Two distinct Dehalobacter metagenome-assembled genomes from anaerobic chloroform and dichloromethane degrading consortia.

Here we present two metagenomes and two Dehalobacter metagenome-assembled genomes from subcultures of an anaerobic chloroform and dichloromethane degrading microbial community used for bioremediation. Our objective was to assemble and curate the genome(s) of Dehalobacter, key biodegraders in the culture, through repeated sequencing and joint assembly with previous datasets.

Changes in microbial communities during high-rate microbial selenate reduction in an up-flow anaerobic fluidized bed reactor.

Biological fluidized bed reactor (FBR) is a promising treatment option for removing selenium oxyanions from wastewater by converting them into elemental selenium. The process can achieve high rates and be efficiently operated at low hydraulic retention times (HRT). However, the effects of HRT on the changes in microbial community in the FBR process have not been previously explored. In this study, dynamic changes of microbial communities both on biofilm carrier and in suspension of a selenate-reducing FBR were explored at various HRTs (0.3-120 h). Based on partial 16S rRNA gene sequencing of the microbial communities, alpha diversity of microbial communities in suspension rather than in the biofilm were impacted by low HRTs (0.3 h-3 h). Members from genera Geobacter, Geoalkalibacter, and Geovibrio were the main selenate-reducing bacteria on carrier throughout the FBR process. Genus Geobacter was dominant in FBR carrier at HRT of 24 h-120 h, whereas Geoalkalibacter and Geovibrio dominated at low HRT of 0.3 h-6 h. Suspended microbial communities detected in the FBR effluent were more sensitive to HRT changes than that in biofilm. "Shock loading" at HRT of 0.3 h had a great impact on microbial community compositions both in the biofilm and effluent. Reactor operation in batch mode and long HRT of 24 h helped recover the community from "shock loading" and improved selenite reduction and ethanol oxidation. Redundancy analysis revealed that HRT, influent pH and selenate loading were key operational parameters impacting both the FBR performance and the composition of microbial communities associated with both the FBR carrier and effluent. Overall, the microbial communities in FBR biofilm flexibly responded to the changes of HRT and showed resilience to the temporary shock loading, enabling efficient selenate removal.

Metagenome from ACT-3/CF: an anaerobic chloroform-degrading microbial community.

Here, we present the metagenome of an anaerobic chloroform respiring mixed microbial community used for bioremediation. Our objective was to obtain draft genomes of key microorganisms in the culture.

Metagenomic and genomic sequences from a nitrate-reducing benzene-degrading enrichment culture.

Metagenome-assembled genomes (MAGs) were recovered from metagenomic assemblies from a nitrate-reducing benzene-degrading enrichment culture. Ten MAGs of high quality or functional interest to benzene degradation are reported, seven of which are single contig genomes.

Draft genome of an anaerobic nitrate-reducing, benzene-degrading member of the order Thermincolales.

We present a metagenome assembled genome (MAG) of an anaerobic bacterium from a nitrate-reducing, benzene-degrading enrichment culture (NRBC). The draft Thermincolales genome consists of 20 contigs with a total length of 4.09 Mbp and includes putative carboxylase genes likely involved in benzene activation.

Vitamin B12 as a source of variability in isotope effects for chloroform biotransformation by Dehalobacter.

Carbon and chlorine isotope effects for biotransformation of chloroform by different microbes show significant variability. Reductive dehalogenases (RDase) enzymes contain different cobamides, affecting substrate preferences, growth yields, and dechlorination rates and extent. We investigate the role of cobamide type on carbon and chlorine isotopic signals observed during reductive dechlorination of chloroform by the RDase CfrA. Microcosm experiments with two subcultures of a Dehalobacter-containing culture expressing CfrA-one with exogenous cobamide (Vitamin B12, B12+) and one without (to drive native cobamide production)-resulted in a markedly smaller carbon isotope enrichment factor (εC, bulk) for B12- (-22.1 ± 1.9‰) compared to B12+ (-26.8 ± 3.2‰). Both cultures exhibited significant chlorine isotope fractionation, and although a lower εCl, bulk was observed for B12- (-6.17 ± 0.72‰) compared to B12+ (-6.86 ± 0.77‰) cultures, these values are not statistically different. Importantly, dual-isotope plots produced identical slopes of ΛCl/C (ΛCl/C, B12+ = 3.41 ± 0.15, ΛCl/C, B12- = 3.39 ± 0.15), suggesting the same reaction mechanism is involved in both experiments, independent of the lower cobamide bases. A nonisotopically fractionating masking effect may explain the smaller fractionations observed for the B12- containing culture.

