Defluorination of Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS) by Acidimicrobium sp. Strain A6.

Incubations with pure and enrichment cultures of Acidimicrobium sp. strain A6 (A6), an autotroph that oxidizes ammonium to nitrite while reducing ferric iron, were conducted in the presence of PFOA or PFOS at 0.1 mg/L and 100 mg/L. Buildup of fluoride, shorter-chain perfluorinated products, and acetate was observed, as well as a decrease in Fe(III) reduced per ammonium oxidized. Incubations with hydrogen as a sole electron donor also resulted in the defluorination of these PFAS. Removal of up to 60% of PFOA and PFOS was observed during 100 day incubations, while total fluorine (organic plus fluoride) remained constant throughout the incubations. To determine if PFOA/PFOS or some of their degradation products were metabolized, and since no organic carbon source except these PFAS was added, dissolved organic carbon (DOC) was tracked. At concentrations of 100 mg/L, PFOA/PFOS were the main contributors to DOC, which remained constant during the pure A6 culture incubations. Whereas in the A6 enrichment culture, DOC decreased slightly with time, indicating that as defluorination of PFOS/PFOA occurred, some of the products were being metabolized by heterotrophs present in this culture. Results show that A6 can defluorinate PFOA/PFOS while reducing iron, using ammonium or hydrogen as the electron donor.

Electrode Colonization by the Feammox Bacterium Acidimicrobiaceae sp. Strain A6.

Acidimicrobiaceae sp. strain A6 (A6), from the Actinobacteria phylum, was recently identified as a microorganism that can carry out anaerobic ammonium (NH4+) oxidation coupled to iron reduction, a process also known as Feammox. Being an iron-reducing bacterium, A6 was studied as a potential electrode-reducing bacterium that may transfer electrons extracellularly onto electrodes while gaining energy from NH4+ oxidation. Actinobacteria species have been overlooked as electrogenic bacteria, and the importance of lithoautotrophic iron reducers as electrode-reducing bacteria at anodes has not been addressed. By installing electrodes in the soil of a forested riparian wetland where A6 thrives, in soil columns in the laboratory, and in A6-bioaugmented constructed wetland (CW) mesocosms and by operating microbial electrolysis cells (MECs) with pure A6 culture, the characteristics and performances of this organism as an electrode-reducing bacterium candidate were investigated. In this study, we show that Acidimicrobiaceae sp. strain A6, a lithoautotrophic bacterium, is capable of colonizing electrodes under controlled conditions. In addition, A6 appears to be an electrode-reducing bacterium, since current production was boosted shortly after the CWs were seeded with enrichment A6 culture and current production was detected in MECs operated with pure A6, with the anode as the sole electron acceptor and NH4+ as the sole electron donor.IMPORTANCE Most studies on electrogenic microorganisms have focused on the most abundant heterotrophs, while other microorganisms also commonly present in electrode microbial communities, such as Actinobacteria strains, have been overlooked. The novel Acidimicrobiaceae sp. strain A6 (Actinobacteria) is an iron-reducing bacterium that can colonize the surface of anodes in sediments and is linked to electrical current production, making it an electrode-reducing bacterium. Furthermore, A6 can carry out anaerobic ammonium oxidation coupled to iron reduction. Therefore, findings from this study open the possibility of using electrodes instead of iron as electron acceptors, as a means to promote A6 to treat NH4+-containing wastewater more efficiently. Altogether, this study expands our knowledge of electrogenic bacteria and opens the possibility of developing Feammox-based technologies coupled to bioelectric systems for the treatment of NH4+ and other contaminants in anoxic systems.

Simultaneous measurements of dissolved CH4 and H2 in wetland soils.

