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To inform the development of global wastewater monitoring systems, we surveyed programmes in 43 countries. Most programmes monitored predominantly urban populations. In high-income countries (HICs), composite sampling at centralised treatment plants was most common, whereas grab sampling from surface waters, open drains, and pit latrines was more typical in low-income and middle-income countries (LMICs). Almost all programmes analysed samples in-country, with an average processing time of 2·3 days in HICs and 4·5 days in LMICs. Whereas 59% of HICs regularly monitored wastewater for SARS-CoV-2 variants, only 13% of LMICs did so. Most programmes share their wastewater data internally, with partnering organisations, but not publicly. Our findings show the richness of the existing wastewater monitoring ecosystem. With additional leadership, funding, and implementation frameworks, thousands of individual wastewater initiatives can coalesce into an integrated, sustainable network for disease surveillance?one that minimises the risk of overlooking future global health threats.

In the case of SARS-CoV-2 pandemic management, wastewater-based epidemiology aims to derive information on the infection dynamics by monitoring virus concentrations in the wastewater. However, due to the intrinsic random fluctuations of the viral signal in the wastewater (due to e.g., dilution; transport and fate processes in sewer system; variation in the number of persons discharging; variations in virus excretion and water consumption per day) the subsequent prevalence analysis may result in misleading conclusions. It is thus helpful to apply data filtering techniques to reduce the noise in the signal. In this paper we investigate 13 smoothing algorithms applied to the virus signals monitored in four wastewater treatment plants in Austria. The parameters of the algorithms have been defined by an optimization procedure aiming for performance metrics. The results are further investigated by means of a cluster analysis. While all algorithms are in principle applicable, SPLINE, Generalized Additive Model and Friedman Super Smoother are recognized as superior methods in this context (with the latter two having a tendency to over-smoothing). A first analysis of the resulting datasets indicates the influence of catchment size for wastewater-based epidemiology as smaller communities both reveal a signal threshold before any relation with infection dynamics is visible and also a higher sensitivity towards infection clusters., Comment: 22 pages 6 Figures

We isolated a filamentous, thermophilic, and first anaerobic representative of the genus Thermoactinomyces, designated strain AMNI-1T, from a biogas plant in Tyrol, Austria and report the results of a phenotypic, genetic, and phylogenetic investigation. Strain AMNI-1T was observed to form a white branching mycelium that aggregates into pellets when grown in liquid medium. Cells could primarily utilize lactose, glucose, and mannose as carbon and energy sources, with acetate accelerating and yeast extract being mandatory for growth. The optimum growth temperature and pH turned out to be 55 °C and pH 7.0, respectively, with an optimum NaCl concentration of 0–2% (w/v). 16S rRNA gene sequence comparison indicated that the genetic relatedness between strain AMNI-1T and Thermoactinomyces intermedius, Thermoactinomyces khenchelensis, and Thermoactinomyces vulgaris was less than 97%. The G + C content of the genomic DNA was 44.7 mol%. The data obtained suggest that the isolate represents a novel and first anaerobic species of the genus Thermoactinomyces, for which the name Thermoactinomyces mirandus is proposed. The type strain is AMNI-1T (= DSM 110094T = LMG 31503T). The description of the genus Thermoactinomyces is emended accordingly.

Wastewater based epidemiology is recognized as one of the monitoring pillars, providing essential information for pandemic management. Central in the methodology are data modelling concepts for both communicating the monitoring results but also for analysis of the signal. It is due to the fast development of the field that a range of modelling concepts are used but without a coherent framework. This paper provides for such a framework, focusing on robust and simple concepts readily applicable, rather than applying latest findings from e.g., machine learning. It is demonstrated that data preprocessing, most important normalization by means of biomarkers and equal temporal spacing of the scattered data, is crucial. In terms of the latter, downsampling to a weekly spaced series is sufficient. Also, data smoothing turned out to be essential, not only for communication of the signal dynamics but likewise for regressions, nowcasting and forecasting. Correlation of the signal with epidemic indicators require multivariate regression as the signal alone cannot explain the dynamics but simple linear regression proofed to be a suitable tool for compensation. It was also demonstrated that short term prediction (7 days) is accurate with simple models (exponential smoothing or autoregressive models) but forecast accuracy deteriorates fast for longer periods., Comment: 27 pages 7 figures