Metagenomic sequences from anaerobic chloroform and dichloromethane degrading microbial communities.

Here, we present metagenomes from two cultures derived from an anaerobic microbial consortium used for bioremediation. One culture dechlorinates chloroform to dichloromethane, which is further mineralized to CO2. A second subculture was amended with only dichloromethane. We sought draft genomes of key microorganisms to identify metabolic potential in these consortia.

SARS-CoV-2 viral titer measurements in Ontario, Canada wastewaters throughout the COVID-19 pandemic.

During the COVID-19 pandemic, the Province of Ontario, Canada, launched a wastewater surveillance program to monitor SARS-CoV-2, inspired by the early work and successful forecasts of COVID-19 waves in the city of Ottawa, Ontario. This manuscript presents a dataset from January 1, 2021, to March 31, 2023, with RT-qPCR results for SARS-CoV-2 genes and PMMoV from 107 sites across all 34 public health units in Ontario, covering 72% of the province's and 26.2% of Canada's population. Sampling occurred 2-7 times weekly, including geographical coordinates, serviced populations, physico-chemical water characteristics, and flowrates. In doing so, this manuscript ensures data availability and metadata preservation to support future research and epidemic preparedness through detailed analyses and modeling. The dataset has been crucial for public health in tracking disease locally, especially with the rise of the Omicron variant and the decline in clinical testing, highlighting wastewater-based surveillance's role in estimating disease incidence in Ontario.

Compound-Specific Carbon, Nitrogen, and Hydrogen Isotope Analysis to Characterize Aerobic Biodegradation of 2,3-Dichloroaniline by a Mixed Enrichment Culture.

Compound-specific isotope analysis (CSIA) is an established tool to track the in situ transformation of organic chemicals at contaminated sites. In this work, we evaluated the potential of multi-element CSIA to assess biodegradation of 2,3-dichloroaniline (2,3-DCA), which is a major industrial feedstock. Using controlled laboratory experiments, we determined, for the first time, negligible carbon (<0.5‰) and hydrogen (<10‰) isotope fractionation and a significant inverse nitrogen isotope fractionation (>10‰) during aerobic 2,3-DCA biodegradation by a mixed enrichment culture. The tentative identification of a glutamate conjugate of 2,3-DCA as a reaction intermediate indicates that the initial multistep enzymatic reaction may be rate-limiting. The formation of the glutamate adduct would increase the bond energy at the N atom, thus likely explaining the observed inverse N isotope fractionation. The corresponding nitrogen enrichment factor was +6.8 ± 0.6‰. This value was applied to investigate the in situ 2,3-DCA biodegradation at a contaminated site where the carbon and nitrogen isotope signatures from field samples suggested similar aerobic processes by native microorganisms. Under the assumption of the applicability of the Rayleigh model in a pilot wetland treating contaminated groundwater, the extent of biodegradation was estimated to be up to 80-90%. This study proposes multi-element CSIA as a novel application to study 2,3-DCA fate in groundwater and surface water and provides insights into biodegradation pathways.

From mec cassette to rdhA: a key Dehalobacter genomic neighborhood in a chloroform and dichloromethane-transforming microbial consortium.

Chloroform (CF) and dichloromethane (DCM) are groundwater contaminants of concern due to their high toxicity and inhibition of important biogeochemical processes such as methanogenesis. Anaerobic biotransformation of CF and DCM has been well documented but typically independently of one another. CF is the electron acceptor for certain organohalide-respiring bacteria that use reductive dehalogenases (RDases) to dechlorinate CF to DCM. In contrast, known DCM degraders use DCM as their electron donor, which is oxidized using a series of methyltransferases and associated proteins encoded by the mec cassette to facilitate the entry of DCM to the Wood-Ljungdahl pathway. The SC05 culture is an enrichment culture sold commercially for bioaugmentation, which transforms CF via DCM to CO2. This culture has the unique ability to dechlorinate CF to DCM using electron equivalents provided by the oxidation of DCM to CO2. Here, we use metagenomic and metaproteomic analyses to identify the functional genes involved in each of these transformations. Though 91 metagenome-assembled genomes were assembled, the genes for an RDase-named acdA-and a complete mec cassette were found to be encoded on a single contig belonging to Dehalobacter. AcdA and critical Mec proteins were also highly expressed by the culture. Heterologously expressed AcdA dechlorinated CF and other chloroalkanes but had 100-fold lower activity on DCM. Overall, the high expression of Mec proteins and the activity of AcdA suggest a Dehalobacter capable of dechlorination of CF to DCM and subsequent mineralization of DCM using the mec cassette. IMPORTANCE: Chloroform (CF) and dichloromethane (DCM) are regulated groundwater contaminants. A cost-effective approach to remove these pollutants from contaminated groundwater is to employ microbes that transform CF and DCM as part of their metabolism, thus depleting the contamination as the microbes continue to grow. In this work, we investigate bioaugmentation culture SC05, a mixed microbial consortium that effectively and simultaneously degrades both CF and DCM coupled to the growth of Dehalobacter. We identified the functional genes responsible for the transformation of CF and DCM in SC05. These genetic biomarkers provide a means to monitor the remediation process in the field.

