Inhibitory mechanisms on dry anaerobic digestion: Ammonia, hydrogen and propionic acid relationship.

Inhibitory pathways in dry anaerobic digestion are still understudied and current knowledge on wet processes cannot be easily transferred. This study forced instability in pilot-scale digesters by operating at short retention times (40 and 33 days) in order to understand inhibition pathways over long term operation (145 days). The first sign of inhibition at elevated total ammonia concentrations (8 g/l) was a headspace hydrogen level over the thermodynamic limit for propionic degradation, causing propionic accumulation. The combined inhibitory effect of propionic and ammonia accumulation resulted in further increased hydrogen partial pressures and n-butyric accumulation. The relative abundance of Methanosarcina increased while that of Methanoculleus decreased as digestion deteriorated. It was hypothesized that high ammonia, total solids and organic loading rate inhibited syntrophic acetate oxidisers, increasing their doubling time and resulting in its wash out, which in turn inhibited hydrogenotrophic methanogenesis and shifted the predominant methanogenic pathway towards acetoclastic methanogenesis at free ammonia over 1.5 g/l. C/N increases to 25 and 29 reduced inhibitors accumulation but did not avoid inhibition or the washout of syntrophic acetate oxidising bacteria.

Looking through the FOG: microbiome characterization and lipolytic bacteria isolation from a fatberg site.

Sewer systems are complex physical, chemical and microbial ecosystems where fats, oils and grease (FOG) present a major problem for sewer management. Their accumulation can lead to blockages ('Fatbergs'), sewer overflows and disruption of downstream wastewater treatment. Further advancements of biological FOG treatments need to be tailored to degrade the FOG, and operate successfully within the sewer environment. In this study we developed a pipeline for isolation of lipolytic strains directly from two FOG blockage sites in the UK, and isolated a range of highly lipolytic bacteria. We selected the five most lipolytic strains using Rhodamine B agar plates and pNP-Fatty acid substrates, with two Serratia spp., two Klebsiella spp. and an environmental Acinetobacter strain that all have the capacity to grow on FOG-based carbon sources. Their genome sequences identified the genetic capacity for fatty acid harvesting (lipases), catabolism and utilization (Fad genes). Furthermore, we performed a preliminary molecular characterization of the microbial community at these sites, showing a diverse community of environmental bacteria at each site, but which did include evidence of sequences related to our isolates. This study provides proof of concept to isolation strategies targeting Fatberg sites to yield candidate strains with bioremediation potential for FOG in the wastewater network. Our work sets the foundation for development of novel bioadditions tailored to the environment with non-pathogenic Acinetobacter identified as a candidate for this purpose.

Divergent evolution toward sex chromosome-specific gene regulation in Drosophila.

The dosage compensation complex (DCC) of Drosophila identifies its X-chromosomal binding sites with exquisite selectivity. The principles that assure this vital targeting are known from the D. melanogaster model: DCC-intrinsic specificity of DNA binding, cooperativity with the CLAMP protein, and noncoding roX2 RNA transcribed from the X chromosome. We found that in D. virilis, a species separated from melanogaster by 40 million years of evolution, all principles are active but contribute differently to X specificity. In melanogaster, the DCC subunit MSL2 evolved intrinsic DNA-binding selectivity for rare PionX sites, which mark the X chromosome. In virilis, PionX motifs are abundant and not X-enriched. Accordingly, MSL2 lacks specific recognition. Here, roX2 RNA plays a more instructive role, counteracting a nonproductive interaction of CLAMP and modulating DCC binding selectivity. Remarkably, roX2 triggers a stable chromatin binding mode characteristic of DCC. Evidently, X-specific regulation is achieved by divergent evolution of protein, DNA, and RNA components.

Achieving drinking water compliance levels for metaldehyde with an acclimated sand bioreactor.

Metaldehyde removal was delivered to below the 0.1 µg L-1 regulatory concentration in a laboratory scale continuous upflow fluidised sand bioreactor that had undergone acclimation through selective enrichment for metaldehyde degradation. This is the first reported case of successful continuous flow biological treatment of metaldehyde from real drinking water sources treating environmentally realistic metaldehyde concentrations. The impact of the acclimation process was impermanent, with the duration of effective treatment directly related to the elevated concentration of metaldehyde used during the enrichment process. The efficacy of the approach was demonstrated in continuous flow columns at both laboratory and pilot scale enabling degradation rates of between 0.1 and 0.2 mg L-1 h-1. Future work needs to focus on optimisation of the sand bioreactor and the acclimation process to ensure viability and feasibility of the approach at full scale.

