Environmental Science: Water Research & Technology

Amplicon sequencing, metagenomics, and quantitative polymerase chain reaction (qPCR) are commonly used techniques to analyse microorganisms and antibiotic resistance genes (ARGs) in activated sludge from wastewater treatment plants (WWTPs). However, the lack of workflow harmonisation poses challenges in comparing measurements across studies and research groups. To address this issue, we examined the impact of DNA extraction procedures on 16S rRNA gene amplicon sequencing, shotgun metagenomics, and qPCR analyses of activated sludge by combining two widely used DNA extraction kits (PowerSoil and FastDNA) and two commonly employed disruption instruments (bead-beater and vortex) through a 2x2 factorial experimental design involving four groups of three analysts performing DNA extractions in triplicates. Our findings revealed significant differences in DNA yield, purity, and reproducibility of amplicon sequencing profiles among the extraction kits. Operator variability also influenced the results. We compared microbiome profiles obtained by amplicon sequencing and metagenomics and observed that bead-beating introduced more variability among triplicates compared to vortexing. The combinations of extraction kits and disruption instruments impacted the relative abundances of specific phyla such as Actinobacteriota, Bacteroidota, and Nitrospirota. For resistome analysis, we employed metagenomics for high-resolution profiling and qPCR for high-sensitivity detection of ARGs. The compositions and diversities of resistome datasets were not significantly affected by the choice of extraction kits and disruption instruments. Although using the same method is ideal for accurate comparisons, our results suggest that acceptable reproducibility can still be achieved when using different methods. This finding encourages the implementation of ARG monitoring in wastewater treatment processes. However, it is important to consider biases introduced by DNA extraction workflows when designing analytical studies, interpreting their results, and comparing their findings. Striving for more harmonised molecular workflows is crucial in the field of wastewater microbiology and engineering. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/546617v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@caa36eorg.highwire.dtl.DTLVardef@1afabfdorg.highwire.dtl.DTLVardef@448b9aorg.highwire.dtl.DTLVardef@296404_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

Despite the wide adoption of wastewater surveillance, more research is needed to understand the fate and transport of viral genetic markers in wastewater. This information is essential for the interpretation of wastewater surveillance data and the development of mechanistic models that link wastewater measurements to the number of individuals shedding virus. In this study, we examined the solid-liquid partitioning behavior of four viruses in wastewater: SARS-CoV-2, respiratory syncytial virus (RSV), rhinovirus (RV), and F+ coliphage/MS2. We used two approaches to achieve this: we (1) conducted laboratory partitioning experiments using lab-grown viruses and (2) examined the distribution of endogenous viruses in wastewater. Partition experiments were conducted at 4{degrees}C and 22{degrees}C; wastewater samples were spiked with varying concentrations of each virus and stored for three hours to allow the system to equilibrate. Solids and liquids were separated via centrifugation and viral RNA concentrations were quantified using reverse-transcription-digital droplet PCR (RT-ddPCR). For the distribution experiment, wastewater samples were collected from six wastewater treatment plants and processed without spiking exogenous viruses; viral RNA concentrations were measured in wastewater solids and liquid. Overall, RNA concentrations were higher in solids than the liquid fraction of wastewater by approximately 3-4 orders of magnitude. Partition coefficients (KF) from laboratory experiments were determined using the Freundlich model and ranged from 2,000-270,000 ml{middle dot}g-1 across viruses and temperature conditions. Distribution coefficients (Kd) determined from endogenous wastewater viruses were consistent with results from laboratory experiments.Further research is needed to understand how virus and wastewater characteristics might influence the partition of viral genetic markers in wastewater. SynopsisWe examined the solid-liquid partitioning behavior of SARS-CoV-2, RSV, RV, and F+coliphage/MS2 RNA in wastewater influent. Overall, partition/distribution coefficients were similar across viruses and temperature conditions.

