Autotrophic community structure in sediments and environmental determinants
A total of 1,926 Gb of metagenomic raw data were obtained for 25 sediment samples, and approximately 10 Gb of scaffolds (≥ 2000 bp) were generated (Additional file 2: Table S1). After binning of these assembled fragments, we identified 117 metagenome-assembled genomes (MAGs) with the capacity of carbon fixation (Additional file 3: Table S2). According to the 95% ANI-based species demarcation [28], these MAGs represented 117 species, and genomic characteristics were summarized in Additional file 3: Table S3. Their genome sizes ranged widely from ~ 1.1 to 7.1 Mb with estimated completeness of 43%-100% and < 3.5% single-gene redundancies, among which approximately 19% and 79% were classified as high-quality and medium-quality drafts, respectively, according to the genomic standards [29]. The result is likely attributed to the high community diversity in Tibetan sediments [26]. These MAGs varied greatly from 30–74% in the GC content, of which ~ 44% had high GC content (≥ 60%). Phylogenomic analysis showed that the 117 MAGs were affiliated with eleven bacterial phyla and one archaeal phylum, including the Proteobacteria (n = 85), Cyanobacteria (n = 7), Firmicutes (n = 5), Bacteroidetes (n = 4), Deinococcus-Thermus (n = 4), Gemmatimonadetes (n = 4), Acidobacteria (n = 2), Verrucomicrobia (n = 1), Chloroflexi (n = 1), Elusimicrobia (n = 1), Nitrospirae (n = 1), and Euryarchaeota (n = 2) (Fig. 1b and Additional file 1: Figure S1), in agreement with the taxonomical information found in Tibetan sediments [26]. Note that approximately 21% of these MAGs (n = 24) are not affiliated with known orders, greatly expanding our knowledge of the autotrophic diversity and physiologies in nature. The findings suggested that the autotrophic community in the studied sediments was taxonomically diverse in phylogeny. Relative abundances of each MAG were estimated for each sample (Fig. 2 and Additional file 5: Table S4), and results revealed their total relative abundance across sample between 1.96% and 11.4%.
Canonical correspondence analysis indicated that pH, salinity, TOC and their interactions explained approximately 15.4%, 12.9%, 10.9% and 5.1% of the observed variation in the autotrophic community, respectively (Fig. 1c). The finding is consistent with numerous studies that salinity and pH are two most important determinants of microbial diversity and community structure in Tibetan sediments [4, 26, 27, 30]. In addition to salinity and pH, TOC also frequently shapes microbial community structure in aquatic ecosystems [31, 32]. Microorganisms surviving under high salinity require more energy for a variety of processes, especially osmotic balance [25]. TOC could facilitate the growth of heterotrophs and facultative autotrophs under high salinity, which is particularly stressful for autotrophic energy generating pathways [33, 34]. A recent study demonstrated that nutrients (TOC and TN) availability contributed to the response of autotrophic microbial community to salinity and mitigated the salinity constraints [4]. Thus, TOC is also an important environmental driver of the autotrophic community structure. In short, the findings indicated that these physicochemical conditions exerted a critical impact on autotrophic microbial community.
Remarkably, canonical correspondence analysis distinguished these sediments into five categories, and this clustering pattern matched the salinity gradient (Fig. 1c, Fig. 1d and Fig. 1e), which was confirmed by the significant correlation (R2 = 0.953, P < 0.001) between the total relative abundance of these MAGs and salinity (Fig. 1d). The results also illustrated that salinity played a more important role in structuring the autotrophic community than pH and TOC, in agreement with previous findings in aquatic ecosystems [4]. Therefore, these sediment samples were divided into five salinity groups: G1 (S01-S06, salinity ~ 0.6‰), G2 (S07-S10, 1.8–5.9‰), G3 (S11-S15, 12.0-43.9‰), G4 (S16-S20, 49.7–70.7‰), and G5 (S21-S25, 75.5–82.6‰).
