Evaluation of different molecular systems for detection and quantification of SARS-CoV-2 RNA from wastewater samples.

Wastewater-based epidemiology has proved to be a suitable approach for tracking the spread of epidemic agents including SARS-CoV-2 RNA. Different protocols have been developed for quantitative detection of SARS-CoV-2 RNA from wastewater samples, but little is known on their performance. In this study we compared three protocols based on Reverse Transcription Real Time-PCR (RT-PCR) and one based on Droplet Digital PCR (ddPCR) for SARS-CoV-2 RNA detection from 35 wastewater samples. Overall, SARS-CoV-2 RNA was detected by at least one method in 85.7 % of samples, while 51.4 %, 22.8 % and 8.6 % resulted positive with two, three or all four methods, respectively. Protocols based on commercial RT-PCR assays and on Droplet Digital PCR showed an overall higher sensitivity vs. an in-house assay. The use of more than one system, targeting different genes, could be helpful to increase detection sensitivity.

Extracellular polymeric substances as paper coating biomaterials derived from anaerobic granular sludge.

Recovering extracellular polymeric substances (EPS) from waste granular sludge offers a cost-effective and sustainable approach for transforming wastewater resources into industrially valuable products. Yet, the application potential of these EPS in real-world scenarios, particularly in paper manufacturing, remains underexplored. Here we show the feasibility of EPS-based biomaterials, derived from anaerobic granular sludges, as novel coating agents in paper production. We systematically characterised the rheological properties of various EPS-based suspensions. When applied as surface sizing agents, these EPS-based biomaterials formed a distinct, ultra-thin layer on paper, as evidenced by scanning electron microscopy. A comprehensive evaluation of water and oil penetration, along with barrier properties, revealed that EPS-enhanced coatings markedly diminished water absorption while significantly bolstering oil and grease resistance. Optimal performance was observed in EPS variants with elevated protein and hydrophobic contents, correlating with their superior rheological characteristics. The enhanced water-barrier and grease resistance of EPS-coated paper can be attributed to its non-porous, fine surface structure and the functional groups in EPS, particularly the high protein content and hydrophobic humic-like substances. This research marks the first demonstration of utilizing EPS from anaerobic granular sludge as paper-coating biomaterials, bridging a critical knowledge gap in the sustainable use of biopolymers in industrial applications.

Recovery of structural extracellular polymeric substances (sEPS) from aerobic granular sludge: Insights on biopolymers characterization and hydrogel properties for potential applications.

Nowadays, wastewater treatment plants (WWTPs) are transforming into water resource recovery facilities (WRRFs) where the resource recovery from waste streams is pivotal. Aerobic granular sludge (AGS) is a novel technology applied for wastewater treatment. Extracellular polymeric substances (EPS) secreted by microorganisms promote the aggregation of bacterial cells into AGS and the structural fraction of EPS (sEPS) is responsible for the mechanical properties of AGS. sEPS can be extracted and recovered from waste AGS by physico-chemical methods and its characterization is to date of relevant concern to understand the properties in the perspective of potential applications. This study reports on: characterization of sEPS extracted and recovered from AGS; - formation and characterization of sEPS-based hydrogels. Briefly, sEPS were extracted by a thermo-alkaline process followed by an acidic precipitation. sEPS-based hydrogels were formed by a cross-linking process with a 2.5% w/w CaCl2 solution. The following key-findings can be drawn: i) hydrogels can be formed starting from 1% w/w sEPS on, by diffusion of Ca2+ into sEPS network; ii) the Ca/C molar ratio of hydrogels decreased with increasing concentration of sEPS from 1 to 10% w/w; iii) the thermogravimetric and spectroscopic behaviours of sEPS show that the cross-linking reaction mainly involves the polysaccharidic fraction of biopolymers; iv) water-holding capacity up to 99 gH2O/gsEPS was registered for 1% w/w sEPS-based hydrogels, suggesting applications in several industrial sectors (i.e. chemical, paper, textile, agronomic, etc.); v) rheological results highlighted a solid-like behaviour (G'≫G") of sEPS-based hydrogels. The power-law fitting of G' vs. sEPS concentration suggests that the expansion of the sEPS network during cross-linking occurs through a percolative mechanism involving the initial formation of sEPS oligomers clusters followed by their interconnection towards the formation of 3D network. These findings provide additional information about the mechanisms of sEPS-based hydrogel formation and reveal the peculiar physico-chemical characteristics of sEPS which nowadays are increasingly gaining interest in the context of resource recovery.

