Heavy Metal Bioaccumulation in Peruvian Food and Medicinal Products.

To better query regional sources of metal(loid) exposure in an under-communicated region, available scientific literature from 50 national universities (undergraduate and graduate theses and dissertations), peer-reviewed journals, and reports published in Spanish and English were synthesized with a focus on metal(loid) bioaccumulation in Peruvian food and medicinal products utilized locally. The study considered 16 metal(loid)s that are known to exert toxic impacts on humans (Hg, Al, Sb, As, Ba, Be, Cd, Cr, Sn, Ni, Ag, Pb, Se, Tl, Ti, and U). A total of 1907 individual analyses contained within 231 scientific publications largely conducted by Peruvian universities were analyzed. These analyses encompassed 239 reported species classified into five main food/medicinal groups-plants, fish, macroinvertebrates and mollusks, mammals, and "others" category. Our benchmark for comparison was the World Health Organization (Codex Alimentarius) standards. The organisms most frequently investigated included plants such as asparagus, corn, cacao, and rice; fish varieties like trout, tuna, and catfish; macroinvertebrates and mollusks including crab and shrimp; mammals such as alpaca, cow, chicken eggs, and milk; and other categories represented by propolis, honey, lichen, and edible frog. Bioaccumulation-related research increased from 2 to more than 25 publications per year between 2006 and 2022. The results indicate that Peruvian food and natural medicinal products can have dangerous levels of metal(loid)s, which can cause health problems for consumers. Many common and uncommon food/medicinal products and harmful metals identified in this analysis are not regulated on the WHO's advisory lists, suggesting the urgent need for stronger regulations to ensure public safety. In general, Cd and Pb are the metals that violated WHO standards the most, although commonly non-WHO regulated metals such as Hg, Al, As, Cr, and Ni are also a concern. Metal concentrations found in Peru are on many occasions much higher than what has been reported elsewhere. We conclude that determining the safety of food/medicinal products is challenging due to varying metal concentrations that are influenced not only by metal type but also geographical location. Given the scarcity of research findings in many regions of Peru, urgent attention is required to address this critical knowledge gap and implement effective regulatory measures to protect public health.

Photosynthetic pretreatment increases membrane-based rejection of boron and arsenic.

The metalloids boron and arsenic are ubiquitous and difficult to remove during water treatment. As chemical pretreatment using strong base and oxidants can increase their rejection during membrane-based nanofiltration (NF), we examined a nature-based pretreatment approach using benthic photosynthetic processes inherent in a unique type of constructed wetland to assess whether analogous gains can be achieved without the need for exogenous chemical dosing. During peak photosynthesis, the pH of the overlying clear water column above a photosynthetic microbial mat (biomat) that naturally colonizes shallow, open water constructed wetlands climbs from circumneutral to approximately 10. This biological increase in pH was reproduced in a laboratory bioreactor and resulted in analogous increases in NF rejection of boron and arsenic that is comparable to chemical dosing. Rejection across the studied pH range was captured using a monoprotic speciation model. In addition to this mechanism, the biomat accelerated the oxidation of introduced arsenite through a combination of abiotic and biotic reactions. This resulted in increases in introduced arsenite rejection that eclipsed those achieved solely by pH. Capital, operation, and maintenance costs were used to benchmark the integration of this constructed wetland against chemical dosing for water pretreatment, manifesting long-term (sub-decadal) economic benefits for the wetland-based strategy in addition to social and environmental benefits. These results suggest that the integration of nature-based pretreatment approaches can increase the sustainability of membrane-based and potentially other engineered treatment approaches for challenging water contaminants.

Transitional dynamics from mercury to cyanide-based processing in artisanal and small-scale gold mining: Social, economic, geochemical, and environmental considerations.

