Rapid cultivation of free-living planktonic anammox cells

Despite that anaerobic ammonium oxidizing (anammox) bacteria are key players in the global nitrogen cycle, no pure cultures are still available. Planktonic cell culture with high purity is, therefore, essential for physiological and biochemical studies of anammox bacteria. However, development of such planktonic cell cultures requires an enormous amount of time and effort. Here we developed a novel rapid method for cultivating free-living planktonic anammox cells. First, anammox granules were physically dispersed, immobilized in 6% polyvinyl alcohol-4% sodium alginate (PVA-SA) gel beads, and then pre-cultured in an up-flow column reactor. Anammox bacteria grew rapidly as loosely flocculated micro-clusters in the gel beads. After 18 days of pre-cultivation, mature gel beads were harvested, physically dispersed by vortex and inoculated into a membrane bioreactor (MBR). The MBR was then continuously operated at a low nitrogen loading rate (<0.9 kg-TN m^-3 d^-1). After 17 days of operation, active free-living planktonic anammox cells with purity >95% were successfully developed in the MBR. Total culture time (gel beads and MBR) to accomplish free-living planktonic anammox cells was only 35 days, which was significantly shorter than the previous reports. This new cultivation technique could greatly facilitate various microbial, physiological and biochemical studies of anammox bacteria.