Very Low Alcohol Consumption Is Associated with Lower Prevalence of Cirrhosis and Hepatocellular Carcinoma in Patients with Non-Alcoholic Fatty Liver Disease.

The role of moderate alcohol consumption in the evolution of NAFLD is still debated. The aim of this study is to evaluate the impact of current and lifelong alcohol consumption in patients with NAFLD. From 2015 to 2020, we enrolled 276 consecutive patients fulfilling criteria of NAFLD (alcohol consumption up to 140 g/week for women and 210 g/week for men). According to their current alcohol intake per week, patients were divided in: abstainers, very low consumers (C1: <70 g/week) and moderate consumers (C2). We created a new tool, called LACU (Lifetime Alcohol Consuming Unit) to estimate the alcohol exposure across lifetime: 1 LACU was defined as 7 alcohol units per week for 1 drinking year. Patients were divided into lifelong abstainers and consumers and the latter furtherly divided into quartiles: Q1-Q4. Stratification according to alcohol intake, both current and cumulative as estimated by LACU, showed that very low consumers (C1 and Q1-Q3) displayed lower frequency of cirrhosis and hepatocellular carcinoma compared to abstainers and moderate consumers (C2 and Q4). We can speculate that up to one glass of wine daily in the context of a Mediterranean diet may be a long-term useful approach in selected NAFLD patients.

Rigid and film bioplastics degradation under suboptimal composting conditions: A kinetic study.

The present research investigates the degradation rate of bioplastics under various composting conditions, including suboptimal ones. Lab-scale tests were carried out setting three variables: temperature (37°C-58°C), humidity (30%-60%) and duration of the thermophilic and the maturation phases (15-60 days). The composting tests were carried out following modified guideline ISO 20200:2015 and lasted for 60 days. Bioplastics in the synthetic waste matrix consisted of Mater-Bi® film biobags and PLA rigid teaspoons. A kinetic study was performed, resulting in faster degradation rates for film bioplastics (first-order kinetics with k = 0.0850-0.1663 d-1) than for rigid (0.0018-0.0136 d-1). Moreover, film bioplastics reached a complete degradation within the 60 days of the test. Concerning the rigid products, 90% degradation would be achieved in 2-3 years for mesophilic conditions. Finally, in the undersieve of 0.5 mm some microplastics were identified with the ImageJ software, mainly relatable to rigid (PLA) bioplastics. Overall, the results disclosed that the combination of mesophilic temperatures and absence of moistening slowed down both the degradation and the disintegration process of bioplastics.

Heavy metal biosorption by Extracellular Polymeric Substances (EPS) recovered from anammox granular sludge.

The recovery and conversion of Extracellular Polymeric Substances (EPS) from sewage sludge into bio-based commodities might improve the economics and environmental sustainability of wastewater treatment. This contribution explores the application of EPS from anammox granular waste sludge as biosorbent for the removal of heavy metals, specifically lead, copper, nickel, and zinc. Adsorption capacities equivalent or higher than well-established adsorbent media emerged from single-metal biosorption studies (up to 84.9, 52.8, 21.7 and 7.4 mg/gTSEPS for Pb2+, Cu2+, Ni2+ and Zn2+, respectively). Combining spectroscopic techniques, a mechanistic hypothesis for metal biosorption, based on a combination of electrostatic interaction, ion exchange, complexation, and precipitation, was proposed. The adsorption mechanisms of extracted EPS and non-extracted EPS in the native biomass were indirectly compared by means of single-metal biosorption studies performed with pristine granules (adsorbing up to 103.7, 36.1, 48.2 and 49.8 mg/gTSgranules of Pb2+, Cu2+, Ni2+, and Zn2+, respectively). In comparison with pristine anammox granules, EPS showed lower adsorption capacities except for copper and different adsorption pathways as postulated based on the adsorption data interpretation via theoretical models. The multi-metal biosorption tests excluded significant competitions among different heavy metals for the EPS binding sites, thus opening further scenarios for the treatment of complex wastewaters.