Two-step mechanism for selective incorporation of lncRNA into a chromatin modifier.

The MLE DExH helicase and the roX lncRNAs are essential components of the chromatin modifying Dosage Compensation Complex (DCC) in Drosophila. To explore the mechanism of ribonucleoprotein complex assembly, we developed vitRIP, an unbiased, transcriptome-wide in vitro assay that reveals RNA binding specificity. We found that MLE has intrinsic specificity for U-/A-rich sequences and tandem stem-loop structures and binds many RNAs beyond roX in vitro. The selectivity of the helicase for physiological substrates is further enhanced by the core DCC. Unwinding of roX2 by MLE induces a highly selective RNA binding surface in the unstructured C-terminus of the MSL2 subunit and triggers-specific association of MLE and roX2 with the core DCC. The exquisite selectivity of roX2 incorporation into the DCC thus originates from intimate cooperation between the helicase and the core DCC involving two distinct RNA selection principles and their mutual refinement.

Assessing the potential of enhanced primary clarification to manage fats, oils and grease (FOG) at wastewater treatment works.

Daily, sewage treatment works (STWs) receive large volumes of fats, oils and greases (FOG), by-products of food preparation. To increase FOG removal at STW, conventional primary sedimentation tanks (PSTs) can be enhanced using chemical coagulant or through dissolved air flotation (DAF) techniques. This work aimed to assess the potential benefits of enhanced primary treatment for FOG removal through an energy and costs analysis. To achieve this, a five-year sampling programme was conducted monthly at 15 STWs measuring FOG concentrations in crude and settled sewage (i.e. after primary treatment). In addition, two DAF pilot systems were trialled for four months and their performance, in terms of FOG removal, was assessed and compared to that of a control primary clarifier. Across the 15 STWs, influent FOG concentrations were found at 57 ±â€¯11 mg.L-1. Chemical coagulants dosed prior to PSTs increased FOG removal rates on average to 71% whilst traditional sedimentation only achieved 50% removal. Effluent FOG concentrations were found between 12-22 mg.L-1 and 19-36 mg.L-1 respectively. By contrast, DAF achieved FOG effluent concentrations on average at 10 ±â€¯4 mg.L-1 corresponding to 74% removal from a relatively low influent concentration of 40 ±â€¯30 mg.L-1. Thus, enhanced primary treatments have the potential to reduce organic load to secondary treatment and increase energy generation through anaerobic digestion. The overall net energy balance was estimated at 2269 MWh.year-1 for the DAF compared to 3445 MWh.year-1 for the chemically-enhanced PST making it a less financially attractive alternative. Yet, in the case where the works require upgrading to accommodate flow or load increases, DAF appeared as a sensible option over sedimentation offering significantly lower capital costs and footprint. In relation to FOG management, upgrading all STWs is not realistic and will require understanding where the benefits would be the highest.

Dry anaerobic digestion of organic waste: A review of operational parameters and their impact on process performance.

Dry digestion is a suitable technology for treating organic wastes with varying composition such as the organic fraction of municipal solids waste. Yet, there is a need for further research to overcome some of the disadvantages associated with the high total solids content of the process. Optimisation of inoculum to substrate ratio, feedstock composition and size, liquid recirculation, bed compaction and use of bulking agents are some of the parameters that need further investigation in batch dry anaerobic digestion, to limit localised inhibition effects and avoid process instability. In addition, further attention on the relation between feedstock composition, organic loading rate and mixing regimes is required for continuous dry anaerobic digestion systems. This paper highlights all the areas where knowledge is scarce and value can be added to increase dry anaerobic digestion performance and expansion.

Characterisation and energy assessment of fats, oils and greases (FOG) waste at catchment level.

