPs. Intracellular metabolites such as amino acids, sugars, volatile fatty acids and small amines were observed as potential storage products, which may serve as energy sources in the aerobic phase. Evidence of the urea cycle was found, which could be involved in reducing the intracellular nitrogen content. This study improves our understanding of how phosphorus is removed in EBPR systems and can enable novel process optimisation strategies.

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 $${\text{X}}_{\text{SS}}$$ ·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 $${\text{X}}_{\text{SS}}$$ ·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.

Clofibric acid (CLF) is the pharmaceutically active metabolite of lipid regulators clofibrate, etofibrate and etofyllinclofibrate, and it is considered both environmentally persistent and refractory. This work studied the biotransformation of CLF in aerobic sequencing batch reactors (SBRs) with mixed microbial cultures, monitoring the efficiency of biotransformation of CLF and the production of metabolites. The maximum removal achieved was 51% biodegradation (initial CLF concentration=2 mg L(-1)), where adsorption and abiotic removal mechanisms were shown to be negligible, showing that CLF is indeed biodegradable. Tests showed that the observed CLF biodegradation was mainly carried out by heterotrophic bacteria. Three main metabolites were identified, including α-hydroxyisobutyric acid, lactic acid and 4-chlorophenol. The latter is known to exhibit higher toxicity than the parent compound, but it did not accumulate in the SBRs. α-Hydroxyisobutyric acid and lactic acid accumulated for a period, where nitrite accumulation may have been responsible for inhibiting their degradation. A metabolic pathway for the biodegradation of CLF is proposed in this study.

Previous studies have shown that polyphosphate-accumulating organisms (PAOs) are able to behave as glycogen-accumulating organisms (GAOs) under different conditions. In this study we investigated the behavior of a culture enriched with Accumulibacter at different levels of polyphosphate (poly-P) storage. The results of stoichiometric ratios Gly degraded/HAc uptake, PHB synthesized/HAc uptake, PHV synthesized/HAc uptake and P release/HAc uptake confirmed a metabolic shift from PAO metabolism to GAO metabolism: PAOs with high poly-P content used the poly-P to obtain adenosine tri-phosphate (ATP), and glycogen (Gly) to obtain nicotinamide adenine dinucleotide (NADH) and some ATP. In a test where poly-P depletion was imposed on the culture, all the acetate (HAc) added in each cycle was transformed into polyhydroxyalkanoate (PHA) despite the decrease of poly-P inside the cells. This led to an increase of the Gly degraded/HAc uptake ratio that resulted from a shift towards the glycolytic pathway in order to compensate for the lack of ATP formed from poly-P hydrolysis. The shift from PAO to GAO metabolism was also reflected in the change in the PHA composition as the poly-P availability decreased, suggesting that polyhydroxyvalerate (PHV) is obtained due to the consumption of excess reducing equivalents to balance the internal NADH, similarly to GAO metabolism. Fluorescence in situ hybridization analysis showed a significant PAO population change from Type I to Type II Accumulibacter as the poly-P availability decreased in short term experiments. This work suggests that poly-P storage levels and GAO-like metabolism are important factors affecting the competition between different PAO Types in enhanced biological phosphorus removal systems. © 2012 Elsevier Ltd., This research work has been supported by the Generalitat Valenciana (GVPRE/2008/044) and the Polytechnic University of Valencia (PAID-06-08-3227), which are gratefully acknowledged. Special acknowledgements to Consejo Nacional de la Ciencia y la Tecnologia de Mexico (CONACYT) No. 207966 and the Fundacao para a Ciencia e Tecnologia for PEst-C/EQB/LA0006/2011, PEst-OE/EQB/LA0004/2011 and SFRH/BPD/30800/2006.

