Assessing wastewater-based epidemiology for the prediction of SARS-CoV-2 incidence in Catalonia.

While wastewater-based epidemiology has proven a useful tool for epidemiological surveillance during the COVID-19 pandemic, few quantitative models comparing virus concentrations in wastewater samples and cumulative incidence have been established. In this work, a simple mathematical model relating virus concentration and cumulative incidence for full contagion waves was developed. The model was then used for short-term forecasting and compared to a local linear model. Both scenarios were tested using a dataset composed of samples from 32 wastewater treatment plants and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) incidence data covering the corresponding geographical areas during a 7-month period, including two contagion waves. A population-averaged dataset was also developed to model and predict the incidence over the full geography. Overall, the mathematical model based on wastewater data showed a good correlation with cumulative cases and allowed us to anticipate SARS-CoV-2 incidence in one week, which is of special relevance in situations where the epidemiological monitoring system cannot be fully implemented.

Removal of nitrate from groundwater by nano-scale zero-valent iron injection pulses in continuous-flow packed soil columns.

Injection of zero-valent iron nanoparticles (nZVI) into aquifers has gained increasing attention of researchers for in-situ treatment of NO3--contaminated groundwater. nZVI has proved efficient in chemically reducing NO3- and, according to recent research efforts, in supporting biological denitrification under favoured conditions. Given the scarce research on nZVI pulsed injection in continuous-flow systems, the objective of this study was to evaluate the effect of nZVI pulses on the removal of NO3- from groundwater in packed soil columns and, more particularly, to elucidate whether or not biotic NO3- removal processes were promoted by nZVI. Three identical columns were filled with aquifer soil samples and fed with the same nitrate polluted groundwater but operated under different conditions: (A) with application of nZVI pulses and biocide spiked in groundwater, (B) without application of nZVI pulses and (C) with application of nZVI pulses. Results showed that the application of nZVI (at 30 mg/L and 78 mg/L doses) resulted in an immediate and sharp removal of NO3- (88-94%), accompanied by an increase in pH (from 7.0 to 9.0-10.0), a drop in redox potential (Eh) (from +420 mV to <100 mV) and a release of Fe(II) and Total Organic Carbon (TOC) in the effluent (to 200 mg/L and 150-200 mg/L, respectively). The released TOC came from the organic polymer used as stabilizer of the nZVI particles. Comparison against the sterilized control column revealed that, under the experimental conditions, no biological denitrification developed and that the removal of NO3- was due to chemical reduction by nZVI. The main by-product of the NO3- removal was NH4+, which at the prevailing pH was partially converted to NH3, which dissipated from the aqueous solution resulting in a net removal of total dissolved N. A mass balance of Fe permitted to quantify the percentage of injected nZVI trapped in the column (>98%) and the NO3- retention capacity of the nZVI particles (13.2-85.5 mg NO3-/g nZVI).

Elimination of SARS-CoV-2 along wastewater and sludge treatment processes.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is shed in the feces of infected people. As a consequence, genomic RNA of the virus can be detected in wastewater. Although the presence of viral RNA does not inform on the infectivity of the virus, this presence of genetic material raised the question of the effectiveness of treatment processes in reducing the virus in wastewater and sludge. In this work, treatment lines of 16 wastewater treatment plants were monitored to evaluate the removal of SARS-CoV-2 RNA in raw, processed waters and sludge, from March to May 2020. Viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in 5 different laboratories. These laboratories participated in proficiency testing scheme and their results demonstrated the reliability and comparability of the results obtained for each one. SARS-CoV-2 RNA was found in 50.5% of the 101 influent wastewater samples characterized. Positive results were detected more frequently in those regions with a COVID-19 incidence higher than 100 cases per 100,000 inhabitants. Wastewater treatment plants (WWTPs) significantly reduced the occurrence of virus RNA along the water treatment lines. Secondary treatment effluents showed an occurrence of SARS-CoV-2 RNA in 23.3% of the samples and no positive results were found after MBR and chlorination. Non-treated sludge (from primary and secondary treatments) presented a higher occurrence of SARS-CoV-2 RNA than the corresponding water samples, demonstrating the affinity of virus particles for solids. Furthermore, SARS-CoV-2 RNA was detected in treated sludge after thickening and anaerobic digestion, whereas viral RNA was completely eliminated from sludge only when thermal hydrolysis was applied. Finally, co-analysis of SARS-CoV-2 and F-specific RNA bacteriophages was done in the same water and sludge samples in order to investigate the potential use of these bacteriophages as indicators of SARS-CoV-2 fate and reduction along the wastewater treatment.

