1 octyne

Soil slurry incubations were performed to evaluate the contributions of AOA and AOB/comammox to the net nitrification potential of both soils. For the assays, fresh soil (4 g) and 30 ml of MilliQ water were weighed in 125 ml Wheaton bottles, and NH₄Cl was added to a final concentration of 1 mM. All bottles were sealed gas tight with 33 mm interlock butyl septa. To differentiate between the contribution of AOA and AOB/comammox to soil nitrification the AOB and comammox specific inhibitors 1-octyne (4 µM) and allyl-2-thiourea (ATU, 100 µM) (Sigma-Aldrich, St. Louis, MO, USA) treatments were included (Taylor et al. 2013 (link), 2015 (link)). The negative control, in which nitrification activity was completely inhibited, received 1 mM phenylacetylene (McCarty and Bremner 1986 (link)). Sealed bottles were shaken at 180 r/m at 23°C for 72 h. Each treatment included four replicate soil slurries and 1 ml subsamples were collected with a 1-ml syringe through the butyl septa daily during the incubation period to measure NO₃⁻ accumulation as described above. NO₂⁻ accumulation was also assessed but was below the limit of quantification of 15 µM in all samples at all timepoints. Soil net nitrification potential (µg N g⁻¹ soil day⁻¹) was calculated by subtracting the initial NO₃⁻ concentration from the accumulated NO₃⁻ concentration, normalized to soil weight and incubation time.

Phenylacetylene (98%) and propyne, 1-pentyne, 1-hexyne, 1-heptyne, and 1-octyne (C₃, C₅, C₆, C₇, and C₈ linear 1-alkynes, respectively, ≥97%) were obtained from Sigma-Aldrich. 1-Butyne was supplied by Apollo Gases Ltd. Acetylene was obtained from BOC, a member of the Linde Group. Protein concentrations were determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) as described by the manufacturer.