Artisanal and small-scale gold mining (ASGM) is the leading global source of anthropogenic mercury (Hg) release to the environment. Top-down mercury reduction efforts have had limited results, but a bottom-up embrace of cyanide (CN) processing could eventually displace mercury amalgamation for gold recovery. However, ASGM transitions to cyanidation nearly always include an overlap phase, with mercury amalgamation then cyanidation being used sequentially. This paper uses a transdisciplinary approach that combines natural and social sciences to develop a holistic picture of why mercury and cyanide converge in gold processing and potential impacts that may be worse than either practice in isolation. We show that socio-economic factors drive the comingling of mercury and cyanide practices in ASGM as much or more so than technical factors. The resultant Hg-CN complexes have been implicated in increasing the mobility of mercury, compared to elemental mercury used in Hg-only processing. To support future inquiry, we identify key knowledge gaps including the role of Hg-CN complexes in mercury oxidation, transport, and fate, and possible links to mercury methylation. The global extent and increase of mercury and cyanide processing in ASGM underscores the importance of further research. The immediacy of the problem also demands interim policy responses while research advances, though ultimately, the well-documented struggles of mercury reduction efforts in ASGM temper optimism about policy responses to the mercury-cyanide transition.

Methane-Oxidizing Activity Enhances Sulfamethoxazole Biotransformation in a Benthic Constructed Wetland Biomat.

Ammonia monooxygenase and analogous oxygenase enzymes contribute to pharmaceutical biotransformation in activated sludge. In this study, we hypothesized that methane monooxygenase can enhance pharmaceutical biotransformation within the benthic, diffuse periphytic sediments (i.e., "biomat") of a shallow, open-water constructed wetland. To test this hypothesis, we combined field-scale metatranscriptomics, porewater geochemistry, and methane gas fluxes to inform microcosms targeting methane monooxygenase activity and its potential role in pharmaceutical biotransformation. In the field, sulfamethoxazole concentrations decreased within surficial biomat layers where genes encoding for the particulate methane monooxygenase (pMMO) were transcribed by a novel methanotroph classified as Methylotetracoccus. Inhibition microcosms provided independent confirmation that methane oxidation was mediated by the pMMO. In these same incubations, sulfamethoxazole biotransformation was stimulated proportional to aerobic methane-oxidizing activity and exhibited negligible removal in the absence of methane, in the presence of methane and pMMO inhibitors, and under anoxia. Nitrate reduction was similarly enhanced under aerobic methane-oxidizing conditions with rates several times faster than for canonical denitrification. Collectively, our results provide convergent in situ and laboratory evidence that methane-oxidizing activity can enhance sulfamethoxazole biotransformation, with possible implications for the combined removal of nitrogen and trace organic contaminants in wetland sediments.

Heavy metal removal by the photosynthetic microbial biomat found within shallow unit process open water constructed wetlands.

Nature-based solutions offer a sustainable alternative to labor and chemical intensive engineered treatment of metal-impaired waste streams. Shallow, unit process open water (UPOW) constructed wetlands represent a novel design where benthic photosynthetic microbial mats (biomat) coexist with sedimentary organic matter and inorganic (mineral) phases, creating an environment for multiple-phase interactions with soluble metals. To query the interplay of dissolved metals with inorganic and organic fractions, biomat was harvested from two distinct systems: the demonstration-scale UPOW within the Prado constructed wetlands complex ("Prado biomat", 88 % inorganic) and a smaller pilot-scale system ("Mines Park (MP) biomat", 48 % inorganic). Both biomats accumulated detectable background concentrations of metals of toxicological concern (Zn, Cu, Pb, and Ni) by assimilation from waters that did not exceed regulatory thresholds for these metals. Augmentation in laboratory microcosms with a mixture of these metals at ecotoxicologically relevant concentrations revealed a further capacity for metal removal (83-100 %). Experimental concentrations encapsulated the upper range of surface waters in the metal-impaired Tambo watershed in Peru, where a passive treatment technology such as this could be applied. Sequential extractions demonstrated that metal removal by mineral fractions is more important in Prado than MP biomat, possibly due to a higher proportion and mass of iron and other minerals from Prado-derived materials. Geochemical modeling using PHREEQC suggests that in addition to sorption/surface complexation of metals to mineral phases (modeled as iron (oxyhydr)oxides), diatom and bacterial functional groups (carboxyl, phosphoryl, and silanol) also play an important role in soluble metal removal. By comparing sequestered metal phases across these biomats with differing inorganic content, we propose that sorption/surface complexation and incorporation/assimilation of both inorganic and organic constituents of the biomat play a dominant role in metal removal potential by UPOW wetlands. This knowledge could be applied to passively treat metal impaired waters in analogous and remote regions.

