Denitrification dominates dissimilatory nitrate reduction across global natural ecosystems.

Denitrification, anaerobic ammonium oxidation (anammox), and dissimilatory nitrate reduction to ammonium (DNRA) are three competing processes of microbial nitrate reduction that determine the degree of ecosystem nitrogen (N) loss versus recycling. However, the global patterns and drivers of relative contributions of these N cycling processes to soil or sediment nitrate reduction remain unknown, limiting our understanding of the global N balance and management. Here, we compiled a global dataset of 1570 observations from a wide range of terrestrial and aquatic ecosystems. We found that denitrification contributed up to 66.1% of total nitrate reduction globally, being significantly greater in estuarine and coastal ecosystems. Anammox and DNRA could account for 12.7% and 21.2% of total nitrate reduction, respectively. The contribution of denitrification to nitrate reduction increased with longitude, while the contribution of anammox and DNRA decreased. The local environmental factors controlling the relative contributions of the three N cycling processes to nitrate reduction included the concentrations of soil organic carbon, ammonium, nitrate, and ferrous iron. Our results underline the dominant role of denitrification over anammox and DNRA in ecosystem nitrate transformation, which is crucial to improving the current global soil N cycle model and achieving sustainable N management.

Type VI secretion system drives bacterial diversity and functions in multispecies biofilms.

Type VI secretion system (T6SS) plays an essential role in interspecies interactions and provides an advantage for a strain with T6SS in multispecies biofilms. However, how T6SS drives the bacterial community structure and functions in multispecies biofilms still needs to be determined. Using gene deletion and Illumina sequencing technique, we estimated bacterial community responses in multispecies biofilms to T6SS by introducing T6SS-containing Pseudomonas putida KT2440. Results showed that the niche structure shifts of multispecies biofilms were remarkably higher in the presence of T6SS than in the absence of T6SS. The presence of T6SS significantly drove the variation in microbial composition, reduced the alpha-diversity of bacterial communities in multispecies biofilms, and separately decreased and increased the relative abundance of Proteobacteria and Bacteroidota. Co-occurrence network analysis with inferred putative bacterial interactions indicated that P. putida KT2440 mainly displayed strong negative associations with the genera of Psychrobacter, Cellvibrio, Stenotrophomonas, and Brevundimonas. Moreover, the function redundancy index of the bacterial community was strikingly higher in the presence of T6SS than in the absence of T6SS, regardless of whether relative abundances of bacterial taxa were inhibited or promoted. Remarkably, the increased metabolic network similarity with T6SS-containing P. putida KT2440 could enhance the antibacterial activity of P. putida KT2440 on other bacterial taxa. Our findings extend knowledge of microbial adaptation strategies to potential bacterial weapons and could contribute to predicting biodiversity loss and change in ecological functions caused by T6SS.

Temperature drives elevational diversity patterns of different types of organisms in Qinghai-Tibetan Plateau wetlands.

The spatial pattern and driving mechanism of biodiversity along elevational gradients are key topics in ecology. However, it is still unclear whether the multidimensional diversity of different types of organisms shows a similar response to elevation changes. Here, we measured the species and phylogenetic diversity of plants, bacteria, fungi, and microbial functional groups (nitrifiers, denitrifiers, methanogens, and methanotrophs) in 36 wetland sites on the Qinghai-Tibetan Plateau. The results showed that both species and phylogenetic diversity of plants, bacteria, and fungi exhibited a significant elevational gradient, in direct contrast to no significant diversity changes observed for denitrifiers, methanogens, and methanotrophs along the same altitude gradient. Our findings suggest that elevation and temperature were more likely to associate with the diversity of plants, bacteria, and fungi than the diversity of microbial functional groups, with important implications for assessing the effect of ongoing climate warming on biodiversity in Qinghai-Tibetan alpine wetlands.

Feammox is more important than anammox in anaerobic ammonium loss in farmland soils around Lake Taihu, China.

