Relevance and Diversity of <i>Nitrospira</i> Populations in Biofilters of Brackish RAS

<div><p>Lithoautotrophic nitrite-oxidizing bacterial populations from moving-bed biofilters of brackish recirculation aquaculture systems (RAS; shrimp and barramundi) were tested for their metabolic activity and phylogenetic diversity. Samples from the biofilters were labeled with ¹³C-bicarbonate and supplemented with nitrite at concentrations of 0.3, 3 and 10 mM, and incubated at 17 and 28°C, respectively. The biofilm material was analyzed by fatty acid methyl ester - stable isotope probing (FAME-SIP). High portions of up to 45% of <i>Nitrospira</i>-related labeled lipid markers were found confirming that <i>Nitrospira</i> is the major autotrophic nitrite oxidizer in these brackish systems with high nitrogen loads. Other nitrite-oxidizing bacteria such as <i>Nitrobacter</i> or <i>Nitrotoga</i> were functionally not relevant in the investigated biofilters. <i>Nitrospira</i>-related 16S rRNA gene sequences were obtained from the samples with 10 mM nitrite and analyzed by a cloning approach. Sequence studies revealed four different phylogenetic clusters within the marine sublineage IV of <i>Nitrospira</i>, though most sequences clustered with the type strain of <i>Nitrospira marina</i> and with a strain isolated from a marine RAS. Three lipids dominated the whole fatty acid profiles of nitrite-oxidizing marine and brackish enrichments of <i>Nitrospira</i> sublineage IV organisms. The membranes included two marker lipids (16∶1 <i>cis</i>7 and 16∶1 <i>cis</i>11) combined with the non-specific acid 16∶0 as major compounds and confirmed these marker lipids as characteristic for sublineage IV species. The predominant labeling of these characteristic fatty acids and the phylogenetic sequence analyses of the marine <i>Nitrospira</i> sublineage IV identified organisms of this sublineage as main autotrophic nitrite-oxidizers in the investigated brackish biofilter systems.</p>