Scalable and customizable parallel flow-through reactors to quantify biological processes related to contaminant attenuation by photosynthetic wetland microbial mats.

Shallow, unit process open water wetlands harbor a benthic microbial mat capable of removing nutrients, pathogens, and pharmaceuticals at rates that rival or exceed those of more traditional systems. A deeper understanding of the treatment capabilities of this non-vegetated, nature-based system is currently hampered by experimentation limited to demonstration-scale field systems and static lab-based microcosms that integrate field-derived materials. This limits fundamental mechanistic knowledge, extrapolation to contaminants and concentrations not present at current field sites, operational optimization, and integration into holistic water treatment trains. Hence, we have developed stable, scalable, and tunable laboratory reactor analogs that offer the capability to manipulate variables such as influent rates, aqueous geochemistry, light duration, and light intensity gradations within a controlled laboratory environment. The design is composed of an experimentally adaptable set of parallel flow-through reactors and controls that can contain field-harvested photosynthetic microbial mats ("biomat") and could be adapted for analogous photosynthetically active sediments or microbial mats. The reactor system is contained within a framed laboratory cart that integrates programable LED photosynthetic spectrum lights. Peristaltic pumps are used to introduce specified growth media, environmentally derived, or synthetic waters at a constant rate, while a gravity-fed drain on the opposite end allows steady-state or temporally variable effluent to be monitored, collected, and analyzed. The design allows for dynamic customization based on experimental needs without confounding environmental pressures and can be easily adapted to study analogous aquatic, photosynthetically driven systems, particularly where biological processes are contained within benthos. The diel cycles of pH and dissolved oxygen (DO) are used as geochemical benchmarks for the interplay of photosynthetic and heterotrophic respiration and likeness to field systems. Unlike static microcosms, this flow-through system remains viable (based on pH and DO fluctuations) and has at present been maintained for more than a year with original field-based materials.•Lab-scale flow-through reactors enable controlled and accessible exploration of shallow, open water constructed wetland function and applications.•The footprint and operating parameters minimize resources and hazardous waste while allowing for hypothesis-driven experiments.•A parallel negative control reactor quantifies and minimizes experimental artifacts.

Pharmaceutical Biotransformation is Influenced by Photosynthesis and Microbial Nitrogen Cycling in a Benthic Wetland Biomat.

In shallow, open-water engineered wetlands, design parameters select for a photosynthetic microbial biomat capable of robust pharmaceutical biotransformation, yet the contributions of specific microbial processes remain unclear. Here, we combined genome-resolved metatranscriptomics and oxygen profiling of a field-scale biomat to inform laboratory inhibition microcosms amended with a suite of pharmaceuticals. Our analyses revealed a dynamic surficial layer harboring oxic-anoxic cycling and simultaneous photosynthetic, nitrifying, and denitrifying microbial transcription spanning nine bacterial phyla, with unbinned eukaryotic scaffolds suggesting a dominance of diatoms. In the laboratory, photosynthesis, nitrification, and denitrification were broadly decoupled by incubating oxic and anoxic microcosms in the presence and absence of light and nitrogen cycling enzyme inhibitors. Through combining microcosm inhibition data with field-scale metagenomics, we inferred microbial clades responsible for biotransformation associated with membrane-bound nitrate reductase activity (emtricitabine, trimethoprim, and atenolol), nitrous oxide reduction (trimethoprim), ammonium oxidation (trimethoprim and emtricitabine), and photosynthesis (metoprolol). Monitoring of transformation products of atenolol and emtricitabine confirmed that inhibition was specific to biotransformation and highlighted the value of oscillating redox environments for the further transformation of atenolol acid. Our findings shed light on microbial processes contributing to pharmaceutical biotransformation in open-water wetlands with implications for similar nature-based treatment systems.

Disinfection byproducts formed during drinking water treatment reveal an export control point for dissolved organic matter in a subalpine headwater stream.

