Comprehensive evaluation of nitrogen contamination in water ecosystems of the Miyun reservoir watershed, northern China: distribution, source apportionment and risk assessment.

Miyun Reservoir plays a vital role as a source of drinking water for Beijing, however it grapples with nitrogen contamination issues that have been poorly understood in terms of their distribution, source, and associated health risks. This study addresses this knowledge gap by employing data on nitrate nitrogen (NO3--N), chloride (Cl-), dual isotopic compositions of NO3- (δ15N-NO3- and δ18O-NO3-) data in water ecosystems, systematically exploring the distribution, source and health risk of nitrogen contaminants in Miyun reservoir watersheds. The results showed that over the past 30 years, surface water runoff has exhibited a notable decrease and periodic fluctuations due to the combined influence of climate and anthropogenic activities, while the total nitrogen (TN) concentration in aquatic ecosystems presented an annual fluctuating upward trend. The TN concentration in the wet season was predominantly elevated because a large amount of nitrogen contaminants migrated into water ecosystems through heavy rainfall or river erosion. The concentration of NO3--N, the main contaminant of the water ecosystems, showed distinct variations across different watersheds, followed as rivers over the Miyun reservoir. Moreover, NO3--N levels gradually increased from upstream to downstream in different basins. NO3--N in surface water was mainly derived from the mixture of agricultural ammonia fertilizer and sewage and manure, with a minority of samples potentially undergoing denitrification. Comparatively, the main sources of NO3--N in groundwater were soil N and sewage and manure, while the denitrification process was inactive. The carcinogenic risks caused by NO3--N in groundwater were deemed either nonexistent or minimal, while the focus should predominantly be on potential non-carcinogenic risks, particularly for infants and children. Therefore, it is crucial to perform proactive measures aimed at safeguarding water ecosystems, guided by an understanding of the distribution, sources, and associated risks of nitrogen contamination.

Inhibition of Extracellular Enzyme Activity by Reactive Oxygen Species upon Oxygenation of Reduced Iron-Bearing Minerals.

The dual roles of minerals in inhibiting and prolonging extracellular enzyme activity in soils and sediments are governed by enzyme adsorption to mineral surfaces. Oxygenation of mineral-bound Fe(II) generates reactive oxygen species (ROS), yet it is unknown whether and how this process alters the activity and functional lifespan of extracellular enzymes. Here, the effect of mineral-bound Fe(II) oxidation on the hydrolytic activity of a cellulose-degrading enzyme ß-glucosidase (BG) was studied using two pre-reduced Fe-bearing clay minerals (nontronite and montmorillonite) and one pre-reduced iron oxide (magnetite) at pH 5 and 7. Under anoxic conditions, BG adsorption to mineral surfaces decreased its activity but prolonged its lifespan. Under oxic conditions, ROS was produced, with the amount of •OH, the most abundant ROS, being positively correlated with the extent of structural Fe(II) oxidation in reduced minerals. •OH decreased BG activity and shortened its lifespan via conformational change and structural decomposition of BG. These results suggest that under oxic conditions, the ROS-induced inhibitory role of Fe(II)-bearing minerals outweighed their adsorption-induced protective role in controlling enzyme activity. These results disclose a previously unknown mechanism of extracellular enzyme inactivation, which have pivotal implications for predicting the active enzyme pool in redox-oscillating environments.

Mineral-Bound Trace Metals as Cofactors for Anaerobic Biological Nitrogen Fixation.

Nitrogenase is the only known biological enzyme capable of reducing N2 to bioavailable NH3. Most nitrogenases use Mo as a metallocofactor, while alternative cofactors V and Fe are also viable. Both geological and bioinformatic evidence suggest an ancient origin of Mo-based nitrogenase in the Archean, despite the low concentration of dissolved Mo in the Archean oceans. This apparent paradox would be resolvable if mineral-bound Mo were bioavailable for nitrogen fixation by ancient diazotrophs. In this study, the bioavailability of mineral-bound Mo, V, and Fe was determined by incubating an obligately anaerobic diazotroph Clostridium kluyveri with Mo-, V-, and Fe-bearing minerals (molybdenite, cavansite, and ferrihydrite, respectively) and basalt under diazotrophic conditions. The results showed that C. kluyveri utilized mineral-associated metals to express nitrogenase genes and fix nitrogen, as measured by the reverse transcription quantitative polymerase chain reaction and acetylene reduction assay, respectively. C. kluyveri secreted chelating molecules to extract metals from the minerals. As a result of microbial weathering, mineral surface chemistry significantly changed, likely due to surface coating by microbial exudates for metal extraction. These results provide important support for the ancient origin of Mo-based nitrogenase, with profound implications for coevolution of the biosphere and geosphere.

