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At least 19 records

Microbial Community Changes across Time and Space in a Constructed Wetland

Constructed wetlands are artificial ecosystems designed to replicate natural wetland processes. Microbial communities play a pivotal role in cycling essential elements, particularly sulfur, which is crucial for trace metal fixation and remobilization in these ecosystems. By their response to their environment, microbial communities act as biological indicators of the wetland performance. To address knowledge gaps pertinent to the changes in trace metal bioavailability in relation to microbial activities in the H-02 constructed wetland, we performed this study to investigate temporal and spatial variations in microbial communities by using molecular biology tools. Quantitative polymerase chain reaction and next generation sequencing techniques were employed to analyze archaeal and bacterial groups associated with sulfur and methane cycling. Alpha diversity indices were used to assess species richness, evenness, and dominance. Results indicated high gene abundance of Desulfuromonas (5.37 × 10 6 g.cell –1 ), methane oxidizing bacteria (6.92 × 10 6 g.cell –1 ), and methanogenic microorganisms (3.02 × 10 5 g.cell –1 ) during cool months. Warm months were marked by sulfate reducing bacteria dominance (3.31 × 10 6 g.cell –1 ), potentially due to competitive interactions and environmental conditions, higher temperatures, and lower redox potential. Spatial variability among microbial groups was insignificant, but trends in gene abundance indicated complex factors influencing these groups. Next generation sequencing data demonstrated Firmicutes as the most abundant phylum with over 50% regardless of the season or sampling location. Cool months exhibited higher alpha diversity than warm months. Overall, this study showed that seasonal changes significantly impacted the microbial communities in the H-02 constructed wetland that are associated with the sulfur cycle and eventually trace metal biogeochemistry, revealing two distinct mechanisms of the sulfur cycle between the two main seasons, whereas spatial variability effects were not conclusive.

54 ENVIRONMENTAL SCIENCES↗

Sink or source? Insights into the behavior of copper and zinc in the sediment porewater of a constructed wetland by peepers

The H-02 free water surface constructed wetland has been applied to remove heavy metals, mainly copper (Cu) and zinc (Zn), from wastewater on the Savannah River Site (Aiken, SC, USA). More and more studies focus on the metal behavior between the sediment and the overlying water, which directly reflects the stability of metals after sedimentation in constructed wetlands. This study focused on the biogeochemical pathways in metal bioavailability and remobilization in the sediment after metals were removed from the overlying water. The dialysis sampling devices (peepers) were used to collect porewater samples from eight depths in the sediment for the measurement of Cu, Zn, dissolved organic carbon (DOC), and major anions (sulfate and chloride). Surface water samples were also collected for the measurement of Cu, Zn, DOC, and anions. Different temporal trends were observed for dissolved Cu between the surface and bottom waters, but not for dissolved Zn. There were no obvious changes in porewater metal concentrations with increasing depths in the sediment. Sediment served as a sink for Cu as only 3% of porewater samples showed higher labile Cu concentrations than the surface water during the entire year. Furthermore, sediment served as a source for Zn in summer and winter as 32% of porewater samples showed higher labile Zn concentrations than the surface water, and these samples were collected at all sediment depths. We think the seasonal changes in the behaviors of Cu and Zn were primarily controlled by the sulfur dynamics and the metal removal processes in the constructed wetland, as well as the different complexing chemistry between Cu and Zn. Also, our study supports that peeper is a powerful tool for studying the biogeochemistry of metals in the sediment.