Changes in climate, season, and vegetation can alter organic export from watersheds. While an accepted tradeoff to protect public health, disinfection processes during drinking water treatment can adversely react with organic compounds to form disinfection byproducts (DBPs). By extension, DBP monitoring can yield insights into hydrobiogeochemical dynamics within watersheds and their implications for water resource management. In this study, we analyzed temporal trends from a water treatment facility that sources water from Coal Creek in Crested Butte, Colorado. These trends revealed a long-term increase in haloacetic acid and trihalomethane formation over the period of 2005-2020. Disproportionate export of dissolved organic carbon and formation of DBPs that exceeded maximum contaminant levels were consistently recorded in association with late spring freshet. Synoptic sampling of the creek in 2020 and 2021 identified a biogeochemical hotspot for organic carbon export in the upper domain of the watershed that contained a prominent fulvic acid-like fluorescent signature. DBP formation potential analyses from this domain yielded similar ratios of regulated DBP classes to those formed at the drinking water facility. Spectrometric qualitative analyses of pre and post-reacted waters with hypochlorite indicated lignin-like and condensed hydrocarbon-like molecules were the major reactive chemical classes during chlorine-based disinfection. This study demonstrates how drinking water quality archives combined with synoptic sampling and targeted analyses can be used to identify and understand export control points for dissolved organic matter. This approach could be applied to identify and characterize analogous watersheds where seasonal or climate-associated organic matter export challenge water treatment disinfection and by extension inform watershed management and drinking water treatment.

Effect of elevation, season and accelerated snowmelt on biogeochemical processes during isolated conifer needle litter decomposition.

Increased drought and temperatures associated with climate change have implications for ecosystem stress with risk for enhanced carbon release in sensitive biomes. Litter decomposition is a key component of biogeochemical cycling in terrestrial ecosystems, but questions remain regarding the local response of decomposition processes to climate change. This is particularly complex in mountain ecosystems where the variable nature of the slope, aspect, soil type, and snowmelt dynamics play a role. Hence, the goal of this study was to determine the role of elevation, soil type, seasonal shifts in soil moisture, and snowmelt timing on litter decomposition processes. Experimental plots containing replicate deployments of harvested lodgepole and spruce needle litter alongside needle-free controls were established in open meadows at three elevations ranging from 2,800-3,500 m in Crested Butte, Colorado. Soil biogeochemistry variables including gas flux, porewater chemistry, and microbial ecology were monitored over three climatically variable years that shifted from high monsoon rains to drought. Results indicated that elevation and soil type influenced baseline soil biogeochemical indicators; however, needle mass loss and chemical composition were consistent across the 700 m elevation gradient. Rates of gas flux were analogously consistent across a 300 m elevation gradient. The additional variable of early snowmelt by 2-3 weeks had little impact on needle chemistry, microbial composition and gas flux; however, it did result in increased dissolved organic carbon in lodgepole porewater collections suggesting a potential for aqueous export. In contrast to elevation, needle presence and seasonal variability of soil moisture and temperature both played significant roles in soil carbon fluxes. During a pronounced period of lower moisture and higher temperatures, bacterial community diversity increased across elevation with new members supplanting more dominant taxa. Microbial ecological resilience was demonstrated with a return to pre-drought structure and abundance after snowmelt rewetting the following year. These results show similar decomposition processes across a 700 m elevation gradient and reveal the sensitivity but resilience of soil microbial ecology to low moisture conditions.

Microbial genetic potential for xenobiotic metabolism increases with depth during biofiltration.

Water infiltration into the subsurface can result in pronounced biogeochemical depth gradients. In this study, we assess metabolic potential and properties of the subsurface microbiome during water infiltration by analyzing sediments from spatially-segmented columns. Past work in these laboratory set-ups demonstrated that removal efficiencies of trace organic pollutants were enhanced by limited availability of biodegradable dissolved organic carbon (BDOC) associated with higher humic ratios and deeper sediment regions. Distinct differences were observed in the microbial community when contrasting shallow versus deeper profile sediments. Metagenomic analyses revealed that shallow sediments contained an enriched potential for bacterial growth and division processes. In contrast, deeper sediments harbored a significant increase in genes associated with the metabolism of secondary metabolites and the biotransformation of xenobiotic water pollutants. Metatranscripts further supported this trend, with increased potential for metabolic attributes associated with the biotransformation of xenobiotics and antibiotic resistance within deeper sediments. Furthermore, increasing ratios of humics in feed solutions correlated to enhanced expression of genes associated with xenobiotic biodegradation. These results provide genetic support for the interplay of dissolved organic carbon limitation and enhanced trace organic biotransformation by the subsurface microbiome.