Deterministic factors modulating assembly of groundwater microbial community in a nitrogen-contaminated and hydraulically-connected river-lake-floodplain ecosystem.

The river-lake-floodplain system (RLFS) undergoes intensive surface-groundwater mass and energy exchanges. Some freshwater lakes are groundwater flow-through systems, serving as sinks for nitrogen (N) entering the lake. Despite the threat of cross-nitrogen contamination, the assembly of the microbial communities in the RLFS was poorly understood. Herein, the distribution, co-occurrence, and assembly pattern of microbial community were investigated in a nitrogen-contaminated and hydraulically-connected RLFS. The results showed that nitrate was widely distributed with greater accumulation on the south than on the north side, and ammonia was accumulated in the groundwater discharge area (estuary and lakeshore). The heterotrophic nitrifying bacteria and aerobic denitrifying bacteria were distributed across the entire area. In estuary and lakeshore with low levels of oxidation-reduction potential (ORP) and high levels of total organic carbon (TOC) and ammonia, dissimilatory nitrate reduction to ammonium (DNRA) bacteria were enriched. The bacterial community had close cooperative relationships, and keystone taxa harbored nitrate reduction potentials. Combined with multivariable statistics and self-organizing map (SOM) results, ammonia, TOC, and ORP acted as drivers in the spatial evolution of the bacterial community, coincidence with the predominant deterministic processes and unique niche breadth for microbial assembly. This study provides novel insight into the traits and assembly of bacterial communities and potential nitrogen cycling capacities in RLFS groundwater.

Distribution, source investigation, and risk assessment of topsoil heavy metals in areas with intensive anthropogenic activities using the positive matrix factorization (PMF) model coupled with self-organizing map (SOM).

Over the past decade, heavy metal (HMs) contamination in soil environments has become severe worldwide. However, their resulting ecological and health risks remained elusive across a variety of soil ecosystems due to the complicated distributions and sources. This study investigated the HMs (Cr, As, Cu, Pb, Zn, Ni, Cd, and Hg) in areas with multi-mineral resources and intensive agricultural activities to study their distribution and source apportionment using a positive matrix factorization (PMF) model coupled with self-organizing map (SOM). The potential ecological and health risks were assessed in terms of distinct sources of HMs. The results disclosed that the spatial distribution of HM contaminations in the topsoil was region-dependent, primarily located in areas with high population intensity. The geo­accumulation index (Igeo) and enrichment factor (EF) values collectively displayed that the topsoils were severely contaminated by Hg, Cu, and Pb, particularly in residential farmland areas. The comprehensive analysis combined with PMF and SOM identified both geogenic and anthropogenic sources of HMs including natural, agricultural, mining, and mixed sources (caused by multi-anthropogenic factors), accounting for 24.9%, 22.6%, 45.9%, and 6.6% contribution rates, respectively. The potential ecological risk was predominantly due to the enrichment of Hg, followed by Cd. The non-carcinogenic risks were mostly below the acceptable risk level, while the potential carcinogenic health risks caused by As and Cr should be paid prime attention to, particularly for children. In addition to the 40% geogenic sources, agricultural activities contributed to 30% of the non-carcinogenic risk, whereas mining activities contributed to nearly half of the carcinogenic health risks.

Disentangling Microbial Syntrophic Mechanisms for Hexavalent Chromium Reduction in Autotrophic Biosystems.