Coagulation, flocculation, and sedimentation (CFS) is widely applied as a combined unit process in the treatment of drinking water, wastewater, and recycled water; however, virus reduction through CFS has not been sufficiently characterized to assign pathogen log reduction value (LRV) credits. This study collected data through a systematic review that yielded over 1000 LRVs from 43 manuscripts covering 46 viruses to characterize virus reduction through CFS. The results demonstrate that CFS is effective at reducing viruses, with 68% of virus LRVs greater than 1. A mixed-effects model was used to identify potential mechanisms of virus reduction with ferric and aluminum coagulants, as well as factors associated with variability in performance. Key insights from the model show that virus reduction is: (1) improved at lower pH, similar to natural organic matter (NOM) reduction, (2) lower in secondary effluent than surface water for drinking water treatment, (3) virus-dependent, and (4) dependent on virus enumeration methods, with lower LRVs observed for molecular techniques. These findings demonstrate the potential for CFS to provide consistent and explainable virus reduction, potentially establishing a foundation for regulatory crediting in potable reuse applications. Future crediting frameworks will need to account for the factors impacting performance to accurately quantify and assign credit for virus reduction.

Noroviruses (NoV) are the leading cause of non-bacterial gastroenteritis across the globe with societal costs of US$60.3 billion per annum. Development of a long amplicon nanopore-based method for dual-typing the RNA-dependent RNA polymerase (RdRp) and major structural protein (VP1) regions from a single RNA fragment could improve existing norovirus typing methods. Its application to wastewater-based epidemiology (WBE) and environmental testing could enable the discovery of novel types and improve tracking throughout the population and into aquaculture and recreational water settings. Here, we develop and optimise such a method for wastewater as the sample matrix. Reverse transcription (RT), PCR and library pooling were optimised and a consensus-based bioinformatics pipeline was developed. Inhibitor removal and LunaScript(R) RT gave robust amplification of the {approx}1000 bp RdRP+VP1 amplicon. Platinum Taq polymerase showed good sensitivity and reduced levels non-specific amplification (NSA) when compared to other polymerases. Optimised PCR annealing temperatures significantly reduced NSA (51.3% and 42.4% for GI and GII), increased yield (86.5% for GII) and increased taxa richness (57.7%) for GII. Analysis of three NoV positive faecal samples showed 100% nucleotide similarity with Sanger sequencing. Eight GI genotypes, 11 polymerase types (p-types) and 13 combinations were detected in wastewater along with 4 GII genotypes, 4 p-types and 8 combinations; highlighting the diversity of norovirus taxa present in wastewater in England. The most common genotypes detected in clinical samples were all detected in wastewater while we also commonly detected several GI genotypes not reported in the clinical data. Application of this method into a WBE scheme, therefore, may allow for more accurate measurement of norovirus diversity within the population.

Interest in reusing treated wastewater drives efforts to eliminate antibiotics from water sources to prevent antibiotic resistance. Micro-aerated anaerobic membrane bioreactors (MA-AnMBR) promote wastewater reuse with high organic matter conversion to biogas, under a small footprint. However, the fates of antibiotics, antibiotic-resistant bacteria (ARB), and their antibiotic-resistance genes (ARGs) are not known in these systems. We studied the effects, conversions, and resistance induction, following the addition of 150 g{middle dot}L-1 of two antibiotics, sulfamethoxazole (SMX) and trimethoprim (TMP), in a laboratory-scale MA-AnMBR. TMP and SMX were removed at 97 and 86%, indicating that micro-aeration did not hamper the removal of the antibiotics. These antibiotics only affected the pH and biogas composition of the process, with a significant change in pH from 7.8 to 7.5, and a decrease in biogas CH4 content from 84 to 78%. TMP was rapidly adsorbed onto the sludge and subsequently degraded during the long retention of the solids of 27 days. SMX adsorption was minimal, but the applied hydraulic retention time of 2.6 days was sufficiently long to biodegrade SMX. The levels of three ARGs (sul1 and sul2 for SMX, dfrA1) and one mobile genetic element biomarker (intI1) were analysed by qPCR, in combination with ARB tracked by plating. Additions of the antibiotics increased the relative abundances of all ARGs and intI1 in the MA-AnMBR sludge, with the sul2 gene folding 15 times after 310 days of operation. The MA-AnMBR was able to reduce the concentration of ARB in the permeate by 3 log. HighlightsO_LIAdditions of SMX and TMP had a negligible effect on the MA-AnMBR performance. C_LIO_LIThe laboratory-scale MA-AnMBR removed 86% of SMX and 97% of TMP. C_LIO_LIA 3 log removal of ARB was achieved between sludge and UF permeate. C_LIO_LIRelative abundances of ARGs were similar in sludge and permeate. C_LIO_LITMP and SMX resistance is better assessed by the heterotrophic plate count of ARB. C_LI