A comparison of lodgepole and spruce needle chemistry impacts on terrestrial biogeochemical processes during isolated decomposition.

This study investigates the isolated decomposition of spruce and lodgepole conifer needles to enhance our understanding of how needle litter impacts near-surface terrestrial biogeochemical processes. Harvested needles were exported to a subalpine meadow to enable a discrete analysis of the decomposition processes over 2 years. Initial chemistry revealed the lodgepole needles to be less recalcitrant with a lower carbon to nitrogen (C:N) ratio. Total C and N fundamentally shifted within needle species over time with decreased C:N ratios for spruce and increased ratios for lodgepole. Differences in chemistry correlated with CO2 production and soil microbial communities. The most pronounced trends were associated with lodgepole needles in comparison to the spruce and needle-free controls. Increased organic carbon and nitrogen concentrations associated with needle presence in soil extractions further corroborate the results with clear biogeochemical signatures in association with needle chemistry. Interestingly, no clear differentiation was observed as a function of bark beetle impacted spruce needles vs those derived from healthy spruce trees despite initial differences in needle chemistry. These results reveal that the inherent chemistry associated with tree species has a greater impact on soil biogeochemical signatures during isolated needle decomposition. By extension, biogeochemical shifts associated with bark beetle infestation are likely driven more by changes such as the cessation of rhizospheric processes than by needle litter decomposition.

Accelerated Snowmelt Protocol to Simulate Climate Change Induced Impacts on Snowpack Dependent Ecosystems.

Field studies that simulate the effects of climate change are important for a predictive understanding of ecosystem responses to a changing environment. Among many concerns, regional warming can result in advanced timing of spring snowmelt in snowpack dependent ecosystems, which could lead to longer snow-free periods and drier summer soils. Past studies investigating these impacts of climate change have manipulated snowmelt with a variety of techniques that include manual snowpack alteration with a shovel, infrared radiation, black sand and fabric covers. Within these studies however, sufficient documentation of methods is limited, which can make experimental reproduction difficult. Here, we outline a detailed plot-scale protocol that utilizes a permeable black geotextile fabric deployed on top of an isothermal spring snowpack to induce advanced snowmelt. The method offers a reliable and cost-effective approach to induce snowmelt by passively increasing solar radiation absorption at the snow surface. In addition, control configurations with no snowpack manipulation are paired adjacent to the induced snowmelt plot for experimental comparison. Past and ongoing deployments in Colorado subalpine ecosystems indicate that this approach can accelerate snowmelt by 14-23 days, effectively mimicking snowmelt timing at lower elevations. This protocol can be applied to a variety of studies to understand the hydrological, ecological, and geochemical impacts of regional warming in snowpack dependent ecosystems.

Hydrogeochemical and microbiological effects of simulated recharge and drying within a 2D meso-scale aquifer.

Oscillating cycles of dewatering (termed drying) and rewetting during managed aquifer recharge (MAR) are used to maintain infiltration rates and could also exert an influence on subsurface microbial structure and respiratory processes. Despite this practice, little knowledge is available about changes to microbial community structure and trace organic chemical biodegradation potential in MAR systems under these conditions. A biologically active two-dimensional (2D) synthetic MAR system equipped with automated sensors (temperature, water pressure, conductivity, soil moisture, oxidation-reduction potential) and embedded water and soil sampling ports was used to test and model these important subsurface processes at the meso-scale. The fate and transport of the antiepileptic drug carbamazepine, the antibiotics sulfamethoxazole and trimethoprim, and the flame retardant tris (2-chloroethyl) phosphate were simulated using the finite element analysis model, FEFLOW. All of these compounds exhibit moderate to poor biodegradability in MAR systems. Within the operational MAR scenario tested, three episodic drying cycles spanning between 18 and 24 days were conducted over a period of 184 days. Notably, cessation of flow and partial dewatering of the 2D synthetic aquifer during dry cycles caused no measurable decrease in soil moisture content beyond the near-surface layer. The episodic flow introduction and dewatering cycles in turn had little impact on overall trace organic chemical biotransformation behavior and soil microbial community structure. However, spatial differences in oxidation-reduction potential and soil moisture were both identified as significant environmental predictors for microbial community structure in the 2D synthetic aquifer.