Several of the waste materials that have a negative impact on the sewer system are produced by fats, oils and greases (FOG) discharged from commercial and domestic kitchens. These materials accumulate at different points in the sewer catchment, from kitchens to pumping stations, sewers and sewage treatment works (STWs), and comprise oily wastewater, floating agglomerates and hard deposits. Despite their detrimental effects, these waste materials have a high calorific content and are an ideal feedstock for energy recovery processes. So far, the overall volume of each type of waste and their physical-chemical properties in relation to their collection point are unknown. However, from a management point of view, knowledge on each feedstock quality and volumes is necessary to develop an economic viable solution for their collection and for energy recovery purposes. In this study, FOG wastes collected from households, food service establishments (FSEs), sewage pumping stations, sewers and STWs, were compared to sewage sludge in terms of organic contents and energy potentials. As expected, FOG recovered at source (households and FSEs) were 'cleaner' and had a higher energy content. Once mixed with wastewater the materials changed in composition and lost some of their energy per unit mass. Our results showed that around 94,730 tonnes.year-1 of these materials could be recovered from the Thames Water Utilities' catchment, one of the most populated in the UK. These materials could produce up to 222 GWh.year-1 as biogas, close to double of what is produced with sewage sludge digestion and around 19% of the company energy needs. Finally, even with over six million households in the catchment, the results showed that most of the FOG waste was produced by FSEs (over 48,000 premises) with an estimated average of 79,810 tonnes.year-1 compared to 14,920 tonnes.year-1 from private households. This is an important outcome as recovery from FSEs will be cheaper and easier if the company decides to implement a collection system for energy recovery.

From full-scale biofilters to bioreactors: Engineering biological metaldehyde removal.

Polar, low molecular weight pesticides such as metaldehyde are challenging and costly to remove from drinking water using conventional treatment methods. Although biological treatments can be effective at treating micropollutants, through biodegradation and sorption processes, only some operational biofilters have shown the ability to remove metaldehyde. As sorption plays a minor role for such polar organic micropollutants, biodegradation is therefore likely to be the main removal pathway. In this work, the biodegradation of metaldehyde was monitored, and assessed, in an operational slow sand filter. Long-term data showed that metaldehyde degradation improved when inlet concentrations increased. A comparison of inactive and active sand batch reactors showed that metaldehyde removal happened mainly through biodegradation and that the removal rates were greater after the biofilm was acclimated through exposure to high metaldehyde concentrations. This suggested that metaldehyde removal was reliant on enrichment and that the process could be engineered to decrease treatment times (from days to hours). Through-flow experiments using fluidised bed reactors, showed the same behaviour following metaldehyde acclimation. A 40% increase in metaldehyde removal was observed in acclimated compared with non-acclimated columns. This increase was sustained for >40 days, achieving an average of 80% removal and compliance (<0.1 µâ€¯L-1) for >20 days. An initial microbial analysis of the acclimated and non-acclimated biofilm from the same filter materials, showed that the microbial community in acclimated sand was significantly different. This work presents a novel conceptual template for a faster, chemical free, low cost, biological treatment of metaldehyde and other polar pollutants in drinking water. In addition, this is the first study to report kinetics of metaldehyde degradation in an active microbial biofilm at a WTW.

Risk assessments for quality-assured, source-segregated composts and anaerobic digestates for a circular bioeconomy in the UK.

A circular economy relies on demonstrating the quality and environmental safety of wastes that are recovered and reused as products. Policy-level risk assessments, using generalised exposure scenarios, and informed by stakeholder communities have been used to appraise the acceptability of necessary changes to legislation, allowing wastes to be valued, reused and marketed. Through an extensive risk assessment exercise, summarised in this paper, we explore the burden of proof required to offer safety assurance to consumer and brand-sensitive food sectors in light of attempts to declassify, as wastes, quality-assured, source-segregated compost and anaerobic digestate products in the United Kingdom. We report the residual microbiological and chemical risks estimated for both products in land application scenarios and discuss these in the context of an emerging UK bioeconomy worth £52bn per annum. Using plausible worst case assumptions, as demanded by the quality food sector, risk estimates and hazard quotients were estimated to be low or negligible. For example, the human health risk of E. coli 0157 illness from exposure to microbial residuals in quality-assured composts, through a ready-to-eat vegetable consumption exposure route, was estimated at ~10-8 per person per annum. For anaerobic digestion residues, 7 × 10-3cases of E. coli 0157 were estimated per annum, a potential contribution of 0.0007% of total UK cases. Hazard quotients for potential chemical contaminants in both products were insufficient in magnitude to merit detailed quantitative risk assessments. Stakeholder engagement and expert review was also a substantive feature of this study. We conclude that quality-assured, source-segregated products applied to land, under UK quality protocols and waste processing standards, pose negligible risks to human, animal, environmental and crop receptors, providing that risk management controls set within the standards and protocols are adhered to.

Potential influence of sewer heat recovery on in-sewer processes.

