On-farm screw press and rotary drum treatment of dairy manure-associated antibiotic residues and resistance.

An on-farm solid-liquid separator (SLS) and rotary drum composter (RD) manure treatment system was monitored for its impact on antibiotic residues and antibiotic resistance genes (ARGs). Administered antibiotics were tracked, and treatment system mass flows were quantified. Total amounts of antibiotic residues and ARGs were calculated from measured concentrations and mass flows. Only oxytetracycline (OTC) and sulfadimethoxine (SDM) were detected in the manure treatment system influent. No ß-lactams were measured despite comprising ∼25% of the antibiotics administered. Nearly 80% of OTC and >90% of SDM partitioned into SLS liquid effluent (SL). The RD reduced the mass of OTC remaining in the SLS solid effluent (SS) significantly by 50%, whereas the mass of SDM appeared to increase after RD treatment. All four ARGs tested were detected in influent, with >70% of the sul1, blaOXA-1 , and intI1 genes (normalized by the 16S ribosomal RNA gene) partitioning into the SL. In contrast, about eight times more normalized tetO gene copies partitioned into the SS than in the SL. All ARGs remaining in the SS were significantly reduced by the RD treatment, with a noteworthy 98% reduction in normalized tetO gene copies. This study provides insight into on-farm levels of antibiotic residues and ARGs in dairy manure, their partitioning during SLS treatment, and their fate after a high-temperature RD treatment reaching 72.2 ± 0.18 °C near the outlet. It also notes the importance of mass-flow standardization of data, and the need to work towards standardization of manure system sampling protocols for antibiotic residues and ARGs.

SARS-CoV-2 RNA in Wastewater Settled Solids Is Associated with COVID-19 Cases in a Large Urban Sewershed.

Wastewater-based epidemiology may be useful for informing public health response to viral diseases like COVID-19 caused by SARS-CoV-2. We quantified SARS-CoV-2 RNA in wastewater influent and primary settled solids in two wastewater treatment plants to inform the preanalytical and analytical approaches and to assess whether influent or solids harbored more viral targets. The primary settled solids samples resulted in higher SARS-CoV-2 detection frequencies than the corresponding influent samples. Likewise, SARS-CoV-2 RNA was more readily detected in solids using one-step digital droplet (dd)RT-PCR than with two-step RT-QPCR and two-step ddRT-PCR, likely owing to reduced inhibition with the one-step ddRT-PCR assay. We subsequently analyzed a longitudinal time series of 89 settled solids samples from a single plant for SARS-CoV-2 RNA as well as coronavirus recovery (bovine coronavirus) and fecal strength (pepper mild mottle virus) controls. SARS-CoV-2 RNA targets N1 and N2 concentrations correlated positively and significantly with COVID-19 clinically confirmed case counts in the sewershed. Together, the results demonstrate that measuring SARS-CoV-2 RNA concentrations in settled solids may be a more sensitive approach than measuring SARS-CoV-2 in influent.

Improving the robustness of beach water quality modeling using an ensemble machine learning approach.

Microbial pollution of beach water can expose swimmers to harmful pathogens. Predictive modeling provides an alternative method for beach management that addresses several limitations associated with traditional culture-based methods of assessing water quality. Widely-used machine learning methods often suffer from high variability in performance from one year or beach to another. Therefore, the best machine learning method varies between beaches and years, making method selection difficult. This study proposes an ensemble machine learning approach referred to as model stacking that has a two-layered learning structure, where the outputs of five widely-used individual machine learning models (multiple linear regression, partial least square, sparse partial least square, random forest, and Bayesian network) are taken as input features for another model that produces the final prediction. Applying this approach to three beaches along eastern Lake Erie, New York, USA, we show that generally the model stacking approach was able to generate reliably good predictions compared to all of the five base models. The accuracy rankings of the stacking model consistently stayed 1st or 2nd every year, with yearly-average accuracy of 78%, 81%, and 82.3% at the three studied beaches, respectively. This study highlights the value of the model stacking approach in predicting beach water quality and solving other pressing environmental problems.

