Anoxic Groundwater Research Articles

Trace element partitioning between natural barite and deep anoxic groundwaters: Implications for radionuclide retention in host rocks of nuclear waste repositories

Safety assessments for deep geological repositories involve risk calculations for the release of radionuclides like Ra, U, Pu and trivalent actinides from the storage containers to the groundwater. The retention of radionuclides through water-mineral interaction along the groundwater flow path could be a crucial factor in case of a repository failure. Barite (BaSO4) assumes significance in this context, as it has the potential to (re)crystallize and incorporate significant quantities of radioactive elements under relevant physico-chemical conditions.The assessment of mineral-fluid partition coefficients provides a means to evaluate the uptake potential of elements into the mineral. Usually, partition coefficients are determined under well-defined and controlled experimental conditions in laboratories. However, these results have shown discrepancies to partitioning coefficients determined from natural systems.Furthermore, effects like diagenesis or changes in the chemical fluid parameters might lead to a secondary alteration of the phases and affecting the retention ability.Here we investigate the incorporation of trace elements in natural barite from a borehole at 415 m depth in the Äspö Hard Rock Laboratory (Sweden). High-resolution LA-ICP-MS enables the quantitative determination of field-based partition coefficients through the integrated pixel average of selected zones in element distribution maps, combined with existing fluid concentration data. Similarities between the solid solution systems (Ra,Ba)SO4 and (Sr,Ba)SO4 allowed the combination of Sr partitioning data with density-functional theory simulations for an estimation of the partition coefficient value for Ra in natural barite. Values in the 10−2 range were determined, showing a deviation from those reported in previous experimental studies in the 10° range.Moreover, lanthanum serves as an analogue element for the radioactive trivalent actinides. The partition coefficient values for La in natural barite were determined in the range of 10−2 and 10−1, aligning well with experimental partition coefficients. Although density functional theory simulations cannot directly convert a La partition coefficient into a partition coefficient for trivalent actinides, it is assumed that these elements exhibit comparable behavior. Besides primary growth zonation, La also exhibits strong secondary enrichment, probably resulting from groundwater mixing and late fluid-mediated element transport through connected intracrystalline pore space oriented at cleavage plane systems. Additional SIMS analysis provides insights into the temporal variation of the sulfate source at the sampling site reflecting the influence of different waters during mineral growth.This study demonstrates a discrepancy between natural and synthetic PRa for barite and emphasizes that secondary processes significantly impact radionuclide retention in barite. Including these effects in reactive transport models will improve the reliability and applicability of integrative risk models.

Promoting the transformation of green rust for As immobilization with Acidovorax sp. strain BoFeN1

Microbially mediated Fe(II) oxidation has a great potential for attenuating arsenic (As) mobility in an anoxic groundwaters. Green rust (GR), a common Fe(II)-bearing phase in such environments, could be easily oxidized into Fe (oxyhydr)oxides through microbial activity. This study focused on Acidovorax sp. strain BoFeN1, an anaerobic nitrate-reducing Fe(II)-oxidizing (NRFO) bacterium, to promote the transformation of GR. In biotic GR transformation experiments, magnetite formation occurred at [As]ini = 5 mg/L while lepidocrocite and goethite were formed at [As]ini = 10 mg/L. In the absence of bacterium, the GR persisted throughout the 120-h experiment. Meanwhile, with the addition of strain BoFeN1, the final aqueous As concentration significantly decreased from 0.237 to 0.004 mg/L (C0 = 5 mg/L) and from 1.457 to 0.096 mg/L (C0 = 10 mg/L) at 120 h. It was indicated that strain BoFeN1 played a crucial role in promoting the GR transformation and enhancing As immobilization. Further investigations revealed that the role of strain BoFeN1 extended beyond Fe-oxidation. With nitrite (the intermediate of nitrate bioreduction) as oxidizer, lepidocrocite/goethite were formed in the chemical-oxidation system, excluding magnetite. In the Bio - [As]ini = 5 mg/L, the occurrence of lepidocrocite via the bio-oxidation of Fe(II) in GR at 24 h, along with the metabolism of strain BoFeN1 reducing nitrate accompanied with H+ consumption, it should be reasonably deduced that the alkaline micro-environment of periplasm induced by strain BoFeN1 were vital for the transformation of lepidocrocite to magnetite triggered by trace Fe(II). However, in the Bio - [As]ini = 10 mg/L, more As adsorbed on GR inhibiting the adsorption of bacterium, so the alkaline micro-environment had no obvious effect on such transformation. This study helps to understand the interdependence between GR and anaerobic NRFO bacterium, and provides a new perspective for more effective As remediation strategies in anoxic groundwaters.

