Species level enrichment of AnAOB and associated growth morphology under the effect of key metabolites

Nineteen anaerobic ammonium oxidizing bacteria (AnAOB) species have been identified, yet the environmental factors that select for each species within a specialized ecological niche have not been described. We enriched AnAOB from a single inoculum under standard enrichment conditions (reactor R1) with stepwise increase in nitrite and ammonia concentration, nitric oxide (NO) supplementation (reactor R2), or with complex organic carbon using wastewater collected from mainstream wastewater treatment facility (reactor R3). AnAOB were enriched up to 80%, 90% and 50% relative abundance in R1, R2 and R3 respectively. Candidatus Brocadia caroliniensis predominated in all reactors, but a shift towards Ca. Brocadia sinica was consistently observed with increasing ammonium and nitrite concentrations beyond 270 mg NH4-N L-1 and 340 mg NO2-N L-1, respectively. In the presence of NO, growth of heterotrophs were inhibited, and Ca. Jettenia could coexist with Ca. B. caroliniensis before diminishing when nitrite increased to 160 mg NO2-N L-1. In contrast, supplementation of organic carbon led to the emergence of heterotrophic communities that coevolved with Ca. B. caroliniensis. Ca. B. caroliniensis and Ca. Jettenia preferentially form biofilms on reactor surfaces, whereas Ca. Brocadia sinica forms granules in suspension. Our results thus indicate that multiple AnAOB species co-exist and occupy sub-niches in anaerobic ammonium oxidation reactors, that the dominant population can be reversibly shifted by, for example, changing the nitrogen load (i.e. high nitrite concentration favors Ca. Brocadia caroliniensis), and that speciation has implications for wastewater process design, with the optimum cell immobilization strategy (i.e. carriers vs granules) dependent on which species dominates.