Denitrification is a crucial process in the global nitrogen cycle, in which two functionally equivalent genes, nirS and nirK, catalyse the critical reaction and are usually used as marker genes. The nirK gene can function independently, whereas nirS requires additional genes to encode nitrite reductase and is more sensitive to environmental factors than nirK. However, the ecological differentiation mechanisms of those denitrifying microbial communities and their adaptation strategies to environmental stresses remain unclear. Here, we conducted metagenomic analysis for sediments and bioreactor samples from Lake Donghu, China. We found that nirS-type denitrifying communities had a significantly lower horizontal gene transfer frequency than that of nirK-type denitrifying communities, and nirS gene phylogeny was more congruent with taxonomy than that of nirK gene. Metabolic reconstruction of metagenome-assembled genomes further revealed that nirS-type denitrifying communities have robust metabolic systems for energy conservation, enabling them to survive under environmental stresses. Nevertheless, nirK-type denitrifying communities seemed to adapt to oxygen-limited environments with the ability to utilize various carbon and nitrogen compounds. Thus, this study provides novel insights into the ecological differentiation mechanism of nirS and nirK-type denitrifying communities, as well as the regulation of the global nitrogen cycle and greenhouse gas emissions.

Denitrification is of great significance for low C/N wastewater treatment. In this study, pyrite autotrophic denitrification (PAD) was coupled with a three-dimensional biofilm electrode reactor (BER) to enhance denitrification. The effect of current on denitrification was extensively studied. The nitrate removal of the PAD-BER increased by 14.90% and 74.64% compared to the BER and the PAD, respectively. In addition, the electron utilization, extracellular polymeric substances secretion, and denitrification enzyme activity (NaR and NiR) were enhanced in the PAD-BER. The microbial communities study displayed that Dokdonella, Hydrogenophaga, Nitrospira, and Terrimonas became the main genera for denitrification. Compared with the PAD and the BER, the abundance of the key denitrification genes narG, nirK, nirS, and nosZ were all boosted in the PAD-BER. This study indicated that the enhanced autotrophic denitrifiers and denitrification genes were responsible for the improved denitrification in the PAD-BER. PRACTITIONER POINTS: PAD-BER displayed higher nitrate removal, EPS, NAR, and NIR activity. The three types of denitrification (HD, HAD, and PAD) and their contribution percentage in the PAD-BER were analyzed. HAD was dominant among the three denitrification processes in PAD-BER. Microbial community composition and key denitrification genes were tested to reveal the denitrification mechanisms.

Nitrogen (N) pollution is a major threat to river ecosystems worldwide. Elucidating the community structure of N-cycling microorganisms in rivers is essential to understanding how ecosystem processes and functions will respond to increasing N inputs. However, previous studies generally focus on limited functional genes through amplicon sequencing or quantitative PCR techniques and cannot cover all N-cycling microorganisms. Here, metagenomic sequencing and genome binning were used to determine N-cycling genes in water, channel sediments, and riparian soils of the Yangtze River, which has been heavily polluted by N. Additionally, the denitrification and anaerobic ammonium oxidation (anammox) rates that reflect N removal potential were measured using 15N isotope pairing technique. Results showed that functional genes involved in organic N metabolism (i.e., organic degradation and synthesis) and nitrate reduction pathways (i.e., dissimilatory and assimilatory nitrate reduction to ammonium and denitrification) were more abundant and diverse than other N-cycling genes. A total of 121 metagenome-assembled genomes (MAGs) were identified to be involved in N-cycling processes, and the key MAGs were mainly taxonomically classified as Alphaproteobacteria and Gammaproteobacteria. The abundance and diversity of most N-cycling genes were higher in soils and sediments than in water, as well as higher in downstream and midstream than in upstream sites. These spatial variations were explained not only by local environment and vegetation but also by geographical and climatic factors. N removal process (i.e., denitrification and anammox) rates were significantly related to the abundance or diversity of several N-cycling genes, and climate and edaphic factors could regulate denitrification and anammox rates directly and indirectly through their effects on functional genes. Overall, these results provide a new avenue for further understanding the biogeographic patterns and environmental drivers of N-cycling microorganisms in rivers from the metagenomic perspective.