Biogeochemical processes in wetland soils are complex and are driven by a microbiological community that competes for resources and affects the soil chemistry. Depending on the availability of various electron acceptors, the high carbon input to wetland soils can make them important sources of methane production and emissions. There are two significant pathways for methanogenesis: acetoclastic and hydrogenotrophic methanogenesis. The hydrogenotrophic pathway is dependent on the availability of dissolved hydrogen gas (H2), and there is significant competition for available H2. This study presents simultaneous measurements of dissolved methane and H2 over a 2-year period at three tidal marshes in the New Jersey Meadowlands. Methane reservoirs show a significant correlation with dissolved organic carbon, temperature, and methane emissions, whereas the H2 concentrations measured with dialysis samplers do not show significant relationships with these field variables. Data presented in this study show that increased dissolved H2 reservoirs in wetland soils correlate with decreased methane reservoirs, which is consistent with studies that have shown that elevated levels of H2 inhibit methane production by inhibiting propionate fermentation, resulting in less acetate production and hence decreasing the contribution of acetoclastic methanogenesis to the overall production of methane.

Unique Organic Matter and Microbial Properties in the Rhizosphere of a Wetland Soil.

Wetlands attenuate the migration of many contaminants through a wide range of biogeochemical reactions. Recent research has shown that the rhizosphere, the zone near plant roots, in wetlands is especially effective at promoting contaminant attenuation. The objective of this study was to compare the soil organic matter (OM) composition and microbial communities of a rhizosphere soil (primarily an oxidized environment) to that of the bulk wetland soil (primarily a reduced environment). The rhizosphere had elevated C, N, Mn, and Fe concentrations and total bacteria, including Anaeromyxobacter, counts (as identified by qPCR). Furthermore, the rhizosphere contained several organic molecules that were not identified in the nonrhizosphere soil (54% of the >2200 ESI-FTICR-MS identified compounds). The rhizosphere OM molecules generally had (1) greater overall molecular weights, (2) less aromaticity, (3) more carboxylate and N-containing COO functional groups, and (4) a greater hydrophilic character. These latter two OM properties typically promote metal binding. This study showed for the first time that not only the amount but also the molecular characteristics of OM in the rhizosphere may in part be responsible for the enhanced immobilization of contaminants in wetlands. These finding have implications on the stewardship and long-term management of contaminated wetlands.

Uranium Redistribution Due to Water Table Fluctuations in Sandy Wetland Mesocosms.

To understand better the fate and stability of immobilized uranium (U) in wetland sediments, and how intermittent dry periods affect U stability, we dosed saturated sandy wetland mesocosms planted with Scirpus acutus with low levels of uranyl acetate for 4 months before imposing a short drying and rewetting period. Concentrations of U in mesocosm effluent increased after drying and rewetting, but the cumulative amount of U released following the dry period constituted less than 1% of the total U immobilized in the soil during the 4 months prior. This low level of remobilization suggests, and XANES analyses confirm, that microbial reduction was not the primary means of U immobilization, as the U immobilized in mesocosms was primarily U(VI) rather than U(IV). Drying followed by rewetting caused a redistribution of U downward in the soil profile and to root surfaces. Although the U on roots before drying was primarily associated with minerals, the U that relocated to the roots during drying and rewetting was bound diffusely. Results show that short periods of drought conditions in a sandy wetland, which expose reduced sediments to air, may impact U distribution without causing large releases of soil-bound U to surface waters.

Spectroscopic evidence of uranium immobilization in acidic wetlands by natural organic matter and plant roots.

Biogeochemistry of uranium in wetlands plays important roles in U immobilization in storage ponds of U mining and processing facilities but has not been well understood. The objective of this work was to study molecular mechanisms responsible for high U retention by Savannah River Site (SRS) wetland sediments under varying redox and acidic (pH = 2.6-5.8) conditions using U L3-edge X-ray absorption spectroscopy. Uranium in the SRS wetland sediments existed primarily as U(VI) bonded as a bidentate to carboxylic sites (U-C bond distance at ∼2.88 Å), rather than phenolic or other sites of natural organic matter (NOM). In microcosms simulating the SRS wetland processes, U immobilization on roots was 2 orders of magnitude higher than on the adjacent brown or more distant white sands in which U was U(VI). Uranium on the roots were both U(IV) and U(VI), which were bonded as a bidentate to carbon, but the U(VI) may also form a U phosphate mineral. After 140 days of air exposure, all U(IV) was reoxidized to U(VI) but remained as a bidentate bonding to carbon. This study demonstrated NOM and plant roots can highly immobilize U(VI) in the SRS acidic sediments, which has significant implication for the long-term stewardship of U-contaminated wetlands.