1.Although cobalt (Co) is widely used in the transition to low-carbon energy technologies, its environmental impact remains almost unknown. This study examines Co impacts on the prokaryotic communities of river biofilms to assess their potential use as bioindicators of Co contamination. To that end, biofilms were grown on blank glass slides placed in artificial streams enriched with Co (0.1, 0.5 and 1 {micro}M Co) for 28 days and prokaryotic abundance and diversity were analyzed using DNA-metabarcoding every 7 days. The resilience of the prokaryotic community was investigated after a further 35 days without Co contamination. Prokaryotic communities were impacted by 0.5 and 1 {micro}M Co from the beginning of the biofilm colonization. Although biofilms reached similar biomasses regardless of Co concentration, control biofilms were dominated by Cyanobacteria and Planctomycetes while Bacteroidetes dominated Co contaminated biofilms. Potential functional redundancy was observed with the implementation of carbon fixation alternatives by non-photosynthetic prokaryotes in biofilms subjected to high Co concentrations. No structural resilience of the biofilms was observed after 35 days without Co contamination. The use of prokaryotic community response measured using molecular approaches appears to be a promising and cost-effective approach for assessing changes in water quality due to metals. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/592147v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@d72d70org.highwire.dtl.DTLVardef@e83c80org.highwire.dtl.DTLVardef@dc002borg.highwire.dtl.DTLVardef@18f481e_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisFew knowledge is available about Co ecotoxicity in freshwaters. This study assess the potential of prokaryotic communities developing in freshwater biofilms to be used as bioindicator of Co contamination.

Antimicrobial resistance (AMR) is a global health challenge and there is increasing recognition of the role of the environment, particularly wastewater, in the development and spread of AMR. Although trace metals are common contaminants in wastewater, the quantitative effects of trace metals on AMR in wastewater settings remain understudied. We experimentally determined the interactions between common antibiotic residues and metal ions found in wastewater and investigated their effects on the development of antibiotic resistance in Escherichia coli over time. These data were then used to expand on a previously developed computational model of antibiotic resistance development in continuous flow settings to incorporate the effects of trace metals acting in combination with multiple antibiotic residues. We found that the common metal ions, copper and iron, interact with both ciprofloxacin and doxycycline at wastewater relevant concentrations. This can significantly affect resistance development due to antibiotic chelation of the metal ions causing a reduction in the antibiotics bioactivity. Furthermore, modeling the effect of these interactions in wastewater systems showed the potential for metal ions in wastewater to significantly increase the development of antibiotic resistant E. coli populations. These results demonstrate the need to quantitatively understand the effects of trace metal-antibiotic interactions on AMR development in wastewater.

This study showcases the beneficial integration of non-sewered sanitation systems (NSSS) with sewered wastewater treatment plants (WWTPs). Treating increasing fractions of influent wastewater loads via six different types of NSSS offered correspondingly increasing savings in operating energy costs at five WWTPs, employing a broad range of typically employed treatment processes. Two NSSS that treat both greywater and blackwater (gb-HRT) and blackwater alone (b-HRT) yielded the highest savings in annual operating energy costs across most WWTPs. Distinctly, NSSS involving urine-separation with and without internal recirculation promoted energy-positive operations, by enhancing anaerobic digestion in selected WWTPs. At the highest NSSS coverage tested (treating 50% of the influent sewage), savings in annual sewered WWTPs operating energy costs ranged from $76k to $800k and increased further to the range $301k to $1.1M annually with process optimization. Therefore, integration of NSSS with sewered WWTPs can improve overall treatment efficiency, while facilitating resilient sanitation practices.