In this study, a novel three-dimensional biofilm electrode reactor (3D-BER) with a graphene oxide (GO)-modified cathode was developed to enhance the denitrification performance of secondary effluent from wastewater treatment plants (SEWTPs). The effects of different hydraulic retention times (HRTs) and currents on the 3D-BER were explored. The results indicated that at the optimal HRT of 4 h and current of 350 mA/m2, the 3D-BER with GO-modified cathode had a higher denitrification rate (2.40 ± 0.1 mg TN/L/h) and less accumulation of intermediate products, especially with 3.34% total nitrogen (TN) molar conversion to N2O. The GO-modified cathode offered a large biocompatible specific surface area and enhanced the conductivity, which favored microbial growth and increased electron transfer efficiency and extracellular enzyme activities. Moreover, the activity of nitrite reductase increased more than that of nitrate reductase to accelerate nitrite reduction, thus facilitating the denitrification process. The proposed 3D-BER provided an effective solution to elevate tertiary denitrification in the SEWTP.

Woodchips are widely used as a low-cost and renewable organic carbon source for denitrifying biofilms in passive nutrient removal systems. One limitation of wood-based biofiltration systems is their relatively poor removal of phosphorus (P) from subsurface drainage and stormwaters, necessitating the use of additional filter media when co-treatment of nitrogen (N) and P is required. Here, we show that anoxic-oxic cycling of woodchip media, which enhances nitrate (NO3-) removal by increasing the mobilization of organic carbon from wood, also improves orthophosphate (Pi) uptake onto woodchips. Orthophosphate removal rates in flow-through woodchip columns ranged from 0 to 34.9 μg PO43- L-1 h-1 under continuously-saturated (anoxic) conditions, and increased to 17.5 to 71.9 μg PO43- L-1 h-1 in columns undergoing drying-rewetting (oxic-anoxic) cycles. The highest Pi removal efficiencies were observed in the first 20 h after reactors were re-flooded, and were concurrent with maxima in polyphosphate kinase (ppk) gene expression by the polyphosphate accumulating organisms (PAOs) Accumulibacter spp. and Pseudomonas spp. Batch experiments confirmed that anoxic-anaerobic-oxic pre-incubation conditions led to orthophosphate uptake onto woodchips as high as 74.9 ± 0.8 mg PO43-/kg woodchip, and batch tests with autoclaved woodchips demonstrated that Pi uptake was due to biological processes and not adsorption. NO3- removal in batch tests was also greatest under oxic incubation conditions, attributed to greater carbon availability in hypoxic to anoxic zones in woodchip biofilms. While further research is needed to elucidate the mechanisms controlling enhanced Pi uptake by woodchip biofilms under anoxic-(anaerobic-)oxic cycling, these results suggest a role for enhanced Pi uptake by PAOs in a nature-based system for treatment of nonpoint source nutrients.

This study uncovered the response of novel micro-granule wastewater treatment technology to different powder carrier materials. Characteristics and distinctions among different systems were assessed based on process performance, sludge aggregation capacity, and microbial metabolism. Zeolite carrier system exhibited remarkable nitrogen removal efficiency of 89.6 ± 0.9 %, while diatomite carriers, in conjunction with intermittent aeration, enhanced simultaneous nitrification and denitrification from 2.6 % to 27.1 %. Iron-based carriers demonstrated efficient phosphorus removal (94.7 ± 1.2 %) through both chemical and microbial pathways. Specific surface area, pore structure and biocompatibility of powder carriers determined the formation and size of micro-granules. Tryptophan-like substances, C-(C/H), and Npr in extracellular polymeric substances strongly correlated with sludge hydrophobicity and granulation. Significant enrichment in norank_Comamonadaceae and Nitrosomonas in zeolite powder carrier system promoted partial nitrification and endogenous denitrification. Differences in metabolic pathways elucidated the up-regulation of amino acid synthesis, energy metabolism, and membrane transport as potential mechanisms driving micro-granule formation and efficient treatment performance.

Denitrification, anammox, and DNRA are three important nitrogen (N) reduction pathways in estuarine sediments. Although salinity is an important variables controlling microbial growth and activities, knowledge about the effects of changing salinity on those three processes in estuarine and coastal wetland sediments are not well understood. Herein, we performed a 60-d microcosms experiment with different salinities (0, 5, 15, 25 and 35 ‰) to explore the vital role of salinity in controlling N-loss and N retention in estuarine wetland sediments. The results showed that sediment organic matter, sulfide, and nitrate (NO3-) were profoundly decreased with increasing salinity, while sediment ammonium (NH4+) and ferrous (Fe2+) varied in reverse patterns. Meanwhile, N-loss and N retention rates and associated gene abundances were differentially inhibited with increasing salinity, while the contributions of denitrification, anammox, and DNRA to total nitrate reduction were apparently unaffected. Moreover, denitrification rate was the most sensitive to salinity, and then followed by DNRA, while anammox was the weakest among these three processes. In other words, anammox bacteria showed a wide range of salinity tolerance, while both denitrification and DNRA reflected a relatively limited dynamic range of it. Our findings could provide insights into temporal interactive effects of salinity on sediment physico-chemical properties, N reduction rates and associated gene abundances. Our findings can improve understanding of the effects of saltwater incursion on the N fate and N balance in estuarine and coastal sediments.

