In-depth profile of NFR and physicochemical characteristics in mangrove sediments
We determined the NFR of 10 depths of the mangrove sediments using acetylene reduction assay (Additional file: Fig. S2). The NFR fluctuated in the range of 0-0.20 nmol/(g*h), and the average NFR was 0.031 nmol/(g*h). Clearly, we observed a depth-related variability of NFR, which reached a maximum at the depth of 90-100cm. Compared to the surficial layers (0–50 cm), the deeper layers (50–100 cm) showed higher NFR and a significantly (R2 = 0.42, p < 0.05) postitive correlation between NFR and depth was detected in mangrove sediments, as revealed by a linear regression analysis (Fig. 1a).
In-depth profile of physicochemical characteristics was also examined in mangrove sediments. Salinity in mangrove sediments ranged from 0.43‰ to 1.54‰, and increased with depth (Additional file: Fig. S2, S3). Conversely, moisture of sediments, with an average of 52%, showed a consistent decreasing with depth (Additional file: Fig. S2, S3). pH and total iron concentration decreased from 0 cm to 50 cm, and then increased from 50 cm to 100 cm (Additional file: Fig. S3). However, the concentrations of NH4+, NO2−, NO3−, available Fe and Fe3+ exhibited no momentous differences with depth (Additional file: Fig. S3). The linear regression analysis showed that moisture had a negative correlation with depth (R2 = 0.61, p < 0.05) (Additional file: Fig. S3d), but salinity had a positive correlation with depth (R2 = 0.93, p < 0.0001) (Additional file: Fig. S3f). It is expected that such changes of physicochemical properies may influence the in-depth profile of diazotrophic communities in mangrove sediments.
In-depth profile of diazotrophic communities in mangrove sediments
To investigate biotic factors contributing to the increased NFR with depth, we analyzed diazotrophic communities in mangrove sediments by sequencing nifH gene amplicons. From all samples, we obtained a total of 2,253,352 high-quality nifH sequences, and the nifH sequences were assigned into 974 operational taxonomic units (OTUs) and 59 genera after trimming (Additional file: Table S1). Notably, we observed the in-depth variation of diazotrophic communities in mangrove sedments. Both Shannon and Chao1 indices showed significant (R2 = 0.47, p < 0.05) negative relationships with depth (Fig. 1a), highlighting the decrease of diazotrophic community diversity and richness with depth. Meanwhile, diazotrophic diversity indices showed negative relationships with NFR (Fig. 1b), namely, diazotrophic community diversity and richness decreased with the increasing of depth. Furthermore, principal coordinates analysis (PCoA) showed that diazotrophic microbial communities in mangrove sediments were well separated by depth, and 50 cm was identified as the partitioning depth based on the Bray-Curtis distance (Additional file: Fig. S4). Two nonparametric tests (ANOSIM and ADONIS) further verified such significant variations (p < 0.001) of diazotrophic communities between sediment layers above and below 50 cm. Together, these findings determined the reduced diversity and varied structure of diazotrophic communities with depth of mangrove sediments.
Key diazotrophs contributing to increased NFR with depth
Taxonomic analysis of diazotrophic communities showed that bacteria (92.53%) dominated biological nitrogen fixation in mangrove sediments, and a few archaea (such as Methanomicrobia belong to Euryarchaeota) (0.18%) had the potential to fix nitrogen. At the phylum level, Proteobacteria was prevalent diazotrophs in mangrove sediments, which is consistent with terrestrial ecosystems [42]. Among Proteobacteria, Deltaproteobacteria occupied the largest proportion with an average relative abundance of 31%, followed by Gammaproteobacteria and Alphaproteobacteria (Additional file: Fig. S5a). At the genus level, aerobic diazotrophs such as Methylomonoas and Heliobacterium had higher relative abundances in the surficial layers (0–50 cm), while diazotrophic members represented by Agrobacterium, Desulfuromonas, Klebsiella and Azospira had higher relative abundances in the deeper layers (50–100 cm) (Additional file: Fig. S5b).
