Presence and diversity of anammox bacteria in cold hydrocarbon-rich seeps and hydrothermal vent sediments of the Guaymas Basin

Abstract

Hydrothermally active sediments are highly productive, chemosynthetic areas which are characterized by the rapid turnover of particulate organic matter under extreme conditions in which ammonia is liberated. These systems might be suitable habitats for anaerobic ammonium oxidizing (anammox) bacteria but this has not been investigated in detail. Here we report the diversity and abundance of anammox bacteria in sediments that seep cold hydrocarbon-rich fluids and hydrothermal vent areas of the Guaymas Basin in the Cortés Sea using the unique functional anammox marker gene, hydrazine synthase (hzsA). All clones retrieved were closely associated to the "Candidatus Scalindua" genus. Phylogenetic analysis revealed two distinct clusters of hzsA sequences (Ca. Scalindua hzsA cluster I and II). Comparison of individual sequences from both clusters showed that several of these sequences had a similarity as low as 76% on nucleotide level. Based on the analysis of this phylomarker, a very high interspecies diversity within the marine anammox group is apparent. Absolute numbers of anammox bacteria in the sediments samples were determined by amplification of a 257 bp fragment of the hszA gene in a qPCR assay. The results indicate that numbers of anammox bacteria are generally higher in cold hydrocarbon-rich sediments compared to the vent areas and the reference zone. Ladderanes, lipids unique to anammox bacteria were also detected in several of the sediment samples corroborating the hzsA analysis. Due to the high concentrations of reduced sulfur compounds and its potential impact on the cycling of nitrogen we aimed to get an indication about the key players in the oxidation of sulfide in the Guaymas Basin sediments using the alpha subunit of the adenosine-5'-phosphosulfate (APS) reductase (aprA). Amplification of the aprA gene revealed a high number of gammaproteobacterial aprA genes covering the two sulfur-oxidizing bacteria aprA lineages as well as sulfate-reducers.

Keywords: anammox; aprA; cold seep; hydrothermal vents; hzsA; sulfide.

Figure 1

Depth profiles of (A) ammonium and (B) sulfide in cold seep and hydrothermal sediments. Sediment cores of the reference zone is depicted in green, cold hydrocarbon-rich seeps in blue and hydrothermal sites in red. Ammonium in hydrothermal sediments was higher and is therefore shown as an inset (the axes have the corresponding units).

Figure 2

Neighbor-joining tree of phylogeny estimated by ClustalW included in the MEGA 5.0 software package, showing >1000 bp fragments of hzsA nucleotide sequences retrieved from the Guaymas Basin sediments. Letters (I or V) of the samples indicate the primer set used for amplification and the number refers to the sample. Samples are color-coded and at collapsed nodes abbreviated: reference zone in green (REF), cold hydrocarbon-rich seeps in blue (CS) and hydrothermal sites in red (V). Values at the internal nodes indicate bootstrap values based on 500 iterations. The bar indicates 5% sequence divergence. The outgroup with other anammox bacteria includes Genbank accession numbers JN703715, JN703714, JN703713, JN703712, AB365070 and CT573071. Accession numbers of individual clones are provided in Supplementary Table 1.

Figure 3

hzsA copy number in different sediment samples (±SD of technical replicates). Sediment cores of the reference zone is depicted in green, cold hydrocarbon-rich seeps in blue and hydrothermal sites in red. Arrows indicate decreasing sediment depth within a single core.

Figure 4

Neighbor-joining tree of phylogeny estimated by ClustalW included in the MEGA 5.0 software package, showing ~400 bp fragments of aprA nucleotide sequences retrieved from the Guaymas Basin sediments. The number refers to the sample and collapsed nodes are abbreviated: reference zone in green (REF), cold hydrocarbon-rich seeps in blue (CS) and hydrothermal sites in red (V). Values at the internal nodes indicate bootstrap values based on 500 iterations. The bar indicates 5% sequence divergence. Accession numbers of individual clones are provided in Supplementary Table 2.

Figure 5

Depth profile of absolute ladderane lipid concentrations in core 1758-14 CT2 including original ladderanes and short chain ladderane fatty acids. Data points represent measurements on the sediment sample pooled from 0 to 3 cm, 6 to 9 cm, and 15 to 18 cm sediment depth.