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Woodchip bioreactors are increasingly used as tools to mitigate nitrogen (N) pollution from agricultural drainage water. They consist of a basin filled with woodchip material through which N contaminated drainage water can flow. During the water transport through the filter matrix, oxygen is rapidly depleted and denitrification removes a fraction of the nitrate N present in the water. However, the N removal efficiency of the bioreactors varies significantly both across systems and seasonally. Furthermore, denitrification can also produce nitrous oxide, which is a potent greenhouse gas. Here, we investigated how variation in hydraulic residence time influenced N removal efficiency and nitrous oxide emissions at eight woodchip bioreactors of different flow designs, monitored for 2–4 years. We also characterised the relative abundance of genes involved in the N cycle at three of the bioreactors using metagenomics. Our results showed that total N removal was 17–73% of the yearly incoming N and that it was influenced by hydraulic residence time and water temperature. Nitrous oxide emissions were variable among the different bioreactors and were higher when the hydraulic residence time was less than 60 h. However, the yearly nitrous oxide release did not exceed 2.4% of the nitrate removal (on N atom basis) and the mean among the bioreactors was 0.6%. Although there were marked differences in nitrate removal and nitrous oxide emissions, there were no clear differences in the relative abundance of N-cycling genes among and within three tested bioreactors. Yet, denitrification genes greatly outnumbered genes related to dissimilatory nitrate reduction to ammonium. Overall, our study showed that all eight bioreactors were effective in removing N from agricultural drainage water and that nitrous oxide emissions were low, especially at hydraulic residence times of 60 h or more.

Bioaugmentation – the addition of specific microorganisms to microbial consortia to obtain specific functions - is a promising method to improve the performance of sub-optimal anaerobic digestion processes and recover methane production. The present study implements for the first time a model with bioaugmentation functionalities, enabling the dynamic microbiological study of augmented anaerobic systems. The model is based on a previous advanced mathematical model focused on ammonia inhibition, which was extended. Three inhibited anaerobic digestion experiments were simulated, where bioaugmentation involved the addition of: (i) hydrogenotrophic methanogenic archaea; (ii) a combination of hydrogenotrophs and syntrophic acetate oxidizing bacteria; and (iii) acetoclastic methanogenic archaea to the inhibited reactors. Methane productivity, pH, and ammonia were correctly predicted by the model, with weighted absolute percentage errors below 10%. Trends in volatile fatty acids accumulation and consumption showed good agreement between in silico and in vivo data. Simulations revealed that bioaugmentation will result in process improvement when specialized hydrogenotrophs are used: not only do they establish themselves in the inhibited environment, but also help in the recovery of acetoclastic methanogens. Overall, the mathematical approach adopted to describe the bioaugmentation phenomena resulted in a robust computational tool for simulation of general bioaugmentation strategies.

Woodchip bioreactors are increasingly used to remove nitrate (NO3–) from agricultural drainage water in order to protect aquatic ecosystems from excess nitrogen. Nitrate removal in woodchip bioreactors is based on microbial processes, but the microbiomes and their role in bioreactor efficiency are generally poorly characterized. Using metagenomic analyses, we characterized the microbiomes from 3 full-scale bioreactors in Denmark, which had been operating for 4–7 years. The microbiomes were dominated by Proteobacteria and especially the genus Pseudomonas, which is consistent with heterotrophic denitrification as the main pathway of NO3– reduction. This was supported by functional gene analyses, showing the presence of the full suite of denitrification genes from NO3– reductases to nitrous oxide reductases. Genes encoding for dissimilatory NO3– reduction to ammonium were found only in minor proportions. In addition to NO3– reducers, the bioreactors harbored distinct functional groups, such as lignocellulose degrading fungi and bacteria, dissimilatory sulfate reducers and methanogens. Further, all bioreactors harbored genera of heterotrophic iron reducers and anaerobic iron oxidizers (Acidovorax) indicating a potential for iron-mediated denitrification. Ecological indices of species diversity showed high similarity between the bioreactors and between the different positions along the flow path, indicating that the woodchip resource niche was important in shaping the microbiome. This trait may be favorable for the development of common microbiological strategies to increase the NO3– removal from agricultural drainage water.

Denitrifying woodchip bioreactors (WBR), which aim to reduce nitrate (NO3−) pollution from agricultural drainage water, are less efficient when cold temperatures slow down the microbial transformation processes. Conducting bioaugmentation could potentially increase the NO3− removal efficiency during these specific periods. First, it is necessary to investigate denitrifying microbial populations in these facilities and understand their temperature responses. We hypothesized that seasonal changes and subsequent adaptations of microbial populations would allow for enrichment of cold-adapted denitrifying bacterial populations with potential use for bioaugmentation. Woodchip material was sampled from an operating WBR during spring, fall, and winter and used for enrichments of denitrifiers that were characterized by studies of metagenomics and temperature dependence of NO3− depletion. The successful enrichment of psychrotolerant denitrifiers was supported by the differences in temperature response, with the apparent domination of the phylum Proteobacteria and the genus Pseudomonas. The enrichments were found to have different microbiomes’ composition and they mainly differed with native woodchip microbiomes by a lower abundance of the genus Flavobacterium. Overall, the performance and composition of the enriched denitrifying population from the WBR microbiome indicated a potential for efficient NO3− removal at cold temperatures that could be stimulated by the addition of selected cold-adapted denitrifying bacteria.

Eutrophication of natural water bodies is moderated by transformation of nitrate (NO 3 −) in riparian wetlands, which serve as filters of infiltrating drain water from upland agricultural areas. The present study comprised field observations, laboratory experiments and metagenomic studies to describe NO 3 − removing transformation pathways and interactions with the cycling of iron (Fe) in a temperate riparian wetland soil profile down to 1 m depth. Water samples from piezometers showed a distinct plume of NO 3 − in the subsurface soil where agricultural drain water was infiltrating. However, within a distance of few meters in the water flow direction, NO 3 − was depleted from the percolating water. Sampling and analyses of soil from the active zone of the biogeochemical NO 3 − removal showed that denitrifying enzyme activity was ~ tenfold higher in the upper 0–25 cm than in the lower 25–100 cm. Yet, net transformation of NO 3 − was substantial also at 25–100 cm when assayed with relatively undisturbed soil samples and by 15N tracer techniques in soil slurries. Transformation pathways of dissimilatory nitrate reduction to ammonium and anaerobic ammonium oxidation were identified, but were quantitatively minor as compared to denitrification. Heterotrophic denitrification and denitrification mediated by oxidation of ferrous iron, Fe(II), were identified as important processes in the wetland soil. The latter was substantiated by geochemical observations, by rates of NO 3 − depletion in slurry incubations with added FeCl 2, and by identification of microorganisms with known capacity of NO 3 − reduction coupled to Fe(II) oxidation (Acidovorax sp.). The transformation pathway of iron-mediated NO 3 − reduction could involve biotic and abiotic reactions, and N 2O, which is a potent greenhouse gas, was a major product of the process. It remains to be seen under field conditions if N 2O emission hotspots are linked to specific sites of dynamic NO 3 − reduction coupled to Fe(II) oxidation.