Design and Validation of Passive Environmental DNA Samplers Using Granular Activated Carbon and Montmorillonite Clay.

Environmental DNA (eDNA) analysis is gaining prominence as a tool for species and biodiversity monitoring in aquatic environments. eDNA shed by organisms is captured in grab samples, concentrated by filtration, extracted, and analyzed using molecular methods. Conventional capture and filtration methods are limited because (1) filtration does not capture all extracellular DNA, (2) eDNA can degrade during sample transport and storage, (3) filters often clog in turbid waters, reducing the eDNA captured, and (4) grab samples are time sensitive due to pulse eDNA inputs. To address these limitations, this work designs and validates Passive Environmental DNA Samplers (PEDS). PEDS consist of an adsorbent-filled sachet that is suspended in water to collect eDNA over time. Both extracellular and cellular DNA are captured, and the extracellular DNA is protected from degradation. The eDNA captured over time may be more representative than a grab sample. Two adsorbents, Montmorillonite Clay (MC) and Granular Activated Carbon (GAC), are tested. In laboratory experiments, MC-PEDS adsorbed five times more extracellular DNA and desorbed up to four times more than GAC-PEDS (despite high levels of eDNA loss during desorption). In microcosm and field experiments, GAC-PEDS captured over an order of magnitude more eDNA than MC-PEDS. Field results further validated PEDS as an effective eDNA capture method compared to conventional methods.

Invited review: Fate of antibiotic residues, antibiotic-resistant bacteria, and antibiotic resistance genes in US dairy manure management systems.

United States dairy operations use antibiotics (primarily ß-lactams and tetracyclines) to manage bacterial diseases in dairy cattle. Antibiotic residues, antibiotic-resistant bacteria (ARB), and antibiotic resistance genes (ARG) can be found in dairy manure and may contribute to the spread of antibiotic resistance (AR). Although ß-lactam residues are rarely detected in dairy manure, tetracycline residues are common and perhaps persistent. Generally, <15% of bacterial pathogen dairy manure isolates are ARB, although resistance to some antibiotics (e.g., tetracycline) can be higher. Based on available data, the prevalence of medically important ARB on dairy operations is generally static or may be declining for antibiotic-resistant Staphylococcus spp. Over 60 ARG can be found in dairy manure (including ß-lactam and tetracycline resistance genes), although correlations with antibiotic usage, residues, and ARB have been inconsistent, possibly because of sampling and analytical limitations. Manure treatment systems have not been specifically designed to mitigate AR, though certain treatments have some capacity to do so. Generally, well-managed aerobic compost treatments reaching higher peak temperatures (>60°C) are more effective at mitigating antibiotic residues than static stockpiles, although this depends on the antibiotic residue and their interactions. Similarly, thermophilic anaerobic digesters operating under steady-state conditions may be more effective at mitigating antibiotic residues than mesophilic or irregularly operated digesters or anaerobic lagoons. The number of ARB may decline during composting and digestion or be enriched as the bacterial communities in these systems shift, affecting relative ARG abundance or acquire ARG during treatment. Antibiotic resistance genes often persist through these systems, although optimal management and higher operating temperature may facilitate their mitigation. Less is known about other manure treatments, although separation technologies may be unique in their ability to partition antibiotic residues based on sorption and solubility properties. Needed areas of study include determining natural levels of AR in dairy systems, standardizing and optimizing analytical techniques, and more studies of operating on-farm systems, so that treatment system performance and actual human health risks associated with levels of antibiotic residues, ARB, and ARG found in dairy manure can be accurately assessed.

Trends in Antimicrobial Resistance Genes in Manure Blend Pits and Long-Term Storage Across Dairy Farms with Comparisons to Antimicrobial Usage and Residual Concentrations.

