Anaerobic Ammonium-Oxidizing (Anammox) Bacteria and Associated Activity in Fixed-Film Biofilters of a Marine Recirculating Aquaculture System

Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems

Statistical analysis of 16S rRNA gene libraries.

<p>Simpson diversity index and Choa₁ nonparametric estimates were used to compare species diversity in the different regions of the GI tract. For Chao₁ phylotype richness estimations, OTUs were binned to genera.</p

Phylogenetic tree showing the relationship of sequences identified in the midgut clone library.

<p>The tree was constructed using near full length (>1200 bp) 16S rRNA sequences using Bayesian inference (ngen = 15 000 000; BI = 12 500). Known sequences sharing highest sequence similarity were included in the phylogeny. Branch labels represent posterior probabilities (* denote branches with ≥97% posterior probability).</p

Mixed diet clone library rarefaction curves with OTUs binned to genus.

<p>Rarefaction curves were calculated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048018#pone.0048018-Dhanasiri1" target="_blank">[36]</a>, allowing comparison of species richness across communities with different sample sizes by plotting the species richness as a function of the number of sequences.</p

Abundance of bacterial genera from the various GI tract regions of <i>P. nigrolineatus</i>.

<p>Relative abundance of bacterial genera identified by 16S rRNA gene cloning recovered from the various GI tract regions of <i>P. nigrolineatus</i>. OTUs were binned to genus according to their closest match in GenBank.</p