Flow cytometry as a tool for the rapid enumeration of 1-µm microplastics spiked in wastewater and activated sludge after coagulation-flocculation-sedimentation.

Considering the limited literature and the difficulty of quantifying 1-µm micro-nanoplastics (1-µm MNP) in complex aqueous matrices such as wastewater and sludge, the removal rate of these very small particles in wastewater treatment plants (WWTP) represents a major challenge. In this study, coagulation-flocculation-sedimentation (CFS) with aluminum salts was investigated to evaluate the removal of 1-µm MNPs spiked in tap water, raw wastewater, pre-settled wastewater, and activated sludge. Quantification of 1-µm MNP was performed using the high-throughput flow cytometry (FCM) analysis which takes only a few minutes and produces results with high accuracy and reproducibly. The results indicated that the 1-µm MNPs were highly stable in pure water and unable to settle rapidly. In raw wastewater, sedimentation without coagulants removed less than 4% of 1-µm MNP. Conversely, CFS treatment showed a significant improvement in the removal of 1-µm MNP from wastewater. At dosages of 0.3-3 mg Al3+/L, the removal of MNPs in wastewater reached 30% and no flocs were observed, while floc formation was visible with increased dosages of 3-12 mg Al3+/L, obtaining MNP removal greater than 90%. CFS in activated sludge with a solids content of 5800 mg MLSS/L registered the highest removal efficiency (95-99%) even for dosages of 0.3-60 mg Al3+/L and pH dropping to 5. However, activated sludge showed extremely high removal efficiency of MNPs (97.3 ± 0.9%) even without coagulants. The large, dense flocs that constitute activated sludge appear particularly efficient in capturing 1-µm MNPs during the sedimentation process even in the absence of coagulants.

Conventional activated sludge vs. photo-sequencing batch reactor for enhanced nitrogen removal in municipal wastewater: Microalgal-bacterial consortium and pathogenic load insights.

Municipal wastewater treatment plants are mostly based on traditional activated sludge (AS) processes. These systems are characterised by major drawbacks: high energy consumption, large amount of excess sludge and high greenhouse gases emissions. Treatment through microalgal-bacterial consortia (MBC) is an alternative and promising solution thanks to lower energy consumption and emissions, biomass production and water sanitation. Here, microbial difference between a traditional anaerobic sludge (AS) and a consortium-based system (photo-sequencing batch reactor (PSBR)) with the same wastewater inlet were characterised through shotgun metagenomics. Stable nitrification was achieved in the PSBR ensuring ammonium removal > 95 % and significant total nitrogen removal thanks to larger flocs enhancing denitrification. The new system showed enhanced pathogen removal, a higher abundance of photosynthetic and denitrifying microorganisms with a reduced emissions potential identifying this novel PSBR as an effective alternative to AS.

Evaluation of concentration procedures, sample pre-treatment, and storage condition for the detection of SARS-CoV-2 in wastewater.

Crucial information on the pandemic's spread has been gathered by monitoring the trend of SARS-CoV-2 in wastewater. This surveillance has highlighted that the initial concentration is a critical step of the analytical procedure due to the low viral titer that may be present in this matrix. This paper presents the results of the evaluation of two different wastewater concentration protocols to determine the most efficient and cost-effective. The two methods tested were the following: (a) a biphasic separation system with PEG-dextran and (b) a PEG/NaCl precipitation protocol. Other aspects of the detection method were also investigated including the influence of storage temperature on virus recovery and the heat treatment of pasteurization, which aims to make samples safer for operators and the environment. The PEG/NaCl precipitation method was found to perform better than the biphasic separation system, allowing for more sensitive identification of the presence of the virus and the detection of a higher viral titer than that identified with the biphasic separation in all results. Storage of the samples at 4.3±0.2°C for up to 3 weeks did not adversely affect the virus titer and the pasteurization pre-treatment increases operator safety and maintains the identification of the viral concentration.

The wave of the SARS-CoV-2 Omicron variant resulted in a rapid spike and decline as highlighted by municipal wastewater surveillance.