Changes in the sediment autotrophic community and carbon fixation pathways along a salinity gradient
Across the five salinity groups, an abundance-salinity correlation was observed that the average autotrophic abundance decreased from G1 to G5 (one-way ANOVA, P < 0.001, Fig. 1e). This inverse correlation is likely due to two reasons: 1) salinity-derived osmotic stress directly limits metabolic activity of carbon fixers, and consumes their energy to maintain cellular osmotic balance, resulting in a decrease in autotrophic biomass; and 2) high salinity decreases the solubility and diffusion coefficient of CO2, thus limiting its availability for carbon fixation, and finally autotrophic biomass decreases [25, 35]. On the other hand, the autotrophic community composition was significantly different among salinity groups (Anosim, Adonis and MRPP, all P = 0.001). The community in the freshwater group G1 (with the lowest salinity) was mainly composed of the Betaproteobacteria (average relative abundance, 7.28%) and Deltaproteobacteria (1.23%), while the Deinococcus-Thermus (2.50%) and Deltaproteobacteria (0.95%) were predominant in the second lowest salinity G2 (Additional file 1: Figure S2). In G3-G5 (from moderate to high salinity), the Deltaproteobacteria (0.86–1.57%) dominated, followed by the Gammaproteobacteria and Cyanobacteria. More specifically, at the species level, no MAGs were shared between G1 and G5 (Additional file 1: Figure S3), in agreement with previous findings that the freshwater taxa could rarely survive under hypersaline conditions [36, 37], implying that the dominant pathway of carbon fixation may be different between the freshwater and hypersaline conditions. Analyses of the top 3 dominant species per salinity group revealed that a specific dominant species from the corresponding group had a higher relative abundance than that in the other groups (one-way ANOVA, all P < 0.01), except the Desulfobacteraceae S20.Bin068, demonstrating that these dominant species have adapted to specific salinity levels (Fig. 2). Previous studies have provided evidences that most known bacteria can growth well under pH 7.5–8.8 instead of a wide range of salinity (freshwater to hypersaline) [26]. Thus, it is reasonable to conclude that salinity has a greater impact than pH on autotrophic microbial community structure in Tibetan sediments.
Among these currently known carbon fixation pathways [5–7], five were identified in the 117 MAGs, of which 35.0%, 32.5%, 17.1%, 12.0% and 3.4% harbored the CBB cycle, WL pathway, rGly pathway, rTCA cycle and 3-HP bicycle, respectively (Fig. 2). Ribulose-1,5-bisphophate carboxylase/oxygenase (RubisCO), which is considered to be the most abundant enzyme on Earth, is integral to carbon fixation via the CBB cycle [11, 12]. However, recent studies reported that forms IV and IV-like RubisCOs might perform other functions instead of carbon fixation [11, 12]. Hence, we constructed a phylogenetic tree of the RubisCO large subunit to determine the RubisCO forms present in the MAGs generated here (Fig. 3a and Additional file 1: Figure S4). This revealed that among 41 MAGs containing the CBB cycle, almost all had the potential to encode forms I or/and II RubisCOs, except Methanothrix sp. S02.Bin032 that possessed forms II/III and III-a RubisCOs-encoding rbcL genes. Forms III and II/III RubisCOs, primarily found in archaea, enable light-independent CO2 incorporation into sugars derived from nucleotides like adenosine monophosphate (AMP) [11, 38]. The findings illustrated that all above 41 MAGs carried the potential to fix atmospheric carbon dioxide via the CBB cycle.