Aerobic Granular Sludge-Membrane BioReactor (AGS-MBR) as a Novel Configuration for Wastewater Treatment and Fouling Mitigation: A Mini-Review.

This mini-review reports the effect of aerobic granular sludge (AGS) on performance and membrane-fouling in combined aerobic granular sludge-membrane bioreactor (AGS-MBR) systems. Membrane-fouling represents a major drawback hampering the wider application of membrane bioreactor (MBR) technology. Fouling can be mitigated by applying aerobic granular sludge technology, a novel kind of biofilm technology characterized by high settleability, strong microbial structure, high resilience to toxic/recalcitrant compounds of industrial wastewater, and the possibility to simultaneously remove organic matter and nutrients. Different schemes can be foreseen for the AGS-MBR process. However, an updated literature review reveals that in the AGS-MBR process, granule breakage represents a critical problem in all configurations, which often causes an increase of pore-blocking. Therefore, to date, the objective of research in this sector has been to develop a stable AGS-MBR through multiple operational strategies, including the cultivation of AGS directly in an AGS-MBR reactor, the occurrence of an anaerobic-feast/aerobic-famine regime in continuous-flow reactors, maintenance of average granule dimensions far from critical values, and proper management of AGS scouring, which has been recently recognized as a crucial factor in membrane-fouling mitigation.

A highly efficient multi-step methodology for the quantification of micro-(bio)plastics in sludge.

The present study develops a multi-step methodology for identification and quantification of microplastics and micro-bioplastics (together called in the current work micro-(bio)plastics) in sludge. In previous studies, different methods for the extraction of microplastics were devised for traditional plastics, while the current research tested the methodology on starch-based micro-bioplastics of 0.1-2 mm size. Compostable bioplastics are expected to enter the anaerobic or aerobic biological treatments that lead to end-products applicable in agriculture; some critical conditions of treatments (e.g. low temperature and moisture) can slow down the degradation process and be responsible for the presence of microplastics in the end-product. The methodology consists of an initial oxidation step, with hydrogen peroxide 35% concentrated to clear the sludge and remove the organic fraction, followed by a combination of flotation with sodium chloride and observation of the residues under a fluorescence microscope using a green filter. The workflow revealed an efficacy of removal from 94% to 100% and from 92% to 96% for plastic fragments, 0.5-2 mm and 0.1-0.5 mm size, respectively. The methodology was then applied to samples of food waste pulp harvested after a shredding pre-treatment in an anaerobic digestion (AD) plant in Italy, where polyethylene, starch-based Mater-Bi® and cellophane microplastics were recovered in amounts of 9 ± 1.3/10 g <2 mm and 4.8 ± 1.2/10 g ⩾2 mm. The study highlights the need to lower the threshold size for the quantification of plastics in organic fertilizers, which is currently set by legislations at 2 mm, by improving the background knowledge about the fate of the micro-(bio)plastics in biological treatments for the organic waste.

Extracellular biopolymers recovered as raw biomaterials from waste granular sludge and potential applications: A critical review.

Granular sludge (GS) is a special self-aggregation biofilm. Extracellular polymeric substances (EPS) are mainly associated with the architectural structure, rheological behaviour and functional stability of fine granules, given that their significance to the physicochemical features of the biomass catalysing the biological purification process. This review targets the EPS excretion from GS and introduces newly identified EPS components, EPS distribution in different granules, how to effectively extract and recover EPS from granules, key parameters affecting EPS production, and the potential applications of EPS-based biomaterials. GS-based EPS components are highly diverse and a series of new contents are highlighted. Due to high diversity, emerging extraction standards are proposed and recovery process is capturing particular attention. The major components of EPS are found to be polysaccharides and proteins, which manifest a larger diversity of relative abundance, structures, physical and chemical characteristics, leading to the possibility to sustainably recover raw materials. EPS-based biomaterials not only act as alternatives to synthetic polymers in several applications but also figure in innovative industrial/environmental applications, including gel-forming materials for paper industry, biosorbents, cement curing materials, and flame retardant materials. In the upcoming years, it is foreseen that productions of EPS-based biomaterials from renewable origins would make a significant contribution to the advancement of the circular economy.