This paper highlights the extraordinarily rapid spread of SARS-CoV-2 loads in wastewater that during the Omicron wave in December 2021-February 2022, compared with the profiles acquired in 2020-21 with 410 samples from two wastewater treatment plants (Trento+suburbs, 132,500 inhabitants). Monitoring of SARS-CoV-2 in wastewater focused on: (i) 3 samplings/week and analysis, (ii) normalization to calculate genomic units (GU) inh-1 d-1; (iii) calculation of a 7-day moving average to smooth daily fluctuations; (iv) comparison with the 'current active cases'/100,000 inh progressively affected by the mass vaccination. The time profiles of SARS-CoV-2 in wastewater matched the waves of active cases. In February-April 2021, a viral load of 1.0E＋07 GU inh-1 d - 1 corresponded to 700 active cases/100,000 inh. In July-September 2021, although the low current active cases, sewage revealed an appreciable SARS-CoV-2 circulation (in this period 2.2E＋07 GU inh-1 d-1 corresponded to 90 active cases/100,000 inh). Omicron was not detected in wastewater until mid-December 2021. The Omicron spread caused a 5-6 fold increase of the viral load in two weeks, reaching the highest peak (2.0-2.2E＋08 GU inh-1 d-1 and 4500 active cases/100,000 inh) during the pandemic. In this period, wastewater surveillance anticipated epidemiological data by about 6 days. In winter 2021-22, despite the 4-7 times higher viral loads in wastewater, hospitalizations were 4 times lower than in winter 2020-21 due to the vaccination coverage >80%. The Omicron wave demonstrated that SARS-CoV-2 monitoring of wastewater anticipated epidemiological data, confirming its importance in long-term surveillance.

Coronaviruses and SARS-CoV-2 in sewerage and their removal: Step by step in wastewater treatment plants.

The fate of Coronaviruses (CoVs) and in particular SARS-CoV-2 in wastewater treatment plants (WWTPs) has not been completely understood yet, but an adequate knowledge on the removal performances in WWTPs could help to prevent waterborne transmission of the virus that is still under debate. CoVs and SARS-CoV-2 are discharged from faeces into the sewer network and reach WWTPs within a few hours. This review presents the fate of SARS-CoV-2 and other CoVs in the primary, secondary and tertiary treatments of WWTPs as well as in sludge treatments. The viral loads decrease progressively along with the treatments from 20 to 3.0E+06 GU/L (Genomic Units/L) in the influent wastewater to concentrations below 2.50E+05 GU/L after secondary biological treatments and finally to negative concentrations (below detection limit) in disinfected effluents. Reduction of CoVs is due to (i) natural decay under unfavourable conditions (solids, microorganisms, temperature) for relatively long hydraulic retention times and (ii) processes of sedimentation, filtration, predation, adsorption, disinfection. In primary and secondary settling, due to the hydrophobic properties, a partial accumulation of CoVs may occur in the separated sludge. In secondary treatment (i.e. activated sludge) CoVs and SARS-CoV-2 loads can be reduced only by about one logarithm (∼90%). To enhance this removal, tertiary treatment with ultrafiltration (Membrane Bioreactors) and chemical disinfection or UV light is needed. CoVs and SARS-CoV-2 in the sludge (1.2E+04-4.6E+08 GU/L) can be inactivated significantly in the thermophilic digestion (55 °C), while mesophilic temperatures (33-37 °C) are not efficient. Additional studies are required to investigate the infectivity of SARS-CoV-2 in WWTPs, especially in view of increasing interest in wastewater reclamation and reuse.

SARS-CoV-2 from faeces to wastewater treatment: What do we know? A review.