Impacts of Groundwater Pumping on Subterranean Microbial Communities in a Deep Aquifer Associated with an Accretionary Prism.

Accretionary prisms are composed mainly of ancient marine sediment scraped from the subducting oceanic plate at convergent plate boundaries. Anoxic groundwater is stored in deep aquifers associated with accretionary prisms and can be collected via deep wells. We investigated how such groundwater pumping affects the microbial community in a deep aquifer. Groundwater samples were collected from a deep well drilled down to 1500 m every six months (five times in total) after completion of deep well construction and the start of groundwater pumping. Next-generation sequencing and clone-library analyses of 16S rRNA genes were used to describe the subterranean microbial communities in the samples. The archaea: the prokaryote ratio in groundwater increased significantly from 1 to 7% (0 and 7 months after initiating groundwater pumping) to 59 to 72% (13, 19, and 26 months after initiating groundwater pumping), and dominant prokaryotes changed from fermentative bacteria to sulfate-reducing archaea. The optimal growth temperature of the sulfate-reducing archaea, estimated based on the guanine-plus-cytosine contents of their 16S rRNA genes, was 48-52 °C, which agreed well with the groundwater temperature at the deep-well outflow. Our results indicated that, in deep aquifers, groundwater pumping enhances groundwater flow, and the supply of sulfate-containing seawater activates the metabolism of thermophilic sulfate-reducing archaea.

Efficient chemical and microbial removal of iron and manganese in a rapid sand filter and impact of regular backwash

Aeration followed by rapid sand filtration is a common method in drinking water treatment to remove iron (Fe) and manganese (Mn) from anoxic groundwater. To ensure the successful removal of Fe and Mn within a single filter, several factors such as raw water characteristics, backwash procedures and chemical and microbial interactions with the filter medium need to be considered. Here, we assess the characteristics of a single medium rapid sand filter with highly efficient removal of Fe and Mn. Using synchrotron X-ray spectroscopy, we show that formation of ferrihydrite-type Fe oxides in the top of the filter (0–50 cm) accounts for >95 % of the removal of dissolved Fe2+ in the filter. Birnessite-type Mn- oxides, which are thought to be biogenic, form over a wider depth interval (0–110 cm). Results of 16S rRNA gene amplicon sequencing indicate a corresponding distinct vertical stratification of the microbial community, with potential iron-oxidizing Gallionella, Leptothrix and Sideroxydans dominating in the upper part of the filter, and nitrifiers being more prevalent deeper in the filter. Besides Fe and Mn-oxide, Fe-flocs and bacteriological hollow sheets form in the upper part of the filter. Both the Fe-flocs, hollow Fe-sheets and part of the Fe and Mn coatings are removed through backwashing, thereby reducing the pressure difference measured over the filter medium linked to clogging of pores (from 14 kPa to 1.5 kPa) and ensuring continued water flow. Backwashing removes part of the Gallionella, but this does not negatively impact the filter performance. Strikingly, SEM imaging with EDS mapping revealed alternating layers of Fe and Mn-oxides on the coated grains throughout the filter. This indicates slow mixing of the filter medium between the upper 30 cm and the rest of the filter during backwashing. Slow mixing likely contributes to continued success of the filter by ensuring homogeneous filter bed growth, while still allowing for stratification of the microbial community.

"Candidatus Siderophilus nitratireducens": a putative nap-dependent nitrate-reducing iron oxidizer within the new order Siderophiliales.

Nitrate leaching from agricultural soils is increasingly found in groundwater, a primary source of drinking water worldwide. This nitrate influx can potentially stimulate the biological oxidation of iron in anoxic groundwater reservoirs. Nitrate-dependent iron-oxidizing (NDFO) bacteria have been extensively studied in laboratory settings, yet their ecophysiology in natural environments remains largely unknown. To this end, we established a pilot-scale filter on nitrate-rich groundwater to elucidate the structure and metabolism of nitrate-reducing iron-oxidizing microbiomes under oligotrophic conditions mimicking natural groundwaters. The enriched community stoichiometrically removed iron and nitrate consistently with the NDFO metabolism. Genome-resolved metagenomics revealed the underlying metabolic network between the dominant iron-dependent denitrifying autotrophs and the less abundant organoheterotrophs. The most abundant genome belonged to a new Candidate order, named Siderophiliales. This new species, "Candidatus Siderophilus nitratireducens," carries genes central genes to iron oxidation (cytochrome c cyc2), carbon fixation (rbc), and for the sole periplasmic nitrate reductase (nap). Using thermodynamics, we demonstrate that iron oxidation coupled to nap based dissimilatory reduction of nitrate to nitrite is energetically favorable under realistic Fe3+/Fe2+ and NO3-/NO2- concentration ratios. Ultimately, by bridging the gap between laboratory investigations and nitrate real-world conditions, this study provides insights into the intricate interplay between nitrate and iron in groundwater ecosystems, and expands our understanding of NDFOs taxonomic diversity and ecological role.