Ammonium (NH4+) oxidation is a key step in nitrogen transformation in ecosystems. Prior to the recent discovery of Feammox (anaerobic NH4+ oxidation coupled with iron reduction), anammox (anaerobic NH4+ oxidation coupled with nitrite reduction) was thought of as the only pathway by which anaerobic NH4+ loss (NH4+ directly to N2) occurs in soils. Experimental evidence has confirmed that both anammox and Feammox contribute to anaerobic NH4+ loss; however, their relative contributions to this process in farmland soils are largely unknown. Therefore, in this study, we examined the seasonal activities of anammox and Feammox in conventional tillage (CT) and no-tillage (NT) soils around Lake Taihu, China. Isotopic tracing experiments showed higher anammox and Feammox rates in summer than in other seasons, and the contribution of Feammox to anaerobic NH4+ loss from the farmland soils (54.6%-69.3%) was higher than that of anammox. Further, the Feammox rates corresponding to the two soil tillage practices were significantly different, whereas their corresponding anammox rates showed no significant differences. Furthermore, molecular analysis showed that the abundance of Geobacteraceae differed significantly with season and tillage practice, whereas the abundance of anammox bacteria showed no significant differences between CT and NT practices. Structural equation modeling also revealed that the anammox rate was directly or indirectly driven by N availability and season, whereas the Feammox rate was driven by soil moisture content, Fe(III) concentration, Fe(III) reduction rates, tillage practice, and season. Overall, this study enhances understanding regarding anaerobic NH4+ oxidation in farmland soils and highlight the importance of Feammox in NH4+ loss in such an ecosystem.

Evidence for the occurrence of Feammox coupled with nitrate-dependent Fe(II) oxidation in natural enrichment cultures.

Feammox is a newly discovered process of anaerobic ammonium oxidation driven by Fe(III) reduction. Nitrate-dependent Fe(II) oxidation (NDFO) is the coupling of Fe(II) oxidation and nitrate reduction to produce N2 under anaerobic conditions. It has not been reported whether the coupling of the two reactions exists in natural enrichment. In this study, enrichment culture experiments were carrired out to prove the occurrence of Feammox with NDFO. The results indicated that the nitrogen and iron cycle were formed during natural enrichment cultures, including Fe(III) reduction and NH4+-N was oxidation to NO3--N, NO2--N and N2, Fe(III) and Fe(II) were cyclically formed, and Fe(II) was oxidized with NO3--N reduced to N2. The removal efficiencies of ammonium nitrogen and total nitrogen in the incubation were about 92.9% and 20% respectively. Organic carbon experiments indicate that sodium acetate can promote the initial NO3--N removal and a low concentration of organic carbon limited the NDFO process because iron-oxidizing bacteria are mixotrophic microorganisms. The added 9,10-anthraquinone-2,6-disulfonate (AQDS) in the later stage can promote NDFO to remove nitrate, thereby increasing the TN removal efficiency to 50%. 15N-isotope tracer incubations provided direct evidence for the occurrence of Feammox coupled to NDFO, with rates producing 30N2 of Feammox (0.024-0.0288 mg N·L-1·d-1) and NDFO (0.0465-0.0833 mg N·L-1·d-1) in three groups (Wetland/Wheat soil/Sediment). 16S rRNA sequencing further demonstrated that Pseudomonas, Rhodanobacter, Acinetobacter and Thermomonas were the dominant generas among the enrichment cultures, and these bacteria belonged to FeOB and FeRB, which may further promote Feammox coupled to NDFO in the cultivation system.

Nitrogen loss through denitrification, anammox and Feammox in a paddy soil.

Since the process of anaerobic ammonium oxidation (anammox) coupled with ferric iron reduction (termed Feammox) was discovered, it has been observed in various natural environments. However, besides the vertical distribution of Feammox in paddy soils, its differences and relationships with traditional nitrogen loss processes, including denitrification and anammox, remain unclear. Here, we studied the distribution of nitrogen loss pathways in different layers (0-50 cm) of paddy soil in southeastern China using 15N isotope tracer technology and molecular analysis. Our study showed that denitrification had a rate of 2.19 ± 0.39 mg N·kg-1·d-1, which was the highest activity in the surface layer (0-10 cm). The activities of anammox and Feammox reached peak values in the 10-20 cm (1.13 ± 0.16 mg N·kg-1·d-1) and 20-30 cm (0.23 ± 0.02 mg N·kg-1·d-1) soil layer, respectively. The nitrogen loss in the surface layer was more serious than that in the deep layer under paddy cultivation. In this study, denitrification was the main nitrogen loss pathway in the surface soil, but Feammox became an important nitrogen loss pathway (up to 26.1%) in the 20-40 cm depth. Overall, our research could improve and perfect the nitrogen cycle pathways in paddy soil.