SARS-CoV-2, the virus that causes COVID-19, has been found in the faeces of infected patients in numerous studies. Stool may remain positive for SARS-CoV-2, even when the respiratory tract becomes negative, and the interaction with the gastrointestinal tract poses a series of questions about wastewater and its treatments. This review aims to understand the viral load of SARS-CoV-2 in faeces and sewage and its fate in wastewater treatment plants (WWTPs). The viral load in the faeces of persons testing positive for SARS-CoV-2 was estimated at between 5·103 to 107.6 copies/mL, depending on the infection course. In the sewerage, faeces undergo dilution and viral load decreases considerably in the wastewater entering a WWTP with a range from 2 copies/100 mL to 3·103 copies/mL, depending on the level of the epidemic. Monitoring of SARS-CoV-2 in sewage, although no evidence of COVID-19 transmission has been found via this route, could be advantageously exploited as an early warning of outbreaks. Preliminary studies on WBE seem promising; but high uncertainty of viral loads in wastewater and faeces remains, and further research is needed. The detection of SARS-CoV-2 in sewage, based on RNA sequences and RT-PCR, requires a shared approach on sample pre-treatment and on-site collection to ensure comparable results. The finding of viral RNA in stools does not imply that the virus is viable and infectious. Viability of CoVs such as SARS-CoV-2 decreases in wastewater - due to temperature, pH, solids, micropollutants - but high inactivation in WWTPs can be obtained only by using disinfection (free chlorine, UVC light). A reduction in the quantity of disinfectants can be obtained by implementing Membrane-Bioreactors with ultrafiltration to separate SARS-CoV-2 virions with a size of 60-140 nm. In sludge treatment, thermophilic digestion is effective, based on the general consensus that CoVs are highly sensitive to increased temperatures.

How inoculation affects the development and the performances of microalgal-bacterial consortia treating real municipal wastewater.

To date, little is known about the start-up of photobioreactors and the progressive development of stable microalgal-bacterial consortia with a view to the full-scale treatment of real wastewater. Two photo-sequencing bioreactors, one inoculated with Chlorella vulgaris (RC) and one with the absence of inoculum (RW), were fed with real municipal wastewater and run in parallel for 101 days. The influence of the inoculation was evaluated in terms of pollutant removal efficiency, excess sludge production, solids settleability and microbial community characteristics. No significant differences were observed in the removal of COD (89 ± 4%; 88 ± 3%) and ammonium (99 ± 1%; 99 ± 1%), mainly associated with bacteria activity. During the first weeks of acclimation, Chlorella vulgaris in RC promoted better P removal and very high variations of DO and pH. Conversely, under steady-state conditions, no significant differences were observed between the performances of RC and RW, showing good settleability and low effluent solids, 7 ± 8 and 13 ± 10 mg TSS/L respectively. Microbiome analysis via 16S rRNA gene sequencing showed that, despite a different evolution, the microbial community was quite similar in both reactors under steady state conditions. Overall, the results suggested that the inoculation of microalgae is not essential to engender a photobioreactor aimed at treating real municipal wastewater.

How suspended solids concentration affects nitrification rate in microalgal-bacterial photobioreactors without external aeration.

The use of microalgae for the treatment of municipal wastewater makes possible to supply oxygen and save energy, but must be coupled with bacterial nitrification to obtain nitrogen removal efficiency above 90%. This paper explores how the concentration of Total Suspended Solids (TSS, from 0.2 to 3.9 g TSS/L) affects the nitrification kinetic in three microalgal-bacterial consortia treating real municipal wastewater. Two different behaviors were observed: (1) solid-limited kinetic at low TSS concentrations, (2) light-limited kinetic at higher concentrations. For each consortium, an optimal TSS concentration that produced the maximum volumetric ammonium removal rate (around 1.8-2.0 mg N L-1 h-1), was found. The relationship between ammonium removal rate and TSS concentration was then modelled considering bacteria growth, microalgae growth and limitation by dissolved oxygen and light intensity. Assessment of the optimal TSS concentrations makes possible to concentrate the microbial biomass in a photobioreactor while ensuring high kinetics and a low footprint.

How far are we from closing the loop of sewage resource recovery? A real picture of municipal wastewater treatment plants in Italy.