Moore swabs have been used extensively for passive sampling in wastewater surveillance, typically yielding presence/absence information for targets of interest. Quantitative analysis of Moore swab data is only possible if target uptake is well characterized, specifically the relationship between quantity of the target in the liquid sample matrix and the quantity of target sorbing to the Moore swab as a function of time. The mechanism of Moore swab absorption remains unclear and is important to understand toward using them more quantitatively. We conducted viral adsorption and desorption experiments using nonpathogenic SARS-CoV-2 surrogates: {Phi}6, MHV, and BCoV as well as heat-inactivated Zika virus (ZIKV). We fit empirical adsorption data from batch experiments to Langmuir, Freundlich and Redlich-Peterson isotherm models. We observed the adsorption behavior of viral targets onto Moore swabs is best characterized by the Redlich-Peterson isotherm model. Moore swabs retained the highest viral RNA concentrations after exposure durations between 9-12 hours in the presence of target microbes during kinetic viral adsorption experiments. The results inform current and future use of Moore swabs to produce quantitative data during wastewater surveillance, especially in settings where composite sampling remains infeasible. ImportanceThis paper describes the adsorption behavior of viruses and bacteriophages to Moore swabs. Passive sampling via Moore swabs is among the most scalable form of passive wastewater sampling, considered critical to advance wastewater surveillance globally. But key unknowns constrain the utility of Moore swabs and all passive sampling approaches, including the quantitative relationship between targets in wastewater and recovery via Moore swabs. Practical questions such as how long they should be deployed and whether they can be interpreted quantitatively really depend on a characterization of viral target loading behaviors on Moore swab material as a function of time and concentration in the wastewater. Here, we use an approach that is seldom applied to microbial targets to examine adsorption behavior of viruses to Moore swabs, deriving isotherms that describe the relationships between concentration of the viral targets in wastewater and time on attachment to swab material. This is a critical step in advancing the application of Moore swab passive sampling for wastewater surveillance, with potential relevance to other microbial targets of interest.

Wastewater-based epidemiology (WBE) has proven to be a powerful tool for tracking the spread of viral pathogens, such as SARS-CoV-2, but as WBE has expanded to include new pathogens, such as mpox virus, more data is needed to guide practitioners on how to design WBE campaigns. Here, we investigated the decay rates of heat-inactivated mpox (HI-MPXV) and attenuated vaccinia virus (VV) in primary influent and settled solids collected from a local POTW at 4{degrees}C, 22{degrees}C, or 35{degrees}C using digital PCR. Subsequently, we studied the solid-liquid partitioning of the viruses in primary influent. Over the 30-day study period, we observed no significant difference in log-linear decay rates between viruses (p=0.5258), with significantly higher decay rates in primary influent (0.109-0.144/day) compared to settled solids (0.019-0.040/day) at both 22{degrees}C (p=0.0030) and 35{degrees}C (p=0.0166). Furthermore, as part of the partitioning experiment, we found that HI-MPXV and VV adsorb to the solids fraction of primary influent at higher intensities than previously studied enveloped viruses (KF = 1,000-31,800 mL/g, n = 1.01-1.41). Likewise, it was determined in the partitioning experiment that a concentration of greater than 103 gc/mL in raw influent was needed for the viable quantification of mpox and vaccinia viruses in the clarified liquid fraction of raw primarily influent. Our study provides essential insights into informative sample collection and storage conditions for the analysis of wastewater and for transport modeling studies. Due to the slow decay observed in settled solids at all tested temperatures in the persistence experiment, this matrix may be most suitable for retrospective analyses of community infection of the mpox virus.

This study focused on the development of machine-learning- (ML) based strategies for mitigating nitrous oxide (N2O) emissions from various wastewater treatment systems in the United States measured using a benchmark USEPA-endorsed protocol. Results revealed that in general, poor process performance correlated with higher N2O emissions. Specifically, local variables including zone-specific dissolved oxygen, ammonia, and nitrite concentrations and global variables including effluent nitrite and nitrate concentrations contributed positively towards N2O emissions from both aerobic and anoxic zones of the process bioreactors. The optimal operational conditions identified for minimizing N2O emissions included operation of aerobic and anoxic zones at DO < 4 mg O2 L-1 and < 1 mg O2 L-1, respectively, coupled with appropriate solids retention times (SRTs) that maximize process performance. Accordingly, our results strongly underscore the utility of ML models in combination with bioprocess fundamentals for predicting and mitigating N2O emissions, while concomitantly optimizing wastewater treatment operations.

