4.2. Environmental variation influences the microbial community composition
In this study, the bacterial community composition was correlated with environmental factors, and was affected by salinity, pH, DO, χlf, Fe and TOC (Fig. 1E and Table 2). These findings suggest that environmental variables may be crucial to shaping the microbial community composition.
Salinity is well known to be a major contributor to microbial community structure and function . The important role of salinity in bacterial communities has been found globally in heterogeneous environments, and in the sedimentary ecosystems of Hypersaline Laguna Tebenquiche . Consistent with those findings, we observed the most significant correlation between salinity and bacterial community structure (Table 2), because salinity is related to osmotic pressure, which changes the intracellular membrane structure and affects metabolic pathways [37, 39]. Previous studies have also shown that bacterial communities in transition zones vary geographically owing to sharp salinity gradients . A possible reason for the influence of salinity on bacterial distribution found in our current study is that our research sites focused on bacterial communities distributed in a geographic area with a wide salinity gradient, including estuaries, coastal margins, and open sea, where salinity values differ significantly.
χlf was correlated with community composition of many phyla, including Alphaproteobacteia, Betaproteobacteia, Planctomycetes, and Cyanobacteria. Cyanobacteria showed a positive correlation with χlf, suggesting that χlf might be related to the growth patterns of ecologically important species such as Cyanobacteria, one of the main participants of primary productivity in the global carbon cycle. In sediment, available iron is momentous for Cyanobacteria growth and Fe oxide minerals have the largest release potential . Therefore, growth of Cyanobacteria is heavily influenced by Fe availability in all water bodies . The organic matter production and the carbon cycle are also affected by Fe availability. The χlf values are coupled to iron-reducing bacterial activity in hydrocarbon contaminated sediments . In our study, we found that iron-reducing bacteria were more abundant in sites with higher χlf values (Fig. 2). A negative relationship between TOC and χlf was observed in the RDA assay, indicating that consumed TOC might be used for iron-reducing bacteria growth, because numerous bacteria including iron-reducing bacteria are dependent on organic matter produced by Cyanobacteria. In the Bohai Sea organic matter could associate microbial iron related metabolism with microbial-driven change in magnetic susceptibility [15, 44] as shown by measurable χlf values.
Sediment DO and water pH were found to be critical in shaping microbial community structure (Fig. 1E; Table 2). The top 10 phyla in this region included Proteobacteria, Chloroflexi, Planctomycetes, and Cyanobacteia (Fig. 1C), which represent the predominant phyla in the sediments of the eastern Mediterranean Sea . In this study, Alphaproteobacteria, Betaproteobacteria, Planctomycetes, and Cyanobacteria were increased with elevated DO and χlf (Figs. S3 and S4) and decreased with increased pH (Fig. S3). This suggests that Betaproteobacteria and Cyanobacteria in the tested sedimentary regions may prefer shallow-estuary sediment (low salinity, high DO, and low pH) with high χlf. pH is one of the most important factors influencing microbial energy respiration, physiology and growth. The intracellular pH is relatively stable, and the extracellular pH depends on the level of cell metabolism. Previous reports confirmed that under low pH and high DO conditions in coal mining-associated lakes, there are high concentrations of Fe (II) and protons because of oxidation of pyrite in mine tailings [46, 47]. The produced Fe (II) is then oxidized by ferroxidans in Betaproteobacteria and precipitates as Fe (III) hydroxysulfate to the sediment . In this study, we found that Betaproteobacteria prefer this kind of environment (low pH, high DO, and high χlf) and this might be related with Fe (II) oxidization.
The RDA assay showed that TOC was positively associated with pH, possibly because surface sediments contain a higher proportion of labile algal derived aliphatic organic matter and more anions [48, 49]. In our study, Proteobacteria was the most abundant group of bacteria. In surface sediments that contain higher proportions of organic matter, Proteobacteria and Bacteroidetes are often prominently detected during the initial degradation of algal derived organic matter in marine sediments [48, 49]. The dominant members of Bacteroidetes in the surface sediments were consistently enriched, similar to reports from previous studies [49, 50]. Because Cyanobacteria is documented to be the main participant and contributor to productivity of the global carbon cycle [51, 52], the occurrence of Bacteroidetes and Cyanobacteria in the surface sediment suggests that Bacteroidetes may survive better in areas rich in fresh organic matter.
Deltaproteobacteria and Gamma proteobacteria declined with increased DO and increased with elevated pH (Fig. S3). This suggests that Deltaproteobacteria and Gamma proteobacteria might prefer to inhabit in deep-marine sediment environments (high salinity, low DO, and high pH). Deltaproteobacteria include many IRB, such as Geobacter, Anaeromyxobacter, Desulfobulbus, Desulfobacter, Desulfuromonas, Desulfuromusa, Pelobacter. In high pH and low DO environment, the increased pH changes the charge on the surface of the trivalent iron oxide changes . The surface organic matter is negatively charged and released and reduction of trivalent iron might occur . These results suggest that sediment salinity, pH, DO, and χlf could be good predictors of bacterial community composition variation.