Effects of the addition of nitrogen and phosphorus on anaerobic ammonium oxidation coupled with iron reduction (Feammox) in the farmland soils.

Anaerobic ammonium oxidation coupled with iron reduction is termed as Feammox, and is a new nitrogen removal process. However, there is a paucity of studies on the response of nutrient additions on Feammox process in farmland ecosystems. In this study, we investigated the shifts of Feammox and iron-reducers under nitrogen (N) and phosphorus (P) applications via isotopic tracing and high-throughput sequencing technology. In the isotopic tracing experiment, Feammox rates was significantly greater in the N and/or P applications soil (0.184-0.239 µg N g-1 day-1) than in the no fertilizer soil (0.172 µg N g-1 day-1). The results indicated that N and P applications could favor the Feammox reaction. Molecular analysis showed that five predominant iron-reducing bacteria, including Geobacter, Anaeromyxobacter, Pseudomonas, Thiobacillus and Bacillus, were detected. Their abundance in the soil with no fertilizer, N, P and N combined with P was 0.93%, 1.11%-1.71%, 0.99%, and 1.40%-1.75%, respectively. This implied that iron-reducing bacteria can be stimulated under N and P applications. Overall, the results of this study demonstrated that N and/or P applications could alter the activity of Feammox, and modulate the potential of IRB in the farmland soils.

Impact of sand mining on the carbon sequestration and nitrogen removal ability of soil in the riparian area of Lijiang River, China.

Riparian areas are widely recognized as the main areas for carbon sequestration and nitrogen pollution removal, while little is known about the effects of the respective sand mining activities on riparian zones. In this study, the effects of sand mining activities on the soil organic carbon (SOC) storage, different N-removal processes (Feammox, anammox, and denitrification), and composition of the relative bacterial community at a depth of 0-40 cm were determined based on investigations in riparian sand mining areas and adjacent forestlands. The SOC density of the sand mining areas (2.59 t ha-1, depth of 0-40 cm) was lower than that of the riparian forestlands (80.42 t ha-1). Compared with those of the riparian forestland, the sand mining area exhibited a dramatic reduction in the CO2-fixed gene abundances (cbbL) and a significant change in the composition of cbbL-containing bacteria. The rates of the Feammox (0.038 ± 0.014 mg N kg-1 d-1), anammox (0.017 ± 0.017 mg N kg-1 d-1), and denitrification (0.090 ± 0.1 mg N kg-1 d-1) processes at a depth of 0-20 cm in the soil layer of the sand mining area were reduced by 70.17%, 91.5%, and 93.62% compared with those of the riparian forestland, respectively. The riparian areas in the study area (approximately 12 ha, depth of 0-40 cm) destroyed by sand mining activities released approximately 933.96 t stored soil carbon, which reduce the annual carbon sequestration potential by 28.8-40.8 t. Moreover, the potential N-removal rates in the riparian forestlands (depth of 0-20 cm) by the Feammox, anammox, and denitrification processes were 1514.21-1530.95 kg N ha-1 year-1, whereas the potential N-removal rates in the sand mining area were only 121.2-126.19 kg N ha-1 year-1. Therefore, more investigations are necessary for comparing the benefits and damage of sand mining activities in riparian areas before more sand mining activities are approved.

Spatial and seasonal distributions of Feammox from ecosystem habitats in the Wanshan region of the Taihu watershed, China.

Anaerobic ammonium oxidation coupled to Fe(III) reduction, termed Feammox, is a newly identified microbial process that occurs in nitrogen and iron cycles. As the seasonal distribution of Feammox in different ecosystem habitats has not been fully explored, this study investigated the potential Feammox rates and the diversity and abundance of iron reducing bacteria (IRB) in three habitats during two seasons by using isotope tracing technique and molecular analysis, respectively. Results showed that potential Feammox rates vary both seasonally and spatially, having relatively higher rates in summer (0.05-0.19 mg N kg-1 d-1) and lower rates in winter (0.02-0.09 mg N kg-1 d-1). In addition, relatively higher and lower rates were observed in farmland soils (0.09-0.19 mg N kg-1 d-1) and river sediments (0.02-0.05 mg N kg-1 d-1), respectively. The abundance and diversity of IRB were also found to vary both spatially and seasonally. Furthermore, the results show that Feammox may transform nitrogen at a rate of approximately 2.4-22.5 kg N ha-1 yr-1 within the investigated area. It is considered that the soil moisture, the Fe(III) content, and the total organic carbon are important factors controlling Feammox and IRB. Overall, these results extend current scientific knowledge about nitrogen and iron cycles in ecosystem habitats.