Identification of environmental and methodological factors driving variability of Pepper Mild Mottle Virus (PMMoV) across three wastewater treatment plants in the City of Toronto.

PMMoV has been widely used to normalize the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, influenza, and respiratory syncytial virus (RSV) to account for variations in the fecal content of wastewater. PMMoV is also used as an internal RNA recovery control for wastewater-based epidemiology (WBE) tests. While potentially useful for the interpretation of WBE data, previous studies have suggested that PMMoV concentration can be affected by various physico-chemical characteristics of wastewater. There is also the possibility that laboratory methods, particularly the variability in centrifugation steps to remove supernatant from pellets can cause PMMoV variability. The goal of this study is to improve our understanding of the main drivers of PMMoV variability by assessing the relationship between PMMoV concentration, the physico-chemical characteristics of wastewater, and the methodological approach for concentrating wastewater samples. We analyzed 24-hour composite wastewater samples collected from the influent stream of three wastewater treatment plants (WWTPs) located in the City of Toronto, Ontario, Canada. Samples were collected 3 to 5 times per week starting from the beginning of March 2021 to mid-July 2023. The influent flow rate was used to partition the data into wet and dry weather conditions. Physico-chemical characteristics (e.g., total suspended solids (TSS), biological oxygen demand (BOD), alkalinity, electrical conductivity (EC), and ammonia (NH3)) of the raw wastewater were measured, and PMMoV was quantified. Spatial and temporal variability of PMMoV was observed throughout the study period. PMMoV concentration was significantly higher during dry weather conditions. Multiple linear regression analysis demonstrates that the number and type of physico-chemical parameters that drive PMMoV variability are site-specific, but overall BOD and alkalinity were the most important predictors. Differences in PMMoV concentration for a single WWTP between two different laboratory methods, along with a weak correlation between pellet mass and TSS using one method may indicate that differences in sample concentration and subjective subsampling bias could alter viral recovery and introduce variability to the data.

Detection of a Human Adenovirus Outbreak, Including Some Critical Infections, Using Multipathogen Testing at a Large University, September 2022-January 2023.

Background: Human adenoviruses (HAdVs) can cause outbreaks of flu-like illness in university settings. Most infections in healthy young adults are mild; severe illnesses rarely occur. In Fall 2022, an adenovirus outbreak was identified in university students. Methods: HAdV cases were defined as university students 17-26 years old who presented to the University Health Service or nearby emergency department with flu-like symptoms (eg, fever, cough, headache, myalgia, nausea) and had confirmed adenovirus infections by polymerase chain reaction (PCR). Demographic and clinical characteristics were abstracted from electronic medical records; clinical severity was categorized as mild, moderate, severe, or critical. We performed contact investigations among critical cases. A subset of specimens was sequenced to confirm the HAdV type. Results: From 28 September 2022 to 30 January 2023, 90 PCR-confirmed cases were identified (51% female; mean age, 19.6 years). Most cases (88.9%) had mild illness. Seven cases required hospitalization, including 2 critical cases that required intensive care. Contact investigation identified 44 close contacts; 6 (14%) were confirmed HAdV cases and 8 (18%) reported symptoms but never sought care. All typed HAdV-positive specimens (n = 36) were type 4. Conclusions: While most students with confirmed HAdV had mild illness, 7 otherwise healthy students had severe or critical illness. Between the relatively high number of hospitalizations and proportion of close contacts with symptoms who did not seek care, the true number of HAdV cases was likely higher. Our findings illustrate the need to consider a wide range of pathogens, even when other viruses are known to be circulating.