Hexavalent chromium [Cr(VI)] is one of the common heavy-metal contaminants in groundwater, and the availability of electron donors is considered to be a key parameter for Cr(VI) biotransformation. During the autotrophic remediation process, however, much remains to be illuminated about how complex syntrophic microbial communities couple Cr(VI) reduction with other elemental cycles. Two series of Cr(VI)-reducing groundwater bioreactors were independently amended by elemental sulfur and iron and inoculated with the same inoculum. After 160 days of incubation, both bioreactors showed similar archaea-dominating microbiota compositions, whereas a higher Cr(VI)-reducing rate and more methane production were detected in the Fe0-driven one. Metabolic reconstruction of 23 retrieved genomes revealed complex symbiotic relationships driving distinct elemental cycles coupled with Cr(VI) reduction in bioreactors. In both bioreactors, these Cr(VI) reducers were assumed to live in syntrophy with oxidizers of sulfur, iron, hydrogen, and volatile fatty acids and methane produced by carbon fixers and multitrophic methanogens, respectively. The significant difference in methane production was mainly due to the fact that the yielded sulfate greatly retarded acetoclastic methanogenesis in the S-bioreactor. These findings provide insights into mutualistic symbioses of carbon, sulfur, iron, and chromium metabolisms in groundwater systems and have implications for bioremediation of Cr(VI)-contaminated groundwater.

Promotion of Microbial Oxidation of Structural Fe(II) in Nontronite by Oxalate and NTA.

Iron redox cycling occurs extensively in soils and sediments. Previous research has focused on microbially mediated redox cycling of aqueous Fe. At circumneutral pH, most Fe occurs in solid phase, where Fe and organic ligands interact closely. However, the role of organic ligands in microbial oxidation of solid-phase Fe(II) is not well understood. Here, we incubated reduced nontronite NAu-2 (rNAu-2) with an iron-oxidizing bacterium and in the presence of oxalate and nitrilotriacetic acid. These ligands significantly enhanced the rate and extent of microbial oxidation of structural Fe(II) in rNAu-2. Aqueous and solid-phase analyses, coupled with biogeochemical modeling, revealed a pathway for ligand-enhanced bio-oxidation of solid-phase Fe(II): (1) dissolution of rNAu-2 to form aqueous Fe(II)-ligand complex; (2) bio-oxidation to Fe(III)-ligand complex; (3) rapid reduction of Fe(III)-ligand complex to Fe(II)-ligand complex by structural Fe(II) in rNAu-2. In this process, the Fe(II)-ligand and Fe(III)-ligand complexes effectively serve as electron shuttle to expand the bioavailable pool of solid-phase Fe(II). These results have important implications for a better understanding of the bioavailability and reactivity of solid-phase Fe pool in the environment.

Distinct functional microbial communities mediating the heterotrophic denitrification in response to the excessive Fe(II) stress in groundwater under wheat-rice stone and rock phosphate amendments.

Denitrifying microbial community can be utilized for eliminating nitrate and Fe(II) combined contamination in groundwater, while excessive amount of Fe(II) limit the process. Natural mineral can be additional substrate for the microbial growth, whereas how it influences the microbial community that mediating the denitrification coupling with Fe(II) oxidation and balancing inhibition of excessive Fe(II) on denitrification remain unclear. In the present study, we conducted a series of microcosm experiments to explore the denitrification and Fe(II) oxidation kinetic, and used RNA-based qPCR and DNA-based high-throughput sequencing to elucidate microbial diversity, co-occurrence and metabolic profiles amended by wheat-rice stone and rock phosphate. The results showed that both minerals could extensively improve and double the denitrification rates (2.0 ± 0.03 to 2.12 ± 0.13 times), decrease the nitrite accumulation and trigger the high resistance of the denitrifiers from the stress of Fe(II), whereas only wheat-rice stone with higher surface area increased the oxidation of Fe(II) (<10%). The addition of both minerals enhanced the microbial alpha-diversity, shaped the beta-diversity and co-occurrence network, and recovered the transcription of nitrate and nitrite reductase (Nar, Nap, NirS, NirK) from the Fe(II) inhibition. Accordingly, heterotroph Methyloversatilis sp., Methylotenra sp. might contribute to the denitrification under wheat-rice stone amendment, Denitratisoma sp. contribute to the denitrification for rock phosphate, and Fe oxidation was partially catalyzed by Dechloromonas sp. or abiotically by the nitrite/nitrous oxide. These findings would be helpful for better understanding the bioremediation of nitrate and Fe contaminated groundwater.

Effect of Fe(II) on reactivity of heterotrophic denitrifiers in the remediation of nitrate- and Fe(II)-contaminated groundwater.

