Wastewater-based prediction of COVID-19 cases using a random forest algorithm with strain prevalence data: A case study of five municipalities in Latvia.

Wastewater-based epidemiology (WBE) is a rapid and cost-effective method that can detect SARS-CoV-2 genomic components in wastewater and can provide an early warning for possible COVID-19 outbreaks up to one or two weeks in advance. However, the quantitative relationship between the intensity of the epidemic and the possible progression of the pandemic is still unclear, necessitating further research. This study investigates the use of WBE to rapidly monitor the SARS-CoV-2 virus from five municipal wastewater treatment plants in Latvia and forecast cumulative COVID-19 cases two weeks in advance. For this purpose, a real-time quantitative PCR approach was used to monitor the SARS-CoV-2 nucleocapsid 1 (N1), nucleocapsid 2 (N2), and E genes in municipal wastewater. The RNA signals in the wastewater were compared to the reported COVID-19 cases, and the strain prevalence data of the SARS-CoV-2 virus were identified by targeted sequencing of receptor binding domain (RBD) and furin cleavage site (FCS) regions employing next-generation sequencing technology. The model methodology for a linear model and a random forest was designed and carried out to ascertain the correlation between the cumulative cases, strain prevalence data, and RNA concentration in the wastewater to predict the COVID-19 outbreak and its scale. Additionally, the factors that impact the model prediction accuracy for COVID-19 were investigated and compared between linear and random forest models. The results of cross-validated model metrics showed that the random forest model is more effective in predicting the cumulative COVID-19 cases two weeks in advance when strain prevalence data are included. The results from this research help inform WBE and public health recommendations by providing valuable insights into the impact of environmental exposures on health outcomes.

Photocatalytic Activity of TiO2 Coatings Obtained at Room Temperature on a Polymethyl Methacrylate Substrate.

Titanium dioxide (TiO2) coatings have a wide range of applications. Anatase exhibits hydrophilic, antimicrobial, and photocatalytic properties for the degradation of organic pollutants or water splitting. The main challenge is to obtain durable anatase nanoparticle coatings on plastic substrates by using straightforward approaches. In the present study, we revealed the preparation of a transparent TiO2 coating on polymethylmethacrylate (PMMA), widely used for organic optical fibres as well as other polymer substrates such as polypropylene (PP), polystyrene (PS), and polycarbonate (PC). The films were spin-coated at room temperature without annealing; therefore, our approach can be used for thermo-sensitive substrates. The deposition was successful due to the use of stripped ultra-small (<4 nm) TiO2 particles. Coatings were studied for the photocatalytic degradation of organic pollutants such as MB, methyl orange (MO), and rhodamine B (RB) under UV light. The TiO2 coating on PMMA degraded over 80% of RB in 300 min under a 365 nm, 100 W mercury lamp, showing a degradation rate constant of 6 × 10−3 min−1. The coatings were stable and showed no significant decrease in degradation activity even after five cycles.

Optimizing phosphorus removal for municipal wastewater post-treatment with Chlorella vulgaris.

The microalgal species Chlorella vulgaris was cultivated in batch conditions to identify the optimum set of initial conditions for the best biomass growth rate, phosphate removal, polyphosphate accumulation, and protein productivity. To study the effect of phosphorus deficiency caused stress, the microalgal biomass was exposed to phosphorus deficiency conditions for periods varying between 1 and 10 days and inoculated at different initial biomass and phosphate concentrations. A 10-day period of phosphate deficiency, supported by low initial biomass concentration (∼0.25 mg DW L-1), increased the phosphate removal by 62-175% when compared to the reference conditions. A 10-day period of biomass P-deficiency also boosted the polyphosphate accumulation and protein productivity, increasing them up to 40 and 46.8 times, respectively, if compared to reference conditions. At the same time, optimization algorithm model results suggested one-day biomass P-starvation with low initial biomass concentration as the optimum combination to achieve the highest performance while the initial phosphate concentration had less impact. The initial conditions suggested by the optimization model were validated in a sequencing batch photobioreactor, giving 101.7 and 138.0% more phosphate removal and polyphosphate accumulation, compared to the reference conditions. The obtained results present microalgae exposure to phosphorus stress as a supplementary tool for wastewater post-treatment targeted on rapid phosphorus removal.