Sulfidogenesis is a promising technology for the selective recovery of chalcophile bulk metals (e.g. Cu, Zn, and Co) from metal-contaminated waters such as acid mine drainage (AMD) and metallurgy waste streams. The use of elemental sulfur (S0) instead of sulfate (SO42-) as electron acceptor reduces electron donor requirements four-fold, lowering process costs, and expands the range of operating conditions to more acidic pH. We previously reported autotrophic S0 reduction using an industrial mesophilic granular sludge as inoculum under thermoacidophilic conditions. Here, we examined the effect of pH on the S0 reduction performance of the same inoculum, in a continuously fed gas-lift reactor run at 30 {degrees}C under neutral (pH 6.9) and acidic (pH 3.8) conditions. Steady-state volumetric sulfide production rates (VSPR) dropped 2.3-fold upon transition to acidic pH, from 1.79 {+/-} 0.18 g{middle dot}L-1{middle dot}d-1 S2-{middle dot}to 0.71 {+/-} 0.07 g{middle dot}L-1{middle dot}d-1 S2-{middle dot} Microbial community analysis via 16S rRNA gene amplicon sequencing showed that at pH 6.9, the S0-reducing genera Sulfurospirillum, Sulfurovum, Desulfurella, and Desulfovibrio were present at the highest relative abundance, while at pH 3.9 Desulfurella dominated the sequenced reads. The detection of acetic acid and the relative abundance of Acetobacterium at pH 6.9 pointed towards acetogenesis, explaining the dominance of the heterotrophic genus Sulfurospirillum in this H2 and CO2-fed bioreactor.

Enteric viruses are a leading cause of waterborne illness worldwide and surveillance studies lack standardization in method selection. The most common and cost-effective approach to concentrating viruses from water samples involves virus adsorption and elution (VIRADEL) procedures, followed by secondary concentration. There is a lack of consistency in how secondary concentration methods are practiced and some methods may have better recovery for particular groups of viruses. Secondary concentration methods typically involve precipitation and the most common methods employ organic flocculation (OF) by acidification at a pH of 3.5, or precipitation by polyethylene glycol (PEG) in combination with the addition of NaCl. In this study, the recovery of coliphage MS2 using the plaque assay and human adenovirus strain 41 (HAdV41) using cell-culture and qPCR assays were evaluated by OF and PEG secondary concentration of spiked samples of wastewater, surface water, and groundwater. The recovery of MS2 and HAdV41 by PEG precipitation was significantly higher than that by OF (p<0.0001) when viruses were detected by culture based methods and marginally better when HAdV41 was enumerated by qPCR (p<0.019). The recovery of HAdV41 by qPCR ranged from 75.3% to 94.4% (n=36). The mean recovery of MS2 by OF was 4.4% (0.9%-7.7%; n=14) and ranged from 57.1% to 87.9% (n=28) for the PEG methods. The poor recovery of MS2 by OF was attributed to inactivation or poor stability at acidic conditions as MS2 were not recovered in the supernatant following OF and centrifugation. The inconsistency and lack of justification for method selection in many studies calls for a systematic study to inform guidance and standardization with respect to the application of concentration methods for various water types and viral pathogens. IMPORTANCEMS2 should not be used as a process control for methods involving acidification and culture-based detection. The dense floc produced by the PEG method may have contributed to higher recoveries as the pellet was more compact and stable than the loose pellet formed by OF. Standard methods for the detection of enteric viruses and surrogates that involve acidification could be modified with PEG precipitation to uphold virus recovery and minimize inactivation.

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proven a practical complement to clinical data for assessing community-scale infection trends. Clinical assays, such as the CDC-promulgated N1, N2, and N3 have been used to detect and quantify viral RNA in wastewater but, to date, have not included estimates of reliability of true positive or true negative. Bayes Theorem was applied to estimate Type I and Type II error rates for detections of the virus in wastewater. Conditional probabilities of true positive or true negative were investigated when one assay was used, or multiple assays were run concurrently. Cumulative probability analysis was used to assess the likelihood of true SARS-CoV-2 detection using multiple samples. Results demonstrate highly reliable positive (>0.86 for priors >0.25) and negative (>0.80 for priors = 0.50) results using a single assay. Using N1 and N2 concurrently caused greater reliability (>0.99 for priors <0.05) when results concurred but generated potentially counterintuitive interpretations when results were discordant. Regional wastewater surveillance data was investigated as a means of setting prior probabilities. Probability of true detection with a single marker was investigated using cumulative probability across all combinations of positive and negative results for a set of three samples. Findings using a low (0.11) and uniformed (0.50) initial prior resulted in high probabilities of detection (>0.95) even when a set of samples included one or two negative results, demonstrating the influence of high sensitivity and specificity values. Analyses presented here provide a practical framework for understanding analytical results generated by wastewater surveillance programs.

