Wastewater-based epidemiology (WBE) can be used to track the spread of SARS-CoV-2 in a population. This study pre-sents the learning outcomes from over two-year long monitoring of SARS-CoV-2 in Stockholm, Sweden. The three main wastewater treatment plants in Stockholm, with a total of six inlets, were monitored from April 2020 until June 2022 (in total 600 samples). This spans five major SARS-CoV-2 waves, where WBE data provided early warning signals for each wave. Further, the measured SARS-CoV-2 content in the wastewater correlated significantly with the level of positive COVID-19 tests (r = 0.86; p << 0.0001) measured by widespread testing of the population. Moreover, as a proof-of-concept, six SARS-CoV-2 variants of concern were monitored using hpPCR assay, demonstrating that var-iants can be traced through wastewater monitoring.During this long-term surveillance, two sampling protocols, two RNA concentration/extraction methods, two calcula-tion approaches, and normalization to the RNA virus Pepper mild mottle virus (PMMoV) were evaluated. In addition, a study of storage conditions was performed, demonstrating that the decay of viral RNA was significantly reduced upon the addition of glycerol to the wastewater before storage at -80 degrees C. Our results provide valuable information that can facilitate the incorporation of WBE as a prediction tool for possible future outbreaks of SARS-CoV-2 and preparations for future pandemics.

An approach based on wastewater epidemiology can be used to monitor the COVID-19 pandemic by assessing the gene copy number of SARS-CoV-2 in wastewater. In the present study, we statistically analyzed such data from six inlets of three wastewater treatment plants, covering six regions of Stockholm, Sweden, collected over an approximate year period (week 16 of 2020 to week 22 of 2021). SARS-CoV-2 gene copy number and population-based biomarker PMMoV, as well as clinical data, such as the number of positive cases, intensive care unit numbers, and deaths, were analyzed statistically using correlations and principal component analysis (PCA). Despite the population differences, the PCA for the Stockholm dataset showed that the case numbers are well grouped across wastewater treatment plants. Furthermore, when considering the data from the whole of Stockholm, the wastewater characteristics (flow rate m3/day, PMMoV Ct value, and SARS-CoV gene copy number) were significantly correlated with the public health agency’s report of SARS-CoV-2 infection rates (0.419 to 0.95, p-value < 0.01). However, while the PCA results showed that the case numbers for each wastewater treatment plant were well grouped concerning PC1 (37.3%) and PC2 (19.67%), the results from the correlation analysis for the individual wastewater treatment plants showed varied trends. SARS-CoV-2 fluctuations can be accurately predicted through statistical analyses of wastewater-based epidemiology, as demonstrated in this study.

The revolutionary transformation from petrol-based production to bio-based production is becoming urgent in line with the rapid industrialization, depleting resources, and deterioration of the ecosystem. Bio-based production from waste-streams is offering a sustainable and environmentally friendly solution. It offers several advantages, such as a longer operation period, less competition for microorganisms, higher efficiency, and finally, lower process costs. In the current study, several bio-based products (organic acids, biomethane, biohydrogen, and metal leachates) produced under acidic conditions are reviewed regarding their microbial pathways, processes, and operational conditions. Furthermore, the limitations both in the production process and in the scale-up are evaluated with future recommendations.

Volatile fatty acids, intermediate products of anaerobic digestion, are one of the most promising biobased products. In this study, the effects of acidic (pH 5), neutral (without pH adjustment) and alkali (pH 10) pH on production efficiency and composition of volatile fatty acids (VFAs) and bacterial community profile were analyzed. The anaerobic sequencing batch reactors were fed cheese production wastewater as substrate and inoculated by anaerobic granular seed sludge. The results showed that acidic pH improved VFA production yield (0.92 at pH 5; 0.42 at pH 10 and 0.21 gCOD/gVS at neutral pH). Furthermore, propionic acid was dominant under both pH 10 (64 +/- 20%) and neutral pH (72 +/- 8%), whereas, acetic acid (23 +/- 20%4), propionic acid (22 +/- 3%), butyric acid (21 +/- 4%) and valeric acid (15 +/- 8%) were almost equally distributed under pH 5. Adaptation of bacterial community to different pH conditions might steer the acid profile: Bacteroidetes (50.07 +/- 2%) under pH 10, Proteobacteria (40.74 +/- 7%) under neutral pH and Firmicutes (47.64 +/- 9%) under pH 5 were the most dominant phylum, respectively. Results indicated pH plays a significant role in VFA production, acid composition, and bacterial community structure. However, in order to gain a concrete understanding effects of pH, characterization of intracellular and extracellular metabolites with dynamics of the microbial community is required.

Wastewater-based epidemiology offers a cost-effective alternative to testing large populations for SARS-CoV-2 virus, and may potentially be used as an early warning system for SARS-CoV-2 pandemic spread. However, viruses are highly diluted in wastewater, and a validated method for their concentration and further processing, and suitable reference viruses, are the main needs to be established for reliable SARS-CoV-2 municipal wastewater detection. For this purpose, we collected wastewater from two European cities during the Covid-19 pandemic and evaluated the sensitivity of RT-qPCR detection of viral RNA after four concentration methods (two variants of ultrafiltration-based method and two adsorption and extraction-based methods). Further, we evaluated one external (bovine corona virus) and one internal (pepper mild mottle virus) reference virus. We found a consistently higher recovery of spiked virus using the modified ultrafiltration-based method. This method also had a significantly higher efficiency (p-value <0.01) for wastewater SARS-CoV-2 detection. The ultracentrifugation method was the only method that detected SARS-CoV-2 in the wastewater of both cities. The pepper mild mottle virus was found to function as a potentially suitable internal reference standard.