Ferric Oxyhydroxylsulfate Precipitation Improves Water Quality in an Acid Mining Lake: A Hydrogeochemical Investigation

Hydrogeochemistry of a lignite pit lake in Lusatia, Germany, was investigated. Anoxic groundwater from the dump aquifer rich in FeII (average ~5911 µmol/L) and SO4 (average ~14,479 µmol/L) contents enter the lake as subsurface inflow; oxidation and subsequent precipitation of poorly crystallized Fe-oxyhydroxysulfate (schwertmannite) occurs and causes acidification (pH~2.8). However, the removal of dissolved loads as solid phases significantly improves the groundwater quality of the downgradient as an outflow. The rainwater isotopic values (δD ~−8.88‰ and δ18O ~−65.86‰) closely matched with the groundwater showing very little isotopic modification, which points to a short residence time of groundwater. The displacement of δD and δ18O values (slope = 5.3) from the meteoric water line reflected the evaporative enrichment of the lake water. The isotopic signature also revealed longer residence times of epilimnion than the hypolimnion waters which are dominated by groundwater. The lake is dimictic and showed abrupt changes in physicochemical parameters along the interface (~0.30 m thick) when separating the epilimnion (upper 4 m) from the hypolimnion (bottom 1.5 m). Lake sediments were found to be dominated by clay size fraction occurring as laminations (thickness: 1~0.5 mm) that reflect seasonal sedimentation. Higher schwertmannite formation in the south as compared to the north (recharge side) also serves as a scavenger of potentially toxic elements which is probably a natural solution to man-made problems. Schwertmannite transformation to goethite releases sulfate which is reduced and fixed as secondary sulfide minerals over time. Overall, waters are of a Ca–SO4 to Ca–Mg–SO4 type with distinct inflow (FeII/FeIII > 2.5) and outflow (FeII/FeIII < 0.5) of groundwater.

Pathogen survival in groundwater: Influence of redox and organic matter

Microbial pathogen survival within the environment can be variable and can depend on many criteria, including environmental conditions e.g. oxygen concentration, temperature, pH, sunlight, etc. (e.g., Horswell et al. (2010)). Groundwater has been shown to enable the prolonged survival of pathogenic organisms due to the absence of sunlight and relatively stable temperatures (Cook and Bolster 2007). In other studies, however, survival has been lower in groundwater when compared with a sterile environment (e.g. sterilised groundwater or artificial groundwater) due to the presence of competing organisms and adverse conditions of pH and redox. To elucidate these discrepancies two experiments were designed: The first, hypothesised that, due to Campylobacters' low tolerance to high oxygen levels, survival in oxic (dissolved oxygen (DO) levels over 5 mg per L) would be less than in anoxic groundwater (DO levels below 2 mg per L). The second hypothesised that the survival of the pathogen Salmonella typhimurium, in groundwater, will be enhanced by organic carbon. METHODS Campylobacter experiment: Campylobacter jejuni isolated from the Havelock North drinking water source was used (designated HN16) (Gilpin et al. 2020). To compare the survival of the outbreak strain with type strain Campylobacter, NCTC 11351 was used. Salmonella experiment: For this experiment, environmental isolates were used rather than laboratory strains. Salmonella, isolated from a stream in Wellington, New Zealand, was identified as Salmonella enterica serovar Typhimurium. The Escherichia coli used was a phylogroup A, isolated from stream sediment in Whangarei Falls, New Zealand. Mesocosm experiments were established containing groundwater (oxic and anoxic for Campylobacter experiment and ultrafiltered, groundwater, groundwater amended with 1% or 10% dissolved organic carbon (DOC). pH, dissolved oxygen (DO), and temperature were monitored over the experimental period. The temperature was maintained at 12-14°C during both experiments. Samples (5 mL) of the groundwater from each jar were taken aseptically at set time points over the experimental period. Samples were then serially diluted in sterile peptone water to give a dilution series from 10-1 to 10-4. Samples were analysed by plating onto selective media. RESULTS Campylobacter experiment: The results presented demonstrated differences in the survival of the two Campylobacter strains tested and differences in survival of Campylobacter HN16 depending on groundwater type. Fig. 1 shows the average concentration of Campylobacter strains in groundwater types over time. The results presented are average of three replicates. Over the whole experimental period survival of Campylobacter HN16 was greatest in anoxic groundwater, and only a 1 log reduction was observed (Fig. 1), equating to a 79.6% survival after 16 days. The die-off rate of Campylobacter HN16 in anoxic groundwater was calculated to be 0.0873 days and T90 6.85 days. Salmonella experiment: Die-of rates for Salmonella were similar over the course of the experiment when no or low levels of DOC were present (Fig. 2a). At high levels of DOC, however, Salmonella showed similar survival to the control. After 84 days only a 1 Log decrease was observed. In comparison, E. coli died off at a faster rate than Salmonella in all mesocosms (Fig. 2). It is interesting to note that in the high DOC mesocosms after day 56 counts of E. coli remained at 103 per mL until the end of the experiment. Conclusions and significance Both experiments demonstrated the survival of pathogenic microorganisms in varying groundwater conditions. The variation in the outbreak strain Campylobacter compared to the type strain indicated variation within species that may lead to enhanced survival in the environment. The Salmonella experiment indicated the presence of additional organic carbon can enhance the survival of pathogens in groundwater. In addition, the variation between the microbial indicator E. coli and Salmonella provides evidence of differences in the survival of microbes in the environment and indicates caution is needed when considering the survival of pathogens in groundwater if reliance is made on microbial indicator organisms.