UV inactivation of Semliki Forest virus and E. coli bacteria by alternative light sources.

The quick spreading of the SARS-CoV-2 virus, initiating the global pandemic with a significant impact on economics and health, highlighted an urgent need for effective and sustainable restriction mechanisms of pathogenic microorganisms. UV-C radiation, causing inactivation of many viruses and bacteria, is one of the tools for disinfection of different surfaces, liquids, and air; however, mainly mercury 254 nm line is commonly used for it. In this paper, we report our results of the experiments with newly elaborated special type polychromatic non-mercury UV light sources, having spectral lines in the spectral region from 190 nm to 280 nm. Inactivation tests were performed with both Escherichia coli (E.coli) bacteria and Semliki Forest virus (SFV) as a representative of human enveloped RNA viruses. In addition, the effect of prepared lamps on virus samples in liquid and dry form (dried virus-containing solution) was tested. Reduction of 4 log10 of E.coli was obtained after 10 min of irradiation with both thallium-antimony and arsenic high-frequency electrodeless lamps. High reduction results for the arsenic light source demonstrated sensitivity of E. coli to wavelengths below 230 nm, including spectral lines around 200 nm. For the Semliki Forest virus, the thallium-antimony light source showed virus inactivation efficiency with a high virus reduction rate in the range of 3.10 to > 4.99 log10 within 5 min of exposure. Thus, the new thallium-antimony light source showed the most promising disinfection effect in bacteria and viruses, and arsenic light sources for bacteria inactivation, opening doors for many applications in disinfection systems, including for pathogenic human RNA viruses.

Detection of SARS-CoV-2 RNA in wastewater and importance of population size assessment in smaller cities: An exploratory case study from two municipalities in Latvia.

Wastewater-based epidemiology (WBE) has regained global importance during the COVID-19 pandemic. The mobility of people and other factors, such as precipitation and irregular inflow of industrial wastewater, are complicating the estimation of the disease prevalence through WBE, which is crucial for proper crisis management. These estimations are particularly challenging in urban areas with moderate or low numbers of inhabitants in situations where movement restrictions are not adopted (as in the case of Latvia) because residents of smaller municipalities tend to be more mobile and less strict in following the rules and measures of disease containment. Thus, population movement can influence the outcome of WBE measurements significantly and may not reflect the actual epidemiological situation in the respective area. Here, we demonstrate that by combining the data of detected SARS-CoV-2 RNA copy number, 5-hydroxyindoleacetic acid (5-HIAA) analyses in wastewater and mobile call detail records it was possible to provide an accurate assessment of the COVID-19 epidemiological situation in towns that are small (COVID-19 28-day cumulative incidence r = 0.609 and 35-day cumulative incidence r = 0.89, p < 0.05) and medium-sized towns (COVID-19 21-day cumulative incidence r = 0.997, 28-day cumulative incidence r = 0.98 and 35-day cumulative incidence r = 0.997, p < 0.05). This is the first study demonstrating WBE for monitoring COVID-19 outbreaks in Latvia. We demonstrate that the application of population size estimation measurements such as total 5-HIAA and call detail record data improve the accuracy of the WBE approach.

Case study on the dairy processing industries and their wastewater generation in Latvia.

The objective of the research presented in this Research Communication was to access the environmental impact of the Latvian dairy industries. Site visits and interviews at Latvian dairy processing companies were done in order to collect site-specific data. This includes the turnover of the dairy industries, production, quality of water in various industrial processes, the flow and capacity of the sewage including their characteristic, existing practices and measures for wastewater management. The results showed that dairy industries in Latvia generated in total approximately 2263 × 103 m3 wastewater in the year 2019. The Latvian dairy effluents were characterized with high chemical oxygen demand (COD), biological oxygen demand (BOD) and total solids (TS). Few dairy plants had pre-treatment facilities for removal of contaminants, and many lacked onsite treatment technologies. Most facilities discharged dairy wastewater to municipal wastewater treatment plants. The current study gives insight into the Latvian dairy industries, their effluent management and pollution at Gulf of Riga due to wastewater discharge.

Increasing Phosphorus Uptake Efficiency by Phosphorus-Starved Microalgae for Municipal Wastewater Post-Treatment.

