Passive sampling, a practical method for wastewater-based surveillance of SARS-CoV-2.

In search of practical and affordable tools for wastewater-based surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), three independent field experiments were conducted using three passive sampler sorbents (electronegative membrane, cotton bud, and gauze) in Guelph, Ontario, Canada. Total daily cases during this study ranged from 2 to 17/100,000 people and 43/54 traditionally collected wastewater samples were positive for SARS-CoV-2 with mean detectable concentrations ranging from 8.4 to 1780 copies/ml. Viral levels on the passive samplers were assessed after 4, 8, 24, 48, 72, and 96 hrs of deployment in the wastewater and 43/54 membrane, 42/54 gauze, and 27/54 cotton bud samples were positive. A linear accumulation rate of SARS-CoV-2 on the membranes was observed up to 48 hours, suggesting the passive sampler could adequately reflect wastewater levels for up to two days of deployment. Due the variability in accumulation observed for the cotton buds and gauzes, and the pre-processing steps required for the gauzes, we recommend membrane filters as a simple cost-effective option for wastewater-based surveillance of SARS-CoV-2.

Identification of degrader bacteria and fungi enriched in rhizosphere soil from a toluene phytoremediation site using DNA stable isotope probing.

Improved knowledge of the ecology of contaminant-degrading organisms is paramount for effective assessment and remediation of aromatic hydrocarbon-impacted sites. DNA stable isotope probing was used herein to identify autochthonous degraders in rhizosphere soil from a hybrid poplar phytoremediation system incubated under semi-field-simulated conditions. High-throughput sequencing of bacterial 16S rRNA and fungal internal transcribed spacer (ITS) rRNA genes in metagenomic samples separated according to nucleic acid buoyant density was used to identify putative toluene degraders. Degrader bacteria were found mainly within the Actinobacteria and Proteobacteria phyla and classified predominantly as Cupriavidus, Rhodococcus, Luteimonas, Burkholderiaceae, Azoarcus, Cellulomonadaceae, and Pseudomonas organisms. Purpureocillium lilacinum and Mortierella alpina fungi were also found to assimilate toluene, while several strains of the fungal poplar endophyte Mortierella elongatus were indirectly implicated as potential degraders. Finally, PICRUSt2 predictive taxonomic functional modeling of 16S rRNA genes was performed to validate successful isolation of stable isotope-labeled DNA in density-resolved samples. Four unique sequences, classified within the Bdellovibrionaceae, Intrasporangiaceae, or Chitinophagaceae families, or within the Sphingobacteriales order were absent from PICRUSt2-generated models and represent potentially novel putative toluene-degrading species. This study illustrates the power of combining stable isotope amendment with advanced metagenomic and bioinformatic techniques to link biodegradation activity with unisolated microorganisms. Novelty statement: This study used emerging molecular biological techniques to identify known and new organisms implicated in aromatic hydrocarbon biodegradation from a field-scale phytoremediation system, including organisms with phyto-specific relevance and having potential for downstream applications (amendment or monitoring) in future and existing systems. Additional novelty in this study comes from the use of taxonomic functional modeling approaches for validation of stable isotope probing techniques. This study provides a basis for expanding existing reference databases of known aromatic hydrocarbon degraders from field-applicable sources and offers technological improvements for future site assessment and management purposes.

Bacillus subtilis Strain DSM 29784 Modulates the Cecal Microbiome, Concentration of Short-Chain Fatty Acids, and Apparent Retention of Dietary Components in Shaver White Chickens during Grower, Developer, and Laying Phases.