Microbial degradation and assimilation of veratric acid in oxic and anoxic groundwaters.

Microbial communities are key players in groundwater ecosystems. In this dark environment, heterotrophic microbes rely on biomass produced by the activity of lithoautotrophs or on the degradation of organic matter seeping from the surface. Most studies on bacterial diversity in groundwater habitats are based on 16S gene sequencing and full genome reconstructions showing potential metabolic pathways used in these habitats. However, molecular-based studies do not allow for the assessment of population dynamics over time or the assimilation of specific compounds and their biochemical transformation by microbial communities. Therefore, in this study, we combined DNA-, phospholipid fatty acid-, and metabolomic-stable isotope probing to target and identify heterotrophic bacteria in the groundwater setting of the Hainich Critical Zone Exploratory (CZE), focusing on 2 aquifers with different physico-chemical conditions (oxic and anoxic). We incubated groundwater from 4 different wells using either 13C-labeled veratric acid (a lignin-derived compound) (single labeling) or a combination of 13CO2 and D-labeled veratric acid (dual labeling). Our results show that heterotrophic activities dominate all groundwater sites. We identified bacteria with the potential to break down veratric acid (Sphingobium or Microbacterium). We observed differences in heterotrophic activities between the oxic and anoxic aquifers, indicating local adaptations of bacterial populations. The dual labeling experiments suggested that the serine pathway is an important carbon assimilation pathway and that organic matter was an important source of hydrogen in the newly produced lipids. These experiments also yielded different labeled taxa compared to the single labeling experiments, showing that there exists a complex interaction network in the groundwater habitats.

Reactive migration mechanism of Fe2+ and As3+ during groundwater discharge into river water: Insight from sand column experiments

During groundwater discharge into river water, the Fe2+ and As3+ in anoxic groundwater are brought into the oxygen-bearing interaction zone. However, the reactive migration and transformation mechanism of Fe2+ and As3+ in the interaction zone are poorly understood. This study is based on sand column experiments to simulate the process of groundwater discharge into river water. Results show that the chemical oxidation and precipitation of Fe2+ in the interaction zone mediated the migration and transformation of Fe2+ and As3+. Fe2+ is oxidized by dissolved oxygen in the pore water of the interaction zone and hydrolyzed to form precipitated Fe oxides, whose 90% were poorly crystalline. The formation of Fe oxides seriously hindered the migration of Fe2+ and As3+ to river water, creating a “Fe and As enriched area” near the side edge of groundwater flow in the interaction zone. About 70% of the As3+ was converted to As(V), and it is primarily retained through adsorption on the surface of Fe oxides or coprecipitation with Fe into the poorly crystalline Fe oxides. At the same time, As3+ significantly promoted the migration of Fe2+ by inhibiting the accumulation of Fe(II) on the surface of secondary minerals and the transformation of poorly crystalline Fe oxides into iron minerals with higher crystallinity. Fe2+ and As3+ concentrations and groundwater velocity impacted the comigration of Fe2+ and As3+ in riparian sediment. This study reflects that Fe2+ controls the fate of As3+ in groundwater during groundwater discharge into river water, which is significant for water quality protection.