Nitrite is known to accumulate in wastewater treatment plants (WWTPs) under certain environmental conditions. The protonated form of nitrite, free nitrous acid (FNA), has been found to cause severe inhibition to numerous bioprocesses at WWTPs. However, this inhibitory effect of FNA may possibly be gainfully exploited, such as repressing nitrite oxidizing bacteria (NOB) growth to achieve N removal via the nitrite shortcut. However, the inhibition threshold of FNA to repress NOB (∼0.02 mg HNO2-N/L) may also inhibit other bioprocesses. This paper reviews the inhibitory effects of FNA on nitrifiers, denitrifiers, anammox bacteria, phosphorus accumulating organisms (PAO), methanogens, and other microorganisms in populations used in WWTPs. The possible inhibition mechanisms of FNA on microorganisms are discussed and compared. It is concluded that a single inhibition mechanism is not sufficient to explain the negative impacts of FNA on microbial metabolisms and that multiple inhibitory effects can be generated from FNA. The review would suggest further research is necessary before the FNA inhibition mechanisms can be more effectively used to optimize WWTP bioprocesses. Perspectives on research directions, how the outcomes may be used to manipulate bioprocesses and the overall implications of FNA on WWTPs are also discussed.

The use of mixed microbial cultures enriched for biological mercury removal is explored in this paper, focusing on the ecological shifts occurring throughout acclimatization to mercury and on the long-term stability of four microbial enrichments. The 16S rRNA genetic profiles obtained by denaturing gradient gel electrophoresis (DGGE) revealed that the glucose and ethanol cultures had similar profiles, whereas the acetate cultures diverged into a totally dissimilar cluster. Quantification of the merA gene copies in each enrichment showed higher values for the glucose culture, followed by the ethanol and then the acetate cultures, which was consistent with the mercury removal performance throughout the study. Isolates were obtained from the four cultures and analyzed with respect to their genetic (16S rRNA) and functional (merA) phylogenies in order to identify mercury-resistant species enriched with different carbon sources. All mercury-resistant isolates obtained from the glucose and ethanol cultures belonged to the Gammaproteobacteria, whereas acetate cultures also contained members of other phyla, with differences in merA sequences. Higher phylogenetic than functional diversity of the isolates, together with increasing merA copies even after culture stabilisation, highlight the role of horizontal gene transfer in the acclimatization process.

In the enhanced biological phosphorus removal (EBPR) process, the competition between polyphosphate accumulating organisms (PAO) and glycogen accumulating organisms (GAO) has been studied intensively in recent years by both microbiologists and engineers, due to its important effects on phosphorus removal performance and efficiency. This study addresses the impact of microbial ecology on assessing the PAO-GAO competition through metabolic modelling, focussing on reviewing recent developments, discussion of how the results from molecular studies can impact the way we model the process, and offering perspectives for future research opportunities based on unanswered questions concerning PAO and GAO metabolism. Indeed, numerous findings that are seemingly contradictory could in fact be explained by the metabolic behaviour of different sub-groups of PAOs and/or GAOs exposed to different environmental and operational conditions. Some examples include the glycolysis pathway (i.e. Embden-Meyerhof-Parnas (EMP) vs. Entner-Doudoroff (ED)), denitrification capacity, anaerobic tricarboxylic acid (TCA) cycle activity and PAOs' ability to adjust their metabolism to e.g. a GAO-like metabolism. Metabolic modelling may further yield far-reaching influences on practical applications as well, and serves as a bridge between molecular/biochemical research studies and the optimisation of wastewater treatment plant operation.

Glycolysis has been generally accepted to be the source of reducing power used for the synthesis of polyhydroxyalkanoates (PHAs) in the anaerobic metabolism of polyphosphate accumulating organisms (PAOs). However, the tricarboxylic acid cycle (TCA) has also been suggested to contribute to the generation of reducing equivalents, creating some controversy in this research field over the last two decades. Various research approaches have been applied in order to clarify this issue, including the analysis of intracellular compounds, labelled carbon substrates and gene expression. However, the conclusions of these studies seem not to be consolidated. The extent of TCA cycle involvement could be related to the experimental methods employed, the community structure of the cultures used, and also the operational conditions employed. This mini-review analyses the historical findings related to the source of reducing power in PAOs, and highlights the different approaches used in the previous studies. Key factors influencing the generation of reducing power through different metabolic pathways are discussed, and further research directions are also proposed.