Understanding the effects of bulk mixing on the determination of the affinity index: consequences for process operation and design.

The main objective of this study is to demonstrate the importance of mixing conditions as a source of inconsistencies between half-saturation indices in comparable systems (e.g. conventional activated sludge, membrane bioreactor) when operated at different conditions or different scales. As proof-of-principle, an exemplary system consisting of the second vessel of a hybrid respirometer has been studied. The system has been modeled both using an integrated computational fluid dynamics (CFD)-biokinetic model (assumed to represent the physical system) and a tanks-in-series, completely stirred tank reactor biokinetic model (representing the applied model). The results show that different mixing conditions cause deviations in the half-saturation indices calculated when matching the applied model to the physical system performance. Additionally, sensor location has been shown to impact the calculation of half-saturation indices in the respirometric system. This will only become more pronounced at larger scales. Thus, mixing conditions clearly affect operation and design of wastewater treatment reactors operated at low substrate concentrations. Both operation and design can be improved with the development and application of integrated CFD-biokinetic or compartmental models.

How well-mixed is well mixed? Hydrodynamic-biokinetic model integration in an aerated tank of a full-scale water resource recovery facility.

Current water resource recovery facility (WRRF) models only consider local concentration variations caused by inadequate mixing to a very limited extent, which often leads to a need for (rigorous) calibration. The main objective of this study is to visualize local impacts of mixing by developing an integrated hydrodynamic-biokinetic model for an aeration compartment of a full-scale WRRF. Such a model is able to predict local variations in concentrations and thus allows judging their importance at a process level. In order to achieve this, full-scale hydrodynamics have been simulated using computational fluid dynamics (CFD) through a detailed description of the gas and liquid phases and validated experimentally. In a second step, full ASM1 biokinetic model was integrated with the CFD model to account for the impact of mixing at the process level. The integrated model was subsequently used to evaluate effects of changing influent and aeration flows on process performance. Regions of poor mixing resulting in non-uniform substrate distributions were observed even in areas commonly assumed to be well-mixed. The concept of concentration distribution plots was introduced to quantify and clearly present spatial variations in local process concentrations. Moreover, the results of the CFD-biokinetic model were concisely compared with a conventional tanks-in-series (TIS) approach. It was found that TIS model needs calibration and a single parameter set does not suffice to describe the system under both dry and wet weather conditions. Finally, it was concluded that local mixing conditions have significant consequences in terms of optimal sensor location, control system design and process evaluation.

Integrated processes for produced water polishing: Enhanced flotation/sedimentation combined with advanced oxidation processes.

In this study, bench scale dissolved air flotation (DAF) and settling processes have been studied and compared to a novel flotation technology based on the use of glass microspheres of limited buoyancy and its combination with conventional DAF, (Enhanced DAF or E-DAF). They were evaluated as pretreatments for advanced oxidation processes (AOPs) to polish produced water (PW) for reuse purposes. Settling and E-DAF without air injection showed adequate turbidity and oil and grease (O&G) removals, with eliminations higher than 87% and 90% respectively, employing 70 mg L-1 of FeCl3 and 83 min of settling time, and 57.9 mg L-1 of FeCl3, 300 mg L-1 of microspheres and a flocculation rate of 40 rpm in the E-DAF process. A linear correlation was observed between final O&G concentration and turbidity after E-DAF. In order to polish the O&G content of the effluent even further, to remove soluble compounds as phenol and to take advantage of residual iron after these treatments, Fenton and photo-Fenton reactions were essayed. After 6 h of the Fenton reaction at pH 3, the addition of 1660 mg L-1 of H2O2 and 133 mg L-1 of iron showed a maximum O&G elimination of 57.6% and a phenol removal up to 80%. Photo-Fenton process showed better results after 3 h, adding 600 mg L-1 of H2O2 and 300 mg L-1 of iron, at pH 3, with a higher fraction of elimination of the O&G content (73.7%) and phenol (95%) compared to the conventional Fenton process.

