Surveillance of SARS-CoV-2 in extensive monitoring of municipal wastewater: key issues to yield reliable results.

Several studies have detected SARS-CoV-2 in the stool of infected people as in urban wastewater. The quantification of SARS-CoV-2 in wastewater appears today as a powerful tool that can help in wastewater-based epidemiology (WBE). The goal is to improve the prediction of new waves of COVID-19 outbreaks and provide an early warning of the evolution of the infection. In this research, we highlighted some practical and scientific aspects that emerged during an extensive ongoing monitoring campaign carried out on a large number of wastewater treatment plants located in the province of Trento (North Italy) and aimed at the detection of SARS-CoV-2 in raw municipal wastewater. The open issues underline are related to the collection and storage (sampling protocol, storage and heat treatment), to the molecular analysis (enrichment phase), and to the mathematical calculation of SARS-CoV-2 load in wastewater, suitable for WBE (standard curve to obtain the concentration of genomic units and flow rate measurements). This study provides some insights that can help in the implementation of surveillance plans in other regions.

The effects of a full-scale anaerobic side-stream reactor on sludge decay and biomass activity.

A full-scale anaerobic side-stream reactor (ASSR) for sludge reduction was monitored in terms of sludge production and compared with the previous conventional activated sludge configuration (CAS). A detailed solid mass balance was calculated on the whole full-scale plant to estimate the sludge reduction associated with the ASSR. The activity of the biomass, which undergoes alternation of aerobic and anaerobic conditions, was investigated by the respirometric test. The ASSR promoted a reduction of heterotrophic biomass activity and the substrate consumption rate in the activated sludge implemented with ASSR (AS + ASSR) was 36% smaller than in the CAS period. The solid mass balance indicated a sludge reduction of 28%. During the 270-day operation, the observed sludge yield passed from 0.438 kgTSS/kgCOD in the CAS to 0.315 in the AS + ASSR configuration. The solubilization of chemical oxygen demand (COD), NH4 +-N and orthophosphate were verified under anaerobic conditions. The results suggest that the possible mechanisms of sludge reduction were the increase of the system sludge retention time (SRT) by ASSR addition, and the reduction in heterotrophic biomass activity added to the organic compounds' hydrolysis.

Laboratory-scale investigation on the role of microalgae towards a sustainable treatment of real municipal wastewater.

Engineered microalgal-bacteria consortia are an attractive solution towards a low-cost and sustainable wastewater treatment that does not rely on artificial mechanical aeration. In the research conducted for this study, a bench-scale photo-sequencing-batch reactor (PSBR) was operated without external aeration. A spontaneous consortium of microalgae and bacteria was developed in the PSBR at a concentration of 0.8-1.7 g TSS/L. The PSBR ensured removal efficiency of 85 ± 8% for chemical oxygen demand (COD) and 98 ± 2% for total Kjeldahl nitrogen (TKN). Nitrogen balance revealed that the main mechanisms for TKN removal was autotrophic nitrification, while N assimilation and denitrification accounted for 4% and 56%, respectively. The development of dense microalgae-bacteria bioflocs resulted in good settleability with average effluent concentration of 16 mgTSS/L. The ammonium removal rate was 2.9 mgN L-1 h-1, which corresponded to 2.4 mgN gTSS-1 h-1. Although this specific ammonium removal rate is similar to activated sludge, the volumetric rate is lower due to the limited total suspended solids (TSS) concentration (three times less than activated sludge). Therefore, the PSBR footprint appears less competitive than activated sludge. However, ammonium was completely removed without artificial aeration, resulting in a very cost-effective process. Only 50% of phosphorus was removed, suggesting that further research on P uptake is needed.

Enhanced nitrogen removal and energy saving in a microalgal-bacterial consortium treating real municipal wastewater.