54 ENVIRONMENTAL SCIENCES↗

Evaluation of changes in the microbial community structure in the sediments of a constructed wetland over the years

This study presents the results of the long term (2007–2014) monitoring of the microbial community structure in the surface sediments of the H-02 constructed wetland system, which was built on the Savannah River Site in Aiken, SC, USA, to treat the waste water generated at the Tritium facility. Microbial community structure provides valuable information about the functioning of constructed wetlands and helps understand the biogeochemical cycling of nutrients and contaminants. Phospholipids fatty acid (PLFA) analysis and qPCR were used to identify major bacterial phyla in the sediments. The physiochemical properties of the sediments were also used to deduce potential effects on the microbial community structure over the years. Sulfate-reducing bacteria (SRB) were the most dominant bacterial groups, and their prevalence was progressively increasing throughout the years most likely on the account of methane producers. Concentrations of trace metals (copper and zinc) were negatively associated with methane producers and oxidizer while positively correlated with SRB. Overall, the H-02 wetland system was efficient in immobilizing copper and zinc through the anaerobic respiration of sulfate by SRB and minimizing methane emission through the progressive elimination of methane producers by SRB and Geobacter. Furthermore, the aim of this study was to monitor the changes in the microbial community structure in the surface sediments of a constructed wetland during the first 7 years of operation.

59 BASIC BIOLOGICAL SCIENCES↗

Metal fluxes at the sediment–water interface in a free water surface constructed wetland

Metal biogeochemistry in the sediment, water, and the sediment–water interface (SWI) was studied in a free water surface constructed wetland. Concentrations of labile copper (Cu), zinc (Zn), sulfate, chloride, and dissolved organic carbon (DOC) were measured with the diffusive gradients in thin film (DGT) and peeper. A good agreement between peeper- and DGT-measured metals was observed for Cu (regression r 2 = 0.3, 95% CI of the slopes > 0), but not for Zn (95% CI of the slopes overlapped with 0), which was attributed to the different complexed compounds between Cu and Zn in porewater. The depth profile of labile metals in sediment porewater varied with time and was consistent with the solid-phase metal deposition, showing higher concentrations in the surface layer (3 to –3 cm) than in the bottom layer (–4 to –13 cm). The depth-averaged labile Cu and Zn concentrations measured by DGT were 1.0 and 3.1 µg/L, and labile sulfate, chloride, and DOC concentrations measured by peeper were 1.8, 3.6, and 2.1 mg/L, respectively. A sharp decrease in sulfate occurred in September when sulfate concentrations became the lowest among sampling months. This was caused by the seasonal sulfur cycles in the wetland, where the dominant sulfur reaction is sulfate reduction in warm seasons and sulfide oxidation in cold seasons. Different metal-removal mechanisms were observed in the two wetland cells; sulfur dynamics controlled the removal processes in the cell without frequent disturbance but failed to influence metal removal in the cell with frequent disturbance due to the interruption of anoxic layers. The flux ratios that compare labile element concentrations between the water column and the SWI (R-Cu, R-Zn, R-DOC, R-sulfate, and R-chloride) were generated to determine metal diffusive fluxes at the interface. Labile Zn was mostly transported from the water to the SWI during all seasons (R-Zn < 1 for all months except January). Labile Cu moved from the SWI to the water during the warm months (R-Cu < 1), which was explained by the bioturbation-induced transport of organic matter based on the positive correlations between R-Cu and R-DOC. In general, sediment can serve either as a sink or a source depending on the environmental conditions, metal speciation, and presence of living organisms. Here, metal flux at the SWI is a key component in the biogeochemical cycling of a constructed wetland.

54 ENVIRONMENTAL SCIENCES↗

Metal Removal by a Free Surface Constructed Wetland and Prediction of Metal Bioavailability and Toxicity with Diffusive Gradients in Thin Films (DGT) and Biotic Ligand Model (BLM)