From the affinity constant to the half-saturation index: understanding conventional modeling concepts in novel wastewater treatment processes.

The "affinity constant" (KS) concept is applied in wastewater treatment models to incorporate the effect of substrate limitation on process performance. As an increasing number of wastewater treatment processes rely on low substrate concentrations, a proper understanding of these so-called constants is critical in order to soundly model and evaluate emerging treatment systems. In this paper, an in-depth analysis of the KS concept has been carried out, focusing on the different physical and biological phenomena that affect its observed value. By structuring the factors influencing half-saturation indices (newly proposed nomenclature) into advectional, diffusional and biological, light has been shed onto some of the apparent inconsistencies present in the literature. Particularly, the importance of non-ideal mixing as a source of variability between observed KS values in different systems has been illustrated. Additionally, discussion on the differences existent between substrates that affect half-saturation indices has been carried out; it has been shown that the observed KS for some substrates will reflect transport or biological limitations more than others. Finally, potential modeling strategies that could alleviate the shortcomings of the KS concept have been provided. These could be of special importance when considering the evaluation and design of emerging wastewater treatment processes.

Implementation of a demand-side approach to reduce aeration requirements of activated sludge systems: directed acclimation of biomass and its effect at the process level.

Conventionally, increased oxygen requirements of biological wastewater treatment systems have been addressed through supply-side approaches that focus on upgrading the oxygen mass transfer capabilities of aeration devices. Such approaches entail high aeration energy penalties for relatively low performance improvements, as well as causing decreased oxygen mass transfer efficiencies. In this study, a nitrifying community acclimated to low dissolved oxygen (DO) conditions has been demonstrated to yield similar treatment performance as a conventional nitrifying process with 20% less aeration requirements and 20% improvement in mass transfer efficiency. Such a demand-side approach has been shown to be successful due to an improvement of the half saturation constant of the acclimated biomass that has, in turn, been linked to the expression of hemoglobin proteins. Directed acclimation of nitrifying communities could help address the operational requirement to carry out ammonia oxidation at consistently low DO conditions in the mainstream anammox and simultaneous nitrification-denitrification processes.

Characterization of heme protein expressed by ammonia-oxidizing bacteria under low dissolved oxygen conditions.

We have recently reported that expression of an unidentified heme protein is enhanced in a nitrifying activated sludge community under low (0.1 mg O2/L) dissolved oxygen (DO) conditions. A preliminary assessment suggested it may be a type of hemoglobin (Hb) or a lesser-known component of the energy-transducing pathways of ammonia-oxidizing bacteria (AOB) (particularly an oxidase or peroxidase). Here, additional work was done to characterize this protein. Due to the unfeasibility of identifying the protein using gene-based methods, our approach was to carry out assays that target the activity and function of the protein, its location in the cell, and determination of the organisms that express it. Using CO-difference spectra, it was shown that the protein is expressed by AOB preferentially in the cytoplasm, while the pyridine hemochromogen method demonstrated that it has heme c as its prosthetic group. Peroxidase and oxidase assays were carried out on the soluble fraction of the low DO-grown cells; neither the peroxidase nor oxidase activities matched those of the CO-binding heme protein detected. Even though it is not possible to conclusively identify the protein detected as a Hb, all other known possibilities have been ruled out. Further work is needed to verify the identity of the heme protein as a Hb and to determine its type and biochemical role under low oxygen conditions.

Enhanced heme protein expression by ammonia-oxidizing communities acclimated to low dissolved oxygen conditions.