In this study, it is proposed that short sequencing batch reactor (SBR) cycles select and maintain a robust and active biomass, able to cope with typical disturbances occurring in wastewater treatment plants. In order to test this hypothesis, an SBR system was subjected to COD, N and P shock loads. It was shown that the sludge enriched in the SBR operated with short cycles was able to rapidly recover from the tested disturbances. COD and N removal recovered within 1-2 days for shock loads of 10 times the standard concentration. The P removal took up to 2-3 sludge ages to fully recover from the COD spike, but the enhanced biological phosphorus removal (EBPR) performance was still able to be totally re-established after each of the tests, even in theoretically adverse conditions for the growth of polyphosphate accumulating organisms.

On-line process control may cause substantial changes to the microbial community in a biological wastewater treatment system. Recent studies have shown such effects can be exploited in control system design to achieve an optimised microbial community. Excellent progress has been made on the elimination of nitrite-oxidising bacteria (NOB) in biological nitrogen removal wastewater treatment systems using on-line aeration control, enabling nitrogen removal via the nitrite pathway. Control methods for eliminating NOB are now available for both continuous systems and sequencing batch reactors, and have been demonstrated with both domestic and various types of industrial wastewaters. The elimination or reduced growth of glycogen accumulating organisms (GAOs), a competitor of polyphosphate accumulating organisms (PAOs), in enhanced biological phosphorus removal (EBPR) systems via pH and carbon source control has been conceptually demonstrated through the use of enriched cultures. However, these strategies are not yet ready for the control of practical EBPR processes. Sludge population optimisation also involves selecting the most desirable organism or a consortium of organisms to perform a required function. This is particularly important for nitrification, one of the most important and delicate steps in modern wastewater treatment plants. Results from both experimental and simulation studies suggest that reactor operation could have a major impact on the nitrifier community structure, which should be further investigated in future studies.

The metabolism of polyphosphate accumulating organisms (PAOs) has been widely studied through the use of lab-scale enrichments. Various metabolic models have been formulated, based on the results from lab-scale experiments using enriched PAO cultures. A comparison between the anaerobic stoichiometry predicted by metabolic models with that exhibited by full-scale sludge in enhanced biological phosphorus removal (EBPR) wastewater treatment plants (WWTPs) was performed in this study. Batch experiments were carried out with either acetate or propionate as the sole carbon source, using sludges from two different EBPR-WWTPs in Australia that achieved different phosphorus removal performances. The results support the hypothesis that the anaerobic degradation of glycogen is the primary source of reducing equivalents generated by PAOs, however, they also suggested a partial contribution of the tricarboxylic acid (TCA) cycle in some cases. The experimental results obtained when acetate was the carbon source suggest the involvement of the modified succinate-propionate pathway for the generation of poly-beta-hydroxyvalerate (PHV). Overall, the batch test results obtained from full-scale EBPR sludge with both substrates were generally well described by metabolic model predictions for PAOs.

Candidatus Accumulibacter Phosphatis is widely considered to be a polyphosphate accumulating organism (PAO) of prime importance in enhanced biological phosphorus removal (EBPR) systems. This organism has yet to be isolated, despite many attempts. Previous studies on the biochemical and physiological aspects of this organism, as well as its response to different EBPR operational conditions, have generally relied on the use of mixed culture enrichments. One frequent problem in obtaining highly enriched cultures of this organism is the proliferation of glycogen accumulating organisms (GAO) that can compete with PAOs for limited carbon sources under similar operational conditions. In this study, Candidatus Accumulibacter Phosphatis has been enriched in a lab-scale bioreactor to a level greater than 90% as quantified by fluorescence in situ hyrbridisation (FISH). This is the highest enrichment of this organism that has been reported thus far, and was obtained by alternating the sole carbon source in the feed between acetate and propionate every one to two sludge ages, and operating the bioreactor within a pH range of 7.0-8.0. Simultaneously, the presence of two known groups of GAOs was eliminated under these operational conditions. Excellent phosphorus removal performance and stability were maintained in this system, where the phosphorous concentration in the effluent was below 0.2 mg/L for more than 7 months. When a disturbance was introduced to this system by adding sludge from an enriched GAO culture, Candidatus Accumulibacter Phosphatis once again became highly enriched, while the GAOs were out-competed. This feeding strategy is recommended for future studies focused on describing the physiology and biochemistry of Accumulibacter, where a highly-enriched culture of this organism is of high importance. (c) 2006 Elsevier Ltd. All rights reserved.