Metabolic versatility enables sulfur-oxidizers to dominate primary production in groundwater

High rates of CO2 fixation and the genetic potential of various groundwater microbes for autotrophic activity have shown that primary production is an important source of organic C in groundwater ecosystems. However, the contribution of specific chemolithoautotrophic groups such as S-oxidizing bacteria (SOB) to groundwater primary production and their adaptation strategies remain largely unknown. Here, we stimulated anoxic groundwater microcosms with reduced S and sampled the microbial community after 1, 3 and 6 weeks. Genome-resolved metaproteomics was combined with 50at-% 13CO2 stable isotope probing to follow the C flux through the microbial food web and infer traits expressed by active SOB in the groundwater microcosms. Already after 7 days, 90% of the total microbial biomass C in the microcosms was replaced by CO2-derived C, increasing to 97% at the end of incubation. Stable Isotope Cluster Analysis revealed active autotrophs, characterized by a uniform 13C-incorporation of 45% in their peptides, to dominate the microbial community throughout incubation. Mixo- and heterotrophs, characterized by 10 to 40% 13C-incorporation, utilized the primarily produced organic C. Interestingly, obligate autotrophs affiliated with Sulfuricella and Sulfuritalea contained traits enabling the storage of elemental S in globules to maintain primary production under energy limitation. Others related to Sulfurimonas seemed to rapidly utilize substrates for fast proliferation, and most autotrophs further maximized their energy yield via efficient denitrification and the potential for H2 oxidation. Mixotrophic SOB, belonging to Curvibacter or Polaromonas, enhanced metabolic flexibility by using organic compounds to satisfy their C requirements. Time series data spanning eight years further revealed that key taxa of our microcosms composed up to 15% of the microbial groundwater community, demonstrating their in-situ importance. This showed that SOB, by using different metabolic strategies, are able to account for high rates of primary production in groundwater, especially at sites limited to geogenic nutrient sources. The widespread presence of SOB with traits such as S storage, H2 oxidation, and organic C utilization in many aquatic habitats further suggested that metabolic versatility governs S-fueled primary production in the environment.

Bioaugmentation has temporary effect on anaerobic pesticide biodegradation in simulated groundwater systems

Groundwater is the most important source for drinking water in The Netherlands. Groundwater quality is threatened by the presence of pesticides, and biodegradation is a natural process that can contribute to pesticide removal. Groundwater conditions are oligotrophic and thus biodegradation can be limited by the presence and development of microbial communities capable of biodegrading pesticides. For that reason, bioremediation technologies such as bioaugmentation (BA) can help to enhance pesticide biodegradation. We studied the effect of BA using enriched mixed inocula in two column bioreactors that simulate groundwater systems at naturally occurring redox conditions (iron and sulfate-reducing conditions). Columns were operated for around 800 days, and two BA inoculations (BA1 and BA2) were conducted in each column. Inocula were enriched from different wastewater treatment plants (WWTPs) under different redox-conditions. We observed a temporary effect of BA1, reaching 100% removal efficiency of the pesticide 2,4-D after 100 days in both columns. In the iron-reducing column, 2,4-D removal was in general higher than under sulfate-reducing conditions demonstrating the influence of redox conditions on overall biodegradation. We observed a temporary shift in microbial communities after BA1 that is relatable to the increase in 2,4-D removal efficiency. After BA2 under sulfate-reducing conditions, 2,4-D removal efficiency decreased, but no change in the column microbial communities was observed. The present study demonstrates that BA with a mixed inoculum can be a valuable technique for improving biodegradation in anoxic groundwater systems at different redox-conditions.Graphical abstract

The effects of arsenic on dechlorination of trichloroethene by consortium DH: Microbial response and resistance