It should be noted that in our study, the correlation analysis with other environmental variables was based on the relative abundance of bacterial community. The relative abundance is not independent data and reflects the mutual restriction between different taxa.
4.3. Environmental variables play a more important role than dispersal limitation (spatial variables) in conditioning bacterial biogeography
Of all measured environmental variables, only pH was correlated with geo-distance (Mantel tests, Table S3). Other drivers (salinity, DO, and χlf) were not significantly correlated spatially. These results indicated that most local environmental conditions were not shaped spatially.
Despite the fact that the magnetite content in the center of the Bohai basin is high, the magnetic susceptibility does not show a correlation with this distance from the magnetite center. One possible explanation is that the sedimentary settling happens in a vertical manner from surface to deep layer, whereas magnetic susceptibility is determined as iron minerals form based on location on earth, in relation to magnetic north. Among the top 15 most dominant genera (Fig. 5B), five genera (Lactococcus, Clostridium, Caulobacter, Gillisia and Sphingomonas) showed a clear correlation with the distance from the center of the magnetite (Table 3). This implies that the exposure of magnetite may shape the geographical distribution of these genera, and most likely by affecting the iron-related geochemical cycle these genera participate in. It is reported that Lactococcus participates in Fe (III) reduction during the external electron transfer mediated by sodium anthraquinone-2,6-disulphonate (AQDS) . It is possible that Lactococcus sp. uses a very small portion of regenerated reducing power NADH for the reduction of external electron acceptor Fe (III) to Fe (II) in anaerobic lactic fermentation . The lactic acid produced by Lactococcus was then used by Clostridium for Fe (III) reduction, because Clostridium could act as a lactic fermenter and Fe reducer [56, 57]. Caulobacter is known to participate in metal oxidation through the biosorption and metabolism of iron . That is one of the reasons that the Caulobacter distribution is associated with the distance from the center of the magnetite. Sphingomonas was identified as a microcystin-degrading bacterium during the decay of Cyanobacteria . Cyanobacteria growth needed available iron . Distribution of Sphingomonas might be adjusted according to iron presence indirectly because of its correlation with Cyanobacteria. Gillisia was detected as siderophore producer in seawater or sand samples . Siderophores are the metal-chelating agents that primarily function to capture the insoluble ferric iron from different habitats [60, 61]. Numerous bacteria cannot produce siderophores but have siderophore acceptors [60, 61]. Gillisia might assist other bacteria such as Cyanobacteria, Lactococcus, Clostridium and Caulobacter that do not have siderophore generation capability for iron release or absorption from Fe-containing minerals. Therefore, the geographical distribution of Gillisia also was impacted by the presence of magnetite (Table 3).
Environmental variables explained 9.80% (P < 0.001) of the total microbial community composition variation, which is higher than spatial factors 6.72% (P < 0.05) in a variation partitioning analysis (Fig. 4). This further suggests that environmental variables play a more important role than spatial variables in shaping the bacterial composition and distribution.
It has been reported that both the environmental and spatial variables play significant roles in influencing the biogeography of total microeukaryotic communities . A different study showed that spatial distance (dispersal limitation) contributed more to bacterial community variation than any other factor . In our current study, we showed that environmental variables were more important than spatial variable for governing bacterial community turnover.
In the present study, OTU patterns and the bacterial community composition were significantly (P < 0.001) correlated with geographical distance (Fig. 3). The results of the distance-decay pattern indicated that dispersal limitation may be another influential factor driving microbial biogeography. Dispersal could eliminate the distance-decay relationship by counteracting microbial compositional differentiation . Limited dispersal should strengthen the distance-decay relationship , and the strength of correlation between dispersal limitation and microbial community composition relies on geographical distance  and organism size . Limitations of microbial dispersal have been demonstrated at large  or intermediate (10-3,000 km) spatial scales . Dispersal limitation may exist in intermediate spatial scale at the Bohai sea (approximately 100 km). Strong dispersal limitations are often associated with increased bacterial size . The bacteria found in the current study are within a relatively narrow size range, from 0.5 -5 µm . This could help explain why the distance-decay curve inclined slightly, which is evidence of community variation purely constrained by spatial factors (6.72%) (Fig. 4). This demonstrated that dispersal limitation was associated with microbial community composition, but was not the dominant factor in shaping microbial biogeography in the Bohai Sea.
A large unexplained fraction (75.49%) was in the variation partitioning analysis. This could be potentially explained by unmeasured environmental variables, local artificial effects, and other factors.
One limitation of the study is that the temporal variation is not considered due to the difficulty of sample collections. Another limitation is that our current experimental approach provides a clear correlation between environmental variables and microbial community composition, but it does not allow us to make a firm causative conclusion. Future studies about the temporal patterns of microbial communities and more controlled experiments to dissect the causative relations are important for understanding the changes in microbial composition and function of microbes in response to environmental factors in coastal areas.