The use of antimicrobials by the livestock industry can lead to the release of unmetabolized antimicrobials and antimicrobial resistance genes (ARG) into the environment. However, the relationship between antimicrobial use, residual antimicrobials, and ARG prevalence within manure is not well understood, specifically across temporal and location-based scales. The current study determined ARG abundance in untreated manure blend pits and long-term storage systems from 11 conventional and one antimicrobial-free dairy farms in the Northeastern U.S. at six times over one-year. Thirteen ARGs corresponding to resistance mechanisms for tetracyclines, macrolides-lincosamides, sulfonamides, aminoglycosides, and ß-lactams were quantified using a Custom qPCR Array or targeted qPCR. ARG abundance differed between locations, suggesting farm specific microbial resistomes. ARG abundance also varied temporally. Manure collected during the winter contained lower ARG abundances. Overall, normalized ARG concentrations did not correlate to average antimicrobial usage or tetracycline concentrations across farms and collection dates. Of the 13 ARGs analyzed, only four genes showed a higher abundance in samples from conventional farms and eight ARGs exhibited similar normalized concentrations in the conventional and antimicrobial-free farm samples. No clear trends were observed in ARG abundance between dairy manure obtained from blend pits and long-term storage collected during two drawdown periods (fall and spring), although higher ARG abundances were generally observed in spring compared to fall. This comprehensive study informs future studies needed to determine the contributions of ARGs from dairy manure to the environment.

Assessing uptake of antimicrobials by Zea mays L. and prevalence of antimicrobial resistance genes in manure-fertilized soil.

Manure-borne antimicrobials and antimicrobial resistance genes (ARGs) are of environmental concern due to their potential to be transferred into the food-web via plant-uptake. In this study, Zea mays L. seeds were grown in three different soil conditions: soil without dairy manure, dairy manure-amended soil, and antimicrobial spiked dairy manure-amended soil, to investigate the potential uptake of antimicrobials and ARGs present in manure. The antimicrobial spiked manure consisted of dairy manure fortified with 1 mg/Kg of each individual antimicrobial compounds belonging to the sulfonamide and tetracycline classes. Samples of the Zea mays L. plants were harvested over the course of three weeks to determine potential uptake of antimicrobials from soil to plant shoots, and to compare prevalence of ARGs in manure amended soils and plant tissue. Antimicrobial analysis was performed using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and ARGs (sul1, tetO, and OXA-1) were analyzed using quantitative polymerase chain reaction (qPCR). The study found that both tetracycline and sulfamerazine antimicrobials bioaccumulated in the Zea mays L., reaching concentrations of nearly 3000 ng/g and 1260 ng/g, respectively. Tetracycline residues predominated in the soil, while sulfonamides had mainly bioaccumulated in Zea mays L. tissue. The greatest average uptake factor within the Zea mays L. tissue was 8 for tetracyclines and 110 for sulfonamides indicating larger bioaccumulation of sulfonamides. Additionally, three ARGs (sul1, tetO, and OXA-1) were detected in the soil, only after manure application. However, ARGs were not detected in any of the plant samples.

Sunlight-mediated inactivation of health-relevant microorganisms in water: a review of mechanisms and modeling approaches.

Health-relevant microorganisms present in natural surface waters and engineered treatment systems that are exposed to sunlight can be inactivated by a complex set of interacting mechanisms. The net impact of sunlight depends on the solar spectral irradiance, the susceptibility of the specific microorganism to each mechanism, and the water quality; inactivation rates can vary by orders of magnitude depending on the organism and environmental conditions. Natural organic matter (NOM) has a large influence, as it can attenuate radiation and thus decrease inactivation by endogenous mechanisms. Simultaneously NOM sensitizes the formation of reactive intermediates that can damage microorganisms via exogenous mechanisms. To accurately predict inactivation and design engineered systems that enhance solar inactivation, it is necessary to model these processes, although some details are not yet sufficiently well understood. In this critical review, we summarize the photo-physics, -chemistry, and -biology that underpin sunlight-mediated inactivation, as well as the targets of damage and cellular responses to sunlight exposure. Viruses that are not susceptible to exogenous inactivation are only inactivated if UVB wavelengths (280-320 nm) are present, such as in very clear, open waters or in containers that are transparent to UVB. Bacteria are susceptible to slightly longer wavelengths. Some viruses and bacteria (especially Gram-positive) are susceptible to exogenous inactivation, which can be initiated by visible as well as UV wavelengths. We review approaches to model sunlight-mediated inactivation and illustrate how the environmental conditions can dramatically shift the inactivation rate of organisms. The implications of this mechanistic understanding of solar inactivation are discussed for a range of applications, including recreational water quality, natural treatment systems, solar disinfection of drinking water (SODIS), and enhanced inactivation via the use of sensitizers and photocatalysts. Finally, priorities for future research are identified that will further our understanding of the key role that sunlight disinfection plays in natural systems and the potential to enhance this process in engineered systems.