SARS-CoV-2 surveillance is crucial to identify variants with altered epidemiological properties. Wastewater-based epidemiology (WBE) provides an unbiased and complementary approach to sequencing individual cases. Yet, national WBE surveillance programs have not been widely implemented and data analyses remain challenging.We deep-sequenced 2,093 wastewater samples representing 95 municipal catchments, covering >57% of Austria’s population, from December 2020 to September 2021. Our Variant Quantification in Sewage pipeline designed for Robustness (VaQuERo) enabled us to deduce variant abundance from complex wastewater samples and delineate the spatiotemporal dynamics of the dominant Alpha and Delta variants as well as regional clusters of other variants of concern. These results were cross validated by epidemiological records of >130,000 individual cases. Finally, we provide a framework to predict emerging variants de novo and infer variant-specific reproduction numbers from wastewater.This study demonstrates the power of national-scale WBE to support public health and promises particular value for countries without dense individual monitoring.Graphical Abstract

The cofactor F420 plays a central role as a hydride carrier in the primary and secondary metabolism of many bacterial and archaeal taxa. The cofactor is best known for its role in methanogenesis, where it facilitates thermodynamically difficult reactions. As the polyglutamate tail varies in length between different organisms, length profile analyses might be a powerful tool for distinguishing and characterizing different groups and pathways in various habitats. Here, the protocol describes the extraction and optimization of cofactor F420 detection by applying solid-phase extraction combined with high-performance liquid chromatography analysis independent of cultural or molecular biological approaches. The method was applied to gain additional information on the expression of cofactor F420 from microbial communities in soils, anaerobic sludge, and pure cultures and was evaluated by spiking experiments. Thereby, the study succeeded in generating different F420 tail-length profiles for hydrogenotrophic and acetoclastic methanogens in controlled methanogenic pure cultures as well as from environmental samples such as anaerobic digester sludge and soils.

Wastewater-based epidemiology is a recognised source of information for pandemic management. In this study, we investigated the correlation between a SARS-CoV-2 signal derived from wastewater sampling and COVID-19 incidence values monitored by means of individual testing programs. The dataset used in the study is composed of timelines (duration approx. five months) of both signals at four wastewater treatment plants across Austria, two of which drain large communities and the other two drain smaller communities. Eight regression models were investigated to predict the viral incidence under varying data inputs and pre-processing methods. It was found that population-based normalisation and smoothing as a pre-processing of the viral load data significantly influence the fitness of the regression models. Moreover, the time latency lag between the wastewater data and the incidence derived from the testing program was found to vary between 2 and 7 days depending on the time period and site. It was found to be necessary to take such a time lag into account by means of multivariate modelling to boost the performance of the regression. Comparing the models, no outstanding one could be identified as all investigated models are revealing a sufficient correlation for the task. The pre-processing of data and a multivariate model formulation is more important than the model structure.

The cofactor F

Anaerobic digestion is a common procedure of treating sewage sludge at wastewater treatment plants. However, plants differ in terms of the number of reactors and, in case of several reactors, their operation mode. To confirm the flexibility of well adapted, full-scale anaerobic digestion plants, we monitored the physicochemical process conditions of two continuously stirred tank reactors over one hydraulic retention time before and after the operation mode was switched from parallel to serial operation. To investigate changes in the involved microbiota, we applied Illumina amplicon sequencing. The rapid change between operation modes did not affect the process performance. In both parallel and serial operation mode, we detected a highly diverse microbial community, in which Bacteroidetes, Firmicutes, Proteobacteria and Claocimonetes were high in relative abundance. While a prominent core microbiome was maintained in both configurations, changes in the involved microbiota were evident at a lower taxonomical level comparing both reactors and operation modes. The most prominent methanogenic Euryarchaeota detected were Methanosaeta and cand. Methanofastidiosum. Volatile fatty acids were degraded immediately in both reactors, suggesting that the second reactor could be used to produce methane on demand, by inserting easily degradable substrates.