Four microalgal species, Chlorella vulgaris, Botryococcus braunii, Ankistrodesmus falcatus, and Tetradesmus obliquus were studied for enhanced phosphorus removal from municipal wastewater after their exposure to phosphorus starvation. Microalgae were exposed to phosphorus starvation conditions for three and five days and then used in a batch experiment to purify an effluent from a small WWTP. After 3-day P-starvation, C. vulgaris biomass growth rate increased by 50% and its PO4 removal rate reached > 99% within 7 days. B. braunii maintained good biomass growth rate and nutrient removal regardless of the P-starvation. All species showed 2-5 times higher alkaline phosphatase activity increase for P-starved biomass than at the reference conditions, responding to the decline of PO4 concentration in wastewater and biomass poly-P content. The overall efficiency of biomass P-starvation on enhanced phosphorus uptake was found to be dependent on the species, N/P molar ratio in the wastewater, as well as the biomass P content.

Characterization of Novel Biopolymer Blend Mycocel from Plant Cellulose and Fungal Fibers.

In this study unique blended biopolymer mycocel from naturally derived biomass was developed. Softwood Kraft (KF) or hemp (HF) cellulose fibers were mixed with fungal fibers (FF) in different ratios and the obtained materials were characterized regarding microstructure, air permeability, mechanical properties, and virus filtration efficiency. The fibers from screened Basidiomycota fungi Ganoderma applanatum (Ga), Fomes fomentarius (Ff), Agaricus bisporus (Ab), and Trametes versicolor (Tv) were applicable for blending with cellulose fibers. Fungi with trimitic hyphal system (Ga, Ff) in combinations with KF formed a microporous membrane with increased air permeability (>8820 mL/min) and limited mechanical strength (tensile index 9-14 Nm/g). HF combination with trimitic fungal hyphae formed a dense fibrillary net with low air permeability (77-115 mL/min) and higher strength 31-36 Nm/g. The hyphal bundles of monomitic fibers of Tv mycelium and Ab stipes made a tight structure with KF with increased strength (26-43 Nm/g) and limited air permeability (14-1630 mL/min). The blends KF FF (Ga) and KF FF (Tv) revealed relatively high virus filtration capacity: the log10 virus titer reduction values (LRV) corresponded to 4.54 LRV and 2.12 LRV, respectively. Mycocel biopolymers are biodegradable and have potential to be used in water microfiltration, food packaging, and virus filtration membranes.

Removal of total phosphorus, ammonia nitrogen and organic carbon from non-sterile municipal wastewater with Trametes versicolor and Aspergillus luchuensis.

Discharge of organic load from treated wastewater may cause environmental eutrophication. Recently, fungi have gained much attention due to their removal of pharmaceutical substances by enzymatic degradation and adsorption. However, the fungal effect in removing nutrients is less investigated. Therefore, two fungal species, the white-rot fungus T. versicolor as a laboratory strain and the mold A. luchuensis as an environmental isolate from the municipal wastewater treatment plant, were studied to determine the fungal potential for phosphorus, nitrogen, and the total organic carbon removal from municipal wastewater, carrying out a batch scale experiment to a fluidized bed pelleted bioreactor. During the batch scale experiment, the total removal (99.9 %) of phosphorus by T. versicolor was attained after a 6 h-long incubation period while the maximal removal efficiency (99.9 %) for phosphorus from A. luchuensis was gained after an incubation period of 24 h. Furthermore, both fungi showed that the pH adjustment to 5.5 kept the concentration of nitrogen constant and stabilized the total organic carbon reduction process for the entire incubation period. The results from the fluidized bed bioreactor demonstrated opposite tendencies on a nutrient removal comparing to a batch experiment where no significant effect on phosphorus, nitrogen, and total organics carbon reduction was observed. The obtained results from this study of batch and fluidized bed bioreactor experiments are a promising starting point for a successful fungal treatment optimization and application to wastewater treatment.

Short-term effect of shock ammonium nitrogen load on activated sludge properties.