A novel universal primer pair for prokaryotes with improved performances for anammox containing communities.

Abundance profiling via 16S rRNA targeted next generation sequencing (NGS) is a common procedure to characterize mixtures of prokaryotic populations inhabiting an environment. Depending on the variable region/s addressed, different maps can be obtained due to their different information content. In this work, we focussed on wastewater microbial communities and we compared several recently developed universal primers that addressed regions V1-V3, V3-V4 and V4. They all proved to have good performance over a wide range of microbial phyla, but the phylum Planctomycetes was not optimally covered, especially for members of the Brocadiales family. Such bacteria are at the basis of the novel nitrogen removal strategy based on anammox process. To overcome this limitation we performed an extensive bioinformatic analysis that allowed the design of a primer (Pro341FB) that shows increased sensitivity for this specific phylum with respect to the previously proposed Pro341F primer. Upon validation using a 16S NGS survey on microbial communities from different wastewater treatment plant (activated sludge systems, anaerobic digesters, aerobic and anaerobic granules) we demonstrated that Pro341FB is able to reveal up to 5 times more members of the Candidatus Brocadiales family (plus many other previously under-covered prokaryotes) than Pro341F, without affecting its excellent previous coverage.

Efficient carbon, nitrogen and phosphorus removal from low C/N real domestic wastewater with aerobic granular sludge.

This work reports on simultaneous nitrification, denitrification and phosphorus removal treating real domestic wastewater with low carbon/nitrogen (C/N) ratio by aerobic granular sludge (AGS). Operations at high sludge retention time (SRT = 61 ± 24 days) resulted in low biomass yield per chemical oxygen demand removed (CODrem) (0.21 ± 0.01 gCODx/gCODrem), lower COD demand for denitrification as well as high effluent quality in terms of total suspended solids (TSS) (22 ± 7 mgTSS/L). The average ratio between the biodegradable soluble COD stored anaerobically as polyhydroxyalkanoates (PHAs) and the N removed was 3.1 ± 0.6 gCODsto/gNrem, suggesting that nitrification/denitrification occurred partly via the nitrite pathway. Results revealed that stable AGS process with high C/N/P removal efficiency of 84/71/96% can be obtained besides a low organic loading rate (0.43 ± 0.11 g COD/L/d) and influent C/N ratio (3.8 ± 1.6 g/g), resulting in a high effluent quality characterized by 25 ± 6 mg sCOD/L, 0.09 ± 0.07 mgPO4-P/L, 9 ± 2 mgTIN/L (10 ± 2 mgTN/L) and 22 ± 7 mgTSS/L.

Monitoring of degradation of starch-based biopolymer film under different composting conditions, using TGA, FTIR and SEM analysis.

This paper presents the results of a composting lab-scale test carried out on Mater-Bi® film, a starch-based biopolymer. The test material is composed by starch, additives and polybutylene adipate terephthalate (PBAT). The test lasted for 45 days and was developed in three replicates under different temperature and moisture conditions, with the aim to assess the influence on Mater-Bi® degradation of less favourable composting conditions as short thermophilic phase, absence of moistening, and a combination of the two factors. The chemical nature and the morphology of the material and of its single components have been investigated before, during and at the end of the composting process, by means of different analytical techniques. ThermoGravimetric Analysis (TGA) allowed to obtain activation energy and weight loss; Fourier Transform InfraRed spectroscopy (FTIR) and Scanning Electron Microscopy (SEM) were used to study changes in the polymeric and morphological structure, and visual analysis provided information on the size of the Mater-Bi® particles. The results show that the biodegradation of PBAT is strongly influenced by the environmental conditions (temperature and moisture); on the contrary, in all the three replicates, both starch and additives are completely biodegraded within the first days of the process.

Anammox-based processes: How far have we come and what work remains? A review by bibliometric analysis.

