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.

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.

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.

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.

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.

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.

Water safety plans and risk assessment: A novel procedure applied to treated water turbidity and gastrointestinal diseases.

Water Safety Plans (WSPs), as recommended by the World Health Organization (WHO), can help drinking water suppliers to identify potential hazards related to drinking water and enable improvements in public health outcomes. In this study we propose a procedure to evaluate the health risk related to turbidity in finished water by determining the cases of drinking water-related gastrointestinal diseases. The results of several epidemiological studies and three-year time series turbidity data, coming from three different drinking water treatment plants (WTPs) located in Tuscany (Italy), have been used to determine the relationship between drinking water turbidity and gastroenteritis incidence and to assess the health risk attributable to the turbidity of tap water. The turbidity variation occurring in the treated water during the monitored period showed an incremental risk compared to the baseline value from 9% to 27% in the three WTPs. Risk reduction due to each treatment step was also evaluated and it was found that a complete treatment train (clari-flocculation, sand filtration, activated carbon filtration and multi-step disinfection) reduces risk by over 600 times. Our approach is a useful tool for water suppliers to quantify health risks by considering time series data on turbidity at WTPs and to make decisions regarding risk management measures.

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.

Methodologies to assess biodegradation of bioplastics during aerobic composting and anaerobic digestion: A review.

Bioplastics are emerging on the market as sustainable materials which rise to the challenge to improve the lifecycle of plastics from the perspective of the circular economy. The article aims at providing a critical insight of research studies carried out in the last 20 years on the degradation of bioplastics under aerobic composting and anaerobic digestion conditions. It mainly focuses on the various and different methodologies which have been proposed and developed to monitor the process of biodegradation of several bioplastic materials: CO2 and CH4 measurements, mass loss and disintegration degree, spectroscopy, visual analysis and scanning electron microscopy. Moreover, across the wide range of studies, the process conditions of the experimental setup, such as temperature, test duration and waste composition, often vary from author to author and in accordance with the international standard followed for the test. The different approaches, in terms of process conditions and monitoring methodologies, are pointed out in the review and highlighted to find significant correlations between the results obtained and the experimental procedures. These observed correlations allow critical considerations to be reached about the efficiency of the methodologies and the influence of the main abiotic factors on the process of biodegradation of bioplastics.

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.

Nitrifying biomass characterization and monitoring during bioaugmentation in a membrane bioreactor.

A membrane bioreactor (MBR), fed with domestic wastewater, was bioaugmented with nitrifying biomass selected in a side-stream MBR fed with a synthetic high nitrogen-loaded influent. Microbial communities evolution was monitored and comparatively analysed through an extensive bio-molecular investigation (16S rRNA gene library construction and terminal-restriction fragment length polymorphism techniques) followed by statistical analyses. As expected, a highly specialized nitrifying biomass was selected in the side-stream reactor fed with high-strength ammonia synthetic wastewater. The bioaugmentation process caused an increase of nitrifying bacteria of the genera Nitrosomonas (up to more than 30%) and Nitrobacter in the inoculated MBR reactor. The overall structure of the microbial community changed in the mainstream MBR as a result of bioaugmentation. The effect of bioaugmentation in the shift of the microbial community was also verified through statistical analysis.

European trends in greenhouse gases emissions from integrated solid waste management.

The European Union (EU) has 28 member states, each with very different characteristics (e.g. surface, population density, per capita gross domestic product, per capita municipal solid waste (MSW) production, MSW composition, MSW management options). In this paper several integrated waste management scenarios representative of the European situation have been generated and analysed in order to evaluate possible trends in the net emission of greenhouse gases and in the required landfill volume. The results demonstrate that an integrated system with a high level of separate collection, efficient energy recovery in waste-to-energy plants and very limited landfill disposal is the most effective according to the indices adopted. Moreover, it is evident that a fully integrated system can make MSW management a carbon sink with a potentiality of up to approximately 40 Mt CO2eq year(-1).

Modeling bioaugmentation with nitrifiers in membrane bioreactors.

Bioaugmentation with nitrifiers was studied using two pilot-scale membrane bioreactors, with the purpose of assessing the suitability of state-of-the-art activated sludge models (ASMs) in predicting the efficiency of bioaugmentation as a function of operating conditions. It was demonstrated that the temperature difference between seeding and seeded reactors (ΔT) affects bioaugmentation efficiency. Experimental data were accurately predicted when ΔT was within a range of up to 10 °C at the higher range, and when the temperature was significantly lower in the seeded reactor compared to the seeding one, standard ASMs overestimated the efficiency of bioaugmentation. A modified ASM, capable of accurately representing the behavior of seeded nitrifying biomass in the presence of high ΔT, would require the inclusion of the effect of temperature time gradients on nitrifiers. A simple linear correlation between ΔT and the Arrhenius coefficient was proposed as a preliminary step.