This study investigated the efficacy of a single strain of Bacillus subtilis (SSB) in modulating the composition of cecal microbiota and its link to the concentration of short-chain fatty acids (SCFA) and apparent retention (AR) of components. A total of 720, 4-week-old Shaver White chicks were allotted to control (CON), 1.1E+08 (low, LSSB), 2.2E+08 (medium, MSSB), or 1.1E+09 (high, HSSB) CFU/kg of diet groups. At grower (10-week), developer (16-week), and laying (28-week) phases, excreta and cecal digesta samples were taken for AR, microbial, and SCFA analyses. Microbial analysis involved high-throughput sequencing of the V3-V4 hypervariable regions of 16S rRNA gene. Bacterial diversity decreased (P < 0.05) at the developer phase as the SSB dose increased; however, a distinct clustering pattern (P < 0.05) of bacterial community was noted. Bacteroides and Faecalibacterium were differentially enriched in the developer for SSB-fed compared to CON-fed birds. Although no differences in microbial diversity were detected in grower and layer phases, different species of Clostridium (XVIII, XIVa, IV, and XIVb)-major butyrate producers-were identified in all phases, with stronger effect sizes for SSB-fed compared to CON-fed birds. Isobutyric acid was elevated in dose response (P = 0.034) in layer phase. In addition, the relative abundances of Alistipes, Lactobacillus, and Bifidobacterium were positively correlated (P < 0.05), with AR of most components for SSB-fed birds in the pullet phase. The results suggested that supplementing chickens' diet with B. subtilis DSM 29784 may selectively enrich beneficial bacterial communities, which in turn are critical in promoting the growth and performance of hens.IMPORTANCE In egg-laying chickens, the trend in the move away from the cage to alternative housing systems and restriction in antimicrobial use requires alternative approaches to maintain health and prevent diseases. There is increased research and commercial interest toward alternative gut health solutions while improving the performance and product safety in poultry production systems. One such approach, in recognition of the importance of the gut microbial community, is the use of microbes as feed supplements (such as probiotics). Unlike meat-type chickens, studies assessing the efficacy of such microbial supplements are limited for egg-laying chickens. Thus, by conducting a comprehensive assessment of the hen microbiota in response to various levels of B. subtilis DSM 29784 during the pullet phase (grower and developer) and the layer phase, the present study demonstrates the importance of direct-fed microbes in modulating gut microbiome, which may relate to improved performance efficiency in the pullet and layer phases.

Targeting Bacteria and Methanogens To Understand the Role of Residual Slurry as an Inoculant in Stored Liquid Dairy Manure.

Microbial communities in residual slurry left after removal of stored liquid dairy manure have been presumed to increase methane emission during new storage, but these microbes have not been studied. While actual manure storage tanks are filled gradually, pilot- and farm-scale studies on methane emissions from such systems often use a batch approach. In this study, six pilot-scale outdoor storage tanks with (10% and 20%) and without residual slurry were filled (gradually or in batch) with fresh dairy manure, and methane and methanogenic and bacterial communities were studied during 120 days of storage. Regardless of filling type, increased residual slurry levels resulted in higher abundance of methanogens and bacteria after 65 days of storage. However, stronger correlation between methanogen abundance and methane flux was observed in gradually filled tanks. Despite some variations in the diversity of methanogens or bacteria with the presence of residual slurry, core phylotypes were not impacted. In all samples, the phylum Firmicutes predominated (∼57 to 70%) bacteria: >90% were members of ClostridiaMethanocorpusculum dominated (∼57 to 88%) archaeal phylotypes, while Methanosarcina gradually increased with storage time. During peak flux of methane, Methanosarcina was the major player in methane production. The results suggest that increased levels of residual slurry have little impact on the dominant methanogenic or bacterial phylotypes, but large population sizes of these organisms may result in increased methane flux during the initial phases of storage.IMPORTANCE Methane is the major greenhouse gas emitted from stored liquid dairy manure. Residual slurry left after removal of stored manure from tanks has been implicated in increasing methane emissions in new storages, and well-adapted microbial communities in it are the drivers of the increase. Linking methane flux to the abundance, diversity, and activity of microbial communities in stored slurries with different levels of residual slurry can help to improve the mitigation strategy. Mesoscale and lab-scale studies conducted so far on methane flux from manure storage systems used batch-filled tanks, while the actual condition in many farms involves gradual filling. Hence, this study provides important information toward determining levels of residual slurry that result in significant reduction of well-adapted microbial communities prior to storage, thereby reducing methane emissions from manure storage tanks filled under farm conditions.

Sodium Persulfate and Potassium Permanganate Inhibit Methanogens and Methanogenesis in Stored Liquid Dairy Manure.