Nitrogen loss through anaerobic ammonium oxidation mediated by Mn(IV)-oxide reduction from agricultural drainage ditches into Jiuli River, Taihu Lake Basin.

Up to date, no great breakthrough has been made in the research of anaerobic ammonium oxidation mediated by Mn(IV)-oxide reduction (termed Mnammox). Recently, the Feammox process has become a hot research topic in the study of nitrogen loss from soils. Interestingly, in this study, an alternative pathway of N loss was proposed in terrestrial ecosystems. Mnammox could produce NO2-, NO3-, and N2 as end products. Here, our study demonstrated the occurrence of Mnammox, and direct evidence for Mnammox in agricultural drainage ditch soils with microbial Mn(IV) and Fe(III) reduction was obtained using the 15NH4+ isotopic tracing technique. The extent and rate of 30N2 and 29N2 production and Mn(IV) reduction were enhanced when amended with 15NH4+ and were further promoted when amended with 15NH4++MnO2. Moreover, although the Fe(III) reduction rate was stimulated with the addition of 15NH4+, the Fe(III) reduction rate greatly decreased when MnO2 was added. Mnammox rates ranged from 0.40 to 0.79 mg N kg-1 d-1, and an estimated 6.57-18.25 kg ha-1 year-1 N loss was associated with Mnammox in the examined soils. We revealed that the Mnammox reaction may be more efficient than the Feammox reaction, and the Feammox rates found in previous studies may have been overestimated. Overall, for the first time, this work provided key evidence for the existence of Mnammox in terrestrial ecosystems and suggested that Mnammox could be an important pathway for nitrogen loss in agricultural drainage ditch soils.

[Denitrification and Anaerobic Ammonium Oxidation in Soil Nitrogen Migration Process in a Farmland of Wanshandang Lake].

To explore the rate variation and contribution to N loss of denitrification and anaerobic ammonia oxidation (ANAMMOX) in the nitrogen migration process of farmland soils in southern China, we assess the physicochemical characteristics soil samples of different soil layers from farmland and different land use types (farmland, river channel, riparian zone, and lake sediment) in a wheat-rice rotation area of Wanshandang Lake. Illumina MiSeq sequencing and quantitative real-time polymerase chain reaction (qPCR) are used to investigate the microbial community composition and functional gene abundances of the samples. The potential denitrification and ANAMMOX rate (calculated by N2) of each sample was determined by an isotope culture experiment. It was demonstrated that the potential denitrification rate was significantly positively correlated with TOC, NH4+-N, and NO3--N (P<0.05), and with the abundances of nirS, nirK, and nosZ (P<0.05). The denitrification rate of surface soils was (11.51±1.04) nmol·(g·h)-1, which was significantly higher than other soil layers and other land use types (P<0.05). While the ANAMMOX rate in farmland soils was the highest in the 20-30 cm layer and reached (0.48±0.07) nmol·(g·h)-1. In addition, denitrification was the main cause of N loss in surface soils of the studied farmland, accounting for 91.9%-99.7% of overall loss, and ANAMMOX played an important role in the production of N2 in deep soils.

[Insight into the Process of Mn-ANAMMOX in Soils of Agricultural Drainage Ditches].

