The fate of severe acute respiratory syndrome coronavirus-2 and pepper mild mottle virus at various stages of wastewater treatment process.

This study investigated the efficiency of the treatment processes of wastewater treatment plants (WWTPs) to remove severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and pepper mild mottle virus (PMMoV) from the wastewater and sewage sludge, as well as the influence of the mode of operation on the quality of the treated wastewater. SARS-CoV-2 and PMMoV were detected and quantified at different stages of the wastewater and sludge treatment process of three major WWTPs in Stockholm, Sweden. The results showed that primary, biological, and advanced membrane treatment processes are effective in removing SARS-CoV-2 from the wastewater with removal efficiencies of 99-100 % for all WWTPs, while the virus was accumulated in the primary and waste-activated sludges due to higher affinity to biosolids. Operation strategies such as bypass reintroduced the virus into the treated wastewater. The WWTPs achieved relatively low PMMoV removal efficiencies (63-87 %) most probably due to the robust capsid structure of the virus. Anaerobic digestion could not completely remove SARS-CoV-2 and PMMoV from the sludge leading to increased levels of SARS-CoV-2 and PMMoV in dewatered sludge. The study gives an overview of WWTPs' role in tackling pathogen spread in society in the event of a pandemic and disease breakout.

Development and implementation of assays to monitor human adenovirus F40/41 in wastewater: Trends preceding, during, and following the non-A-to-E hepatitis outbreak in Stockholm.

Human adenovirus (HAdV) type F41 has been identified as a possible cause of the non-A-to-E hepatitis outbreak. This study uses wastewater monitoring to track HAdV F40 and F41, supporting clinical investigations and providing insights into the pathogen's role in the outbreak. Given the limited clinical monitoring in Sweden of HAdV-F40/41, this approach also helps estimate the true infection burden of this pathogen during the outbreak. This study developed three qPCR assays for the hexon, penton, and fiber genes of HAdV F40 and F41. The hexon assay was F41-specific, while the fiber assay detected multiple HAdV-F strains. Comprehensive monitoring of HAdV-F40/41 levels in Stockholm's wastewater was conducted over 1.5 years, capturing the period before, during, and after the outbreak. A significant infection wave was observed in spring 2022, with strains beyond lineage 2 contributing to the outbreak. Moreover, simultaneous SARS-CoV-2 surveillance revealed that HAdV-F infections peaked at different times from COVID-19, but the HAdV-F wave aligned with the relaxation of pandemic restrictions. These findings offer valuable insights for future HAdV-F investigations and confirm its role in the non-A-to-E hepatitis outbreak.

Discovery of sulfonamide resistance genes in deep groundwater below Patna, India.

Global usage of pharmaceuticals has led to the proliferation of bacteria that are resistant to antimicrobial treatments, creating a substantial public health challenge. Here, we investigate the emergence of sulfonamide resistance genes in groundwater and surface water in Patna, a rapidly developing city in Bihar, India. We report the first quantification of three sulfonamide resistance genes (sulI, sulII and sulIII) in groundwater (12-107 m in depth) in India. The mean relative abundance of gene copies was found to be sulI (2.4 × 10-2 copies/16S rRNA gene) > sulII (5.4 × 10-3 copies/16S rRNA gene) > sulIII (2.4 × 10-3 copies/16S rRNA gene) in groundwater (n = 15) and surface water (n = 3). A comparison between antimicrobial resistance (AMR) genes and wastewater indicators, particularly tryptophan:fulvic-like fluorescence, suggests that wastewater was associated with AMR gene prevalence. Urban drainage channels, containing hospital and domestic wastes, are likely a substantial source of antimicrobial resistance in groundwater and surface water, including the Ganges (Ganga) River. This study is a reference point for decision-makers in the fight against antimicrobial resistance because it quantifies and determines potential sources of AMR genes in Indian groundwater.

How to develop a bio-based phosphorus mining strategy for eutrophic marine sediments: Unlocking native microbial processes for anaerobic phosphorus release.