The microbial processes occurring in constructed wetlands (CWs) are difficult to understand owing to the complex interactions occurring between a variety of substrates, microorganisms, and plants under the given physicochemical conditions. This frequently leads to very large unexplained nitrogen losses in these systems. In continuation of our findings on Anammox contributions, our research on full-scale field CWs has suggested the significant involvement of the sulfur cycle in the conventional C-N cycle occurring in wetlands, which might closely explain the nitrogen losses in these systems. This paper explored the possibility of the sulfur-driven autotrophic denitrification (SDAD) pathway in different types of CWs, shallow and deep and passive and aerated systems, by analyzing the metagenomic bacterial communities present within these CWs. The results indicate a higher abundance of SDAD bacteria (Paracoccus and Arcobacter) in deep passive systems compared to shallow systems and presence of a large number of SDAD genera (Paracoccus, Thiobacillus, Beggiatoa, Sulfurimonas, Arcobacter, and Sulfuricurvum) in aerated CWs. The bacteria belonging to the functional category of dark oxidation of sulfur compounds were found to be enriched in deep and aerated CWs hinting at the possible role of the SDAD pathway in total nitrogen removal in these systems. As a case study, the percentage nitrogen removal through SDAD pathway was calculated to be 15-20% in aerated wetlands. The presence of autotrophic pathways for nitrogen removal can prove highly beneficial in terms of reducing sludge generation and hence reducing clogging, making aerated CWs a sustainable wastewater treatment solution.

In this paper, a biological aerated filter (BAF) based on ferromanganese oxide-biochar (FMBC) was constructed to investigated the removal performance and mechanism for conventional pollutants and four kinds of antibiotic, in contrast of conventional zeolite loaded BAF (BAF-A) and bamboo biochar filled BAF (BAF-B). Results showed that the average removal efficiency of total nitrogen (TN), total phosphorus (TP) and antibiotics in a FMBC-BAF (named by BAF-C) were 52.97 ± 2.27%, 51.58 ± 1.92% and 70.36 ± 1.00% ~ 81.65 ± 0.99% respectively in running period (39-100 d), which were significantly higher than those of BAF-A and BAF-B. In the BAF-C, the expression of denitrification enzyme activities and the secretion of extracellular polymeric substance (EPS) especially polyprotein (PN) were effectively stimulated, as well as accelerated electron transfer activity (ETSA) and lower electrochemical impedance spectroscopy (EIS) were acquired. After 100 days of operation, the abundance of nitrogen, phosphorus and antibiotic removal functional bacteria like Sphingorhabdus (4.52%), Bradyrhizobium (1.98%), Hyphomicrobium (2.49%), Ferruginibacter (7.80%), unclassified_f_Blastoca tellaceae (1.84%), norank_f_JG30-KF-CM45 (6.82%), norank_f_norank_o_SBR1031 (2.43%), Nitrospira (2.58%) norank_f_Caldilineaceae (1.53%) and Micropruina (1.11%) were enriched. Mechanism hypothesis of enhanced performances of nutrients and antibiotics removal pointed that: The phosphorus was removed by adsorption and precipitation, antibiotics removal was mainly achieved through the combined action of adsorption and biodegradation, while nitrogen removal was realized by biologic nitrification and denitrification in a FMBC-BAF for aquaculture wastewater treatment.

A novel A. pittii J08 with heterotrophic nitrification and aerobic denitrification (HN-AD) isolated from pond sediments could rapidly degrade inorganic nitrogen (N) and total nitrogen (TN-N) with ammonium (NH4+-N) preference. N degradation rate of NH4+-N, nitrite (NO2--N) and nitrate (NO3--N) were 3.9 mgL-1h-1, 3.0 mgL-1h-1 and 2.7 mgL-1h-1, respectively. In addition, strain J08 could effectively utilize most of detected low-molecular-weight carbon (LMWC) sources to degrade inorganic N with a wide adaptability to various culture conditions. Whole genome sequencing (WGS) analysis revealed that assembled genome of stain J08 possessed the crucial genes involved in dissimilatory/assimilatory NO3--N reduction and NH4+-N assimilation. These results indicated that strain J08 could be applied to wastewater treatment in aquaculture.