Exploring relationships between taxa and carbon fixation pathways showed that all MAGs affiliated with the Deinococci (n = 4) and Epsilonproteobacteria (n = 5) harbored the rTCA cycle, while MAGs belonging to the Cyanobacteria (n = 7) and Gemmatimonadetes (n = 4) exclusively coded for CBB cycle and 3-HP bicycle, respectively (Fig. 2 and Fig. 3c). Meanwhile, ~ 91% of the MAGs belonging to the Betaproteobacteria (n = 22) and Gammaproteobacteria (n = 11) contained the CBB cycle with the WL pathway identified in ~ 88% of the 27 deltaproteobacterial MAGs (Fig. 2). There was a significant negative correlation (P < 0.001) between the number of the CBB cycle-containing MAGs and salinity (Fig. 4), supporting the view that the diversity of the CBB-performing autotrophic prokaryotes decreases with enhancing salinity in aquatic ecosystems [4]. For the main carriers of the CBB cycle, taxonomy shifted from the Betaproteobacteria in G1 to the Cyanobacteria and Gammaproteobacteria in G2 and G3, to the Cyanobacteria in G4, and finally to the Gammaproteobacteria in G5, which is consistent with the conclusion of a recent study that the Betaproteobacteria dominated the CBB-performing autotrophic community in the freshwater lake and the Gammaproteobacteria dominated the hypersaline lake [4]. Moreover, for the rTCA pathway, the dominant carriers belonged to the Epsilonproteobacteria in G1 (the average relative abundance: ~0.47%) and the Deinococci in G2-G5 (0.18–2.50%), respectively. The WL pathway were mainly encoded by the Deltaproteobacteria in all five salinity groups.
Comparison of carbon fixation pathways among the five salinity groups revealed that the CBB cycle was predominant in the freshwater G1 samples, with the rTCA cycle dominating in the second lowest salinity G2 (t test, all P < 0.001). Comparative analysis showed that the CBB cycle-containing MAGs in G1 had a higher abundance in comparison with that in the other groups (one-way ANOVA, P < 0.01). The top 3 dominant MAGs (Rhodoferax sp. S03.Bin061 and Hydrogenophaga sp. S05.Bin024 and S04.Bin021) in G1 encoded the CBB cycle and the top 2 dominant MAGs (Trueperaceae sp. S10.Bin132 and S23.Bin013) in G2 encoded rTCA cycle. The discovery of the potential autotrophic Rhodoferax bacterium was inconsistent with previous results that members of the Rhodoferax could not fix CO2 [39, 40], expanding the autotrophic diversity and the niche of the Rhodoferax. Besides, previous studies also gave evidences that members of the genus Hydrogenophaga (e.g., Hydrogenophaga pseudoflava) could use the CBB cycle for carbon fixation [41]. In saline to hypersaline sediments (G3-G5), autotrophs mainly contained the CBB and WL pathways, and the WL pathway became the most important in the G5 samples (Fig. 3b). Statistical analysis indicated a significantly positive (P < 0.001) correlation between the fraction of MAGs with the WL pathway and salinity (Fig. 4). There may be two explanations for this: 1) autotrophs need to reallocate energy to accumulate inorganic and organic osmoregulators (e.g., K+, sugars, polyols and quaternary amines) to deal with intracellular osmotic stress caused by salinity [42, 43]. In this instance perhaps the more energy-efficient (no ATP consumption) WL pathway provides an advantage for survival in comparison to the energy-demanding (9 ATP consuming) CBB cycle [44]. A recent study indicated that the CO2-reduction via WL is more ATP-efficient and yields more biomass than CO2-carboxylation dependent pathways (including CCB, rTCA and 3-HP) [7]. 2) Primordial life on earth may have started in hypersaline environments [45], and the WL pathway is considered to be the most ancient pathway for carbon fixation [46, 47], so microbes containing the WL pathway may be more suitable for living in high salinity environments. The inference of an operative WL pathway in the G5 dominant Desulfotignum sp. S23.Bin123 and S15.Bin083 was supported by previous research [48–50]. Overall, these pathway shifts along the salinity gradient suggest that it provides a selective advantage between different mechanisms for carbon fixation.
