Epidemiology Update of Hepatitis E Virus (HEV) in Uruguay: Subtyping, Environmental Surveillance and Zoonotic Transmission.

Hepatitis E Virus (HEV) infection is an emergent zoonotic disease of increasing concern in developed regions. HEV genotype 3 (HEV-3) is mainly transmitted through consumption of contaminated food in high-income countries and is classified into at least 13 subtypes (3a-3n), based on p-distance values from complete genomes. In Latin America, HEV epidemiology studies are very scant. Our group has previously detected HEV3 in clinical cases, swine, wild boars, captive white-collared peccaries, and spotted deer from Uruguay. Herein, we aimed to provide novel insights and an updated overview of the molecular epidemiology of zoonotic HEV in Uruguay, including data from wastewater-based surveillance studies. A thorough analysis of HEV whole genomes and partial ORF2 sequences from Uruguayan human and domestic pig strains showed that they formed a separate monophyletic cluster with high nucleotide identity and exhibited p-distance values over the established cut-off (0.093) compared with reference subtypes' sequences. Furthermore, we found an overall prevalence of 10.87% (10/92) in wastewater, where two samples revealed a close relationship with humans, and animal reservoirs/hosts isolates from Uruguay. In conclusion, a single, new HEV-3 subtype currently circulates in different epidemiological settings in Uruguay, and we propose its designation as 3o along with its reference sequence.

Optimization of anaerobic digestion and solubilization of biosludges from the kraft cellulose industry using thermal hydrolysis as pretreatment.

The management of secondary sludge from aerobic treatment of effluents from the cellulose industry is a current problem. The usual ways of disposal do not provide added value to the waste as they assume an economy based on "take-make-waste" (linear economy). In this work, thermal hydrolysis (TH) and anaerobic digestion (AD) are proposed to valorize this biosludge. Based on a Doehlert experimental plan, a response surface methodology (RSM) defined by seven different TH conditions is proposed. After TH, biomethanation potential (BMP) tests were performed to evaluate the AD possibilities. The TH conditions cover a temperature range between 125 °C and 205 °C and a reaction time from 15 min to 45 min. The TH process was successful in enhancing the bioavailability of the waste, increasing the concentration of soluble organic matter quantified by chemical oxygen demand of the soluble fraction (CODs), and decreasing the concentration of volatile suspended solids (VSS). However, response surfaces performed for CODs and VSS revealed the existence of optimums, which demonstrated the adverse effects of the more severe TH conditions. Organic matter solubilization was confirmed by microscopic observations. The amount of suspended organic matter after TH is reduced by two to three times compared to the untreated value. The subsequent BMP of the hydrolyzed waste increases between 100% and 220% compared to the untreated condition, wich had a BMP value of 84 NmL CH4 gVS-1. The response surface determined for the BMP reveals the presence of a maximum point of methane production at 202 °C for 31 min, which differs from the maximum CODs value observed at 196 °C for 40 min.

Wastewater surveillance of SARS-CoV-2 genomic populations on a country-wide scale through targeted sequencing.

SARS-CoV-2 surveillance of viral populations in wastewater samples is recognized as a useful tool for monitoring epidemic waves and boosting health preparedness. Next generation sequencing of viral RNA isolated from wastewater is a convenient and cost-effective strategy to understand the molecular epidemiology of SARS-CoV-2 and provide insights on the population dynamics of viral variants at the community level. However, in low- and middle-income countries, isolated groups have performed wastewater monitoring and data has not been extensively shared in the scientific community. Here we report the results of monitoring the co-circulation and abundance of variants of concern (VOCs) of SARS-CoV-2 in Uruguay, a small country in Latin America, between November 2020-July 2021 using wastewater surveillance. RNA isolated from wastewater was characterized by targeted sequencing of the Receptor Binding Domain region within the spike gene. Two computational approaches were used to track the viral variants. The results of the wastewater analysis showed the transition in the overall predominance of viral variants in wastewater from No-VOCs to successive VOCs, in agreement with clinical surveillance from sequencing of nasal swabs. The mutations K417T, E484K and N501Y, that characterize the Gamma VOC, were detected as early as December 2020, several weeks before the first clinical case was reported. Interestingly, a non-synonymous mutation described in the Delta VOC, L452R, was detected at a very low frequency since April 2021 when using a recently described sequence analysis tool (SAM Refiner). Wastewater NGS-based surveillance of SARS-CoV-2 is a reliable and complementary tool for monitoring the introduction and prevalence of VOCs at a community level allowing early public health decisions. This approach allows the tracking of symptomatic and asymptomatic individuals, who are generally under-reported in countries with limited clinical testing capacity. Our results suggests that wastewater-based epidemiology can contribute to improving public health responses in low- and middle-income countries.