Stored liquid dairy manure is a hotspot for methane (CH) emission, thus effective mitigation strategies are required. We assessed sodium persulfate (NaSO), potassium permanganate (KMnO), and sodium hypochlorite (NaOCl) for impacts on the abundance of microbial communities and CH production in liquid dairy manure. Liquid dairy manure treated with different rates (1, 3, 6, and 9 g or mL L slurry) of these chemicals or their combinations were incubated under anoxic conditions at 22.5 ± 1.3°C for 120 d. Untreated and sodium 2-bromoethanesulfonate (BES)-treated manures were included as negative and positive controls, respectively, whereas sulfuric acid (HSO)-treated manure was used as a reference. Quantitative real-time polymerase chain reaction was used to quantify the abundances of bacteria and methanogens on Days 0, 60, and 120. Headspace CH/CO ratios were used as a proxy to determine CH production. Unlike bacterial abundance, methanogen abundance and CH/CO ratios varied with treatments. Addition of 1 to 9 g L slurry of NaSO and KMnO reduced methanogen abundance (up to ∼28%) and peak CH/CO ratios (up to 92-fold). Except at the lowest rate, chemical combinations also reduced the abundance of methanogens (up to ∼17%) and CH/CO ratios (up to ninefold), although no impacts were observed when 3% NaOCl was used alone. With slurry acidification, the ratios reduced up to twofold, whereas methanogen abundance was unaffected. Results suggest that NaSO and KMnO may offer alternative options to reduce CH emission from stored liquid dairy manure, but this warrants further assessment at larger scales for environmental impacts and characteristics of the treated manure.

Evaluation of Legume-Rhizobial Symbiotic Interactions Beyond Nitrogen Fixation That Help the Host Survival and Diversification in Hostile Environments.

Plants often experience unfavorable conditions during their life cycle that impact their growth and sometimes their survival. A temporary phase of such stress, which can result from heavy metals, drought, salinity, or extremes of temperature or pH, can cause mild to enormous damage to the plant depending on its duration and intensity. Besides environmental stress, plants are the target of many microbial pathogens, causing diseases of varying severity. In plants that harbor mutualistic bacteria, stress can affect the symbiotic interaction and its outcome. To achieve the full potential of a symbiotic relationship between the host and rhizobia, it is important that the host plant maintains good growth characteristics and stay healthy under challenging environmental conditions. The host plant cannot provide good accommodation for the symbiont if it is infested with diseases and prone to other predators. Because the bacterium relies on metabolites for survival and multiplication, it is in its best interests to keep the host plant as stress-free as possible and to keep the supply stable. Although plants have developed many mitigation strategies to cope with stress, the symbiotic bacterium has developed the capability to augment the plant's defense mechanisms against environmental stress. They also provide the host with protection against certain diseases. The protective features of rhizobial-host interaction along with nitrogen fixation appear to have played a significant role in legume diversification. When considering a legume-rhizobial symbiosis, extra benefits to the host are sometimes overlooked in favor of the symbionts' nitrogen fixation efficiency. This review examines all of those additional considerations of a symbiotic interaction that enable the host to withstand a wide range of stresses, enabling plant survival under hostile regimes. In addition, the review focuses on the rhizosphere microbiome, which has emerged as a strong pillar of evolutionary reserve to equip the symbiotic interaction in the interests of both the rhizobia and host. The evaluation would draw the researchers' attention to the symbiotic relationship as being advantageous to the host plant as a whole and the role it plays in the plant's adaptation to unfavorable environmental conditions.

Burden of disease from contaminated drinking water in countries with high access to safely managed water: A systematic review.