Inorganic arsenic and organochlorines are frequently co-occurring contaminants in anoxic groundwater environments, and the bioremediation of their composite pollution has long been a rigorous predicament. Currently, the dechlorination behaviors and stress responses of microbial dechlorination consortia to arsenic are not yet fully understood. This study assessed the reductive dechlorination performance of a Dehalococcoides-bearing microcosm DH under gradient concentrations of arsenate [As(V)] or arsenite [As(III)] and investigated the response patterns of different functional microorganisms. Our results demonstrated that although the dechlorination rates declined with increasing arsenic concentrations in both As(III/V) scenarios, the inhibitory impact was more pronounced in As(III)-amended groups compared to As(V)-amended groups. Moreover, the vinyl chloride (VC)-to-ethene step was more susceptible to arsenic exposure compared to the trichloroethene (TCE)-to-dichloroethane (DCE) step, while high levels of arsenic exposure [e.g. As(III) > 75 μM] can induce significant accumulation of VC. Functional gene variations and microbial community analyses revealed that As(III/V) affected reductive dechlorination by directly inhibiting organohalide-respiring bacteria (OHRB) and indirectly inhibiting synergistic populations such as acetogens. Metagenomic results indicated that arsenic metabolic and efflux mechanisms were identical among different Dhc strains, and variations in arsenic uptake pathways were possibly responsible for their differential responses to arsenic exposures. By comparison, fermentative bacteria showed high potential for arsenic resistance due to their inherent advantages in arsenic detoxification and efflux mechanisms. Collectively, our findings expanded the understanding of the response patterns of different functional populations to arsenic stress in the dechlorinating consortium and provided insights into modifying bioremediation strategies at co-contaminated sites for furtherance.

Reference conditions and threshold values for nitrate-nitrogen in New Zealand groundwaters

ABSTRACT Management of groundwater quality is assisted by an understanding of reference conditions, which describe the concentration ranges expected for key substances in the absence of human impact. This study evaluates reference conditions for NO3–N in New Zealand groundwater based on three complementary methods: hierarchical cluster analysis, relationships to groundwater age, and regression against a measure of land-use impact. The three methods result in very similar national-scale estimates of reference conditions for NO3–N concentration in oxic, minimally impacted groundwater, with the 80th, 90th and 95th percentiles found to be 1.65 ± 0.12, 1.97 ± 0.14 and 2.32 ± 0.14 mg/l, respectively (weighted average ± 95% confidence level), in good general agreement with previous studies from New Zealand and overseas. Anoxic groundwaters were treated separately for definition of reference conditions, with the 80th and 90th percentiles of NO3–N found to be 0.04 ± 0.01 and 0.16 ± 0.01, respectively (the 95th percentile could not be estimated reliably). For both oxic and anoxic groundwater, where a site-specific investigation has not been conducted to estimate reference conditions at a local scale, we suggest that the 80th percentile is an appropriate national-scale default threshold, to match the thresholds used for surface waters under the Australian and New Zealand Guidelines for Fresh and Marine Water Quality.

Influence of filter age on Fe, Mn and NH4+ removal in dual media rapid sand filters used for drinking water production

Rapid sand filtration is a common method for removal of iron (Fe), manganese (Mn) and ammonium (NH4+) from anoxic groundwaters used for drinking water production. In this study, we combine geochemical and microbiological data to assess how filter age influences Fe, Mn and NH4+ removal in dual media filters, consisting of anthracite overlying quartz sand, that have been in operation for between ∼2 months and ∼11 years. We show that the depth where dissolved Fe and Mn removal occurs is reflected in the filter medium coatings, with ferrihydrite forming in the anthracite in the top of the filters (< 1 m), while birnessite-type Mn oxides are mostly formed in the sand (> 1 m). Removal of NH4+ occurs through nitrification in both the anthracite and sand and is the key driver of oxygen loss. Removal of Fe is independent of filter age and is always efficient (> 97% removal). In contrast, for Mn, the removal efficiency varies with filter age, ranging from 9 to 28% at ∼2–3 months after filter replacement to 100% after 8 months. After 11 years, removal reduces to 60–80%. The lack of Mn removal in the youngest filters (at 2–3 months) is likely the result of a relatively low abundance of mineral coatings that adsorb Mn2+ and provide surfaces for the establishment of a microbial community. 16S rRNA gene amplicon sequencing shows that Gallionella, which are known Fe2+ oxidizers, are present after 2 months, yet Fe2+ removal is mostly chemical. Efficient NH4+ removal (> 90%) establishes within 3 months of operation but leakage occurs upon high NH4+loading (> 160 µM). Two-step nitrification by Nitrosomonas and Candidatus Nitrotoga is likely the most important NH4+ removal mechanism in younger filters during ripening (2 months), after which complete ammonia oxidation by Nitrospira and canonical two-step nitrification occur simultaneously in older filters. Our results highlight the strong effect of filter age on especially Mn2+but also NH4+ removal. We show that ageing of filter medium leads to the development of thick coatings, which we hypothesize leads to preferential flow, and breakthrough of Mn2+. Use of age-specific flow rates may increase the contact time with the filter medium in older filters and improve Mn2+ and NH4+ removal.