The cofactor F420 is synthesized by many different organisms and as a redox cofactor, it plays a crucial role in the redox reactions of catabolic and biosynthetic metabolic pathways. It consists of a deazaflavin structure, which is linked via lactate to an oligoglutamate chain, that can vary in length. In the present study, the methanogenic Archaea Methanosarcina thermophila and Methanoculleus thermophilus were cultivated on different carbon sources and their coenzyme F420 composition has been assayed by reversed-phase ion-pair high-performance liquid chromatography with fluorometric detection regarding both, overall cofactor F420 production and distribution of F420 glutamyl tail length. In Methanosarcina thermophila cultivated on methanol, acetate, and a mixture of acetate and methanol, the most abundant cofactors were F420-5 and F420-4, whereby the last digit refers to the number of expressed glutamyl rests. By contrast, in the obligate CO2 reducing Methanoculleus thermophilus the most abundant cofactors were F420-3 and F420-4. In Methanosarcina thermophila, the relative proportions of the expressed F420 tail length changed during batch growth on all three carbon sources. Over time F420-3 and F420-4 decreased while F420-5 and F420-6 increased in their relative proportion in comparison to total F420 content. In contrast, in Methanoculleus thermophilus the relative abundance of the different F420 cofactors remained stable. It was also possible to differentiate the two methanogenic Archaea based on the glutamyl tail length of the cofactor F420. The cofactor F420-5 in concentrations >2% could only be assigned to Methanosarcina thermophila. In all four variants a trend for a positive correlation between the DNA concentration and the total concentration of the cofactor could be shown. Except for the variant Methanosarcina thermophila with acetate as sole carbon source the same could be shown between the concentration of the mcrA gene copy number and the total concentration of the cofactor.

Die moderne Abwasserbehandlung weist ein groses Potenzial zur Bereitstellung erneuerbarer Energien auf. Auf Klaranlagen kann die chemische Energie im Abwasser genutzt werden, indem in den Faulturmen Biomethan produziert wird. Diese Biomethanproduktion kann durch die gezielte Zugabe organischer Abfalle in die Faulturme gesteigert werden (= Co-Vergarung). Abfalle, die in den Faulturm als Co-Substrate zugegeben werden, mussen pumpfahig und in den Faulschlamm einmischbar sein. Typische Abfallstrome, die dafur in Frage kommen sind z. B. Speisereste aus Groskuchen oder Molkereiabfalle. Derartige Abfalle konnen meist nach geringer mechanischer Aufbereitung dem Faulturm beigemischt werden. Diese Abfalle sind begehrte Reststoffe der Klaranlage, da sie im Allgemeinen weitgehend frei von Storstoffen wie Glas, Steinen oder Kunststoffen sind. Die begrenzte Verfugbarkeit von Speiseresten oder Molkereiabfallen fuhrt zunehmend dazu, dass auch Bioabfalle aus der Getrenntsammlung oder uberlagerte Lebensmittel mechanisch aufbereitet und in den Klaranlagen mitverarbeitet werden. Bei der Nutzung derartiger Substrate ist vermehrt mit Folgeproblemen auf der Klaranlage zu rechnen, da sie starker mit Storstoffen belastet sind. Durch Abrasion an Aggregaten, verlegte Leitungen oder Sedimenten im Faulturm ergeben sich Mehrkosten fur die Klaranlage. Bei der Betrachtung der Mehrkosten durch die Co-Vergarung von mechanisch aufbereitetem Bioabfall mussen neben den Aufwendungen, die auf Storstoffe zuruckzufuhren sind, auch Kosten fur den Schlammanfall aus den Co-Substraten und der Ruckbelastung, insbesondere von Stickstoff, in die Abwasserreinigung der Klaranlage berucksichtigt werden. In dieser Arbeit wird eine okonomische Betrachtung aufgestellt, die die relevanten Kosten der Co-Vergarung erfasst und den Erlosen aus der Verwertung des zusatzlich produzierten Biomethans gegenuberstellt.

pH is a central environmental factor influencing CH4 production from organic substrates, as every member of the complex microbial community has specific pH requirements. Here, we show how varying pH conditions (5.0&ndash, 8.5, phosphate buffered) and the application of a phosphate buffer per se induce shifts in the microbial community composition and the carbon flow during nine weeks of thermophilic batch digestion. Beside monitoring the methane production as well as volatile fatty acid concentrations, amplicon sequencing of the 16S rRNA gene was conducted. The presence of 100 mM phosphate resulted in reduced CH4 production during the initial phase of the incubation, which was characterized by a shift in the dominant methanogenic genera from a mixed Methanosarcina and Methanoculleus to a pure Methanoculleus system. In buffered samples, acetate strongly accumulated in the beginning of the batch digestion and subsequently served as a substrate for methanogens. Methanogenesis was permanently inhibited at pH values &le, 5.5, with the maximum CH4 production occurring at pH 7.5. Adaptations of the microbial community to the pH variations included shifts in the archaeal and bacterial composition, as less competitive organisms with a broad pH range were able to occupy metabolic niches at unfavorable pH conditions.