Anaerobic ammonium oxidation mediated by MnO2 (termed Mn-ANAMMOX) is a newly discovered microbial nitrogen removal pathway. However, few studies have reported on the Mn-ANAMMOX process and related microbial communities in agricultural drainage ditches. In this study, Mn(Ⅳ)-reducing bacteria (MnBR) enrichment cultivation was carried out for 340 days and an isotope tracing technique and high-throughput sequencing technology were used to provide convincing evidence of the occurrence of Mn-ANAMMOX. The results showed that simultaneous NH4+ oxidation and MnO2 reduction occurred during the reaction, and the production of NO2-, NO3-, 30N2, and Mn2+ was detected. Additionally, the average Mn-ANAMMOX rate, ammonium removal rate, and total nitrogen removal rate were 2.88 mg·(kg·d)-1, 20%, and 15%, respectively. Moreover, high-throughput sequencing results showed that after 340 d in the enrichment cultivation experiments, the abundance of MnBR increased from 27% to 70% at the phylum level, and the major genera of MnBR were determined as Acinetobacter and Geothrix, with relative abundances of 26.63% and 4.07%, respectively. Overall, the occurrence of Mn-ANAMMOX was directly proven during the MnBR enrichment cultivation experiments, and it might play an essential role in the pathway of microbial nitrogen removal.

Variation of Feammox following ammonium fertilizer migration in a wheat-rice rotation area, Taihu Lake, China.

Feammox is a newly discovered and important anaerobic nitrogen (N) loss pathway, and its variation and role in removing N following the application of N fertilizer and its migration from paddies to other land use types and from surface soils to deep soils have not been thoroughly elucidated to date. In this study, field sampling and slurry incubation experiments were performed to evaluate the Feammox rate between different land use types (paddy, irrigation ditch, riparian zone and lake, 0-10 cm) and different paddy soil depths (0-70 cm) in a wheat-rice rotation area in China. Based on a 15N-labelled isotope-tracing technique and analysis of microbial communities, it was estimated that the potential Feammox rate ranged from 0.031 to 0.42 mg N kg-1 d-1 in this area. In the soil profile of the paddy, the depth of 20-30 cm was the active region of Feammox, with a value of 0.37 ±â€¯0.057 mg N kg-1 d-1. Compared with the surface soil (0-10 cm) of the paddy (0.18 ±â€¯0.031 mg N kg-1 d-1), the potential Feammox rate of the irrigation ditch soil was not significantly different, but that of the lake riparian soil and lake sediment were decreased by 27.27% and 32.11%, respectively (p < 0.01). Fe(III) content was the best predictor of the Feammox rate and explained the variation of the Feammox rate by 36.00% in the surface soil. At the genus level, the paddy soil at a depth of 20-30 cm had the greatest abundance of the genera in which the Fe reduction bacteria were distributed; and where Bacillus, Geobacter and Anaeromyxobacter had higher proportions. It was estimated that the potential N loss by Feammox was in the range of 7.36 (the lake) ∼43.35 (the paddy) kg N ha-1 year-1 in the surface soil of this area. Considering denitrification and the Feammox rate as a whole, we found that denitrification remained to be the main contributor to N loss in the surface soil (94.72-96.89% of N loss), although Feammox dominated N loss in the deep soil (below 0-10 cm).

Nitrogen loss through anaerobic ammonium oxidation coupled to Iron reduction from ecosystem habitats in the Taihu estuary region.

Anaerobic ammonium oxidation coupled to iron reduction, termed Feammox, is a new microbial process linked the nitrogen cycles. However, the nitrogen losses through Feammox from different ecosystem habitats remain unclear. In this study, isotope tracing technology and molecular microbial analysis were used to investigate the Feammox and its contribution to the nitrogen loss in the farmland and riparian soils, and river sediments. The potential Feammox rates were detected, which varied from 0.07 to 0.15mgNkg-1d-1 among the three ecosystem habitats. Feammox rates were significantly higher in the farmlands or riparian soils than in the river sediments. Feammox, denitrification and anaerobic ammonium oxidation (anammox) were estimated to account for approximately 3.5-4.2%, 92.6-93.1% and 2.8-3.9% of the total nitrogen losses respectively, while a significant correlation was observed between the Feammox rates and the denitrification rates (r=0.72, P<0.05). In addition, a nitrogen loss at 8.3-17.8kgNha-1yr-1 was linked with Feammox in the examined soils. This study demonstrated that Feammox could be a potential pathway of nitrogen loss from ecosystem habitats.

[Insight into the Mechanism of Feammox in the Surface Soils of a Riparian Zone].