We performed Linear discriminant analysis Effect Size (LEfSe) on diazotrophic communities in mangrove sediments. Interestingly, diazotrophs presented manifest hierarchical clustering when 50 cm was set as the partitioning depth (Additional file: Fig. S6). The LEfSe results showed that, most of the diazotrophs enriched in surficial sediments (above 50 cm) belonged to Proteobacteria, such as Burkholderiales, Nitrosomonadales, Desulfarculales, Myxococcales and Methylococcales. Conversely, Agrobacterium affiliated to Alphaproteobacteria, Azotobacter affiliated to Deltaproteobacteria, and Dehalococcoides affiliated to Chloroflexi appeared conspicuous clustering in sediments below 50 cm (Additional file: Fig. S6). The diazotrophs specific for deep sediments tended to be crucial for biological nitrogen fixation in mangrove sediments because their higher relative abundances in deeper sediments corresponded with stronger NFR (Fig. 2b).
To further examine whether these deep sediment-specific diazotrophs played major roles in biological nitrogen fixation, we performed Pearson’s correlation analysis between NFR and abundances of diazotrophic genera in mangrove sediment profiles (Fig. 2, Additional file: Table S2). Among 11 diazotrophic genera having negative correlations with NFR, Desulfovibrio (R2 = 0.56, p < 0.05) and Anaeromyxobacter (R2 = 0.53, p < 0.05) had higher relative abundances, accounting for 5.2% and 2.9% of all diazotrophs, respectively (Fig. 2b, Additional file: Table S3). However, we also observed two diazotrophic genera that have positive correlations with NFR, namely Agrobacterium (R2 = 0.73, p < 0.05) and Azotobacter (R2 = 0.48, p < 0.05) (Fig. 2b). Azotobacter had a higher average relative abundance (11.9%) than Agrobacterium (3.3%) (Additional file: Table S3). Given their positive correlations with depth and their functional roles previously reported [4, 55], we assumed that both of them (especially Azotobacter) played a decisive role for the increased NFR with depth of mangrove sedments.
Relationships among sediment physicochemical characteristics, diazotrophic communities and NFR
To reveal the relationship between diazotrophic communities and environmental factors, we performed RDA to estimate the factors that had significant (p < 0.01) influences on diazotrophic communities (Fig. 3a). The results showed that nitrate, ammonia, moisture and salinity were the important driving factors of diazotrophic communities in mangrove sediments. Specially, moisture and salinity were the only two environmental characteristics significantly related to depth (Additional file: Fig. S3d, f).Through all detected depths, Pearson’s correlation analysis revealed that diazotrophic communities in terms of Chao 1 index and PCoA1 of diazotrophic communities were significantly (p < 0.05) negative with salinity (Additional file: Table S4, S5), suggesting that salinity and moisture were the main environmental factors driving the in-depth variation of diazotrophic communities in mangrove sediments.
To further evaluate the direct and indirect effects of depth, sediment properties (including moisture and salinity), and diazotrophic community richness and structure on NFR, we conducted SEM analysis based on known relationships among these observed variables (Fig. 3b). Consistent with the linear regression (Additional file: Fig. S3d, f) and RDA results (Fig. 3a), depth showed a directly positive effect on salinity and a directly negative effect on moisture, and salinity exerted significant effects on the diazotrophic community structure (Fig. 3a). Among all the observed variables in the model, only the diazotrophic community structure had a direct effect on NFR (Fig. 3c) although depth could indirectly influence NFR by strongly affecting sediment salinity (Fig. 3b). Collectively, these results indicated that salinity-driven diazotrophic community structure played a critical role in determining the in-depth profile of NFR in mangrove sediments.
In-depth profile of biological nitrogen fixation and its downstream processes in mangrove sediments
Samples from three depths (M1: 0–10 cm, M2: 50–60 cm, M3: 90–100 cm) of mangrove sediments were selected for metagenomic sequencing analysis of N-cycling gene profiles across the surficial, middle and deep sediments. We first proposed an in-depth schema to illustrate metabolic potentials for various nitrogen cycling processes based on key N-cycling functional genes (Fig. 4a-c). Notably, a total of eight pathways consistently revealed depth-related variations in terms of functional gene abudances (Fisher’s exact test, p < 0.05), including nitrogen fixation, nitrification, denitrification, dissimilatory nitrate reduction to ammonium (DNRA), assimilatory nitrate reduction, ammonia assimilation, nitrate assimilation and organic N decomposition.