Biological reduction of uranium coupled with oxidation of ammonium by Acidimicrobiaceae bacterium A6 under iron reducing conditions.

This study investigated the possibility of links between the biological immobilization of uranium (U) and ammonium oxidation under iron (Fe) reducing conditions. The recently-identified Acidimicrobiaceae bacterium A6 (ATCC, PTA-122488) derives energy from ammonium oxidation coupled with Fe reduction. This bacterium has been found in various soil and wetland environments, including U-contaminated wetland sediments. Incubations of Acidimicrobiaceae bacteria A6 with nontronite, an Fe(III)-rich clay, and approximately 10 µM U indicate that these bacteria can use U(VI) in addition to Fe(III) as an electron acceptor in the presence of ammonium. Measurements of Fe(II) production and ammonium oxidation support this interpretation. Concentrations of approximately 100 µM U were found to entirely inhibit Acidimicrobiaceae bacteria A6 activity. These results suggest that natural sites of active ammonium oxidation under Fe reducing conditions by Acidimicrobiaceae bacteria A6 could be hotspots of U immobilization by bioreduction. This is the first report of biological U reduction that is not coupled to carbon oxidation.

Uranium immobilization in an iron-rich rhizosphere of a native wetland plant from the Savannah River Site under reducing conditions.

The hypothesis of this study was that iron plaques formed on the roots of wetland plants and their rhizospheres create environmental conditions favorable for iron reducing bacteria that promote the in situ immobilization of uranium. Greenhouse microcosm studies were conducted using native plants (Sparganium americanum) from a wetland located on the Savannah River Site, Aiken, SC. After iron plaques were established during a 73-day period by using an anoxic Fe(II)-rich nutrient solution, a U(VI) amended nutrient solution was added to the system for an additional two months. Compared to plant-free control microcosms, microcosms containing iron plaques successfully stimulated the growth of targeted iron reducing bacteria, Geobacter spp. Their population continuously increased after the introduction of the U(VI) nutrient solution. The reduction of some of the U(VI) to U(IV) by iron reducing bacteria was deduced based on the observations that the aqueous Fe(II) concentrations increased while the U(VI) concentrations decreased. The Fe(II) produced by the iron reducing bacteria was assumed to be reoxidized by the oxygen released from the roots. Advanced spectroscopic analyses revealed that a significant fraction of the U(VI) had been reduced to U(IV) and they were commonly deposited in association with phosphorus on the iron plaque.

Laboratory study of nitrification, denitrification and anammox processes in membrane bioreactors considering periodic aeration.

The possibility of using membrane bioreactors (MBRs) in simultaneous nitrification-anammox-denitrification (SNAD) by considering periodic aeration cycles was investigated. Two separate reactors were operated to investigate the effect of different anammox biomass in the presence of nitrifying and denitrifying biomass on the final nitrogen removal efficiency. The results illustrated that the reactor with higher anammox biomass was more robust to oxygen cycling. Around 98% Total Nitrogen (TN) and 83% Total Organic Carbon (TOC) removal efficiencies were observed by applying one hour aeration over a four-hour cycle. Decreasing the aeration time to 30, 15, and 2 min during a four-hour cycle affected the final TN removal efficiencies. However, the effect of decreasing aeration on the TN removal efficiencies in the reactor with higher anammox biomass was much lower compared to the regular reactor. The nitrous oxide (N2O) emission was a function of aeration as well, and was lower in the reactor with higher anammox biomass. The results of q-PCR analysis confirmed the simultaneous co-existence of nitrifiers, anammox, and denitrifiers in both of the reactors. To simulate the TN removal in these reactors as a function of the aeration time, a new model, based on first order reaction kinetics for both denitrification and anammox was developed and yielded a good agreement with the experimental observations.

Defluorination of PFAS by Acidimicrobium sp. strain A6 and potential applications for remediation.