Underestimation of SARS-CoV-2 in wastewater due to single or double mutations in the N1 qPCR probe binding region.

Wastewater surveillance using RT-qPCR has now been widely adopted to track circulating levels of SARS-CoV-2 virus in many sewersheds. The CDC qPCR assays targeting two regions (N1 and N2) within the N gene are commonly used, but a discrepancy between the two biomarkers has been noticed by independent studies using these methods since late 2021. The reason is presumed to be due to mutations in regions targeted by the N1 qPCR probe. In this study, we systematically investigated and unequivocally confirmed that the underlying reason for this discrepancy was mutations in the N1 probe target, and that a single mutation could cause a significant drop in signal. We first confirmed the proportion of related mutations in wastewater samples (Jan 2021-Dec 2022) using nested PCR and LC-MS. Based on relative proportions of N1 alleles, we separated the wastewater data into four time periods corresponding to different variant waves: Period I (Alpha and Delta waves with 0 mutation), Period II (BA.1/BA.2 waves with a single mutation found in all Omicron strains), Period III (BA.5.2* wave with two mutations), and Period IV (BQ.1* wave with two mutations). Significantly lower N1 copies relative to N2 copies in samples from Periods II-IV compared to those from Period I was observed in wastewater. To further pinpoint the extent to which each mutation impacted N1 quantification, we compared the qPCR response among different synthetic oligomers with corresponding mutations. This study highlighted the impact of even just one or two mutations on qPCR-based wastewater surveillance of SARS-CoV-2.

Inhibitory Control Training for Anxiety and Math Achievement in Primary School Children: Protocol for a Proof-of-Concept Study.

BACKGROUND: Cognitive control training (CCT) has shown potential to reduce emotional vulnerability in adults and adolescents. However, there is scant literature testing the efficacy of CCT for the reduction of anxiety and transferring the effects to educational outcomes in children. Building on the evidence that a greater ability to suppress a prepotent response (inhibitory control) is associated with higher math achievement in children, it is plausible that training inhibitory processes using a CCT paradigm may be beneficial for reducing anxiety, improving inhibitory control, and in turn increasing math achievement. OBJECTIVE: This proof-of-concept study aims to investigate the efficacy of 15 sessions of inhibitory control training for reduction in anxiety and improvement in math achievement in primary school children. METHODS: We will use a 2 (group: CCT, adaptive Go/No-Go vs active control, low-load task) multiplied by 4 (time: pre- vs posttraining vs 1-month vs 3-month follow-up) randomized design in a nonselected sample of 100 children aged 8-10 years. Both groups will complete 10 minutes of daily training for 3 weeks at school. The dependent variables will be anxiety and correlates (Spence Children's Anxiety Scale, Penn State Worry Questionnaire for Children, Revised Children's Anxiety and Depression Scale, Child Response Style Questionnaire, and Modified Abbreviated Math Anxiety Scale), inhibitory control (Go/No-Go task), shifting (color-shape shifting task), updating (n-back task), and math achievement (Applied Problems, Calculation, and Math Facts Fluency subtests from the Woodcock-Johnson IV Tests of Achievement). RESULTS: We opened enrollment in September 2023. The initial results are expected to be published in late 2024. We predict that children in the CCT group will show a reduction in emotional symptoms; improvements in inhibition, shifting, and updating performance; and advances in math achievement from pre- to posttraining, and that these effects will be maintained at 1- and 3-month follow-ups, compared to children in the active control group. CONCLUSIONS: The CCT paradigm used in our study will provide a greater understanding of the emotional and cognitive transfer effects on children and inform future work. Specifically, the findings will advance the knowledge of deploying inhibitory control training with children and provide valuable insights into its use for reducing anxiety and advancing math achievement. TRIAL REGISTRATION: Open Science Framework ofs.io/de2qa; https://doi.org/10.17605/OSF.IO/DE2QA. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/52929.

Functional screening pipeline to uncover laccase-like multicopper oxidase enzymes that transform industrial lignins.

Laccase-like multicopper oxidases are recognized for their potential to alter the reactivity of lignins for application in value-added products. Typically, model compounds are employed to discover such enzymes; however, they do not represent the complexity of industrial lignin substrates. In this work, a screening pipeline was developed to test enzymes simultaneously on model compounds and industrial lignins. A total of 12 lignin-active fungal multicopper oxidases were discovered, including 9 enzymes active under alkaline conditions (pH 11.0). Principal component analysis revealed the poor ability of model compounds to predict enzyme performance on industrial lignins. Additionally, sequence similarity analyses grouped these enzymes with Auxiliary Activity-1 sub-families with few previously characterized members, underscoring their taxonomic novelty. Correlation between the lignin-activity of these enzymes and their taxonomic origin, however, was not observed. These are critical insights to bridge the gap between enzyme discovery and application for industrial lignin valorization.