Heat recovery from combined sewers has a significant potential for practical renewable energy provision as sources of heat demand and sewer pipes are spread across urban areas. Sewers are continuously recharged with relatively hot wastewater, as well as interacting with heat sources from surrounding air and soil. However, the potential effects of modifying sewage temperature on in-sewer processes have received little attention. The deposition of fats, oils and greases (FOGs) and hydrogen sulphide formation are biochemical processes and are thus influenced by temperature. This paper utilises a case study approach to simulate anticipated temperature reductions in a sewer network due to heat recovery. A laboratory investigation into the formation of FOG deposits at temperatures varying between 5 °C and 20 °C provided mixed results, with only a weak temperature influence, highlighting the need for more research to fully understand the influence of the wastewater composition as well as temperature on FOG deposit formation. A separate modelling investigation into the formation of hydrogen sulphide when inflow temperature is varied between 5 °C and 20 °C showed considerable reductions in hydrogen sulphide formation. Hence, heat extraction from sewers could be a promising method for managing some in-sewer processes, combined with traditional methods such as chemical dosing.

Understanding microbial ecology can help improve biogas production in AD.

454-Pyrosequencing and lipid fingerprinting were used to link anaerobic digestion (AD) process parameters (pH, alkalinity, volatile fatty acids (VFAs), biogas production and methane content) with the reactor microbial community structure and composition. AD microbial communities underwent stress conditions after changes in organic loading rate and digestion substrates. 454-Pyrosequencing analysis showed that, irrespectively of the substrate digested, methane content and pH were always significantly, and positively, correlated with community evenness. In AD, microbial communities with more even distributions of diversity are able to use parallel metabolic pathways and have greater functional stability; hence, they are capable of adapting and responding to disturbances. In all reactors, a decrease in methane content to <30% was always correlated with a 50% increase of Firmicutes sequences (particularly in operational taxonomic units (OTUs) related to Ruminococcaceae and Veillonellaceae). Whereas digesters producing higher methane content (above 60%), contained a high number of sequences related to Synergistetes and unidentified bacterial OTUs. Finally, lipid fingerprinting demonstrated that, under stress, the decrease in archaeal biomass was higher than the bacterial one, and that archaeal Phospholipid etherlipids (PLEL) levels were correlated to reactor performances. These results demonstrate that, across a number of parameters such as lipids, alpha and beta diversity, and OTUs, knowledge of the microbial community structure can be used to predict, monitor, or optimise AD performance.

Aged-engineered nanoparticles effect on sludge anaerobic digestion performance and associated microbial communities.

To investigate the potential effect of aged engineered nanoparticles (a-ENPs) on sludge digestion performance, 150L pilot anaerobic digesters (AD) were fed with a blend of primary and waste activated sludge spiked either with a mixture of silver oxide, titanium dioxide and zinc oxide or a mixture of their equivalent bulk metal salts to achieve a target concentration of 250, 2000, and 2800mgkg-1 dry weight, respectively. Volatile fatty acids (VFA) were 1.2 times higher in the spiked digesters and significantly different (p=0.05) from the control conditions. Specifically, isovaleric acid concentration was 2 times lower in the control digester compared to the spiked digesters, whereas hydrogen sulfide was 2 times lower in the ENPs spiked digester indicating inhibitory effect on sulfate reducing microorganisms. Based on the ether-linked isoprenoids concentration, the total abundance of methanogens was 1.4 times lower in the ENPs spiked digester than in the control and metal salt spiked digesters. Pyrosequencing indicated 80% decrease in abundance and diversity of methanogens in ENPs spiked digester compared to the control digester. Methanosarcina acetivorans and Methanosarcina barkeri were identified as nano-tolerant as their relative abundance increased by a factor of 6 and 11, respectively, compared to the other digesters. The results further provide compelling evidence on the resilience of Fusobacteria, Actinobacteria and the Trojan horse-like effect of ENPs which offered a competitive advantage to some organisms while reducing microbial abundance and diversity.

Ubiquitylation of the acetyltransferase MOF in Drosophila melanogaster.