Improvements in the anaerobic digestion of biological sludge from pulp and paper mills using thermal pretreatment.

The current disposal of biosludge generated in wastewater treatment has high costs and causes environmental problems, anaerobic digestion (AD) of solid waste is a promising alternative. Thermal hydrolysis (TH) is an accepted technology to enhance anaerobic biodegradability of sewage sludge, but this technology has not been developed to be used with biological sludge from industrial wastewater treatment. In this work, the improvements to the AD of biological sludge from cellulose industry when thermal pretreatment is carried out were experimentally determined. The experimental conditions for TH were 140 °C and 165 °C for 45 minutes. Batch tests were carried out to quantify methane production evaluated as biomethane potential (BMP), anaerobic biodegradability according to volatile solids (VS) consumption and kinetic adjustments. An innovative kinetic model based on the serial mechanism of fast and slow biodegradation fractions was tested for untreated waste, and parallel mechanism was also evaluated. Increases in BMP and biodegradability values according to VS consumption were determined with increasing TH temperature. The results of 241 NmL CH4 gVS substrate-1 for BMP and 65% biodegradability are reported for the 165 °C treatment. AD rate increased for the TH waste compared to the untreated biosludge. Improvements of up to 159% for BMP and 260% for biodegradability according to VS consumption were quantified for TH biosludge compared to untreated biosludge.

Need of adjustment of methanogenic activities from solids digester sludge: Modelling dilution effects on micro-organisms biomass concentration.

Anaerobic digestion is a widespread technology used for organic-based solid waste management. Specific methanogenic activity tests are simple and cost-effective tools for sludge characterization and system diagnosis. However, in solid digesters, substrate and organic inert material dilute micro-organisms, enlarging the activity tests and distorting experimental results. To correct this situation, correction factors, defined as the ratio of micro-organisms concentration to total volatile solids content, are considered. Due to the impossibility to have a quick measurement of the mass of micro-organisms in the digester content, correction factors were evaluated based on the simulation of a simple model. To verify the importance of the correction, hydrogenotrophic and acetoclastic methanogenic activities were studied, involving a stage for sludge acclimatization and subsequent processing in a continuous digester. This situation was compared with a wastewater up-flow anaerobic reactor where no dilution effects are presented. A correcting factor of 0.79 was obtained for the acclimatization period, whereas correcting factors of 0.25-0.30 were estimated for the two periods of the digester. Tendencies shown for raw activities differed from those observed after using correcting factors to adjust activity values for periods 1 and 2 of the digester; also, the gap between the up-flow anaerobic reactor (without solids dilution effects) and the digester activities was reduced from sixfold to double, evidencing the relevance of this correction tool. Additionally, correcting factors also enabled a reasonable calculation of the inoculum size during the design of the activity tests.

A simple kinetic model applied to anaerobic digestion of cow manure.

A simple model of anaerobic degradation in a continuous stirred digester is presented. The hydrolysis of cow manure was modelled as consisting of two fractions, one rapidly degradable and the other more slowly degradable, and both processes were represented by first-order kinetics in a two-substrate first-order (TSFO) model. The fractions were separated by water flushing. Biomethane potential (BMP) tests were performed to determine the hydrolysis constant and biodegradability of each fraction. The hydrolysis constants of the rapidly and slowly degradable fractions were 0.278 and 0.069 d-1, respectively. Coupled with a simple anaerobic digestion model, the TSFO model was used to simulate the digester behaviour and predict methane production. Experiments in a 3.0 L digester were used to determine the decay constant and yield values and to validate the model. Two solid loads (2.9 and 4.4 gVS/L.d) were applied to the digester, and the dynamics of both biodegradable fractions, the non-biodegradable fraction and the microorganism concentration were reproduced by the model. These results approximate the actual biodegradable solids removal to within 85%. A parametric sensitivity study was performed, and the results show that the hydrolysis constant mainly influences the biodegradable fractions and that the decay and yield parameters mainly influence the microorganism concentration.

Hydrogenotrophic activity: A tool to evaluate the kinetics of methanogens.