Microbial community structure of sea spray aerosols at three California beaches.

We characterized the microbial communities in sea spray aerosols (SSA), water and sand of three beaches in central California (Cowell Beach, Baker Beach and Lovers Point) by sequencing the V4 region of the 16S rRNA gene. Average concentrations of 16S rRNA genes in SSA ranged from 2.4 × 104 to 1.4 × 105 gene copies per m3 of air. A total of 9781 distinct OTUs were identified in SSA and of these, 1042 OTUs were found in SSA of all beaches. SSA microbial communities included marine taxa, as well as some associated with the terrestrial environment. SSA taxa included organisms that play important roles in biogeochemical cycling of elements such as Planctomyces and Synechococcus, as well as those representing potential pathogens and fecal indicator bacteria including Staphylococcus epidermidis and Enterococcus spp. There were a large number of shared OTUs among SSA and water, and there was relatively high similarity between SSA and water communities. Results are consistent with a conceptual model where SSA is generated by breaking waves and bubble bursting in marine waters and that enables the transport of microorganisms from the sea to sand or other environments.

Environmental DNA (eDNA) Shedding and Decay Rates to Model Freshwater Mussel eDNA Transport in a River.

Freshwater mussels are vital components of stream ecosystems, yet remain threatened. Thus, timely and accurate species counts are critical for proper conservation and management. Mussels live in stream sediments and can be challenging to survey given constraints related to water depth, flow, and time of year. The use of environmental DNA (eDNA) to monitor mussel distributions and diversity is a promising tool. Before it can be used as a monitoring tool, however, we need to know how much eDNA mussels shed into their environment and how long the eDNA persists. Here, we present a novel application of eDNA to estimate both the presence/absence and abundance of a freshwater mussel species, Lampsilis siliquoidea. The eDNA shedding and decay rates reported within are the first for freshwater mussels. We determined that eDNA shedding was statistically similar across mussel densities, but that first-order decay constants varied between experimental treatments. Finally, we effectively modeled downstream transport of eDNA and present a model that can be used as a complementary tool to estimate mussel density. Our results suggest that eDNA has the potential to be a complementary tool to survey mussels and enhance current efforts to monitor and protect freshwater mussel biodiversity.

Persistence of marine fish environmental DNA and the influence of sunlight.

Harnessing information encoded in environmental DNA (eDNA) in marine waters has the potential to revolutionize marine biomonitoring. Whether using organism-specific quantitative PCR assays or metabarcoding in conjunction with amplicon sequencing, scientists have illustrated that realistic organism censuses can be inferred from eDNA. The next step is establishing ways to link information obtained from eDNA analyses to actual organism abundance. This is only possible by understanding the processes that control eDNA concentrations. The present study uses mesocosm experiments to study the persistence of eDNA in marine waters and explore the role of sunlight in modulating eDNA persistence. We seeded solute-permeable dialysis bags with water containing indigenous eDNA and suspended them in a large tank containing seawater. Bags were subjected to two treatments: half the bags were suspended near the water surface where they received high doses of sunlight, and half at depth where they received lower doses of sunlight. Bags were destructively sampled over the course of 87 hours. eDNA was extracted from water samples and used as template for a Scomber japonicus qPCR assay and a marine fish-specific 12S rRNA PCR assay. The latter was subsequently sequenced using a metabarcoding approach. S. japonicus eDNA, as measured by qPCR, exhibited first order decay with a rate constant ~0.01 hr -1 with no difference in decay rate constants between the two experimental treatments. eDNA metabarcoding identified 190 organizational taxonomic units (OTUs) assigned to varying taxonomic ranks. There was no difference in marine fish communities as measured by eDNA metabarcoding between the two experimental treatments, but there was an effect of time. Given the differences in UVA and UVB fluence received by the two experimental treatments, we conclude that sunlight is not the main driver of fish eDNA decay in the experiments. However, there are clearly temporal effects that need to be considered when interpreting information obtained using eDNA approaches.

