Construction of a Consortium Comprising Ammonia-oxidizing Bacteria and Denitrifying Bacteria Isolated from Marine Sediment

Abstract

A unique consortium of ammonia-oxidizing bacteria (AOB) and denitrifying bacteria was obtained via a long-term, 3-step cultivation of isolates from organically-enriched marine sediment. We developed this microbial consortium for possible applications in the remediation of degraded habitats in closed aquaculture or other aquatic environments via microbial degradation. Analysis of media components found definitive evidence of nitrogen removal via the coupling of ammonia-oxidation and denitrification. The phylogenetic diversity of the consortium was investigated by performing polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) that targeted the 16S rRNA gene, and the functional genes involved in ammonia-oxidation (amoA) and denitrification (nirS, nirK, norB and nosZ). Consequently, no significant divergence was observed, and thus it is suggested microbial populations were selected via a long-term, 3-step incubation process. All of the 16S rRNA clones fell into three phylogenetic groups, namely, gamma-proteobacteria, Actinobacteria and Flavobacteria. For almost half of the clones, the closest relatives in the database were identified as Alcanivorax spp. and these clones were present at all cultivation stages. The presence of these species as the dominant clones is significant since these bacterial species are known to reduce nitrate to nitrite. Accordingly, their abundance in our microbial consortium may have been responsible for the observed stepwise denitrification. All sequences of the amoA gene were identified to be Nitrosomonas lineage. Half of the nirS clones were identified to be from one major group of well-known denitrifying bacteria, Pseudomonas sp. Furthermore, 70% of the nirK clones were closely related to the nirK sequences of uncultured bacterial clones isolated from arable soil. The qnorB clones consisted of clusters exclusively, and formed a distinct cluster from the novel sequences of cultivated species. The nosZ clones also were not found in any of the closest relatives in the database including the uncultured bacterium from marine sediment. The unique clones obtained from the functional genes were related to each denitrification step.