Metabolic potential of sediment autotrophs in response to salinity
We examined the potential of these microbes for driving biogeochemical cycles. To do this, the important pathways and related key genes in carbon, nitrogen, sulfur and iron metabolisms and respiration were searched (Fig. 2 and Additional files: Tables S7-S10). This revealed that these MAGs possessed the potential to perform central carbohydrate metabolism such as glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathway (PPP). Approximately 56% of these MAGs contained glycolysis pathways including Embden-Meyerhof-Parnas (EMP, n = 51) and Entner-Doudoroff (ED, n = 19), of which five (Hydrogenophaga S05.Bin024 and S07.Bin178, Maritimibacter S23.Bin089, Sulfurovum S06.Bin221, and Rhodobacteraceae S06.Bin070) contained both pathways. It is not unprecedented to have both catabolic pathways in the same organism, such as Polaromonas sp. JS666 and Hydrogenophaga b174 [51, 52]. Interestingly, previous reports revealed that this kind of bacteria usually possessed an incomplete PPP [51, 52], but Hydrogenophaga S07.Bin178 contained a complete PPP. These findings greatly increase our knowledge of catabolism mechanisms of these genera. Further analysis showed a significant positive trend (P < 0.001) between the fraction of MAGs containing the EMP pathway and salinity (Fig. 4). In the highest salinity G5 sites, the top 3 dominants all carried the EMP pathway. This correlation is likely due to that the fermentative EMP pathway yields more energy from C6 and C5 sugars than the ED pathway [53], and the additional energy generated is beneficial for autotrophs to resist environmental stresses, especially salinity stress. Furthermore, roughly 41% of the 117 MAGs might produce energy via the TCA cycle, along with the gluconeogenesis pathway identified in ~ 50%. Notably, the fraction of MAGs with gluconeogenesis potential showed a significant linear positive relationship (P < 0.001) with TOC, whereas it was significantly negatively correlated with salinity (P < 0.001). It could be explained that TOC and increased salinity inhibited carbon fixation, resulting in a decline in related glucose yield [4, 33, 34, 54], while facultative autotrophs could utilize organic matter to produce glycose [4, 33, 34] and possibly use gluconeogenesis to maintain energy homeostasis (ATP synthesis) and glucose production. Meanwhile, autotrophs with the EMP pathway would be selected and enriched under high salinity conditions as they had a competitive advantage in glucose utilization over autotrophs with the ED pathway [53]. In addition, although more than a half (~ 58%) of MAGs harbored the oxidative or/and non-oxidative branches of PPP, a major source of NADPH and metabolic intermediates for biosynthetic processes [55], only ~ 14% had the ability to perform the complete PPP.
Then we identified the coxLMS genes encoding aerobic CO dehydrogenase in 11 MAGs (such as the dominant Hydrogenophaga S05.Bin024, Desulfotignum S14.Bin031, S15.Bin083 and S23.Bin123, and Maritimibacter S23.Bin089), suggesting they are capable of CO oxidization. Previous studies also confirmed that members of the Hydrogenophaga and Desulfotignum genera could oxidize CO and generate equivalents for nitrate reduction [49, 52]. The 11 species may be oxidizing CO formed from photochemical degradation of dissolved organic carbon in aquatic environments and then use the generated electrons to support ATP generation and CO2 fixation [56, 57]. This CO oxidation potential showed the highest number in the freshwater G1 (t test, all P < 0.05), and the highest fraction in the highest salinity G5 sites (t test, all P < 0.01). Besides, all seven cyanobacterial MAGs code for oxygenic photosynthesis (psa or psb), evidenced by previous findings [58]. The pufABLM genes were detected in several MAGs affiliated with Alphaproteobacteria (n = 3), Betaproteobacteria (n = 3) and Gemmatimonadetes (n = 3), indicating they are potential aerobic anoxygenic photosynthetic (AAP) bacteria [59]. The three betaproteobacterial MAGs (S04.Bin010, Burkholderiales S01.Bin058, and Hydrogenophaga S07.Bin178) with the potential to perform the CBB cycle and aerobic anoxygenic photosynthesis suggests that RuBisCO in AAP bacteria was involved in both CO2 fixation and the central redox cofactor recycling, because CO2 fixation could be used to maintain redox balance by recycling reduced redox cofactors for photoheterotrophic metabolism [60, 61].