In the microbial competition observed in enhanced biological phosphorus removal (EBPR) systems, an undesirable group of micro-organisms known as glycogen-accumulating organisms (GAOs) compete for carbon in the anaerobic period with the desired polyphosphate-accumulating organisms (PAOs). Some studies have suggested that a propionate carbon source provides PAOs with a competitive advantage over GAOs in EBPR systems; however, the metabolism of GAOs with this carbon source has not been previously investigated. In this study, GAOs were enriched in a laboratory-scale bioreactor with propionate as the sole carbon source, in an effort to better understand their biochemical processes. Based on comprehensive solid-, liquid- and gas-phase chemical analytical data from the bioreactor, a metabolic model was proposed for the metabolism of propionate by GAOs. The model adequately described the anaerobic stoichiometry observed through chemical analysis, and can be a valuable tool for further investigation of the competition between PAOs and GAOs, and for the optimization of the EBPR process. A group of Alphaproteobacteria dominated the biomass (96 % of Bacteria) from this bioreactor, while post-fluorescence in situ hybridization (FISH) chemical staining confirmed that these Alphaproteobacteria produced poly-β-hydroxyalkanoates (PHAs) anaerobically and utilized them aerobically, demonstrating that they were putative GAOs. Some of the Alphaproteobacteria were related to Defluvicoccus vanus (16 % of Bacteria), but the specific identity of many could not be determined by FISH. Further investigation into the identity of other GAOs is necessary.

The cause of Huntington disease (HD) is a polyglutamine repeat expansion of more than 36 units in the huntingtin protein, which is inversely correlated with the age at onset of the disease. However, additional genetic factors are believed to modify the course and the age at onset of HD. Recently, we identified the V471A polymorphism in the autophagy-related gene ATG7, a key component of the autophagy pathway that plays an important role in HD pathogenesis, to be associated with the age at onset in a large group of European Huntington disease patients. To confirm this association in a second independent patient cohort, we analysed the ATG7 V471A polymorphism in additional 1,464 European HD patients of the "REGISTRY" cohort from the European Huntington Disease Network (EHDN). In the entire REGISTRY cohort we could not confirm a modifying effect of the ATG7 V471A polymorphism. However, analysing a modifying effect of ATG7 in these REGISTRY patients and in patients of our previous HD cohort according to their ethnic origin, we identified a significant effect of the ATG7 V471A polymorphism on the HD age at onset only in the Italian population (327 patients). In these Italian patients, the polymorphism is associated with a 6-years earlier disease onset and thus seems to have an aggravating effect. We could specify the role of ATG7 as a genetic modifier for HD particularly in the Italian population. This result affirms the modifying influence of the autophagic pathway on the course of HD, but also suggests population-specific modifying mechanisms in HD pathogenesis.

Enhanced biological phosphorus removal (EBPR) performance is directly affected by the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). This study investigates the effects of carbon source on PAO and GAO metabolism. Enriched PAO and GAO cultures were tested with the two most commonly found volatile fatty acids (VFAs) in wastewater systems, acetate and propionate. Four sequencing batch reactors (SBRs) were operated under similar conditions and influent compositions with either acetate or propionate as the sole carbon source. The stimulus for selection of the PAO and GAO phenotypes was provided only through variation of the phosphorus concentration in the feed. The abundance of PAOs and GAOs was quantified using fluorescence in situ hybridisation (FISH). In the acetate fed PAO and GAO reactors, "Candidatus Accumulibacter phosphatis" (a known PAO) and "Candidatus Competibacter phosphatis" (a known GAO) were present in abundance. A novel GAO, likely belonging to the group of Alphaproteobacteria, was found to dominate the propionate fed GAO reactor. The results clearly show that there are some very distinctive differences between PAOs and GAOs in their ability to take up acetate and propionate. PAOs enriched with acetate as the sole carbon source were immediately able to take up propionate, likely at a similar rate as acetate. However, an enrichment of GAOs with acetate as the sole carbon source took up propionate at a much slower rate (only about 5% of the rate of acetate uptake on a COD basis) during a short-term switch in carbon source. A GAO enrichment with propionate as the sole carbon source took up acetate at a rate that was less than half of the propionate uptake rate on a COD basis. These results, along with literature reports showing that PAOs fed with propionate (also dominated by Accumulibacter) can immediately switch to acetate, suggesting that PAOs are more adaptable to changes in carbon source as compared to GAOs. This study suggests that the PAO and GAO competition could be influenced in favour of PAOs through the provision of propionate in the feed or even by regularly switching the dominant VFA species in the wastewater. Further study is necessary in order to provide greater support for these hypotheses.