This paper presents the results of a broad-scale survey of resource recovery implementation in Italian wastewater treatment plants (WWTPs). To the best of our knowledge, this is the first survey comprising a large number of WWTPs done in Europe: more than 600 plants were investigated, representing a treated load of around 20 million population equivalent (≈25% of the total in Italy). Conventional and innovative options for both material and energy recovery along the water and sludge line were surveyed, in order to i) offer a real and complete picture of the current state of resource recovery in WWTPs, and ii) underline key aspects and potential areas for improvements, as a baseline for future developments in the direction of more sustainable plants. Survey outcomes showed that resource recovery is just in its infancy in sewage treatment: only 40% of plants perform at least one option for material/energy recovery. The action most often implemented is recovery of material from surplus sludge for agricultural purposes and the internal reuse of treated effluent as water for various types of plant maintenance. The production of energy from biogas also occurs frequently but only in large plants. On the other hand, some well-known options, such as external reuse of treated effluent or nutrients recovery, were implemented only in a minority of plants: this is likely due to limitations resulting either from strict regulation or difficulty placing recovered products on the market. In conclusion, an overall explanation of these driving forces within the system is explored.

Evaluation of zosteric acid for mitigating biofilm formation of Pseudomonas putida isolated from a membrane bioreactor system.

This study provides data to define an efficient biocide-free strategy based on zosteric acid to counteract biofilm formation on the membranes of submerged bioreactor system plants. 16S rRNA gene phylogenetic analysis showed that gammaproteobacteria was the prevalent taxa on fouled membranes of an Italian wastewater plant. Pseudomonas was the prevalent genus among the cultivable membrane-fouler bacteria and Pseudomonas putida was selected as the target microorganism to test the efficacy of the antifoulant. Zosteric acid was not a source of carbon and energy for P. putida cells and, at 200 mg/L, it caused a reduction of bacterial coverage by 80%. Biofilm experiments confirmed the compound caused a significant decrease in biomass (-97%) and thickness (-50%), and it induced a migration activity of the peritrichous flagellated P. putida over the polycarbonate surface not amenable to a biofilm phenotype. The low octanol-water partitioning coefficient and the high water solubility suggested a low bioaccumulation potential and the water compartment as its main environmental recipient and capacitor. Preliminary ecotoxicological tests did not highlight direct toxicity effects toward Daphnia magna. For green algae Pseudokirchneriella subcapitata an effect was observed at concentrations above 100 mg/L with a significant growth of protozoa that may be connected to a concurrent algal growth inhibition.

Recirculation or artificial aeration in vertical flow constructed wetlands: a comparative study for treating high load wastewater.

Vertical subsurface-flow constructed wetlands at pilot-scale have been applied to treat high hydraulic and organic loads by implementing the following configurations: (1) intermittent recirculation of the treated wastewater from the bottom to the top of the bed, (2) intermittent artificial aeration supplied at the bottom of the bed and (3) the combination of both. These configurations were operated with a saturated bottom layer for a 6h-treatment phase, followed by a free drainage phase prior to a new feeding. COD removal efficiency was 85-90% in all the configurations and removed loads were 54-70 gCOD m(-2)d(-1). The aerated and recirculated wetland resulted in a higher total nitrogen removal (8.6 gN m(-2)d(-1)) due to simultaneous nitrification/denitrification, even in the presence of intermittent aeration (6.8 Nm(3)m(-2)d(-1)). The extra investment needed for implementing aeration/recirculation would be compensated for by a reduction of the surface area per population equivalent, which decreased to 1.5m(2)/PE.

Comparison of quantitative PCR and flow cytometry as cellular viability methods to study bacterial membrane permeabilization following supercritical CO2 treatment.