Structure and composition of natural ferrihydrite nano-colloids in anoxic groundwater

Fe-rich mobile colloids play vital yet poorly understood roles in the biogeochemical cycling of Fe in groundwater by influencing organic matter (OM) preservation and fluxes of Fe, OM, and other essential (micro-)nutrients. Yet, few studies have provided molecular detail on the structures and compositions of Fe-rich mobile colloids and factors controlling their persistence in natural groundwater. Here, we provide comprehensive new information on the sizes, molecular structures, and compositions of Fe-rich mobile colloids that accounted for up to 72% of aqueous Fe in anoxic groundwater from a redox-active floodplain. The mobile colloids are multi-phase assemblages consisting of Si-coated ferrihydrite nanoparticles and Fe(II)-OM complexes. Ferrihydrite nanoparticles persisted under both oxic and anoxic conditions, which we attribute to passivation by Si and OM. These findings suggest that mobile Fe-rich colloids generated in floodplains can persist during transport through redox-variable soils and could be discharged to surface waters. These results shed new light on their potential to transport Fe, OM, and nutrients across terrestrial-aquatic interfaces.

Deep anoxic aquifers could act as sinks for uranium through microbial-assisted mineral trapping

Uptake of uranium (U) by secondary minerals, such as carbonates and iron (Fe)-sulfides, that occur ubiquitously on Earth, may be substantial in deep anoxic environments compared to surficial settings due to different environment-specific conditions. Yet, knowledge of U reductive removal pathways and related fractionation between 238U and 235U isotopes in deep anoxic groundwater systems remain elusive. Here we show bacteria-driven degradation of organic constituents that influences formation of sulfidic species facilitating reduction of geochemically mobile U(VI) with subsequent trapping of U(IV) by calcite and Fe-sulfides. The isotopic signatures recorded for U and Ca in fracture water and calcite samples provide additional insights on U(VI) reduction behaviour and calcite growth rate. The removal efficiency of U from groundwater reaching 75% in borehole sections in fractured granite, and selective U accumulation in secondary minerals in exceedingly U-deficient groundwater shows the potential of these widespread mineralogical sinks for U in deep anoxic environments.

Virus-Host Dynamics in Archaeal Groundwater Biofilms and the Associated Bacterial Community Composition.

Spatial and temporal distribution of lytic viruses in deep groundwater remains unexplored so far. Here, we tackle this gap of knowledge by studying viral infections of Altivir_1_MSI in biofilms dominated by the uncultivated host Candidatus Altiarchaeum hamiconexum sampled from deep anoxic groundwater over a period of four years. Using virus-targeted direct-geneFISH (virusFISH) whose detection efficiency for individual viral particles was 15%, we show a significant and steady increase of virus infections from 2019 to 2022. Based on fluorescence micrographs of individual biofilm flocks, we determined different stages of viral infections in biofilms for single sampling events, demonstrating the progression of infection of biofilms in deep groundwater. Biofilms associated with many host cells undergoing lysis showed a substantial accumulation of filamentous microbes around infected cells probably feeding off host cell debris. Using 16S rRNA gene sequencing across ten individual biofilm flocks from one sampling event, we determined that the associated bacterial community remains relatively constant and was dominated by sulfate-reducing members affiliated with Desulfobacterota. Given the stability of the virus-host interaction in these deep groundwater samples, we postulate that the uncultivated virus-host system described herein represents a suitable model system for studying deep biosphere virus-host interactions in future research endeavors.