The optimization of total nitrogen removal from municipal wastewater was investigated in a laboratory-scale photo-sequencing batch reactor (PSBR) operated with a mixed microalgal-bacterial consortium spontaneously acclimatized to real wastewater. No external aeration was provided in the PSBR to reduce energy consumption: oxygen was only supplied by the microalgal photosynthesis. The enhancement of total nitrogen removal was achieved through: (1) feeding of wastewater in the dark phase to provide readily biodegradable COD when oxygen was not produced, promoting denitrification; (2) intermittent use of the mixer to favor simultaneous nitrification-denitrification inside the dense flocs and to achieve 41% energy saving with respect to continuous mixing. Efficient COD removal (86 ± 2%) was observed, obtaining average effluent concentrations of 37 mg/L and 22 mg/L of total COD and soluble COD, respectively. TKN removal was 97 ± 3%, with an average effluent concentration of 0.5 ± 0.7 mg NH4 +-N/L. Assimilation of nitrogen by heterotrophic bacteria accounted only for 20% of TKN removal, whilst the major part of TKN was nitrified. In particular, the nitrification rate was 1.9 mgN L-1 h-1 (specific rate 2.4 mgN gTSS-1 h-1), measured with dissolved oxygen near zero, when the oxygen demand was higher than the oxygen produced by photosynthesis. Total nitrogen of 6.3 ± 4.4 mgN/L was measured in the effluent after PSBR optimization.

Benchmarking of energy consumption in municipal wastewater treatment plants - a survey of over 200 plants in Italy.

One of the largest surveys in Europe about energy consumption in Italian wastewater treatment plants (WWTPs) is presented, based on 241 WWTPs and a total population equivalent (PE) of more than 9,000,000 PE. The study contributes towards standardised resilient data and benchmarking and to identify potentials for energy savings. In the energy benchmark, three indicators were used: specific energy consumption expressed per population equivalents (kWh PE-1 year-1), per cubic meter (kWh/m3), and per unit of chemical oxygen demand (COD) removed (kWh/kgCOD). The indicator kWh/m3, even though widely applied, resulted in a biased benchmark, because highly influenced by stormwater and infiltrations. Plants with combined networks (often used in Europe) showed an apparent better energy performance. Conversely, the indicator kWh PE-1 year-1 resulted in a more meaningful definition of a benchmark. High energy efficiency was associated with: (i) large capacity of the plant, (ii) higher COD concentration in wastewater, (iii) separate sewer systems, (iv) capacity utilisation over 80%, and (v) high organic loads, but without overloading. The 25th percentile was proposed as a benchmark for four size classes: 23 kWh PE-1 y-1 for large plants > 100,000 PE; 42 kWh PE-1 y-1 for capacity 10,000 < PE < 100,000, 48 kWh PE-1 y-1 for capacity 2,000 < PE < 10,000 and 76 kWh PE-1 y-1 for small plants < 2,000 PE.

Anaerobic side-stream reactor for excess sludge reduction: 5-year management of a full-scale plant.

The long-term performances of a full-scale anaerobic side-stream reactor (ASSR) aimed at sludge reduction have been monitored for the first time, in comparison with a conventional activated sludge process (CAS). The plant was integrated with an ASSR treatment of 2293-3293 m(3). Operational parameters in the ASSR were: ORP -250 mV, interchange ratio of 7-10%, hydraulic retention time of 7 d. No worsening of effluent quality was observed in the ASSR configuration and removal efficiency of COD and NH4 was above 95%. A slight increase in the Sludge Volume Index did not cause worsening in effluent solids concentration. The observed sludge yield (Yobs) passed from 0.44 kgTSS/kgCOD in the CAS to 0.35 in the ASSR configuration. The reduction of Yobs by 20% is lower than expected from the literature where sythetic wastewater is used, indicating that sludge reduction efficiency is largely affected by inert mass fed with influent real wastewater. An increase by 45% of the ASSR volume did not promote a further reduction of Yobs, because sludge reduction is affected not solely by endogenous decay but also by other factors such as interchange ratio and aerobiosis/anaerobiosis alternation.

Energy audit in small wastewater treatment plants: methodology, energy consumption indicators, and lessons learned.

Energy audits in wastewater treatment plants (WWTPs) reveal large differences in the energy consumption in the various stages, depending also on the indicators used in the audits. This work is aimed at formulating a suitable methodology to perform audits in WWTPs and identifying the most suitable key energy consumption indicators for comparison among different plants and benchmarking. Hydraulic-based stages, stages based on chemical oxygen demand, sludge-based stages and building stages were distinguished in WWTPs and analysed with different energy indicators. Detailed energy audits were carried out on five small WWTPs treating less than 10,000 population equivalent and using continuous data for 2 years. The plants have in common a low designed capacity utilization (52% on average) and equipment oversizing which leads to waste of energy in the absence of controls and inverters (a common situation in small plants). The study confirms that there are several opportunities for reducing energy consumption in small WWTPs: in addition to the pumping of influent wastewater and aeration, small plants demonstrate low energy efficiency in recirculation of settled sludge and in aerobic stabilization. Denitrification above 75% is ensured through intermittent aeration and without recirculation of mixed liquor. Automation in place of manual controls is mandatory in illumination and electrical heating.