The activated sludge process suffers from rapid load changes of ammonium (NH4), which may result in process failure during wastewater treatment. In this study, the response of activated sludge properties in terms of microfauna composition and sludge volume index (SVI5 and SVI30) on short-term increase of NH4 concentration (from 55 mg/l to 105 mg/l) was evaluated in batch scale reactors over 72 h. The results show that the first-step nitrification (NH4 transformation to nitrite (NO2)) was inhibited after 24 h, whereas the second-step nitrification (NO2 transformation to nitrate (NO3)) was not significantly affected. Sludge volume indices (sedimentation ability characteristics) SVI5 and SVI30 in the reactor with NH4-N shock concentration increased, whereas microfauna diversity decreased (Shannon-Weaver index decreased from 2.12 at 48 h to 1.23 at 72 h) leading to dominance of stalked ciliate Epistylis sp. Notable changes in inorganic carbon concentration (IC) were observed, indicating that rapid decrease of IC concentration leads to nitrification inhibition and challenges the overall process recovery. To conclude, short-term exposure of about two times higher concentration of NH4 caused significant changes in activated sludge properties by inhibiting NH4-oxidizing bacteria, reducing sludge microfauna diversity and deteriorating sludge sedimentation ability.

Identifying the underlying causes of biological instability in a full-scale drinking water supply system.

Changes in bacterial concentration and composition in drinking water during distribution are often attributed to biological (in)stability. Here we assessed temporal biological stability in a full-scale distribution network (DN) supplied with different types of source water: treated and chlorinated surface water and chlorinated groundwater produced at three water treatment plants (WTP). Monitoring was performed weekly during 12 months in two locations in the DN. Flow cytometric total and intact cell concentration (ICC) measurements showed considerable seasonal fluctuations, which were different for two locations. ICC varied between 0.1-3.75 × 105 cells mL-1 and 0.69-4.37 × 105 cells mL-1 at two locations respectively, with ICC increases attributed to temperature-dependent bacterial growth during distribution. Chlorinated water from the different WTP was further analysed with a modified growth potential method, identifying primary and secondary growth limiting compounds. It was observed that bacterial growth in the surface water sample after chlorination was primarily inhibited by phosphorus limitation and secondly by organic carbon limitation, while carbon was limiting in the chlorinated groundwater samples. However, the ratio of available nutrients changed during distribution, and together with disinfection residual decay, this resulted in higher bacterial growth potential detected in the DN than at the WTP. In this study, bacterial growth was found to be higher (i) at higher water temperatures, (ii) in samples with lower chlorine residuals and (iii) in samples with less nutrient (carbon, phosphorus, nitrogen, iron) limitation, while this was significantly different between the samples of different origin. Thus drinking water microbiological quality and biological stability could change during different seasons, and the extent of these changes depends on water temperature, the water source and treatment. Furthermore, differences in primary growth limiting nutrients in different water sources could contribute to biological instability in the network, where mixing occurs.

A pipeline for developing and testing staining protocols for flow cytometry, demonstrated with SYBR Green I and propidium iodide viability staining.

The increasing use of flow cytometry (FCM) for analyses of environmental samples has resulted in a large variety of staining protocols with varying results and limited comparability. Viability assessment with FCM is in this context of particular interest because incorrect staining could severely affect the outcome/interpretation of the results. Here we propose a pipeline for the development and optimization of staining protocols for environmental samples, demonstrated with the common viability dye combination of SYBR Green I (SG) and propidium iodide (PI). Optimization steps included the assessment of dye solvents, determination of suitable PI concentration, and determining the optimal staining temperature and staining time. We demonstrated that dimethyl sulfoxide (DMSO) could impair membrane integrity, when used for SGPI stock solution preparation, and TRIS buffer was chosen as an alternative. Moreover we selected 6µM as optimal PI final concentration: less than 3µM resulted in incomplete staining of damaged cells, while concentrations higher that 12µM resulted in false PI-positive staining of intact cells. Low temperatures (25°C) resulted in a slow reaction and did not enable the staining of all bacteria, while high temperatures (44°C) caused damage to cells and false PI-positive results. Hence, 35°C was selected as optimal staining temperature. We further showed that a minimum of 15min were necessary to obtain stable staining results. Moreover, we showed that addition of EDTA resulted in 1-39% more PI-positive results compared to an EDTA-free sample, and argue that insufficient evidence currently exist in favor of adding EDTA to all samples in general. Altogether, the data clearly shows the need to be careful, precise and reproducible when staining cells for flow cytometric analyses, and the need to assess and optimize staining protocols with both viable and non-viable bacteria.