The vast majority of residents of high-income countries (≥90%) reportedly have high access to safely managed drinking water. Owing perhaps to the widely held perception of near universal access to high-quality water services in these countries, the burden of waterborne disease in these contexts is understudied. This systematic review aimed to: identify population-scale estimates of waterborne disease in countries with high access to safely managed drinking water, compare methods to quantify disease burden, and identify gaps in available burden estimates. We conducted a systematic review of population-scale disease burden estimates attributed to drinking water in countries where ≥90% of the population has access to safely managed drinking water per official United Nations monitoring. We identified 24 studies reporting estimates for disease burden attributable to microbial contaminants. Across these studies, the median burden of gastrointestinal illness risks attributed to drinking water was ∼2,720 annual cases per 100,000 population. Beyond exposure to infectious agents, we identified 10 studies reporting disease burden-predominantly, cancer risks-associated with chemical contaminants. Across these studies, the median excess cancer cases attributable to drinking water was 1.2 annual cancer cases per 100,000 population. These median estimates slightly exceed WHO-recommended normative targets for disease burden attributable to drinking water and these results highlight that there remains important preventable disease burden in these contexts, particularly among marginalized populations. However, the available literature was scant and limited in geographic scope, disease outcomes, range of microbial and chemical contaminants, and inclusion of subpopulations (rural, low-income communities; Indigenous or Aboriginal peoples; and populations marginalized due to discrimination by race, ethnicity, or socioeconomic status) that could most benefit from water infrastructure investments. Studies quantifying drinking water-associated disease burden in countries with reportedly high access to safe drinking water, focusing on specific subpopulations lacking access to safe water supplies and promoting environmental justice, are needed.

Understanding methane emission from stored animal manure: A review to guide model development.

National inventories of methane (CH4 ) emission from manure management are based on guidelines from the Intergovernmental Panel on Climate Change using country-specific emission factors. These calculations must be simple and, consequently, the effects of management practices and environmental conditions are only crudely represented in the calculations. The intention of this review is to develop a detailed understanding necessary for developing accurate models for calculating CH4 emission from liquid manure, with particular focus on the microbiological conversion of organic matter to CH4 . Themes discussed are (a) the liquid manure environment; (b) methane production processes from a modeling perspective; (c) development and adaptation of methanogenic communities; (d) mass and electron conservation; (e) steps limiting CH4 production; (f) inhibition of methanogens; (g) temperature effects on CH4 production; and (h) limits of existing estimation approaches. We conclude that a model must include calculation of microbial response to variations in manure temperature, substrate availability and age, and management system, because these variables substantially affect CH4 production. Methane production can be reduced by manipulating key variables through management procedures, and the effects may be taken into account by including a microbial component in the model. When developing new calculation procedures, it is important to include reasonably accurate algorithms of microbial adaptation. This review presents concepts for these calculations and ideas for how these may be carried out. A need for better quantification of hydrolysis kinetics is identified, and the importance of short- and long-term microbial adaptation is highlighted.

A mechanistic model of methane emission from animal slurry with a focus on microbial groups.

Liquid manure (slurry) from livestock releases methane (CH4) that contributes significantly to global warming. Existing models for slurry CH4 production-used for mitigation and inventories-include effects of organic matter loading, temperature, and retention time but cannot predict important effects of management, or adequately capture essential temperature-driven dynamics. Here we present a new model that includes multiple methanogenic groups whose relative abundance shifts in response to changes in temperature or other environmental conditions. By default, the temperature responses of five groups correspond to those of four methanogenic species and one uncultured methanogen, although any number of groups could be defined. We argue that this simple mechanistic approach is able to describe both short- and long-term responses to temperature where other existing approaches fall short. The model is available in the open-source R package ABM (https://github.com/sashahafner/ABM) as a single flexible function that can include effects of slurry management (e.g., removal frequency and treatment methods) and changes in environmental conditions over time. Model simulations suggest that the reduction of CH4 emission by frequent emptying of slurry pits is due to washout of active methanogens. Application of the model to represent a full-scale slurry storage tank showed it can reproduce important trends, including a delayed response to temperature changes. However, the magnitude of predicted emission is uncertain, primarily as a result of sensitivity to the hydrolysis rate constant, due to a wide range in reported values. Results indicated that with additional work-particularly on the magnitude of hydrolysis rate-the model could be a tool for estimation of CH4 emissions for inventories.

Dairy manure acidification reduces CH4 emissions over short and long-term.

