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Typically, nitrification is a two-stage microbial process and is key in wastewater treatment and nutrient recovery from waste streams. Changes in salinity represent a major stress factor that can trigger response mechanisms, impacting the activity and the physiology of bacteria. Despite its pivotal biotechnological role, little information is available on the specific response of nitrifying bacteria to varying levels of salinity. In this study, synthetic communities of ammonia-oxidizing bacteria (AOB  Nitrosomonas europaea  and/or  Nitrosomonas ureae ) and nitrite-oxidizing bacteria (NOB  Nitrobacter winogradskyi  and/or  Nitrobacter vulgaris ) were tested at 5, 10, and 30 mS cm -1  by adding sodium chloride to the mineral medium (0, 40, and 200 mM NaCl, respectively). Ammonia oxidation activity was less affected by salinity than nitrite oxidation. AOB, on their own or in combination with NOB, showed no significant difference in the ammonia oxidation rate among the three conditions. However,  N. winogradskyi  improved the absolute ammonia oxidation rate of both  N. europaea  and  N. ureae .  N. winogradskyi ’s nitrite oxidation rate decreased to 42% residual activity upon exposure to 30 mS cm -1 , also showing a similar behavior when tested with  Nitrosomonas  spp. The nitrite oxidation rate of  N. vulgaris , as a single species, was not affected when adding sodium chloride up to 30 mS cm -1 , however, its activity was completely inhibited when combined with  Nitrosomonas  spp. in the presence of ammonium/ammonia. The proteomic analysis of a co-culture of  N. europaea  and  N. winogradskyi  revealed the production of osmolytes, regulation of cell permeability and an oxidative stress response in  N. europaea  and an oxidative stress response in  N. winogradskyi , as a result of increasing the salt concentration from 5 to 30 mS cm -1 . A specific metabolic response observed in  N. europaea  suggests the role of carbon metabolism in the production of reducing power, possibly to meet the energy demands of the stress response mechanisms, induced by high salinity. For the first time, metabolic modifications and response mechanisms caused by the exposure to salinity were described, serving as a tool toward controllability and predictability of nitrifying systems exposed to salt fluctuations.

Metabolic and Proteomic Responses to Salinity in Synthetic Nitrifying Communities of Nitrosomonas spp. and Nitrobacter spp.