Biodegradation of naphthalenesulphonate polymers: the potential of a combined application of fungi and bacteria.

The potential of several fungi and their synergy with bacterial biomasses were evaluated as a solution for the removal of 2-naphthalensulphonic acid polymers (2-NSAPs) from petrochemical wastewater, characterized by a chemical oxygen demand (COD) greater than 9000 mg/L. The ability of fungi to grow on 2-NSAP mixtures was preliminarily investigated using a solid medium, and then the action of the selected strains, both in suspended and immobilized form, was evaluated in terms of degradation, depolymerization, sorption and an increase in biodegradability of 2-NSAP. Among the 25 fungi evaluated two, in particular, Bjerkandera adusta and Pleurotus ostreatus, have been found to significantly depolymerize 2-NSAP yielding to the corresponding monomer (2-naphthalenesulphonic acid, 2-NSA), which has been further degraded by a bacterial consortia selected in a wastewater treatment plant (WWTP). The fungal treatment alone was able to reduce the COD value up to 44%, while activated sludge removed only 9% of the initial COD. In addition, the combined treatment (fungi and bacteria) allowed an increase in the COD removal up to 62%.

Monitoring the oxygen transfer efficiency of full-scale aeration systems: investigation method and experimental results.

This paper reports the results of a series of off-gas tests aimed at monitoring the evolution of the oxygen transfer efficiency in an urban wastewater treatment plant (3,500 population equivalent) located in Tuscany (Italy). The tests were conducted over a 2-year period starting with the testing of the aeration system. It was found that in the absence of membrane-panel cleaning operations, the oxygen transfer efficiency under standard conditions in process water (αSOTE) dropped from 18 to 9.5% in 2 years. This gives rise to a 40% increase in the wastewater treatment plant annual energy costs. The on-site chemical cleaning of the diffusers allowed for an almost total recovery of the transfer efficiency (αSOTE equal to 16%). The use of the off-gas method for monitoring the oxygen transfer efficiency over time is therefore essential for enabling correct planning of the cleaning operations of the diffusers and for cutting the energy consumption and operating costs of the aeration system.

Characterization and comparison of bacterial communities selected in conventional activated sludge and membrane bioreactor pilot plants: a focus on Nitrospira and Planctomycetes bacterial phyla.

A pilot-scale membrane bioreactor (MBR) and a conventional activated sludge system (CAS) were in parallel operated to investigate the impact of the separation technology on the structure and functionality of the selected microbial community. Microbial communities as well as nitrogen removal efficiency of the biomass were characterized. Kinetics and microbial community structure turned out to be duly correlated. The impact of the separation technology on selective conditions and, in particular, the higher variability of solid separation efficiency in CAS with respect to MBR pilot plant possibly represented the main factor influencing the selection of bacterial communities. Concerning nitrifiers, bacteria of the genus Nitrospira were predominant in the MBR. This was in accordance with kinetics of nitrite-oxidizing bacteria that suggested the presence of k-strategists, while r-strategists were selected in the CAS plant, possibly because of the presence of transient higher concentrations of nitrite (in the range of 0.05-0.18 and of 0.05-4.4 mg [Formula: see text]-N L(-1) in the MBR and CAS effluents, respectively). An unexpectedly high presence of bacteria belonging to two specific phylogenetic clades of Planctomycetes was found in both reactors.

Techno-economic evaluation of the application of ozone-oxidation in a full-scale aerobic digestion plant.

This paper deals with the application of the ozone-oxidation in a full scale aerobic sludge digester. Ozonation was applied continuously to a fraction of the biological sludge extracted from the digestion unit; the ozonated sludge was then recirculated to the same digester. Three different ozone flow rates were tested (60,500 and 670g O3 h(-1)) and their effects evaluated in terms of variation of the total and soluble fractions of COD, nitrogen and phosphorous, of total and volatile suspended solids concentrations and Sludge Volume Index in the aerobic digestion unit. During the 7-month operation of the ozonation process, it was observed an appreciable improvement of the aerobic digestion efficiency (up to about 20% under the optimal conditions) and of the sludge settleability properties. These results determined an average reduction of about 60% in the biological sludge extracted from the plant and delivered to final disposal. A thorough economic analysis showed that this reduction allowed to achieve a significant cost saving for the plant with respect to the previous years operated without ozonation. Furthermore, it was determined the threshold disposal cost above which implementation of the ozone oxidation in the aerobic digestion units of similar WWTPs becomes economically convenient (about 60€t(-1) of sludge).