The nuclear acetyltransferase MOF (KAT8 in mammals) is a subunit of at least two multi-component complexes involved in transcription regulation. In the context of complexes of the 'Non-Specific-Lethal' (NSL) type it controls transcription initiation of many nuclear housekeeping genes and of mitochondrial genes. While this function is conserved in metazoans, MOF has an additional, specific function in Drosophila in the context of dosage compensation. As a subunit of the male-specific-lethal dosage compensation complex (MSL-DCC) it contributes to the doubling of transcription output from the single male X chromosome by acetylating histone H4. Proper dosage compensation requires finely tuned levels of MSL-DCC and an appropriate distribution of MOF between the regulatory complexes. The amounts of DCC formed depends directly on the levels of the male-specific MSL2, which orchestrates the assembly of the DCC, including MOF recruitment. We found earlier that MSL2 is an E3 ligase that ubiquitylates most MSL proteins, including MOF, suggesting that ubiquitylation may contribute to a quality control of MOF's overall levels and folding state as well as its partitioning between the complex entities. We now used mass spectrometry to map the lysines in MOF that are ubiquitylated by MSL2 in vitro and identified in vivo ubiquitylation sites of MOF in male and female cells. MSL2-specific ubiquitylation in vivo could not be traced due to the dominance of other, sex-independent ubiquitylation events and conceivably may be rare or transient. Expressing appropriately mutated MOF derivatives we assessed the importance of the ubiquitylated lysines for dosage compensation by monitoring DCC formation and X chromosome targeting in cultured cells, and by genetic complementation of the male-specific-lethal mof2 allele in flies. Our study provides a comprehensive analysis of MOF ubiquitylation as a reference for future studies.

PionX sites mark the X chromosome for dosage compensation.

The rules defining which small fraction of related DNA sequences can be selectively bound by a transcription factor are poorly understood. One of the most challenging tasks in DNA recognition is posed by dosage compensation systems that require the distinction between sex chromosomes and autosomes. In Drosophila melanogaster, the male-specific lethal dosage compensation complex (MSL-DCC) doubles the level of transcription from the single male X chromosome, but the nature of this selectivity is not known. Previous efforts to identify X-chromosome-specific target sequences were unsuccessful as the identified MSL recognition elements lacked discriminative power. Therefore, additional determinants such as co-factors, chromatin features, RNA and chromosome conformation have been proposed to refine targeting further. Here, using an in vitro genome-wide DNA binding assay, we show that recognition of the X chromosome is an intrinsic feature of the MSL-DCC. MSL2, the male-specific organizer of the complex, uses two distinct DNA interaction surfaces-the CXC and proline/basic-residue-rich domains-to identify complex DNA elements on the X chromosome. Specificity is provided by the CXC domain, which binds a novel motif defined by DNA sequence and shape. This motif characterizes a subclass of MSL2-binding sites, which we name PionX (pioneering sites on the X) as they appeared early during the recent evolution of an X chromosome in D. miranda and are the first chromosomal sites to be bound during de novo MSL-DCC assembly. Our data provide the first, to our knowledge, documented molecular mechanism through which the dosage compensation machinery distinguishes the X chromosome from an autosome. They highlight fundamental principles in the recognition of complex DNA elements by protein that will have a strong impact on many aspects of chromosome biology.

Anaplastic lymphoma kinase aberrations correlate with metastatic features in pediatric rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most frequent soft tissue tumor in childhood and arises from immature mesenchymal cells committed to skeletal muscle differentiation. Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in several cancers. Moreover, ALK full-length receptor protein has been observed in RMS, although its clinical and functional significance is yet controversial. The role of ALK and its clinical relevance were investigated in a selected cohort of 74 FFPE pediatric RMS and a panel of RMS cell lines, evaluating its gene and protein status, utilizing Fluorescent In Situ Hybridization (FISH), immunohistochemistry (IHC) and Western blot approaches. Moreover, to get insight into its possible therapeutic relevance, effects of ALK silencing on cell proliferation, invasion and apoptosis were studied in RMS cells. ALK IHC positivity was significantly correlated with gene copy number gain, the alveolar subtype, PAX3/7-FOXO1 rearrangements, the presence of metastasis at diagnosis and a worse overall outcome. Furthermore, EML4-ALK fusion gene associated with higher protein expression was identified in an embryonal RMS. ALK silencing in RH30 ALK positive cells strongly inhibited invasion capability. Overall, our data suggest a potential role of ALK in pediatric RMS.

Organic loading rate: A promising microbial management tool in anaerobic digestion.