In assessing the potential for respiration, genes encoding the aa3- and cbb3-type cytochrome c oxidases (coxABCD and ccoNOQP, respectively) and cytochrome bd ubiquinol oxidase (cydAB) were found in these MAGs (n = 14, 33 and 59, respectively) (Additional file 6: Table S5), indicating that most (~ 68%) putative autotrophs likely utilize oxygen as a terminal electron acceptor in the studied sediments. Note that in comparison with the low-oxygen-affinity aa3-type oxidase induced under oxic conditions, the cbb3-type and bd oxidases are high-affinity terminal oxygen reductases capable of functioning under microoxic to anoxic conditions [62], and the bd oxidase has a lower energetic efficiency than the heme-copper oxidases (aa3- and cbb3-type) as it does not pump protons [63]. Given the presence of the ccoNOQP or/and cydAB genes, more than a half (~ 56%) of these potential autotrophs were adapted to microoxic to anoxic conditions. Meanwhile, given the occurrence of genes (coxABCD and ccoNOQP, coxABCD and cydAB, or coxABCD, ccoNOQP and cydAB) encoding the low- and high-affinity oxidases, ~ 11% of the MAGs affiliated with the Betaproteobacteria (n = 6), Gammaproteobacteria (n = 5), Alphaproteobacteria (n = 1) and Epsilonproteobacteria (n = 1) could survive under fully aerobic to anoxic conditions, according to previous reports [63–65]. Further statistics revealed the highest species number and fraction of putative autotrophic MAGs with the ccoNOQP genes in the freshwater G1 (Fig. 4), while no significant difference was observed in the MAGs with the cydAB genes among the five salinity groups.
Autotrophic sulfur cycling in sediments of different salinities
Recent studies have revealed the important roles of autotrophs in the biogeochemical sulfur cycle [14, 66]. We found 49 MAGs with dissimilatory sulfite reductase genes (dsrAB) (Additional file 7: Table S6). Phylogeny of concatenated DsrAB proteins indicates 31 reductive and 18 oxidative bacterial-types (Fig. 5a and Additional file 1: Figure S5). Deltaproteobacteria S05.Bin005 likely gained the dsrAB genes by lateral gene transfer (LGT), supporting the fact that this genes in organisms from Deltaproteobacteria may be acquired in multiple LGT events [66, 67], as shown in Fig. 5a. Besides, we summarized seven types of dsr operons in 48 of the 49 MAGs (Fig. 5b). A recent study reported that the gene composition (dsrAB and dsrD/dsrEFH) of the dsr operon may determine the direction of the dissimilatory pathway between sulfite and sulfide [66, 67]. Therefore, 23 deltaproteobacterial MAGs were inferred to reduce sulfate to sulfide via the dissimilatory sulfate reduction (dsr) pathway and 13 MAGs affiliated with the Gammaproteobacteria (n = 7), Betaproteobacteria (n = 5) and Deltaproteobacteria (n = 1) likely oxidize sulfide to sulfate through the reverse dsr (rdsr) pathway. Only Desulfotignum S25.Bin111 had the potential to perform both functions, likely depending on oxygen concentration and/or oxidation reduction potential, that increased the number of this especial genomes to 14 (the Deltaproteobacteria n = 5, Ca. Lambdaproteobacteria n = 4, Actinobacteria n = 4, and Nitrospirae n = 1) [66, 67]. Desulfotignum species were previously inferred to be sulfate-reducing bacteria [50], but our findings suggested that some specific Desulfotignum species could be both sulfate reducers and sulfide oxidizers, increasing our understanding of ecological roles of these bacteria. In the freshwater G1 samples, MAGs with dsr genes were the least prominent, while rdsr (sulfide oxidation) were the highest (t test, all P < 0.05), in good agreement with the lowest concentration of sulfate and that the most dominant species Rhodoferax S03.Bin061 and Hydrogenophaga S05.Bin024 and S04.Bin021 had the capability to oxidize sulfide via the rdsr pathway. Besides, although the Rhodoferax and Hydrogenophaga species were evidenced to oxidize reduced sulfur compounds in many studies [52, 68], our findings provide a new oxidation mechanism in these genera and strengthen their importance in the sulfur cycle.