Acidimicrobium sp. strain A6 is a recently discovered autotrophic bacterium that is capable of oxidizing ammonium while reducing ferric iron and is relatively common in acidic iron-rich soils. The genome of Acidimicrobium sp. strain A6 contains sequences for several reductive dehalogenases, including a gene for a previously unreported reductive dehalogenase, rdhA. Incubations of Acidimicrobium sp. strain A6 in the presence of perfluorinated substances, such as PFOA (perfluorooctanoic acid, C8HF15O2) or PFOS (perfluorooctane sulfonic acid, C8HF17O3S), have shown that fluoride, as well as shorter carbon chain PFAAs (perfluoroalkyl acids), are being produced, and the rdhA gene is expressed during these incubations. Results from initial gene knockout experiments indicate that the enzyme associated with the rdhA gene plays a key role in the PFAS defluorination by Acidimicrobium sp. strain A6. Experiments focusing on the defluorination kinetics by Acidimicrobium sp. strain A6 show that the defluorination kinetics are proportional to the amount of ammonium oxidized. To explore potential applications for PFAS bioremediation, PFAS-contaminated biosolids were augmented with Fe(III) and Acidimicrobium sp. strain A6, resulting in PFAS degradation. Since the high demand of Fe(III) makes growing Acidimicrobium sp. strain A6 in conventional rectors challenging, and since Acidimicrobium sp. strain A6 was shown to be electrogenic, it was grown in the absence of Fe(III) in microbial electrolysis cells, where it did oxidize ammonium and degraded PFAS.

A push-pull test to measure root uptake of volatile chemicals from wetland soils.

This paper introduces a novel modification of the single-well "push-pull" test that uses nonvolatile and multiple volatile tracers to investigate the transport and root uptake kinetics of volatile chemicals in saturated soils. This technique provides an estimate of potential volatilization fluxes without relying on enclosure-based measurements. The new push-pull methodology was validated with mesocosm experiments, and bench-scale hydroponic measurements were performed to develop an empirical relationship for scaling root uptake rates between chemicals. A new modeling approach to interpret data using sulfur hexafluoride and helium as dual volatile tracers was developed and shown to decrease errors relative to existing analytical techniques that utilize bromide as a conservative tracer. Root uptake of the volatile tracers was diffusion-limited, and uptake rate constants (kv) in vegetated experimental mesocosms ranged from 0.021 ± 9.0 × 10(-4) h(-1) for CFC-12 to 2.41 ± 0.98 h(-1) for helium. Hydroponic and mesocosm experiments demonstrate that the molecular diameter is a robust empirical predictor of kv.

Enhanced semipermanent dialysis samplers for long-term environmental monitoring in saturated sediments.

The ability to sample in situ natural environmental processes has proven to be challenging when working with redox-sensitive contaminates in saturated sediments in wetland systems, especially within the rhizosphere, where sharp redox gradients are common. Many traditional approaches are invasive and disturb natural sediment chemistry. Through laboratory and field studies, the work presented in this study demonstrates a novel semipermanent dialysis sampler that allows for long-term, anaerobic monitoring of shallow sediments. Dialysis samplers were deployed and tested for over 1 year while being exposed to extremes in climate. These newly designed devices produce statistically reproducible data and capture sensitive redox trends. Results from the newly designed samplers were compared to conventional samplers. Initially, both the new and old designs yielded statistically similar data, but these data diverged over a period of months. The new devices are less invasive, so data gathered from these devices are more likely to be a closer representation of true conditions in the subsurface. By giving reliable data from a consistent location in space, these new samplers represent a significant step forward in capturing spatial and temporal variability in wetland redox chemistry during long-term monitoring.

Modeling the kinetics of perfluorooctanoic and perfluorooctane sulfonic acid biodegradation by Acidimicrobium sp. Strain A6 during the feammox process.