The H-02 constructed wetland is a free water surface wetland to remove copper (Cu) and zinc (Zn) from the industrial wastewater. In this study, we evaluated the performance of the wetland from 2018 to 2019 and coupled the diffusive gradients in thin films (DGTs) and biotic ligand model (BLM) to explore metal speciation and bioavailability in wetland waters. Surface water samples were collected and piston DGTs were deployed in different sites of the wetland. The H-02 wetland functioned well during the sampling period with high removal efficiencies (Cu: 73.8 ± 1.2% and Zn: 75.2 ± 16.0%). In our study, with the assumption that the combination of BLM predicted inorganic metals species, BLM Cu(II) and BLM Zn(II), were the bioavailable and toxic species, DGT-Cu did not correlate to BLM Cu(II) (P = 0.47), but DGT-Zn positively correlated to BLM Zn(II) (R 2 = 0.35, P < 0.001). Compared to the modeling results of BLM, DGT-indicated labile and/or bioavailable Cu included not only free Cu ions and inorganic Cu complexes but also a high percentage of Cu-labile organic matter complexes. DGT-indicated Zn included free Zn ion, inorganic Zn, and only a low percentage of Zn-labile organic matter complexes. Our findings illustrated the appropriate use of passive sampling techniques and geological modeling when biomonitoring could be substituted. Furthermore, the close monitoring of metal concentrations, speciation, and bioavailability helps us understand metal biogeochemistry and metal removal processes and ensure the long-term sustainability of the constructed wetland.

54 ENVIRONMENTAL SCIENCES↗

Bioremediation of copper in sediments from a constructed wetland ex situ with the novel bacterium Cupriavidus basilensis SRS

The H-02 constructed wetland was designed to remove metals (primarily copper and zinc) to treat building process water and storm water runoff from multiple sources associated with the Tritium Facility at the DOE-Savannah River Site, Aiken, SC. The concentration of Cu and Zn in the sediments has increased over the lifetime of the wetland and is a concern. A bioremediation option was investigated at the laboratory scale utilizing a newly isolated bacterium of the copper metabolizing genus Cupriavidus isolated from Tim’s Branch Creek, a second-order stream that eventually serves as a tributary to the Savannah River, contaminated with uranium and other metals including copper, nickel, and mercury. Cupriavidus basilensis SRS is a rod-shaped, gram-negative bacterium which has been shown to have predatory tendencies. The isolate displayed resistance to the antibiotics ofloxacin, tetracycline, ciprofloxacin, select fungi, as well as Cu 2+ and Zn 2+ . Subsequent ribosomal sequencing demonstrated a 100% confidence for placement in the genus Cupriavidus and a 99.014% match to the C. basilensis type strain. When H-02 wetland samples were inoculated with Cupriavidus basilensis SRS samples showed significant (p < 0.05) decrease in Cu 2+ concentrations and variability in Zn 2+ concentrations. Over the 72-h incubation there were no significant changes in the inoculate densities (10 6 –10 8 cells/ML) indicating Cupriavidus basilensis SRS resiliency in this environment. This research expands our understanding of the Cupriavidus genus and demonstrates the potential for Cupriavidus basilensis SRS to bioremediate sites impacted with heavy metals, most notably copper.

54 ENVIRONMENTAL SCIENCES↗

Seasonal Sulfur Redox Cycling in Two Constructed Wetlands with Insight on How They Age

Long-term metal remediation in wetland treatment systems (WTSs) involves facilitating dissimilatory sulfate reduction to produce sulfide and mineralize metals in deep sediments. We evaluated seasonal sulfur cycling in two constructed wetlands (Maintained WTS constructed in 2007, and the Unmaintained WTS constructed in 2000) on the Savannah River Site in Aiken, South Carolina, USA. Significant interactions in sulfide concentration were observed between sediment depth, season, and wetland (F = 4.64, df = 11, P = 3.28 × 10 − 5). In the Maintained WTS, dissimilatory sulfate reduction dominated the surface sediments during the warm season (0–2 cm depth, t=-2.66, P = 9.70 × 10 − 3), unlike the Unmaintained system. Sulfate concentrations in pore waters increased in the warm season (F = 7.84, df = 1, P = 6.50 × 10 − 3), contrary to expectations. Sulfur limitation in the Unmaintained WTS during the warm season correlated with increased sulfur assimilation in giant bulrush. Lower sulfide concentrations in surface sediments of the Unmaintained WTS illustrated aging effects. The Maintained WTS shows potential for managing erosion, pH reduction, and sulfur limitation observed in the older Unmaintained WTS.

