Metagenome of the Microbial Community of Anammox Granules in a Nitritation/Anammox Wastewater Treatment System

ABSTRACT

We sequenced the metagenome of a granular sludge in a nitritation/anammox bioreactor used for the treatment of ammonium-rich wastewater. Proteobacteria, Planctomycetes, Bacteroidetes, Chloroflexi, Ignavibacteriae, and Acidobacteria were the predominant phyla in the studied bioreactor. Binning of contigs yielded a near-complete genome of the dominant anammox bacterium assigned to the candidate genus Brocadia.

GENOME ANNOUNCEMENT

Partial nitritation/anammox (N/A) technologies are promising for the treatment of ammonium-rich wastewater. However, the anammox bacteria are characterized by very low growth rates, which limits the scale-up of the anammox process, while the absence of pure cultures of these bacteria complicates an investigation of their genetic and physiological characteristics. Moreover, anammox bacteria are highly sensitive to a number of environmental factors (temperature, pH, etc.) that cause instability of the anammox process (1). The implementation of N/A technologies is also impeded by the complexity of microbial communities of bioreactors comprising not only anammox bacteria, but also a number of other physiological groups, such as fermentative bacteria, nitrifiers, denitrifiers, methanogens, and others (2–4).

In this study, a 100-liter hybrid completely mixed constant-flow N/A reactor with suspended and immobilized activated sludge operating at a Moscow wastewater treatment plant (5) was analyzed. The 50-g sample of firmly attached sludge, representing anammox granules, was isolated on day 120 of the reactor operation when it stably removed about 81% of the total nitrogen with an influent ammonium concentration of 270 to 300 mg NH₄-N/liter (5). Metagenomic DNA was isolated using the PowerSoil DNA isolation kit (Mo Bio, Inc. Laboratories, Carlsbad, CA) and sequenced with a Roche Genome Sequencer FLX (GS FLX) using the Titanium XL+ protocol for a shotgun library and paired-end library, with an average size of 8 kbp. About 312 Mb and 61 Mb of sequences were obtained for the shotgun and the paired-end libraries, respectively. To characterize the taxonomic composition of the microbial community, we identified raw pyrosequencing reads representing 16S rRNA genes using a BLASTN search against the SILVA SEED database (6). The RDP Classifier (7) was used for the taxonomic classification of these sequences. Proteobacteria, mostly represented by the class Betaproteobacteria, was the most abundant lineage (26.8% of all reads classified at the phylum level), followed by Planctomycetes (20.0%), Bacteroidetes (14%), Chloroflexi (11%), Ignavibacteriae (6%), and Acidobacteria (5%). The presence of these bacterial lineages reflects the complexity of microbial processes in the studied N/A bioreactor performed by anammox Planctomycetes, fermentative bacteria, and denitrifiers. Archaea accounted for only 1% of 16S rRNA reads and were represented by methanogens of the order Methanobacteriales.

The reads were de novo assembled into contigs using the Newbler Assembler version 2.9 (454 Life Sciences, Branford, CT). A total of 7,798 contigs with an N₅₀ contig size of 2,137 bp were obtained. Binning of contigs using the CONCOCT tool (8) allowed us to obtain a near-complete composite genome of the dominant anammox bacterium. A BLASTN search against the NCBI database revealed that 16S rRNA sequence from this genome is 97% identical to that of Candidatus “Brocadia caroliniensis” (2). The obtained genomic data will be useful for further studies of microbial processes in wastewater treatment systems and genome-based analysis of anammox bacteria.

Accession number(s).

The sequences obtained in this project have been deposited in the NCBI Sequence Read Archive under the accession numbers SRX3153926, SRX3153925, and SRX3153924.