Nitrogen contamination remains a severe environmental problem and a major threat to sustainable development worldwide. A systematic analysis of the literature indicates that the partial nitritation-anammox (PN/AMX) process is still actively studied as a viable option for energy-efficient and feasible technology for the sustainable treatment of N- rich wastewaters, since its initial discovery in 1990. Notably, the mainstream PN/AMX process application remains the most challenging bottleneck in AMX technology and fascinates the world's attention in AMX studies. This paper discusses the recent trends and developments of PN/AMX research and analyzes the results of recent years of research on the PN/AMX from lab-to full-scale applications. The findings would deeply improve our understanding of the major challenges under mainstream conditions and next-stage research on the PN/AMX process. A great deal of efforts has been made in the process engineering, PN/AMX bacteria populations, predictive modeling, and the full-scale implementations during the past 22 years. A series of new and excellent experimental findings at lab, pilot and full-scale levels including good nitrogen removal performance even under low temperature (15-10 °C) around the world were achieved. To date, pilot- and full-scale PN/AMX have been successfully used to treat different types of industrial sewage, including black wastewater, sludge digester liquids, landfill leachate, monosodium glutamate wastewater, etc. Supplementing the qualitative analysis, this review also provides a quantitative bibliometrics study and evaluates global perspectives on PN/AMX research published during the past 22 years. Finally, general trends in the development of PN/AMX research are summarized with the aim of conveying potential future trajectories. The current review offers a valuable orientation and global overview for scientists, engineers, readers and decision makers presently focusing on PN/AMX processes.

Two-stage granular sludge partial nitritation/anammox process for the treatment of digestate from the anaerobic digestion of the organic fraction of municipal solid waste.

The increasing amount of source separated organic fraction of municipal solid wastes (OFMSW) treated by anaerobic digestion for energy recovery requires the implementation of cost-efficient processes for the treatment of the produced digestate, especially in terms of nitrogen removal. The autotrophic nitrogen removal process, based on the coupling of two biological processes, partial nitritation (PN) and anammox (A), appears as a suitable solution due to important savings in operational costs compared to conventional treatment processes. However, its application could be hampered by the high salinity and inhibitory potential of this kind of digestate. In this contribution, two lab-scale granular sludge reactors performing the PN and anammox processes, respectively, were used to treat (opportunely diluted) real OFMSW digestate originating from full-scale biogas plants with the aim of assessing their treatment feasibility in a two-stage PN/A configuration. The PN process was implemented in an air-lift granular sludge reactor and was able to treat a nitrogen loading rate of about 1 g N L-1 d-1 at 30 ±â€¯0.5 °C; moreover, its effluent was suitable for the subsequent anammox treatment, with an appropriate effluent NO2-/NH4+ ratio and marginal inhibiting effects. In the anammox granular sludge reactor, the anammox activity was affected by high salinity levels, nonetheless a stable reactor performance at a nitrogen removing rate of 0.83 ±â€¯0.20 and 0.31 ±â€¯0.04 g N L-1 d-1 at 35 ±â€¯0.5 °C, were achieved when treating 50% and 30% diluted real wastewaters at a conductivity in the reactor of 9.1 and 11.2 mS cm-1, respectively.

Hydrogels formed by anammox extracellular polymeric substances: structural and mechanical insights.

The recovery of biopolymers from the waste sludge produced in wastewater treatments and their application in other industrial sectors, would substantially increase the environmental and economical sustainability of the process, promoting the development of a circular economy. In this study, extracellular polymeric substances (EPS) extracted from anammox granular waste sludge, were investigated and characterized. Rheological and differential scanning calorimetry measurements on EPS aqueous dispersions indicate the formation of an extended 3-D network above a threshold concentration, with a clear dependence of the mechanical and water retention properties on EPS content. The structural characterization, performed with transmission electron microscopy and small angle X-ray scattering, reveals the presence of functional amyloids as putative structural units, observed for the first time in an EPS-based hydrogel. As a proof of concept of the applicative potential, we explored the water and grease resistance provided to paper by an EPS coating. These results shed light on the structural details of EPS-based hydrogels, and pave the way for the possible use of EPS-based materials as a cheap, eco-friendly alternative to commonly adopted paper coatings, in line with a circular economy pattern for wastewater treatment.

Extraction, recovery and characterization of structural extracellular polymeric substances from anammox granular sludge.

