Stable isotope probing and dynamic loading experiments provide insight into the ecophysiology of novel ammonia oxidizers in rapid gravity sand filters

Nitrification is often the dominant microbial process in rapid gravity sand filters (RSF), used to treat aerated groundwater to produce drinking water. RSFs harbor diverse microbial communities including a range of ammonia oxidizing clades; Betaproteobacteria (Nitrosomonas, Nitrosospira), Archaea, diverse potentially ammonia oxidizing heterotrophs and abundant Nitrospira spp., recently shown to comprise both canonical nitrite oxidizing as well as complete ammonium oxidizing (comammox) types. We examined the contributions of the different ammonia oxidizers to in situ ammonia oxidation, and aimed to elucidate the differences in ecophysiology between the ammonia oxidizing clades that enable them to co-exist in this unique environment. Experiments were conducted using sand columns designed and operated to mimic the conditions in the full-scale parent RSF. RNA and DNA stable isotope probing based on 13C-bicarbonate incorporation during continuous feeding with either ammonium or nitrite as sole energy source implicated Nitrospira spp. and certain ‘heterotrophic’ bacteria in addition to Nitrosomonas spp. in autotrophy during ammonium oxidation in RSFs. Further experimentation aimed to elucidate the ecophysiology of each ammonia oxidizing clade in RSFs, in particular comammox Nitrospira for which little is currently known. Columns were fed with RSF effluent spiked with various concentrations of ammonium ranging from 0.1- 5.0 mg/L delivered at different loading rates to examine the effects of both ammonium loading and oxygen limitation on ammonia oxidizers. Our observations indicate that the native conditions in the RSF used in this study foster the enrichment of comammox Nitrospira, which provides a preliminary step in the description of their ecophysiology.