Establishment and convergence of photosynthetic microbial biomats in shallow unit process open-water wetlands.

The widespread adoption of engineered wetlands designed for water treatment is hindered by uncertainties in system reliability, resilience and management associated with coupled biological and physical processes. To better understand how shallow unit process open-water wetlands self-colonize and evolve, we analyzed the composition of the microbial community in benthic biomats from system establishment through approximately 3 years of operation. Our analysis was conducted across three parallel demonstration-scale (7500 m2) cells located within the Prado Constructed Wetlands in Southern California. They received water from the Santa Ana River (5.9 ±â€¯0.2 mg/L NO3-N), a water body where the flow is dominated by municipal wastewater effluent from May to November. Phylogenetic inquiry and microscopy confirmed that diatoms and an associated aerobic bacterial community facilitated early colonization. After approximately nine months of operation, coinciding with late summer, an anaerobic community emerged with the capability for nitrate attenuation. Varying the hydraulic residence time (HRT) from 1 to 4 days the subsequent year resulted in modest ecological changes across the three parallel cells that were most evident in the outlet regions of the cells. The community that established at this time was comparatively stable for the remaining years of operation and converged with one that had previously formed approximately 550 km (350 miles) away in a pilot-scale (400 m2) wetland in Northern California. That system received denitrified (20.7 ±â€¯0.7 mg/L NO3-N), secondary treated municipal wastewater for 5 years of operation. Establishment of a core microbiome between the two systems revealed a strong overlap of both aerobic and anaerobic taxa with approximately 50% of the analyzed bacterial sequences shared between the two sites. Additionally the same species of diatom, Stauirsa construens var. venter, was prolific in both systems as the putative dominant primary producer. Our results indicate that despite differences in scale, geographic location and source waters, the shallow open-water wetland design can select for a rapid convergence of microbial structure and functionality associated with the self-colonizing benthic biomat. This resulting biomat matures over the first growing season with operational parameters such as HRT further exerting a modest selective bias on community succession.

Ecosystem Resilience and Limitations Revealed by Soil Bacterial Community Dynamics in a Bark Beetle-Impacted Forest.

Forested ecosystems throughout the world are experiencing increases in the incidence and magnitude of insect-induced tree mortality with large ecologic ramifications. Interestingly, correlations between water quality and the extent of tree mortality in Colorado montane ecosystems suggest compensatory effects from adjacent live vegetation that mute responses in less severely impacted forests. To this end, we investigated whether the composition of the soil bacterial community and associated functionality beneath beetle-killed lodgepole pine was influenced by the extent of surrounding tree mortality. The most pronounced changes were observed in the potentially active bacterial community, where alpha diversity increased in concert with surrounding tree mortality until mortality exceeded a tipping point of ~30 to 40%, after which diversity stabilized and decreased. Community structure also clustered in association with the extent of surrounding tree mortality with compositional trends best explained by differences in NH4+ concentrations and C/N ratios. C/N ratios, which were lower in soils under beetle-killed trees, further correlated with the relative abundance of putative nitrifiers and exoenzyme activity. Collectively, the response of soil microorganisms that drive heterotrophic respiration and decay supports observations of broader macroscale threshold effects on water quality in heavily infested forests and could be utilized as a predictive mechanism during analogous ecosystem disruptions.IMPORTANCE Forests around the world are succumbing to insect infestation with repercussions for local soil biogeochemistry and downstream water quality and quantity. This study utilized microbial community dynamics to address why we are observing watershed scale biogeochemical impacts from forest mortality in some impacted areas but not others. Through a unique "tree-centric" approach, we were able to delineate plots with various tree mortality levels within the same watershed to see if surviving surrounding vegetation altered microbial and biogeochemical responses. Our results suggest that forests with lower overall tree mortality levels are able to maintain "normal" ecosystem function, as the bacterial community appears resistant to tree death. However, surrounding tree mortality influences this mitigating effect with various linear and threshold responses whereupon the bacterial community and its function are altered. Our study lends insight into how microscale responses propagate upward into larger-scale observations, which may be useful for future predictions during analogous disruptions.