The complete SOX (sulfur oxidation) system was detected in 16 MAGs (Additional file 7: Table S6), mainly in the G1 sites (t test, all P < 0.001), suggesting they are able to oxidize S2O32− to SO42−. Meanwhile, we observed partial SOX system (lacking soxCD genes) in another 11 MAGs, meaning the transformation from S2O32− to S(0) only without going all the way to SO42− [69]. Among the above-mentioned 11 MAGs, four gammaproteobacterial MAGs including Thioalkalivibrio paradoxus S10.Bin039, Thioalkalivibrio nitratireducens S13.Bin160, Thioalkalivibrio S25.Bin012 and Thiotrichales S21.Bin127 contain the versatility of mechanisms of sulfur cycling. They are able to convert S(0) to S2− via oxygenase/reductases (Sor) and sulfhydrogenases (HydGBAD), and also may oxidize S(0) to SO32− due to the identified sdo (encoding sulfur dioxygenase) and sor genes, depending on environmental conditions. Inconsistent with previous conclusions that the Thioalkalivibrio species are sulfur-oxidizing bacteria [70, 71], our findings inferred that these organisms might be sulfur reducers as well, suggesting their great adaptability to environmental change. Furthermore, eight MAGs belonging to the Deltaproteobacteria (n = 3), Deinococcus-Thermus (n = 3), Gammaproteobacteria (n = 1) and Acidobacteria (n = 1) might also perform S(0) reduction via Sor, HydGBAD and/or sulfur reductase (sreAB). Interestingly, more than a half (~ 52%) of the 117 MAGs contained sqr or/and fccAB genes (encoding sulfide:quinone oxidoreductase and sulfide dehydrogenase, respectively) and thus might be able to directly oxidize sulfide to sulfur. Additionally, around 38% of the MAGs, mainly in G1 sites (t test, all P < 0.05), likely utilize S(0) as an electron source and produce SO32− by sulfur dioxygenase (sdo). In brief, these findings indicated a coordination in sulfur cycling among sediment autotrophs.
Autotrophic nitrogen-cycling capabilities along a salinity gradient
To better assess the role of sediment autotrophic microbes in nutrient cycling, we reconstructed key nitrogen utilization pathways (Additional file 8: Table S7). This revealed that ~ 15% of the MAGs, most prevalent in the moderate salinity G3 sites (t test, all P < 0.05), possessed nitrogenase (nifDKH) genes, suggesting they are autotrophic diazotrophic bacteria, an important nutrient source in the lake. In previous studies, the fixation of inorganic carbon and nitrogen by such microorganisms was recognized as a crucial process to the development of life in extreme environments [72, 73]. In the G3 sediments, autotrophic microbial communities were dominated by diazotrophs, which was also found in some Tibetan soils [74], and the reason for this phenomenon is still mysterious. Dissimilatory nitrate reduction pathway was detected in a few MAGs affiliated with the Betaproteobacteria (n = 7), Deltaproteobacteria (n = 2) and Gammaproteobacteria (n = 1), with the highest species number and fraction in the freshwater G1 sites (Fig. 4). More than a half of the individuals (n = 59) identified here involved in denitrification, but only Rhodocyclaceae S06.Bin133 is capable of complete denitrification (to N2) and considered as an autotrophic sulfide-oxidizing denitrifier, and four species affiliated with the Alphaproteobacteria (Rhodobacteraceae S24.Bin137) and Gammaproteobacteria (S13.Bin138, S22.Bin169 and Thiotrichaceae S06.Bin195) code a partial pathway for nitrite to nitrogen. This suggests a coordination in denitrification among sediment autotrophs. Autotrophic nitrate-reducing and denitrifying bacteria are commonly found in sediments [75, 76], and the coupling of carbon fixation, nitrate