This study investigated the effect of changes in organic loading rate (OLR) and feedstock on the volatile fatty acids (VFAs) production and their potential use as a bioengineering management tool to improve stability of anaerobic digesters. Digesters were exposed to one or two changes in OLR using the same or different co-substrates (Fat Oil and Grease waste (FOG) and/or glycerol). Although all the OLR fluctuations produced a decrease in biogas and methane production, the digesters exposed twice to glycerol showed faster recovery towards stable conditions after the second OLR change. This was correlated with the composition of the VFAs produced and their mode of production, from parallel to sequential, resulting in a more efficient recovery from inhibition of methanogenesis. The change in acids processing after the first OLR increase induced a shift in the microbial community responsible of the process optimisation when the digesters were exposed to a subsequent OLR increase with the same feedstock. When the digesters were exposed to an OLR change with a different feedstock (FOG), the recovery took 7d longer than with the same one (glycerol). However, the microbial community showed functional resilience and was able to perform similarly to pre-exposure conditions. Thus, changes in operational conditions can be used to influence microbial community structure for anaerobic digestion (AD) optimisation. Finally, shorter recovery times and increased resilience of digesters were linked to higher numbers of Clostridia incertae sedis XV, suggesting that this group may be a good candidate for AD bioaugmentation to speed up recovery after process instability or OLR increase.

Influence of microalgal N and P composition on wastewater nutrient remediation.

Microalgae have demonstrated the ability to remediate wastewater nutrients efficiently, with methods to further enhance performance through species selection and biomass concentration. This work evaluates a freshwater species remediation characteristics through analysis of internal biomass N:P (nitrogen:phosphorus) and presents a relationship between composition and nutrient uptake ability to assist in species selection. Findings are then translated to an optimal biomass concentration, achieved through immobilisation enabling biomass intensification by modifying bead concentration, for wastewaters of differing nutrient concentrations at hydraulic retention times (HRT) from 3 h to 10 d. A HRT <20 h was found suitable for the remediation of secondary effluent by immobilised Scenedesmus obliquus and Chlorella vulgaris at bead concentrations as low as 3.2 and 4.4 bead·mL(-1). Increasing bead concentrations were required for shorter HRTs with 3 h possible at influent concentrations <5 mgP L(-1).

Microbial production of succinic acid using crude and purified glycerol from a Crotalaria juncea based biorefinery.

Microbial conversion of crude and purified glycerol obtained in the process of biorefining Crotalaria juncea is carried out to produce succinic acid using Escherichia coli. Batch tests are performed for nine different substrate concentrations of commercial, purified and crude glycerol, in order to observe cell growth and substrate utilization rate. Inhibitory (Halden-Andrew, Aiba-Edward, Tessier type and Andrews) as well as non-inhibitory (Monod, Moser and Tessier) models are fitted to the relationship between specific growth rate and substrate concentration obtained from the growth curves. Considering the inhibition effect, Aiba-Edward model ranked 1 out of 7 in case of two samples and Haldane-Andrew model ranked 1 in case of one sample. Aiba-Edward model gave the best fitment for a large range of concentrations of all the three types of glycerol, crude, purified and laboratory grade. Maximum production of succinic acid is obtained from commercial glycerol at pH 7 and 37.5 °C.

Insights into the effect of mixed engineered nanoparticles on activated sludge performance.

In this study, the effects, fate and transport of ENPs in wastewater treatment plants (WWTP) were investigated using three parallel pilot WWTPs operated under identical conditions. The WWTPs were spiked with (i) an ENP mixture consisting of silver oxide, titanium dioxide and zinc oxide, and (ii) bulk metal salts. The third plant served as control (unspiked). ENP effects were evaluated for (i) bulk contaminant removal, (ii) activated sludge (AS) process performance, (iii) microbial community structure and dynamics and (iv) microbial inhibition. ENPs showed a strong affinity for biosolids and induced a specific oxygen uptake rate two times higher than the control. The heterotrophic biomass retained its ability to nitrify and degrade organic matter. However, non-recovery of ammonia- and nitrite-oxidizing bacteria such as Nitrosomonas, Nitrobacter or Nitrospira in the ENP spiked reactors suggests selective inhibitory effects. The results further suggest that ENPs and metal salts have antimicrobial properties which can reduce synthesis of extracellular polymeric substances and therefore floc formation. Scanning electron microscopy evidenced selective damage to some microbes, whereas lipid fingerprinting and 454 pyrosequencing indicated a temporal shift in the microbial community structure and diversity. Acidovorax, Rhodoferax, Comamonas and Methanosarcina were identified as nano-tolerant species. Competitive growth advantage of the nano-tolerant species influenced the removal processes and unlike other xenobiotic compounds, ENPs can hasten the natural selection of microbial species in AS.