Foodborne illness due to bacterial pathogens is increasing worldwide as a consequence of the higher consumption of fresh and minimally processed food products, which are more easily cross-contaminated. The efficiency of food pasteurization methods is usually measured by c.f.u. plate counts, a method discriminating viable from dead cells on the basis of the ability of cells to replicate and form colonies on standard growth media, thus ignoring viable but not cultivable cells. Supercritical CO2 (SC-CO2) has recently emerged as one of the most promising fresh food pasteurization techniques, as an alternative to traditional, heat-based methods. In the present work, using three SC-CO2-treated foodborne bacteria (Listeria monocytogenes, Salmonella enterica and Escherichia coli) we tested and compared the performance of alternative viability test methods based on membrane permeability: propidium monoazide quantitative PCR (PMA-qPCR) and flow cytometry (FCM). Results were compared based on plate counts and fluorescent microscopy measurements, which showed that the former dramatically reduced the number of cultivable cells by more than 5 log units. Conversely, FCM provided a much more detailed picture of the process, as it directly quantifies the number of total cells and distinguishes among three categories, including intact, partially permeabilized and permeabilized cells. A comparison of both PMA-qPCR and FCM with plate count data indicated that only a fraction of intact cells maintained the ability to replicate in vitro. Following SC-CO2 treatment, FCM analysis revealed a markedly higher level of bacterial membrane permeabilization of L. monocytogenes with respect to E. coli and S. enterica. Furthermore, an intermediate permeabilization state in which the cellular surface was altered and biovolume increased up to 1.5-fold was observed in L. monocytogenes, but not in E. coli or S. enterica. FCM thus compared favourably with other methods and should be considered as an accurate analytical tool for applications in which monitoring bacterial viability status is of importance, such as microbiological risk assessment in the food chain or in the environment.

Use of silica microspheres having refractive index similar to bacteria for conversion of flow cytometric forward light scatter into biovolume.

This research describes an alternative approach for the rapid conversion of flow cytometric Forward Angle Light Scattering (FALS) into bacterial biovolume. The Rayleigh-Gans theory was considered for explaining the main parameters affecting FALS intensity: sensitivity analysis of the model was carried out, taking into account the parameters characteristic of bacterial cells and characteristics of the flow cytometer. For particles with size in the typical range of bacteria, the FALS intensity is affected mainly by volume and refractive index of bacterial cells and is approximately independent of the shape of the cells. The proposed conversion from FALS intensity into bacterial biovolume is based on a calibration curve determined by using silica microspheres having relative refractive index as far as possible similar to that of bacteria. The approach was validated for two different flow cytometers (the first equipped with an arc lamp and the second with a laser) by comparing the biovolume distribution obtained from FALS conversion with the biovolume measured conventionally under epifluorescence microscopy. The specific case of bacteria taken from a WWTP was addressed. Compared to the time-consuming conventional microscopic approach, the application of FALS for sizing bacterial biovolume could be a very promising tool being completed in few minutes, simultaneously to the enumeration of bacteria during the flow cytometric analysis.

Effects of sonication on bacteria viability in wastewater treatment plants evaluated by flow cytometry--fecal indicators, wastewater and activated sludge.

The application of sonication to wastewater or sludge contributes to the dispersion of aggregates, the solubilisation of particulate matter with an increase in its biodegradability, the damage of microorganisms due to the loss of cellular membrane integrity. This research is aimed at investigating the effects of sonication at 20kHz frequency on viability of microorganisms present in raw wastewater and activated sludge taken from a municipal wastewater treatment plant, as well as pure strains of Escherichia coli and E. faecalis. Flow cytometry was applied for the identification and quantification of viable and dead bacteria free in the bulk liquid, after the fluorescent staining of cellular nucleic acids. The main results showed that: (i) cells of E. coli were highly sensitive to sonication, even at low specific ultrasonic energy (E(s)), and disintegration of a large amount of cells was observed; (ii) on the contrary E. faecalis were more resistant than E. coli, even if high levels of E(s) were applied; (iii) bacteria in raw wastewater exhibited a dynamic of viable and dead bacteria similar to E. coli; (iv) in activated sludge samples, low levels of E(s) produced a prevalent disaggregation of flocs releasing single cells in the bulk liquid, while disruption of bacteria was induced only by very high levels of E(s).

A review and assessment of emerging technologies for the minimization of excess sludge production in wastewater treatment plants.