This study examined the anaerobic release of phosphorus (P) from two different Baltic Sea sediments (B and F), focusing on the impact of initial concentration of externally introduced waste-derived volatile fatty acids (VFA) as the carbon source, temperature, pH, and mixing conditions. The first batch bioreactor set was operated to demonstrate the effect of VFA on anaerobic P release at different concentrations (1000-10000 mg/L as COD) at 20 °C. A notable P release of up to 15.85 mg/L PO4-P was observed for Sediment B at an initial carbon concentration of 10000 mg COD/L. However, VFA consumption in the bioreactors was minimal or no subsequent. The second batch bioreactor set was carried out to investigate the effect of temperature (20 °C-35 °C), pH (5.5, 7.0 and 8.5) and mixing conditions on P release by introducing lower initial carbon concentration (1000 mg COD/L) considering the potential risk for VFA accumulation in the bioreactors. Maximum P releases of 4.4 mg/L and 3.5 mg/L were for Sediment B and Sediment F, respectively. Two-way ANOVA tests revealed that the operation time and pH and their interactions were statistically significant (p < 0.05) for both sediments while the effect of mixing was not statistically significant. Most of the sulfate was reduced during batch bioreactor operation and Desulfomicobiaceae became dominant among other sulfate-reducing bacteria (SRB) possibly shows the importance of SRB in terms of anaerobic P release. This study gives an insight into future implementations of phosphorus mining from eutrophic environment under anaerobic conditions.

Phosphorus mining from marine sediments adopting different carbon/nitrogen strategies driven by anaerobic reactors: The exploration of potential mechanism and microbial activities.

To investigate the possibility of phosphorus (P) recovery from marine sediment and explore the role of the carbon: nitrogen ratio in affecting the internal P release under anaerobic conditions, we experimented with the external addition of carbon (acetic acid and glucose) and ammonia nitrogen (NH4-N) to expose P release mechanisms. The 24-day anaerobic incubations were conducted with four different carbon: nitrogen dosing groups including no NH4-N addition and COD/N ratios of 100, 50, and 10. The P release showed that extra NH4-N loading significantly suppressed the decomposition of P (p < 0.05) from the marine sediment, the maximum P release was 4.07 mg/L and 7.14 mg/L in acetic acid- and glucose-fed systems, respectively, without extra NH4-N addition. Additionally, the results exhibited that the imbalance of carbon: nitrogen not only failed to induce the production of organic P mineralization enzyme (alkaline phosphatase) in the sediment but also suppressed its activity under anaerobic conditions. The highest enzyme activity was observed in the group without additional NH4-N dosage, with rates of 1046.4 mg/(kg∙h) in the acetic acid- and 967.8 mg/(kg∙h) in the glucose-fed system, respectively. Microbial data analysis indicated that a decrease in the abundance of P release-regulating bacteria, including polyphosphate-accumulating organisms (Rhodobacteraceae) and sulfate-reducing bacteria (Desulfosarcinaceae), was observed in the high NH4-N addition groups. The observed reduction in enzyme activity and suppression of microbial activity mentioned above could potentially account for the inhibited P decomposition in the presence of high NH4-N addition under anaerobic conditions. The produced P-enriched solution from the bioreactors may offer a promising source for future recovery endeavors.

Influence of pH, Heat Treatment of Inoculum, and Selenium Oxyanions on Concomitant Selenium Bioremediation and Volatile Fatty Acid Production from Food Waste.

Developing novel strategies to enhance volatile fatty acid (VFA) yield from abundant waste resources is imperative to improve the competitiveness of biobased VFAs over petrochemical-based VFAs. This study hypothesized to improve the VFA yield from food waste via three strategies, viz., pH adjustment (5 and 10), supplementation of selenium (Se) oxyanions, and heat treatment of the inoculum (at 85 °C for 1 h). The highest VFA yield of 0.516 g COD/g VS was achieved at alkaline pH, which was 45% higher than the maximum VFA production at acidic pH. Heat treatment resulted in VFA accumulation after day 10 upon alkaline pretreatment. Se oxyanions acted as chemical inhibitors to improve the VFA yield at pH 10 with non-heat-treated inoculum (NHT). Acetic and propionic acid production was dominant at alkaline pH (NHT); however, the VFA composition diversified under the other tested conditions. More than 95% Se removal was achieved on day 1 under all the conditions tested. However, the heat treatment was detrimental for selenate reduction, with less than 15% Se removal after 20 days. Biosynthesized Se nanoparticles were confirmed by transmission and scanning electron microscopy and and energy dispersive X-ray analyses. The heat treatment inhibited the presence of nonsporulating bacteria and methanogenic archaea (Methanobacteriaceae). High-throughput sequencing also revealed higher relative abundances of the bacterial families (such as Clostridiaceae, Bacteroidaceae, and Prevotellaceae) that are capable of VFA production and/or selenium reduction.

Conceptual system for sustainable and next-generation wastewater resource recovery facilities.