The effectiveness of enhanced biological phosphorus removal (EBPR) systems is directly affected by the competition of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). This study investigated the short-term effects of carbon source on PAO and GAO performance. The tests were designed to clearly determine the impact of volatile fatty acid (VFA) composition on the performance of two types of biomass, one enriched for PAOs and the other for GAOs. The two populations were enriched in separate reactors using identical operating conditions and very similar influent compositions with acetate as the sole carbon source. The only difference was that a very low level of phosphorus was present in the influent to the GAO reactor. The abundance of PAOs and GAOs was quantified using fluorescence in-situ hybridisation. The results clearly show that there are some very distinctive differences between PAOs and GAOs in their ability to utilise different carbon substrates. While both are able to take up acetate rapidly and completely, the GAOs are far slower at consuming propionate than the PAOs during short-term substrate changes. This provides a potentially highly valuable avenue to influence the competition between PAOs and GAOs. Other VFAs studied seem to be less usable in the short term by both PAOs and GAOs, as indicated by their much lower uptake rates.

The type of carbon source present in the wastewater is one factor that affects the competition between polyphosphate accumulating organisms (PAO) and glycogen accumulating organisms (GAO) and therefore, the efficiency of the enhanced biological phosphorus removal (EBPR) process. This study investigated the impact of the carbon source composition on the anaerobic and aerobic kinetics of PAOs and the EBPR performance of an 85% PAO enrichment. When both acetate (HAc) and propionate (HPr) were present, propionate was depleted more quickly, with a constant uptake rate of 0.18 ± 0.02 C-mol/(C-mol biomass·h), while the acetate uptake rate decreased with an increase in propionate concentration, due to the substrate competition between acetate and propionate. The metabolic model for PAOs was modified to incorporate the anaerobic substrate competition effect. The aerobic rates for phosphorus (P) uptake, glycogen production and polyhydroxyalkanoates (PHA) degradation were within the same range for all tests, indicating that these rates are essentially independent of the acetate and propionate concentration, simplifying the calibration procedure for metabolic models. The metabolic model applied to describe the anaerobic and aerobic activity agreed well with the experimental data of HAc, HPr, P, PHA and biomass growth. The low glycogen consumption observed suggest that some reducing equivalents were generated anaerobically through the TCA cycle. The results of this work suggest that the propionate uptake kinetics by PAOs can provide them an advantage over GAOs in EBPR systems, even when the propionate fraction of the influent is relatively low.