Acidification with sulphuric acid and cleaning residual manure in tanks are promising practices for reducing methane (CH4), which is a potent greenhouse gas. To date, no data are available on CH4 reductions from acidifying only residual manure (rather than all manure). Moreover, long-term effects of manure acidification (i.e. inoculating ability of previously acidified residual manure in the subsequent storages) are not known. To address these gaps, fresh manure (FM; 150 mL) combined with treated or untreated inoculum (30 mL) were anaerobically incubated at 17°C, 20°C, and 23°C for 116 d. Acidified treatments, regardless of location of acid addition, reduced CH4 production by 81% at 17°C, 78% at 20°C, and 19% at 23°C compared to the control (untreated FM and untreated inoculum). To test long-term acidification effects, FM was inoculated with manure that had been acidified 6-months prior. This created comparable CH4 production to FM with no inoculum and reduced CH4 production by 99% at 17°C and 20°C, and 49% at 23°C compared to the control. Results indicate that residual slurries of acidified manure become poor inoculants in subsequent storage, hence manure acidification has a long-term treatment effect in reducing CH4 production. This could reduce how often acidification is needed in dairy manure tanks and also increasing its cost-effectiveness for farmers.

Intermittent agitation of liquid manure: effects on methane, microbial activity, and temperature in a farm-scale study.

Liquid manure storages are a significant source of methane (CH4) emissions. Farmers commonly agitate (stir) liquid manure prior to field application to homogenize nutrients and solids. During agitation, manure undergoes mechanical stress and is exposed to the air, disrupting anaerobic conditions. This on-farm study aimed to better understand the effects of agitation on CH4 emissions, and explore the potential for intentional agitation (three times) to disrupt the exponential increase of CH4 emissions in spring and summer. Results showed that agitation substantially increased manure temperature in the study year compared to the previous year, particularly at upper- and mid-depths of the stored manure. The temporal pattern of CH4 emissions was altered by reduced emissions over the subsequent week, followed by an increase during the second week. Microbial analysis indicated that the activity of archaea and methanogens increased after each agitation event, but there was little change in the populations of methanogens, archaea, and bacteria. Overall, CH4 emissions were higher than any of the previous three years, likely due to warmer manure temperatures that were higher than the previous years (despite similar air temperatures). Therefore, intermittent manure agitation with the frequency, duration, and intensity used in this study is not recommended as a CH4 emission mitigation practice. Implications: The potential to mitigate methane emissions from liquid manure storages by strategically timed agitation was evaluated in a detailed farm-scale study. Agitation was conducted with readily-available farm equipment, and targeted at the early summer to disrupt methanogenic communities when CH4 emissions increase exponentially. Methane emissions were reduced for about one week after agitation. However, agitation led to increased manure temperature, and was associated with increased activity of methanogens. Overall, agitation was associated with similar or higher methane emissions. Therefore, agitation is not recommended as a mitigation strategy.

Reduction in Methane Emissions From Acidified Dairy Slurry Is Related to Inhibition of Methanosarcina Species.

Liquid dairy manure treated with sulfuric acid was stored in duplicate pilot-scale storage tanks for 120 days with continuous monitoring of CH4 emissions and concurrent examination of changes in the structure of bacterial and methanogenic communities. Methane emissions were monitored at the site using laser-based Trace Gas Analyzer whereas quantitative real-time polymerase chain reaction and massively parallel sequencing were employed to study bacterial and methanogenic communities using 16S rRNA and methyl-coenzyme M Reductase A (mcrA) genes/transcripts, respectively. When compared with untreated slurries, acidification resulted in 69-84% reductions of cumulative CH4 emissions. The abundance, activity, and proportion of bacterial communities did not vary with manure acidification. However, the abundance and activity of methanogens (as estimated from mcrA gene and transcript copies, respectively) in acidified slurries were reduced by 6 and 20%, respectively. Up to 21% reduction in mcrA transcript/gene ratios were also detected in acidified slurries. Regardless of treatment, Methanocorpusculum predominated archaeal 16S rRNA and mcrA gene and transcript libraries. The proportion of Methanosarcina, which is the most metabolically-diverse methanogen, was the significant discriminant feature between acidified and untreated slurries. In acidified slurries, the relative proportions of Methanosarcina were ≤ 10%, whereas in untreated slurries, it represented up to 24 and 53% of the mcrA gene and transcript libraries, respectively. The low proportions of Methanosarcina in acidified slurries coincided with the reductions in CH4 emissions. The results suggest that reduction of CH4 missions achieved by acidification was due to an inhibition of the growth and activity of Methanosarcina species.