Aromatic compounds like phenyl acids derived from lignocellulose degradation have been suspected to negatively influence biogas production processes. However, results on this topic are still inconclusive. To study phenyl acid formation in batch reactors during the start-up phase of anaerobic degradation, different amounts of straw from grain were mixed with mesophilic and thermophilic sludge, respectively. Molecular biological parameters were assessed using next-generation sequencing and qPCR analyses. Metagenomic predictions were done via the program, piphillin. Methane production, concentrations of phenylacetate, phenylpropionate, phenylbutyrate, and volatile fatty acids were monitored chromatographically. Methanosarcina spp. was the dominant methanogen when high straw loads were effectively degraded, and thus confirmed its robustness towards overload conditions. Several microorganisms correlated negatively with phenyl acids; however, a negative effect, specifically on methanogens, could not be proven. A cascade-like increase/decrease from phenylacetate to phenylpropionate, and then to phenylbutyrate could be observed when methanogenesis was highly active. Due to these results, phenylacetate was shown to be an early sign for overload conditions, whereas an increase in phenylbutyrate possibly indicated a switch from degradation of easily available to more complex substrates. These dynamics during the start-up phase might be relevant for biogas plant operators using complex organic wastes for energy exploitation.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

Biogas from anaerobic digestion (AD) may contribute towards a green energy scene in the future. Despite many benefits, AD plant operators are still challenged by limitations of the technology. This study aimed at evaluating the effect of biochar addition to AD reactors operated at 45°C to optimize productivity, efficiency and stability of the process. Biochar is characterized by a large surface area and may therefore facilitate the creation of biofilms on the one hand and remove environmental contaminants on the other hand. Moreover, biochar may serve as pH control agent due to its alkaline properties, counteracting reactor acidification. Our results showed that the addition of biochar reduced efficiently the amount of H2S that initially occurred in the headspace during reactor start-up. Nevertheless, no impact on biogas- and methane yields was observed. This might be explained with an already well-working AD system without any severe disturbances, where the balancing nature of biochar did hardly count. Future studies, however, need to evaluate the effect on stressed systems. The microbiome analysis provided a detailed view of the microbial dynamics during the start-up phase, revealing Methanosarcina as dominant methanogen in the end. The addition of biochar did not alter the microbial community composition.

Six methodologically different approaches were evaluated and compared regarding their suitability to quantify and characterise granular anammox biomass. The investigated techniques were gravimetric analysis (GA), activity measurements (AM), Coulter counter analysis (CC), quantitative PCR (qPCR), heme protein quantification (HQ) and the novel image analysis technique Particle Tracking (PT). The focus was set on the development of fast, economic and user-friendly approaches for potential implementation in regular wastewater treatment plant (WWTP) monitoring. To test the effectiveness of each technique, two sample matrices were chosen at the WWTP Strass (Austria): i) sludge liquor of the DEMON ® tank, treating ammonium-rich reject water of anaerobic digestion via the deammonification process and rich in anammox biomass (SL), and ii) the mainstream biological stage, that has been enriched with anammox biomass for more than two years (B). In both of these plants hydro-cyclones are installed for density-fractioning of the sludge into a low- and a high-density fraction, thus leading to a characteristic anammox distribution in the investigated sample set. All investigated methods could statistically discriminate the SL samples. Heme quantification and qPCR were also able to correctly classify the B-samples and both methods showed a Pearson's correlation coefficient of 0.81. An asset of the PT and CC method is the additional qualitative characterization of granule size distribution that can help to better understand and optimise general process operation (cyclone operation duration and construction characteristics). In combination these two methods were able to elucidate the relationship of gross granule volume and actual biomass, excluding the dead volume of inner cavities and exopolymers. We found a linear sphere-equivalent-radius correction factor (3.96 ± 0.15) for investigated anammox granules, that can be used for the fast and reliable PT technique to avoid biomass overestimation. We also recommend routine HQ and PT analysis as ideal monitoring strategy for anammox abundance in wastewater facilities with the HQ technique entailing the further advantage of being also suited for non-granular anammox biomass.

