(ii) Growth experiments of Nitrosomonas sp. Is79 with heterotrophic bacteria and N. winogradskyi.

All growth experiments were conducted in AOB-MS medium with 1 mM NH₄⁺. Cultures were inoculated with 1% (vol/vol) late logarithmic phase Nitrosomonas sp. Is79 and one or more of the following: 0.2% (vol/vol) R2A medium, 0.2% (vol/vol) culture of heterotrophic bacteria cultivated in R2A, or 1% (vol/vol) N. winogradskyi culture. In addition, we used the enrichment culture G5-7. Heterotrophic bacterial cultures were precultured in liquid R2A for 2 days prior to inoculation. All 10 heterotrophic isolates were combined into a mixed culture, which was added to Nitrosomonas sp. Is79 to resemble as well as possible the original G5-7 heterotrophic population. When the cultures had consumed the NH₄⁺, they were transferred to fresh AOB-MS medium (1% vol/vol). This transfer was repeated once more, resulting in three successive growth cycles. Samples were taken regularly during the third growth cycle, stored at −20°C, and later used to determine NO₂⁻/NO₃⁻ concentrations. Following the third growth cycle, co-cultures were plated on R2A agar. Colony PCR and sequencing were used to confirm the identity of the heterotrophic bacteria.