Sulfide-Induced Dissimilatory Nitrate Reduction to Ammonium Supports Anaerobic Ammonium Oxidation (Anammox) in an Open-Water Unit Process Wetland.

Open-water unit process wetlands host a benthic diatomaceous and bacterial assemblage capable of nitrate removal from treated municipal wastewater with unexpected contributions from anammox processes. In exploring mechanistic drivers of anammox, 16S rRNA gene sequencing profiles of the biomat revealed significant microbial community shifts along the flow path and with depth. Notably, there was an increasing abundance of sulfate reducers (Desulfococcus and other Deltaproteobacteria) and anammox microorganisms (Brocadiaceae) with depth. Pore water profiles demonstrated that nitrate and sulfate concentrations exhibited a commensurate decrease with biomat depth accompanied by the accumulation of ammonium. Quantitative PCR targeting the anammox hydrazine synthase gene, hzsA, revealed a 3-fold increase in abundance with biomat depth as well as a 2-fold increase in the sulfate reductase gene, dsrA These microbial and geochemical trends were most pronounced in proximity to the influent region of the wetland where the biomat was thickest and influent nitrate concentrations were highest. While direct genetic queries for dissimilatory nitrate reduction to ammonium (DNRA) microorganisms proved unsuccessful, an increasing depth-dependent dominance of Gammaproteobacteria and diatoms that have previously been functionally linked to DNRA was observed. To further explore this potential, a series of microcosms containing field-derived biomat material confirmed the ability of the community to produce sulfide and reduce nitrate; however, significant ammonium production was observed only in the presence of hydrogen sulfide. Collectively, these results suggest that biogenic sulfide induces DNRA, which in turn can explain the requisite coproduction of ammonium and nitrite from nitrified effluent necessary to sustain the anammox community.IMPORTANCE This study aims to increase understanding of why and how anammox is occurring in an engineered wetland with limited exogenous contributions of ammonium and nitrite. In doing so, the study has implications for how geochemical parameters could potentially be leveraged to impact nutrient cycling and attenuation during the operation of treatment wetlands. The work also contributes to ongoing discussions about biogeochemical signatures surrounding anammox processes and enhances our understanding of the contributions of anammox processes in freshwater environments.

Spatial impacts of inorganic ligand availability and localized microbial community structure on mitigation of zinc laden mine water in sulfate-reducing bioreactors.

Sulfate-reducing bioreactors (SRBRs) represent a passive, sustainable, and long-term option for mitigating mining influenced water (MIW) during release. Here we investigate spatial zinc precipitation profiles as influenced by substrate differentiation, inorganic ligand availability (inorganic carbon and sulfide), and microbial community structure in pilot-scale SRBR columns fed with sulfate and zinc-rich MIW. Through a combination of aqueous sampling, geochemical digests, electron microscopy and energy-dispersive x-ray spectroscopy, we were able to delineate zones of enhanced zinc removal, identify precipitates of varying stability, and discern the temporal and spatial evolution of zinc, sulfur, and calcium associations. These geochemical insights revealed spatially variable immobilization regimes between SRBR columns that could be further contrasted as a function of labile (alfalfa-dominated) versus recalcitrant (woodchip-dominated) solid-phase substrate content. Both column subsets exhibited initial zinc removal as carbonates; however precipitation in association with labile substrates was more pronounced and dominated by metal-sulfide formation in the upper portions of the down flow columns with micrographs visually suggestive of sphalerite (ZnS). In contrast, a more diffuse and lower mass of zinc precipitation in the presence of gypsum-like precipitates occurred within the more recalcitrant column systems. While removal and sulfide-associated precipitation were spatially variable, whole bacterial community structure (ANOSIM) and diversity estimates were comparatively homogeneous. However, two phyla exhibited a potentially selective relationship with a significant positive correlation between the ratio of Firmicutes to Bacteroidetes and sulfide-bound zinc. Collectively these biogeochemical insights indicate that depths of maximal zinc sulfide precipitation are temporally dynamic, influenced by substrate composition and broaden our understanding of bio-immobilized zinc species, microbial interactions and potential operational and monitoring tools in these types of passive bioreactors.

Effect of advanced oxidation on N-nitrosodimethylamine (NDMA) formation and microbial ecology during pilot-scale biological activated carbon filtration.

