Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/5/2011
Publication Date: 9/14/2011
Citation: Rothrock Jr, M.J., Vanotti, M.B., Szogi, A.A., Gonzalez, M.G., Fujii, T. 2011. Long-term preservation of Anammox bacteria. Applied Microbiology and Biotechnology. 92:147-157. Interpretive Summary: ARS scientists have discovered a novel anammox bacteria isolate Candidatus “Brocadia caroliniensis” that oxidizes ammonia and releases di-nitrogen under anaerobic conditions. The novel anammox bacterium is useful for wastewater treatment applications. The bacterial isolate is capable of reactivation after lyophilization. Long-term preservation and successful reactivation was a requirement to deposit the novel bacterium in culture collections. The goal of this study was to develop a simple long-term preservation protocol to allow for the reactivation of this novel anammox bacterium. This is the first report on the successful reactivation of anammox biomass preserved via sub-zero freezing followed by lyophilization (freeze drying).
Technical Abstract: Deposit of useful microorganisms in culture collections requires long-term preservation and successful reactivation techniques. The goal of this study was to develop a simple preservation protocol for the long-term storage and reactivation of the anammox biomass. To achieve this, anammox biomass was frozen or lyophilized at two different freezing temperatures (-60 deg C and in liquid nitrogen (-200 deg C)) in skim milk media (with and without glycerol), and the reactivation of anammox activity was monitored after a 4 month storage period. Of the different preservation treatments tested, only anammox biomass preserved via freezing in liquid nitrogen followed by lyophilization in skim milk media without glycerol achieved stoichiometric ratios for the anammox reaction similar to the biomass in both the parent bioreactor and in the freshly-harvested control treatment. A freezing temperature of -60 deg C alone, or in conjunction with lyophilization, resulted in the partial recovery of the anammox bacteria, with an equal mixture of anammox and nitrifying bacteria in the reactivated biomass. To our knowledge, this is the first report of the successful reactivation of anammox biomass preserved via sub-zero freezing and/or lyophilization. The simple preservation protocol developed from this study could be beneficial to accelerate the integration of anammox-based processes into current treatment systems through a highly-efficient starting anammox biomass.