Aim The aim of this study was to develop a vaginal self-emulsifying delivery system for curcumin being capable of spreading, of permeating the mucus gel layer and of protecting the drug being incorporated in oily nanodroplets towards mucus interactions and immobilization. Methods The emulsifying properties of curcumin loaded SEDDS containing 30% Cremophor RH40, 20% Capmul PG-8, 30% Captex 300, 10% DMSO and 10% tetraglycol (SEDD formulation A) as well as 25% PEG 200, 35% Cremophor RH40, 20% Captex 355, 10% Caprylic acid and 10% Tween 80 (SEDD formulation B) after diluting 1 + 2 with artificial vaginal fluid were characterized regarding droplet size and zeta potential. Collagen swelling test was used to examine the irritation potential of SEDDS. Additionally to mucus binding studies, permeation studies in the mucus were performed. Furthermore, spreading potential of the novel developed formulations was compared with a commercial available o/w cream (non-ionic hydrophilic cream) on vaginal mucosa. Results SEDDS displayed a mean droplet size between 38 and 141 nm and a zeta potential of −0.3 to −1.6 mV. The collagen swelling test indicated no significant irritation potential of both formulations over 24 h. An immediate interaction of unformulated curcumin with the mucus was determined, whereas both SEDDS facilitated drug permeation through the mucus layer. Formulation B showed a 2.2-fold improved transport ratio of curcumin compared to SEDD formulation A. In comparison to the vaginal cream, SEDD formulation A and B were able to spread over the vaginal mucosa and cover the tissue to a 17.8- and 14.8-fold higher extent, respectively. Conclusion According to these results, SEDDS seems to be a promising tool for vaginal application.

Volatile fatty acids (VFA) represent short-chain fatty acids consisting of six or fewer carbon atoms that can be distilled at atmospheric pressure. In anaerobic digestion processes VFAs are of central importance for maintaining stable reactor performance and biogas production, are used as indicators for arising problems and are important process monitoring parameters. In the present study, sludge derived form a full-scale anaerobic digester of a wastewater treatment plant was spiked with formate, acetate, propionate, and butyrate in order to evaluate various commonly used techniques for VFA extraction, preservation, and storage. It was shown that VFA extraction after centrifugation warranted the highest recovery rates for spiked VFAs. Moreover, experiments clearly indicated the importance of a fast sample handling, including the necessity of immediate cooling of the samples. Chemical sample preservation within a narrow time frame or deep freezing emerged as an alternative to instant VFA extraction. Short-time storage of extracted VFA samples at + 4°C is an option for up to 7 days, for longer periods storage at –20°C was found to be applicable.

Co-digestion of organic waste and sewage sludge enhances biogas production and reduces the mass of remaining solids. This phenomenon of enhanced organic matter decomposition by adding labile substrate is known from other habitats like soils and sediments where it is called priming effect. It is thus suggested to adopt the term priming effect also in environmental biotechnology, and in particular for biomethanisation of wastewater sludges by the addition of energy-rich co-substrates.

SARS-CoV-2 surveillance by wastewater-based epidemiology is poised to provide a complementary approach to sequencing individual cases. However, robust quantification of variants and de novo detection of emerging variants remains challenging for existing strategies. We deep sequenced 3,413 wastewater samples representing 94 municipal catchments, covering59% of the population of Austria, from December 2020 to February 2022. Our system of variant quantification in sewage pipeline designed for robustness (termed VaQuERo) enabled us to deduce the spatiotemporal abundance of predefined variants from complex wastewater samples. These results were validated against epidemiological records of311,000 individual cases. Furthermore, we describe elevated viral genetic diversity during the Delta variant period, provide a framework to predict emerging variants and measure the reproductive advantage of variants of concern by calculating variant-specific reproduction numbers from wastewater. Together, this study demonstrates the power of national-scale WBE to support public health and promises particular value for countries without extensive individual monitoring.