Staphylococcus aureus Strain Newman Photoinactivation and Cellular Response to Sunlight Exposure.

Sunlight influences microbial water quality of surface waters. Previous studies have investigated photoinactivation mechanisms and cellular photostress responses of fecal indicator bacteria (FIB), including Escherichia coli and enterococci, but further work is needed to characterize photostress responses of bacterial pathogens. Here we investigate the photoinactivation of Staphylococcus aureus (strain Newman), a pigmented, waterborne pathogen of emerging concern. We measured photodecay using standard culture-based assays and cellular membrane integrity and investigated photostress response by measuring the relative number of mRNA transcripts of select oxidative stress, DNA repair, and metabolism genes. Photoinactivation experiments were performed in both oxic and anoxic systems to further investigate the role of oxygen-mediated and non-oxygen-mediated photoinactivation mechanisms. S. aureus lost culturability much faster in oxic systems than in anoxic systems, indicating an important role for oxygen in photodecay mechanisms. S. aureus cell membranes were damaged by sunlight exposure in anoxic systems but not in oxic systems, as measured by cell membrane permeability to propidium iodide. After sunlight exposure, S. aureus increased expression of a gene coding for methionine sulfoxide reductase after 12 h of sunlight exposure in the oxic system and after 6 h of sunlight exposure in the anoxic system, suggesting that methionine sulfoxide reductase is an important enzyme for defense against both oxygen-dependent and oxygen-independent photostresses. This research highlights the importance of oxygen in bacterial photoinactivation in environmentally relevant systems and the complexity of the bacterial photostress response with respect to cell structure and transcriptional regulation.IMPORTANCEStaphylococcus aureus is a pathogenic bacterium that causes gastrointestinal, respiratory, and skin infections. In severe cases, S. aureus infection can lead to life-threatening diseases, including pneumonia and sepsis. Cases of community-acquired S. aureus infection have been increasing in recent years, pointing to the importance of considering S. aureus transmission pathways outside the hospital environment. Associations have been observed between recreational water contact and staphylococcal skin infections, suggesting that recreational waters may be an important environmental transmission pathway for S. aureus However, prediction of human health risk in recreational waters is hindered by incomplete knowledge of pathogen sources, fate, and transport in this environment. This study is an in-depth investigation of the inactivation of a representative strain of S. aureus by sunlight exposure, one of the most important factors controlling the fate of microbial contaminants in clear waters, which will improve our ability to predict water quality changes and human health risk in recreational waters.

Biomonitoring of marine vertebrates in Monterey Bay using eDNA metabarcoding.

Molecular analysis of environmental DNA (eDNA) can be used to assess vertebrate biodiversity in aquatic systems, but limited work has applied eDNA technologies to marine waters. Further, there is limited understanding of the spatial distribution of vertebrate eDNA in marine waters. Here, we use an eDNA metabarcoding approach to target and amplify a hypervariable region of the mitochondrial 12S rRNA gene to characterize vertebrate communities at 10 oceanographic stations spanning 45 km within the Monterey Bay National Marine Sanctuary (MBNMS). In this study, we collected three biological replicates of small volume water samples (1 L) at 2 depths at each of the 10 stations. We amplified fish mitochondrial DNA using a universal primer set. We obtained 5,644,299 high quality Illumina sequence reads from the environmental samples. The sequence reads were annotated to the lowest taxonomic assignment using a bioinformatics pipeline. The eDNA survey identified, to the lowest taxonomic rank, 7 families, 3 subfamilies, 10 genera, and 72 species of vertebrates at the study sites. These 92 distinct taxa come from 33 unique marine vertebrate families. We observed significantly different vertebrate community composition between sampling depths (0 m and 20/40 m deep) across all stations and significantly different communities at stations located on the continental shelf (<200 m bottom depth) versus in the deeper waters of the canyons of Monterey Bay (>200 m bottom depth). All but 1 family identified using eDNA metabarcoding is known to occur in MBNMS. The study informs the implementation of eDNA metabarcoding for vertebrate biomonitoring.