Shifting the concept of municipal wastewater treatment to recover resources is one of the key factors contributing to a sustainable society. A novel concept based on research is proposed to recover four main bio-based products from municipal wastewater while reaching the necessary regulatory standards. The main resource recovery units of the proposed system include upflow anaerobic sludge blanket reactor for the recovery of biogas (as product 1) from mainstream municipal wastewater after primary sedimentation. Sewage sludge is co-fermented with external organic waste such as food waste for volatile fatty acids (VFAs) production as precursors for other bio-based production. A portion of the VFA mixture (product 2) is used as carbon sources in the denitrification step of the nitrification/denitrification process as an alternative for nitrogen removal. The other alternative for nitrogen removal is the partial nitrification/anammx process. The VFA mixture is separated with nanofiltration/reverse osmosis membrane technology into low-carbon VFAs and high-carbon VFAs. Polyhydroxyalkanoate (as product 3) is produced from the low-carbon VFAs. Using membrane contactor-based processes and ion-exchange techniques, high-carbon VFAs are recovered as one-type VFA (pure VFA) and in ester forms (product 4). The nutrient-rich fermented and dewatered biosolid is applied as a fertilizer. The proposed units are seen as individual resource recovery systems as well as a concept of an integrated system. A qualitative environmental assessment of the proposed resource recovery units confirms the positive environmental impacts of the proposed system.

Statistical Analysis of SARS-CoV-2 Using Wastewater-Based Data of Stockholm, Sweden.

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.

Long-term SARS-CoV-2 surveillance in the wastewater of Stockholm: What lessons can be learned from the Swedish perspective?

Wastewater-based epidemiology (WBE) can be used to track the spread of SARS-CoV-2 in a population. This study presents 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 variants can be traced through wastewater monitoring. During this long-term surveillance, two sampling protocols, two RNA concentration/extraction methods, two calculation 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 °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.

A COST Action on microbial responses to low pH: Developing links and sharing resources across the academic-industrial divide.

We present work of our COST Action on "Understanding and exploiting the impacts of low pH on micro-organisms". First, we summarise a workshop held at the European Federation of Biotechnology meeting on Microbial Stress Responses (online in 2020) on "Industrial applications of low pH stress on microbial bio-based production", as an example of an initiative fostering links between pure and applied research. We report the outcomes of a small survey on the challenging topic of developing links between researchers working in academia and industry that show that, while people in different sectors strongly support such links, barriers remain that obstruct this process. We present the thoughts of an expert panel held as part of the workshop above, where people with experience of collaboration between academia and industry shared ideas on how to develop and maintain links. Access to relevant information is essential for research in all sectors, and because of this we have developed, as part of our COST Action goals, two resources for the free use of all researchers with interests in any aspects of microbial responses to low pH. These are (1) a comprehensive database of references in the literature on different aspects of acid stress responses in different bacterial and fungal species, and (2) a database of research expertise across our network. We invite the community of researchers working in this field to take advantage of these resources to identify relevant literature and opportunities for establishing collaborations.

The effects of pH on the production of volatile fatty acids and microbial dynamics in long-term reactor operation.

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.

Phosphorus mining from eutrophic marine environment towards a blue economy: The role of bio-based applications.

Global phosphorus reserves are under pressure of depletion in the near future due to increased consumption of primary phosphorus reservoirs and improper management of phosphorus. At the same time, a considerable portion of global marine water bodies has been suffering from eutrophication due to excessive nutrient loading. The marine environment can be considered as a valuable phosphorus source due to nutrient rich eutrophic seawater and sediment which could potentially serve as phosphorus mines in the near future. Hence, sustainable phosphorus recovery strategies should be adapted for marine systems to provide phosphorus for the growing market demand and simultaneously control eutrophication. In this review, possible sustainable strategies for phosphorus removal and recovery from marine environments are discussed in detail. Bio-based strategies relying on natural phosphorus uptake/release metabolism of living organisms are suggested as promising options that can provide both phosphorus removal and recovery from marine waters for achieving a sustainable marine ecosystem. Among them, the utilization of microorganisms seems promising to develop novel strategies. However, the research gap for the technical applicability of these strategies is still considerably big. Therefore, future research should focus on the technical development of the strategies through laboratory and/or field studies. Coupling phosphorus mining with other valorisation pathways (i.e., metal recovery, energy production) is also suggested to improve overall sustainability and economic viability. Environmental, economic and societal challenges should altogether be well addressed prior to real scale applications.

Long-term alkaline volatile fatty acids production from waste streams: Impact of pH and dominance of Dysgonomonadaceae.