The Effect of Varying Fatty Acid Composition on Postprandial Airway Inflammation, Pulmonary Function, and Airway Resistance in Healthy, Young Adults.

OBJECTIVE: To examine the effect of varying fatty acid composition in a HFM on eNO, pulmonary function, and airway resistance. METHODS: Fifteen individuals [6 M/9 F; 21.9 ± 1.5 years old] each completed three HFM conditions {SF, O6FA, and O3FA; 12 kcal/kg body weight, 63% total fat, and 0.72 g/kg sugar smoothies} in random order separated by at least 48 h. Airway inflammation assessed via eNO, pulmonary function measured using the maximum flow volume loop (MFVL) and airway resistance measured using impulse oscillometry (iOS) were taken at baseline, 2h and 4h postprandially. RESULTS: There was no difference in eNO or iOS across time in any condition or between conditions (p > 0.05). There was a significant time by condition effect for FEV1 post-HFM in the SF and O6FA conditions (p < 0.05). CONCLUSION: Different fatty acid compositions do not increase eNO or iOS in healthy, college-aged participants after consumption of a HFM, though the minimally processed meals with fruit added may contribute to these findings.

Metagenomes and metagenome-assembled genomes from a sequentially fed anaerobic digester treating solid organic municipal waste.

We present a data set of four metagenomes and 281 metagenome-assembled genomes describing the microbial community of a laboratory-scale high solids anaerobic digester. Our objective was to obtain information on the coding potential of the microbial community and draft genomes of the most abundant organisms in the digester.

Depression Moderates the Relationship between Trait Anxiety, Worry and Attentional Control in Melanoma Survivors.

Cancer survivors commonly contend with concurrent cognitive difficulties such as problems with attention and concentration, and psychological distress, including anxiety and depression. However, the associations between attentional and emotional difficulties within the specific context of melanoma survivors remain relatively unexplored. Premised on attentional control theory, the current study employed a cross-sectional design to explore the interplay among trait anxiety (dispositional) and situational anxiety (cancer-related worry), depression and attentional control (ability to inhibit distractors and flexibly shift within and between tasks) in a sample of 187 melanoma survivors aged 18 to 58 years (Mage = 36.83 years, SDage = 5.44 years; 93% female). Data were analyzed using a moderated multiple regression, with anxiety, cancer worry and depression as predictors, and attentional control as the criterion variable. After statistically controlling for the variance of chemotherapy, we found that individuals with higher trait anxiety and higher cancer-related worry reported greater attentional control at low levels of depression, yet poorer attentional control at high depression, relative to individuals with low anxiety. Our findings suggest that anxiety and depression are differentially related to attentional control in melanoma survivors. The results provide a marker for clinicians addressing anxiety and depression in this population. Implications for primary healthcare are discussed.

A Multifunctional Dehalobacter? Tandem Chloroform and Dichloromethane Degradation in a Mixed Microbial Culture.

Chloroform (CF) and dichloromethane (DCM) contaminate groundwater sites around the world but can be cleaned up through bioremediation. Although several strains of Dehalobacter restrictus can reduce CF to DCM and multiple Peptococcaceae can ferment DCM, these processes cannot typically happen simultaneously due to CF sensitivity in the known DCM-degraders or electron donor competition. Here, we present a mixed microbial culture that can simultaneously metabolize CF and DCM and create an additional enrichment culture fed only DCM. Through genus-specific quantitative polymerase chain reaction, we find that Dehalobacter grows while either CF alone or DCM alone is converted, indicating its involvement in both metabolic steps. Additionally, the culture was maintained for over 1400 days without the addition of an exogenous electron donor, and through electron balance calculations, we show that DCM metabolism would produce sufficient reducing equivalents (likely hydrogen) for CF respiration. Together, these results suggest intraspecies electron transfer could occur to continually reduce CF in the culture. Minimizing the addition of electron donor reduces the cost of bioremediation, and "self-feeding" could prolong bioremediation activity long after donor addition ends. Overall, understanding this mechanism informs strategies for culture maintenance and scale-up and benefits contaminated sites where the culture is employed for remediation worldwide.