Concerning the role of cell lysis-cryptic growth in anaerobic side-stream reactors: the single-cell analysis of viable, dead and lysed bacteria.

In the Anaerobic Side-Stream Reactor (ASSR), part of the return sludge undergoes alternating aerobic and anaerobic conditions with the aim of reducing sludge production. In this paper, viability, enzymatic activity, death and lysis of bacterial cells exposed to aerobic and anaerobic conditions for 16 d were investigated at single-cell level by flow cytometry, with the objective of contributing to the understanding of the mechanisms of sludge reduction in the ASSR systems. Results indicated that total and viable bacteria did not decrease during the anaerobic phase, indicating that anaerobiosis at ambient temperature does not produce a significant cell lysis. Bacteria decay and lysis occurred principally under aerobic conditions. The aerobic decay rate of total bacteria (bTB) was considered as the rate of generation of lysed bacteria. Values of bTB of 0.07-0.11 d(-1) were measured in anaerobic + aerobic sequence. The enzymatic activity was not particularly affected by the transition from anaerobiosis to aerobiosis. Large solubilisation of COD and NH4(+) was observed only under anaerobic conditions, as a consequence of hydrolysis of organic matter, but not due to cell lysis. The observations supported the proposal of two independent mechanisms contributing equally to sludge reduction: (1) under anaerobic conditions: sludge hydrolysis of non-bacterial material, (2) under aerobic conditions: bacterial cell lysis and oxidation of released biodegradable compounds.

Toxicant inhibition in activated sludge: fractionation of the physiological status of bacteria.

In wastewater treatment plants the sensitivity of activated sludge to a toxicant depends on the toxicity test chosen, and thus the use of more than one test is suggested. The physiological status of bacteria in response to toxicants was analysed by flow cytometry to distinguish intact, permeabilised, active cells and cells disrupted. Results were compared with respirometry and bioluminescence bioassay (Vibrio fischeri). 3,5-Dichlorophenol (DCP) was used as reference xenobiotic. DCP has a strong effect on cellular integrity, causing an increase in permeabilised and disrupted cells. A reduction of 44-80% of intact cells with 6-30 mgDCP/L for 5h was found. Inhibition of active cells was 25-49%, at 6-30 mgDCP/L for 5h. The bioluminescence bioassay resulted oversensitive to DCP compared to tests based on activated sludge, while oxygen uptake rate was affected similarly to intact cells measured by flow cytometry. Landfill leachate was tested: a detrimental impact on both cellular integrity and enzymatic activity was observed. Reduction of intact cells and active cells was by 32% and 61% respectively after addition of 50% (v/v) of leachate for 5h. The flow cytometry analysis proposed here might be widely applicable in the monitoring of various toxicants and in other aquatic biosystems.

Accurate flow cytometric monitoring of Escherichia coli subpopulations on solid food treated with high pressure carbon dioxide.

AIMS: Evaluation of flow cytometry coupled with viability markers to monitor the inactivation of Escherichia coli cells spiked on solid food following High Pressure Carbon Dioxide (HPCD), a mild processing technology. METHODS AND RESULTS: Flow cytometry (FCM) coupled with SYBR-Green I and Propidium Iodide was applied to monitor the effect of HPCD treatment on E. coli cells spiked on fresh cut carrots, therefore mimicking contamination of food products by faecal coliforms. FCM allowed to distinguish E. coli cells from carrot debris and natural flora, to evaluate the reduction of total cells, and to quantify viable and dead cells based on their membrane integrity after HPCD treatment. The comparison of FCM results with conventional cultivation methods revealed that HPCD treatments performed at 120 bar, 22 or 35°C for 5 min disrupted 43 and 53% of bacterial cells, respectively, and produced a large percentage of partially permeabilized (96·5 and 98%) cells. CONCLUSIONS: Although treatments at 22°C for 10 min and at 35°C for 7 min were sufficient to inhibit the ability of all E. coli cells to replicate with an inactivation of 8 Log, FCM analysis showed that the inactivation of intact cells was only 2-2·5 Log. A fraction of HPCD-treated cells maintained their metabolic activity and re-growth capacity, indicating that the treatment induces a transitory Viable But Not Cultivable (VNBC) state. SIGNIFICANCE AND IMPACT OF THE STUDY: Under stress conditions many pathogens enter in a VBNC state, thus escaping detection by traditional cultivation methods. We provide the first evaluation of HPCD-mediated bacterial inactivation on fresh food using FCM coupled with viability markers, which should assist in the prevention of food-associated health risks.

