Residence of habitat-specific anammox bacteria in the deep-sea subsurface sediments of the South China Sea: analyses of marker gene abundance with physical chemical parameters

Abstract

Anaerobic ammonium oxidation (anammox) has been recognized as an important process for the global nitrogen cycle. In this study, the occurrence and diversity of anammox bacteria in the deep-sea subsurface sediments of the South China Sea (SCS) were investigated. Results indicated that the anammox bacterial sequences recovered from this habitat by amplifying both 16S rRNA gene and hydrazine oxidoreductase encoding hzo gene were all closely related to the Candidatus Scalindua genus. A total of 96 16S rRNA gene sequences from 346 clones were grouped into five subclusters: two subclusters affiliated with the brodae and arabica species, while three new subclusters named zhenghei-I, -II, and -III showed ≤ 97.4% nucleic acid sequence identity with other known Candidatus Scalindua species. Meanwhile, 88 hzo gene sequences from the sediments also formed five distant subclusters within hzo cluster 1c. Through fluorescent real-time PCR analysis, the abundance of anammox bacteria in deep-sea subsurface sediment was quantified by hzo genes, which ranged from 1.19 × 10(4) to 7.17 × 10(4) copies per gram of dry sediments. Combining all the information from this study, diverse Candidatus Scalindua anammox bacteria were found in the deep-sea subsurface sediments of the SCS, and they could be involved in the nitrogen loss from the fixed inventory in the habitat.

Figure 1

Phylogenetic tree based on an alignment of 16S rRNA gene sequences showing the phylogenetic affiliations of anammox bacterial sequences from the different subseafloor sediment of South China Sea. The numbers in parentheses refer to how many clones retrieved by the amplification with the primer sets Brod541F/Amx820R and Amx368/ Amx820R were assigned to an individual phylotype. The consensus phylogenetic tree constructed based on neighbor-joining analysis. The numbers at the nodes are percentages that indicate the levels of bootstrap support based on 500 resampled data sets. Branch lengths correspond to sequence differences as indicated by the scale bar

Figure 2

Phylogenetic tree constructed from alignments of Hzo protein sequences deduced from deposited hzo gene sequences in the GenBank and experimental sequences obtained by amplifying the community DNA sampled from the subseafloor sediments of the South China Sea at different depths. The numbers in parentheses refer to how many hzo gene clones retrieved using the respective primer set were assigned to an individual phylotype. The numbers at the nodes are percentages that indicate the levels of bootstrap support based on 500 resampled data sets. Branch lengths correspond to sequence differences as indicated by the scale bar. Major clades were named following a proposal of Schmid et al. [40]

Figure 3

Abundance of hzo gene of anammox bacteria in sediments of selective sampling sites at the South China Sea. The experiment was performed in triplicate. Error bars represent standard deviations of triplicate analysis

Figure 4

Weighted UniFrac PCA analyses for (a) 16S rRNA gene sequences and (b) deduced amino acid sequences of Hzo protein in the context of physical and chemical conditions