Behavior and stability of adenosine triphosphate (ATP) during chlorine disinfection.

Adenosine triphosphate (ATP) analysis is a cultivation-independent alternative method for the determination of bacterial viability in both chlorinated and non-chlorinated water. Here we investigated the behavior and stability of ATP during chlorination in detail. Different sodium hypochlorite doses (0-22.4 mg-Cl2 L(-1); 5 min exposure) were applied to an Escherichia coli pure culture suspended in filtered river water. We observed decreasing intracellular ATP with increasing chlorine concentrations, but extracellular ATP concentrations only increased when the chlorine dose exceeded 0.35 mg L(-1). The release of ATP from chlorine-damaged bacteria coincided with severe membrane damage detected with flow cytometry (FCM). The stability of extracellular ATP was subsequently studied in different water matrixes, and we found that extracellular ATP was stable in sterile deionized water and also in chlorinated water until extremely high chlorine doses (≤11.2 mg-Cl2 L(-1); 5 min exposure). In contrast, ATP decreased relatively slowly (k = 0.145 h(-1)) in 0.1 µm filtered river water, presumably due to degradation by either extracellular enzymes or the fraction of bacteria that were able to pass through the filter. Extracellular ATP decreased considerably faster (k = 0.368 h(-1)) during batch growth of a river water bacterial community. A series of growth potential tests showed that extracellular ATP molecules were utilized as a phosphorus source during bacteria proliferation. From the combined data we conclude that ATP released from bacteria at high chlorine doses could promote bacteria regrowth, contributing to biological instability in drinking water distribution systems.

Biological instability in a chlorinated drinking water distribution network.

The purpose of a drinking water distribution system is to deliver drinking water to the consumer, preferably with the same quality as when it left the treatment plant. In this context, the maintenance of good microbiological quality is often referred to as biological stability, and the addition of sufficient chlorine residuals is regarded as one way to achieve this. The full-scale drinking water distribution system of Riga (Latvia) was investigated with respect to biological stability in chlorinated drinking water. Flow cytometric (FCM) intact cell concentrations, intracellular adenosine tri-phosphate (ATP), heterotrophic plate counts and residual chlorine measurements were performed to evaluate the drinking water quality and stability at 49 sampling points throughout the distribution network. Cell viability methods were compared and the importance of extracellular ATP measurements was examined as well. FCM intact cell concentrations varied from 5×10(3) cells mL(-1) to 4.66×10(5) cells mL(-1) in the network. While this parameter did not exceed 2.1×10(4) cells mL(-1) in the effluent from any water treatment plant, 50% of all the network samples contained more than 1.06×10(5) cells mL(-1). This indisputably demonstrates biological instability in this particular drinking water distribution system, which was ascribed to a loss of disinfectant residuals and concomitant bacterial growth. The study highlights the potential of using cultivation-independent methods for the assessment of chlorinated water samples. In addition, it underlines the complexity of full-scale drinking water distribution systems, and the resulting challenges to establish the causes of biological instability.

Minimum information about a biofilm experiment (MIABiE): standards for reporting experiments and data on sessile microbial communities living at interfaces.

The minimum information about a biofilm experiment (MIABiE) initiative has arisen from the need to find an adequate and scientifically sound way to control the quality of the documentation accompanying the public deposition of biofilm-related data, particularly those obtained using high-throughput devices and techniques. Thereby, the MIABiE consortium has initiated the identification and organization of a set of modules containing the minimum information that needs to be reported to guarantee the interpretability and independent verification of experimental results and their integration with knowledge coming from other fields. MIABiE does not intend to propose specific standards on how biofilms experiments should be performed, because it is acknowledged that specific research questions require specific conditions which may deviate from any standardization. Instead, MIABiE presents guidelines about the data to be recorded and published in order for the procedure and results to be easily and unequivocally interpreted and reproduced. Overall, MIABiE opens up the discussion about a number of particular areas of interest and attempts to achieve a broad consensus about which biofilm data and metadata should be reported in scientific journals in a systematic, rigorous and understandable manner.