Water treatment combining advanced oxidative processes with subsequent exposure to biological activated carbon (BAC) holds promise for the attenuation of recalcitrant pollutants. Here we contrast oxidation and subsequent biofiltration of treated wastewater effluent employing either ozone or UV/H2O2 followed by BAC during pilot-scale implementation. Both treatment trains largely met target water quality goals by facilitating the removal of a suite of trace organics and bulk water parameters. N-nitrosodimethylamine (NDMA) formation was observed in ozone fed BAC columns during biofiltration and to a lesser extent in UV/H2O2 fed columns and was most pronounced at 20 min of empty bed contact time (EBCT) when compared to shorter EBCTs evaluated. While microbial populations were highly similar in the upper reaches, deeper samples revealed a divergence within and between BAC filtration systems where EBCT was identified to be a significant environmental predictor for shifts in microbial populations. The abundance of Nitrospira in the top samples of both columns provides an explanation for the oxidation of nitrite and corresponding increases in nitrate concentrations during BAC transit and support interplay between nitrogen cycling with nitrosamine formation. The results of this study demonstrate that pretreatments using ozone versus UV/H2O2 impart modest differences to the overall BAC microbial population structural and functional attributes, and further highlight the need to evaluate NDMA formation prior to full-scale implementation of BAC in potable reuse applications.

Pilot-scale Columns Equipped with Aqueous and Solid-phase Sampling Ports Enable Geochemical and Molecular Microbial Investigations of Anoxic Biological Processes.

Column studies can be employed to query systems that mimic environmentally relevant flow-through processes in natural and built environments. Sampling these systems spatially throughout operation, while maintaining the integrity of aqueous and solid-phase samples for geochemical and microbial analyses, can be challenging particularly when redox conditions within the column differ from ambient conditions. Here we present a pilot-scale column design and sampling protocol that is optimized for long-term spatial and temporal sampling. We utilized this experimental set-up over approximately 2 years to study a biologically active system designed to precipitate zinc-sulfides during sulfate reducing conditions; however, it can be adapted for the study of many flow-through systems where geochemical and/or molecular microbial analyses are desired. Importantly, these columns utilize retrievable solid-phase bags in conjunction with anoxic microbial techniques to harvest substrate samples while minimally disrupting column operation.

Rare taxa maintain microbial diversity and contribute to terrestrial community dynamics throughout bark beetle infestation.

A global phenomenon of increasing bark beetle-induced tree mortality has heightened concern regarding ecosystem response and biogeochemical implications. Here we explore microbial dynamics under lodgepole pines through the analysis of bulk (16S rDNA) and potentially active (16S rRNA) communities to understand terrestrial ecosystem responses associated with this form of large-scale tree morality. We found that the relative abundance of bulk and potentially active taxa was correlated across taxonomic levels, but at lower levels cladal differences became more apparent. Despite this correlation, there was a strong differentiation of community composition between bulk and potentially active taxa, with further clustering in association with stages of tree mortality. Surprisingly, community clustering as a function of tree phase had limited correlation to soil water content and total nitrogen concentrations, which were the only two measured edaphic parameters to differ in association with tree phase. Bacterial clustering is more readily explained by the observed decrease in abundance of active, rare microorganisms after tree death in conjunction with stable alpha diversity measurements. This enables the rare fraction of the terrestrial microbial community to maintain metabolic diversity by transitioning between metabolically active and dormant states during this ecosystem disturbance and contributes disproportionately to community dynamics and archived metabolic capabilities. These results suggest that analyzing the bulk and potentially active communities after beetle infestation might be a more sensitive indicator of disruption than measuring local edaphic parameters. IMPORTANCE: Forests around the world are experiencing unprecedented mortality due to insect infestations fueled in part by a changing climate. While above-ground processes have been explored, changes at the terrestrial interface relevant to microbial biogeochemical cycling remain largely unknown. In this study, we investigated the changing bulk and potentially active microbial communities beneath healthy and beetle-killed trees. We found that even though few edaphic parameters were altered from beetle infestation, the rare microbes were more likely to be active and fluctuate between dormancy and metabolic activity. This indicates rare as opposed to abundant taxa contribute disproportionately to microbial community dynamics and presumably biogeochemical cycling within these types of perturbed ecosystems.