The composition and colloidal properties of extracellular polymeric substances (EPS) from anammox granular sludge were investigated through a complete set of spectroscopic and scattering techniques. To fully characterize EPS, we developed a robust and reproducible extraction/recovery protocol specific for anammox biofilms, based on the change of water affinity under alternated alkaline and acidic conditions, each monitored with Z-potential and dynamic light scattering analysis. This method enabled both extraction as a colloidal suspension and recovery as a solid of large amounts of EPS (0.38 ±â€¯0.04 and 0.21 ±â€¯0.02 g/g, respectively), including for the first time its structural components. The dominance of the proteinaceous fraction was revealed by all methods tested, resulting in the highest protein/carbohydrates ratio reported for biofilms applied in the wastewater sector. The abundance of proteinaceous ordered structures and in particular of cross-ß motifs was detected, indicating for the first time the presence of amyloid-like aggregates in anammox EPS, and suggesting the key role of the protein fraction in determining the mechanical properties of the parent biofilm. The robustness and reproducibility of the proposed method fill the current gap towards a reliable full-scale recovery as well as towards an accurate and meaningful investigation of anammox EPS and pave the way for further exploration of their applicative potential thus stimulating the desirable shift from the current wastewater treatment perspective towards biorefinery in a circular economy context.

Extracellular polymeric substances of biofilms: Suffering from an identity crisis.

Microbial biofilms can be both cause and cure to a range of emerging societal problems including antimicrobial tolerance, water sanitation, water scarcity and pollution. The identities of extracellular polymeric substances (EPS) responsible for the establishment and function of biofilms are poorly understood. The lack of information on the chemical and physical identities of EPS limits the potential to rationally engineer biofilm processes, and impedes progress within the water and wastewater sector towards a circular economy and resource recovery. Here, a multidisciplinary roadmap for addressing this EPS identity crisis is proposed. This involves improved EPS extraction and characterization methodologies, cross-referencing between model biofilms and full-scale biofilm systems, and functional description of isolated EPS with in situ techniques (e.g. microscopy) coupled with genomics, proteomics and glycomics. The current extraction and spectrophotometric characterization methods, often based on the principle not to compromise the integrity of the microbial cells, should be critically assessed, and more comprehensive methods for recovery and characterization of EPS need to be developed.

Distribution and microbial community structure analysis of a single-stage partial nitritation/anammox granular sludge bioreactor operating at low temperature.

In the last decade, autotrophic nitrogen removal technologies based on anammox metabolism have become state of the art in urban and industrial wastewater treatment systems, due to their advantages over traditional nitrogen removal processes. However, their application is currently limited to the treatment of warm wastewater (25-40°C) mainly due to the low growth rate of the anammox bacteria. The extension of the application field to wastewater characterized by lower temperatures (8-20°C), such as those typical for municipal sewage, allows the design of treatment systems with a net energy production. In this study, the distribution and bacterial community structure of a lab-scale single-stage partial nitritation/anammox (PN/A) granular sludge bioreactor operating at low temperatures was analysed using next-generation sequencing techniques. The presence of ammonium-oxidizing bacteria and anammox bacteria was found, but the appearance of other bacterial species shows a complex microbial ecosystem. Evaluation of ecological roles of representative species inside the single-stage PN/A bioreactor was accomplished. Results obtained will be helpful for the future design and operation of PN/A systems performing at low temperatures.

Comparison of bacterial communities of conventional and A-stage activated sludge systems.

The bacterial community structure of 10 different wastewater treatment systems and their influents has been investigated through pyrosequencing, yielding a total of 283486 reads. These bioreactors had different technological configurations: conventional activated sludge (CAS) systems and very highly loaded A-stage systems. A-stage processes are proposed as the first step in an energy producing municipal wastewater treatment process. Pyrosequencing analysis indicated that bacterial community structure of all influents was similar. Also the bacterial community of all CAS bioreactors was similar. Bacterial community structure of A-stage bioreactors showed a more case-specific pattern. A core of genera was consistently found for all influents, all CAS bioreactors and all A-stage bioreactors, respectively, showing that different geographical locations in The Netherlands and Spain did not affect the functional bacterial communities in these technologies. The ecological roles of these bacteria were discussed. Influents and A-stage bioreactors shared several core genera, while none of these were shared with CAS bioreactors communities. This difference is thought to reside in the different operational conditions of the two technologies. This study shows that bacterial community structure of CAS and A-stage bioreactors are mostly driven by solids retention time (SRT) and hydraulic retention time (HRT), as suggested by multivariate redundancy analysis.