reduction and oxidation of reduced sulfur compounds performed by such microorganisms is successfully applied to wastewater treatment [77, 78]. Urease genes (ureDABCEFG) genes were identified in 14 MAGs (Fig. 2), suggesting these species could acquire C and N by hydrolyzing urea to ammonia and carbon dioxide [79], which then could be fixed to yield glucose via a carbon fixation pathway. The number of species and the fraction of these potential urea-utilizers declined with increased salinity (both P < 0.001, Fig. 4). Two abundant species Hydrogenophaga S05.Bin024 and S04.Bin021 in the freshwater G1 sites possess the capability of urea utilization, evidenced by previous research [80]. To our surprise, genes encoding ammonia monooxygenase, which is responsible for the transformation from ammonia to hydroxylamine [81], were not detected in any of the MAGs. However, 42 MAGs likely participated in a partial process of nitrification (converting hydroxylamine to nitrate). In general, autotrophic microorganisms in Xiaochaidan Lake sediments likely use multiple strategies to gain nitrogen resources for growth, illustrating that they are important participants in the nitrogen cycle.
Iron metabolism by the sediment autotrophs
Iron is a ubiquitously abundant redox active transition metal in sedimentary systems [82], serving as an essential nutrient and an electron donor or acceptor to many prokaryotes [83]. More than a half (~ 56%) of the MAGs encode proteins with similarity to iron oxidase (iro) or/and sulfocyanin and the cbb3-type oxidase (ccoNOQP) (Additional file 9: Table S8), suggesting they are capable of transferring electrons to oxidases during iron oxidation [84]. This suggests that many autotrophic microbes in Xiaochaidan Lake sediments are also oxidizing iron, consistent with that abundant bacteria could oxidize iron in lake sediments [85]. Among them, eight were inferred to catalyze nitrate-dependent Fe(II) oxidation under anaerobic conditions, which have been observed in several different sediments [86, 87], suggesting a tight coupling of Fe and N redox cycles in anaerobic sedimentary environments that has significant implications for mechanisms of NO3− removal and the regeneration of reactive Fe(III) oxides in hydromorphic sediments, as well as the transformation of various natural and contaminant organic and inorganic compounds [83]. It was notable that the species number and fraction of the MAGs with the sulfocyanin-encoding gene were significantly negatively associated with salinity (both P < 0.001). In the freshwater G1 sites, the most dominant species Rhodoferax S03.Bin061 codes for sulfocyanin to oxidize iron, inconsistent with the fact that the Rhodoferax species are Fe(III)-reducers [88, 89]. In addition to iron oxidation, 10 MAGs possessed ferric-chelate reductase (feR-like) gene, potentially catalyzing ferric iron reduction with NAD(P)H as the electron donor [90]. FeR was first characterized in a strictly anaerobic sulfate-reducing archaeon, Archaeoglobus fulgidus [91]. Intriguingly, among the above-mentioned 10 ferR-like gene-containing MAGs, four deltaproteobacterial species (Desulfotignum S09.Bin188 and S14.Bin031, and Desulfobacteraceae S24.Bin046 and S24.Bin080) also had the potential to perform sulfate reduction. In addition, the mtrB/pioB-like genes encoding decaheme-associated outer membrane proteins of MtrB/PioB family were identified in 19 MAGs, which are common among Fe(II) oxidizers and Fe(III) reducers [92]. These findings implied that autotrophic microorganisms likely be important players in the biogeochemical cycle of iron (especially iron oxidation) in sedimentary systems.