This paper focuses on the most promising technologies, available for full-scale applications, aimed to the on-site reduction of the excess sludge produced in municipal wastewater treatment plants. New techniques are added to the conventional stages of wastewater treatment, both integrated in the activated sludge bioreactors or applied as pretreatment for the enhancement of anaerobic digestion. A concise review about the alternatives based on physical, chemical or biological mechanisms is described. The present work highlights the efficiency of two such techniques, sonolysis and alkaline thermolysis integrated on the return flow from the secondary settler into the activated sludge bioreactors. The investigation on the effect of sonolysis and alkaline thermolysis on activated sludge samples was carried out by evaluating the COD concentration released in soluble and colloidal form and biodegradability measured by respirometry. The physicochemical treatments of sludge have several advantages (easy management, stability in performances and flexibility), but are associated with high operational costs that often limit the wide-scale applications. The application of hybrid methods, that couple almost two techniques for the enhancement of efficiency with respect to a single one, could optimise the sludge reduction, giving a significant saving in energy consumption for large-scale operations, but further research is needed.

Método respirométrico para o monitoramento de processos biológicos / Respirometric method for the monitoring of biological processes

Este trabalho apresenta os atuais métodos respirométricos voltados ao controle biológico em geral, com técnicas desenvolvidas para a aplicação em tratamentos de águas residuárias, principalmente no controle de processos por lodos ativados e recentes desenvolvimentos do método para a aplicação em sistemas de tratamento por fitodepuração (Wetlands). Descreve novos métodos que foram desenvolvidos no Laboratorio di Ingegneria Sanitaria-Ambientale (LISA) da Università di Trento, Itália. Estes métodos baseiam-se em duas metodologias de laboratório para a aplicação da respirometria em fitoderuração: o primeiro caracteriza a atividade bacteriológica do solo com a finalidade de medir a concentração de O2 na fase gasosa, o outro avalia a concentração de O2 na fase aquosa.

Effects of the metalloid oxyanion tellurite (TeO32-) on growth characteristics of the phototrophic bacterium Rhodobacter capsulatus.

This work examines the effects of potassium tellurite (K2TeO3) on the cell viability of the facultative phototroph Rhodobacter capsulatus. There was a growth mode-dependent response in which cultures anaerobically grown in the light tolerate the presence of up to 250 to 300 microg of tellurite (TeO3(2-)) per ml, while dark-grown aerobic cells were inhibited at tellurite levels as low as 2 microg/ml. The tellurite sensitivity of aerobic cultures was evident only for growth on minimal salt medium, whereas it was not seen during growth on complex medium. Notably, through the use of flow cytometry, we show that the cell membrane integrity was strongly affected by tellurite during the early growth phase (< or =50% viable cells); however, at the end of the growth period and in parallel with massive tellurite intracellular accumulation as elemental Te0 crystallites, recovery of cytoplasmic membrane integrity was apparent (> or =90% viable cells), which was supported by the development of a significant membrane potential (Deltapsi = 120 mV). These data are taken as evidence that in anaerobic aquatic habitats, the facultative phototroph R. capsulatus might act as a natural scavenger of the highly soluble and toxic oxyanion tellurite.

Assessment of activated sludge viability with flow cytometry.

The aim of the study was to evaluate the applicability of fluorescent dyes and multiparameter flow cytometry for the rapid and direct viability/activity assessment of activated sludge samples taken from wastewater treatment plants. Viability and activity of the biomass were estimated respectively through cellular membrane integrity, staining with SYBR Green I and Propidium Iodide, and through fluorogenic dyes capable of detecting enzymatic activity, as FDA and BCECF-AM. A procedure has been developed to disaggregate sludge flocs before dyes staining and cytometric analysis. The developed procedure allows a high recovery of bacteria with good accuracy and repeatability, and minimize the damage of the cells suspension obtained from the disaggregation of the flocs. These measurements were applied to estimate the two main parameters required to define the biological activated sludge process: the endogenous decay rate and the specific growth rate in exponential phase with high F/M ratio. Oxygen utilization rate measurements (OUR) were conducted to conventionally monitor the activity of the biomass. The preliminary data are encouraging and support the possibility to investigate bacteria dynamics on wastewater treatment plants.