Decay of sewage-sourced microbial source tracking markers and fecal indicator bacteria in marine waters.

The decay of sewage-sourced enterococci, Escherichia coli, three human-associated microbial source tracking (MST) markers, Salmonella, Campylobacter, and norovirus GII was measured in situ in coastal, marine waters. Experiments examined the effects of sunlight intensity and season on decay. Seawater was seeded with untreated sewage, placed into permeable dialysis bags, and deployed in the coastal ocean near the water surface, and at 18 cm, and 99 cm depths, to vary solar intensity, during winter and summer seasons. Microbial decay was modeled using a log-linear or shoulder log-linear decay model. Pathogen levels were too low in sewage to obtain kinetic parameters. Human-associated MST markers all decayed with approximately the same rate constant (k âˆ¼ 1.5 d-1) in all experimental treatments, suggesting markers could be detectable up to ∼6 days after a raw sewage spill. E. coli and enterococci (culturable and molecular marker) k significantly varied with season and depth; enterococci decayed faster at shallow depths and during the summer, while E. coli decayed faster at shallow depths and during the winter. Rate constants for MST markers and culturable FIB diverged except at the deepest depth in the water column potentially complicating the use of MST marker concentrations to allocate sources of FIB contamination.

Quantification of Environmental DNA (eDNA) Shedding and Decay Rates for Three Marine Fish.

Analysis of environmental DNA (eDNA) to identify macroorganisms and biodiversity has the potential to significantly augment spatial and temporal biological monitoring in aquatic ecosystems. Current monitoring methods relying on the physical identification of organisms can be time consuming, expensive, and invasive. Measuring eDNA shed from organisms provides detailed information on the presence and abundance of communities of organisms. However, little is known about eDNA shedding and decay in aquatic environments. In the present study, we designed novel Taqman qPCR assays for three ecologically and economically important marine fish-Engraulis mordax (Northern Anchovy), Sardinops sagax (Pacific Sardine), and Scomber japonicas (Pacific Chub Mackerel). We subsequently measured fish eDNA shedding and decay rates in seawater mesocosms. eDNA shedding rates ranged from 165 to 3368 pg of DNA per hour per gram of biomass. First-order decay rate constants ranged from 0.055 to 0.101 per hour. We also examined the size fractionation of eDNA and concluded eDNA is both intra- and extracellular. Finally, we derived a simple mass-balance model to estimate fish abundance from eDNA concentration. The mesocosm-derived shedding and decay rates inform the interpretation of eDNA concentrations measured in environmental samples and future use of eDNA as a monitoring tool.

Solar Inactivation of Enterococci and Escherichia coli in Natural Waters: Effects of Water Absorbance and Depth.

The decay of sewage-sourced Escherichia coli and enterococci was measured at multiple depths in a freshwater marsh, a brackish water lagoon, and a marine site, all located in California. The marine site had very clear water, while the waters from the marsh and lagoon contained colored dissolved organic matter that not only blocked light but also produced reactive oxygen species. First order decay rate constants of both enterococci and E. coli were between 1 and 2 d(-1) under low light conditions and as high as 6 d(-1) under high light conditions. First order decay rate constants were well correlated to the daily average UVB light intensity corrected for light screening incorporating water absorbance and depth, suggesting endogenous photoinactivation is a major pathway for bacterial decay. Additional laboratory experiments demonstrated the presence of colored dissolved organic matter in marsh water enhanced photoinactivation of a laboratory strain of Enterococcus faecalis, but depressed photoinactivation of sewage-sourced enterococci and E. coli after correcting for UVB light screening, suggesting that although the exogenous indirect photoinactivation mechanism may be active against Ent. faecalis, it is not for the sewage-source organisms. A simple linear regression model based on UVB light intensity appears to be a useful tool for predicting inactivation rate constants in natural waters of any depth and absorbance.

