Rapid and simple cryopreservation of anaerobic ammonium-oxidizing bacteria

Abstract

A quick and simple protocol for long-term cryopreservation of anaerobic ammonium-oxidizing bacteria (anammox bacteria) was developed. After 29 weeks of preservation at -80°C, activity recovery for all tested cultures under at least one of the applied sets of preservation conditions was observed. Growth recovery was also demonstrated for a single-cell culture of "Candidatus Kuenenia stuttgartiensis."

Fig 1

Percent recovery of specific activity of anammox biomass in batch tests after preservation for 29 weeks at −80°C under the indicated storage conditions. Specific anammox activity was based on ²⁹N-N₂ production from ¹⁵N-nitrite and unlabeled ammonium. Preservation experiments and batch tests were performed in duplicate (error bars represent variations in activity between individual cryopreserved tubes). No variation in activity was observed for aggregated “Candidatus Kuenenia stuttgartiensis” and single-cell “Candidatus Scalindua” sp. without CPA.

Fig 2

Resuscitation of preserved planktonic cells of “Candidatus Kuenenia stuttgartiensis.” The two-liter bioreactor was operated as a membrane bioreactor (MBR) as described elsewhere (8, 24). Open circles, optical density; closed circles, nitrogen removal.