Alcohols as inhibitors of ammonia oxidizing archaea and bacteria

Ammonia oxidizers are key players in the global nitrogen cycle and are responsible for the oxidation of ammonia to nitrite, which is further oxidized to nitrate by other microorganisms. Their activity can lead to adverse effects on some human-impacted environments, including water pollution through leaching of nitrate and emissions of the greenhouse gas nitrous oxide (N2O). Ammonia monooxygenase (AMO) is the key enzyme in microbial ammonia oxidation and shared by all groups of aerobic ammonia oxidizers. The AMO has not been purified in an active form, and much of what is known about its potential structure and function comes from studies on its interactions with inhibitors. The archaeal AMO is less well studied as ammonia oxidizing archaea were discovered much more recently than their bacterial counterparts. The inhibition of ammonia oxidation by aliphatic alcohols (C1-C8) using the model terrestrial ammonia oxidizing archaeon 'Candidatus Nitrosocosmicus franklandus' C13 and the ammonia oxidizing bacterium Nitrosomonas europaea was examined in order to expand knowledge about the range of inhibitors of ammonia oxidizers. Methanol was the most potent specific inhibitor of the AMO in both ammonia oxidizers, with half-maximal inhibitory concentrations (IC50) of 0.19 and 0.31 mM, respectively. The inhibition was AMO-specific in 'Ca. N. franklandus' C13 in the presence of C1-C2 alcohols, and in N. europaea in the presence of C1-C3 alcohols. Higher chain-length alcohols caused non-specific inhibition and also inhibited hydroxylamine oxidation. Ethanol was tolerated by 'Ca. N. franklandus' C13 at a higher threshold concentration than other chain-length alcohols, with 80 mM ethanol being required for complete inhibition of ammonia oxidation. Short-chain alcohols specifically inhibit the archaeal and bacterial ammonia monooxygenase, a key enzyme in nitrification, whereas higher chain-length alcohols are non-specific inhibitors of the model ammonia oxidizers 'Candidatus Nitrosocosmicus franklandus' and Nitrosomonas europaea.