Here we examined the impact of a commonly employed method used to measure nitrogen fixation, the acetylene reduction assay (ARA), on a marine sediment community. Historically, the ARA technique has been broadly employed for its ease of use, in spite of numerous known artifacts. To gauge the severity of these effects in a natural environment, we employed high-throughput 16S rRNA gene sequencing to detect differences in acetylene-treated sediments vs. non-treated control sediments after a 7 h incubation. Within this short time period, significant differences were seen across all activity of microbes identified in the sediment, implying that the changes induced by acetylene occur quickly. The results have important implications for our understanding of marine nitrogen budgets. Moreover, because the ARA technique has been widely used in terrestrial and freshwater habitats, these results may be applicable to other ecosystems.

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24Water column nitrification is a key process in the nitrogen cycle as it links reduced and 25 oxidized forms of nitrogen and also provides the substrate (nitrate) needed for reactive nitrogen 26 removal by denitrification. We measured potential water column ammonium and nitrite 27 oxidation rates at four sites along an estuary to continental shelf gradient over two summers. In 28 most cases, nitrite oxidation rates outpaced ammonium oxidation rates. Overall, ammonium and 29 nitrite oxidation rates were higher outside of the estuary, and this trend was primarily driven by 30 higher oxidation rates in deeper waters. Additionally, both ammonium and nitrite oxidation rates 31 were impacted by different in situ variables. Ammonium oxidation rates throughout the water 32 column as a whole were most positively correlated to depth and salinity and negatively 33 correlated to dissolved oxygen and light. In contrast, nitrite oxidation rates throughout the water 34 column were negatively correlated with light and pH. Multivariate regression analysis revealed 35 that while both surface (< 20m) and deep (> 20m) ammonium oxidation rates were most strongly 36 predicted by depth and light, surface rates were also regulated by salinity and deep rates by 37 temperature. Surface (< 20m) nitrite oxidation rates were best explained by [H + ] (i.e. pH) alone, 38 while salinity, [H + ], temperature, and depth all played a role in predicting deep (> 20m) nitrite 39 oxidation rates. These results support the growing body of evidence that ammonium oxidation 40 and nitrite oxidation are not always coupled, should be measured separately, and are influenced 41 by different environmental conditions. 42 43 44 45 46 Ammonium oxidizers compete with phytoplankton and heterotrophic bacteria for substrate 62

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