Biogeochemistry↗

Temporal deposition of copper and zinc in the sediments of metal removal constructed wetlands

The objective of this study was to explore the effects of time, seasons, and total carbon (TC) on Copper (Cu) and Zinc (Zn) deposition in the surface sediments. This study was performed at the H-02 constructed wetland on the Savannah River Site (Aiken, SC, USA). Covering both warm (April-September) and cool (October-March) seasons, several sediment cores were collected twice a year from the H-02 constructed wetland cells from 2007 to 2013. Total concentrations of Cu and Zn were measured in the sediments. Concentrations of Cu and Zn (mean ± standard deviation) in the surface sediments over 7 years of operation increased from 6.0 ± 2.8 and 14.6 ± 4.5 mg kg -1 to 139.6 ± 87.7 and 279.3 ± 202.9 mg kg -1 dry weight, respectively. The linear regression model explained the behavior and the variability of Cu deposition in the sediments. On the other hand, using the generalized least squares extension with the linear regression model allowed for unequal variance and thus produced a model that explained the variance properly, and as a result, was more successful in explaining the pattern of Zn deposition. Total carbon significantly affected both Cu ( p = 0.047) and Zn ( p < 0.001). Time effect on Cu deposition was statistically significant ( p = 0.013), whereas Zn was significantly affected by the season ( p = 0.009).

54 ENVIRONMENTAL SCIENCES↗

Spatiotemporal Changes in Trace Metal Bioavailability in the Sediment Pore water of a Constructed Wetland Using Passive Pore water Samplers

Sediments in aquatic systems often act as a major sink for contaminants. Diffusive gradient in thin films (DGTs) and in situ equilibrium dialysis samplers (peepers) are two major in situ pore water sampling devices that overcome the problems associated with conventional pore water sampling methods. In the present study, DGTs and peepers were used to study the spatial and seasonal effects (cool months, October–February; warm months, May–September) on metal bioavailability in the H‐02 constructed wetland and the sink versus source role of the sediments by calculating the metal resupply capacity. Data showed similar seasonal trends in metal concentrations using passive samplers, peepers, and DGTs. Pooled Cu and Zn concentrations measured using DGTs were lower in warm months (1.67 ± 1.50 and 2.62 ± 0.68 μg L –1 , respectively, p < 0.001) versus in cool months (2.12 ± 0.65 and 5.58 ± 1.33 μg L –1 , respectively, p < 0.001; mean ± 95% confidence interval). Sulfate (SO 4 2− ) concentrations were significantly (p = 0.0139) lower in warm months (averaged at 0.22 ± 0.05 mg L –1 ) compared to in cool months (0.16 ± 0.05 mg L –1 ). The increase in SO 4 2− concentration is an indicator of the lower activity of sulfate‐reducing bacteria, which need SO 4 2− during anaerobic respiration, in which SO 4 2− is reduced to sulfide (S 2– ) that forms insoluble salts with Cu and Zn, which could partially explain the higher bioavailability of these metals in the cool season. Metal resupply capacity of the sediments was mostly <0.2 for Cu and Zn. Taken together, the H0‐2 wetland sediments mostly acted as a sink to both Cu and Zn over the course of the present study. Environ Toxicol Chem 2023;42:2726–2736. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

54 ENVIRONMENTAL SCIENCES↗

Removal, distribution and retention of metals in a constructed wetland over 20 years

The A-01 wetland treatment system (WTS) was designed to remove metals (primarily copper) from the effluent at the A-01 National Pollution Discharge Elimination System (NPDES) outfall at the Savannah River Site, Aiken, SC. This research investigated metal removal, distribution and retention in the A-01 WTS over a period of 20 years. The findings are important for ensuring continued metal sequestration in the A-01 WTSs over time, providing management guidance for constructed wetlands, and investigating changes in metal remediation effectiveness as a wetland ages. During 20 years of operation, systematic water and sediment sampling validated the wetlands' performance. After passage through the treatment cells, Cu concentrations were well below permit limits during all years of operation, often falling below 10 μg L -1 . Cu removal has been consistent over time, averaging about 80% despite large changes in influent Cu concentrations. Most divalent metals were rapidly removed from the water and held in the sediments shortly after the water entered the treatment wetland. Average removal of Pb from water by the wetland system was 67 and 74% in 2004 and 2020, respectively. Comparable values for Zn were 52 and 65%, respectively. Generally, the highest concentrations of Cu, Pb, and Zn were found in the sediment from the first cell in each pair of cells suggesting that most of the Cu, Pb, and Zn in the A-01 effluent was bound to the sediment quickly. Diffusive gradients in thin films (DGT) measurements of Cu and Zn in the sediments were much lower than bulk sediment concentrations. These results suggest that most of the Cu and Zn in the A-01 WTS sediments was not bioavailable, hence not toxic to aquatic organisms, as a likely consequence of adsorption to sediment particles and complexation with organic and inorganic substances.

