Prevalence of SARS-CoV-2 variants in Prague wastewater determined by nanopore-based sequencing.

The early detection of upcoming disease outbreaks is essential to avoid both health and economic damage. The last four years of COVID-19 pandemic have proven wastewater-based epidemiology is a reliable system for monitoring the spread of SARS-CoV-2, a causative agent of COVID-19, in an urban population. As this monitoring enables the identification of the prevalence of spreading variants of SARS-CoV-2, it could provide a critical tool in the fight against this viral disease. In this study, we evaluated the presence of variants and subvariants of SARS-CoV-2 in Prague wastewater using nanopore-based sequencing. During August 2021, the data clearly showed that the number of identified SARS-CoV-2 RNA copies increased in the wastewater earlier than in clinical samples indicating the upcoming wave of the Delta variant. New SARS-CoV-2 variants consistently prevailed in wastewater samples around a month after they already prevailed in clinical samples. We also analyzed wastewater samples from smaller sub-sewersheds of Prague and detected significant differences in SARS-CoV-2 lineage progression dynamics among individual localities studied, e.g., suggesting faster prevalence of new variants among the sites with highest population density and mobility.

Monitoring of monkeypox viral DNA in Prague wastewater.

Monkeypox virus (Mpxv) is a dsDNA virus that has become a global concern for human health in 2022. As both infected people and non-human hosts can shed the virus from their skin, faeces, urine and other body fluids, and the resulting sewage contains viral load representative of the whole population, it is highly promising to detect the spread of monkeypox virus in municipal wastewater. We established a methodology for sewage-based monitoring of Mpxv in Prague and analysed samples (n = 24) already early August-October of 2022 in a municipality with 1.4 million inhabitants that only reported 29 cumulative cases in this period. We isolated Mpxv DNA with the Wizard Enviro Total Nucleic Acid Kit, and thereafter detected Mpxv DNA using the EliGene® Monkeypox RT-PCR Kit. Prague wastewater was positive for Mpxv (in total 9 positive samples in periods with 1-9 new cases per week, coinciding with a weekly incidence of 0.07-0.64 per 100,000 inhabitants. The method for confirmation of wastewater positivity via semi-nested PCR and Sanger sequencing was successfully confirmed on positive controls including Mpxv particles and Mpxv-positive wastewater from the Netherlands. However, for Prague wastewater samples, amplification of Mpxv DNA via semi-semi-nested PCR was unsuccessful. This was probably due to extremely low case count, leading to the amplification of non-target bacterial DNA. Compared to other studies with much higher Mpxv prevalence, we show the outstanding sensitivity of our approach for monitoring the spread of monkeypox using wastewater.

Synergistic removal of pharmaceuticals and antibiotic resistance from ultrafiltered WWTP effluent: Free-floating ARGs exceptionally susceptible to degradation.

To protect the environment and human health, antibiotic resistance genes (ARGs) and persistent pharmaceuticals need to be removed from WWTP effluent prior to its reuse. However, an efficient process for removing free-floating extracellular DNA (exDNA) in combination with a wide range of pharmaceuticals is yet to be reported for real process conditions. As a possible solution, we treated real ultrafiltered WWTP effluent with UV/H2O2 and combined GAC and zeolite sorption. In terms of exDNA, sequencing and high-throughput quantitative PCR (HT-qPCR) showed that exDNA is a potent carrier of numerous ARGs in ultrafiltered WWTP effluent (123 ARGs), including multi-drug efflux pump mexF that became the dominant exARG in GAC effluent over time. Due to the exposure to degradation agents, exDNA was reduced more efficiently than intracellular DNA, and overall levels of ARGs were substantially lowered. Moreover, GAC sorption was particularly effective in the removal of almost all the 85 detected pharmaceutical residues, with fresh GAC demonstrating an efficiency of up to 100%. However, zeolite (Si/Al 0.8) addition was needed to enhance the removal of persistent pollutants such as gabapentin and diclofenac to 57% and up to 100%, respectively. Our combined approach eminently decreases the hazardous effects of pharmaceuticals and antibiotic resistance in the ultrafiltered WWTP effluent, producing effluent suitable for multiple reuse options according to the latest legislation. In addition, we provided similarly promising but less extensive data for surface water and treated greywater.

Effect of temperature on the compositions of ladderane lipids in globally surveyed anammox populations.

The adaptation of bacteria involved in anaerobic ammonium oxidation (anammox) to low temperatures will enable more efficient removal of nitrogen from sewage across seasons. At lower temperatures, bacteria typically tune the synthesis of their membrane lipids to promote membrane fluidity. However, such adaptation of anammox bacteria lipids, including unique ladderane phospholipids and especially shorter ladderanes with absent phosphatidyl headgroup, is yet to be described in detail. We investigated the membrane lipids composition (UPLC-HRMS/MS) and dominant anammox populations (16S rRNA gene amplicon sequencing, Fluorescence in situ hybridization) in 14 anammox enrichments cultivated at 10-37 °C. "Candidatus Brocadia" appeared to be the dominant organism in all but two laboratory enrichments of "Ca. Scalindua" and "Ca. Kuenenia". At lower temperatures, the membranes of all anammox populations were composed of shorter [5]-ladderane ester (reduced chain length demonstrated by decreased fraction of C20/(C18 + C20)). This confirmed the previous preliminary evidence on the prominent role of this ladderane fatty acid in low-temperature adaptation. "Ca. Scalindua" and "Ca. Kuenenia" had distinct profile of ladderane lipids compared to "Ca. Brocadia" biomasses with potential implications for adaptability to low temperatures. "Ca. Brocadia" membranes contained a much lower amount of C18 [5]-ladderane esters than reported in the literature for "Ca. Scalindua" at similar temperature and measured here, suggesting that this could be one of the reasons for the dominance of "Ca. Scalindua" in cold marine environments. Furthermore, we propose additional and yet unreported mechanisms for low-temperature adaptation of anammox bacteria, one of which involves ladderanes with absent phosphatidyl headgroup. In sum, we deepen the understanding of cold anammox physiology by providing for the first time a consistent comparison of anammox-based communities across multiple environments.