BackgroundWastewater monitoring is increasingly used for community surveillance of infectious diseases, especially after the COVID-19 pandemic as the genomic footprints of pathogens shed by infected individuals can be traced in the environment. However, detection and concentration of pathogens in the environmental samples and their efficacy in predicting infectious diseases can be influenced by meteorological conditions and quality of samples. ObjectivesThis research examines whether meteorological conditions and sample pH affect SARS-CoV-2 concentrations in wastewater samples, and whether the association of SARS-CoV-2 with COVID-19 cases and mortality improves when adjusted for meteorological conditions and sample pH value in Miami-Dade County, FL. MethodsDaily wastewater samples were collected from Miami-Dade Wastewater Treatment Plant in Key Biscayne, Florida from August 2021 to August 2022. The samples were analyzed for pH and spiked with OC43. RNA was extracted from the concentrated wastewater sample and SARS-CoV-2 was quantified using qPCR. COVID-19 and mortality data were acquired from the Centers for Disease Control and Prevention (CDC) and meteorological data from the National Climatic Data Center. COVID-19 case and mortality rates were modelled with respect to time-lagged wastewater SARS-CoV-2 adjusting for meteorological conditions, and sample pH value and OC43 recovery. ResultsTemperature, dew point, pH values and OC43 recovery showed significant associations with wastewater SARS-CoV-2. Time-lagged wastewater SARS-CoV-2 showed significant associations with COVID-19 case and mortality incidence rates. This association improved when wastewater SARS-CoV-2 levels were adjusted for (or instrumented on) meteorological conditions, OC43 recovery, and sample pH. A 0.47% change in COVID-19 case incidence rate was associated with 1% change in wastewater SARS-CoV-2 ({beta} [~] 0.47; 95% CI = 0.29 - 0.64; p < 0.001). A 0.12 % change in COVID-19 mortality rate was associated with 1 % change in SARS-CoV-2 in wastewater 44 days prior. A 0.07% decline in COVID-19 mortality rate was associated with a unit increase in ambient temperature 28 days prior. DiscussionTime lagged wastewater SARS-CoV-2 (and its adjustment for sample pH and RNA recovery) and meteorological conditions can be used for the surveillance of COVID-19 case and mortality. These findings can be extrapolated to improve the surveillance of other infectious diseases by proactive measurements of infectious agent(s) in the wastewater samples, adjusting for meteorological conditions and sample pH value.

Subtropical climate in Florida offers a unique opportunity for sustainable biofuel production using native microalgae cultivated in untreated wastewater. This study bioprospected oleaginous microalgal consortia from a wastewater holding tank at the Thomas P. Smith Water Reclamation Facility in Tallahassee, Florida, aiming to identify strains capable of both nutrient remediation and lipid accumulation. Using Fluorescence Activated Cell Sorting (FACS), chlorophyll-containing cells were isolated and cultured on BG-11 media. Shotgun metagenomics revealed that the most robust consortia- labeled C3, C4, and C9, were dominated by Chlamydomonas, Acutodesmus, and Volvox spp., alongside diverse bacterial, fungal, and archaeal communities. Functional gene analysis indicated active pathways for photosynthesis, lipid biosynthesis, and nutrient assimilation. In microcosm experiments, these consortia achieved up to 100% ammonia, 95% phosphorus, and 89% nitrate removal, outperforming control treatments. Lipid screening confirmed significant accumulation, with consortium C9 showing the highest yield. These findings underscore the potential of native microalgal consortia for integrated wastewater treatment and biofuel production, advancing circular bioeconomy strategies for subtropical regions.

Coffee processing wastewater (CPW), a byproduct of agro-industrial operations, contains high organic loads alongside recalcitrant and potentially inhibitory compounds such as caffeine and tannins. This study evaluated the performance of micro-aeration-enhanced anaerobic digestion (MA-AD) for the treatment and valorization of CPW to promote a more sustainable approach to coffee production. Oxygen was intermittently introduced via oxidation-reduction potential-controlled dosing, allowing for comparative assessment across anaerobic and micro-aerobic redox regimes. While both conventional anaerobic digestion (AD) and MA-AD achieved comparable reductions in total and volatile solids (>48% and >60%, respectively) and total and soluble chemical oxygen demand (>66% and >86%, respectively), MA-AD exhibited significantly higher total suspended solids concentrations and turbidity in later phases, likely due to gas sparging-induced floc disruption and particulate release. pH profiles indicated a shift toward increased acidification under MA-AD, without compromising process stability, with both reactors stabilizing between pH 6.8-7.1. Caffeine degradation was accelerated under MA-AD in the first dosing phase (>85% removal in 28 h), though long-term degradation efficiency converged with the control. Methane production was consistently lower in MA-AD (up to 43% reduction), attributed to the oxygen sensitivity of methanogens and possible substrate competition. These results underscore the importance of oxygen dose regulation, redox control, and microbial adaptation in optimizing MA-AD performance. The findings support MA-AD as a promising strategy for enhancing hydrolysis and partial removal of recalcitrant compounds in CPW. However, further refinement is required to sustain biogas quality and yield at scale.

