Cultivation of ammonia-oxidising archaea on solid medium

Abstract

Ammonia-oxidising archaea (AOA) are environmentally important microorganisms involved in the biogeochemical cycling of nitrogen. Routine cultivation of AOA is exclusively performed in liquid cultures and reports on their growth on solid medium are scarce. The ability to grow AOA on solid medium would be beneficial for not only the purification of enrichment cultures but also for developing genetic tools. The aim of this study was to develop a reliable method for growing individual colonies from AOA cultures on solid medium. Three phylogenetically distinct AOA strains were tested: 'Candidatus Nitrosocosmicus franklandus C13', Nitrososphaera viennensis EN76 and 'Candidatus Nitrosotalea sinensis Nd2'. Of the gelling agents tested, agar and Bacto-agar severely inhibited growth of all three strains. In contrast, both 'Ca. N. franklandus C13' and N. viennensis EN76 tolerated Phytagel™ while the acidophilic 'Ca. N. sinensis Nd2' was completely inhibited. Based on these observations, we developed a Liquid-Solid (LS) method that involves immobilising cells in Phytagel™ and overlaying with liquid medium. This approach resulted in the development of visible distinct colonies from 'Ca. N. franklandus C13' and N. viennensis EN76 cultures and lays the groundwork for the genetic manipulation of this group of microorganisms.

Keywords: Phytagel; ammonia-oxidising archaea; colonies; cultivation; solid medium; solidifying agent.

Figure 1.

Effects of different solidifying agents on the growth of ‘Ca. N. franklandus C13’: (A) Phytagel™ and agarose (B) agar and Bacto-agar (C) Noble-agar and (D) Phytagel™ and Bacto-agar (50:50) mixture. The control cultures were grown in the absence of a solidifying agent. Nitrite concentrations plotted represent the average of three replicate cultures. Error bars are standard errors of the means and may be smaller than the size of the symbol.

Figure 2.

‘Ca. N.  franklandus C13’ colonies developing in Phytagel™. Colonies are grown and maintained in 100 ml Duran glass bottles, and measure <1 mm in diameter.

Figure 3.

Fluorescent micrographs of cells growing with the Phytagel^TM. Cells growing within Phytagel^TM (left column) and cells from a pure liquid culture of ‘Ca. N. franklandus C13’ as a control (right column). Cells from a pure culture of Nitrosomonas europaea ATCC 19178 were used as a control (not shown). Cells are stained with FISH probes for archaea (ARCH-915, violet), bacteria (EUB338 mix, green) and DAPI (blue). Images were viewed at 1000 X magnification.

Figure 4.

Transmission electron microscopy of cells growing within Phytagel^TM (left column) and cells from a pure liquid batch grown culture of ‘Ca. N. franklandus C13’ (right column).

Figure 5.

Fluorescent micrographs of cells grown from a colony of ‘Ca. N.  franklandus C13’. Cells were stained with FISH probes for archaea (Arch-915, violet), bacteria (EUB338 mix, green) and DAPI (blue). Images were viewed at 1000 X magnification. The graph shows nitrite accumulation from the subcultured colonies. Nitrite concentrations were measured in triplicate and error bars are standard errors of the means and may be smaller than the size of the symbol.