Per- and polyfluoroalkyl substances (PFAS) are emerging contaminants of concern due to their health effects and persistence in the environment. Although perfluoroalkyl acids (PFAAs), such as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are very difficult to biodegrade because they are completely saturated with fluorine, it has recently been shown that Acidimicrobium sp. A6 (A6), which oxidizes ammonium under iron reducing conditions (Feammox process), can defluorinate PFAAs. A kinetic model was developed and tested in this study using results from previously published laboratory experiments, augmented with results from additional incubations, to couple the Feammox process to PFAS defluorination. The experimental results show higher Feammox activity and PFAS degradation in the A6 enrichment cultures than in the highly enriched A6 cultures. The coupled experimental and modeling results show that the PFAS defluorination rate is proportional to the rate of ammonium oxidation. The ammonium oxidation rate and the defluorination rate increase monotonically, but not linearly, with increasing A6 biomass. Given that different experiments had different level of Feammox activity, the parameters required to simulate the Feammox varied between A6 cultures. Nonetheless, the kinetic model was able to simulate an anaerobic incubation system and show that PFAS defluorination is proportional to the Feammox activity.

Enhanced Feammox activity and perfluorooctanoic acid (PFOA) degradation by Acidimicrobium sp. Strain A6 using PAA-coated ferrihydrite as an electron acceptor.

Acidimicrobium sp. Strain A6 (A6) can degrade perfluoroalkyl acids (PFAAs) by oxidizing NH4+ while reducing Fe(Ⅲ). However, supplying and distributing Fe(III) phases in sediments is challenging since surface charges of Fe(III)-phases are typically positive while those of sediments are negative. Therefore, ferrihydrite particles were coated with polyacrylic acid (PAA) with four different molecular weights, resulting in a negative zeta potential on their surface. Zeta potential was determined as a function of pH and PAA loading, with the lowest value observed when the PAA/ferrihydrite ratio was > 1/5 (w/w) at a pH of 5.5. Several 50-day incubations with an A6-enrichment culture were conducted to determine the effect of PAA-coated ferrihydrite as the electron acceptor of A6 on the Feammox activity and PFOA degradation. NH4+ oxidation, PFOA degradation, production of shorter-chain PFAS, and F- were observed in all PAA-coated samples. The 6 K and 450 K treatments exhibited significant reductions in PFOA concentration and substantial F- production compared to incubations with bare ferrihydrite. Electrochemical impedance spectroscopy showed lowered charge transfer resistance in the presence of PAA-coated ferrihydrite, indicating that PAAs facilitated electron transfer to ferrihydrite. This study highlights the potential of PAA-coated ferrihydrite in accelerating PFAS defluorination, providing novel insights for A6-based bioremediation strategies.

Gas-phase and transpiration-driven mechanisms for volatilization through wetland macrophytes.

Natural and constructed wetlands have gained attention as potential tools for remediation of shallow sediments and groundwater contaminated with volatile organic compounds (VOCs). Wetland macrophytes are known to enhance rates of contaminant removal via volatilization, but the magnitude of different volatilization mechanisms, and the relationship between volatilization rates and contaminant physiochemical properties, remain poorly understood. Greenhouse mesocosm experiments using the volatile tracer sulfur hexafluoride were conducted to determine the relative magnitudes of gas-phase and transpiration-driven volatilization mechanisms. A numerical model for vegetation-mediated volatilization was developed, calibrated with tracer measurements, and used to predict plant-mediated volatilization of common VOCs as well as quantify the contribution of different volatilization pathways. Model simulations agree with conclusions from previous work that transpiration is the main driver for volatilization of VOCs, but also demonstrate that vapor-phase transport in wetland plants is significant, and can represent up to 50% of the total flux for compounds with greater volatility like vinyl chloride.

Uranium reduction and microbial community development in response to stimulation with different electron donors.

Stimulating microbial reduction of soluble U(VI) to less soluble U(IV) shows promise as an in situ bioremediation strategy for uranium contaminated groundwater, but the optimal electron donors for promoting this process have yet to be identified. The purpose of this study was to better understand how the addition of various electron donors to uranium-contaminated subsurface sediments affected U(VI) reduction and the composition of the microbial community. The simple electron donors, acetate or lactate, or the more complex donors, hydrogen-release compound (HRC) or vegetable oil, were added to the sediments incubated in flow-through columns. The composition of the microbial communities was evaluated with quantitative PCR probing specific 16S rRNA genes and functional genes, phospholipid fatty acid analysis, and clone libraries. All the electron donors promoted U(VI) removal, even though the composition of the microbial communities was different with each donor. In general, the overall biomass, rather than the specific bacterial species, was the factor most related to U(VI) removal. Vegetable oil and HRC were more effective in stimulating U(VI) removal than acetate. These results suggest that the addition of more complex organic electron donors could be an excellent option for in situ bioremediation of uranium-contaminated groundwater.