Influence of high organic loads during the summer period on the performance of hybrid constructed wetlands (VSSF + HSSF) treating domestic wastewater in the Alps region.

One of the limits for the application of constructed wetlands (CWs) in mountain regions (such as the Alps) is associated with the considerable land area requirements. In some mountain areas, the treatment of domestic wastewater at popular tourist destinations is particularly difficult during the summer, when the presence of visitors increases hydraulic and organic loads. This paper aims to evaluate whether a hybrid CW plant designed on the basis of the resident population only, can treat also the additional load produced by the floating population during the tourist period (summer, when temperatures are favourable for biological treatment), without a drastic decrease of efficiency and without clogging problems. The research was carried out by considering two operational periods: the first one was based on literature indications (3.2 m(2)/PE in the VSSF unit) and the second one assumed higher hydraulic and organic loads (1.3 m(2)/PE in the VSSF unit). The removal efficiency in the hybrid CW system decreased slightly from 94 to 88% for COD removal and from 78 to 75% for total N removal, even after applying a double hydraulic (from 55 to 123 L m(-2) d(-1)) and organic load (from 37 to 87 g COD m(-2) d(-1) and from 4.4 to 10.3 g TKN m(-2) d(-1)). The results showed that in the summer period the application of high loads did not affect the efficiency of the hybrid CW plant significantly, suggesting that it is possible to refer the CW design to the resident population only, with subsequent considerable savings in superficial area.

Kinetics of heterotrophic biomass and storage mechanism in wetland cores measured by respirometry.

Although oxygen uptake rate has been widely used in activated sludge for measuring kinetic and stoichiometric parameters or for wastewater characterization, its application in constructed wetlands (CWs) cores has been recently proposed. The aim of this research is to estimate the kinetic and stoichiometric parameters of the heterotrophic biomass in CW cores. Respirometric tests were carried out with pure carbonaceous substrate and real wastewater. Endogenous respiration was about 2 gO2 m(-3) h(-1) (per unit of bed volume), while the kinetic parameters obtained for COD oxidation were very high (maximum rate per unit of bed volume of 10.7-26.8 gCOD m(-3) h(-1)) which indicates high biodegradation potential in fully aerobic environment. Regarding to stoichiometric parameter, the maximum growth yield, Y(H), was 0.56-0.59 mgCOD/mgCOD, while the storage yield, Y(STO), was 0.75-0.77 mgCOD/mgCOD. The storage mechanism was observed in CW cores during COD oxidation, which leads to the transformation of the external soluble substrate in internal storage products, probably as response to intermittent loads applied in CW systems, transient concentrations of readily biodegradable substrate and alternance of feast/famine periods.

Bacteria permeabilization and disruption caused by sludge reduction technologies evaluated by flow cytometry.

Technologies proposed in the last decades for the reduction of the sludge production in wastewater treatment plants and based on the mechanism of cell lysis-cryptic growth (physical, mechanical, thermal, chemical, oxidative treatments) have been widely investigated at lab-, pilot- and, in some cases, at full-scale but the effects on cellular lysis have not always been demonstrated in depth. The research presented in this paper aims to investigate how these sludge reduction technologies affect the integrity and permeabilization of bacterial cells in sludge using flow cytometry (FCM), which permits the rapid and statistically accurate quantification of intact, permeabilised or disrupted bacteria in the sludge using a double fluorescent DNA-staining instead of using conventional methods like plate counts and microscope. Physical/mechanical treatments (ultrasonication and high pressure homogenisation) caused moderate effects on cell integrity and caused significant cell disruption only at high specific energy levels. Conversely, thermal treatment caused significant damage of bacterial membranes even at moderate temperatures (45-55 °C). Ozonation significantly affected cell integrity, even at low ozone dosages, below 10 mgO(3)/gTSS, causing an increase of permeabilised and disrupted cells. At higher ozone dosages the compounds solubilised after cell lysis act as scavengers in the competition between soluble compounds and (particulate) bacterial cells. An original aspect of this paper, not yet reported in the literature, is the comparison of the effects of these sludge reduction technologies on bacterial cell integrity and permeabilization by converting pressure, temperature and ozone dosage to an equivalent value of specific energy. Among these technologies, comparison of the applied specific energy demonstrates that achieving the complete disruption of bacterial cells is not always economically advantageous because excessive energy levels may be required.

