Molecular detection methods and characterization of anammox bacteria from different ecological niches

Abstract

Anaerobic ammonium oxidation (anammox), one of the latest discoveries to the nitrogen cycle has been a very active focal research area on biochemistry, ecophysiology and ecology. Anammox biochemical process, the oxidation of ammonium with nitrite as electron acceptor under anoxic conditions, is mediated by a group of bacteria affiliated within a deep branch of Planctomycetes. Although the existence of anammox bacteria has been established in a number of ecosystems, its activity and ecological distribution pattern are still not well understood. The objectives of this research were to: compare different gene markers for effectively detection of them from various ecological niches, evaluate the recently developed 16S rRNA gene-based PCR primers for niche specificity, elucidate the bias of anammox bacterial diversity caused by inefficient DNA extraction in preparation of DNA templates for PCR reaction, and delineate the ecological distribution pattern of anammox bacteria along an anthropogenic nitrogen input gradient from a polluted coastal wetland to the pristine South China Sea (SCS).

Several PCR primer sets for genes encoding 16S rRNA, hydrazine oxidoreductase (HZO), cytochrome cd1-containing nitrite reductase (NirS), and hydrazine synthase subunit A (HzsA) were assessed with sediments from coastal wetland, SCS, and granules from wastewater treatment plant. Selective primer sets were effective in recovering anammox bacteria from these samples, while nirS gene-based PCR primer sets were sample specific. In addition, two recently reported and widely applied 16S rRNA gene-based PCR primer sets (Brod541F/Amx820R and A438f/A684r) were further evaluated in profiling anammox bacteria from various sediment samples, showing more favorable results by A438f/A684r than Brod541F/Amx820R.

Bias in anammox bacteria diversity was investigated by comparative analysis of coastal wetland sediment and rice paddy soil with PCR-denaturing gradient gel electrophoresis (DGGE) technique. Successive extractions of DNAs from coastal wetland in Hong Kong and Baijiang rice paddy soils in Northeast China were carried out to compare the diversity of ammonia/ammonium-oxidizing microorganisms, including both anammox bacteria, and ammonia-oxidizing archaea and bacteria. Results showed that anammox bacterial diversity was seriously biased due to inefficient DNA extraction of the rice paddy soil from the first extraction, resulting in lower diversity of anammox bacteria in the samples.

Changes in community composition of anammox bacteria were analyzed using sediment samples along an anthropogenic nitrogen gradient and results showed variations of anammox bacteria community structures from the upstream of Pearl River to deep sediment of the SCS. Mai Po wetlands, dominated by Kuenenia and Scalindua, contained the highest diversity of anammox bacteria followed by the shallow SCS (Scalindua zhenghei-I and S3), deep SCS (Scalindua zhenghei-I, S2 and S. arabica) and riparian sediments of Pearl River (Kuenenia and Brocadia). Salinity, inorganic nitrogen species [〖NH4〗^+, Σ(〖〖NO〗_2〗^_) + (〖〖NO〗_3〗^_)], and ratio of 〖NH4〗^+/Σ(〖〖NO〗_2〗^_) + (〖〖NO〗_3〗^_)] significantly affected the community compositions of anammox bacteria. Data collectively indicated that optimization of PCR primers enables unraveling new anammox bacterial phylotypes from the environments and current experimental procedures used in extraction of template DNA from samples seriously lower the diversity and composition of anammox bacteria retrieved.