This study aimed to develop a novel strategy for tailor-made volatile fatty acid (VFA) composition. For this purpose, the mixed microbial culture was bioaugmented by Propionibacterium acidipropionici. Anaerobic sequencing batch reactors were operated with cheese wastewater under alkali pH. While the maximum propionic acid production almost four times increased (3779 +/- 201 mgCODeq propionic acid/L in the bioaugmented reactor and 942 +/- 172 mgCODeq propionic acid/L in the control reactor), there was no significant difference in VFA composition. The gene copy number of P.acidipropionici increased 20 times after the bioaugmentation. Furthermore, the gene copy number of P.acidipropionici was positively correlated with total VFA and isovaleric acid concentration. The relative abundance of family Flavobacteriaceae increased in the bioaugmented reactor, which might be caused by the syntrophic relation between Flavobacteriaceae and P. acidipropionici. The cycle analysis results showed that the shorter cycle (6h) could ensure the same efficiency.

Production of targeted volatile fatty acid (VFA) composition by fermentation is a promising approach for upstream and post-stream VFA applications. In the current study, the bioaugmented mixed microbial culture by Clostridium aceticum was used to produce an acetic acid dominant VFA mixture. For this purpose, anaerobic sequencing batch reactors (bioaugmented and control) were operated under pH 10 and fed by cheese processing wastewater. The efficiency and stability of the bioaugmentation strategy were monitored using the production and composition of VFA, the quantity of C. aceticum (by qPCR), and bacterial community profile (16S rRNA Illumina Sequencing). The bioaugmented mixed culture significantly increased acetic acid concentration in the VFA mixture (from 1170 +/- 18 to 122 +/- 9 mgCOD/L) compared to the control reactor. Furthermore, the total VFA production (from 1254 +/- 11 to 5493 +/- 36 mgCOD/L) was also enhanced. Nevertheless, the bioaugmentation could not shift the propionic acid dominancy in the VFA mixture. The most significant effect of bioaugmentation on the bacterial community profile was seen in the relative abundance of the Thermoanaerobacterales Family III. Incertae sedis, its relative abundance increased simultaneously with the gene copy number of C. aceticum during bioaugmentation. These results suggest that there might be a syntropy between species of Thermoanaerobacterales Family III. Incertae sedis and C. aceticum. The cycle analysis showed that 6 h (instead of 24 h) was adequate retention time to achieve the same acetic acid and total VFA production efficiency. Biobased acetic acid production is widely applicable and economically competitive with petroleum-based production, and this study has the potential to enable a new approach as produced acetic acid dominant VFA can replace external carbon sources for different processes (such as denitrification) in WWTPs. In this way, the higher treatment efficiency for WWTPs can be obtained by recovered substrate from the waste streams that promote a circular economy approach.

Production of polyhydroxyalkanoates is an important field in the biorefinery as bio-alternative to conventional plastics. However, its commercialization is still limited by high production cost. In this study, a process with the potential to reduce the production cost of polyhydroxyalkanoates was proposed. Mixed cultures accumulated polyhydroxyalkanoates using volatile fatty acid-rich effluents from waste streams, without pH and temperature control. In addition, the impact of two types of carbon sources was investigated by analyzing the microbial community as well as the polyhydroxyalkanoate accumulation capacity. Mixed cultures successfully adapted to different substrates, consuming the volatile fatty acids in their totality. The phyla Proteobacteria, Bacteroidetes and Firmicutes dominated the bacterial community. The highest polyhydroxyalkanoate content was 43.5% w/w, which is comparable to contents reported from mixed cultures using synthetic carbon sources. The biopolymer consisted of (R)-3-hydroxybutyrate 94.8 ± 1.7% w/w and (R)-3-hydroxyvaletare 5.2 ± 1.7% w/w.

Bio-based production of materials from waste streams is a pivotal aspect in a circular economy. This study aimed to investigate the influence of inoculum (three different sludge taken from anaerobic digestors), pH (5 & 10) and retention time on production of total volatile fatty acids (VFAs), VFA composition as well as the microbial community during anaerobic digestion of food waste. The highest VFA production was ∼22000 ± 1036 mg COD/L and 12927 ± 1029 mg COD/L on day 15 using the inoculum acclimated to food waste at pH 10 and pH 5, respectively. Acetic acid was the dominant VFA in the batch reactors with initial alkaline conditions, whereas both propionic and acetic acids were the dominant products in the acidic condition. Firmicutes, Chloroflexi and Bacteroidetes had the highest relative abundance in the reactors. VFA generation was positively correlated to the relative abundance of Firmicutes.

The key factor for promoting bioeconomy and circularity in the cities is to examine waste streams such as municipal wastewater, sludge, solid waste, food waste, bakery industry waste, and pulp and paper industry waste as raw materials rather than wastes to be disposed. In this regard, biorefinery concept is promising for converting biomass into valuable products. Biomethane, high market value volatile fatty acids, and polyhydroxyalkanoates are some of the main outstanding products for waste biorefineries. Algal biorefinery concept is also promising as serving as a multiple product factory that can also be integrated with municipal wastewater systems. Phosphorus recovery is also essential to control eutrophication in the receiving water bodies and reversing phosphorus back to the market. Therefore, future management of cities should include these specified approaches to support circularity and minimize possible environmental problems.