Heterotrophic denitrifiers, capable of simultaneous nitrate reduction and Fe(II) oxidation, can be applied for the remediation of nitrate and Fe(II) combined contamination in groundwater. Under strictly anaerobic condition, denitrifying microbial communities were enriched with the coexistence of soluble nitrate, Fe(II) and associated nutrient elements to monitor the denitrification process. Low abundance of Fe(II) (e.g., 10 mg L-1 in this study) tended to stimulate the activity of denitrifying microbial communities. However, elevated Fe(II) concentration (50 and 100 mg L-1 in this study), acted as a stress, strongly inhibited the activity and reproduction of denitrifiers. Besides, through thermodynamics calculations, methanol rather than Fe(II) was proved to be the preferable electron donors for both energy metabolism and anabolism. Betaproteobacteria was found to be the most predominant (sub)phylum in all enriched microbial assemblages. Methylovesartilis was the most predominant group mainly catalyzed for methanol based denitrification, and others denitrifiers included Methylophilaceae, Dechloromonas and Denitratisoma. Excessive Fe(II) in the solution greatly reduced the proportions of these denitrifying groups, while the influence seemed to be less apparent on functional genes composition. As such, a conceptional metabolism pathway of the most dominant genus (i.e., Methylovesartilis) for nitrate reducing as well as methanol and Fe(II) oxidation confirmed that biotic nitrate reducing and Fe(II) oxidizing were potentially proceeded in cytoplasm by enzymes such as NarGHI. The Fe(II) oxidation rate depended on the rate of Fe(II) entering into the cell. These findings provide a clear mechanistic understanding of heterotrophic denitrification coupling with Fe(II) oxidation, and environmental implication for the bioremediation of nitrate and Fe(II) contaminated groundwater.

Geochemical and Temporal Influences on the Enrichment of Acidophilic Iron-Oxidizing Bacterial Communities.

UNLABELLED: Two acid mine drainage (AMD) sites in the Appalachian bituminous coal basin were selected to enrich for Fe(II)-oxidizing microbes and measure rates of low-pH Fe(II) oxidation in chemostatic bioreactors. Microbial communities were enriched for 74 to 128 days in fed-batch mode, then switched to flowthrough mode (additional 52 to 138 d) to measure rates of Fe(II) oxidation as a function of pH (2.1 to 4.2) and influent Fe(II) concentration (80 to 2,400 mg/liter). Biofilm samples were collected throughout these operations, and the microbial community structure was analyzed to evaluate impacts of geochemistry and incubation time. Alpha diversity decreased as the pH decreased and as the Fe(II) concentration increased, coincident with conditions that attained the highest rates of Fe(II) oxidation. The distribution of the seven most abundant bacterial genera could be explained by a combination of pH and Fe(II) concentration. Acidithiobacillus, Ferrovum, Gallionella, Leptospirillum, Ferrimicrobium, Acidiphilium, and Acidocella were all found to be restricted within specific bounds of pH and Fe(II) concentration. Temporal distance, defined as the cumulative number of pore volumes from the start of flowthrough mode, appeared to be as important as geochemical conditions in controlling microbial community structure. Both alpha and beta diversities of microbial communities were significantly correlated to temporal distance in the flowthrough experiments. Even after long-term operation under nearly identical geochemical conditions, microbial communities enriched from the different sites remained distinct. While these microbial communities were enriched from sites that displayed markedly different field rates of Fe(II) oxidation, rates of Fe(II) oxidation measured in laboratory bioreactors were essentially the same. These results suggest that the performance of suspended-growth bioreactors for AMD treatment may not be strongly dependent on the inoculum used for reactor startup. IMPORTANCE: This study showed that different microbial communities enriched from two sites maintained distinct microbial community traits inherited from their respective seed materials. Long-term operation (up to 128 days of fed-batch enrichment followed by up to 138 days of flowthrough experiments) of these two systems did not lead to the same, or even more similar, microbial communities. However, these bioreactors did oxidize Fe(II) and remove total iron [Fe(T)] at very similar rates. These results suggest that the performance of suspended-growth bioreactors for AMD treatment may not be strongly dependent on the inoculum used for reactor startup. This would be advantageous, because system performance should be well constrained and predictable for many different sites.