Biodegradation of PFOA in microbial electrolysis cells by Acidimicrobiaceae sp. strain A6.

Acidimicrobiaceae sp. strain A6 (A6), is an anaerobic autotrophic bacterium capable of oxidizing ammonium (NH4+) while reducing ferric iron and is also able to defluorinate PFAS under these growth conditions. A6 is exoelectrogenic and can grow in microbial electrolysis cells (MECs) by using the anode as the electron acceptor in lieu of ferric iron. Therefore, cultures of A6 amended with perfluorooctanoic acid (PFOA) were incubated in MECs to investigate its ability to defluorinate PFAS in such reactors. Results show a significant decrease in PFOA concentration after 18 days of operation, while producing current and removing NH4+. The buildup of fluoride and shorter chain perfluorinated products was detected only in MECs with applied potential, active A6, and amended with PFOA, confirming the biodegradation of PFOA in these systems. This work sets the stage for further studies on the application of A6-based per- and polyfluorinated alkyl substances (PFAS) bioremediation in microbial electrochemical systems for water treatment.

Anaerobic degradation of perfluorooctanoic acid (PFOA) in biosolids by Acidimicrobium sp. strain A6.

Anaerobic incubations were performed with biosolids obtained from an industrial wastewater treatment plant (WWTP) that contained perfluorooctanoic acid (PFOA), and with per- and polyfluoroalkyl substances- (PFAS) free, laboratory-generated, biosolids that were spiked with PFOA. Biosolid slurries were incubated for 150 days as is, after augmenting with either Acidimicrobium sp. Strain A6 or ferrihydrite, or with both, Acidimicrobium sp. Strain A6 and ferrihydrite. Autoclaved controls were run in parallel. Only the biosolids augmented with both, Acidimicrobium sp. Strain A6 and ferrihydrite showed a decrease in the PFOA concentration, in excess of 50% (total, dissolved, and solid associated). Higher concentrations of PFOA in the biosolids spiked with PFOA and no previous PFAS exposure allowed to track the production of fluoride to verify PFOA defluorination. The buildup of fluoride over the incubation time was observed in these biosolid incubations spiked with PFOA. A significant increase in the concentration of perfluoroheptanoic acid (PFHpA) over the incubations of the filter cake samples from the industrial WWTP was observed, indicating the presence of a non-identified precursor in these biosolids. Results show that anaerobic incubation of PFAS contaminated biosolids, after augmentation with Fe(III) and Acidimicrobium sp. Strain A6 can result in PFAS defluorination.

Monitoring nitrogen loading and retention in an urban stormwater detention pond.

Stormwater detention ponds have become ubiquitous in urbanized areas and have been suggested as potential hotspots of N transformation within urban watersheds. As a result, there is a great deal of interest in their use as structural best management practices to reduce the excessive N export from these watersheds. We conducted continuous monitoring of the influent and effluent N loads of a stormwater detention pond located on the Princeton University campus in Princeton, New Jersey. Our monitoring was conducted during four 21-d periods representing the four seasons of the northeastern United States. Water quality samples were collected and analyzed for nitrate (NO3-) during all four monitoring periods. During two of these periods, loads of ammonium (NH4+), dissolved organic N, and particulate N (PN) were measured. Our results show that NO3- dominated the influent N load, particularly in dry weather inflows to the detention pond. However, PN, which is often neglected in stormwater quality monitoring, made up as much as 30% of the total load and an even greater fraction during storm events. The results of our monitoring suggest that seasonal variation may play an important role in N retention within the detention pond. Although retention of NO3-, the most dominant fraction of N in the influent stormwater, was observed during the summer sampling period, no significant NO3- retention was observed during the spring or the two cold-weather sampling periods.