Direct quantification of bacterial biomass in influent, effluent and activated sludge of wastewater treatment plants by using flow cytometry.

A rapid multi-step procedure, potentially amenable to automation, was proposed for quantifying viable and active bacterial cells, estimating their biovolume using flow cytometry (FCM) and to calculate their biomass within the main stages of a wastewater treatment plant: raw wastewater, settled wastewater, activated sludge and effluent. Fluorescent staining of bacteria using SYBR-Green I + Propidium Iodide (to discriminate cell integrity or permeabilisation) and BCECF-AM (to identify enzymatic activity) was applied to count bacterial cells by FCM. A recently developed specific procedure was applied to convert Forward Angle Light Scatter measured by FCM into the corresponding bacterial biovolume. This conversion permits the calculation of the viable and active bacterial biomass in wastewater, activated sludge and effluent, expressed as Volatile Suspended Solids (VSS) or particulate Chemical Oxygen Demand (COD). Viable bacterial biomass represented only a small part of particulate COD in raw wastewater (4.8 +/- 2.4%), settled wastewater (10.7 +/- 3.1%), activated sludge (11.1 +/- 2.1%) and effluent (3.2 +/- 2.2%). Active bacterial biomass counted for a percentage of 30-47% of the viable bacterial biomass within the stages of the wastewater treatment plant.

Biological treatment of winery wastewater: an overview.

The treatment of winery wastewater can realised using several biological processes based both on aerobic or anaerobic systems using suspended biomass or biofilms. Several systems are currently offered by technology providers and current research envisages the availability of new promising technologies for winery wastewater treatment. The present paper intends to present a brief state of the art of the existing status and advances in biological treatment of winery wastewater in the last decade, considering both lab, pilot and full-scale studies. Advantages, drawbacks, applied organic loads, removal efficiency and emerging aspects of the main biological treatments were considered and compared. Nevertheless in most treatments the COD removal efficiency was around 90-95% (remaining COD is due to the un-biodegradable soluble fraction), the applied organic loads are very different depending on the applied technology, varying for an order of magnitude. Applied organic loads are higher in biofilm systems than in suspended biomass while anaerobic biofilm processes have the smaller footprint but in general a higher level of complexity.

Membrane bioreactors for winery wastewater treatment: case-studies at full scale.

The membrane bioreactor technology (MBR) is nowadays a suitable alternative for winery wastewater treatment, thanks to low footprint, complete suspended solids removal, high efficiency in COD abatement and quick start-up. In this paper, data from two full-scale MBRs equipped with flat-sheet membranes (plant A and plant B) are presented and discussed. COD characterisation by respirometry pointed out the high biodegradability degree of both wastewater, with a strong prevalence of the readily biodegradable fraction. An extended version of Activated Sludge Model No. 3 was used to fit the experimental OUR profiles and to assess the maximum growth rate of heterotrophic biomass on sludge samples collected at both sites; the stoichiometric yield coefficients were also calculated. Sludge filterability and dewaterability were investigated with batch tests; laboratory results confirmed the field observations. Finally, some considerations are listed, aimed at defining possible key-issues for optimal process design and operation.

Detection and elimination of cyanobacteria from frescoes: the case of the St. Brizio Chapel (Orvieto Cathedral, Italy).