This study has investigated the acclimation of ammonia-oxidizing communities (AOC) to low dissolved oxygen (DO) concentrations. Under controlled laboratory conditions, two sequencing batch reactors seeded with activated sludge from the same source were operated at high DO (near saturation) and low DO (0.1 mg O2/L) concentrations for a period of 220 days. The results demonstrated stable and complete nitrification at low DO conditions after an acclimation period of approximately 140 days. Acclimation brought about increased specific oxygen uptake rates and enhanced expression of a particular heme protein in the soluble fraction of the cells in the low DO reactor as compared to the high DO reactor. The induced protein was determined not to be any of the enzymes or electron carriers present in the conventional account of ammonia oxidation in ammonia-oxidizing bacteria (AOB). Further research is required to determine the specific nature of the heme protein detected; a preliminary assessment suggests either a type of hemoglobin protein or a lesser-known component of the energy-transducing pathways of AOB. The effect of DO on AOC dynamics was evaluated using the 16S rRNA gene as the basis for phylogenetic comparisons and organism quantification. Ammonium consumption by ammonia-oxidizing archaea and anaerobic ammonia-oxidizing bacteria was ruled out by fluorescent in situ hybridization in both reactors. Even though Nitrosomonas europaea was the dominant AOB lineage in both high and low DO sequencing batch reactors at the end of operation, this enrichment could not be linked in the low DO reactor to acclimation to oxygen-limited conditions.

Measurement of organic nitrogen and phosphorus fractions at very low concentrations in wastewater effluents.

The purpose of this study was to develop simple, accurate, and inexpensive measurement protocols for dissolved organic nitrogen (DON) and dissolved non-reactive phosphorus (DNRP) at low levels in wastewater effluents. Two protocols are presented--one to measure DON exclusively, and the other to measure DON and DNRP simultaneously. Currently, DON and DNRP are calculated indirectly by subtracting the dissolved inorganic fractions from the total dissolved concentration, resulting in significant errors. To increase the accuracy of DON measurements, effluent sample pretreatment using ion exchange to remove nitrate was applied. Spectrometric methods were selected to measure the inorganic fractions-the second derivative UV spectroscopy method for nitrate, and the malachite green method for orthophosphate. These methods, combined with the optimized persulfate digestion of the samples, can be used to measure total dissolved nitrogen and phosphorus accurately. The measurement ranges attained were 0.05 to 3 mg N/L for DON and 0.01 to 0.5 mg P/L for DNRP.

Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose.

Escherichia coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol, was further engineered, using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The three resulting strains expressed Vitreoscilla hemoglobin (VHb) at various levels, and the production of ethanol was inversely proportional to the VHb level. High levels of VHb were correlated with an inhibition of ethanol production; however, the strain (TS3) with the lowest VHb expression (approximately the normal induced level in Vitreoscilla) produced, under microaerobic conditions in shake flasks, more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the predominant carbon source. Ethanol production was dependent on growth conditions, but increases were as high as 30%, 119%, and 59% for glucose, xylose, and corn stover hydrolysate, respectively. Only in the case of glucose, however, was the theoretical yield of ethanol by TS3 greater than that achieved by others with FBR5 grown under more closely controlled conditions. TS3 had no advantage over FBR5 regarding ethanol production from arabinose. In 2 L fermentors, TS3 produced about 10% and 15% more ethanol than FBR5 for growth on glucose and xylose, respectively. The results suggest that engineering of microorganisms with vgb/VHb could be of significant use in enhancing biological production of ethanol.

Effluent dissolved organic nitrogen and dissolved phosphorus removal by enhanced coagulation and microfiltration.

Plants aiming to achieve very low effluent nutrient levels (<3 mg N/L for N, and <0.1 mg P/L for P) need to consider removal of effluent fractions hitherto not taken into account. Two of these fractions are dissolved organic nitrogen (DON) and dissolved non-reactive phosphorus (DNRP) (mainly composed of organic phosphorus). In this research, enhanced coagulation using alum (at doses commonly employed in tertiary phosphorus removal) followed by microfiltration (using 0.22 µm pore size filters) was investigated for simultaneous effluent DON and dissolved phosphorus (DP) fractions removal. At an approximate dose of 3.2 mg Al(III)/L, corresponding to 1.5 Al(III)/initial DON-N and 3.8 Al(III)/initial DP-P molar ratios, maximum simultaneous removal of DON and DP was achieved (69% for DON and 72% for DP). At this dose, residual DON and DP concentrations were found to be 0.3 mg N/L and 0.25 mg P/L, respectively. Analysis of the trends of removal revealed that the DNRP removal pattern was similar to that commonly reported for dissolved reactive phosphorus. Since this study involved intensive analytical work, a secondary objective was to develop a simple and accurate measurement protocol for determining dissolved N and P species at very low levels in wastewater effluents. The protocol developed in this study, involving simultaneous digestion for DON and DNRP species, was found to be very reliable and accurate based on the results.