Reductive dehalogenation in groundwater by Si-Fe(II) co-precipitates enhanced by internal electric field and low vacancy concentrations

Fe(II) and silicate can form Si-Fe(II) co-precipitates in anoxic groundwater and sediments, but their phase composition and reactivity towards subsurface pollutants are largely unknown. Three types of Si-Fe(II) co-precipitations with the same chemical composition, namely Si-Fe(II)-I, Si-Fe(II)-II, and Si-Fe(II)-III, have been synthesized by different hydroxylation sequences in this work. It was found that Si-Fe(II)-III reduce carbon tetrachloride (CT) much faster (k1=0.04419 min−1) than Si-Fe(II)-I (0 min−1) and Si-Fe(II)-II (7.860 × 10−4 min−1). XRD results show that the main component of Si-Fe(II)-III is ferrous silicate (FeSiO3), which is quite different from that of Si-Fe(II)-I and Si-Fe(II)-II. The unique arrangement of hydroxyl coordination, the less distorted octahedral structure, the polyhedral morphology and the absence of Si-A center vacancies in Si-Fe(II)-III are responsible for its high reductive dehalogenation reactivity. The highest redox activity of Si-Fe(II)-III was shown by electrochemical characterization. The [FeII-O-Si]+ in Si-Fe(II)-III may stabilize the dichlorocarbene anion (˸CCl2−), which favors the transformation of CT to methane (9.2%). The Si-Fe(II) co-precipitates consist of countless internal electric fields, and the transformation of hydroxyl and CT both consumed electrons. The coexistence of hydroxyl and CT increases the electron density in the electron-rich region due to their electronegativity, enhancing their electron-accepting capabilities. This study deepens our understanding of the phase composition and electronic structure of Si-Fe(II) co-precipitates, which fills the gap in the reductive dehalogenation of halides by Si-Fe(II) co-precipitates.

Insights into pH-Dependent CuII-Mediated Transformation of Oxytetracycline: Evidence from CuI Formation Kinetics

As animal feed additives, both veterinary tetracycline antibiotics (TCs) and heavy metals, such as CuII ions, often coexisted in aquaculture wastewater and could cotransport as CuII–TCs complexes into groundwater. Previous study emphasized the oxidative role of CuII on the transformation of TCs in the presence of oxygen and the excess CuII, but the transformation of TCs with limited CuII in dark and anoxic groundwater remained unclear. Hence, this study systematically investigated the CuII-mediated transformation of oxytetracycline (OTC), as the most widely used veterinary TCs, under limited CuII and anoxic conditions. The results indicated that OTC degradation was significantly pH-dependent in relation with the speciation of CuII–OTC complexes at pH 3–8.5, and the presence of humic acid (1–10 mg/L) or CaII (10–50 times of CuII) did not change the transformation rate of OTC with CuII significantly. Linking OTC degradation kinetics and CuI formation kinetics, the oxidative role of CuII could be quantitatively evaluated to 6.99–20.2% at varying pH and CuII/OTC ratios from 1:1 to 4:1. A redox site model was built to describe the kinetics of CuI formation, which indicated that CuII-mediated oxidative transformation of OTC is subject to nonidentical specific CuII-binding sites at varying pH. This study is among the first to focus on the CuI formation kinetics to further evaluate the oxidative role of CuII in the catalyzed transformation of TCs, which provides novel insights into the natural attenuation of CuII–TCs complexes as an overlooked class of emerging contaminants in anoxic groundwater.

Sulfide perturbation of NOM-bearing oxic environments induces dark •OH production

The photo-irradiation of natural organic matter (NOM) is a well-documented pathway of •OH production in surface waters. Using humic acid (HA) as a model compound, we show that the perturbation of NOM-bearing oxic environments by naturally occurring reduced species, sulfide, contributes to •OH production under dark conditions. Sulfide concentration had a more prominent influence on •OH production than HA. When HA was controlled at 16 mM C under oxic and neutral pH conditions, the increase of sulfide concentration from 0 to 5 mM increased the cumulative •OH concentration from <2 to 72.2 μM within 120 min. When sulfide was maintained at 2.5 mM, the increase of HA concentration from 0 to 16 mM C increased the cumulative •OH concentration from <0.6 to 44.8 μM. •OH accumulation showed good consistency with sulfide removal and O2 consumption. O2 consumption increased with increasing HA concentration at a fixed sulfide concentration, and also increased with increasing sulfide concentration at a fixed HA concentration. UV–Vis and FTIR characterizations suggested that the reduced HA moieties resulting from sulfide interaction with HA acted as the redox-active moieties responsible for •OH production. Additionally, 3DEEM fluorescence suggested the reaction of sulfide with oxic HA as a partially reversible reduction-oxidation process with production of labile HA fluorophoric intermediates. Our findings implicate that the perturbation of NOM-bearing oxic environments by sulfide, i.e., from the discharge of anoxic groundwater, springs, seafloor black smokers, and hydrothermal vents, is another important source of •OH in natural environments in addition to photo-irradiation.