The biomass of an oxygen-limited autotrophic nitrification/denitrification (OLAND) biofilm reactor was preserved in various ways to find a storage method for both aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). Storage occurred at −20°C with and without glycerol as cryoprotectant and at 4 and 20°C with and without nitrate as redox buffer. After 2 and 5 months, reactivation of AerAOB and AnAOB was achieved with the biomass stored at 4°C with and without nitrate and at 20°C with nitrate. Moreover, the presence of the AerAOB and AnAOB was confirmed with fluorescent in situ hybridization (FISH). Preservation in a nitrate environment resulted in a lag phase for the AnAOB reactivation. The supplied nitrate was denitrified during storage, and a real-time polymerase chain reaction with nitrifying and denitrifying genes allowed to estimate that at least 1.0 to 6.0% of the OLAND biofilm consisted of denitrifiers. It was concluded that reactivation after long-term storage is possible and that preservation at 4°C without nitrate addition is the recommended storage technique. The possibility to store OLAND biomass will facilitate research on AnAOB and can overcome larger-scale start-up and inhibition problems of novel nitrogen processes involving AnAOB.
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This research was funded by a Ph.D. grant (aspirant) for Siegfried E. Vlaeminck from the Fund of Scientific Research-Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen). Part of the work also derives from project grant GOA 1205073 (2003–2008) of the Ministerie van de Vlaamse Gemeenschap, Bestuur Wetenschappelijk Onderzoek (Belgium).
The authors gratefully thank Greet Van de Velde for technical support, Pascal Boeckx for kind assistance with N2O analyses and Loïs Maignien, Kim Heylen, Peter Deschryver, Ilse Forrez, Lieven Wittebolle and the anonymous reviewers for critically reviewing this manuscript.
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Vlaeminck, S.E., Geets, J., Vervaeren, H. et al. Reactivation of aerobic and anaerobic ammonium oxidizers in OLAND biomass after long-term storage. Appl Microbiol Biotechnol 74, 1376–1384 (2007). https://doi.org/10.1007/s00253-006-0770-2