Consistent with the trend of diazotrophic activity (NFR), the gene clusters for nitrogen fixing (nifH/D/K) increased in abundance with depth. Compared to the surficial layer (M1: 0–10 cm), the abundance of nitrogen fixation genes in deep sediment increased by 41.9% (Fig. 4c). Such increasing trend also occurred in ammonia assimilation and assimilatory nitrate reduction (Fig. 4). Particularly, the functional genes (nasA, narB and nirA) involved in assimilatory nitrate reduction remarkably increased 1.5, 17.6 and 9.3 times from the surficial layer to the deep sediment (Fig. 4c). By contrast, the abundance of functional genes involved in nitrification (aomA, amoB, amoC and hao), denitrification (nirK, nirS, norB and norC), DNRA (nrfA, nrfH, nirB and nirD) and organic N decomposition (ureA, ureB and ureC) significantly (p < 0.05) decreased with depth (Fig. 4). Taking the rate-limiting process of denitrification as example, the abundance of napA/B decreased by 21.5% from the surficial layer to the deep sediment (Fig. 4c). Overall, these functional gene patterns showed that both biological nitrogen fixation and its downstream processes in mangrove sediments underwent the depth-related variation with divergent trends.
Versatile functions and adaptation strategies of diazotrophic MAGs
De novo assembly and binning of metagenomic sequencing data from three depths of mangrove sediments allowed the reconstruction of 3 archaeal and 64 bacterial MAGs (completeness > 50%, contaminated < 10%; Additional file: Supplementary Data 1). Given that metagenomic sequencing generated enormous data accompanied by tremendous undiscovered information, we inferred their potential physiological capabilities by annotating genes using the KAAS and TIFRFAM databases. Among all 67 high-quality and high-completion MAGs, three MAGs possessed genes for nitrogen fixation (nifH/D/K), namely M2.bin.35, M2.bin.46 and M3.bin.42, which represented one Chloroflexi and two Desulfurmonadales (Fig. 5, Additional file: Supplementary Data 2). Interestingly, these three MAGs consistently contained genes associated with other nitrogen cycling processes, such as ammonia assimilation and the complete DNRA pathway (Fig. 5, Additional file: Supplementary Data 2). Additionally, M2.bin.35 had the genes involved with entire denitrification except for converting NO3− to N2O (Fig. 5, Additional file: Supplementary Data 2). However, genes related to nitrate assimilation, assimilatory nitrate reduction, organic N decomposition or nitrification were absent in these three diazotrophic MAGs (Fig. 5, Additional file: Supplementary Data 2).
Further functional annotation showed many potentials of these diazotrophic MAGs. From the perspective of energy metabolism, three MAGs contained genes involved in the complete or nearly complete carbon fixation pathways (such as Wood-Ljungdahl pathway) (Fig. 5, Additional file: Supplementary Data 2), so they would have the potential to convert inorganic carbon into organic molecules such as acetyl-CoA. Via TCA cycle, acetyl-CoA could be further utilized by these diazotrophs to generate energy for microbial metabolism (Fig. 5, Additional file: Supplementary Data 2). Combined with the detection of genes enconding lactate dehydrogenase (ldh), pyruvate oxidoreductase (porA/C) and formate dehydrogenase (fodG) (Fig. 5, Additional file: Supplementary Data 2), our results suggest that these diazotrophs showed an amphitrophic lifestyle in mangrove sediments. From the perspective of adaptive strategies, diazotrophs from middle or deep mangrove sediments contained NiFe-hydrogenase genes for anaerobic respiration, oxidative stress responses (hyaB, hybC), sulfur reduction and anaerobic cobalamin biosynthesis (Fig. 5, Additional file: Supplementary Data 2). The adaptation of diazotrophs to the low-oxygen deep sediments was further supported by the occurrence of genes related to pyruvate oxidoreductase (porA/C), thioredoxin peroxidase, cytochrome c oxidases (coxA/B), ccb3-type cytochrome c oxidases (ccoN/O/P/Q) and a cytochrome bd ubiquinol oxidase (cydA) (Fig. 5, Additional file: Supplementary Data 2). More importantly, we also observed that these diazotrophic MAGs contained glycine betaine reductase and glucose/sorbosone dehydrogenase to support their adaptation to hypersaline deep sediments. Together, these results indicated that the halotolerant diazotrophs in deep mangrove sediments were functionally versatile and facultative anaerobes.