Fermentative chemoorganoheterotrophic bacteria (FCB) and purple photoorganoheterotrophic bacteria (PPB) are two interesting microbial guilds to process carbohydrate-rich wastewaters. Their interaction has been studied in axenic pure cultures or co-cultures. Little is known about their metabolic interactions in open cultures. We aimed to harness the competitive and syntrophic interactions between PPB and FCB in mixed cultures. We studied the effect of reactor regimes (batch or continuous, CSTR) and illumination modes (continuous irradiation with infrared light, dark, or light/dark diel cycles) on glucose conversions and the ecology of the process. In batch, FCB outcompeted (>80%) PPB, under both dark and infrared light conditions. In CSTR, three FCB populations of Enterobacteriaceae, Lachnospiraceae and Clostridiaceae were enriched (>70%), while Rhodobacteraceae relatives of PPB made 30% of the community. Fermentation products generated from glucose were linked to the dominant FCB. Continuous culturing at a dilution rate of 0.04 h-1 helped maintain FCB and PPB in syntrophy: FCB first fermented glucose into volatile fatty acids and alcohols, and PPB grew on fermentation products. Direct supply of carboxylates like acetate under infrared light enriched for PPB (60%) independent of reactor regimes. Ecological engineering of FCB- and PPB-based biorefineries can help treat and valorize carbohydrate-based waste feedstocks. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/444055v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1ee41forg.highwire.dtl.DTLVardef@12d74aorg.highwire.dtl.DTLVardef@f46351org.highwire.dtl.DTLVardef@a47706_HPS_FORMAT_FIGEXP M_FIG C_FIG

Digital PCR (dPCR) shows great promise for precise, sensitive, and inhibition-resilient measurement of nucleic acids in surface water, providing an advantage for applications such as microbial source tracking (MST). Herein, we describe our empirical optimization of two triplex format dPCR reactions (Triplex 1 - Cow M2, Rum2Bac, Cow M3; Triplex 2 - HF183/BacR287, Pig2Bac, Entero1a) on the QIAcuity One system for MST. Each triplex produced gene copy measurements similar to single-plex format assays for a standard reference material (SRM 2917) at a fixed concentration and along a concentration gradient. For achieved water samples previously tested by single-plex assays, each triplex also produced MST marker measurements comparable to the single-plex results. The triplexes described here can be directly adopted for MST on the QIAcuity, or the optimization protocol we demonstrate can be used to develop additional multiplex assays on the QIAcuity system.

Concentrations of nucleic acids from a range of respiratory viruses including human influenza A and B, respiratory syncytial virus (RSV), metapneumovirus, parainfluenza virus, rhinovirus, and seasonal coronaviruses in wastewater solids collected from wastewater treatment plants correlate to clinical data on disease occurrence in the community contributing to the wastewater. Viral nucleic acids enter wastewater from various excretions including stool, urine, mucus, sputum, and saliva deposited in toilets or other drains in buildings. In order to relate the measured concentrations in wastewater at a treatment plant to actual number of infections in a community, concentrations of the viral nucleic acids in these human excretions are needed as inputs to a mass balance model. In this study, we carried out a systematic review and meta-analysis to characterize the concentrations and presence of influenza A and B, respiratory syncytial virus (RSV), metapneumovirus, parainfluenza virus, rhinovirus, and seasonal coronaviruses in stool, urine, mucus, sputum, and saliva. The systematic review protocol can be accessed at https://doi.org/10.17605/OSF.IO/ESVYC. We identified 220 data sets from 50 unique articles that met inclusion criteria and reported information on viral concentrations and presence in these excretions. Data were unevenly distributed across virus type (with the most available for influenza) and excretion type (with the most available for respiratory excretions). The majority of data sets only reported the presence or absence of the virus in an excretion in a cross-sectional study design. There is a need for more concentration data, including longitudinal data, across all respiratory virus and excretion types. Such data would allow quantitatively linking virus wastewater concentrations to numbers of infected individuals.