Anaerobic ammonium oxidation coupled to iron (Ⅲ) reduction (termed Feammox) is a recently discovered pathway of nitrogen cycling. However, little is known about the pathways of N transformation via the Feammox process in riparian zones. In this study, evidence of Feammox in the riparian zone soil layers (0-20 cm) was demonstrated using the isotope tracing technique and a high-throughput sequencing technology. The results showed that Feammox occurred in the riparian zones in four different soil layers (A:0-5 cm, B:5-10 cm, C:10-15 cm, D:15-20 cm) and the Feammox rates ranged from 0.25 mg·(kg·d)-1 to 0.29 mg·(kg·d)-1. In the B soil sample, the Feammox rate was significantly higher than in the other soil samples (P<0.05). In addition, iron reducing bacteria played an essential role in the Feammox process, and Anaeromyxobacter and Geobacter were detected in all the soil samples. In the B soil sample, the abundance of iron reducing bacteria was significantly higher than in the other soil samples (P<0.05). Overall, the co-occurrence of ammonium oxidation and iron reduction suggest that Feammox can play an essential role in the pathway of nitrogen removal in riparian zones.

Nitrogen loss from anaerobic ammonium oxidation coupled to Iron(III) reduction in a riparian zone.

Anaerobic ammonium oxidation coupled to iron(III) reduction (termed Feammox) is a recently discovered pathway of nitrogen cycling. However, little is known about the pathways of N transformation via Feammox process in riparian zones. In this study, evidence for Feammox in riparian zones with or without vegetation cover was demonstrated using isotope tracing technique and high-throughput sequencing technology. The results showed that Feammox could occur in riparian zones, and demonstrated that N2 directly from Feammox was dominant Feammox pathway. The Feammox rates in vegetated soil samples was 0.32-0.37 mg N kg-1 d-1, which is higher than that in un-vegetated soil samples (0.20 mg N kg-1 d-1). Moreover, the growth of vegetation led to a 4.99-6.41% increase in the abundance of iron reducing bacteria (Anaeromyxobacter, Pseudomonas and Geobacter) and iron reducing bacteria play an essential role in Feammox process. An estimated loss of 23.7-43.9 kg N ha-1 year-1 was associated with Feammox in the examined riparian zone. Overall, the co-occurrence of ammonium oxidation and iron reduction suggest that Feammox can play an essential role in the pathway of nitrogen removal in riparian zones.

[Effect of Elodea nuttallii-Immobilized Nitrogen Cycling Bacteria on the Mechanism of Nitrogen Removal in Polluted River Water].

Surface water, Elodea nuttallii and undisturbed sediment cores from the Qinshui River in Gonghu Bay were collected to carry out a simulation experiment in a laboratory to study the effect of Elodea nuttallii-immobilized nitrogen-cycling bacteria on nitrogen removal mechanisms from the river water. In this study, the transformation and fate of ammonium among four different treatment groups were investigated by using a stable 15 N isotope pairing technique combined with high-throughput sequencing technology[Treatment A:bare sediment, Treatment B:sediment+immobilized nitrogen cycling bacteria (INCB), Treatment C:sediment+E. nuttallii, Treatment D:sediment+INCB+E. nuttallii]. The results of the 15 N mass-balance model showed that there were three pathways to the ultimate fate of nitrogen:precipitated with the sediments, absorbed by E. nuttallii, and consumed by microbial processes[denitrification and anaerobic ammonium oxidation (ANAMMOX)]. The percentages of E. nuttallii assimilated in the 15 NH4+ were 25.44% and 19.79% for treatments C and D. The sediment storage ratio of 15 NH4+ accounted for 7.94%, 5.52%, 6.47% and 4.86% in treatments A, B, C, and D, respectively. The proportion of 15 NH4+ lost as 15 N-labelled gas were 16.06%, 28.86%, 16.93% and 33.09% in the four different treatment groups, respectively. Denitrification and anammox were the bacterial primary processes in N2 and N2O production. The abundance and diversity of microorganisms was relatively higher in the treatment with E. nuttallii-immobilized nitrogen cycling bacteria (E-INCB) assemblage technology applied. Furthermore, the removal rates of 15 NH4+ were 24%, 34.38%, 48.84% and 57.74% in treatments A, B, C and D, respectively. These results show that the E-INCB assemblage technology may improve the capacity for nitrogen removal from the river water.