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Denitrifying woodchip bioreactors are used at field-scale to remove nitrate from agricultural drainage water before it contaminates aquatic ecosystems. However, low microbial activity at cold temperatures during the winter half-year may cause the efficiency of nitrate removal to drop below 20%. Here, we isolated 134 strains of denitrifying bacteria from woodchips and wetland soil to search for candidates for bioaugmentation to improve nitrate removal at cold temperatures. The strains were identified by analyses of 16S rRNA genes and were dominated by Pseudomonadaceae (n = 63), notably the genus Pseudomonas (n = 58). Two candidates for bioaugmentation, P. proteolytica WBRS105 and P. chlororaphis PSS4, were selected among six strains that were characterized in detail by assays of growth and denitrification at 5 and 10 °C, combined with genome analysis. Both strains possessed the genes for complete denitrification and degradation of carbon substrates such as cellulose, hemicellulose and lignin. Also, both strains were able to grow with extracts from woodchips as sole sources of energy and carbon. Bioaugmentation under laboratory conditions indicated that the two strains were able to compete with an established microbial community in enrichment cultures at 5 °C and enhance nitrate removal rates by 27–37% when applied to woodchips from an operating full-scale bioreactor. The successful bioaugmentation of woodchip microbiomes at low temperature is encouraging, although the increased rate obtained under laboratory conditions should be further tested, e.g., under pilot-scale conditions, to verify the potential of the technology to increase the efficiency of bioreactors treating agricultural drainage water.

Denitrifying woodchip bioreactors (WBRs) remove nitrate (NO3−) from agricultural drainage water at field-scale, but their efficacy at cold temperatures remains uncertain. This study shows how hydraulic residence time (HRT) controls NO3− removal and environmental side-effects of WBRs at low water temperature under pilot-scale conditions with controlled operation of nine WBRs (94 dm3). Hydraulic properties were assessed by a bromide tracer test, and NO3− removal, emissions of nitrous oxide (N2O) and methane (CH4), and losses of dissolved organic carbon (DOC) were measured at HRTs of 5–30 h. Inlet NO3− concentrations were increasingly reduced at higher HRTs. The relationship between HRT and the efficiency (%) of NO3− removal was linear (Radj2 = 0.94), while the relationship between HRT and NO3− reduction rates (NRR) was logistic (Radj2 = 0.88). Gaseous emissions of N2O were equally low at HRTs of 10–30 h, but higher at 5 h (P < 0.05). Methane fluxes were small, but with consistent emissions at HRTs of 20–30 h and uptake at 5–15 h. HRT had limited effect on effluent DOC concentrations, but strong effect on mass losses that were five-fold higher (320 mg L−1) at the HRT of 5 h than at 30 h. In summary, at cold temperatures HRTs of ≤ 20 h resulted in suboptimal NRR, accelerating DOC losses, and increased risk of N2O losses at least below a threshold HRT of 5–10 h. HRTs of 20–30 h gave maximal NRR, smallest losses of DOC and N2O, but an increased risk of CH4 emissions.

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.

Bioaugmentation with specialized inocula has been proven a feasible way to remediate under-performing anaerobic digestion (AD) processes. However, a major bottleneck for successful and cost-effective bioaugmentation is the lack of ready-to-use, specialized methanogenic cultures when required. The reason is the slow growth of the anaerobic consortia and the high cost of maintaining them active in the necessary amounts for successful bioaugmentation applications. This study offers an effective procedure where customized AD inocula could be preserved and used on-demand for remediation of ammonia inhibited AD reactors. Additionally, it introduces the biological and physicochemical mechanisms that render the long-term preservation of the AD inocula possible. Specifically, two different preservation carriers (i.e. agar gel and liquid basic anaerobic medium) were assessed at two different temperatures (i.e. 4 °C and 24 °C) using an ammonia tolerant methanogenic consortium. The results from methane production, lag-phase, maximum methane production rate and cell viability indicate that the consortium preserved for 168 days in agar gel at 24 °C performed best compared to the other tested preservation conditions. Meanwhile, 16S rRNA sequencing analysis indicated that Methanosarcina soligelidi and Methanoculleus palmolei shown a high revival rate and metabolic activity after long-term preservation. Thus, this successful long-term preservation method of ready-to-use AD consortia could render the successful bioaugmentation in full-scale biogas reactors economically possible in the near future.

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.