Anaerobic-digestion-based technology is key to achieving sustainable water management and resource recovery. It is essential to understand the material flux and kinetics involved in methanogenesis to optimize the organic matter removal and methane production. In this sense, specific methanogenic activity is a cost-effective tool to characterize the biological activity of anaerobic biosludge, to monitor the performance of reactors, and study the kinetics of acetate and H2 conversion to methane. Established protocols are applied for the acetoclastic activity test. However, hydrogenotrophic activity assay remains less widespread and is not standardized. In this work, the assay design for hydrogenotrophic activity is discussed and full calculation is presented, based on the kinetics for the H2/CO2 conversion to methane. An equation to calculate the inoculum size is proposed, suitable for a wide variety of types of biosludge: from a wastewater treatment plant to solid digesters, from a high-rate reactor to lagoons. The applied zero-order model fitted adequately to data for pilot-scale and full-scale anaerobic reactors: the p-values from the ANOVA F-test were below 1E-03; standard deviations for triplicate experiments were between 3 and 12%, coherent with the values found in the literature. Microbial growth during the test was negligible, below 1.2% of the biomass dosed in the vial. As a complement, acetoclastic activity was determined for each sample. The use of both acetoclastic and hydrogenotrophic activity is relevant for the study of the methanogenesis and gives a better characterization of the performance of the biosludge in anaerobic reactors rather than only using the specific acetoclastic methanogenic activity.

Microbiota adaptation after an alkaline pH perturbation in a full-scale UASB anaerobic reactor treating dairy wastewater.

The aim of this study was to understand how the microbial community adapted to changes, including a pH perturbation, occurring during the start-up and operation processes in a full-scale methanogenic UASB reactor designed to treat dairy wastewater. The reactor performance, prokaryotic community, and lipid degradation capacity were monitored over a 9-month period. The methanogenic community was studied by mcrA/mrtA gene copy-number quantification and methanogenic activity tests. A diverse prokaryotic community characterized the seeding sludge as assessed by sequencing the V4 region of the 16S rRNA gene. As the feeding began, the bacterial community was dominated by Firmicutes, Synergistetes, and Proteobacteria phyla. After an accidental pH increase that affected the microbial community structure, a sharp increase in the relative abundance of Clostridia and a decrease in the mcrA/mrtA gene copy number and methanogenic activity were observed. After a recovery period, the microbial population regained diversity and methanogenic activity. Alkaline shocks are likely to happen in dairy wastewater treatment because of the caustic soda usage. In this work, the plasticity of the prokaryotic community was key to surviving changes to the external environment and supporting biogas production in the reactor.

Microbial communities from 20 different hydrogen-producing reactors studied by 454 pyrosequencing.

To provide new insight into the dark fermentation process, a multi-lateral study was performed to study the microbiology of 20 different lab-scale bioreactors operated in four different countries (Brazil, Chile, Mexico, and Uruguay). Samples (29) were collected from bioreactors with different configurations, operation conditions, and performances. The microbial communities were analyzed using 16S rRNA genes 454 pyrosequencing. The results showed notably uneven communities with a high predominance of a particular genus. The phylum Firmicutes predominated in most of the samples, but the phyla Thermotogae or Proteobacteria dominated in a few samples. Genera from three physiological groups were detected: high-yield hydrogen producers (Clostridium, Kosmotoga, Enterobacter), fermenters with low-hydrogen yield (mostly from Veillonelaceae), and competitors (Lactobacillus). Inocula, reactor configurations, and substrates influence the microbial communities. This is the first joint effort that evaluates hydrogen-producing reactors and operational conditions from different countries and contributes to understand the dark fermentation process.

Modelling of an EGSB treating sugarcane vinasse using first-order variable kinetics.

An expanded granular sludge bed (EGSB) anaerobic reactor treating sugar cane vinasse was modelled using a simple model with two steps (acidogenesis and methanogenesis), two populations, two substrates and completely mixed conditions. A first-order kinetic equation for both steps with time-variant kinetic coefficients was used. An observer system was used to estimate the evolution of kinetic constants over time. The model was validated by comparing methane flow predictions with experimental values. An estimation of evolution of populations of microorganisms was also performed. This approach allows calculation of specific kinetic constants that reflect biological activity of microorganisms. Variation of specific kinetic constants reflects the influence of the fraction of raw vinasse in the feed. High salt concentrations in the reactor may have inhibited the process.

Modelling of slaughterhouse solid waste anaerobic digestion: determination of parameters and continuous reactor simulation.

