Cadmium effects on net N2O production by the deep-sea isolate Shewanella loihica PV-4

Abstract

Deep-sea mining may lead to the release of high concentrations of metals into the surrounding seabed, which can disturb important ecosystem functions provided by microbial communities. Among these, the production of N2O and its reduction to N2 is of great relevance since N2O is an important greenhouse gas. Metal impacts on net N2O production by deep-sea bacteria are, however, currently unexplored. Here, we evaluated the effects of cadmium (Cd) on net N2O production by a deep-sea isolate, Shewanella loihica PV-4. We performed a series of Cd exposure incubations in oxic conditions and determined N2O fluxes during induced anoxic conditions, as well as the relative expression of the nitrite reductase gene (nirK), preceding N2O production, and N2O reductase gene (nosZ), responsible for N2O reduction. Net N2O production by S. loihica PV-4 exposed to Cd was strongly inhibited when compared to the control treatment (no metal). Both nirK and nosZ gene expression were inhibited in reactors with Cd, but nirK inhibition was stronger, supporting the lower net N2O production observed with Cd. The Cd inhibition of net N2O production observed in this study poses the question whether other deep-sea bacteria would undergo the same effects. Future studies should address this question as well as its applicability to complex communities and other physicochemical conditions, which remain to be evaluated.

Keywords: deep-sea; denitrification; gene expression; metal contamination; mining; nitrous oxide.

Figure 1.

Optical density at 600 nm in the bioreactors during the exposure experiments with S. loihica PV-4 in oxic conditions. Each point represents the OD600 measurement from a single reactor at a given time point. The shaded area represents the 95% confidence interval of the locally estimated scatterplot smoothing (solid line). The dotted vertical lines represent the period when anoxia was induced for N₂O and gene expression measurements.

Figure 2.

Nitrous oxide amount in the bioreactors during the exposure experiments with S. loihica PV-4. Each point represents the N₂O measurement from a single reactor at a given time point during the anoxic period. The solid line represents the linear regression per reactor and the shaded area represents the 95% confidence interval. Calculated slopes and fluxes are presented in Table S4.

Figure 3.

Relative expression of nirK and nosZ genes in S. loihica PV-4 grown with Cd at 0.01 mM (red) and without the metal (blue). Relative expression was estimated by normalizing gene transcript copy numbers by the average of recA and rpoB transcript copy numbers in each sample. The boxes represent the first and third quartiles, with median value bisecting each box. The whiskers extend to the largest/smallest value, excluding outliers (data beyond 1.5 x interquartile range).