Hydrogen-producing Escherichia coli strains overexpressing lactose permease: FT-IR analysis of the lactose-induced stress.

The lactose permease gene (lacY) was overexpressed in the septuple knockout mutant of Escherichia coli, previously engineered for hydrogen production from glucose. It was expected that raising the lactose transporter activity would elevate the intracellular lactose concentration, inactivate the lactose repressor, induce the lactose operon, and as a result stimulate overall lactose consumption and conversion. However, overexpression of the lactose transporter caused a considerable growth delay in the recombinant strain on lactose, resembling to some extent the "lactose killing" phenomenon. Therefore, the recombinant strain was subjected to selection on lactose-containing media. Selection on plates with 3% lactose yielded a strain with a decreased content of the recombinant plasmid but with an improved ability to grow and produce hydrogen on lactose. Macromolecular analysis of its biomass by means of Fourier transform-infrared spectroscopy demonstrated that increase of the cellular polysaccharide content might contribute to the adaptation of E. coli to lactose stress.

Evaluating the potential of biofilm control in water supply systems by removal of phosphorus from drinking water.

Effect of microbially available phosphorus (MAP) on biofilm development in drinking water systems was investigated at the pilot-scale experiments over 3 years. Completely mixed biofilm reactors Propella (water detention time 24 h, flow rate 0.25 m s(-1), PVC pipe coupons) were used as water distribution network models. Four experimental runs were carried out with water containing different levels of phosphorus which was limiting nutrient for bacterial growth. Positive correlation between MAP in the inlet water and heterotrophic plate count (correlation coefficient 0.95) in biofilm, as well as for the total bacteria number (correlation coefficient 0.71), was observed. However, our experiments showed that removal of phosphorus down to very low levels (below detection limits of chemical method and MAP < 1 microg L(-1) was not an efficient strategy to eliminate bacterial regrowth and biofilm formation (< 51,00,000 cells/cm2) in drinking water supply systems.

Effect of Phosphorus on survival of Escherichia coli in drinking water biofilms.

The effect of phosphorus addition on survival of Escherichia coli in an experimental drinking water distribution system was investigated. Higher phosphorus concentrations prolonged the survival of culturable E. coli in water and biofilms. Although phosphorus addition did not affect viable but not culturable (VBNC) E. coli in biofilms, these structures could act as a reservoir of VBNC forms of E. coli in drinking water distribution systems.

Formation of biofilms in drinking water distribution networks, a case study in two cities in Finland and Latvia.

The formation of biofilms in drinking water distribution networks is a significant technical, aesthetic and hygienic problem. In this study, the effects of assimilable organic carbon, microbially available phosphorus (MAP), residual chlorine, temperature and corrosion products on the formation of biofilms were studied in two full-scale water supply systems in Finland and Latvia. Biofilm collectors consisting of polyvinyl chloride pipes were installed in several waterworks and distribution networks, which were supplied with chemically precipitated surface waters and groundwater from different sources. During a 1-year study, the biofilm density was measured by heterotrophic plate counts on R2A-agar, acridine orange direct counting and ATP-analyses. A moderate level of residual chlorine decreased biofilm density, whereas an increase of MAP in water and accumulated cast iron corrosion products significantly increased biofilm density. This work confirms, in a full-scale distribution system in Finland and Latvia, our earlier in vitro finding that biofilm formation is affected by the availability of phosphorus in drinking water.

Sorption of humic substances on aquifer material at artificial recharge of groundwater.

Experiments in batch equilibrium system were carried out to evaluate the importance of physical and chemical factors determining the sorption efficiency of humic substances (HS) on aquifer material, which has been used for artificial recharge of groundwater (ARG) in drinking water production. Results showed that an increase of the amount of clay in the aquifer material and a decrease of pH in water increased the sorption efficiency. The sorption of higher molecular weight, more hydrophobic and aromatic HS (Aldrich and forest soil humic acids) were greater than the sorption of acidic HS (river fulvic acids), either on the aquifer material or to its representative sorbing phases, clay and organic matter. The sorption on the aquifer material was largely due to physical sorption (hydrophobic attractions). This study showed the importance of HS composition on their removal during ARG and contributed to an understanding of the HS sorption mechanisms in this process.