Enhanced biological phosphorus removal (EBPR) is one of the most important methods of phosphorus removal in municipal wastewater treatment plants, having been described by different modelling approaches. In this process, the PAOs (polyphosphate accumulating organisms) and GAOs (glycogen accumulating organisms) compete for volatile fatty acids uptake under anaerobic conditions. Recent studies have revealed that the metabolic pathways used by PAOs in order to obtain the energy and the reducing power needed for polyhydroxyalkanoates synthesis could change depending on the amount of polyphosphate stored in the cells. The model presented in this paper extends beyond previously developed metabolic models by including the ability of PAO to change their metabolic pathways according to the content of poly-P available. The processes of the PAO metabolic model were adapted to new formulations enabling the change from P-driven VFA uptake to glycogen-driven VFA uptake using the same process equations. The stoichiometric parameters were changed from a typical PAO coefficient to a typical GAO coefficient depending on the internal poly-P with Monod-type expressions. The model was calibrated and validated with seven experiments under different internal poly-P concentrations, showing the ability to correctly represent the PAO metabolic shift at low poly-P concentrations. The sensitivity and error analysis showed that the model is robust and has the ability to describe satisfactorily the change from one metabolic pathway to the other one, thereby encompassing a wider range of process conditions found in EBPR plants. © 2014 Elsevier Ltd. All rights reserved., This research work has been supported by the Generalitat Valenciana (GVPRE/2008/044) and the Polytechnic University of Valencia (PAID-06-08-3227), which are gratefully acknowledged. Special acknowledgements to Consejo Nacional de la Ciencia y la Tecnologia de Mexico (CONACYT) No. 207966. Fundacao para a Ciencia e Tecnologia, Portugal is acknowledged through projects PTDC/AAC-AMB/120581/2010 and PEst-C/EQB/LA0006/2013.

This study investigates, for the first time, the application of metabolic models incorporating polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) towards describing the biochemical transformations of full-scale enhanced biological phosphorus removal (EBPR) activated sludge from wastewater treatment plants (WWTPs). For this purpose, it was required to modify previous metabolic models applied to lab-scale systems by incorporating the anaerobic utilisation of the TCA cycle and the aerobic maintenance processes based on sequential utilisation of polyhydroxyalkanoates, followed by glycogen and polyphosphate. The abundance of the PAO and GAO populations quantified by fluorescence in situ hybridisation served as the initial conditions of each biomass fraction, whereby the models were able to describe accurately the experimental data. The kinetic rates were found to change among the four different WWTPs studied or even in the same plant during different seasons, either suggesting the presence of additional PAO or GAO organisms, or varying microbial activities for the same organisms. Nevertheless, these variations in kinetic rates were largely found to be proportional to the difference in acetate uptake rate, suggesting a viable means of calibrating the metabolic model. The application of the metabolic model to full-scale sludge also revealed that different Accumulibacter clades likely possess different acetate uptake mechanisms, as a correlation was observed between the energetic requirement for acetate transport across the cell membrane with the diversity of Accumulibacter present. Using the model as a predictive tool, it was shown that lower acetate concentrations in the feed as well as longer aerobic retention times favour the dominance of the TCA metabolism over glycolysis, which could explain why the anaerobic TCA pathway seems to be more relevant in full-scale WWTPs than in lab-scale systems.

This study analysed the enhanced biological phosphorus removal (EBPR) microbial community and metabolic performance of five full-scale EBPR systems by using fluorescence in situ hybridisation combined with off-line batch tests fed with acetate under anaerobic–aerobic conditions. The phosphorus accumulating organisms (PAOs) in all systems were stable and showed little variability between each plant, while glycogen accumulating organisms (GAOs) were present in two of the plants. The metabolic activity of each sludge showed the frequent involvement of the anaerobic tricarboxylic acid cycle (TCA) in PAO metabolism for the anaerobic generation of reducing equivalents, in addition to the more frequently reported glycolysis pathway. Metabolic variability in the use of the two pathways was also observed, between different systems and in the same system over time. The metabolic dynamics was linked to the availability of glycogen, where a higher utilisation of the glycolysis pathway was observed in the two systems employing side-stream hydrolysis, and the TCA cycle was more active in the A 2 O systems. Full-scale plants that showed higher glycolysis activity also exhibited superior P removal performance, suggesting that promotion of the glycolysis pathway over the TCA cycle could be beneficial towards the optimisation of EBPR systems.