A model based on the work of Angelidaki et al. (1993) was applied to simulate the anaerobic biodegradation of ruminal contents. In this study, two fractions of solids with different biodegradation rates were considered. A first-order kinetic was used for the easily biodegradable fraction and a kinetic expression that is function of the extracellular enzyme concentration was used for the slowly biodegradable fraction. Batch experiments were performed to obtain an accumulated methane curve that was then used to obtain the model parameters. For this determination, a methodology derived from the "multiple-shooting" method was successfully used. Monte Carlo simulations allowed a confidence range to be obtained for each parameter. Simulations of a continuous reactor were performed using the optimal set of model parameters. The final steady-states were determined as functions of the operational conditions (solids load and residence time). The simulations showed that methane flow peaked at a flow rate of 0.5-0.8 Nm(3)/d/m(reactor)(3) at a residence time of 10-20 days. Simulations allow the adequate selection of operating conditions of a continuous reactor.

Integrated anaerobic treatment of dairy industrial wastewater and sludge.

Performance parameters were studied in an alternative full-scale dairy effluent treatment system comprising two anaerobic sludge-blanket reactors in parallel arrangement with upward flow, internal fat-separation by flotation, external lamella settler and floated material digester. Reactors were initially inoculated with flocculent sludge and granulated in a high-load stage. Using loading rates up to a maximum 5.5 kg COD/m(3) x d-hydraulic residence time of 17 hours- reactor efficiency was found to remain stable around 90% of COD. Average sludge digester efficiency using a loading rate of 3.5 kgVS/m(3) x d with a lipid content of 47% of COD amounted to 78% of VS (87% of lipid removal). LCFA inhibition as assayed using palmitate was found to depend not only on the palmitate concentration but also on the palmitate-to-biomass concentration ratio.

Assessment on the performance of a series of two UASB reactors compared against one of the same total volume using Anaerobic Digestion Model No 1 (ADM1).

The ADM1 is applied to simulate the behaviour of an Upflow Anaerobic Sludge Blanket Reactor (UASB) treating blood powder (spray dried blood meal) wastewater. Considering a completely mixed liquid phase, the model is defined by a set of differential equations which can be numerically solved. Experimental data allows validating the output of the model which is then used for performing numerous simulations. A single reactor is compared to a series of two identical reactors of half the volume each. For the same total hydraulic retention time, the series reaches an outlet concentration 74% inferior to that of the single reactor. Furthermore, for the same total conversion the volume required by the series is 43% of that of the single reactor.

Relevamiento de reactores anaerobios en América Latina / Anaerobic reactors survey in Latin America

Se realiza un relevamiento de los reactores anaerobios construídos en América Latina. Se procesan los distintos aspectos de la información, a saber, cantidades, volúmenes, tipos de reactores, evolución en el tiempo, tipo de efluente y origen de la tecnología. La participación de la región en el desarrollo de la tecnología de la digestión anaerobia para el tratamiento de efluentes resulta ser importante y en franco desarrollo, presentando además una importante base tecnológica nacional. Se presentan asimismo datos de parámetros de diseño para los principales tipos de efluentes que surgen de los reactores en funcionamiento

Transporte de contaminante en la zona no saturada de un relleno sanitario / Transport of the pollutant in unsaturated zone of a sanitary landfill

Se estudia el transporte de contaminante (materia orgánica) en la zona no saturada de un Relleno Sanitario mediante modelos físicos, datos de un Relleno y un modelo numérico que incluye la generación del mismo a partir de la materia particulada y la degradación biológica en el líquido. La ecuación de transporte se resuelve mediante el uso de una malla espacial móvil y deformable que tiene en cada punto la misma velocidad que la de transporte. Se realiza asimismo un estudio de sensibilidad paramétrica que permite identificar los parámetros con mayor incidencia y seguir profundizando su estudio en las siguientes etapas. Esta primer etapa en el estudio de un Relleno Sanitario será continuada posteriormente por la investigación del flujo en la zona saturada y en el suelo, completando así la consideración del proceso global

Tratamiento de aguas residuales de frigorífico / Treatment of cold-storage plant sewage

El presente trabajo consiste en el estudio de una planta de tratamiento de aguas residuales de frigorífico donde se procesan 650 bovinos por día. Se evaluó un sistema de tratamiento realizado en base a instalaciones existentes, sin introducir modificaciones significativas. Las instalaciones existentes permiten realizar tratamientos primarios (zarandas y flotación) y secundarios (reactor anaerobio y lagunas). En base a la evaluación primaria se concluyó que la presencia de sólidos suspendidos y grasas ocasiona problemas en los tratamientos biológicos. Para mejorar la eficiencia de los tratamientos primarios se realizaron ensayos de flotación con aire a presión obteniendo importantes mejoras