Adaptation of flocculent anammox culture to low temperature by cold shock: long-term response of the microbial population.

Partial nitritation-anammox (PN/A) process will substantially reduce the costs for the removal of nitrogen in the mainstream of municipal sewage. However, one of the mainstream PN/A challenges is to reduce the time necessary for the adaptation of anammox bacteria to lower temperatures in mild climates. In this study, we exposed anammox flocculent culture to cold shocks [35°C → 5°C (8 h) → 15°C] and evaluated long-term cold shock response. Over a post-shock period of 40 d at 15°C, the nitrogen removal rates in the shocked culture were significantly higher compared to control, with maximum rates up to 0.082 and 0.033 kg-N/kg-VSS/d or 0.164 and 0.076 kg-N/m3/d, for shocked culture and control, respectively. In the corresponding semi-batch cycles, the shocked culture was on average 136 ± 101% more active than the control, due to the negative effect of cold shock on side populations and more active anammox cells. Per FISH, Ca. Brocadia anammoxidans and Ca. Scalindua survived the shock and remained present throughout. Thus, both anammox microorganisms seem to respond favourably to cold shocks. In sum, we provide further evidence that cold shocks accelerate the adaptation of anammox to the mainstream of municipal WWTPs. Further, for the first time, we report the long-term adaptive response of anammox to cold shocks.

High-Rate Partial Nitritation of Municipal Wastewater after Psychrophilic Anaerobic Pretreatment.

Partial nitritation/anammox can provide energy-efficient nitrogen removal from the main stream of municipal wastewater. The main bottleneck is the growth of nitrite oxidizing bacteria (NOB) at low temperatures (<15 °C). To produce effluent suitable for anammox, real municipal wastewater after anaerobic pretreatment was treated by enriched ammonium oxidizing bacteria (AOB) in suspended sludge SBR at 12 °C. NOB were continually washed out using aerobic duration control strategy (ADCS). Solids retention time was set to 9-16 days. Using this approach, average ammonia conversion higher than 57% at high oxidation rate of 0.4 ± 0.1 kg-N kg-VSS-1 d-1 was achieved for more than 100 days. Nitrite accumulation (N-NO2-/N-NOX) of 92% was maintained. Thus, consistently small amounts of present NOB were efficiently suppressed. Our mathematical model explained how ADCS enhanced the inhibition of NOB growth via NH3 and HNO2. This approach will produce effluent suitable for anammox even under winter conditions in mild climates.

Applicability of one-stage partial nitritation and anammox in MBBR for anaerobically pre-treated municipal wastewater.

Energy consumption of municipal wastewater treatment plants can be reduced by the anaerobic pre-treatment of the main wastewater stream. After this pre-treatment, nitrogen can potentially be removed by partial nitritation and anammox (PN/A). Currently, the application of PN/A is limited to nitrogen-rich streams (>500 mg L(-1)) and temperatures 25-35 °C. But, anaerobically pretreated municipal wastewater is characterized by much lower nitrogen concentrations (20-100 mg L(-1)) and lower temperatures (10-25 °C). We operated PN/A under similar conditions: total ammonium nitrogen concentration 50 mg L(-1) and lab temperature (22 °C). PN/A was operated for 342 days in a 4 L moving bed biofilm reactor (MBBR). At 0.4 mg O2 L(-1), nitrogen removal rate 33 g N m(-3) day(-1) and 80 % total nitrogen removal efficiency was achieved. The capacity of the reactor was limited by low AOB activity. We observed significant anammox activity (40 g N m(-3) day(-1)) even at 12 °C, improving the applicability of PN/A for municipal wastewater treatment.

The impact of influent total ammonium nitrogen concentration on nitrite-oxidizing bacteria inhibition in moving bed biofilm reactor.

The application of nitrification-denitrification over nitrite (nitritation-denitritation) with municipal (i.e. diluted and cold (or low-temperature)) wastewater can substantially improve the energy balance of municipal wastewater treatment plants. For the accumulation of nitrite, it is crucial to inhibit nitrite-oxidizing bacteria (NOB) with simultaneous proliferation of ammonium-oxidizing bacteria (AOB). The present study describes the effect of the influent total ammonium nitrogen (TAN) concentration on AOB and NOB activity in two moving bed biofilm reactors operated as sequencing batch reactors (SBR) at 15 °C (SBR I) and 21 °C (SBR II). The reactors were fed with diluted reject water containing 600, 300, 150 and 75 mg TAN L(-1). The only factor limiting NOB activity in these reactors was the high concentrations of free ammonia and/or free nitrous acid (FNA) during the SBR cycles. Nitrite accumulation was observed with influents containing 600, 300 and 150 mg TAN L(-1) in SBR I and 600 and 300 in SBR II. Once nitrate production established in the reactors, the increase of influent TAN concentration up to the original 600 mg TAN L(-1) did not limit NOB activity. This was due to the massive development of NOB clusters throughout the biofilm that were able to cope with faster formation of FNA. The results of the fluorescence in situ hybridization analysis preliminarily showed the stratification of bacteria in the biofilm.