Polyhydroxyalkanoates (PHAs) are polyesters that can be produced from industrial wastewater or surplus products by mixed microbial cultures (MMC). To optimise PHA production by MMCs, the link between the microbial structure and function of these enrichments must be better established. This study investigates, for the first time, the impact of operational changes on the microbial community and the associated process performance of PHA producing MMCs. It was found that a PHA producing community fed with fermented molasses was dominated by a combination of Azoarcus, Thauera and Paracoccus, where the former two groups were present in highest abundance. Dominance of either Thauera or Azoarcus seemed to be determined by the organic loading rate imposed in the selection reactor. While higher Azoarcus enrichments led to higher PHA production yields and lower biomass growth yields as compared to Thauera, the Thauera abundance was strongly linked to higher hydroxyvalerate (HV) fractions. Paracoccus abundance was correlated with a lower PHA production capacity as compared to Azoarcus, and produced lower HV fractions than Thauera and Azoarcus. The findings of this study suggest that MMCs targeting the enrichment of Azoarcus as the primary biomass fraction with Thauera as a minor fraction lead to optimal specific PHA production and polymers with high HV content, which is likely to improve their mechanical properties.

The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small interfering RNA (siRNA) (DC-3) targeting the highly conserved 5′ cyclization sequence (5′CS) region of the DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype in vitro . To determine if DC-3 siRNA could inhibit DENV in vivo , an “ in vivo -ready” version of DC-3 was synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups in several tissues at various time points postinfection. These results suggest that exogenously introduced siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses.

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).

In this review, we provide an overview of the methods employed in four recent studies that described novel methods for computational prediction of secreted effectors from type III and IV secretion systems in Gram-negative bacteria. We present the results of these studies in terms of performance at accurately predicting secreted effectors and similarities found between secretion signals that may reflect biologically relevant features for recognition. We discuss the Web-based tools for secreted effector prediction described in these studies and announce the availability of our tool, the SIEVE server ( http://www.sysbep.org/sieve ). Finally, we assess the accuracies of the three type III effector prediction methods on a small set of proteins not known prior to the development of these tools that we recently discovered and validated using both experimental and computational approaches. Our comparison shows that all methods use similar approaches and, in general, arrive at similar conclusions. We discuss the possibility of an order-dependent motif in the secretion signal, which was a point of disagreement in the studies. Our results show that there may be classes of effectors in which the signal has a loosely defined motif and others in which secretion is dependent only on compositional biases. Computational prediction of secreted effectors from protein sequences represents an important step toward better understanding the interaction between pathogens and hosts.

In this study, enhanced biological phosphorus removal (EBPR) metabolic models are expanded in order to incorporate the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) under sequential anaerobic/anoxic/aerobic conditions, which are representative of most full-scale EBPR plants. Since PAOs and GAOs display different denitrification tendencies, which is dependent on the phylogenetic identity of the organism, the model was separated into six distinct biomass groups, constituting Accumulibacter Types I and II, as well as denitrifying and non-denitrifying Competibacter and Defluviicoccus GAOs. Denitrification was modelled as a multi-step process, with nitrate (NO3), nitrite (NO2), nitrous oxide (N2O) and di-nitrogen gas (N2) being the primary components. The model was calibrated and validated using literature data from enriched cultures of PAOs and GAOs, obtaining a good description of the observed biochemical transformations. A strong correlation was observed between Accumulibacter Types I and II, and nitrate-reducing and non-nitrate-reducing PAOs, respectively, where the abundance of each PAO subgroup was well predicted by the model during an acclimatisation period from anaerobic–aerobic to anaerobic–anoxic conditions. Interestingly, a strong interdependency was observed between the anaerobic, anoxic and aerobic kinetic parameters of PAOs and GAOs. This could be exploited when metabolic models are calibrated, since all of these parameters should be changed by an identical factor from their default value. Factors that influence these kinetic parameters include the fraction of active biomass, relative aerobic/anoxic fraction and the ratio of acetyl-CoA to propionyl-CoA. Employing a metabolic approach was found to be advantageous in describing the performance and population dynamics in such complex microbial ecosystems.

The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately in dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. Here we identify specific miRNAs that function at synapses to control dendritic spine structure by performing a functional screen. One of the identified miRNAs, miR-138, is highly enriched in the brain, localized within dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons. miR-138 controls the expression of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the alpha(13) subunits of G proteins (G alpha(13)). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized G alpha(13) both suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of G alpha(13) might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized complexity of miRNA-dependent control of dendritic spine morphogenesis.