Improvement of urban lake water quality by removal of Escherichia coli through the action of the bivalve Anodonta californiensis.

High levels of fecal indicator bacteria, such as Escherichia coli, can be indicative of poor water quality. The use of shellfish to reduce eutrophication has been proposed, but application of bivalves to reduce bacterial levels has not been extensively reported. Removal of E. coli by the native freshwater mussel Anodonta californiensis was studied using laboratory batch systems and field-based flow-through systems. Batch systems were utilized to determine the fate and inactivation of E. coli after uptake by the mussel. Batch experiments demonstrated that uptake patterns followed first order kinetics and E. coli was inactivated with less than 5% of the initial colonies recoverable in fecal matter or tissue. Flow-through systems located at an urban impaired lake in San Francisco, CA were utilized to determine uptake kinetics under environmentally relevant conditions. The bivalves maintained a 1-log removal of E. coli for the duration of exposure. The calculated uptake rates can be used in conjunction with hydrologic models to determine the number of bivalves needed to maintain removal of E. coli in different freshwater systems. The outcomes of this study support the use of native freshwater bivalves to achieve the co-benefits of rehabilitating a freshwater ecosystem and improving water quality via reduction of E. coli in contaminated freshwater systems.

Temporal stability of the microbial community in sewage-polluted seawater exposed to natural sunlight cycles and marine microbiota.

Billions of gallons of untreated wastewater enter the coastal ocean each year. Once sewage microorganisms are in the marine environment, they are exposed to environmental stressors, such as sunlight and predation. Previous research has investigated the fate of individual sewage microorganisms in seawater but not the entire sewage microbial community. The present study used next-generation sequencing (NGS) to examine how the microbial community in sewage-impacted seawater changes over 48 h when exposed to natural sunlight cycles and marine microbiota. We compared the results from microcosms composed of unfiltered seawater (containing naturally occurring marine microbiota) and filtered seawater (containing no marine microbiota) to investigate the effect of marine microbiota. We also compared the results from microcosms that were exposed to natural sunlight cycles with those from microcosms kept in the dark to investigate the effect of sunlight. The microbial community composition and the relative abundance of operational taxonomic units (OTUs) changed over 48 h in all microcosms. Exposure to sunlight had a significant effect on both community composition and OTU abundance. The effect of marine microbiota, however, was minimal. The proportion of sewage-derived microorganisms present in the microcosms decreased rapidly within 48 h, and the decrease was the most pronounced in the presence of both sunlight and marine microbiota, where the proportion decreased from 85% to 3% of the total microbial community. The results from this study demonstrate the strong effect that sunlight has on microbial community composition, as measured by NGS, and the importance of considering temporal effects in future applications of NGS to identify microbial pollution sources.

Diversity and transport of microorganisms in intertidal sands of the California coast.

Forced by tides and waves, large volumes of seawater are flushed through the beach daily. Organic material and nutrients in seawater are remineralized and cycled as they pass through the beach. Microorganisms are responsible for most of the biogeochemical cycling in the beach; however, few studies have characterized their diversity in intertidal sands, and little work has characterized the extent to which microbes are transported between different compartments of the beach. The present study uses next-generation massively parallel sequencing to characterize the microbial community present at 49 beaches along the coast of California. In addition, we characterize the transport of microorganisms within intertidal sands using laboratory column experiments. We identified extensive diversity in the beach sands. Nearly 1,000 unique taxa were identified in sands from 10 or more unique beaches, suggesting the existence of a group of "cosmopolitan" sand microorganisms. A biogeographical analysis identified a taxon-distance relationship among the beaches. In addition, sands with similar grain size, organic carbon content, exposed to a similar wave climate, and having the same degree of anthropogenic influence tended to have similar microbial communities. Column experiments identified microbes readily mobilized by seawater infiltrating through unsaturated intertidal sands. The ease with which microbes were mobilized suggests that intertidal sands may represent a reservoir of bacteria that seed the beach aquifer where they may partake in biogeochemical cycling.