Molecular detection of Candidatus Scalindua pacifica and environmental responses of sediment anammox bacterial community in the Bohai Sea, China

Abstract

The Bohai Sea is a large semi-enclosed shallow water basin, which receives extensive river discharges of various terrestrial and anthropogenic materials such as sediments, nutrients and contaminants. How these terrigenous inputs may influence the diversity, community structure, biogeographical distribution, abundance and ecophysiology of the sediment anaerobic ammonium oxidation (anammox) bacteria was unknown. To answer this question, an investigation employing both 16S rRNA and hzo gene biomarkers was carried out. Ca. Scalindua bacteria were predominant in the surface sediments of the Bohai Sea, while non-Scalindua anammox bacteria were also detected in the Yellow River estuary and inner part of Liaodong Bay that received strong riverine and anthropogenic impacts. A novel 16S rRNA gene sequence clade was identified, putatively representing an anammox bacterial new candidate species tentatively named "Ca. Scalindua pacifica". Several groups of environmental factors, usually with distinct physicochemical or biogeochemical natures, including general marine and estuarine physicochemical properties, availability of anammox substrates (inorganic N compounds), alternative reductants and oxidants, environmental variations caused by river discharges and associated contaminants such as heavy metals, were identified to likely play important roles in influencing the ecology and biogeochemical functioning of the sediment anammox bacteria. In addition to inorganic N compounds that might play a key role in shaping the anammox microbiota, organic carbon, organic nitrogen, sulfate, sulfide and metals all showed the potentials to participate in the anammox process, releasing the strict dependence of the anammox bacteria upon the direct availability of inorganic N nutrients that might be limiting in certain areas of the Bohai Sea. The importance of inorganic N nutrients and certain other environmental factors to the sediment anammox microbiota suggests that these bacteria were active for the in situ N transforming process and maintained a versatile life style well adapted to the varying environmental conditions of the studied coastal ocean.

Figure 1. Map of the Bohai Sea and the sites of sampling stations.

The insert map shows the geographical location of the Bohai Sea in the western Pacific Ocean. Abbreviation and numerical symbols: BS, Bohai Sea; 1, Haihe River; 2, Luanhe River; 3, Dalinghe River; 4, Liaohe River; and 5, Daliaohe River.

Figure 2. Phylogenetic analysis of representative Ca. Scalindua 16S rRNA gene sequences obtained from the Bohai Sea.

The tree branch distances represent nucleotide substitution rate, and the scale bar represents the expected number of changes per homologous position. The Aquifex pyrophilus 16S rRNA gene sequence was used as outgroup. Bootstrap values (100 resamplings) higher than 70% are shown with solid circle symbols and those less than 70% but greater or equal to 50% are shown with open circle symbols on the corresponding nodes. The Ca. Scalindua 16S rRNA gene sequences obtained in this study are shown in bold.

Figure 3. Distance neighbor-joining phylogenetic tree of nearly full-length anammox bacterial and environmental 16S rRNA gene sequences.

The tree branch distances represent nucleotide substitution rate and scale bar represents expected number of changes per homologous position. A. pyrophilus 16S rRNA gene sequence was used as outgroup. Bootstrap values (100 resamplings) are shown near the corresponding nodes. Sequences shown in red form a monophyletic cluster and putatively define the new anammox bacterium candidate species, “Ca. Scalindua pacifica”.

Figure 4. Phylogenetic analysis of representative Hzo protein sequences deduced from obtained Bohai Sea hzo gene sequences.

The tree branch distances represent amino acid substitution rate, and the scale bar represents the expected number of changes per homologous position. The cluster 2 Hzo sequence (GenBank accession CAJ70788) was used as outgroup. Bootstrap values higher than 70% of 100 resamplings are shown with solid circle symbols, and those less than 70% but greater or equal to 50% are shown with open circle symbols on the corresponding nodes. The anammox bacterial Hzo sequences obtained in this study are shown in bold.

Figure 5. Dendrograms of hierarchical clustering analyses of the Bohai Sea sediment anammox bacterial assemblages.

(a) Dendrogram of the hierarchical clustering analysis based on the Ca. Scalindua 16S rRNA gene sequences, and (b) dendrogram of the hierarchical clustering analysis based on the Hzo protein sequences. Both clustering dendrograms were obtained by using the Fast UniFrac normalized and weighted Jackknife Environment Clusters statistical method. The percentage supports of the classification tested with sequence jackknifing resamplings are shown near the corresponding nodes.

Figure 6. CCA ordination plots of the relationship between Bohai Sea sediment anammox bacterial assemblages and environmental factors.

Only the first two principal dimensions of the CCA results were shown using the data of (a) the Ca. Scalindua 16S rRNA gene sequence OTUs and (b) the anammox bacteria Hzo sequence OTUs. Correlations between the Bohai Sea environmental factors and CCA axes are represented by the length and angle of arrows (environmental factor vectors). Covarying environmental variables (defined as r≥0.950), such as surface seawater salinity and EC25 (r = 0.994) and bottom seawater salinity and EC25 (r = 0.958), were checked to minimize collinearity in the analyses.

Figure 7. Abundances of the Bohai Sea sediment total bacteria, Ca. Scalindua bacteria and total anammox bacteria.

These data were determined via qPCR specific to the respective target genes. The means and standard errors were calculated with three replicate qPCR measurements. The numerical values in the graph show the ratios of the total anammox bacterial hzo gene abundance to the Ca. Scalindua bacterial 16S rRNA gene abundance at each specific sampling station.