Immunohistochemical localization of sulfhydryloxidase (SOx) has been examined in the testis of the Axolotl (Ambystoma mexicanum). The urodelan testis contains germ cells in various phases of differentiation from primordial germ cells to mature spermatozoa. SOx immunoreactivity is present in mitochondria of primordial germ cells and primary spermatogonia and declines within the population of secondary spermatogonia, suggesting, that the antibody used to localize SOx may serve to estimate the developmental stage of spermatogonia towards meiosis, since more undifferentiated cells react positively. Intensity of immunostaining increases again in spermatocytes and becomes most intense in early round spermatids correlating on ultrastructural level with an accumulation of numerous mitochondria in that part of the cytoplasm, where the acrosome vesicle is formed. Mature sperm are immunonegative. Additionally, Leydig cells within the glandular tissue are stained by the antibody. Thus the distribution pattern of SOx immunoreactivity principally resembles that in the mammalian testis found during ontogenesis or in the adult seminiferous epithelium. The possible functional significance of mitochondrial SOx in germ cells and Leydig cells is discussed. These results suggest, that the amphibian testis is a model for experimental problems dealing with the investigation of germ cells in various developmental phases including very undifferentiated premeiotic germ cells. The cystic testis may be of value in studying influences of various experimental conditions on varied homogeneous populations of germ cells.

Background This paper presents a metabolic model describing the production of polyhydroxyalkanoate (PHA) copolymers in mixed microbial cultures, using mixtures of acetic and propionic acid as carbon source material. Material and energetic balances were established on the basis of previously elucidated metabolic pathways. Equations were derived for the theoretical yields for cell growth and PHA production on mixtures of acetic and propionic acid as functions of the oxidative phosphorylation efficiency, P/O ratio. The oxidative phosphorylation efficiency was estimated from rate measurements, which in turn allowed the estimation of the theoretical yield coefficients. Results The model was validated with experimental data collected in a sequencing batch reactor (SBR) operated under varying feeding conditions: feeding of acetic and propionic acid separately (control experiments), and the feeding of acetic and propionic acid simultaneously. Two different feast and famine culture enrichment strategies were studied: (i) either with acetate or (ii) with propionate as carbon source material. Metabolic flux analysis (MFA) was performed for the different feeding conditions and culture enrichment strategies. Flux balance analysis (FBA) was used to calculate optimal feeding scenarios for high quality PHA polymers production, where it was found that a suitable polymer would be obtained when acetate is fed in excess and the feeding rate of propionate is limited to ~0.17 C-mol/(C-mol.h). The results were compared with published pure culture metabolic studies. Conclusion Acetate was more conducive toward the enrichment of a microbial culture with higher PHA storage fluxes and yields as compared to propionate. The P/O ratio was not only influenced by the selected microbial culture, but also by the carbon substrate fed to each culture, where higher P/O ratio values were consistently observed for acetate than propionate. MFA studies suggest that when mixtures of acetate and propionate are fed to the cultures, the catabolic activity is primarily guaranteed through acetate uptake, and the characteristic P/O ratio of acetate prevails over that of propionate. This study suggests that the PHA production process by mixed microbial cultures has the potential to be comparable or even more favourable than pure cultures.

Mercury (Hg) is the most highly toxic heavy metal, and must be removed from waterways to very low levels. Biologically mediated mercury removal is an emerging technology that has the potential to be robust, efficient and cost-effective. In this study, the impact of carbon source on the behaviour and microbial community composition of mixed microbial cultures was evaluated, and their performance was compared with a pure culture of Pseudomonas putida spi3. Glucose and acetate, two carbon sources that are commonly present in wastewaters, were chosen for this study. Distinct microbial populations were enriched with each carbon source. Glucose led to a more suitable microbial culture for Hg(2+) bioreduction that was able to reduce Hg(2+) at faster rates when compared to acetate. Furthermore, acetate consistently led to poorer process performance, irrespective of the microbial culture, possibly due to the formation of mercuric acetate complexes. It is proposed that glucose can be a more beneficial carbon source than acetate for the successful operation of Hg bioremediation systems.