A rosy discoloration partly masking the Luca Signorelli frescoes in St. Brizio Chapel (Orvieto Cathedral, Italy) for many years proved to be a biological alteration, so the present research focused on investigating biodeteriogens and selecting an appropriate biocide to treat them. Optical epifluorescence and electronic microscopic observations of the rosy powder revealed a prevalent autofluorescent coccoid form with a diameter bigger than 5 microm. Chlorophylls a and b were extracted, suggesting the presence of cyanobacteria, a thesis subsequently confirmed by flow cytometry. Cultural media were inoculated with the rosy powder, and microorganisms grew as a green patina in phototrophic conditions and as a rosy patina when organic compounds were added to the mineral medium. The rosy discoloration was most likely caused by the presence of phycoerythrin. The sequencing of the cyanobacteria-specific polymerase chain reaction (PCR)-DGGE bands matched, with a similarity percentage >94, uncultured cyanobacteria, and the sequences were deposited in the GenBank under EU874241, EU874242, EU874243, EU874244, EU874245, EU874246, and EU874247. Finally, the efficiency of the two biocides Neo Desogen and Metatin 5810-101, both based on benzalkonium chloride, was evaluated using adenosine triphosphate measurements and PCR-based detection of cyanobacteria. Metatin, used in situ at 2% of the trade product, proved to be the better biocide, no cyanobacteria being detected after the Metatin treatment.

Respirometric techniques for assessment of biological kinetics in constructed wetland.

The aim of this research was the development of a procedure to measure biological kinetics of organic matter oxidation and nitrification in constructed wetland, by using respirometric techniques. Columns simulating cores of vertical subsurface flow systems were investigated. The oxygen uptake rate (OUR) of the columns was calculated on the basis of the difference of DO concentrations measured continuously at the top and at the bottom of the column. From the respirogram, the following kinetic parameters have been evaluated: maximum rate of oxidation of readily biodegradable COD, maximum rate of nitrification, endogenous respiration of the biomass grown inside the bed. In order to improve the interpretation of the respirograms, additional respirometric tests were carried out on the wetland columns by using pure substrates, such as acetate (carbon source) and ammonium (substrate for nitrification). The kinetic parameters obtained from respirograms can be useful for control and design of constructed wetlands or for improving nutrient and carbon mass balances.

Heavy metal removal from winery wastewater in the case of restrictive discharge regulation.

In most cases of winery effluent, the heavy metal content, especially zinc (Zn) and copper (Cu), does not meet the limits for the discharge as imposed by the most restrictive regulations at international level (0.4 mgCu/L and 1.0 mgZn/L in the Italian regulations). An alternative for the reduction of Cu and Zn is the on-site pre-treatment of wastewater at the winery in order to meet the limits for the discharge into the public sewerage. The purpose of this study is to evaluate the effectiveness of a pre-treatment based on chemical precipitation with chelating agents (TMT: 2,4,6-trimercaptotriazine), for the reduction of Cu and Zn from raw winery wastewater. The chemical precipitation was optimised at lab-scale through jar tests in order to evaluate the optimal dosages. An average dosage equal to 0.84 mL of TMT (15%) for 1 mg of Cu removed was estimated. Furthermore, the efficiency of the on-site chemical pre-treatment was investigated at pilot scale. The results confirmed the feasibility of using TMT treatment for the reduction of Cu and Zn in order to meet the limits for discharge into the sewerage. Contextually to the removal of heavy metals, the chemical pre-treatment allowed us to obtain the reduction of particulate COD and TSS. Finally, the costs for the operation and the management of the on-site pre-treatment were evaluated.

Treatment of winery wastewater in a full-scale fixed bed biofilm reactor.

The treatment of winery wastewater was performed at full-scale applying a two-stage fixed bed biofilm reactor (FBBR) system for the discharge in the sewerage. The results of the first year of operation at the full-scale plant are presented. Values of removed organic loads and effluent concentrations were interpreted on the basis of the COD fractionation of influent wastewater assessed through respirometric tests. The average removal efficiency of total COD was 91 %. It was not possible to reach an higher efficiency because of the unbiodegradable soluble fraction of COD (about 10% of total COD on average during the whole year), that cannot be removed by biological process or settling. Due to the high empty space offered by the plastic carriers, FBBRs did not require backwashing during the seasonal operationing period of the plant (September-March). In comparison with other treatment systems the FBBR configuration allows one to ensure a simple management, to obtain high efficiency also in the case of higher fluctuations of flow and loads and to guarantee a good settleability of the sludge, without bulking problems.