Alkaline co-fermentation of primary sludge and external organic waste (OW) was studied to elucidate the influence of substrate ratios and long-term system robustness and microbial community dynamics using batch and semi-continuous reactors. Volatile fatty acid (VFA) production increased with increasing OW fraction in the substrate due to synergistic effects of co-degradation. VFA production at pH 10 increased up to 30,300 mgCOD/L (yield of 630 mg COD/gVSfed) but reduced over time to ≈10,000 mgCOD/L. Lowering pH to 9 led to the restoration of VFA production with a maximum of 32,000 mg COD/L (676 mg COD/g VSfed) due to changes in microbial structure. VFA was composed mainly of acetic acid, but propionic acid increased at pH 9. The microbial community was dominated by Bacillaceae (34 ± 10%) and Proteinivoracales_uncultured (16 ± 11%) at pH 10, while Dysgonomonadaceae (52 ± 8%) was enriched at pH 9. The study demonstrated a zero-waste strategy that turns organic wastes into bio-based products.

Pure cultures for synthetic culture development: Next level municipal waste treatment for polyhydroxyalkanoates production.

Polyhydroxyalkanoates (PHAs), as bio-based plastics, promise a transition from petroleum products to green and sustainable alternatives. However, their commercial production is yet impeded by high production costs. In this study, we assessed synthetic culture in mono and co-culture modes for bacterial PHA production. It was demonstrated that volatile fatty acids (VFAs) derived from food waste and primary sludge are cheap carbon sources for maintaining high production yields in the synthetic cultures. The maximum obtained PHA was 77.54 ± 5.67% of cell dried weight (CDW) (1.723 g/L) from Cupriavidus necator and 54.9 ± 3.66% of CDW (1.088 g/L) from Burkholderia cepacia. The acquired results are comparable to those in literature using sugar substrates. Comparatively, lower PHA productions were obtained from the co-cultivations ranging between 36-45 CDW% (0.39-0.48 g/L). Meanwhile, the 3-hydroxyvalerate content in the biopolymers were increased up to 21%.

Bioaugmented Mixed Culture by Clostridium aceticum to Manipulate Volatile Fatty Acids Composition From the Fermentation of Cheese Production Wastewater.

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.

A pilot-scale study of granule-based anaerobic reactors for biogas recovery from municipal wastewater under sub-mesophilic conditions.

The influence of hydraulic retention time (HRT of 3-5 h) and temperature (20-25 °C) on performance and microbial dynamics of two pilot-scale upflow anaerobic sludge blanket (UASB) reactors with different granule size distribution (UASB1 = 3-4 mm and UASB2 = 1-2 mm) were investigated for 217 days. Increasing the HRT to 5 h even at a lower temperature of 20 °C enhanced COD removal and biogas production with average of 59 ± 16% (up to 85%) and 73 ± 9 L/(m3·d) (up to 102 L/(m3·d)) for UASB1; 63 ± 16% (up to 85%) and 75 ± 9 L/(m3·d) (up to 90 L/(m3·d)) for UASB2, respectively. This is explained by sufficient contact time between microorganisms and substrate. Acetoclastic methanogenic activity was higher in UASB1 because Methanosaetaceae (produces methane from acetate) dominated (64 ± 4%). However, Methanoregulaceae (29 ± 3%) and Methanomicrobiales_unassigned (20 ± 6%) which produce methane from H2/CO2 and formate were significant in UASB2. The extent of change in the microbial dynamics with HRT and temperature was more obvious in the smaller granule reactor.

Bioaugmentation as a strategy for tailor-made volatile fatty acid production.

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.

Prevalence of SARS-CoV-2 in Communities Through Wastewater Surveillance-a Potential Approach for Estimation of Disease Burden.

The episodic outbreak of COVID-19 due to SARS-CoV-2 is severely affecting the economy, and the global count of infected patients is increasing. The actual number of patients had been underestimated due to limited facilities for testing as well as asymptomatic nature of the expression of COVID-19 on individual basis. Tragically, for emerging economies with high population density, the situation has been more complex due to insufficient testing facilities for diagnosis of the disease. However, the recent reports about persistent shedding of viral RNA of SARS-CoV-2 in the human feces have created a possibility to track the prevalence and trends of the disease in communities, known as wastewater-based epidemiology (WBE). In this article, we highlight the current limitations and future prospects for WBE to manage pandemics.

Bioconversion of food waste to volatile fatty acids: Impact of microbial community, pH and retention time.

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.