54 ENVIRONMENTAL SCIENCES↗

Performance of the A-01 Constructed Wetland over 20 Years

The A-01 wetland treatment system (WTS) was designed to remove metals from the effluent at the A-01 National Pollution Discharge Elimination System (NPDES) outfall at the Savannah River Site, Aiken, SC. The A-01 effluent flows from a retention basin to a splitter box, where it is distributed to four pairs of cells in series before discharge to a stream. Each treatment cell is a one-acre wetland that contains Schoenoplectus californicus (giant bulrush) and has a nominal 24-hour retention time. The purpose of this research was to investigate metal removal, distribution and retention in the A-01 WTS over a period of 20 years. During 20 years of operation, systematic water and sediment sampling validated the wetlands' performance. After passage through the treatment cells, Cu concentrations were well below permit limits (22 pg L -1 ) during all years of operation, often falling below 10 pg L -1 . Cu removal has been consistent over time, averaging about 80% despite large changes in influent Cu concentrations. Most divalent metals were rapidly removed from the water and held in the sediments shortly after the water entered the treatment wetlands. Average removal of Pb from water by the wetland system was 67 and 74%, respectively, in 2004, and 2020. Comparable values for Zn were 52 and 65%, respectively. Generally, the highest concentrations of Cu, Pb, and Zn were found in the sediment from the first cell in each pair of cells suggesting that most of the Cu, Pb, and Zn in the A-01 effluent was bound to the sediment quickly. Diffusive gradients in thin films (DGT) measurements of Cu and Zn in the sediments were much lower than bulk sediment concentrations. These results suggest that most of the Cu and Zn in the A-01 WTS sediments was not bioavailable, hence not toxic to aquatic organisms, as a likely consequence of adsorption to sediment particles and complexation with organic and inorganic substances.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

Integrated Anaerobic Membrane Bioreactor (AnMBR) – electro assisted fermentation platform for total resource recovery from diverse wastewaters

The overall objective of the project was to demonstrate a successful wastewater resource recovery platform comprising an Anaerobic Membrane Bioreactor (AnMBR) to achieve >50% Carbon sequestration efficiency as Volatile Fatty Acids (VFAs) or as methane gas from agricultural (animal feeding operations) wastewater along with the generation of final water exceeding Biological Nutrient Removal (BNR) standards for indirect potable reuse by coupling with constructed wetlands has been successfully accomplished. The AnMBR achieved stable performance over 350 days, removing 80–90% of COD and BOD₅ and recovering methane (0.17±0.07 L CH4/g COD at 66.5±0.03% v/v) at 3-5 LMH flux and 5-9 days HRT with 3-5% w/v total solids. Phosphorus recovery via CaO addition in an 80-L coagulation-flocculation-sedimentation unit ranged from 40.6% to 99.7%, yielding products with 11.4–13.6% P content and citric acid solubility of 32–38.6% P, comparable to rock phosphate mineral. Ammonium adsorption achieved ~94.5% recovery with exchange capacity of 10 – 16 g NH4-N/kg clinoptilolite. The CW polishing step met Kansas discharge standards for BOD5 and TN (<30 and <10 mg /L,) and approached the TP standard (~2.5 – 4 mg P/L). These findings have either already resulted in two peer reviewed publications, two patent applications, and one publication in conference proceedings.

42 ENGINEERING↗